Volledig proefschrift (2 134 kB)

In vitro characterization of the human biotransformation

of marine derived anti-cancer drugs

Esther F.A. Brandon

CIP-GEGEVENS KONINKLIJKE BIBLIOTHEEK, DEN HAAG Brandon, Esther Fleur Annette In vitro characterization of the human biotransformation of marine derived anti-cancer drugs 2004 Dissertation Utrecht University, Faculty of Pharmaceutical Sciences (with summary in Dutch) ISBN 90-393-3714-4 Printed by Ponsen en Looijen B.V., Wageningen, The Netherlands Cover design by Conny Groenendijk, Utrecht University, Utrecht, The Netherlands The cover images represent Aplidium albicans, the source of the novel marine anti-cancer drug Aplidine (figures donated by PharmaMar SA, Tres Cantos, Madrid, Spain).

In vitro characterization of the human biotransformation of marine derived anti-cancer drugs

In vitro opheldering van de menselijke biotransformatie van uit zeeorganismen verkregen antikankergeneesmiddelen

(met een samenvatting in het Nederlands)

Proefschrift

Ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de Rector Magnificus, Prof. Dr. W.H. Gispen,

ingevolge het besluit van het College voor Promoties in het openbaar te verdedigen op

woensdag 16 juni 2004 des middags te 12.45 uur

door

Esther Fleur Annette Brandon geboren op 3 juli 1977, te Leiderdorp

Promotoren: Prof. Dr. J.H.M. Schellens

Department of Biomedical Analysis - Division of Clinical Drug Toxicology Faculty of Pharmaceutical Sciences, Utrecht University Utrecht, The Netherlands

Department of Medical Oncology and Experimental Therapy Antoni van Leeuwenhoek Hospital - The Netherlands Cancer Institute Amsterdam, The Netherlands

Prof. Dr. J.H. Beijnen

Department of Biomedical Analysis - Division of Analytical Drug Toxicology Faculty of Pharmaceutical Sciences, Utrecht University Utrecht, The Netherlands

Department of Pharmacy and Pharmacology Slotervaart Hospital - The Netherlands Cancer Institute Amsterdam, The Netherlands

Co-promotor: Dr. Ir. I. Meijerman

Department of Biomedical Analysis - Division of Clinical Drug Toxicology Faculty of Pharmaceutical Sciences, Utrecht University Utrecht, The Netherlands

The research described in this thesis was carried out at the Department of Biomedical Analysis, Division of Clinical and Analytical Drug Toxicology of the Faculty of Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands. Financial support in printing expenses was kindly provided by: Faculty of Pharmaceutical Sciences, Utrecht University Department of Biomedical Analysis, Division of Drug Toxicology BD Biosciences - Gentest, Erembodegem, Belgium

Voor Patrick, mijn ouders en mijn oma die de afronding helaas niet meer heeft mogen meemaken

Table of contents

List of abbreviations 1

General introduction 3

Chapter 1 In vitro test methods in human hepatic drug biotransformation research:

pros and cons

9

Chapter 2

In vitro characterization of the biotransformation of thiocoraline, a novel marine anti-cancer drug

33

Chapter 3

In vitro characterization of the human biotransformation pathways of aplidine, a novel marine anti-cancer drug

47

Chapter 4

Structure elucidation of aplidine metabolites formed in vitro by human liver microsomes using triple quadrupole mass spectrometry

67

Chapter 5

In vitro characterization of the human biotransformation and CYP reaction phenotype of ET-743 (Trabectidin®, Yondelis®), a novel marine anti-cancer drug

81

Chapter 6

In vitro cytotoxicity of ET-743 (Trabectidin®, Yondelis®), a marine anti-cancer drug, in the Hep G2 cell line; influence of cytochrome P450 and phase II inhibition

and cytochrome P450 induction

99

Chapter 7

Validation of in vitro cell models used in drug metabolism studies; genotyping of cytochrome P450 and phase II enzyme polymorphisms in

the human hepatoma (Hep G2) and ovarian carcinoma (IGROV-1) cell lines

115

General discussion and conclusions 137

Summary 147

Samenvatting 149

List of publications 153

Curriculum Vitae 155

Dankwoord 157

1

List of abbreviations A aplidine Acetyl CoA acetyl coenzyme A ADME adsorption disposition metabolism elimination BNPP or B bis(p-nitrophenyl)phosphate bp base pairs C coumarin CID collision-induced dissociation CL clearance CLint intrinsic clearance Cmax maximum concentration CYP cytochrome P450 CZ chlorzoxazone DM dexamethasone DMSO dimethyl sulfoxide DNP 2,6-dichloro-4-nitrophenol DOC dynamic organ culture DNA deoxyribonucleic acid DTT dithiothreitol ET-743 or ET ecteinascidin 743 F furafylline FAM phosphoramidite FCS fetal calf serum fu free fraction (unbound fraction) GAPDH glyceraldehydes-3’-phosphatedehydrogenase GST glutathione-S-transferase GT glutathione h hour Hip hydroxyisovalerylpropionyl HPLC high pressure liquid chromatography HLM human liver microsomes IC lethal concentration in cell culture IC5 concentration of compound giving 50 % cell death IC50 concentration of compound giving 5 % cell death Ist isostatine i.v. intravenous KC ketoconazole Km Michaelis-Menten constant Km(app) apparent Michaelis-Menten constant LDH lactate dehydrogenase Leu leucine Me methyl min or ’ minute(s) MGB major groove binder MLM mouse liver microsomes MR metyrapone MS mass spectrometry NAT N-acetyltransferase NADP or NADPH nicotinamide adenine dinucleotide phosphate

List of abbreviations

2

NRS NADPH regenerating system 17-ODA or O 17-octadecynoic acid p.a. pro analysis PA proadifen PAPS adenosine-3’-phosphate-5’-phosphosulphate PB piperonyl butoxide PBS phosphate buffered saline PCR polymerase chain reaction PMSF or P phenylmethylsulfonyl fluoride Pro proline PT phenanthrene Pyr pyruvoyl RAF relative activity factor ref. reference RFLP restiction fragment length polymorphism RN ritonavir RNA ribonucleic acid mRNA messenger RNA RT-PCR reverse transcriptase PCR s or ’’ second(s) SD standard deviation SNP single nucleotide polymorphism SP sulfaphenazole SRB sulforhodamine B SULT sulfotransferase SXR steroid and xenobiotic receptor Thr threonine Tyr tyrosine UDPGA uridine diphosphoglucuronic acid UGT uridine diphosphoglucuronosyl transferase UV ultraviolet Vmax maximum biotransformation rate WF warfarin

3

General introduction

Cancer is the second cause of death in the Netherlands. The treatment of cancer has been improved, however, overall the chances for patients with advanced cancer are still poor. Therefore, new anti-cancer drugs are very much needed to improve treatment outcome of patients and decrease treatment related side effects. Many of the active cytotoxic agents originate from natural resources, mainly plants and terrestrial microorganisms. The search for marine derived products started 50 years ago and over the past few years, about 3000 new compounds from various marine sources, e.g. sponge, mollusk, and sea hare, have been described as anti-cancer agent. Some have already entered clinical trials [1-3]. In this study, three novel marine derived anti-cancer drugs were investigated, namely: thiocoraline, aplidine (Aplidin®), and ET-743 (Yondelis®, Trabectedin®).

Thiocoraline, a thiodepsipeptide, is derived from the actinomycete Micromonospora marina living in the Mozambique strait. Thiocoraline causes an arrest in the G1 phase of the cell cycle and a decrease in the progression towards the G2 phase. The mode of action is inhibition of DNA polymerase α, which results in the inhibition of DNA elongation, cell cycle progression, and clonogenicity [4]. However, it does not inhibit DNA topoisomerase I or II, nor does it induce DNA breakage [4, 5]. Thiocoraline has shown anti-proliferative activity against several cancer cell lines in the in vitro screening program of the National Cancer Institute [4] and against human carcinoma xenografts in vivo [5]. Thiocoraline is about to enter phase I clinical trials.

Aplidine (Aplidin®), a cyclic depsipeptide, is derived from the Mediterranean tunicate Aplidium albicans (figures on thesis cover) [6]. It has been shown to inhibit the DNA synthesis, with modest to no effect on RNA synthesis [6]. Furthermore, aplidine is a potent inhibitor of protein synthesis in tumor cells [7]. Its mode of action is also believed to involve down-regulation of the flt-1 receptor for the vascular endothelial growth factor (involved in angiogenesis and tumor vascularization), induction of apoptosis in cancer cells, and arrest of the cell cycle in the G1 phase [8-11]. Aplidine demonstrated broad in vitro and in vivo activity against various tumor types [8, 12]. Phase I clinical trials with aplidine have been completed in Europe and Canada with more than 200 patients and the activity and safety of various i.v. infusion schedules have been assessed [11]. Aplidine treatment showed clinical benefit in patients with various tumor types, including renal, bronchial carcinoid and medullary thyroid carcinoma’s, and with non-Hodgkin’s lymphoma [13]. Phase II studies assessing the dose-response relationship in renal and colorectal cancer are in progress and additional phase II studies are planned [11].

Ecteinascidin-743 (Yondelis®, Trabectedin®) is a tetrahydroisoquinoline and is like aplidine derived from a tunicate, in this case the Caribbean tunicate Ecteinascidia turbinata. The mode of action of Ecteinascidin-743 (ET-743) has not been completely elucidated, but several mechanisms have been proposed. It is believed to involve binding to the minor groove of the DNA, interactions with transcription factors, and DNA binding proteins, disorganization of the microtubule network, inhibition of topoisomerase I, pertubation of the cell cycle, and interference with DNA repair mechanisms [14, 15]. ET-743 is also an inhibitor and antagonist of the steroid and xenobiotic receptor (SXR), involved in the induction of several cytochrome P450s (CYP), phase II enzymes, and drug transporters [16, 17]. However, ET-743 inhibits only activated transcription of these enzymes and transporters and not the constitutive transcription [18]. Because ET-743 is a transcription interfering agent, it could therefore be useful to treat multi-drug resistant tumors that have induced transcription of drug transporters [18-23].


4

ET-743 exhibited in vitro activity at nanomolar concentrations against various solid tumor cell lines [14] and it appears effective against human xenografts in vivo [24, 25]. Multiple infusion schedules were investigated in phase I clinical trials and clinical studies investigating the effects of combination therapy of ET-743 with other anti-cancer drugs are currently in progress or preparation [14]. Thus far, phase II clinical trials with ET-743 showed activity against soft-tissue sarcomas and breast, endometrial, and ovarian cancer [11, 26-30].

Although, aplidine and ET-743 are already being tested in clinical trials and thiocoraline will enter phase I trials in the near future, little is known about the biotransformation in humans. Knowledge about biotransformation is important in order to interpret the pharmacological properties of these compounds observed in clinical trials and to predict possible drug-drug interactions with other (anti-cancer) drugs.

Biotransformation pathways can be divided into two categories: phase I (oxidation, reduction, and hydrolysis) and phase II (conjugation) reactions (figure 1) [31, 33]. The CYP superfamily is the largest and most important group of the phase I enzymes, to which also esterases, alcohol dehydrogenases and other enzyme systems belong [34]. A classification system has been developed in which each cytochrome P450 enzyme is assigned a family, subfamily and number based on their similarity in DNA sequence. The CYP3A, CYP2D, and CYP2C subfamilies are of great importance and are responsible for respectively 50%, 25%, and 20% of the biotransformation of all drugs [35, 36]. Furthermore, the CYP3A subfamily is also the most abundant CYP enzyme, accounting for 40-60% of the hepatic CYP enzymes. The majority of the metabolites formed by biotransformation are inactive, but sometimes bioactivation by CYP occurs and this can play an important role in human drug toxicology [37]. Phase II reactions increase the water solubility of a drug thereby making the drug more readily excretable. Uridine diphosphoglucuronosyl transferase (UGT) is one of the major phase II enzyme families involved, but also sulfotransferase (SULT), N-acetyltransferase (NAT), glutathione-S-transferase (GST), and others belong to this group of conjugating enzymes [34, 38, 39]. Figure 1. Drug metabolism. In general, a parent compound is converted into an intermediate metabolite which then forms conjugates, but it may involve only one of these reaction. Some metabolites are more toxic than the parent compound, called bioactivation [based on 33].

Both CYP and UGT isozymes show pronounced inter-individual variability. These variabilities are caused by variation in enzyme activities resulting from differences in gender, age, genetic polymorphisms, disease, and food or drug intake [37, 40-42]. Inter-individual variability may lead to inter-individual variation in biotransformation. Therefore, it is important to elucidate the contribution of the different phase I and II enzymes to the biotransformation of each novel drug. Different in vitro methods can be used to investigate which enzymes are involved in the biotransformation of a compound and the importance of these enzymes in the metabolic pathway. These are summarized in Chapter 1.

Phase I Drug Often

reactive intermediate

Often inactive product

Phase II

Oxidation Reduction Hydrolysis Conjugation


5

The elucidation of the enzymes involved in the biotransformation of thiocoraline and aplidine are described in Chapter 2 and 3, respectively. The metabolites of aplidine formed by cytochrome P450 isozymes could be isolated and their structure was elucidated using triple quadrupole mass spectrometry, as described in Chapter 4. Chapter 5 is focused on the biotransformation and enzyme kinetics of ET-743, whereas, Chapter 6 deals with the influence of cytochrome P450 and phase II inhibition and cytochrome P450 induction on the cytotoxicity of ET-743. Chapter 7 describes the evaluation of the genetic polymorphisms in cytochrome P450 and phase II enzymes in the cell systems used in the studies: the human Hep G2 and IGROV-1 cancer cell lines.


6

References 1. Munro M.H.G., Blunt J.W., Dumdei E.J., Hickford S.J.H., Lill R.E., Li S., Battershill

C.N., and Duckworth A.R. (1999). The discovery and development of marine compounds with pharmaceutical potential. J. Biotechnol. 70: 15-25.

2. Cragg G.M., Newman D.J., and Weiss R.B. (1997). Coral reefs, forests, and thermal vents: the worldwide exploration of nature for novel antitumor agents. Semin. Oncol. 24: 156-163.

3. Schwartsmann G., Brondani da Rocha A., Berlinck R.G.S., and Jimeno J. (2001). Marine organisms as a source of new anticancer agents. Lancet Oncol. 2: 221-225.

4. Erba E., Bergamaschi D., Ronzoni S., Faretta M., Taverna S., Bonafanti M., Catapano C.V., Faircloth G., Jimeno J., and D’Incalci M. (1999). Mode of action of thiocoraline, a natural marine compound with anti-tumour activity. Br. J. Cancer 80: 971-980.

5. Faircloth G., Jimeno J., and D’Incalci M. (1997). Biological activity of thiocoraline, a novel marine depsipeptide (abstract). Eur. J. Cancer 33: 175.

6. Schwartsmann G., Brondani da Rocha A., Mattei J., and Lopes R. (2003). Marine-derived anticancer agents in clinical trials. Expert. Opin. Investig. Drugs 12: 1367-1383.

7. Urdiales J.L., Morata P., Nunez De Castro I., and Sanchez-Jimenez F. (1996). Antiproloferative effect of dehydrodidemnin B (DDB), a depsipeptide isolated from Mediterranean tunicates. Cancer Lett. 102: 31-37.

8. Faircloth G., Hanauske A., Depenbrock H., Peter R., Crews C.M., Manzanares I., Meely K., Grant W., and Jimeno J.M. (1997). Pre-clinical characterization of aplidine, a new marine anticancer depsipeptide (abstract). Proc. Am. Assoc. Cancer Res. 38: 692.

9. Erba E., Ronzoni S., Bergamaschi D., Bassano L., Desiderio M.A., Faircloth G., Jimeno J., and D’Incalci M. (1999). Mechanism of antileukemic activity of apldine (abstract). Proc. Am. Assoc. Cancer Res. 40: 3.

10. Broggini M., Marchini S., D’Incalci M., Faircloth G.T., and Jimeno J. (1999). Changes in gene expression in tumor cells exposed to the two marine compounds, ET-743 and aplidine, by using cDNA microarrays (abstract). Proc. Am. Assoc. Cancer Res. 10: 310.

11. Jimeno J.M. (2002). A clinical armamentarium of marine-derived anti-cancer compounds. Anti-Cancer Drugs 13 (suppl 1): S15-S19.

12. Depenbrock H., Peter R., Faircloth G.T., Manzanares I., Jimeno J., and Hanauske A.R. (1998). In vitro activity of aplidine, a new marine-derived anti-cancer compound, on freshly explanted clonogenic human tumour cells and haematopoietic precursor cells. Br. J. Cancer 78: 739-744.

13. Raymond E., Paz-Ares L., Izquierdo M., Belanger K., Maroun J., Bowman A., Anthoney A., Jodrell D., Armand J.P., Cortes-Funes H., Germa-Lluch J., Twelves C., Celli N., Guzman C., and Jimeno J. (2001). Phase I trials with aplidine, a new marine derived anticancer compound (abstract). Eur. J. Cancer 37 (suppl 6), S32.

14. van Kesteren Ch., de Vooght M.M.M., López-Lázaro L., Mathôt R.A.A., Schellens J.H.M., Jimeno J.M., and Beijnen J.H. (2003). Yondelis® (trabectedin, ET-743): the development of an anticancer agent of marine origin. Anti-Cancer Drugs 14: 487-502.

15. D’Incalci M., Erba E., Damia G., Galliera E., Carassa L., Marchini S., Mantovani R., Tognon G., Fruscio R., Jimeno J., and Faircloth G.T. (2002). Unique features of the mode of action of ET-743. The Oncologist 7: 201-216.

16. Rinehart K.L., Gravalos L.G., Faircloth G., and Jimeno J. (1995). Ecteinascidin (ET-743): Preclinical antitumor development of a marine derived natural product (abstract). Proc. Am. Assoc. Cancer Res. 36: 2322.


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17. Synold T.W., Dussault I., and Forman B.M. (2001). The orphan nuclear receptor SXR coordinately regulates drug metabolism and efflux. Nat. Med. 7: 584-590.

18. Friedman D., Hu Z., Kolb E.A., Gorfajn B., and Scotto K.W. (2002). Ecteinascidin-743 inhibits activated but not constitutive transcription. Cancer Res. 62: 3377-3381.

19. Jin S., Gorfajn B., Faircloth G., and Scotto K.W. (2000). Ecteinascidin 743, a transcription-targeted chemotherapeutic that inhibits MDR1 activation. Proc. Natl. Acad. Sci. USA 97: 6775-6779.

20. Minuzzo M., Marchini S., Broggini M., Faircloth G., D’Incalci M., and Mantovani R. (2000). Interference of transcriptional activation by the antineoplastic drug ecteinascidin-743. Proc. Natl. Acad. Sci. USA 97: 6780-6784.

21. Takebayashi Y., Pourquier P., Zimonjic D.B., Nakayama K., Emmert S., Ueda T., Urasaki Y., Kanzaki A., Akiyama S.-L., Popescu N., Kreamer K.H., and Pommier Y. (2001). Antiproliferative activity of ecteinascidin 743 is dependent upon transcription-coupled nucleotide-excision repair. Nat. Med. 7: 961-966.

22. Donald S., Verschoyle R.D., Edwards R., Judah D.J., Davies R., Riley J., Dinsdale D., Lòpez-Làzaro L., Smith A.G., Gant T.W., Greaves P., and Gescher A.J. (2002). Hepatobiliary damage and changes in hepatic gene expression caused by the antitumor drug ecteinascidin-743 (ET-743) in the female rat. Cancer Res. 62: 4256-4262.

23. Louneva N., Saitta B., Herrick D.J., and Jimenez S.A. (2003). Transcriptional inhibition of type I collagen gene expression in scleroderma fibroblasts by the antineoplastic drug ecteinascidin 743. J. Biol. Chem. 278: 40400-40407.

24. Sparfel L., Payen L., Gilot D., Sidaway J., Morel F., Guillouzo A., and Fardel O. (2003). Pregnane X receptor-dependent and -independent effects of 2-acetylaminofluorene on cytochrome P450 3A23 expression and liver cell proliferation. Biochem. Biophys. Res. Commun. 300: 278-284.

25. Jimeno J.M., Faircloth G., Cameron L., Meely K., Vega E., Gómez A., Fernández Sousa-Faro J.M., and Rinehart K. (1996). Progress in the acquisition of new marine-derived anticancer compounds: development of Ecteinascidin-743 (ET-743). Drugs of the Future 21:1155-1165.

26. Demetri G.D., Manola J., Harmon D., Maki R.G., Seiden M.V., Supko J.G., Ryan D.P., Puchlaski T.A., Goss G., Merriam P., Waxman A., Slater S., Potter A., Quigley M.T., Lopez T., Sancho M.A., Guzman C., Jimeno J., and Garcia-Carbonero R. (2001). Ecteinascidin-743 (ET-743) induces durable responses and promising 1-year survival rates in soft tissue sarcomas (STS): Final results of phase II and pharmacokinetic studies in the U.S.A. (abstract). Proc. Am. Soc. Clin. Oncol. 20: 1406.

27. Yovine A., Riofrio M., Brain E., Blay J.Y., Kahatt C., Delaloge S., Bautier L., Coffu P., Jimeno J., Cvitkovic E., and Misset J.L. (2001). Ecteinascidin (ET-743) given as a 24 hour (H) intravenous continuous infusion (IVCI) every 3 weeks: results of a Phase II trial in patients (pts) with pretreated soft tissue sarcomas (PSTS) (abstract). Proc. Am. Soc. Clin. Oncol. 20: 36.

28. Le Cesne A., Blay J., Judson I., van Oosterom A., Verweij J., Radford J., Lorigan P., Rodenhuis S., Di Paola E.D., van Glabbeke M., Jimeno J., and Nielsen O. (2001). ET-743 is an active drug in adult soft-tissue sarcoma (STS): a STBSG-EORTC phase II trial (abstract). Proc. Am. Soc. Clin. Oncol. 20: 1407.

29. Zelek L., Yovine A., Brain E., Turpin F., Taamma A., Riofrio M., Spielmann M., Jimeno J., and Cvitkovic E. (2000). Preliminary results of phase II study of Ecteinascidin-743 with the 24 hour continuous infusion Q3 weeks schedule in pretreated advanced/metastatic breast cancer patients (abstract). Clin. Cancer Res. 6: 85.

30. Aune G.J., Furuta T., and Pommier Y. (2002). Ecteinascidin-743: a novel anticancer drug with a unique mechanism of action. Anti-Cancer Drugs 13: 545-555.


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31. Derelanko M.J. and Hollinger M.A. (1995). Handbook of toxicology. CRC press (West Palm Beach, USA): 539-579.

32. Crommentuyn K.M.L., Schellens J.H.M., van den Berg J.D., and Beijnen J.H. (1998). In-vitro metabolism of anti-cancer drugs, methods and applications: paclitaxel, docetaxel, tamoxifen and ifosfamide. Cancer Treat. Rev. 24: 345-366.

33. Oesch F., Herrero M.E., Hengstler J.G., Lohmann M., and Arand M. (2000). Metabolic detoxification: implications for thresholds. Toxicol. Pathol. 28: 382-387.

34. Lu F.C. (1996). Basic Toxicology – Fundamentals, Target Organs and Risk Assessment. Taylor and Francis (Washington DC, USA), 3rd edition: 27-39.

35. Wrighton S.A. and Stevens J.C. (1992). The human hepatic cytochromes P450 involved in drug metabolism. Crit. Rev. Toxicol. 22: 1-21.

36. Smith D.A. and Jones B.C. (1991). Commentary: speculations on the structure-activity relationship (SSAR) of cytochrome P450 enzymes. Biochem. Pharmacol. 44: 2089-2098.

37. Wormhoudt L.W., Commandeur J.N.M., and Vermeulen N.P.E. (1999). Genetic polymorphisms of human N-acetyltransferase, cytochrome P450, glutathione-S-transferase, and epoxide hydrolase enzymes: relevance to xenobiotic metabolism and toxicity. Crit. Rev. Toxicol. 29: 59-124.

38. Range H.P., Dale M.M., and Ritter J.M. (1996). Pharmacology. Churchill Livingstone (Edinburgh, UK), 3rd edition: 83.

39. Ritter J.K. (2000). Roles of glucuronidation and UDP-glucuronosyltransferase in xenobiotic bioactivation reactions. Chem. Biol. Interact. 129: 171-193.

40. Tanaka E. (1999). Gender-related differences in pharmacokinetics and their clinical significance. J. Clin. Pharm. Ther. 24: 339-346.

41. MacKenzie P.I., Miners J.O., and McKinnon R.A. (2000). Polymorphisms in UDP glucuronosyltransferase genes: functional consequences and clincial relevance. Clin. Chem. Lab. Med. 38: 889-892.

42. Tukey R.H. and Strassburg C.P. (2000). Human UDP-glucuronosyltraferases: metabolism, expression, and disease. Annu. Rev. Pharmacol. Toxicol. 40: 581-616.

9

CHAPTER In vitro test methods in human hepatic drug biotransformation research: pros and cons. Esther F.A. Brandon, Christiaan D. Raap, Irma Meijerman, Jos H. Beijnen, and Jan H.M. Schellens. Abstract

The liver is the predominant organ where biotransformation of foreign compounds takes place, although, other organs may also be involved in drug biotransformation. Ideally, an in vitro model for drug biotransformation should accurately resemble biotransformation in vivo in the liver. Several in vitro human liver models have been developed in the past few decades, including supersomes, microsomes, cytosol, S9 fraction, cell lines, transgenic cell lines, primary hepatocytes, liver slices, and perfused liver. A general advantage of these models is a reduced complexity of the study system. On the other hand, there are several more or less serious specific drawbacks for each model, which prevents their widespread use and acceptance by the regulatory authorities as an alternative for in vivo screening. This chapter describes the practical aspects of selected in vitro human liver models with comparisons between the methods.

Chapter 1

10

Introduction

The development of a new therapeutic agent always involves a preclinical screening stage. During this stage the main pharmacokinetic, pharmacodynamic, and toxicological properties of the candidate drug are investigated. The preclinical investigation is based on both in vitro models and in vivo experiments in various animal species [1].

Drug biotransformation is one of the most important factors that can affect the overall therapeutic and toxic profile of a drug. It can lead to detoxification and excretion of the drug, but also to bioactivation. For this reason, drug biotransformation is a pivotal factor in the early developmental stage of new drugs. Biotransformation occurs in many tissues with the liver as the most important organ, but also the kidneys, skin, lungs, and intestine can be involved [2]. The liver is the largest internal organ of the human body and is strategically located between the digestive tract and the other parts of the body [3].

Drug biotransformation is divided into two types of reactions, namely phase I (hydrolysis, oxidation, and reduction) and phase II reactions (conjugation). The biotransformation pathway of a drug is mediated by phase I, phase II, or a combination of both. The cytochrome P450 (CYP) enzyme superfamily plays a dominating role in the phase I biotransformation and is mainly present in the liver [4, 5]. Many different CYP isoforms have been characterized, which are categorized in families based on their sequence. Table 1A gives an overview of the human CYP families 1 to 4, which are the main CYPs involved in drug biotransformation and lists the various model substrates, which are used to quantify specific CYP activity [6-8]. Table 1B lists the various inducers and inhibitors of the different isozymes [9].

It is becoming increasingly apparent that also drug transporters (phase III) influence the adsorption, distribution, and elimination of a drug and not only the therapeutic efficacy [10]. The drug transporters are located in epithelial and endothelial cells of the liver, gastrointestinal tract, kidney, blood-brain barrier, and other organs. They are responsible for the transport of most of the commonly prescribed drugs across cellular barriers and thus for the concentration at the target or biotransformation site. Multidrug resistance proteins (p-glycoprotein and MRPs) have been shown to be important to explain the pharmacokinetics of a drug in man [11]. Thus, the elucidation of the influence of drug transporters on the ADME (adsorption disposition metabolism elimination) of a drug is essential in the early developmental stage of new drugs.

In vitro test methods: pros and cons

11

Table 1A. An overview of the main human CYP isoforms (CYP1 to 4) involved in drug biotransformation and their occurrence [6-8]. Also an overview of the model substrates used to quantify the CYP isozyme activity and a recommendation according to the FDA, American Association of Pharmaceutical Sciences (AAPS), and the European Federation of Pharmaceutical Sciences (Basel conference 2000) [6-9]. isoform occurrence model substrates recommendation CYP1A1 mainly

extrahepatic 7-ethoxyresorufin O-deethylation

CYP1A2 liver phenacetin O-deethylation caffeine N3-demethylation

preferred acceptable

CYP2A6 liver coumarin C7-hydroxylation preferred CYP2B1/2 pentoxyresorufin O-dealkylation CYP2B6 liver (S)-mephenytoin N-demethylation

Bupropion hydroxylation preferred acceptable

CYP2C8 liver intestine

paclitaxel C6-α-hydroxylation preferred

CYP2C9 liver intestine

(S)-warfarin C6-, C7 hydroxylation diclofenac 4’-hydroxylation tolbutamide para CH3-hydroxylation

preferred acceptable acceptable

CYP2C18/19 liver (S)-mephenytoin C4’-hydroxylation preferred CYP2D6

liver intestine kidney

bufuralol C1’-hydroxylation dextromorphan O-demethylation codeine O-demethylation

preferred preferred acceptable

CYP2E1 liver intestine leukocytes

chlorzoxazone C6-hydroxylation lauric acid C(ω1)-hydroxylation

preferred acceptable

CYP3A4

liver gastro-intestinal tract

midazolam C1’-hydroxylation testosterone C6-β-hydroxylation

preferred preferred

CYP4A11 liver kidney

lauric acid ω-hydroxylation

Chapter 1

12

Table 1B. An overview of commonly known inhibitors and inducers of the CYP isozyme activity [9]. isoform inhibitor inducer CYP1A1 α-naphtoflavone polycyclic hydrocarbons CYP1A2 furafylline

smoking 3-methylcholanthrene char-grilled meat rifampicin

CYP2A6 sulfaphenazole pyrazole barbiturates

CYP2B1/2 CYP2B6 sertraline CYP2C8 glitazones rifampicin

barbiturates CYP2C9 sulfaphenazole rifampicin

phenobarbital CYP2C18/2C19 ticlopidine

ketoconazole rifampicin carbamazepine

CYP2D6

quinidine haloperidol

CYP2E1 diethyl-dithiocarbamate ethanol CYP3A4

ketoconazole grapefruit juice

rifampicin barbiturates

CYP4A11 17-octadecynoic acid Key question in human drug biotransformation research is how to make reliable

extrapolations from the in vitro or in vivo model to the clinical practice. Thus, the objective is to establish a useful model system with a strong predictive power for human biotransformation. Several in vitro models have been developed in the past, ranging from (recombinant) isolated enzymes to the intact perfused liver (see figure 1). They are used to obtain early information about biotransformation pathways and to predict drug-drug interactions at the metabolic level [12]. The quality of the human liver for the preparations of the different in vitro methods described, is a dominant factor in the outcome of the in vitro studies, especially in precision-cut liver slices and isolated hepatocytes [13]. Livers which are not suitable for transplantation or liver sections from biopsies are used and in order to ensure a viable cell yield as high as possible or, in case of cell fractions, the highest enzyme activity, the liver or liver section needs to be processed as soon as possible after the resection.


13

Figure 1. In vitro and in vivo models used in the development of new drugs, ranging from man to isolated enzymes, in order of in vivo resemblance.

The optimal model system in a given situation depends on a number of factors, such as in vivo resemblance, expense, availability of the model, and ethical considerations. In vitro data from human and animal models can be used to choose the best in vivo model (e.g. mouse, rat, dog) for further testing. In conclusion, it can be stated that an in vitro model is always a compromise between convenience and relevance.

Current guidelines for human drug development allow that in vitro systems are used in supportive studies, and therefore in vitro data should be used mainly qualitatively [14, 15]. For example, when in vitro data show a lack of drug-drug interaction, no in vivo experiments have to be performed, but when a drug-drug interaction is demonstrated then in vivo experiments have to follow [15]. A complete replacement of animal experiments by in vitro models in the near future seems to be an unrealistic scenario, because a lack of validation prevents acceptance by regulatory authorities [16, 17].

In this chapter, an overview of different in vitro models (supersomes, microsomes, cytosol, S9 fraction, cell lines, transgenic cell lines, primary hepatocytes, liver slices, and perfused liver) for human biotransformation is given with their advantages and disadvantages. The phase III in vitro models (e.g. confluent cell monolayers) are beyond the scope of this review and are already extensively described by Zhang et al. (2003) with their advantages and disadvantages [10]. Human CYP and UGT supersomes (Baculovirus-insect-cell-expressed)

Insect cells lack endogenous cytochrome P450 (CYP) and uridine diphosphoglucuronosyl transferase (UGT) activity and therefore microsomes, which consist of vesicles of the hepatocyte endoplasmic reticulum, of human CYP or UGT transfected insect cells can be a useful tool in human biotransformation studies. Since the expression is baculovirus mediated, microsomes of these cells are sometimes referred to as baculosomes, but more often as supersomes (Gentest (Becton Dickinson Company, Woburn, MA, USA) offers them under this trade name) [18].

The availability of specifically expressed human CYPs and UGTs in supersomes allows the investigation of the contribution of a single metabolic enzyme to the biotransformation pathway of the compound under investigation. At present all common human CYPs, co-expressed with NADPH-cytochrome P450 reductase and optionally cytochrome b5, and

supers

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ethically acceptableresemblance of true in vivo situation

Chapter 1

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UGTs are offered in supersomes. A control experiment, an incubation with non-transfected supersomes, must always be conducted [19]. A NADPH-regenerating system (which consists of β-NADP, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase) or NADPH is required to supply the energy demand of the CYPs [20]. For the UGT activity uridine diphosphoglucuronic acid (UDPGA) has to be added as co-factor.

The specific CYP and UGT activity can be measured with various model substrates, e.g. midazolam C1’-hydroxylation for CYP3A4 and estradiol 3-glucuronidation for UGT1A [6-8, 21]. The CYP and UGT activity is usually provided by the supplier of the supersomes. Supersomes are commercially available from different companies.

A major advantage of supersomes is that they can be used to study isozyme specific drug biotransformation, but also drug-drug interactions (e.g. fluvoxamine-theophylline interaction at CYP1A2) [22]. In the last few years the development of new CYP and UGT supersomes has increased considerably. The different genotypes of the CYP isozymes (e.g. CYP2C9*1, 2C9*2, and 2C9*3) are now also commercially available [21]. Hence, also the influence of different polymorphisms on the drug biotransformation pattern can be studied. A disadvantage is that, in UGT supersomes, the UGT active site is shielded behind a hydrophobic barrier resulting in latency of glucuronidations. However, this advantage can be overcome by using a pore forming agent, e.g. alamethicin [23].

They are a valuable supplement to human liver microsomes and therefore it is likely that their application will increase in the future [24-26]. More detailed information will become available in the future about the specific advantages and disadvantages of this model compared to the other in vitro models. Human liver microsomes

Human liver microsomes (HLM) still account for the most popular in vitro model, providing an affordable way to give a good indication of the CYP and UGT metabolic profile. Also the influence of specific isozymes can be studied in the presence of specific inhibitors [6]. The large availability of human microsomes and their simplicity in use contribute to the popularity of this in vitro model.

Liver microsomes consist of vesicles of the hepatocyte endoplasmic reticulum and are prepared by differential centrifugation and thus contain almost only CYP and UGT enzymes [27]. Liver preparations, other than from fresh human liver, can also be used (e.g. liver slices, liver cell lines, primary hepatocytes) for preparation of microsomes [28, 29]. The CYP and UGT enzyme activity can be measured by various model substrates. In commercially available HLM, the CYP activity is already characterized by the supplier [30-32]. Like in supersomes, NADPH regenerating system or NADPH is required to supply the energy demand of the CYPs and UDPGA and alamethicin for UGT activity.

The activity of HLMs can vary substantially between individuals. This problem, however, can be successfully solved by the application of pooled microsomes, which results in a representative enzyme activity [19]. These pools can be purchased from different companies. Individual human liver microsomes can also be used to screen for the inter-individual variability in the biotransformation of a drug. It is also possible to identify the critical CYP involved in the biotransformation of the drug using individual HLMs by correlating the enzyme activity of a particular CYP, using a bank of human donors, to the metabolism of the drug. The influence of gender on drug biotransformation can be investigated with gender specific HLM pools. Also different animal liver microsomes (e.g. mouse, rat, monkey) can be purchased from different companies. The results obtained can be used to screen for the best in vivo model for human drug biotransformation.


15

The major advantages of microsomes are low costs, simplicity in use, and they are one of the best-characterized in vitro systems for drug biotransformation research. However, some major drawbacks exist (see also table 2). First of all, it should be noted that results obtained with microsomes cannot be used for quantitative estimations of in vivo human biotransformation, because CYPs and UGTs are enriched in the microsomal fraction and there is no competition with other enzymes. This results in higher biotransformation rates in microsomes compared to the human in vivo situation, but also compared to primary hepatocytes and liver slices [33]. Additionally, the absence of other enzymes (e.g. N-acetyltransferase (NAT), glutathione-S-transferase (GST), and sulfotransferase (SULT)) and cytosolic co-factors can leave metabolites formed in intact liver cells unnoticed [7].

Table 2. The advantages and disadvantages of human liver microsomes, the most popular model in human drug biotransformation research. advantages disadvantages easily applicable affordable well established inter-individual variation can be studied

unsuitable for quantitative measurements incomplete representation of in vivo situation only CYP and UGT enzymes

The limitations make microsomes only useful for qualitative routine CYP and UGT

screening in the early phase of drug development, and not for quantitative prediction of human biotransformation. However, some equations exist to extrapolate the in vitro HLM data to in vivo pharmacokinetics values for humans, but there is disagreement about the best equations to be used. Human liver cytosol fractions

The liver cytosolic fraction contains the soluble phase II enzymes, e.g. NAT, GST, and SULT [21]. It is obtained by differential centrifugation of whole-liver homogenate, like microsomes. For the catalytic activity of the phase II enzymes addition of exogenous cofactors, e.g. acetyl coenzyme A (acetyl CoA), dithiothreitol (DTT), and acetyl CoA regenerating system for NAT, adenosine-3’-phosphate-5’-phosphosulfate (PAPS) for SULT, and glutathione (GT) for GST, is necessary [21]. Human liver cytosol is commercially available from different companies, like Gentest Corporation (Becton Dickinson Company, Woburn, MA, USA) and XenoTech (Kansas City, KS, USA).

The specific NAT isozymes are also commercially available in cytosol without the other soluble phase II enzymes. These enzymes are prepared from the cytosolic (soluble) fraction of insect cells infected with recombinant baculovirus. The influence of both NAT1 and NAT2 isozymes on the biotransformation pathway can be studied with this system [21].

The main advantage is the presence of only 3 enzymes in the cytosolic fraction at higher concentrations compared to human liver S9 fraction. The biotransformation capacity of NAT, SULT, and GST can be studied separately or in combination depending on the cofactors added. A disadvantage is that only the soluble phase II enzymes are present in the liver cytosol fraction and that therefore the UGT, which is located on the endoplasmic reticulum, metabolic pathways cannot be investigated with this model.

So far, cytosol has not been used very often in drug biotransformation research [34-36]. It will probably play a more important role in the future, because researchers are more and more aware that knowledge of the entire biotransformation pathway is of importance and not only knowledge of biotransformation by CYP.

Chapter 1

16

Human liver S9 fractions

The liver S9 fraction contains both microsomal and cytosolic fractions. Similar as with supersomes and microsomes, a NADPH regenerating system or NADPH solution is required to supply the energy demand of the CYP enzymes [21]. For the catalytic activity of phase II enzymes addition of exogenous cofactors is necessary, UDPGA and alamethicin for UGT, acetyl CoA, DTT, and acetyl CoA regenerating system for NAT, PAPS for SULT, and GT for GST.

Human liver S9 fraction is mainly used in combination with the Ames test, which is a simple and rapid in vitro method for detecting the mutagenicity of chemicals [37]. The Ames test plays a critical role in development of new drugs and is used for predicting possible mutagenicity of a compound. However, many procarcinogens remain inactive until enzymatic transformation and thus a metabolic activation system, e.g. human liver S9 fraction, is necessary for testing not only the genotoxicity of a drug, but also of its metabolites in humans [38].

Compared with microsomes and cytosol, S9 fractions offer a more complete representation of the metabolic profile, as they contain both phase I and phase II activity. In some cases with S9 fractions metabolites are formed, which are not produced by either the cytosolic fraction or the microsomal fraction alone, due to a phase I reaction followed by phase II biotransformation. However, a disadvantage is the overall lower enzyme activity in the S9 fraction compared to microsomes or cytosol, which may leave some metabolites unnoticed.

Liver S9 fractions have been used since the 1970’s, but not as extensively as microsomes [39-41]. It is a useful tool to study the human biotransformation, but the best option is to use it in addition to microsomes and cytosol. It is likely that the S9 fraction is going to be more widely used in the future, because of the importance to elucidate the entire biotransformation pathway and not only metabolites produced by phase I or phase II, but also by a combination of both. Liver cell lines

Liver cell lines as in vitro model are less popular compared to other described models. This is mainly due to their dedifferentiated cellular characteristics and incomplete expression of all families of metabolic enzymes. Human liver cell lines can be isolated from primary tumors of the liver parenchyma, seen after chronic hepatitis or cirrhosis [42].

An important requirement of cell lines as a model is that they must resemble the normal physiology of human hepatocytes in vivo. The usefulness of hepatoma cell lines as in vitro model therefore falls or stands with their ability to express human phase I and phase II enzymes. Currently available human liver cell lines are presented in table 3 [43]. Only a few are used in biotransformation studies. There are also different animal hepatoma cell lines, but they are unpopular in human drug biotransformation research where human systems are preferred [43]. Established cell lines can be readily obtained from specialized companies like ATCC (Manassas, VA, USA).


17

Table 3. An overview of the human hepatoma cell lines known [43]; the Hep G2 cell line is the most frequently applied cell line in human drug biotransformation studies [44-52]. designation origin enzymes active (constitutive) Hep G2 hepatocellular

carcinoma CYP1A and 3A and UGT

BC2 hepatoma CYP1A1/2, 2A6, 2B6, 2C9, 2E1, 3A4 and GST and UGT

Hep 3B hepatocellular carcinoma

CYP1A1

C3A hepatoblastoma CYP3A PLC/PRF/5 hepatoma GST SNU-398 hepatocellular

carcinoma

SNU-449 hepatocellular carcinoma



SK-Hep-1

The most frequently used and best-characterized human hepatoma cell line is the Hep G2 cell line [44-49]. This cell line, established in 1979, still has a variety of liver specific metabolic functions. Under standard culturing conditions, the cell line shows nearly undetectable levels of functional CYP. However, various isoforms can be induced by pretreatment with inducing agents. Exposure to 3-methylcholanthrene and rifampicin, results in higher levels of CYP1A and 3A respectively, compared to untreated cells. Compared to freshly isolated human hepatocytes however, the overall CYP-activity remains low [45]. Also the composition of the culture medium has a significant effect on the metabolic enzyme activity in Hep G2 cells. Earle’s medium gives a strong increase of the activity of CYP1A and 2B, compared to Dulbecco’s medium and Williams’ E medium [44]. In a direct comparison between Hep G2 cells, human liver slices and human liver microsomes, the Hep G2 cells showed to be an unsuitable model for biotransformation of cyclosporin A, which is mainly metabolized by CYP3A. In contrast to human liver slices and microsomes, the cells generated only one out of three primary metabolites of cyclosporin A. This underlines again that outcomes of experiments with human cell lines should be interpreted with caution [48].

The cell line BC2 has been established more recently (1998) and has been shown to express many drug metabolizing enzymes, especially the most important drug metabolizing CYPs (1A1/2, 2A6, 2B6, 2C9, 2E1, and 3A4) and the phase II enzymes GST and UGT [50]. It should be noted, however, that basal activities of these enzymes are very low and remain low after induction compared to freshly isolated human hepatocytes. It is the first cell line which combines the convenience of stable phenotypic expression in culture of a large set of drug metabolizing enzymes, however it has not been used by other research groups other than the group which isolated the cell line [50].

Chapter 1

18

Other human hepatoma cell lines that have been used for cytotoxicity studies are the PLC/PRF/5 and the Hep 3B cell lines [51, 52]. These cell lines have hardly been used for drug biotransformation studies, because their enzyme expression levels are very low and are difficult to induce.

Compared to primary hepatocytes, cell lines are generally easier to culture and have relatively stable enzyme concentrations. However, an important disadvantage is the absence or low expression level of most important phase I and phase II drug metabolizing enzymes, which limits its application. It is difficult to detect metabolites in cell lines and it is also difficult to investigate the individual CYPs or other enzymes due to their low expression levels.

It is likely that the cell lines will only be used in the enzyme induced state for drug biotransformation studies and most likely in combination with cytotoxicity studies of the drug and its metabolites. Transgenic cell lines

Another approach to obtain a cell line expressing phase I and/or phase II enzymes is the recombinant expression of the human enzyme in a cell line. At present all known human CYPs involved in drug biotransformation have successfully been over-expressed in cells and these cell lines are available for research [26]. Also human UGTs have been successfully transfected into the V79 cell line [53].

Cell lines may be transfected at high efficiency using protoplast fusion, centrifugation of lysozyme treated bacteria bearing the desired vector with the parent cells in the presence of polyethylene glycol. Stable expression of human CYPs in a human cell line has first been achieved in 1993 by Crespi et al. [54]. The V79 Chinese hamster cell line and the Hep G2 cell line have since then been engineered for stable expression of one or more CYPs and UGTs, but also other transfected cell lines have been used to study human biotransformation [53-67]. Gentest developed the MLC-5 human lymphoblast cell line, which stably expresses CYP1A2, 2A6, 2E1, and 3A4 and microsomal epoxide hydrolase [21].

Transfected cell lines are often as easy to culture as non-transfected cell lines and their main advantage over non-transfected cells is the higher expression of CYP and UGT isozymes. The expression levels are high enough to perform biotransformation experiments. The transgenic cell lines can be readily obtained from specialized companies, like ATCC and Gentest. Unfortunately, transgenic cell lines can be very expensive compared to the other in vitro models.

Transgenic cell lines, like supersomes, allow the study of single enzyme reactions. It is possible to elucidate the influence of one isoenzyme or a combination of a number of isoenzymes on the biotransformation and to screen for differences in cytotoxicity of the metabolites. Transfected cell lines can also be used to generate metabolites for structure elucidation and pharmacological characterization and to assess potential drug-drug interactions at the metabolic level. A limitation is that only one or a few isozymes are expressed, which is not a complete reflection of the in vivo situation.

In the future, it is likely that more transgenic cell lines will be used to study human biotransformation, because the enzymatic levels are high and stable enough to study the biotransformation. Hopefully, more and more cell lines which express several (iso)enzymes will become available at moderate costs.


19

Hepatocytes

Primary hepatocytes are a popular in vitro system for drug biotransformation research due to their good resemblance of in vivo human liver. Detailed reviews dealing with this model have been published previously and, therefore, primary hepatocytes will be discussed here only briefly [68-70].

Hepatocytes of various animal species can be isolated from the liver by the traditional collagenase perfusion operation developed by Howard and Pesch (1967) [71]. This method requires the whole liver, which is not available in the case of human liver. Human liver is mainly obtained from patients that undergo partial liver resection, e.g. because of liver metastasis. Therefore, a method for human liver parts has been developed and this is a modification of the traditional collagenase perfusion [70, 72]. Preferably, the perfusion takes place immediately after resection. When this is not possible, the tissue can be stored at 4°C for up to 48 hours in “University of Wisconsin” (UW) solution, without relevant loss of viability [73].

Once isolated, hepatocytes can be held in suspension, in which case they remain viable for only a few hours, or they can be maintained in monolayer culture for a maximum of 4 weeks. Both cultured hepatocytes and suspensions of primary hepatocytes have repeatedly proven to be powerful tools to analyze the specific metabolic profile of a variety of drugs with good in vitro - in vivo correlations [74-78]. However, it has been widely recognized that cultured hepatocytes are subject to a gradual loss of liver specific functions, with special reference to a decreased CYP expression. This loss is different for the specific CYP isoforms; for some isoforms it becomes evident after a few days of culture (CYP2E1 and 3A4), while others remain nearly unaffected by the isolation and culturing processes (CYP1A2 and 2C9) [79]. Various culturing methods have been explored in an effort to maintain the liver specific characteristics of hepatocytes during prolonged culture. These include the application of culturing matrices (e.g. double layer collagen gel sandwich; this culture method can be used to study biotransformation, but also transporter mediated biliary excretion), the addition of specific nutrients, hormones, and inducers to the culture medium, and also the co-culturing of hepatocytes with other cell types (e.g. the hepatic Kupffer cells) [80-88].

An advantage of isolated hepatocytes compared to liver slices and perfused liver is the possibility of cryopreservation. Cryopreserved hepatocytes have been shown to retain the activity of most phase I and phase II enzymes [89, 90]. Due to successful cryopreservation techniques, human hepatocytes are now commercially available [69]. Due to their widespread use in drug biotransformation research, isolated hepatocytes have become a well-established and well-characterized in vitro model and with special techniques isolated hepatocytes can be made viable for up to 4 weeks. However, it should be noted that prolonged culture conditions result in a more complex data interpretation, since outcomes partly depend on culture system factors. A disadvantage is the lack of liver non-hepatocyte cells. Although hepatocytes account for the vast majority of the liver volume (about 80%), other cells like Kupffer cells may be necessary for co-factor supply. Another problem encountered with human hepatocytes, like with human liver microsomes, is the considerable inter-individual variation. This problem can be overcome by using mixtures of hepatocytes from multiple donors to mimic an average enzyme content. Also animal primary hepatocytes are used in human biotransformation studies and they can be used, like HLM, to choose the best animal in vivo model, the in vivo model that has the most resemblance with the human biotransformation pathway.

Despite the disadvantages (see table 4), isolated hepatocytes are a useful tool to predict human biotransformation, and are therefore often used in human drug biotransformation studies and will be in the future.

Chapter 1

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Table 4. Primary hepatocytes and their advantages and disadvantages in human biotransformation studies. advantages disadvantages well established, well characterized viability for up to 4 weeks study of mediators and enzyme inducers possible viable cell enrichment possible cryopreservation possible drug transporters still present and operational

isolation can be complicated and time consuming only preselected cells can be studied cell damage during isolation cellular interactions more difficult to study

Liver slices

Cultures of tissue slices have already been developed in the 1920’s by Otto Heinrich Wartburg. His technique was adopted by later workers, including Hans Adolf Krebs who used razor blade cut tissue slices to study the biotransformation of amino acids in a variety of organs of different species [91]. Today, the incubation of liver slices in nutrient enriched media offers a powerful tool to study biotransformation in vitro.

Initially, it proved to be difficult to produce uniform slices, which led to irreproducible results. Even after the development of more precise slicing devices (e.g. the McIlwain tissue chopper) in the early 1970’s, the technique remained unpopular. This was partially due to good results obtained with primary hepatocytes, which became the model of choice for drug biotransformation studies. The liver slices as model for human drug biotransformation studies fell further into disuse [92, 93].

The development of high precision tissue slicers however, set the stage for the ‘renaissance’ of liver slices in in vitro biotransformation studies. The Krumdieck tissue slicer for example, allows the rapid production of equally sized slices of less than 250 µm thickness [94]. The Brendel-Vitron is essentially a simpler slicing device but gives access to slices of equal quality [95, 96]. The thin slices obtained with the Krumdieck and the Brendel-Vitron slicers realistically and reliably represent the in vivo situation, and have been used to study the biotransformation of many compounds [92].

The resected tissue can be stored at 4°C in UW-solution up to 48 hours without loss of phase I and phase II enzyme activity [97]. However, the long-term storage of liver slices in liquid nitrogen has shown to be complicated and there is no optimal cryopreservation protocol. No commercially human liver slices are yet available. The duration of the CYP activity is short and this is probably due to impaired diffusion of nutrients and oxygen in the tissue slice. A recent study with rat liver slices, showed that the amount of many CYPs drops below half of the initial value within 24 hours [98]. However, CYP induction has been reported, e.g. rifampicin has been reported to induce CYP3A and Aroclor 1254, omeprazole, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and 3,3’-diindolylmethane induce CYP1A [99-101].

Dynamic organ culturing (DOC) was developed to prolong the limited viability period of liver slices. In this technique, the slice is continuously exposed to both culture medium and gas atmosphere [102]. Some variants of the DOC have been developed later [103, 104].


21

In terms of qualitative and quantitative biotransformation properties, slices have been shown to be comparable to perfused liver, a more complicated model that is discussed in the next paragraph.

The advantages and disadvantages of using liver slices in human drug biotransformation studies are summarized in table 5. One of the main advantages is the non-requirement for digestive enzymes and thus the intact cellular tissue architecture, allowing for observing biotransformation in non-hepatocytes. Also, the possibility to study the induction of CYP isoforms by new drugs is a main advantage. The most prominent disadvantages include inadequate penetration of the medium into the inner part of the slice, damaged cells on the slice outer edges with impaired biotransformation and the short viability time-period of 5 days. Also the optimal incubation method is highly dependent on the applications of the liver slices.

Table 5. Comparison of advantages and disadvantages of precision cut liver slices used in human drug research. advantages disadvantages

non-requirement of harmful proteases intact cellular interactions normal spatial arrangement morphological studies possible

inadequate penetration of the medium damaged cells on the outer edges limited viable period technology still being developed and optimized viable cell enrichment not possible non-inducible by CYP-inducers cryopreservation needs further optimization expensive equipment necessary

It is a powerful tool to study biotransformation in vitro, but the drawbacks mentioned still

prevent its large-scale application. Isolated perfused liver

Although an isolated perfused liver is considered to be the best representation of the in vivo situation, it has never been used with human liver and only on small scale with animal livers. Many specific drawbacks make the animal perfused liver less attractive as model for biotransformation studies.

The procedure for perfusion of intact rat liver has been described in detail in 1959 by Brauer et al. [105]. The perfusion is carried out with Krebs-Henseleit buffer as perfusate, while other media like diluted blood solutions have also been reported [106]. The test compound under investigation should be dissolved in the medium and the viability period of the liver is only 3 hours [107].

There are several reasons why the perfused liver has not been widely used in human biotransformation studies. There are no human livers available for such studies and animal livers are not always the correct model for human drug biotransformation. Also the perfused liver method is labor-intensive, has a poor reproducibility, and the functional integrity is limited to 3 hours. An overview of the advantages and disadvantages is given in table 6. Since an intact perfused liver has only slight advantages over precision cut liver slices, the latter will be preferentially used for practical reasons. In view of animal welfare, it is an inappropriate model as well, as the ratio animal to experiment is 1:1.

Chapter 1

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Table 6. Advantages and disadvantages of the isolated perfused liver model in human biotransformation research. advantages Disadvantages best representation of in vivo situation bile can be collected and analyzed three dimensional architecture all cell types, so also biotransformation by non-hepatocytes

delicate model, difficult to handle limited experimental viable period poor reproducibility no human liver available

The perfused animal liver is only a useful model in cases where bile secretion is of

importance, or when validation of other in vitro methods is required. Conclusion

Although the isolated perfused liver gives an excellent representation of the in vivo situation, practical inconveniences like unavailability of human liver, poor reproducibility, and test limitation of 3 hours prevents the method from being used on a large scale. Liver slices only have slight disadvantages over the isolated perfused liver and liver slices are thus preferred over the perfused liver model. Therefore perfused animal liver is only the model of choice in biotransformation studies when bile excretion is necessary.

Liver slices and primary hepatocyte suspensions also give a good picture of the in vivo metabolic profile, and offer a more efficient use of tissue. A disadvantage of these models, however, is a rapid decline of viability and metabolic capacity within hours after isolation. Cultures of primary hepatocytes have a longer viability period, but the decline of some enzymes is still rapid. Methods to prolong the viability period with maintenance of hepatospecific functions have been developed for liver slices and cultured primary hepatocytes, but can complicate data interpretation. A disadvantage of the primary hepatocytes compared to the liver slices is that the normal liver integrity is not maintained. However, an advantage of the cultured primary hepatocytes is that the decline of the enzymes can be reduced by adding inducers of these enzymes to the culture medium, which is not possible in liver slices.

Established cell lines have a relatively stable phenotype in culture compared to primary hepatocytes, liver slices, and perfused liver, but they usually lack or overexpress many essential enzymes specific for the liver which limits their use. Transgenic cell lines, with an established CYP expression, are a better option, but there is no transgenic cell line at this moment that represents the true in vivo human hepatocyte. Established and transgenic cell lines offer a model to study a combination of biotransformation and cytotoxicity of the drug and its metabolites.

Subcellular liver fractions are widely used to characterize the metabolic profile of novel compounds. Microsomes can be used to obtain information on CYP and UGT mediated biotransformation (phase I), while cytosol can be used to study phase II biotransformation of the soluble phase II enzymes (NAT, SULT, and GST). CYP and UGT supersomes and NAT cytosol provide information about CYP, UGT, or NAT isozymes. S9 fractions can be used to study both phase I and phase II biotransformation at the same time. Supersomes and other sources of artificially expressed human CYPs are valuable to identify new metabolites and to elucidate the contribution of individual CYPs, UGTs, and NATs to the biotransformation of the compound under investigation. However, a disadvantage of the subcellular fractions is


23

that the drug biotransformation is not influenced by drug transporters, which is normally the case in intact cells and organs.

Biotransformation research of a new drug can start with a simple model while the model can become more complex at later stages. The best is to start with microsomes and cytosol, then CYP and UGT supersomes and NAT cytosol, the S9 fraction, followed by (transfected) cell lines and primary hepatocytes, and finally liver slices. Also drug-drug interactions and the influence of polymorphisms can be studied using different in vitro techniques. Table 7 summarizes the different in vitro techniques with their main advantages and disadvantages. An overview of the different models of choice and their preference in use is shown in table 8. The perfused liver should only be used in case of bile excretion study and is not a good model for biotransformation research.

Although at present, in vitro models are unable to replace in vivo screening completely, they offer promising features. They can reduce the number of animals needed and offer a less complex way to elucidate the human biotransformation pathway of a new drug. It is likely that the different in vitro techniques to study human biotransformation, except perfused liver, will become increasingly important in the early development stage of a new drug before starting in vivo experiments. So, that the most promising drugs are selected and in vivo testing can be performed as efficient as possible.

Chapter 1

24

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tiviti

es

stud

y of

indi

vidu

al;,

gend

er a

nd sp

ecie

s spe

cific

bio

trans

form

atio

n

both

pha

se I

and

II

stud

y of

indi

vidu

al, g

ende

r and

spec

ies s

peci

fic b

iotra

nsfo

rmat

ion

easy

to c

ultu

re

rela

tivel

y st

able

enz

yme

expr

essi

on le

vels

C

YPs

indu

cibl

e

easy

to c

ultu

re

high

er e

xpre

ssio

n le

vels

st

udy

of o

ne is

ozym

e or

com

bina

tion

of C

YPs

wel

l est

ablis

hed

and

char

acte

rized

st

udy

of m

edia

tors

and

enz

yme

indu

cers

pos

sibl

e dr

ug tr

ansp

orte

rs st

ill p

rese

nt a

nd o

pera

tiona

l

inta

ct c

ellu

lar i

nter

actio

ns

Mor

phol

ogic

al st

udie

s pos

sibl

e in

ter-

indi

vidu

al v

aria

tion

can

be st

udie

d

bile

form

atio

n th

ree

dim

ensi

onal

arc

hite

ctur

e

Tabl

e 7.

An

over

view

of d

iffer

ent i

n vi

tro

mod

els a

nd th

eir a

dvan

tage

s and

dis

adva

ntag

es

in v

itro

tech

niqu

es

hum

an C

YP

and

UG

T su

pers

omes

hum

an li

ver m

icro

som

es

hum

an li

ver c

ytos

ol

hum

an li

ver S

9 fr

actio

n

hum

an li

ver c

ell l

ines

trans

geni

c ce

ll lin

es

prim

ary

hepa

tocy

tes

liver

slic

es

isol

ated

per

fuse

d liv

er


25

argu

men

ts

1)

com

bina

tion

of e

nzym

es in

one

mod

el, C

YPs

and

UG

Ts in

m

icro

som

es a

nd so

lubl

e ph

ase

II in

cyt

osol

2)

on

e sp

ecifi

c is

ozym

e pr

esen

t 3)

co

mbi

natio

n of

pha

se I

and

II e

nzym

es

4)

inta

ct c

ell t

o st

udy

5)

inta

ct li

ver s

truct

ure

6)

only

for b

ile e

xcre

tion

1)

met

abol

ite o

f one

spec

ific

isoz

yme

(hig

her y

ield

) 2)

on

ly w

hen

isoz

yme

is n

ot p

rese

nt a

s iso

late

d en

zym

e 3)

on

ly w

hen

met

abol

ite is

com

bina

tion

of p

hase

I an

d II

bi

otra

nsfo

rmat

ion

1)

com

bina

tion

of e

nzym

es in

one

mod

el

2)

inte

ract

ion

at o

ne sp

ecifi

c is

ozym

e 3)

in

tact

cel

l

1)

CY

P po

lym

orph

ism

s in

supe

rsom

es a

vaila

ble

2)

from

one

pat

ient

that

show

s tha

t pol

ymor

phis

m

3)

from

one

pat

ient

that

show

s tha

t pol

ymor

phis

m

1)

inta

ct c

ell a

nd c

ytot

oxic

ity is

mea

sura

ble

2)

influ

ence

of o

ne o

r com

bina

tion

of e

nzym

es in

cyt

otox

icity

ca

n be

stud

ied

3)

inta

ct li

ver s

truct

ure

1)

diff

eren

t ani

mal

mod

els a

re a

vaila

ble

2)

rat C

YP

supe

rsom

es a

re a

vaila

ble

3)

diff

eren

t ani

mal

mod

els a

re a

vaila

ble

4)

inta

ct c

ell a

nd d

iffer

ent a

nim

al m

odel

s are

ava

ilabl

e

in v

itro

mod

el

1)

pool

ed m

icro

som

es a

nd c

ytos

ol (g

ende

r spe

cific

fr

actio

ns c

an b

e us

ed)

2)

supe

rsom

es a

nd N

AT

cyto

sol

3)

hum

an li

ver S

9 fr

actio

n 4)

(tr

ansg

enic

) cel

l lin

es a

nd p

rimar

y he

pato

cyte

s 5)

liv

er sl

ices

6)

pe

rfus

ed a

nim

al li

ver

1)

CY

P or

UG

T su

pers

omes

or N

AT

cyto

sol

2)

mic

roso

mes

or c

ytos

ol

3)

S9 fr

actio

n

1)

mic

roso

mes

or c

ytos

ol

2)

CY

P or

UG

T su

pers

omes

or N

AT

cyto

sol

3)

prim

ary

hepa

tocy

tes

1)

supe

rsom

es

2)

mic

roso

mes

or c

ytos

ol

3)

prim

ary

hepa

tocy

tes

1)

prim

ary

hepa

tocy

tes

2)

(tran

sgen

ic) c

ell l

ines

3)

liv

er sl

ices

1)

mic

roso

mes

and

/or c

ytos

ol

2)

supe

rsom

es

3)

S9 fr

actio

n 4)

pr

imar

y he

pato

cyte

s

Tabl

e 8.

An

over

view

of d

iffer

ent i

n vi

tro

mod

els t

o be

use

d at

diff

eren

t sta

ges i

n dr

ug b

iotra

nsfo

rmat

ion

rese

arch

drug

rese

arch

drug

bio

trans

form

atio

n

isol

atio

n m

etab

olite

s

drug

-dru

g in

tera

ctio

ns

influ

ence

of p

olym

orph

ism

s

toxi

city

of d

rugs

and

its m

etab

olite

s

choo

se a

nim

al a

s in

vivo

mod

el

Chapter 1

26

References 1. Koster A.S., de Mol N.J., Storm G., van Heuven-Nolsen D., van den Brink G., and

Perquin J. (1997). Introduction to Pharmacogenesis. Faculty of Pharmaceutical Sciences (Utrecht, The Netherlands), 1st edition.

2. Lu F.C. (1996). Toxicology - Fundamentals, Target Organs and Risk Assessment. Taylor and Francis (Washington DC, USA), 3rd edition: 3-39.

3. Moffett D.F., Moffett S.B., and Schauf C.L. (1993). Human Physiology. Mosby-Year Book Inc. (St. Louis, USA), 2nd edition: 626-629

4. Derelanko M.J. and Hollinger M.A. (1995). Handbook of Toxicology. CRC Press (New York, USA), 1st edition: 539-579.

5. Rang H.P., Dale M.M., and Ritter J.M. (1996). Pharmacology. Churchill Livingstone (London, UK), 3rd edition: 82.

6. Birkett D.J., MacKenzie P.I., Veronese M.E., and Miners J.O. (1993). In vitro approaches can predict human drug metabolism. Trends Pharmacol. Sci. 14: 292-294.

7. Crommentuyn K.M.L., Schellens J.H.M., van den Berg J.D., and Beijnen J.H. (1998). In-vitro metabolism of anti-cancer drugs, methods and applications: paclitaxel, docetaxel, tamoxifen and ifosfamide. Cancer Treat. Rev. 24: 345-366.

8. Schenkman J.B. and Greim H. (1993). Cytochrome P450. Springer Verlag (Berlin-Heidelberg, Germany), 1st edition.

9. Tucker G.T., Houston J.B., and Huang S.M. (2001). Optimizing drug development: strategies to assess drug metabolism/transporter interaction potential towards a consensus. Br. J. Clin. Pharmacol. 52: 107-117.

10. Zhang Y., Bachmeier C., and Miller D.W. (2003). In vitro and in vivo models for assessing drug efflux transporter activity. Adv. Drug Deliv. Rev. 55: 31-51.

11. Fricker G. and Miller D.S. (2002). Relevance of multidrug resistance proteins for intestinal drug absorption in vitro and in vivo. Pharmacol. Toxicol. 90: 5-13.

12. Ekins S., Ring B.J., Grace J., McRobie-Belle J., and Wrighton S.A. (2000). Present and future in vitro approaches for drug metabolism. J. Pharmacol. Toxicol. Methods 44, 313-324.

13. Fisher R.L., Gandolfi A.J., and Brendel K. (2001). Human liver quality is a dominant factor in the outcome of in vitro studies. Cell Biol. Toxicol. 17: 179-189.

14. http://www.eudra.org/emea.html. ICH Guideline S7A. The European Agency for the Evaluation of Medicinal Products. Human Medicines Evaluation Unit: London, UK (accessed March 2002).

15. http://www.fda.gov. U.S. Food and Drug Administration. Center for Drug Evaluation and Research (accessed March 2002).

16. Anderson D. and Russell T. (1995). The status of alternative methods in toxicology. The Royal Society of Chemistry (Cambridge, UK), 1st edition: 47.

17. Clark D.G. (1994). Barriers to the acceptance of in vitro alternatives. Toxicol. In Vitro 8: 907-909

18. Chen L., Buters J.T.M., Hardwick J.P., Tamura S., Penman B.W., Gonzalez F.J., and Crespi C.L. (1997). Coexpression of cytochrome P4502A6 and human NADPH-P450 oxidoreductase in the baculovirus system. Drug Metab. Dispos. 25: 399-405.

19. Araya Z. and Wikwall K. (1999). 6α-Hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes. Biochim. Biophys. Acta 1438: 47-54.

20. Taavitsainen P., Anttila M., Nyman L., Karnani H., Salonen J.S., and Pelkonen O. (2000). Selegeline metabolism and cytochrome P450 enzymes: in vitro study in human liver microsomes. Pharmacol. Toxicol. 86: 215-221.


27

21. Gentest, a BD Biosciences Company. http://www.gentest.com (accessed May 2002). 22. Yoa C., Kunze K.L., Kharasch E.D., Wang Y., Trager W.F., Ragueneau L., and

Levy R.H. (2001). Fluvoxamine-theophylline interaction: gap between in vitro and in vivo constants toward cytochrome P4501A2. Clin. Pharmacol. Ther. 70: 415-424.

23. Fisher M.B., Paine M.F., Strelevitz T.J., and Wrighton S.A (2001). The role of hepatic and extrahepatic UDP-glucuronosyltransferases in human drug metabolism. Drug Metab. Rev. 33: 273-297.

24. Huang Z., Roy P., and Waxman D.J. (2000). Role of human liver microsomal CYP 3A4 and CYP 2B6 in catalyzing N-dichloroethylation of cyclophosphamide and ifosfamide. Biochem. Pharmacol. 59: 961-972.

25. Rendic S., Nolteernsting E., and Schänzer W. (1999). Metabolism of anabolic steroids by recombinant human cytochrome P450 enzymes. Gas chromatographic-mass spectrometric determination of metabolites. J. Chromatogr. B 735: 73-83.

26. Gasser R., Funk C., Matzinger P., Klemisch W., and Vigerchouqnet A. (1999). Use of transgenic cell lines in mechanistic studies of drug metabolism. Toxicol.In Vitro 13: 625-632.

27. Pelkonen O., Kaltiala E.H., Larmi T.K.I., and Kärki N.T. (1974). Cytochrome P-450-linked monooxygenase system and drug-induced spectral interactions in human liver microsomes. Chem. Biol. Interact. 9: 205-216.

28. Olsen A.K., Hansen K.T., and Friis C. (1997). Pig hepatocytes as an in vitro model to study the regulation of human CYP 3A4: prediction of drug-drug interactions with 17α-ethynylestradiol. Chem. Biol. Interact. 107: 93-108.

29. Skaanild M.T. and Friis C. (2000). Expression changes of CYP2A and CYP3A in microsomes from pig liver and cultured hepatocytes. Pharmacol. Toxicol. 87: 174-178.

30. Bradford M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 7: 248-254.

31. Lowry O.H., Rosebrough N.J., Farr A.L., and Randall R.J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275.

32. Peterson G.L. (1977). A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83: 346-356.

33. Sidelmann U.G., Cornett C., Tjornelund J., and Hansen S.H. (1996). A comparative study of precision cut liver slices, hepatocytes and liver microsomes from the Wistar rat using metronidazole as a model substance. Xenobiotica 26: 709-722.

34. Favetta P., Guitton J., Degoute C.S., Van Deale L., and Boulieu R. (2000). High-performance liquid chromatographic assay to detect hydroxylate and conjugate metabolites of propofol in human urine. J. Chromatogr. B 742: 25-35.

35. Frandsen H. and Alexander J. (2000). N-acetyltransferase-dependent activation of 2-hydroxyamino-1-methyl-6-phenylimidazol[4,5-b]pyridine: formation of 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine, a possible biomarker for the reactive dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Carcinogenesis 21: 1197-1203.

36. Long L., Moschel R.C., and Dolan M.E. (2001). Debenzylation of O6-benzyl-8-oxoguanine in human liver: implications for O6-benzylguanine metabolism. Biochem. Pharmacol. 61: 721-726.

37. Maron D.M. and Ames B.N. (1983). Revised methods for the Salmonella mutagenicity test. Mutat. Res. 113: 173-215.

38. Hakura A., Suzuki S., and Satoh T. (1999). Advantage of the use of human liver S9 in the Ames test. Mutat. Res. 438: 29-36.

Chapter 1

28

39. Mae T., Inaba T., Konishi E., Hosoe K., and Hidaka T. (2000). Identification of enzymes responsible for rifalazil metabolism in human liver microsomes. Xenobiotica 30: 565-574.

40. Mandagere A.K., Thompson T.N., and Hwang K.-K. (2002). Graphical model for estimating oral bioavailability of drugs in humans and other species from their Caco-2 permeability and in vitro liver enzyme metabolic stability rates. J. Med. Chem. 45: 304-311.

41. Sumida K., Ooe N., Nagahori H., Saito K., Isobe N., Kaneko H., and Nakatsuka I. (2001). An in vitro reporter gene assay method incorporating metabolic activation with human and rat S9 or liver microsomes. Biochem. Biophys. Res. Commun. 280: 85-91.

42. Crommelin D.J.A., and Sindelar R.D. (1997). Pharmaceutical Biotechnology: an introduction for pharmacists and pharmaceutical scientists. Harwood Academic Publishers (New York, USA), 1st edition.

43. http://www.atcc.org. American Type Culture Collection (ATCC) (accessed June 2002). 44. Doostdar H., Duthie S.J., Burke M.D., Melvin W.T., and Grant M.H. (1988). The

influence of culture medium composition on drug metabolising enzyme activities of the human liver derived Hep G2 cell line. FEBS Letters 241: 15-18.

45. Fardel O., Morel F., Ratanasavanh D., Fautrel A., Beaune P., and Guillouzo A. (1992). Expression of drug metabolizing enzymes in human Hep G2 hepatoma cells. Cell. Molec. Aspects Cirrhosis 216: 327-330.

46. Galijatovic A., Otake Y., Walle U.K., and Walle T. (1999). Extensive metabolism of the flavonoid chrysin by human Caco-2 and Hep G2 cells. Xenobiotica 29: 1241-1256.

47. Urani C., Doldi M., Crippa S., and Camatini M. (1998). Human-derived cell lines to study xenobiotic metabolism. Chemosphere 37: 2785-2795.

48. Vickers A.E., Fischer V., Connors S., Fisher R.L., Baldeck J.P, Maurer G., and Brendel K. (1992). Cyclosporin A metabolism in human liver, kidney and intestine slices. Comparison to rat and dog slices and human cell lines. Drug Metab. Dispos. 20: 802-809.

49. Walle T., Otake Y., Galijatovic A., Ritter J.K., and Walle U.K. (2000). Induction of UDP-glucuronosyltransferase UGT1A1 by the flavonoid chrysin in the human hepatoma cell line Hep G2. Drug Metab. Dispos. 28: 1077-1082.

50. Gomez-Lechon M.J., Donato T., Jover R., Rodriguez C., Ponsoda X., Glaise D., Castell J.V., and Guguen-Guillouzo C. (2001). Expression and induction of a large set of drug-metabolizing enzymes by the highly differentiated human hepatoma cell line BC2. Eur. J. Biochem. 268: 1448-1459.

51. Almar M.M. and Dierickx P.J. (1990). In vitro interaction of mercury, copper (II) and cadmium with human glutathione transferase pi. Res. Commun. Chem. Pathol. Pharmacol. 69: 99-102.

52. Ricci M.S., Toscano D.G., Mattingly C.J., and Toscano W.A. (1999). Estrogen receptor reduces CYP1A1 induction in cultured human endometrial cells. J. Biol. Chem. 274: 3430-3438.

53. Wooster R., Ebner T., Sutherland L., Clarke D., and Burchell B. (1993). Drug and xenobiotic glucuronidation catalysed by cloned human liver UDP-glucuronosyltransferases stably expressed in tissue culture cell lines. Toxicology 82: 119-129.

54. Crespi C.L., Langenbach R., and Penman B.W. (1993). Human cell lines, derived from AHH-1 TK+/- human lymphoblasts, genetically engineerd for expression of cytochromes P450. Toxicology 82: 89-104.

55. Doehmer J. (1993). V79 Chinese Hamster cells genetically engineered for cytochrome P450 and their use in mutagenicity and metabolism studies. Toxicology 82: 105-118.


29

56. Philip P.A., Ali-Sadat S., Doehmer J., Kocarek T., Akhtar A., Lu H., and Chan K.K. (1999). Use of V79 cells with stably transfected cytochrome P450 cDNAs in studying the metabolism and effects of cytotoxic drugs. Cancer Chemother. Pharmacol. 43: 59-67.

57. Caro A.A. and Cederbaum A.I. (2001). Synergistic toxicity of Iron and arachidonic acid in HepG2 cells overexpressing CYP2E1. Molec. Pharmacol. 60: 742-752.

58. Dai Y. and Cederbaum A.I. (1995). Cytotoxicity of acetaminophen in human cytochrome P4502E1-transfected Hep G2 cells. J. Pharmacol. Exp. Ther. 273: 1497-1505.

59. Delescluse C., Ledirac N., Li R., Piechocki M.P., Hines R.N., Gidrol X., and Rahmani R. (2001). Induction of cytochrome P450 1A1 gene expression, oxidative stres, and genotoxicity by carbaryl and thiabendazole in transfected human HepG2 and lymphoblatoid cells. Biochem. Pharmacol. 61: 399-407.

60. Feierman D.E., Melnikov Z., and Zhang J. (2002). The paradoxical effect of acetaminophen on CYP3A4 activity and content in transfected Hep G2 cells. Arch. Biochem. Biophys. 398: 109-117.

61. Jover R., Bort R., Gomez-Lechon M.J., and Castell J.V. (1998). Re-expression of C/EBPα induces CYP2B6, CYP2C9 and CYP2D6 genes in HepG2 cells. FEBS Letters 431: 227-230.

62. Ono S., Tsutsui M., Gonzalez F.J., Satoh T., Masubuchi Y., Horie T., Suzuki T., and Narimatsu S. (1995). Oxidative metabolism of bunitrolol by complementary DNA-expressed cytochrome P450 isoenzymes in a human hepatoma cell line (Hep G2) using recombinant vaccinia virus. Pharmacogenetics 5: 97-102.

63. Cavin C., Mace K., Offord E.A., and Schilter B. (2001). Protective effects of coffee diterpenes against aflatoxin B1-induced genotoxicity: mechanisms in rat and human cells. Food Chem. Toxicol. 39: 549-556.

64. Hu M., Li Y., Davitt C.M., Huang S.-M., Thummel K., Penman B.W., and Crespi C.L. (1999). Transport and metabolic characterization of Caco-2 cells expressing CYP3A4 and CYP3A4 plus oxidoreductase. Pharm. Res. 16: 1352-1359.

65. Kawahara I., Kato Y., Suzuki H., Achira M., Ito K., Crespi C.L., and Sugiyama Y. (2000). Selective inhibition of human cytochrome P450 3A4 by N-[2(R)-hydroxy-1(S)-indanyl]-5-[2(S)-(1,1-dimethylethylaminocarbonyl)-4-[(furo[2,3-B]pyridin-5-yl)methyl] piperazin-1-yl]-4(S)-hydroxy-2(R)-phenylmethylpentanamide and P-glycoprotein by valspodar in gene transfectant systems. Drug Metab. Dispos. 28: 1238-1243.

66. Lewis C.W., Smith J.E., Anderson J.G., and Freshney R.I. (1999). Increase cytotoxicity of food-borne mycotoxins toward human cell lines in vitro via enhanced cytochrome p450 expression using the MTT bioassay. Mycopathologia 148: 97-102.

67. van Vleet T.R., Macé K., and Coulombe Jr. R.A. (2002). Comparative aflatoxin B1 activation and cytoxicity in human broncial cells expressing cytochromes P450 1A2 and 3A4. Cancer Res. 62: 105-112.

68. Cross D.M. and Bayliss M.K. (2000). A commentary on the use of hepatocytes in drug metabolism studies during drug discovery and development. Drug Metab. Rev. 32: 219-240.

69. Hengstler J.G., Utesch D., Steinberg P., Platt K.L., Diener B., Ringel M., Swales N., Fischer T., Biefang K., Gerl M., Böttger T., and Oesch F. (2000). Cryopreserved primary hepatocytes as a constantly available in vitro model for the evaluation of human and animal drug metabolism and enzyme induction. Drug Metab. Rev. 32: 81-118.

70. Puviani A.C., Ottolenghi C., Tassinari B., Pazzi P., and Morsiani E. (1998). An update on high-yield isolation methods and on the potential clinical use of isolated liver cells. Comp. Biochem. Physiol. A 121: 99-109.

Chapter 1

30

71. Howard R.B., Christensen A.K., Gibbs F.A., and Pesch L.A. (1967). The enzymatic preparation of isolated intact parenchymal cells from rat liver. J. Cell Biol. 35: 675-684.

72. Berry M.N., Edwards A.M., and Barritt G.J. (1991). Laboratory techniques in biochemistry and molecular biology: isolated hepatocytes, preparation, properties and applications. Burdon R.H. and P.H. van Knippenberg. Elsevier (Amsterdam, The Netherlands), 1st edition, volume 21.

73. Guyomard C., Chesne C., Meunier B., Fautrel A., Clerc C., Morel F., Rissel M., Campion J.P., and Guillouzo A. (1990). Primary culture of adult rat hepatocytes after 48-hour preservation of the liver with cold UW solution. Hepatology 12: 1329-1336.

74. Chenery R.J., Ayrton A., Oldham H.G., Standring P., Norman S.J., Seddon T., and Kirby R. (1987). Diazepam metabolism in cultured hepatocytes from rat, rabbit, dog, guinea pig, and man. Drug Metab. Dispos. 15: 312-317.

75. Le Bigot J.F., Begue J.M., Kiechel J.R., and Guillouzo A. (1987). Species differences in metabolism of ketotifen in rat, rabbit and man: demonstration of similar pathways in vivo and in cultured hepatocytes. Life Sci. 40: 883-890.

76. Bayliss M.K., Bell J.A., Jenner W.N., Park G.R., and Wilson K. (1999). Utility of hepatocytes to model species in the metabolism of loxtidine and to predict pharmacokinetic parameters in rat, dog and man. Xenobiotica 29: 253-268.

77. Berry M.N., Halls H.J., and Grivell M.B. (1992). Techniques for pharmacological and toxicological studies with isolated hepatocyte suspensions. Life Sci. 51: 1-16.

78. Cross D.M., Bell J.A., and Wilson K. (1995). Kinetics of ranitidine metabolism in dog and rat isolated hepatocytes. Xenobiotica 25: 367-375.

79. George J., Goodwin B., Liddle C., Tapner M., and Farrel G.C. (1997). Time-dependant expression of cytochrome P450 genes in primary cultures of well-differentiated human hepatocytes. J. Lab. Clin. Med. 129: 638-648.

80. Ammann P. and Maier P. (1997). Preservation and inducibility of xenobiotic metabolism in long-term cultures of adult rat liver cell aggregates. Toxicol. In Vitro 11: 43-56.

81. De Smet K., Brüning T., Blaszkewicz M., Bolt H.M., Vercruysse A., and Rogiers V. (2000). Biotransformation of trichloroethylene in collagen gel sandwich cultures of rat hepatocytes. Arch. Toxicol. 74: 587-592.

82. Jauregui H.O., Naik S., Santangini H., Pan J., Trenkler D., and Mullon C. (1994). Primary cultures of rat hepatocytes in hollow fiber chambers. In Vitro Cell. Dev. Biol. Anim. 30: 23-29.

83. Koebe H.G., Deglmann C.J., Metzger R., Hoerrlein S., and Schildberg F.W. (2000). In vitro toxicology in hepatocyte bioreactors-extracellular acidification rate (EAR) in a target cell line indicates hepato-activated transformation of substrates. Toxicology 154: 31-44.

84. Block G.D., Locker J., Bowen W.C., Petersen B.E., Katyal S., Strom S.C., Riley T., Howard T.A., and Michalopoulos G.K. (1996). Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF alpha in a chemically defined (HGM) medium. J. Cell Biol. 132: 1133-1149.

85. ‘t Hoen P.A.C., Commandeur J.N.M., Vermeulen N.P.E., van Berkel T.J.C., and Bijsterbosch M.K. (2000). Selective induction of cytochrome P450 3A1 by dexamethasone in cultured rat hepatocytes. Biochem. Pharmacol. 60: 1509-1518.

86. Sewer M.B. and Morgan E.T. (1997). Nitric oxide-independant suppresion of P450 2C11 expression by interleukin-1β and endotoxin in primary rat hepatocytes. Biochem. Pharmacol. 54: 729-737.


31

87. Vernia S., Beaune P., Coloma J., and López-García M.P. (2001). Differential sensitivity of rat hepatocyte CYP isoforms to self-generated nitric oxide. FEBS Letters 488: 59-63.

88. Milosevic N., Schawalder H., and Maier P. (1999). Kupffer cell-mediated differential down-regulation of cytochrome P450 metabolism in rat hepatocytes. Eur. J. Pharmacol. 368: 75-87.

89. Annaert P.P., Turncliff R.Z., Booth C.L., Thakker D.R., and Brouwer K.L. (2001). P-glycoprotein-mediated in vitro biliary excretion in sandwich-cultured rat hepatocytes. Drug Metab. Dispos. 29: 1277-1283.

90. Silva J.M., Day, S.H. and Nicoll-Griffith D.A. (1999). Induction of cytochrome-P450 in cryopreserved rat and human hepatocytes. Chem. Biol. Interact. 121: 49-63.

91. Krebs H.A. (1933). Untersuchungen über den Stoffwechsel der Aminosäuren im Tierkörper. Hoppe-Seyler’s Zeitschrift für physiologische Chemie 217: 190-227.

92. Ekins S. (1996). Past, present, and future applications of precision-cut liver slices for in vitro xenobiotic metabolism. Drug Metab. Rev. 28: 591-623.

93. Fell H.B. (1976) The development of organ culture. Organ Culture in Biomedical Research. Cambridge University Press (Cambridge, UK), 1st edition: 1-13.

94. Krumdieck C.L., dos Santos J.E., and Ho K.J. (1980). A new instrument for the rapid preparation of tissue slices. Anal. Biochem. 104: 118-23.

95. Bach P.H., Vickers A.E.M., Fisher R., Baumann A., Brittebo E., Carlile D.J., Koster H.J., Lake B.G., Salmon F., Sawyer T.W., and Skibinski G. (1996). The use of tissue slices for pharmacotoxicology studies. The report and recommendations of ECVAM Workshop 20. ATLA 24: 893-923.

96. Price R.J., Ball S.E., Renwick A.B., Barton P.T., Beamand J.A., and Lake B.G. (1998). Use of precision-cut rat liver slices for studies of xenobiotic metabolism and toxicity: comparison of the Krumdieck and Brendel tissue slicers. Xenobiotica 28: 361-371.

97. Olinga P., Merema M., Slooff M.J., Meijer D.K., and Groothuis G.M. (1997). Influence of 48 hours of cold storage in University of Wisconsin organ preservation solution on metabolic capacity of rat hepatocytes. J. Hepatol. 27: 738-743.

98. Hashemi E., Till, C. and Ioannides C. (2000). Stability of cytochrome P450 proteins in cultured precision-cut rat liver slices. Toxicology 149: 51-61.

99. Lake B.G., Ball S.E., Renwick A.B., Tredger J.M., Kao J., Beamand J.A., and Price R.J. (1997). Induction of CYP3A isoforms in cultured precision-cut human liver slices. Xenobiotica 27: 1165-1173.

100. Lake B.G., Charzat C., Tredger J.M., Renwick A.B., Beamand J.A., and Price R.J. (1996). Induction of cytochrome P450 isoenzymes in cultured precision-cut rat and human liver slices. Xenobiotica 26: 297-306.

101. Lake B.G., Tredger J.M., Renwick A.B., Barton P.T., and Price R.J. (1998). 3,3'-Diindolylmethane induces CYP1A2 in cultured precision-cut human liver slices. Xenobiotica 28: 803-811.

102. Smith P.F., Gandolfi A.J., Krumdieck C.L., Putnam C.W., Zukoski C.F., Davis W.M., and Brendel K. (1985). Dynamic organ culture of precision liver slices for in vitro toxicology. Life Sci. 36: 1367-1375.

103. Brendel K., Gandolfi A.J., Krumdieck C.L., and Smith P.F. (1987). Tissue slicing and culturing revisited. Trends Pharmacol. Sci. 8: 11-15.

104. Olinga P., Meijer D.K.F., Slooff M.J.H., and Groothuis G.M.M. (1997). Liver slices in in vitro pharmacotoxicology with special reference to the use of human liver tissue. Toxicol. In Vitro 12: 77-100.

105. Brauer R.W., Pessotti R.L., and Pizzolato P. (1959). Isolated rat liver preparation. Bile production and other basic properties. Proc. Soc. Expo. Biol. 78: 174-181.

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106. Alexander B., Aslam, M. and Benjamin I.S. (1998). The dependance of hepatic function upon sufficient oxygen supply during prolonged isolated rat liver perfusion. J. Pharmacol. Toxicol. Methods 39: 185-192.

107. Wu W.N., McKown L.A., Yorgey K.A., and Pritchard J.F. (1999). In vitro metabolic products of RWJ-34130, an antiarrythmic agent, in rat liver preparations. J. Pharm. Biomed. Anal. 20: 687-695.

33

CHAPTER In vitro characterization of the biotransformation of thiocoraline, a novel marine anti-cancer drug. Esther F.A. Brandon, Rolf W. Sparidans, Irma Meijerman, Ignasio Manzanares, Jos H. Beijnen, and Jan H.M. Schellens. Abstract

Thiocoraline is a potent new marine anti-cancer drug in vitro, which will be tested in phase I clinical studies shortly. To assess the biotransformation and the potential implications for human pharmacology and toxicology, the in vitro metabolism of thiocoraline was characterized using human plasma, human liver preparations, cytochrome P450 (CYP) and uridine diphosphoglucuronosyl transferase (UGT) supersomes, and human cell lines.

Thiocoraline was significantly metabolized by enzymes present in human plasma; t½ shifted from 25.2 h in phosphate buffered saline to 4.3 h in human plasma. Using CYP supersomes, it was shown that thiocoraline was mainly metabolized by CYP3A4, with CYP1A1, 2C8, and 2C9 playing a minor role in the biotransformation (< 3%). Only minor glucuronidation was observed for thiocoraline by UGT1A1 and UGT1A9 and no glucuronidation was observed in human liver S9 fraction. In addition, no glucosidation and sulfation were observed for thiocoraline in human liver cytosol and S9 fraction. However, the metabolites formed by cytochrome P450 were further conjugated by UGT, glutathione-S-transferase (GST), and sulfotransferase (SULT). In contrast to the CYP metabolism observed in supersomes, no effect could be observed from the CYP3A4 inhibitors on the cytotoxicity of thiocoraline in Hep G2 cells. However, this could be due to low CYP expression levels in the Hep G2 and IGROV-1 cell line.

These results provide evidence that human CYP3A4 plays a major role in the metabolism of thiocoraline in vitro and that the metabolites formed by CYP are conjugated by the phase II enzymes UGT, SULT, and GST.

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Introduction

Thiocoraline, a cyclic thiodepsipeptide, isolated from the marine microorganism Micromonospora marina L-13-ACM2-092, is a potent anti-tumor antibiotic (figure 1) [1, 2]. It has shown anti-proliferative activity against human non-small cell lung, colon, breast, renal, and melanoma cancer cell type subpanels in the in vitro screening program of the National Cancer Institute [3]. Further, thiocoraline is an effective antitumour agent against human carcinoma xenografts in vivo [4]. Thiocoraline causes an arrest in the G1 phase of the cell cycle and a decrease in the progression into the G2 phase. The mode of action is inhibition of DNA polymerase α, which results in the inhibition of DNA elongation at concentrations that inhibit cell cycle progression and clonogenicity [3]. Thiocoraline does not inhibit DNA topoisomerase I or II, nor does it induce DNA breakage [3, 4]. The biotransformation of thiocoraline has not yet been identified, but two potential degradation products or metabolites in plasma were observed during validation of a quantitative HPLC assay for thiocoraline [5]. Phase I clinical trials will soon be started. However, assessment of the biotransformation of thiocoraline is important in order to determine its pharmacological properties and the formation of metabolites with possible toxic or cytotoxic potential prior to execution of clinical anti-cancer studies. In this respect, it is also important to determine the thermal and chemical stability under physiological conditions in appropriate matrices.

NHN

OH

N

OH

NH

O

S S

O

N

N

NH

O

O

NH

N

O

N

O

S

O

S

O

SS

O O

M.W. = 1156 g/mol

= hydrolysis ofthioester

= hydroxylation

= conjugation

= S-glucuronidationC48H56N10O12S6

Figure 1. Chemical structure of thiocoraline [1]. The different circles and squares indicate potential sites for biotransformation based on Curry (1974) and Gibson and Skett (1995) [27, 28].

The biotransformation of thiocoraline is influenced by the presence of several potential sites for degradation, oxidation, hydrolysis, and conjugation (figure 1). The chemical degradation of thiocoraline was studied in aqueous solution and human plasma. The biotransformation of thiocoraline was evaluated using pooled human liver microsomes, human liver cytosol and S9 fraction, and human cancer cell lines. The contribution of the various isoforms of human cytochrome P450 (CYP) and uridine diphosphoglucuronosyl transferase (UGT) to the biotransformation of thiocoraline was also determined using supersomes. Furthermore, the cytotoxicity of the different metabolites formed was studied in inhibition studies of CYPs and esterases in the human cell lines Hep G2 and IGROV-1 (an ovarian carcinoma cell line, which is often used in cytotoxicity screening). The Hep G2 cell line, a hepatoma cell line, expresses several enzymes, namely CYP1A, 2B1, 2B2, 2B6, and

In vitro characterization of the thiocoraline biotransformation

35

3A, glutathione-S-transferase (GST), and UGT, but some expression levels are nearly undetectable. The cell line can be used for biotransformation studies, but is used more often for cytotoxicity studies [6-10]. The cytotoxicity of thiocoraline and the metabolites formed in human liver microsomes was studied in the Hep G2 and IGROV-1 cell line to screen for cytotoxic metabolites. Materials and methods

Materials. Thiocoraline was kindly donated by PharmaMar (Tres Cantos, Madrid, Spain). Acetonitrile (gradient grade) was purchased from Biosolve (Valkenswaard, The Netherlands) and formic acid (p.a.), MgCl2

.6H2O (p.a.) and dimethyl sulfoxide (DMSO, synthesis grade) from Merck (Darmstadt, Germany). Water was purified on a multi-laboratory scale by reversed osmosis. Pooled human liver microsomes, pooled human liver cytosol, pooled human liver S9 fraction, and human CYP and UGT supersomes (Baculovirus-insect-cell expressed) were provided by Gentest (Becton Dickinson, Woburn, MA, USA). Male CD-1 mice liver microsomes were obtained from XenoTech LLC (Kansas City, KS, USA) and normal mouse serum from CLB (Amsterdam, The Netherlands). Pooled human plasma was pooled from 3 different blank, drug-free human plasma donations obtained from Bloedbank Midden Nederland (Utrecht, The Netherlands). RPMI-1640 medium (with l-glutamine and 25 mM HEPES), heat-inactivated fetal calf serum, penicillin/streptomycin, and Hanks’ Balanced Salt Solution (pH 7.4) were all obtained from Gibco BRL (Breda, The Netherlands). All other chemicals were purchased from Sigma Chemical Company (St. Louis, MO, USA) and were of analytical grade.

Thiocoraline degradation in aqueous solution. Thiocoraline stock solution in DMSO was 1:1000 (v:v) diluted in phosphate buffered saline (PBS, pH 7.4) to yield a final concentration of 1 µg/ml. The aqueous solution was vortex-mixed and incubated at 37°C in a shaking water bath for 0 to 24 h. The solution was diluted 1:1 (v/v) with acetonitrile and the samples were then subjected to HPLC analysis. Control experiments were performed without thiocoraline.

Pooled human plasma and normal mouse serum incubations with thiocoraline in the absence and presence of esterase inhibitors. One µl PBS (1% (v/v) DMSO) or esterase inhibitor solution in PBS (1% (v/v) DMSO, final concentration of 200 µM) was pipetted into a polypropylene micro tube and 89 µl pooled human plasma or normal mouse serum were added. The following esterase inhibitors were examined for their effect on the metabolism of thiocoraline in human plasma: bis(p-nitrophenyl)phosphate (BNPP, a carboxyl esterase inhibitor) and phenylmethylsulfonyl fluoride (PMSF, a cholesterol esterase inhibitor). After vortex-mixing, the tubes were incubated at 37°C in a shaking water bath for 1 h. Next, 10 µl of an aqueous dilution of a thiocoraline stock solution (10 µg/ml, 1% (v/v) DMSO) were added. The tube was vortex-mixed briefly and incubated further at 37°C in a shaking water bath for 0 to 6 h and 0 to 4 h for human plasma and mouse serum, respectively. The reaction was terminated by adding 200 µl acetonitrile and vortex-mixing. The removal of proteins was accomplished by centrifuging the samples at approximately 15,000 g and 4°C for 5 min followed by injection of the supernatant into the HPLC system. Control experiments were performed without substrate and without human plasma or normal mouse serum using PBS as a substitute.

Chapter 2

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Thiocoraline incubations with pooled human liver microsomes and male CD-1 mice liver microsomes. The incubation procedure of thiocoraline with human and mice liver microsomes was a modification of the method described by Sparidans et al. (2001) [11]. Twenty-five µl of 0.5 M potassium phosphate buffer (pH 7.4) were pipetted into a polypropylene micro tube on ice and 50 µl NADP regenerating system (NRS: 1.5 U/ml glucose-6-phosphate dehydrogenase, 0.5 mg/ml β-NADP, 4.0 mg/ml D-glucose-6-phosphate in 0.6 % (w/v) NaHCO3), and 7.5 µl of 20 mg/ml MgCl2

.6H2O solution were added. After brief vortex-mixing, the tubes were incubated at 37°C in a shaking water bath for 2 min. Next, 5 µl of a pooled human liver microsomes suspension (lot number 18, pooled from 21 individuals) or male CD-1 mice liver microsomes (lot number 0010134, pooled from 400 mice) were added. The tube was vortex-mixed briefly again and 50 µl of an aqueous dilution of thiocoraline (1% (v/v) DMSO, final concentration range of 0.02-10 µg/ml in the microsomes suspension) were added. The mixture was then incubated at 37°C in a shaking water bath for 3 h. The reaction was terminated by adding 125 µl acetonitrile and vortex-mixing and the sample was centrifuged at approximately 15,000 g and 4°C for 1 min to remove proteins. The supernatant was injected for HPLC analysis. Control experiments were performed without substrate and without NRS, respectively.

Thiocoraline incubated with human CYP supersomes. Incubation with human CYP supersomes was performed according to the incubation method with liver microsomes. Instead of liver microsomes, 5 µl of the supersomes suspension were added. The following human CYP supersomes were tested: CYP1A1 (lot number 15), CYP1A2 (lot number 20), CYP2A6 (lot number 6), CYP2B6 (lot number 8), CYP2C8 (lot number 11), CYP2C9*1(Arg144) (lot number 17), CYP2C19 (lot number 12), CYP2D6*1 (lot number 27), CYP2E1 (lot number 9), CYP3A4 (lot number 40), and CYP4A11 (lot number 7). All CYPs were co-expressed with P450 reductase and CYP2A6, 2B6, 2C8, 2C9, 2C19, 2E1, and 3A4 were also co-expressed with cytochrome b5. The incubation was terminated after 3 h by adding 125 µl acetonitrile and vortex-mixing. The sample was centrifuged at approximately 15,000 g and 4°C for 1 min for the removal of proteins and the supernatant was injected for HPLC analysis. Control experiments were performed without substrate and without NRS or with insect cell control supersomes (lot number 22).

Human UGT supersomes incubations with thiocoraline. The incubation of thiocoraline with human UGT supersomes was a modification of the method described by Gentest [12]. Twenty µl of 0.5 M potassium phosphate buffer (pH 7.4) were pipetted into a polypropylene micro tube on ice and 10 µl of 0.5 mg/ml alamethicin, 20 µl of 20 mg/ml MgCl2

.6H2O, 20 µl of 20 mM uridine diphosphoglucuronic acid (UDPGA), and 70 µl water were added. After vortex-mixing briefly, the tubes were incubated at 37°C in a shaking water bath for 2 min. Next, 10 µl of the supersomes suspension were added. The following human UGT supersomes were tested: UGT1A1 (lot number 8), UGT1A3 (lot number 8), UGT1A9 (lot number 6), and UGT2B15 (lot number 5). After vortex-mixing briefly, 50 µl of an aqueous dilution of thiocoraline (final concentration of 2 µg/ml in the supersomes suspension) were added. The supersomes mixture was vortex-mixed and then incubated at 37°C in a shaking water bath for 3 h. The reaction was terminated by adding 200 µl acetonitrile and vortex-mixing. Next, the sample was centrifuged at approximately 15,000 g and 4°C for 1 min to remove proteins. Finally, the supernatant was subjected to HPLC analysis. Control experiments were performed without substrate and without UDPGA or with UGT insect cell control supersomes (lot number 5).


37

Glucuronidation of thiocoraline with UGT from rabbit liver. The glucuronidation of thiocoraline with rabbit UGT was a modification of the method described by Sparidans et al. (2001) [11]. Forty µl of 0.1 M magnesium dichloride, 10 µl of 0.5 mg/ml alamethicin, 50 µl rabbit UGT (lot 39H7848) in 0.5 M potassium phosphate buffer (pH 7.4), and 50 µl of 20 mM UDPGA were pipetted into a polypropylene micro tube on ice. After vortex-mixing briefly, 50 µl thiocoraline in water (1% (v/v) DMSO, final concentration of 2 µg/ml) were added. The tube was vortex-mixed again and incubated at 37°C for 4 h. The reaction was terminated by adding 200 µl acetonitrile and vortex-mixed briefly. Proteins were removed by centrifugation for 1 min at approximately 15,000 g and 4˚C and the supernatant was subjected to HPLC analysis. Individual control experiments were performed without substrate, without UDPGA and without UGT, respectively.

Pooled human liver cytosol incubations with thiocoraline. The incubation of thiocoraline with pooled human liver cytosol was a modification of the method described by Gentest [12]. Equal volumes (20 µl) of 1 M potassium phosphate buffer (pH 7.4), 1 mM acetyl-coenzyme A (acetyl-CoA), 45 mM acetyl-DL-carnitine, 80 units/ml carnitine acetyl transferase (from pigeon breast muscle), 1 mM adenosine 3’-phosphate 5’-phosphosulfate (PAPS), and 10 mM glutathione were pipetted into a polypropylene micro tube on ice. Twenty-six µl H2O and 4 µl human liver cytosol (lot number 2) were added and vortex-mixed briefly. Next, 50 µl of an aqueous dilution of thiocoraline (1% (v/v) DMSO), final concentration of 2 µg/ml in the cytosol suspension) were added and, after vortex-mixing briefly, the mixture was incubated at 37°C in a shaking water bath for 3 h. The reaction was terminated by adding 200 µl acetonitrile and vortex-mixing. Samples were centrifuged at approximately 15,000 g and 4°C for 1 min to remove proteins and the supernatant was analyzed by HPLC. Individual control experiments were performed without substrate, only acetyl-CoA, acetyl-DL-carnitine and carnitine acetyl transferase (only N-acetyltransferase (NAT) activity), only PAPS (only sulfotransferase (SULT) activity), only glutathione (only glutathione-S-transferase (GST) activity), and without all co-factors for enzyme activity. In addition, all three substrates of NAT were individually tested as control.

Human liver S9 fraction incubations with thiocoraline. The incubation of thiocoraline with pooled human liver S9 fraction was a modification of the method described by Gentest [12]. Equal volumes (10 µl) of 1 M potassium phosphate buffer (pH 7.4), 15 mg/ml UDPGA, 1 mM PAPS, and 10 mM glutathione were pipetted into a polypropylene micro tube on ice. Twelve µl NRS (5 U/ml glucose-6-phosphate dehydrogenase, 1.67 mg/ml β-NADP, and 13.33 mg/ml D-glucose-6-phosphate in 2% (w/v) NaHCO3), 6 µl of 20 mg/ml MgCl2

.6H2O, and 12 µl H2O were added and vortex-mixed briefly. Subsequently, the tubes were incubated at 37°C in a shaking water bath for 2 min. Five µl pooled human liver S9 fraction (lot number 5) were added and vortex-mixed. Next, 25 µl of an aqueous dilution of thiocoraline (1% (v/v) DMSO, final concentration of 2 µg/ml in the S9 suspension) were added and, after vortex-mixing briefly, the mixture was incubated at 37°C in a shaking water bath for 3 h. The reaction was terminated by adding 100 µl acetonitrile and vortex-mixing. The sample was centrifuged at approximately 15,000 g and 4°C for 1 min and the supernatant was then injected for HPLC analysis. Individual control experiments were performed without substrate, without all co-factors for enzyme activity and with only one or two co-factors present (so only one or a combination of two enzymes was active), respectively.

Analysis of thiocoraline and potential metabolites by HPLC with fluorescence detection. The chromatographic assay was a modification of the method described by Sparidans et al. (1999) [5]. The supernatant of the incubated mixtures was analyzed on an HPLC system consisting of two LC-10ATVP pumps, a SIL-10ADVP autoinjector (equipped with a 500 µl sample loop), a SCL-10AVP system controller (all from Shimadzu, Kyoto, Japan), and a FP-920 fluorescence detector (Jasco, Tokyo, Japan). The column was

Chapter 2

38

thermostated by a Waters temperature control module and a Waters column heater module (Milford, MA, USA). Data were recorded on a Hermac Pentium 440, 122 MB personal computer (Scherpenzeel, The Netherlands) equipped with the Class-VP 5.032 software (Shimadzu). The eluent flow rate was 1.0 ml/min and the fluorescence detection wavelengths were 365 nm for excitation and 540 nm for emission. Injections (50 µl) were made on a Symmetry C18 column (4.6 x 100 mm, dp=3.5 µm, Waters Chromatography, Milford, MA, USA) with a Sentry Guard Symmetry C18 pre-column (3.9 x 20 mm, dp=5 µm, Waters). The column temperature was maintained at 40°C. A gradient program was used with eluent A comprising 10 mM formic acid in water and eluent B comprising 10 mM formic acid in acetonitrile. After injection, elution started with 20% B for 2 min. Next, the eluent composition was raised linearly to 80% B during 21 min. This percentage was the maintained for 2 min before conditioning with 20% B for 15 min.

Cell culture growth. The human hepatic carcinoma cell line (Hep G2) and the human ovarian adenocarcinoma cell line (IGROV-1) were kindly donated by the Netherlands Cancer Institute (Amsterdam, The Netherlands). Routine cultivation of these monolayer cells was performed in RPMI-1640 medium (with L-glutamine and 25 mM HEPES) supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were sub-cultured weekly (ratio of 1:5 (v/v) and 1:25 (v/v) for Hep G2 and IGROV-1 cells, respectively) and the medium was refreshed after 3 days.

Cytotoxicity of thiocoraline in Hep G2 and IGROV-1 cells in the absence and presence of esterase inhibitors or cytochrome P450 inhibitors. The cytotoxicity of the esterase and CYP inhibitors was determined for both cell lines and inhibitor concentrations below the IC5 were used in further experiments. For the determination of the cytotoxicity of thiocoraline in the absence and presence of inhibitors, cells were seeded onto 96-well microtitre plates at a concentration of 4000 and 1250 cells/well (volume is 200 µl/well), for the Hep G2 cell line and IGROV-1 cell line, respectively. After 48 h, the cells were incubated with inhibitor. The esterase inhibitors BNPP and PMSF and the following CYP inhibitors were examined for their effect on the cytotoxicity of thiocoraline: furafylline (CYP1A2), ketoconazole (CYP1A1, 2A6, 2C8, 2C19, 2D6, and 3A4), metyrapone (CYP2A6 and 3A4), piperonyl butoxide (CYP3A), proadifen (CYP2A6, 2B6, 2C9, 2E1, and 3A4), and sulfaphenazole (CYP2C9) [13]. For the incubation with inhibitors 20 µl/well of cell culture medium was removed and replaced by 20 µl inhibitor solution (in medium with 1% (v/v) DMSO). The cells were incubated with 10 µM furafylline, 5 µM ketoconazole, 200 µM metyrapone, 10 µM piperonyl butoxide, 10 µM proadifen, 200 µM sulfaphenazole, 75 µM BNPP, or 200 µM PMSF in Hep G2 respectively. The same concentrations were employed in the IGROV-1 cells except for proadifen (2.5 µM) and BNPP (100 µM). The cells were incubated for 1 h at 37°C and 5 % CO2. Next, the cells were exposed to thiocoraline at concentrations of 0-500 ng/ml and 0-333.3 ng/ml for Hep G2 and IGROV-1, respectively, for 5 days. Therefore, 100 µl of a 1000 or 1500 ng/ml thiocoraline solution with inhibitor was added to the wells in one column and from this column serial dilutions were made into the microtitre plate. Cell growth was determined at day 5 using the sulforhodamine B assay (SRB assay). Cell survival (%) was calculated relatively to control cells and 100% killed cells (killed with 10% Triton-X-100 1 h prior to the SRB assay). Concentration-viability curves were constructed from this data and the IC50 (concentration of compound giving 50 % survival) was calculated by the Softmax®Pro 3.1 software (Molecular Devices, Sunnyvale, CA, USA). Control experiments were performed without thiocoraline and without inhibitor, respectively.


39

Sulforhodamine B assay (SRB assay). The SRB assay was a modification of the method described by Higgins et al. (1993) [14]. The cell culture medium was removed and the cells were fixed in 100 µl of 10% (w/v) trichloroacetic acid for 60 min at 4°C. The wells were rinsed three times with tap water to remove solutes and cells were stained with 50 µl of 0.4% (w/v) sulforhodamine B (SRB) in 1% (v/v) acetic acid for 15 min. The cells were washed three times with 1% (v/v) acetic acid and air-dried. After drying, 120 µl of 10 M Tris in Hanks’ Balanced Salt Solution (pH 7.4) were added to solubilize the protein bound SRB. After mixing, the absorbance was measured at 540 nm using a Versamax microtitre plate reader (Molecular Devices, Sunnyvale, CA, USA). Data were recorded and analyzed on a Hermac Pentium 440, 122 MB personal computer (Scherpenzeel, The Netherlands) equipped with the Softmax®Pro 3.1 software (Molecular Devices).

Data analysis. The results are expressed as mean ± standard deviation (SD). Differences between the results were analyzed by the student t-test for paired and unpaired observations, when appropriate. P < 0.05 was considered as statistically significant. Results

Half-life of thiocoraline in PBS, pooled human plasma and normal mouse serum. The data in figure 2 show that thiocoraline has a half-life (t½) of 25.2 ± 1.5 hours in PBS and only of 4.31 ± 0.30 h and 0.61 ± 0.02 h in respectively pooled human plasma and normal mouse serum. Degradation products of thiocoraline in PBS could be observed after 24 h, but were not identified because the isolation using HPLC was not successful. The t½ of thiocoraline in pooled human plasma in the presence of the carboxyl esterase inhibitor BNPP and the cholesterol esterase PMSF did not significantly increase compared to pooled human plasma without esterase inhibitors. However, in mouse serum, BNPP was able to increase the t½ of thiocoraline from 0.61 ± 0.02 h to 1.18 ± 0.06 h (p < 0.05). Incubations of thiocoraline with mouse serum in the presence of PMSF did not show a difference in the t½ of thiocoraline. In addition to chemical degradation, carboxyl esterases are probably responsible for the conversion of thiocoraline in mouse serum.

Figure 2. The half-life of thiocoraline (1 µg/ml) in PBS, pooled human plasma, and normal mouse serum in the absence and presence of the esterase inhibitors BNPP or PMSF at 37°C. The half-life (t½) was determined from 10 time points in five-fold for PBS and 4 time points in five-fold for human plasma in both the absence and presence of BNPP and PMSF, respectively. The calculation of t½ in mouse serum was based on 3 time points in the absence of esterase inhibitors and in the presence of PMSF and from 5 time points in the presence of BNPP (all time points were in triplicate); bars indicate the SD. The amount of thiocoraline was measured using HPLC analysis with fluorescence detection and the thiocoraline peak area at 0 hours was set as 100%. * significantly different (p < 0.05) compared to normal mouse serum.

PBS human plasma mouse serum0123456

no inhibitor+ BNPP+ PMSF

2022242628

half

-lif

e (h

)

*

Chapter 2

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Comparison of the biotransformation in pooled human liver microsomes and male CD-1 mice liver microsomes. Figure 3A shows the % biotransformation of thiocoraline at different concentrations in incubations with pooled human liver microsomes after 3 h at 37°C. A concentration of 2 µg/ml thiocoraline showed to be the appropriate concentration for further experiments (50% metabolized after 3 h). The difference between biotransformation of 2 µg/ml of thiocoraline in pooled human liver microsomes and male CD-1 mice liver microsomes after 3 h at 37°C is shown in figure 3B. Pooled human liver microsomes metabolized 59.4 ± 0.3 % of the thiocoraline in 3 h and male CD-1 mice liver microsomes only 35.5 ± 5.2 %. Figure 3. Percentages biotransformation at different thiocoraline concentrations (0.1-10 µg/ml) after incubation with pooled human liver microsomes for 3 h at 37°C (A) and the percentages biotransformation of thiocoraline after 3 h at 37°C of 2 µg/ml thiocoraline by pooled human liver microsomes (HLM) or mouse CD-1 male liver microsomes (MLM) (B). The amount of thiocoraline was analyzed using HPLC with fluorescence detection. The percentage biotransformation was determined using an incubation of thiocoraline with HLM or MLM without NRS (only degradation). Each point/column is the mean of 3 replicates; bars indicate the SD. * significantly different (p < 0.05).

Thiocoraline biotransformation by human CYP supersomes. Incubation of thiocoraline with human CYP3A4 supersomes at 37°C for 3 h reduced the amount of thiocoraline significantly with 44.0 ± 2.3 %. Furthermore, incubations with CYP1A1, 2C8, and 2C9 showed a slight decrease in the thiocoraline amount, respectively 98.5 ± 1.9 %, 97.8 ± 1.3 %, and 97.0 ± 3.1 % of the thiocoraline were recovered after 3 h, but only the decrease after incubation with CYP2C8 showed to be significant.

A

0

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40

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concentration thiocoraline (µg/ml)

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% b

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41

Glucuronidation of thiocoraline by human UGT supersomes and rabbit UGT. Thiocoraline was not glucuronidated by human UGT1A1, 1A9, and rabbit UGT during 3 h at 37°C. However, incubations of thiocoraline with UGT1A3 and 2B15 supersomes showed a slight decrease in the thiocoraline concentrations; 91.1 ± 1.8% and 94.0 ± 8.2% were respectively recovered after 3 h at 37°C, but this was not significant.

Biotransformation of thiocoraline in pooled human liver cytosol and pooled human liver S9 fraction. Thiocoraline was metabolized by cytochrome P450 in pooled human liver S9 fraction, followed by phase II metabolism (figure 4). The enzymes sulfotransferase and glutathione-S-transferase did not metabolize thiocoraline directly in pooled human liver cytosol and pooled human liver S9. The CYPs present in the pooled human liver S9 fraction metabolized 38.3 ± 3.3 % of the thiocoraline during 3 h at 37°C. CYP activity in combination with the individual phase II enzymes UGT, SULT, and GST resulted in a reduction of thiocoraline with 52.0 ± 3.5, 50.7 ± 2.1, and 56.3 ± 4.9 %, respectively. When all the enzyme substrates were present, 57.6 ± 4.8 % of the thiocoraline was metabolized by pooled human liver S9 fraction. Thus, thiocoraline was metabolized by CYP and the formed thiocoraline metabolites were conjugated by the phase II enzymes UGT, SULT, and GST. The phase II enzymes studied did not directly metabolize thiocoraline. The metabolism of thiocoraline or its phase I metabolites by N-acetyl-transferase could not be studied due to insolubility of thiocoraline in the presence of the NAT substrates carnitine acetyl transferase and acetyl CoA (results not shown).

0

20

40

60

80

100

120

cytosolcontrol

SULT incytosol

GST incytosol

S9control

CYP inS9

UGT inS9

SULT +GST in

S9

CYP +UGT in

S9

CYP +SULT in

S9

CYP +GST in

S9

CYP +UGT +SULT +GST in

S9

% th

ioco

ralin

e re

mai

ning

**! *! *! *!

Figure 4. Comparison of the biotransformation of thiocoraline by SULT and GST in human liver cytosol and S9 fraction and CYP in combination with UGT, SULT, and GST in human liver S9 fraction. The amount of thiocoraline was analyzed using HPLC with fluorescence detection. The percentage remaining was determined using a thiocoraline incubation with cytosol or S9 fraction without enzyme substrates. Each column is the mean of 3 replicates; bars indicate the SD. * significantly different (p < 0.05) compared to S9 control and ! significantly different (p < 0.05) compared to CYP in S9.

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Cytotoxicity of thiocoraline in Hep G2 and IGROV-1 cells in the absence and presence of esterase and CYP inhibitors. Thiocoraline has an IC50 value of 11 ± 9 ng/ml in the Hep G2 cell line and 2.9 ± 1.6 ng/ml in the IGROV-1 cell line (IC50 values were significantly different (p < 0.05)). No effect of the different (iso)enzyme inhibitors could be observed in both the Hep G2 and the IGROV-1 cell line. Discussion and conclusions

Thiocoraline is a novel anti-cancer agent with promising activity in a range of preclinical models. Thiocoraline has been selected for clinical testing and therefore we conducted studies to unravel its degradation and biotransformation in humans. This information is indispensable before starting clinical studies with the drug. From these results predictions about pharmacokinetics and drug-drug interaction may be made. In the present investigation the degradation at 37°C was quantified and the enzymatic break-down of thiocoraline, using different in vitro techniques, was elucidated.

This study showed that the half-life of thiocoraline in pooled human plasma and normal mouse serum is much shorter than in PBS. Also the half-life of thiocoraline in normal mouse serum is much shorter compared to pooled human plasma. In vivo experiments with thiocoraline showed a short plasma half-life in mice compared to other species (“PharmaMar, personal communication”). The presented in vitro results are comparable to these previous in vivo results. The difference between PBS and plasma/serum is due to the presence of enzymes in the plasma/serum. The enzyme concentrations and composition differs between mouse serum and human plasma, explaining the differences between mouse serum and human plasma. The carboxyl esterase inhibitor BNPP increases the half-life of thiocoraline in mouse serum, while the cholesterol esterase inhibitor PMSF had no influence. This indicates that carboxyl esterases are partly responsible for the clearance of thiocoraline from mouse serum. However, both BNPP and PMSF had no influence on the half-life of thiocoraline in human plasma. The ester-bond in thiocoraline is most likely subject to de-esterification, but the esterases responsible for the clearance in human plasma could not be identified. It is possible that the carboxyl esterases play a role in the clearance in human plasma, but also other esterases could be responsible. Lower carboxyl esterase levels in human plasma compared to mouse serum could explain the lack of effect of BNPP. In addition, a too high concentration of thiocoraline (several times Km) could result in low biotransformation percentages in human plasma, which were too small to be observed.

The pre-dominant cytochrome P450 isozymes in the human liver are CYP2C9 and 3A4 [15-17]. Based on the results found in CYP supersomes, it is most likely that CYP3A4 is the main CYP isozyme responsible for the biotransformation of thiocoraline in the liver. CYP2C8 plays a minor role in the biotransformation of thiocoraline.

The differences in biotransformation percentages between pooled human liver microsomes, pooled human liver S9 fraction, human CYP3A4 supersomes and male CD-1 mice liver microsomes are the result from the differences in CYP3A activity (testosterone 6β-hydroxylase activity, which was provided by Gentest). The CYP3A enzyme activities in order of activity were: pooled human liver microsomes > human CYP3A4 supersomes > male CD-1 mice liver microsomes > pooled human liver S9 fraction respectively.

The risk of drug-drug interactions with thiocoraline and other CYP3A4 substrates is present [18]. The clinical relevance should be determined in patients. Gender differences are observed in CYP3A4 activity; it exhibits higher activity in female than in male [19]. Nonetheless, this is not always of clinical importance [17, 19], but attention should be paid to side-effects and toxicity arising from gender-differences during clinical trials. Also other


43

factors play a role in the individual CYP3A4 activity like aging, disease, and genetic polymorphisms. Finally, human cancers, including colon, breast, lung, liver, kidney and prostate, are known to express cytochrome P450 isoforms including CYP3A [20]. This may result in a decreased anti-tumor activity of thiocoraline if the thiocoraline metabolites are not active. Therefore, in clinical trials with cancer patients attention should be paid to the individual pharmacokinetics and toxicity of thiocoraline.

Thiocoraline itself is only slightly metabolized by UGT1A3 and 2B15 in human UGT supersomes and no glucuronidation could be observed in pooled human liver S9 fraction and rabbit UGT. The other phase II enzymes studied, sulfotransferase and glutathione-S-transferase, did not directly metabolize thiocoraline in pooled human liver cytosol and S9 fraction. However, the thiocoraline metabolites formed by cytochrome P450 were further conjugated by all phase II enzymes studied.

In patients, inter-individual variability in conjugation is high for the different phase II enzymes, due to aging, gender, disease, drug or food intake and genetic polymorphisms. Polymorphisms have been identified for all the phase II enzymes studied and these polymorphisms can lead to significant lower activity levels of the different enzymes [21-23]. There are also a few reports of gender-differences in conjugation pathways [19]. Pharmacokinetics and toxicity of thiocoraline in cancer patients caused by phase II enzymes should be carefully studied during clinical trials. However, the CYP-mediated step is probably the rate-limiting elimination step and potential differences in glucuronidation, sulfation and glucosidation activities may not be detected [19]. This should be assessed during clinical phase I studies. It could be that N-acetyl transferase also plays a role in the biotransformation of thiocoraline or one of its metabolites, however, this could not be studied due to insolubility of thiocoraline in the presence of the substrates necessary for NAT activity in cell fractions.

The IC50 value of thiocoraline was higher in Hep G2 compared to IGROV-1 cells. No significant influence of the different esterase and CYP inhibitors on the cytotoxicity could be observed in both cell lines. This could be caused by the high standard deviation due to the differences in cytotoxicity between cell passages, but also by low enzyme levels or lack of enzymes in both cell lines. Hep G2 cells are known to express CYP1A, 2B6, 2C9, 2E1, and 3A4, but under standard culturing conditions they are nearly undetectable [8, 10]. However, other compounds tested showed differences in IC50 values in the presence and absence of CYP3A4 inhibitors (results not shown) indicating that CYP3A4 activity is present. Also, Hep G2 cells are known to express UGT, NAT, GST, and SULT, but at low activity levels [8, 24]. There is no literature describing the presence of CYP, UGT, or SULT enzymes in the IGROV-1 cell line, only GST was reported [25, 26]. Probably CYP isozymes are not present or at very low levels in IGROV-1 cells. The presence of the different CYP enzymes and their activity will be determined and validated in future studies.

No effect could be observed from BNPP, a carboxyl esterase inhibitor, and PMSF, a cholesterol esterase inhibitor, in both cell lines. Carboxyl esterases most likely have only a minor role in the biotransformation of thiocoraline, however nothing is known about the esterase activity in the Hep G2 and IGROV-1 cell line. Therefore, low activity levels or absence of esterases could result in a lack of effect of esterase inhibitor on the cytotoxicity of thiocoraline in both cell lines.

The cytotoxicity of the thiocoraline metabolites formed in pooled human liver microsomes could not be studied, because the thiocoraline could not be obtained sterile and without pooled human liver proteins after incubation with liver microsomes using ultra-filtration due to 100% protein binding (results not shown). However, the cytotoxicity of the metabolites could be studied if metabolites are isolated using the chromatographic assay after incubation with pooled human liver microsomes or S9 fraction. Some thiocoraline

Chapter 2

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metabolites were observed after incubation with pooled human liver microsomes, but concentrations were too low for correct isolation for off-line MS analysis using the current chromatographic assay. HPLC with online MS analysis may be a better choice for the identification of metabolites planned in the near future.

In conclusion, thiocoraline has a short half-life in human plasma of 4.3 h. Thiocoraline metabolism in human liver microsomes and S9 fractions is catalyzed by cytochrome P450. CYP3A4 is the predominant CYP metabolizing thiocoraline in human CYP supersomes. The thiocoraline metabolites formed by CYP are further metabolized by phase II enzymes, namely UGT, SULT, and GST. Thiocoraline showed high cytotoxicity in Hep G2 and IGROV-1 cell lines, which could not be altered using CYP and esterase inhibitors. These results may form a good basis for assessment of the pharmacokinetics and biotransformation of thiocoraline in patients.


45

References 1. Perez Baz J., Canedo L.M., Fernandez Puentes J.L., and Silva Elipe M.V. (1997).

Thiocoraline, a novel depsipeptide with antitumor activity produced by a marine micromonospora II. Physico-chemical properties and structure determination. J. Antibiotics 50: 738-741.

2. Romero F., Espliego F., Perez Baz J., Garcia De Quesada T., Gravalos D., De La Calle F., and Fernandez Puentes J.L. (1997). Thiocoraline, a new depsipeptide with antitumor activity produced by a marine micromonospora I. Taxonomy, fermentation, isolation and biological activities. J. Antibiotics 50: 734-737.

3. Erba E., Bergamaschi D., Ronzoni S., Faretta M., Taverna S., Bonafanti M., Catapano C.V., Faircloth G., Jimeno J., and D’Incalci M. (1999). Mode of action of thiocoraline, a natural marine compound with anti-tumour activity. Br. J. Cancer 80: 971-980.

4. Faircloth G., Jimeno J., and D’Incalci M. (1997). Biological activity of thiocoraline, a novel marine depsipeptide (abstract). Eur. J. Cancer 33: 175.

5. Sparidans R.W., Henrar R.E.C., Jimeno J.M., Faircloth G., Floriano P., and Beijnen J.H. (1999). Bioanalysis of thiocoraline, a new marine antitumoral depsipeptide, in plasma by high-performance liquid chromatography and fluorescence detection. J. Chromatogr. B 726: 255-260.

6. Sassa S., Sugita O., Galbraith R.A., and Kappas A. (1987). Drug metabolism by the human hepatoma cell, Hep G2. Biochem. Biophys. Res. Commun. 143: 52-57.

7. Doostdar H., Burke M.D., Melvin W.T., and Grant M.H. (1991). The effects of dimethylsulphoxide and 5-aminolevulinic acid on the activities of cytochrome P450-dependent mixed function oxidase and UDP-glucuronosyltransferase activities in human Hep G2 hepatoma cells. Biochem. Pharmacol. 42: 1307-1313.

8. Fardel O., Morel F., Ratanasanh D., Fautrel A., Beaune P., and Guillouzo A. (1992). Expression of drug metabolizing enzymes in human HepG2 hepatoma cells. Cell.Molec. Aspects Cirrhosis 216: 327-330.

9. Rueff J., Chiapella C., Chipman J.K., Darroudi F., Duarte Silva I., Duverger-van Bogaert M., Fonti E., Glatt H.R., Isern P., Laires A., Leonard A., Llagostera M., Mossesso P., Natarajan A.T., Palitti F., Rodrigues A.S., Schinoppi A., Turchi G., and Werle-Schneider G. (1996). Development and validation of alternative metabolic systems for mutagenicity testing in short-term assays. Mutat. Res. 353: 151-176.

10. Sumida A., Fukuen S., Yamamoto I., Matsuda H., Naohara M., and Azuma J. (2000). Quantative analysis of constitutive and inducable CYPs mRNA expression in the HepG2 cell line using reverse transcription-competitive PCR. Biochem. Biophys. Res. Commun. 267: 756-760.

11. Sparidans R.W., Rosing H., Hillebrand M.J.X., López-Lázaro L., Jimeno J.M., Manzanares I., van Kesteren Ch., Cvitkovic E., van Oosterom A.T., Schellens J.H.M., and Beijnen J.H. (2001). Search for metabolites of ecteinascidin 743, a novel, marine-derived, anticancer agent, in man. Anti-Cancer Drugs 12: 653-666.

12. Gentest, a BD Biosciences Company. http://www.gentest.com (accessed March 2002). 13. Gentest Cytochrome P450 database. http://www.gentest.com/human_p450_database

(accessed January 2001). 14. Higgins III J.D., Neely L., and Fricker S. (1993). Synthesis and cytotoxicity of some

cyclometallated palladium complexes. J. Inorg. Biochem. 49: 149-156. 15. Whrighton S.A. and Stevens J.C. (1992). The human hepatic cytochromes P450 involved

in drug metabolism. Crit. Rev. Toxicol. 22: 1-21.

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17. Wilkinson G.R. (1996). Cytochrome P4503A (CYP3A) metabolism: prediction of in vivo activity in humans. J. Pharmacokinet. Biopharm. 24: 475-490

18. MacLeod S.L., Nowell S., Massengill J., Jazieh A., McClure G., Plaxco J., Kadlubar F.F., and Lang N.P. (2000). Cancer therapy and polymorphisms of cytochromes P450. Clin. Chem. Lab. Med. 38: 883-887.

19. Tanaka E. (1999). Gender-related differences in pharmacokinetics and their clinical signifficance. J. Clin. Pharm. Ther. 24: 339-346.

20. Patterson L.H., McKeown S.R., Robson T., Gallagher R., Raleigh S.M., and Orr S. (1999). Antitumour prodrug develepment using cytochrome P450 (CYP) mediated activation. Anticancer Drug Des. 14: 473-486.


22. Nishiyama T., Ogura K., Nakano H., Ohnuma T., Kaku T., Hiratsuka A., Muro K., and Watabe T. (2002). Reverse geometrical selectivity in glucuronidation and sulfation of cis- and trans-4-hydroxytamoxifens by human liver UDP-glucuronosyltransferases and sulfotransferases. Biochem. Pharmacol. 63: 1817-1830.

23. Miners J.O. and MacKenzie P.I. (1991). Drug glucuronidation in humans. Pharmacol. Ther. 51: 347-369.

24. Grant H., Duthie S.J., Gray A.G., and Burke D. (1988). Mixed function oxidase and UDP-glucuronyltransferase activities in the human Hep G2 hepatoma cell line. Biochem. Pharmacol. 37: 4111-4116.

25. Ferretti A., D'Ascenzo S., Knijn A., Iorio E., Dolo V., Pavan A., and Podo F. (2002). Detection of polyol accumulation in a new ovarian carcinoma cell line, CABA I: a 1H NMR study. Br. J. Cancer 86: 1180-1187.

26. Perego P., Paolicchi A., Tongiani R., Pompella A., Tonarelli P., Carenni N., Romanelli S., and Zunino F. (1997). The cell-specific anti-proliferative effect of reduced glutathione is mediated by gamma-glutamyl transpeptidase-dependent extracellular pro-oxidant reactions. Int. J. Cancer 71: 246-250.

27. Curry S.H. (1974). Drug disposition and pharmaconkinetics with a consideration of pharmacological and clinical relationships. Blackwell Scientific Publications (Oxford, UK): 42-48.

28. Gibson G.G. and Skett P. (1995). Introduction to drug metabolism. Blackie Academic and Professional (London, UK): 1-34.

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CHAPTER In vitro characterization of the human biotransformation pathways of aplidine, a novel marine anti-cancer drug. Esther F.A. Brandon, Rolf W. Sparidans, Ronald D. van Ooijen, Irma Meijerman, Luis López Lázaro, Jos H. Beijnen, and Jan H.M. Schellens Abstract

Aplidine is a potent marine anti-cancer drug and is currently being investigated in phase II clinical trials. To assess the biotransformation pathways and their potential implications for human pharmacology and toxicology, the in vitro metabolism of aplidine was characterized using incubations with human plasma, liver preparations, cytochrome P450 (CYP) and uridine diphosphoglucuronosyl transferase (UGT) supersomes, and cell lines.

Aplidine is metabolized by carboxyl esterases in human plasma. Using CYP supersomes and liver microsomes, it was shown that aplidine is metabolized mainly by CYP3A4 and also by CYP2A6, 2E1, and 4A11. Four metabolites were observed after incubation with human liver microsomes, one formed by CYP2A6 (C-demethylation) and three by CYP3A4 (hydroxylation and/or C-dealkylation), identified by tandem-mass spectrometry. Only minor glucuronidation, catalyzed by UGT1A3 and 1A9, was observed for aplidine in liver microsomes and no glucuronidation was observed in liver S9 fraction. In addition, no glucosidation and sulfation were observed for aplidine in human liver cytosol and S9 fraction. However, the aplidine metabolites formed by CYP were further conjugated by the phase II enzymes UGT, glutathione-S-transferase (GST), and sulfotransferase (SULT). In accordance with the findings in microsomes and CYP supersomes, a significant effect of specific CYP2A6, 2E1, 3A4, and 4A11 inhibitors on the cytotoxicity of aplidine in Hep G2 and IGROV-1 cells could be observed.

These results provide evidence that CYP3A4 has a major role in the metabolism of aplidine in vitro, with involvement of CYP2A6, 2E1, and 4A11. The metabolites formed by CYP are conjugated by UGT, SULT, and GST.

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Introduction

Aplidine (Aplidin®) is a cyclic depsipeptide isolated from the Mediterranean tunicate Aplidium albicans. The compound exhibited in vitro activity against solid tumor cell lines obtained from melanoma and ovarian, renal, prostate, and breast cancer, but especially against non-small cell lung and colon cancer cells [1]. In addition, aplidine appears effective against human xenografts of lymphomas and gastric, prostate, and bladder tumors in vivo [2]. Phase I clinical trials with aplidine have been completed in Europe and Canada with more than 200 participating patients and the treatment showed clinical benefit in various tumor types, e.g. neuroendocrine tumors [3]. Phase II studies assessing the dose-response relationship in renal and colorectal cancer are in progress and further phase II studies including a large list of solid tumors are planned [4]. Aplidine showed a high variable clearance, an extensive distribution volume, and an intermediate elimination half-life in phase I clinical trials. Thus far, no data describing the pharmacokinetics of aplidine in patients from phase I or II clinical trials have been published. The dose-limiting toxicity of aplidine is fatigue, which affects 33% of all patients. The toxicity is primarily muscular, characterized by muscle pain and weakness. No haematological [4] and bone marrow (“PharmaMar, personal communication”) toxicities have been observed. The biotransformation pathways of aplidine, however, have not been identified. Knowledge about metabolism is important in order to interpret the pharmacokinetic properties found in phase I and II clinical trials and to predict possible drug-drug interactions with other (anti-cancer) drugs.

Aplidine is a potent inhibitor of protein synthesis and the ornithine decarboxylase activity is reduced drastically [5]. Its mode of action is believed to involve down-regulation of the flt-1 receptor for the vascular endothelial growth factor (involved in angiogenesis and tumor vascularization), induction of apoptosis in cancer cells, and arrest of the cell cycle in the G1 phase [1, 4, 6, 7].

The biotransformation of aplidine by human phase I, phase II enzymes and cytochrome P450 (CYP) in combination with phase II enzymes or the elucidation of CYP metabolites structures have not yet been reported. In this explorative investigation, different in vitro methods were therefore used including pooled human plasma, pooled human liver fractions in combination with HPLC-UV analysis. The involvement of various isoforms of cytochrome P450 and uridine diphosphoglucuronosyl transferase (UGT) to the biotransformation was investigated using pooled human liver microsomes in combination with specific CYP inhibitors and CYP and UGT supersomes. The human cancer cell lines Hep G2 and IGROV-1 were used to study the cytotoxicity of aplidine and its metabolites. Furthermore, the cytotoxicity of the aplidine metabolites formed by CYPs was studied in both cell lines, using aplidine incubated with pooled human liver microsomes. The elucidation of the biotransformation products of aplidine may be complicated by the presence of several potential sites for phase I and II reactions and the formation of degradation products (figure 1) [8, 9].

In vitro characterization of the aplidine biotransformation

49

N

O

OH

NH

OO

N

NNO

O

NO

O

O

O

N

OO

OOO

C57H87N7O15

M.W. = 1110 g/mol

[(S)-Me2-Tyr]

[(S)-Pro]

[(S)-Leu]

[2S,4S-Hip][3S,4R,5S-Ist]

[(R)-N(Me)-Leu]

[(S)-Pro]

[1S,2R-Thr]

= oxidation and conjugation

= hydroxylation

= conjugation

[Pyr]

Figure 1. Chemical structure of aplidine [10]. The different squares and ovals indicate potential sites for biotransformation [8, 9]. In addition, all the ester and amide bonds are potential sites for hydrolysis. Hip - hydroxyisovalerylpropionyl, Ist - isostatine, Leu - leucine, Me – methyl, Pro - proline, Pyr - pyruvoyl, Thr - threonine, Tyr - tyrosine. Materials and methods

Materials. Aplidine was kindly donated by PharmaMar (Tres Cantos, Madrid, Spain). Acetonitrile (gradient grade) was purchased from Biosolve (Valkenswaard, The Netherlands) and formic acid (p.a.), MgCl2

.6H2O (p.a.) and dimethyl sulfoxide (DMSO, synthesis grade) from Merck (Darmstadt, Germany). Water was purified on a multi-laboratory scale by reversed osmosis. Pooled human liver microsomes, pooled human liver cytosol, pooled human liver S9 fraction, and human CYP and UGT supersomes (Baculovirus-insect-cell expressed) were provided by Gentest (Becton Dickinson, Woburn, MA, USA). Pooled human plasma, with citrate, phosphate, and dextrose as anti-coagulants, was pooled from 4 different blank, drug-free human plasma donations obtained from the Saquin Bloedbank (Utrecht, The Netherlands). RPMI-1640 medium (with l-glutamine and 25 mM HEPES), heat-inactivated fetal calf serum, penicillin/streptomycin, and Hanks’ Balanced Salt Solution (pH 7.4) were all obtained from Gibco BRL (Breda, The Netherlands). All other chemicals were purchased from Sigma Chemical Company (St. Louis, MO, USA) and were of analytical grade.

Aplidine degradation in aqueous solution and pooled human plasma and the effect of esterase inhibitors. One µl of 200 mM esterase inhibitor solution in DMSO or DMSO (control) was pipetted into a polypropylene micro tube and 89 µl phosphate buffered saline (pH 7.4) or pooled human plasma (pooled from 4 different blank, drug-free human plasma donations) were added. The following esterase inhibitors were examined for their effect on the metabolism of aplidine in human plasma: bis(p-nitrophenyl)phosphate (BNPP), a carboxyl esterase inhibitor, and phenylmethylsulfonyl fluoride (PMSF), a cholesterol esterase inhibitor. After vortex-mixing, the tubes were incubated at 37°C in a shaking water bath for 15 min. Then, 10 µl of an aqueous dilution of an aplidine stock solution (100 µg/ml, 1% (v/v) DMSO) was added (final aplidine concentration of 10 µg/ml). The tube was briefly

Chapter 3

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vortex-mixed and further incubated at 37°C in a shaking water bath for 0 to 6 h. The reaction was terminated by adding 200 µl acetonitrile and vortex-mixing. The removal of proteins was established by centrifuging the samples at 15,000 g and 4°C for 5 min. The supernatant was injected for HPLC analysis. Control experiments were performed without substrate, respectively.

Aplidine incubations with pooled human liver microsomes. The incubation procedure of aplidine with human liver microsomes was a modification of the method described by Sparidans et al. (2001) for ecteinascidin 743 [10]. Twenty-five µl of 0.5 M potassium phosphate buffer (pH 7.4) were pipetted into a polypropylene micro tube on ice and 50 µl NADP regenerating system (NRS: 1.5 U/ml glucose-6-phosphate dehydrogenase, 0.5 mg/ml β-NADP, 4.0 mg/ml D-glucose-6-phosphate in 0.6 % (w/v) NaHCO3), 7.5 µl of 20 mg/ml MgCl2

.6H2O solution, and 50 µl of an aqueous aplidine solution (final concentration range of 1-100 µg/ml in the microsomes suspension) were added. After briefly vortex-mixing, the tubes were incubated for 2 min at 37°C in a shaking water bath. Next, 5 µl of pooled human liver microsomes (mixed gender, lot number 21) were added. The tube was vortex-mixed briefly again and the mixture was incubated at 37°C in a shaking water bath for 3 h. The reaction was terminated and proteins removed as previously described. The supernatant was injected for HPLC analysis. Control experiments were performed without aplidine and without liver microsomes, respectively.

Aplidine incubated with pooled mixed gender, female and male human liver microsomes and CYP supersomes. Aplidine incubations with mixed gender, female and male microsomes (lot number 21, 1, and 2, respectively) were performed as with the liver microsomes. Five µl of the CYP supersomes suspension were added to incubations with human CYP. The following human CYP supersomes were tested: CYP1A1 (lot number 15), CYP1A2 (lot number 20), CYP2A6 (lot number 6), CYP2B6 (lot number 8), CYP2C8 (lot number 11), CYP2C9*1(Arg144) (lot number 17), CYP2C19 (lot number 12), CYP2D6*1 (lot number 27), CYP2E1 (lot number 9), CYP3A4 (lot number 40), and CYP4A11 (lot number 7). All CYPs were co-expressed with P450 reductase and CYP2A6, 2B6, 2C8, 2C9, 2C19, 2E1, and 3A4 were also co-expressed with cytochrome b5 in the insect cells. A final concentration of 30 µg/ml of aplidine (concentration and time leading to 50% biotransformation in pooled mixed gender microsomes in previous experiment) was incubated and after 3 h the reaction was terminated and proteins were removed as previously described. The supernatant was injected for HPLC analysis. Control experiments were performed without substrate and without microsomes or with insect cell control supersomes (lot number 22), respectively.

Aplidine incubated with pooled human liver microsomes in the absence and presence of CYP inhibitors. Aplidine incubations with liver microsomes in the absence and presence of CYP inhibitors were performed according to the method described for liver microsomes with slight modifications. Twelve and a half µl of 1 M potassium phosphate buffer (pH 7.4) were pipetted into a polypropylene micro tube on ice and 50 µl NRS, 7.5 µl of 20 mg/ml MgCl2

.6H2O solution, and 10 µl of an aqueous CYP inhibitor solution (1% (v/v) DMSO) were added. Aplidine was incubated with microsomes and the following inhibitors based on the results found with CYP supersomes: 200 µM coumarin (CYP2A6), 200 µM chlorzoxazone (CYP2E1), 100 µM ritonavir (CYP3A4), or 25 µM 17-octadecynoic acid (17-ODA) (CYP4A11). After briefly vortex-mixing, the tubes were incubated at 37°C in a shaking water bath for 2 min. Next, 5 µl of pooled human liver microsomes (mixed gender, lot number 21) were added. The tube was briefly vortex-mixed again and the mixture was then incubated at 37°C in a shaking water bath for 5 min. Fifty µl of a aqueous aplidine solution (final concentration of 30 µg/ml, 1% (v/v) DMSO) were added and briefly vortex-mixed. The tube was incubated further at 37°C in a shaking water bath for 3 h. The


51

reaction was terminated and proteins removed as previously described. The supernatant was injected for HPLC analysis. Control experiments as described for microsomes.

Glucuronidation of aplidine in pooled human liver microsomes. The glucuronidation of aplidine with human UGT was a modification of the method described by Sparidans et al. (2001) for rabbit UGT [10]. Thirty µl of 0.1 M magnesium dichloride, 10 µl of 0.5 mg/ml alamethicin, 50 µl aplidine in water with 1% (v/v) DMSO (final concentration of 10 µg/ml), 25 µl of 1 M potassium phosphate buffer (pH 7.4), 25 µl water, and 50 µl of 15 mg/ml uridine diphosphoglucuronic acid (UDPGA) were pipetted into a polypropylene micro tube on ice. After briefly vortex-mixing, the tube was incubated at 37°C in a shaking water bath for 2 min. Next, 10 µl of pooled human liver microsomes (lot number 21) were added. The tube was vortex-mixed briefly again and the mixture was incubated at 37°C in a shaking water bath for 3 h. The reaction was terminated and proteins removed as described. The supernatant was injected for HPLC analysis. Individual control experiments were performed without aplidine and without pooled human liver microsomes, respectively.

Aplidine incubated with human UGT supersomes. The incubation of aplidine with human UGT supersomes was performed according to a modification of the method described by Gentest [11]. Twenty µl of 1 M potassium phosphate buffer (pH 7.4) were pipetted into a polypropylene micro tube on ice and 10 µl of 0.5 mg/ml alamethicin, 20 µl of 0.1 M MgCl2

.6H2O, 20 µl of 20 mM UDPGA, 50 µl aplidine in water with 1% (v/v) DMSO (final concentration of 30 µg/ml), and 70 µl H2O were added. After briefly vortex-mixing, the tubes were incubated at 37°C in a shaking water bath for 2 min. Next, 10 µl of the supersomes suspension were added. The following human UGT supersomes were tested: UGT1A1 (lot number 8), UGT1A3 (lot number 8), UGT1A9 (lot number 6), and UGT2B15 (lot number 5). The supersomes mixture was vortex-mixed and then incubated at 37°C in a shaking water bath for 3 h. The reaction was terminated and proteins removed as described. The supernatant was injected for HPLC analysis. Control experiments were performed without substrate and without UDPGA or with UGT insect cell control supersomes (lot number 5).

Conjugation of aplidine in pooled human liver cytosol. The incubation procedure for aplidine with pooled human liver cytosol was a modification of the method described by Gentest [11]. Equal volumes (20 µl) of 1 M potassium phosphate buffer (pH 7.4), 10 mM dithiotreitol (DTT), 1 mM acetyl-coenzyme A (acetyl-CoA), 45 mM acetyl-DL-carnitine, 80 units/ml carnitine acetyl transferase (from pigeon breast muscle, lot nr. 90K7400), 1 mM adenosine 3’-phosphate 5’-phosphosulfate (PAPS), and 10 mM glutathione were pipetted into a polypropylene micro tube on ice. Six µl H2O and 50 µl aplidine in water with 1% (v/v) DMSO (final concentration of 30 µg/ml) were added and briefly vortex-mixed. Next, 4 µl human liver cytosol (lot number 2) were added and, after briefly vortex-mixing, the mixture was incubated at 37°C in a shaking water bath for 3 h. The reaction was terminated by adding 200 µl acetonitrile and vortex-mixing. The proteins were removed as previously described and the supernatant was injected for chromatographic analysis. Individual control experiments were performed without aplidine, only DTT, acetyl-CoA, acetyl-DL-carnitine, and carnitine acetyl transferase (only N-acetyltransferase (NAT) activity), only PAPS (only sulfotransferase (SULT) activity), only glutathione (only glutathione-S-transferase (GST) activity), without all co-factors for enzyme activity and without cytosol, respectively. In addition, all four substrates of NAT were individually tested as controls.

Aplidine incubations with pooled human liver S9 fraction. The incubation of aplidine with pooled human liver S9 fraction was a modification of the method described by Gentest [11]. Equal volumes (10 µl) of 75 mg/ml UDPGA, 1 mM PAPS, and 10 mM glutathione were pipetted into a polypropylene micro tube on ice. Twenty-four µl NRS (3.3x more concentrated compared to microsomes), 50 µl aplidine in water with 1% (v/v) DMSO (final concentration of 30 µg/ml), 12 µl of 20 mg/ml MgCl2

.6H2O, 20 µl of 1 M potassium

Chapter 3

52

phosphate buffer (pH 7.4), and 54 µl water were added and briefly vortex-mixed. Subsequently, the tubes were incubated at 37°C in a shaking water bath for 2 min. Next, 10 µl pooled human liver S9 fraction (lot number 6) were added and vortex-mixed. The mixture was incubated at 37°C in a shaking water bath for 3 h. The reaction was terminated and proteins removed as previously described. The supernatant was then injected for chromatographic analysis. Individual control experiments were performed without substrate, without all co-factors for enzyme activity, and with co-factors present for only one or two enzymes (only one enzyme or a combination of CYP with one phase II enzyme were active), respectively.

Protein binding of aplidine in pooled human liver microsomes. Aplidine (final concentration of 5, 10, and 20 µg/ml) was pre-incubated with human microsomes for 15 min at room temperature. The reaction was terminated by removing proteins using ultra-centrifugation with Micronon YM-10 ultra-centrifuge tubes (cut-off filter of 10 kDa) (Millipore, Bedford, MA, USA) for 90 min at 14,000 g. The protein binding was estimated by quantification of aplidine in the ultra-filtrate using a liquid chromatographic assay. Calibration was performed using a standard curve of aplidine in 1:1 (v/v) water:acetonitrile.

Analysis of aplidine and metabolites by HPLC with ultra-violet (UV) detection and off-line triple quadrupole mass spectrometry (MSMS). The chromatographic assay was a modification of the method described by Waterval et al. (2001) [12]. The supernatants of the incubated mixtures were analyzed on an HPLC system consisting of two LC-10ATVP pumps, a SIL-10ADVP autoinjector (equipped with a 500 µl sample loop), a SCL-10AVP system controller, and a SPD-M10AVP photodiode array detector (all from Shimadzu, Kyoto, Japan). Data were recorded on a Hermac Pentium 440, 122 MB personal computer (Scherpenzeel, The Netherlands) equipped with the Class-VP 5.032 software (Shimadzu). Injections (50 µl) were made on a Symmetry C18 column (4.6 x 100 mm, dp=3.5 µm, Waters Chromatography, Milford, MA, USA) with a Sentry Guard Symmetry C18 pre-column (3.9 x 20 mm, dp=5 µm, Waters). The column temperature was maintained at room temperature. A gradient program was used with eluent A comprising 10 mM formic acid in water and eluent B comprising 10 mM formic acid in acetonitrile. After injection, elution started with 45% B and the eluent composition was raised linearly to 75% B during 20 min. This percentage was maintained for 2 min before conditioning with 45% B for 8 min. The eluent flow rate was 1.0 ml/min, the UV detection array was between 190 and 300 nm (resolution is 2 nm) and the peak areas were determined at 225 nm.

Structure elucidations were performed on a triple quadrupole instrument (Quattro Ultima, Micromass, Manchester, UK) equipped with a Z-nano electrospray source, according to the method described by Brandon et al. (Chapter 4). Data acquisition and processing were performed using MassLynx NT 3.5 data system (Micromass). The protonated aplidine and metabolite ions [M+H]+ or the sodium adduct ions [M+Na]+ were formed via nano-electrospray ionization (ESI+). The MS spectra, recorded between m/z 500 – 1200, were obtained at a scan speed of ~350 m/z units s-1 with a mass resolution corresponding to 0.5 m/z unit at half peak height. The product ion spectra, recorded between 100 and 1200 m/z (varying depending on the compound), were obtained at a scan speed of ~550 m/z units s-1, with mass resolution corresponding to 0.5 m/z unit at half peak height. The instrument was previously calibrated with sodium iodide. The in vitro metabolites of aplidine were identified after liquid chromatographic isolation using similar structure assignments by MS/MS previously reported by Ngoka et al. (1999) for the structure elucidation of didemnin B and its sodium adduct using collision-induced dissociation (CID) mass spectrometry [13].


53

Cell culture growth. The human hepatic carcinoma cell line (Hep G2) and the human ovarian adenocarcinoma cell line (IGROV-1) were kindly donated by the Netherlands Cancer Institute (Amsterdam, The Netherlands). Routine cultivation of these monolayer cells was performed in RPMI-1640 medium (with L-glutamine and 25 mM HEPES) supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were sub-cultured weekly (ratio of 1:5 (v/v) and 1:25 (v/v) for Hep G2 and IGROV-1 cells, respectively) and the medium was refreshed after 3 days.

Cytotoxicity of aplidine in Hep G2 and IGROV-1 cells in the absence and presence of esterase inhibitors or cytochrome P450 inhibitors. The cytotoxicity of the esterase and CYP inhibitors was determined for both cell lines and inhibitor concentrations below the IC5 were used in further experiments. For the determination of the cytotoxicity of aplidine in the absence and presence of inhibitors, cells were seeded onto 96-well microtitre plates at a concentration of 4,000 and 1,250 cells/well (200 µl/well) for the Hep G2 cell line and IGROV-1 cell line, respectively. After 48 h, the cells were exposed to the CYP and esterase inhibitor. The cells were incubated with 50 µM chlorzoxazone (CYP2E1), 50 µM coumarin (CYP2A6), 10 µM furafylline (CYP1A2), 5 µM ketoconazole (CYP1A1, 2A6, 2C8, 2C19, 2D6, and 3A4), 200 µM metyrapone (CYP2A6 and 3A4), 10 µM 17-ODA (CYP4A11), 10 µM piperonyl butoxide (CYP3A), 10 µM proadifen (CYP2A6, 2B6, 2C9, 2E1, and 3A4), 10 µM ritonavir (CYP3A4), 200 µM sulfaphenazole (CYP2C9), 75 µM BNPP (carboxyl esterase), or 200 µM PMSF (cholesterol esterase) in Hep G2, respectively. The same concentrations were employed for the IGROV-1 cells except for proadifen (2.5 µM) and BNPP (100 µM). The cells were pre-incubated for 1 h at 37°C and 5% (v/v) CO2. Next, the cells were exposed to aplidine at concentrations of 0.025-500 ng/ml for Hep G2 and 0.017-333.3 ng/ml for IGROV-1 and inhibitors, for 5 days. Therefore, 100 µl of a 1000 or 1500 ng/ml aplidine solution with inhibitor was added to the wells, containing 200 µl medium, and from this serial dilutions were made into the microtitre plate. Cell growth was determined at day 5 using the sulforhodamine B assay (SRB assay). Cell survival (%) was calculated relative to control cells and 100% killed cells (killed with 10% Triton-X100, 1 h prior to the SRB assay). Concentration-viability curves were constructed from these data and the IC50 (concentration of compound giving 50 % survival) was calculated by the Softmax®Pro 3.1 software (Molecular Devices, Sunnyvale, CA, USA). Control experiments were performed without aplidine and without inhibitor, respectively.

Cytotoxicity of aplidine metabolites formed by pooled human liver microsomes. Aplidine (final concentration of 10 µg/ml) was pre-incubated with pooled human liver microsomes (lot number 21) for 30 min as previously described. The reaction was terminated by removing proteins with ultra-centrifugation (using Micronon YM-10 ultra-centrifuge tubes for 90 min at 14,000 g). The polypropylene micro tubes used to collect the filtrate were sterile and the filtrate was handled aseptically. A sample of the ultra-filtrate was injected for HPLC analysis to quantify the percentage of biotransformation. For the cell culture experiment, the ultra-filtrate was diluted 1:20 (v/v) in RPMI-1640 medium. The Hep G2 and IGROV-1 cells were seeded and treated as previously described and the cytotoxicity was determined after 5 days using the SRB assay. Control experiments were performed with aplidine without pooled human liver microsomes, with only microsomes and with aplidine with microsomes incubated at 4°C, respectively.

Chapter 3

54

Sulforhodamine B assay (SRB assay). The SRB assay was a modification of the method described by Higgins et al. (1993) [14]. The cell culture medium was removed and the cells were fixed in 100 µl of 10% (w/v) trichloroacetic acid for 60 min at 4°C. The wells were rinsed three times with tap water to remove solutes and cells were stained with 50 µl of 0.4% (w/v) sulforhodamine B (SRB) in 1% (v/v) acetic acid for 15 min. The cells were washed three times with 1% (v/v) acetic acid and air-dried. After drying, 120 µl of 10 M Tris in Hanks’ Balanced Salt Solution (pH 7.4) were added to solubilize the protein bound SRB. After mixing, the absorbance was measured at 540 nm using a Versamax microtitre plate reader (Molecular Devices) and the data were analyzed using the Softmax®Pro 3.1 software.

Data analysis. The results are expressed as mean ± standard deviation (SD). Differences between the results were analyzed by the student t-test for unpaired observations. Results

Half-life of aplidine in PBS and pooled human plasma. Aplidine has a half-life (t½) of in 8.76 ± 0.98 h in PBS and of only 4.19 ± 0.77 h in pooled human plasma. Degradation products or metabolites were not observed in the HPLC chromatograms after aplidine incubation in PBS and pooled human plasma. The t½ of aplidine in pooled human plasma in the presence of the cholesterol esterase inhibitor PMSF (t½ is 3.26 ± 0.85 h) did not significantly change (p > 0.05) compared to pooled human plasma without esterase inhibitors (t½ is 4.19 ± 0.77 h). However, the carboxyl esterase inhibitor BNPP was able to increase the t½ of aplidine in plasma from 4.19 ± 0.77 h to 10.94 ± 0.96 h (p < 0.05). The half-life of aplidine in pooled human plasma in the presence of BNPP did not significantly differ from the half-life in PBS. In addition to chemical degradation, carboxyl esterases may thus be responsible for the clearance of aplidine from human plasma in vitro.

Biotransformation of aplidine in pooled human liver microsomes. Microsomal incubations of aplidine resulted in the formation of five chromatographic peaks corresponding to 4 metabolites of aplidine (figure 2). After off-line structure elucidation using triple quadrupole MS two peaks (apli-h1 and apli-h2) showed to be two conformers of apli-h (hydroxylated at the isopropyl-group) (Chapter 4). The other 3 metabolites were apli-da (dealkylated at the (R)-N(methyl)-leucine group), apli-da/h (dealkylated at the (R)-N(methyl)-leucine and hydroxylated at the isopropyl group), and apli-dm (demethylated at the C-atom in the threonine group) (Chapter 4). In figure 3, an overview of the proposed metabolite structures is shown. The metabolites apli-da and apli-h were immediately metabolized to apli-da/h if the aplidine concentration was below 10 µg/ml. Above this concentration, the metabolites apli-da and apli-h can be observed. Apli-dm is the main metabolite formed at low concentrations of aplidine (< 10 µg/ml) and is directly formed following zero order kinetics. Most likely, more metabolites than the 4 observed have been formed because the sum of aplidine remaining and the observed metabolites seemed to be lower than 100% in the concentration range from 2 to 20 µg/ml (table 1) (UV absorption at 225 nm, spectra of aplidine and all metabolites are almost identical (Figure 4).


55

Figure 2. Chromatogram at 225 nm of aplidine after incubation without pooled human liver microsomes (1) and aplidine after incubation with microsomes (2).

N

O

OH

NH

OO

NH

NH

NO

O

NO

O

O

O

N

OO

OOO

OH

N

O

OH

NH

O

NH

NH

NO

O

NO

O

O

O

N

OO

OOO

O

OH

N

O

OH

NH

OO

NH

NH

NO

O

NO

O

O

O

N

OO

OOO

N

O

OH

NH

OO

NH

NH

NO

O

NO

O

O

O

N

OO

OOO

(B)(A)

(C) (D)

Figure 3. Structures of discovered metabolites of aplidine: apli-da/h (A), apli-h (B), apli-da (C), and apli-dm (D).

2

1

Time (min)

Abs

orba

nce

(mA

U)

Minutes0 2 4 6 8 10 12 14 16 18 20

mA

U

-5

0

5

10

15

20

25

30

35

40

45

apli-

da/h ap

li-h1

apli-

da apli-

h2

apli-

dm

aplid

ine

A

aplid

ine

B

Chapter 3

56

tota

l

95.7

38.8

45.0

72.3

44.7

92.5

98.3

SD

15.8

2.0

1.1

0.8

0.7

0.4

0.2

% a

pli-d

m

76.5

28.2

20.2

12.0

3.7

1.9

1.0

SD 0.

8

0.9

0.3

0.3

% a

pli-d

a

n.d.

n.d.

n.d.

5.4

4.8

2.9

1.3

SD 1.

4

1.9

1.2

0.6

% a

pli-h

n.d.

n.d.

n.d.

28.4

12.2

6.7

2.9

SD

3.8

1.1

0.6

1.4

0.5

0.1

0.1

% a

pli-

da/h

19.2

10.7

16.2

11.0

2.3

0.9

0.3

SD 2.

4

0.5

4.1

16.4

25.5

%

aplid

ine

n.d.

*

n.d.

8.6

15.6

21.8

80.1

92.9

Tab

le 1

: Per

cent

ages

reco

vere

d of

apl

idin

e an

d m

etab

olite

s afte

r inc

ubat

ion

with

hum

an li

ver m

icro

som

es d

urin

g 3h

. Q

uant

ified

usi

ng U

V a

t 225

nm

. co

ncen

trat

ion

aplid

ine

(µg/

ml)

1 2 5 10

20

50

100

* n.

d. =

not

det

ecte

d


57

190 200 210 220 230 240 250 260 270 280 290 300

wavelenght (nm)

rela

tive

abso

rban

ce

2345

67

1

Figure 4. UV spectra (190-300 nm) of the aplidine metabolites apli-da/h (1), apli-h (2 and 3), apli-da (4), and apli-dm (5) and aplidine (6 and 7).

Comparison of the aplidine biotransformation in male, female, and mixed gender pooled human liver microsomes and protein binding in mixed gender microsomes. Incubation of aplidine with mixed gender human liver microsomes at 37°C for 3 h reduced the amount of aplidine by 46.7 ± 4.5 %. Incubations of aplidine with female human liver microsomes did not show a difference in percentage aplidine remaining (45.5 ± 8.3 %). However, male human liver microsomes resulted in a significant decrease of the percentage aplidine metabolized compared to mixed gender to 27.4 ± 2.7 % after incubation. The formation of the aplidine metabolites apli-da/h, apli-da, and apli-h was significantly impaired during incubation with male human liver microsomes (results not shown). The formation of the apli-dm metabolite was not significantly different between mixed gender, male, and female liver microsomes. Aplidine has a protein binding of less than 1% in human liver microsomes in the concentration range of 5-20 ng/ml (results not shown).

Aplidine biotransformation by human CYP supersomes. Aplidine was significantly metabolized by CYP2A6, 2E1, 3A4, and 4A11 supersomes during 3 h at 37°C, 35.2 ± 0.2 %, 20.5 ± 2.6 %, 28.2 ± 4.4 %, and 35.4 ± 2.4 % of the aplidine was metabolized, respectively. The other CYP supersomes did not metabolize aplidine. The aplidine metabolites apli-da/h, apli-da, and apli-h were formed specifically during aplidine incubation with CYP3A4 supersomes and apli-dm was formed only after incubation of aplidine with CYP2A6 supersomes (results not shown). No metabolites were observed after incubation with CYP2E1 and 4A11 supersomes.

Aplidine biotransformation by pooled human liver microsomes in the absence and presence of CYP inhibitors. Based on the result found in CYP supersomes, aplidine was incubated with human liver microsomes (mixed gender) in the presence of several CYP inhibitors. All the tested inhibitors could significantly decrease the aplidine biotransformation by pooled human liver microsomes. CYP3A4 was the main CYP isozyme responsible for the conversion of aplidine in pooled human liver microsomes; the percentage aplidine metabolized in the presence of the CYP3A4 inhibitor ritonavir decreased from 70.0 ± 4.2 %

Chapter 3

58

to 25.8 ± 1.5 %. Coumarin, chlorzoxazone, and 17-ODA reduced the aplidine percentage metabolized to 52.3 ± 3.0 %, 40.4 ± 1.2 %, and 58.3 ± 7.8 % respectively. After aplidine incubation with pooled human liver microsomes in combination with ritonavir, only the apli-dm metabolite could be observed. The other three CYP inhibitors showed no effect on the formation of the aplidine metabolites (results not shown). Most likely, other metabolites than the ones observed with the used method are formed by cytochrome P450.

Table 2: Percentage aplidine and metabolites after incubation with mixed gender pooled human liver microsomes in the presence 200 µM coumarin (CYP2A6 inhibitor), 200 µM chlorzoxazone (CYP2E1 inhibitor), 100 µM ritonavir (CYP3A4 inhibitor), or 25 µM 17-ODA (CYP4A11 inhibitor) for 3h at 37°C.

CYP inhibitor

% apli-da/h

SD % apli-h

SD % apli-da

SD % apli-dm

SD % aplidine

SD

no inhibitor 1.3 0.2 9.9 1.6 3.0 0.4 1.8 0.2 30.0 1.8 coumarin 1.3 0.1 11.5 0.2 4.2 0.5 2.3 0.1 47.7 2.7

chlorzoxazone 1.0 0.3 9.9 2.9 4.0 0.7 2.0 0.1 59.6 1.7 ritonavir n.d.* n.d. n.d. 2.1 0.1 74.2 4.4 17-ODA 2.2 0.2 12.2 0.9 4.0 0.1 1.6 0.1 41.7 5.6

* n.d.= not detected.

Glucuronidation of aplidine by pooled human liver microsomes and UGT supersomes. Thirty µg/ml aplidine was significantly glucuronidated by UGT1A3 and 1A9 supersomes; 13.9 ± 1.0 % and 21.2 ± 0.9 % for UGT1A3 and 1A9, respectively. A significant glucuronidation could also be observed for aplidine by pooled human liver microsomes (HLM), 82.2 ± 6.3 % of the 10 µg/ml aplidine was recovered after 3 h. Furthermore, incubations with UGT2B15 showed a slight aplidine conversion, but this was not significant. Unfortunately, no glucuronidation product could be observed for aplidine after incubation with UGT supersomes nor with pooled human liver microsomes.

Aplidine biotransformation by pooled human liver cytosol. Aplidine was not metabolized by the phase II enzymes sulfotransferase and glutathione-S-transferase present in pooled human liver cytosol (results not shown). The metabolism of aplidine by N-acetyltransferase could not be studied due to degradation of aplidine in the presence of the NAT substrates acetyl-CoA and carnitine acetyl transferase (results not shown).

Biotransformation of aplidine and its metabolites by pooled human liver S9 fraction. Aplidine was significantly metabolized by cytochrome P450 in pooled human liver S9 fraction, followed by phase II metabolism (figure 5). The CYPs present in the S9 fraction metabolized 34.1 ± 2.7 % of the aplidine during 3 h at 37°C. CYP activity in combination with the individual phase II enzymes UGT, SULT, and GST resulted in a further reduction of aplidine to 43.7 ± 3.5 %, 73.5 ± 7.5%, and 50.6 ± 5.8 %, respectively. When all the enzyme substrates were present, 60.5 ± 4.4 % of the aplidine was converted by pooled human liver S9 fraction. The phase II enzymes UGT, SULT, and GST did not metabolize aplidine directly. Thus, aplidine needed to be metabolized first by CYP and next the formed metabolites could be conjugated by the phase II enzymes UGT, SULT, and GST. The identified aplidine metabolites apli-da/h, apli-da, apli-h, and apli-dm were conjugated by SULT and apli-dm was also conjugated by UGT (results not shown). Therefore, other aplidine metabolites have probably also been formed by CYP which are then conjugated.


59

0

20

40

60

80

100

120

140

control CYP UGT SULT GST CYP +UGT

CYP +SULT

CYP +GST

CYP +UGT +SULT +

GST

% a

plid

ine

rem

aini

ng

*

*!

*!

*!*!

Figure 5. Comparison of the biotransformation of aplidine in human liver S9 fraction by CYP and by CYP and a phase II enzyme (UGT, SULT, and GST). The percentage remaining was determined using an aplidine incubation without S9 fraction as control (= 100 %). Each column is the mean of 3 replicates; bars indicate the SD. * significantly different (P < 0.05) compared to control and ! significantly different (p < 0.05) compared to CYP.

Cytotoxicity of aplidine in Hep G2 and IGROV-1 cells in the absence and presence of CYP and esterase inhibitors. Aplidine was found to have an IC50 value of 11.2 ± 4.2 ng/ml in the Hep G2 cell line and 1.7 ± 0.9 ng/ml in the IGROV-1 cell line (figure 6). A significant decrease in IC50 value in the presence of the CYP inhibitors chlorzoxazone, coumarin, 17-ODA, and ritonavir could be observed in the Hep G2 cell line. Chlorzoxazone, coumarin, ketoconazole, 17-ODA, and ritonavir significantly decreased the IC50 value of aplidine in the IGROV-1 cell line. The CYP3A4 inhibitor ritonavir showed the highest decrease in the IC50 value of aplidine in both cell lines.

Chapter 3

60

Figure 6. Aplidine cytotoxicity in Hep G2 and IGROV-1 cells in the absence and presence of CYP or esterase inhibitors: A – aplidine, CZ – chlorzoxazone, C – coumarin, F – furafylline, KC – ketoconazole, MR – metyrapone, O – 17-octadecynoic acid, PB – piperonyl butoxide, PA – proadifen, RN – ritonavir, SP – sulfaphenazole, B – BNPP, P – PMSF. Each column is the mean of 7 replicates for aplidine (7 different passages) and 3 replicates for aplidine in combination with the inhibitors (3 different passages); bars indicate the SD. * significantly different (p < 0.05) compared to aplidine without inhibitor.

Cytotoxicity of aplidine pre-incubated with pooled human liver microsomes in the Hep G2 and IGROV-1 cell line. The IC50 of aplidine pre-incubated with pooled human liver microsomes did not significantly differ from aplidine after correction for the difference in aplidine concentration present (results not shown). Therefore, the aplidine metabolites are not cytotoxic or to a much lesser extent than aplidine itself.

A

0

2

4

6

8

10

12

14

16

A + CZ + CM + F + KC + MR + O + PB + PA + RN + SP + B + P

IC50

val

ue

**

**

B

0.0

0.5

1.0

1.5

2.0

2.5

3.0

A + CZ + CM + F + KC + MR + O + PB + PA + RN + SP + B + P

IC50

val

ue

* * **

*


61

Discussion and conclusions

Aplidine is a novel anti-cancer agent and phase II studies with this drug have been started [3-4]. The degradation and biotransformation pathways of aplidine in humans were investigated in vitro to support the pharmacokinetic and toxicological findings of clinical trials.

Enzymes in human plasma contributed significantly to the conversion of aplidine at 37°C. The carboxyl esterase inhibitor BNPP increased the half-life of aplidine indicating that carboxyl esterases were among the enzymes responsible for the clearance of aplidine from human plasma. Most circulating aplidine is present within the blood cells and not in the plasma [15] (“PharmaMar, personal communication”) and therefore degradation kinetics of aplidine in blood in vivo could differ from in vitro data. However, based on the results, BNPP could be added to aplidine containing plasma samples from clinical trials in order to stabilize aplidine, thus preventing degradation by carboxyl esterases.

Coumarin, chlorzoxazone, ritonavir were able to inhibit the biotransformation of aplidine by microsomes and to decrease the cytotoxicity of aplidine in cell lines. Ketoconazole also significantly decreased the IC50 value in the IGROV-1 cell line. Based on these results together with the results from the CYP supersomes, CYP2A6, 2E1, 3A4, and 4A11 may be involved in the biotransformation of aplidine in the liver. The lack of significant effect of ketoconazole in Hep G2 cells and metyrapone, piperonyl butoxide, and proadifen in both cell lines, may be caused by incomplete inhibition of the isozyme due to limitations of the concentration (below the IC5 value). The effect of the different CYP inhibitors on the cytotoxicity in the Hep G2 and IGROV-1 cell line and on the biotransformation by human liver microsomes, indicates that CYP3A4 was the main CYP responsible for the biotransformation of aplidine in vitro.

The validity of the method using CYP inhibitors combined with microsomes and cell lines was already proved by others [16, 17]. The CYP inhibitors tested were selected using the human cytochrome P450 database from Gentest and the drug interaction table of the Indiana University School of Medicine [16, 17]. The inhibitor concentrations used with human liver microsomes and cell lines in this study were within the range or above the concentrations to inhibit CYPs used by others, who also investigated the effect of CYP inhibitors on the biotransformation of (novel) compounds. The CYP inhibitors used in the cell culture experiments were all used at concentrations below their IC5 value, thus the inhibitor had no direct influence on the viability of the cells. However, due to the limitations of the IC5, the inhibitor concentrations could be lower than the concentration needed to obtain complete inhibition. The observation that there was a significant decrease in the IC50 value of aplidine in combination with CYP inhibitors indicates that these CYPs are involved in the biotransformation of aplidine. However, the lack of effect of some CYP inhibitors on the cytotoxicity of aplidine might be because the activity levels in the cell lines are possibly to low to cause a significant difference in biotransformation of aplidine or the inhibitor concentration is to low for complete inhibition.

The biotransformation of aplidine was not significantly different for mixed gender and female microsomes, male showed a significant lower aplidine biotransformation percentage. This is probably caused by the significantly lower amount of CYP3A4 in the male liver microsomes, which was almost equal in mixed gender and female microsomes. The pooled mixed gender human liver microsomes were formulated from material derived from at least 21 individuals and the single gender pools were derived from 5 or more male or female donors [11]. The pooled microsomes have been designed for a profile of catalytic activities representative for many individuals and for minimal lot-to-lot variability. However, variance between the different lot numbers may occur, but the pools are large enough to examine

Chapter 3

62

gender-related differences in metabolism [11]. As the different pooled single gender microsomes are representative for the whole male or female population, this indicates that on average the amount of CYP3A4 in the male liver is lower compared to female liver. The differences in biotransformation of aplidine between females and males microsomes indicate that there is a gender-related difference in metabolism for aplidine.

The observed products are undoubtedly metabolites of aplidine, because they are only formed in the presence of the specific CYP isoenzymes. Apli-da/h, apli-h, and apli-da are formed by CYP3A4 and apli-dm formation is catalyzed by CYP2A6. According to mass spectrometric and spectrophotometric data, the metabolites are aplidine hydroxylated at the isopropyl group (apli-h), aplidine dealkylated at (R)-N(methyl)-leucine (apli-da), aplidine dealkylated at the (R)-N(methyl)-leucine group and hydroxylated at the isopropyl group (apli-da/h), and aplidine demethylated at the C-atom in the threonine group (apli-dm) (Chapter 4). The hydroxylation of the isopropyl group is a common reaction catalyzed by cytochrome P450 [8, 9]. N- and O-dealkylation have repeatedly been reported [8, 9], but the C-dealkylation has not been described previously. Though unlikely, based on the MSMS data C-dealkylation of aplidine has occurred catalyzed by cytochrome P450.

The metabolites of aplidine formed by cytochrome P450 in human liver microsomes, were found to be less toxic compared to aplidine, indicating that aplidine is detoxified in the liver. This is supported by the decrease in IC50 value of aplidine in combination with some CYP inhibitors. Thus, most likely CYPs in the liver are involved in the bioinactivation of aplidine. However, aplidine metabolites formed in vivo and not in vitro could have a higher cytotoxicity compared to aplidine itself and could influence the (hepato)toxicity in patients.

All results indicated an important role for CYP3A4 in the metabolism aplidine. Therefore, the risk of in vivo drug-drug interaction, when aplidine is combined with other CYP3A4 substrates, is present [18, 19]. Consideration is warranted when aplidine is given in combination with other anti-cancer drugs that are metabolized by CYP3A4 or drugs that influence its activity [20, 21]. Furthermore, the data obtained with microsomes indicated that gender can play a role in the biotransformation and metabolic clearance in patients. However, due to high within-gender differences existing in CYP3A4 activity, gender differences are not always of clinical importance [22, 23]. Thus far, no gender differences have been described for patients treated with aplidine, but in phase I clinical trials; aplidine showed a large inter-individual variability (“PharmaMar, personal communication”). This emphasizes the influence of the high inter-individual variance in CYP3A4 activity on the pharmacokinetics of aplidine. Food components, aging, disease, and genetic polymorphisms also influence the individual CYP isozyme activity [22-24]. The genetic component in the inter-individual variability in CYP3A4 activity has been estimated to be high, but the underlying genetic factors are largely unknown [25]. Furthermore, it is most likely that CYP3A5 (same substrates as CYP3A4) is capable of metabolizing aplidine. In less than 9% of the Caucasians, CYP3A5 is functional and they will probably show a higher biotransformation rate for aplidine compared to patients without functional CYP3A5 [26]. Thus, genotyping patients for CYP3A5 may contribute to the safety of the patients treated with aplidine. When aplidine is given, attention should be paid to toxicity arising from gender, genetic polymorphisms, food components (e.g. grapefruit juice inhibits CYP3A and St. John’s wort induces CYP3A), and drug-drug interactions [17].

Aplidine was glucuronidated in vitro by UGT1A3 and 1A9 in human UGT supersomes and by the UGT isozymes present in liver microsomes. However, no glucuronidation was observed in liver S9 fraction, probably because of lower UGT enzyme activities compared to UGT supersomes and human liver microsomes. The other phase II enzymes studied, SULT and GST, did not directly metabolize aplidine in pooled human liver cytosol and S9 fraction.


63

However, the metabolites formed by CYP were further conjugated by all phase II enzymes studied.

Gender differences have been observed with glucuronidation activity being higher in man than in women [22]. Furthermore, the individual glucuronidation activity is also influenced by aging, disease, and genetic polymorphisms [27, 28]. Inter-individual variability in SULT and GST activity has been observed [23, 29, 30], influenced by aging, disease, food or drug intake, and genetic polymorphisms. However, the rate-limiting step in the aplidine metabolism is most likely the CYP-mediated step and inter-individual differences in glucuronidation, sulfation, and glucosidation activity may not be of clinical importance.

In conclusion, aplidine has a short half-life in human plasma. The half-life of aplidine could be prolonged by addition of a carboxyl esterase inhibitor. Thus, carboxyl esterases can play an important role in the metabolism of aplidine in plasma. Aplidine metabolism in human liver microsomes is catalyzed by CYP and UGT. CYP3A4 is most likely the predominant CYP metabolizing aplidine and CYP2A6, 2E1, and 4A11 play a minor role, and glucuronidation is mediated by UGT1A3 and 1A9. The metabolites formed by CYP (identified and unobserved metabolites) are further metabolized by phase II enzymes, namely UGT, SULT, and GST. Further, aplidine is detoxified by the CYPs in the liver. These findings can be used for the interpretation of pharmacokinetic and toxicological data obtained from clinical trials with aplidine in patients.

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References 1. Faircloth G., Hanauske A., Depenbrock H., Peter R., Crews C.M., Manzanares I.,

Meely K., Grant W., and Jimeno J.M. (1997). Pre-clinical characterization of aplidine, a new marine anticancer depsipeptide (abstract). Proc. Am. Assoc. Cancer Res. 38: 692.

2. Depenbrock H., Peter R., Faircloth G.T., Manzanares I., Jimeno J., and Hanauske A.R. (1998). In vitro activity of aplidine, a new marine-derived anti-cancee compound, on freshly explanted clonogenic human tumour cells and haematopoietic precursor cells. Br. J. Cancer 78: 739-744.

3. Raymond E., Paz-Ares L., Izquierdo M., Belanger K., Maroun J., Bowman A., Anthoney A., Jodrell D., Armand J.P., Cortes-Funes H., Germa-Lluch J., Twelves C., Celli N., Guzman C., and Jimeno J. (2001). Phase I trials with aplidine, a new marine derived anticancer compound (abstract). Eur. J. Cancer 37 (suppl. 6): S32.

4. Jimeno J.M. (2002). A clinical armamentarium of marine-derived anti-cancer compounds. Anti-Cancer Drugs 13 (suppl. 1): S15-S19.

5. Urdiales J.L., Morata P., Nunez De Castro I., and Sanchez-Jimenez F. (1996). Antiproloferative effect of dehydrodidemnin B (DDB), a depsipeptide isolated from Mediterranean tunicates. Cancer Lett. 102: 31-37.

6. Erba E., S. Ronzoni, Bergamaschi D., Bassano L., Desiderio M.A., Faircloth G., Jimeno J., and D’Incalci M. (1999). Mechanism of antileukemic activity of aplidine (abstract). Proc. Am. Assoc. Cancer Res. 40: 3.


8. Curry S.H. (1974). Drug disposition and pharmacokinetics with a consideration of pharmacological and clinical relationships. Blackwell Scientific Publications (Oxford, UK): 42-48.

9. Gibson G.G. and Skett P. (1994). Introduction to drug metabolism. Blackie Academic and Professional (London, UK), 2nd edition: 1-34.

10. Sparidans R.W., Rosing H., Hillebrand M.J.X., Lopez-Lazaro L., Jimeno J.M., Manzanares I., van Kesteren Ch., Cvitkovic E., van Oosterom A.T., Schellens J.H.M., and Beijnen J.H. (2001). Search for metabolites of ecteinascidin 743, a novel, marine-derived, anti-cancer agent, in man. Anti-Cancer Drugs 12: 653-666.

11. Gentest, a BD Biosciences Company. http://www.gentest.com (accessed March 2002). 12. Waterval J.C.M., Bloks J.C., Sparidans R.W., Beijnen J.H., Rodriguez-Campos I.M.,

Bult A., Lingeman H., and Underberg W.J.M. (2001). Degradation kinetics of aplidine, a new marine antitumoural cyclic peptide, in aqueous solution. J. Chromatogr. B 754: 161-168.

13. Ngoka L.C.M., Gross M.L., and Toogood P.L. (1999). Sodium-directed selective cleavage of lactones: a method for structure determination of cyclodepsipetides. Int. J. Mass Spectrom. 182/183: 289-298.

14. Higgins III J.D., Neely L., and Fricker S. (1993). Synthesis and cytotoxicity of some cyclometallated palladium complexes. J. Inorg. Biochem. 49: 149-156.

15. Zucchetti M., Lopez-Lazaro L., Celli N., Cicchella B., Twelves C., Paz-Agres L., Izquierdo M., Bowman A., Raymond E., Maroun E., Belanger K., and D’Inclaci M. (2000). Clinical pharmacokinetics (PK) of aplidine (APL) in patients with solid tumors and non-Hodgkin lymphomas (abstract). Proc. Am. Assoc. Cancer Res. 30: 4932.

16. Gentest Cytochrome P450 database. http://www.gentest.com/human_p450_database (accessed January 2003).


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17. Drug interaction table. http://medicine.iupui.edu/flockhart/table.htm (accessed January 2003).

18. Levy R.H., Thummel K.E., Trager W.F., Hansten P.D., and Eichelbaum M. (2000). Metabolic drug interactions. Lippincott Williams and Wilkins (Philadelphia, USA).

19. Tucker G.T. (1992). The rational selection of drug interaction studies: implications of recent advances in drug metabolism. Int. J. Clin. Pharmacol. Ther. Toxicol. 30: 550-553.

20. Desai P.B., Duan J.Z., Zhu Y.W., and Kouzi S. (1998). Human liver microsomal metabolism of paclitaxel and drug interactions. Eur. J. Drug Metab. Pharmacokinet. 23: 417-424.

21. Vecht C.J., Wagner G.L., and Wilms E.B. (2003). Interactions between antiepileptic and chemotherapeutic drugs. Lancet Neurol. 2: 404-409.



24. MacLeod S.L., Nowell S., Massengill J., Jazieh A., McClure G., Plaxco J., Kadlubar F.F., and Lang N.P. (2000). Cancer therapy and polymorphisms of cytochrome P450. Clin. Chem. Lab. Med. 38: 883-887.

25. Eiselt R., Domanski T.L., Zibat A., Mueller R., Presecan-Siedel E., Hustert E., Zanger U.M., Brockmoller J., Klenk H.P., Meyer U.A., Khan K.K., He Y.A., Halpert J.R., and Wojnowski L. (2001). Identification and functional characterization of eight CYP3A4 protein variants. Pharmacogenetics 11:447-58.

26. van Schaik R.H., van der Heiden I.P., van den Anker J.N., and Lindemans J. (2001). CYP3A5 variant allele frequencies in Dutch Caucasians. Clin. Chem. 48: 1668-1671.

27. Tukey R.H. and Strassburg C.P. (2000). Human UDP-glucuronosyltransferases: metabolism, expression, and disease. Annu. Rev. Pharmacol. Toxicol. 40: 581-616.

28. MacKenzie P.I., Miners J.O., and McKinnon R.A. (2000). Polymorphisms in UDP glucuronosyltransferase genes: functional consequences and clinical relvance. Clin. Chem. Lab. Med. 38: 889-892.

29. Glatt H., Boeing H., Engelke C.E.H., Ma L., Kuhlow A., Pabel U., Pomplun D., Teubner W., and Meinl W. (2001). Human cytosolic sulphotransferases: genetics, characterization, toxicological aspects. Mutat. Res. 482: 27-40.

30. Coughtrie M.W.H. (2002). Sulfation trough the looking glass – recent advances in sulfotransferase research for the curious. Pharmacogenomics J. 2: 297-308.

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67

CHAPTER Structure elucidation of aplidine metabolites formed in vitro by human liver microsomes using triple quadrupole mass spectrometry. Esther F.A. Brandon, Ronald D. van Ooijen, Rolf W. Sparidans, Luis López Lázaro, Albert J.R. Heck, Jos H. Beijnen, and Jan H.M. Schellens. Abstract

The cyclic depsipeptide aplidine is a new anti-cancer drug of marine origin. Four metabolites of this compound were found after incubation with pooled human microsomes using gradient HPLC with ultraviolet detection. After chromatographic isolation, the metabolites have been identified using nano-electrospray triple quadrupole mass spectrometry. A highly specific sodium-ion interaction with the cyclic structure opens the depsipeptide ring and cleavage of the amino acid residues gives sequence information when activated by collision-induced dissociation in the second quadrupole. The aplidine molecule could undergo the following metabolic reactions: hydroxylation at the isopropyl group (metabolites apli-h 1 and apli-h 2); C-dealkylation at the N(Me)-leucine group (metabolite apli-da); hydroxylation at the isopropyl group and C-dealkylation at the N(Me)-leucine group (metabolite apli-da/h), and C-demethylation at the threonine group (metabolite apli-dm). The identification of these metabolites formed in vitro may greatly aid the elucidation of the metabolic pathways of aplidine in humans.

Chapter 4

68

Introduction

Aplidine (figure 1) is a cyclic depsipeptide isolated from the Mediterranean tunicate Aplidium albicans. It was previously named dehydrodidemnin B, because of its structural relation to didemnin B (figure 1) and other didemnins harvested from the tunicate Trididemnum solidum. Aplidine showed the strongest in vitro anti-tumor activity of 42 tested didemnins [1]. Aplidine has demonstrated in vitro anti-proliferative activity against a variety of human tumor cell lines, but specifically against non-small cell lung and colon tumor cells [2]. In in vivo studies in mice significant tumor growth inhibition by aplidine was observed in human lymphomas and gastric, prostate, and bladder tumors [3, 4]. Aplidine is believed to down-regulate the flt-1 receptor for the vascular endothelial growth factor, which is involved in angiogenesis and tumor vascularization, arrest of the cell cycle in the G1 phase, and induction of apoptosis in cancer cells [2, 5-7]. It is also a potent inhibitor of the protein synthesis [8].

N

O

OH

NH

O

O

N

N

NO

O

N

O

O

O

N

OO

OOO

R

[(S)-Me2-Tyr]

[(S)-Pro]

[(S)-Leu]

[2S,4S-Hip][3S,4R,5S-Ist]

[1S,2R-Thr]

[(R)-N(Me)-Leu]

[(S)-Pro]

[Pyr]

aplidine R =Odidemnin B R -OH

Figure 1. Chemical structure of aplidine and didemnin B [10, 11]. Hip - hydroxyisovalerylpropionyl, Ist - isostatine, Leu - leucine, Me – methyl, Pro - proline, Pyr - pyruvoyl, Thr - threonine, Tyr - tyrosine.

Four phase I metabolites were discovered by us after recent in vitro investigations of the aplidine biotransformation using pooled mixed gender human liver microsomes. This chapter presents the mass spectrometric investigations for the identification of the four metabolites of aplidine. The structure elucidation of didemnin B and its sodium adduct using collision-induced dissociation (CID) mass spectrometry was previously reported by Ngoka et al. (1999) [9]. The in vitro metabolites of aplidine were identified using similar structure assignments by MS/MS after liquid chromatographic isolation.

Structure elucidation of aplidine metabolites

69

Materials and methods

Chemicals. Aplidine was kindly donated by PharmaMar (Tres Cantos, Madrid, Spain). Acetonitrile (gradient grade) was purchased from Biosolve (Valkenswaard, The Netherlands) and formic acid (p.a.), MgCl2

.6H2O (p.a.) and dimethyl sulfoxide (DMSO, synthesis grade) from Merck (Darmstadt, Germany). Water was purified on a multi-laboratory scale by reversed osmosis. Pooled human liver microsomes (mixed gender) were provided by Gentest (Becton Dickinson, Woburn, MA, USA). All other chemicals were purchased from Sigma Chemical Company (St. Louis, MO, USA) and were of analytical grade.

Sample Preparation. The incubation procedure of aplidine with human liver microsomes was a modification of the method described by Sparidans et al. (2001) for ecteinascidin 743 [10]. Twenty-five µl of 0.5 M potassium phosphate buffer (pH 7.4) were pipetted into a polypropylene micro tube on ice and 50 µl NADP regenerating system (NRS: 1.5 U/ml glucose-6-phosphate dehydrogenase, 0.5 mg/ml β-NADP, 4.0 mg/ml D-glucose-6-phosphate in 0.6 % (w/v) NaHCO3), 7.5 µl of 20 mg/ml MgCl2

.6H2O solution, and 50 µl of an aqueous aplidine solution (final concentration of 30 µg/ml in the microsomes suspension) were added. After briefly vortex-mixing, the tubes were incubated for 2 min at 37°C in a shaking water bath. Next, 5 µl pooled human liver microsomes (mixed gender, lot number 21) were added. The tube was vortex-mixed briefly again and the mixture was incubated at 37°C in a shaking water bath for 3 h. The reaction was terminated by adding 125 µl acetonitrile and vortex-mixing. The sample was centrifuged at approximately 15,000 g and 4°C for 1 min to remove proteins and the supernatant was injected for HPLC analysis. Control experiments were performed without aplidine and without liver microsomes, respectively. The extracts were purified by injection of 100 µl of the sample into the chromatographic system and manual collection of the individual metabolites. Pooled and single fractions of each metabolite were evaporated by a Gyrovap centrifugal evaporator (Howe, Banburry, UK) under vacuum (approximately 32 Pa) and temperature was kept at -50°C by a Cryocool CC-100 II (Neslab Instuments Inc., Portsmouth, NH, USA). For mass spectrometric analysis the residues were dissolved in 5 to 10 µl of 0.1% (v/v) formic acid in water/acetonitrile (1/1 (v/v)).

High performance liquid chromatography. The chromatographic assay was a modification of the method described by Waterval et al. (2001) [11]. The supernatants of the incubated mixtures were analyzed on an HPLC system consisting of two LC-10ATVP pumps, a SIL-10ADVP autoinjector (equipped with a 500 µl sample loop), a SCL-10AVP system controller, and a SPD-M10AVP photodiode array detector (all from Shimadzu, Kyoto, Japan). Data acquisition and processing were performed using a Class-VP 5.032 data system (Shimadzu). Injections were made on a Symmetry C18 column (4.6 x 100 mm, dp=3.5 µm, Waters Chromatography, Milford, MA, USA) with a Sentry Guard Symmetry C18 pre-column (3.9 x 20 mm, dp=5 µm, Waters). The column temperature was kept at room temperature. A gradient program was used with eluent A comprising 10 mM formic acid in water and eluent B comprising 10 mM formic acid in acetonitrile. After injection, elution started with 45% B and the eluent composition was raised linearly to 75% B during 20 min. This percentage was maintained for 2 min before conditioning with 45% B for 8 min. The eluent flow rate was 1.0 ml/min, the UV detection array was operated between 190 and 300 nm (resolution is 2 nm) and the peak areas were determined at 225 nm.

Chapter 4

70

Mass spectrometry. Mass spectrometric measurements were performed on a triple quadrupole instrument (Quattro Ultima, Micromass, Manchester, UK) equipped with a Z-nano electrospray source. Data acquisition and processing were performed using MassLynx NT 3.5 data system (Micromass). The protonated aplidine and metabolite ions [M+H]+ or the sodium adduct ions [M+Na]+ were formed via nano-electrospray ionization (ESI+). The compounds entered the mass spectrometer through an electrospray capillary set at 1.1 - 1.2 kV and nitrogen was used as nebulizing gas at a flow rate of 70 l/h. The cone voltage was set at 20 to 30 V and the ion-source parameters were optimized with respect to the analyzed ions. The MS spectra, recorded between m/z 500 – 1200, were obtained at a scan speed of ~350 m/z units s-1 with a mass resolution corresponding to 0.5 m/z unit at half peak height. Argon was used as collision gas for CID at a pressure of 2.3 * 10-3 mbar and the collision energy was set at 20 to 30 V. The product ion spectra, recorded between 100 and 1200 m/z (varied depending on the compound), were obtained at a scan speed of ~550 m/z units s-1, with mass resolution corresponding to 0.5 m/z unit at half peak height. The instrument was previously calibrated with sodium iodide. Results and discussion

Chromatograms of aplidine incubated without and with pooled human liver microsomes are shown in figure 2. Two conformations of aplidine (aplidine A and B) can be observed. The two conformers are the cis and trans isomers of the pyruvoyl - proline amide bond and the plateau between the peaks is caused by the inter-conversion of the aplidine isomers during the chromatographic run [11-14]. The trans conformer of aplidine is eluted before the cis conformer [13]. The observed products are metabolites of aplidine, because they are only formed in the presence of human liver microsomes with the drug. Furthermore, the spectra of aplidine and the observed products recorded online with a photodiode array detector are almost identical (figure 3).


71

Figure 2. Chromatogram at 225 nm of aplidine after incubation without pooled human liver microsomes (1) and aplidine after incubation with microsomes (2).

190 200 210 220 230 240 250 260 270 280 290 300

wavelenght (nm)

rela

tive

abso

rban

ce

2345

67

1

Figure 3. UV spectra (190-300 nm) of the aplidine metabolites apli-da/h (1), apli-h 1 and 2 (2 and 3 respectively), apli-da (4), and apli-dm (5) and aplidine A and B (6 and 7).

2

1

Minutes0 2 4 6 8 10 12 14 16 18 20

mA

U

-5

0

5

10

15

20

25

30

35

40

45

apli-

da/h ap

li-h1

apli-

da apli-

h2

apli-

dm

aplid

ine

A

aplid

ine

B

Time (min)

Abs

orba

nce

(mA

U)

Chapter 4

72

The structural assignments of aplidine (dehyrodidemnin B) and its metabolites, are based on the work of Ngoka et al. (1999) on the MS/MS identification of cyclodepsipeptides, e.g. didemnin B [9]. The protonated ions are very stable under our (CID) conditions where only the side chain fragment (296 m/z) and the cyclic fragment (817 m/z) are found for didemnin B. However, the sodium ion selectively binds to ester bonds causing ring opening. In didemnins, it exclusively binds to the ester group at threonine and not at hydroxyisovalerylpropionyl group. The selective cleavage (figure 4) leads to a linear acylium ion and under CID conditions a sequence specific fragmentation occurs from the C-terminus. However, the ester linkage between leucine and hydroxyisovalerylpropionyl group is not broken and they will be ejected simultaneous. To elucidate the structure of the aplidine metabolites, the atomic mass unit (amu) gains or losses were compared with the [M+H]+ (changes in the side chain) and [M+Na]+ (changes in the ring structure) product ion CID spectra of aplidine.

N

O

OH

NH

OO

N

NO

O

N

OO

OOO

RNa

+

NO

O

NO

O

A

R =

N

OC

+

O

N

O

N

OOO

OOH

NH O

NR O

O

Na+

B942.1845.0575.7

418.4

295.41133.4

ring opening

Figure 4. The sodium adduct of aplidine (A) and its selective scission of the depsipeptide ring (included in the figure are fragment ions that may be observed under MS/MS conditions after collision-induced dissociation) (B). R = side chain.


73

Aplidine A and B. The product ion spectra for aplidine A and B as [M+H]+ and [M+Na]+ in the mass range of m/z 200 – 1200 amu are illustrated in figure 5A and B, respectively. In the [M+H]+ product ion spectra, the side chain (295 m/z) and the ring (815 m/z) are found (table 1). The first fragmentation step for aplidine in the [M+Na]+ product ion spectra is the loss of the (S)-Me2-tyrosine moiety (-191.2 m/z) and the fragmentation cascade is followed by the proline (-97.1 m/z), leucine and hydroxyisovalerylpropionyl (- 269.3 m/z), and the isostatine (-157.2 m/z) moieties (table 2) and finally the threonine group (-123.1 m/z). Sequence specific fragmentation occurs and aplidine behaves the same as didemnin B. Therefore, the [M+Na]+ spectra can be used to elucidate the metabolite structures. Aplidine A and B have identical MS spectra, which agrees with their isomeric structures. Figure 5. Product ion CAD spectra of aplidine A and B as [M+H]+ (A) and [M+Na]+ (B) generated by ESI+ on the mass spectrometer. In the [M+H]+ spectrum the 1083 m/z is [M+H]+ with loss of C2H5 from isostatine and the 799 m/z is the ring with loss of H2O.

200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150m /z0

100

%

1111

295

220

1083799

307 349

817

A

B

100 200 300 400 500 600 700 800 900 1000 1100 1200m /z0

100

%

x10 941.7

210.2

194.3

113.1

97.1

140.1

175.3

575.4

407.3

374.3226.2365.2

238.3336.3292.3

418.3

487.4443.4557.4515.4

576.4844.6

798.6713.5

1132.8

942.6

943.7

1133.8

1134.8

Chapter 4

74

Metabolite apli-h 1 and 2. The product ion spectra for apli-h 1 and 2 as [M+H]+ and [M+Na]+ are illustrated in figure 6A and B respectively. The [M+H]+ exhibits a 16 amu higher mass in the ring compared to aplidine (table 1). This indicates the gain of an OH-group in the ring. From the product ions of the [M+Na]+ it can be seen that the OH-group is attached to the leucine-hydroxyisovalerylpropionyl group (table 2). The product ion of the leucine-hydroxyisovalerylpropionyl group was further investigated with MSMS of the product ion. The fragmentation pattern showed that the OH-group was attached to the isopropyl group of hydroxyisovalerylpropionyl (results not shown). Apli-h 1 and 2 have identical MS spectra, which can be explained by the cis and trans isomerism as for aplidine. Figure 6. Product ion CAD spectra of apli-h 1 and 2 as [M+H]+ (A) and [M+Na]+ (B) generated by ESI+ on the mass spectrometer. In the [M+H]+ spectrum the 1099 m/z is [M+H]+ with loss of C2H5 from isostatine and the 815 m/z is the ring with loss of H2O. In the [M+Na]+ spectrum the 1091 m/z and 900 m/z are losses of C4H9 from leucine.

200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150m /z0

100

%

1127

295

220

833815

307365

535 557 797684 735707

1099

859

10691013917

A

B

200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200m /z0

100

%

aplidine305 53 (4.543) Cm (37:146) TOF MSMS 1148.70ES+ 2.20e3x10 899.6

365.3

210.3

268.3226.2 277.3

347.3310.3

575.4

400.3

418.3

487.4

443.5

469.4

557.4515.4

671.4576.4

645.5802.5

756.6

672.4 757.6 803.6

1148.8

1090.7

957.7

900.6

901.7

958.7

959.7

1091.7

1092.7

1149.8

1150.7


75

Metabolite apli-da. The [M+H]+ and [M+Na]+ product ion spectra, illustrated in figure 7A and B respectively, show the loss of 56 amu units compared to aplidine. From the [M+H]+ spectra, it appears that the loss is specifically in the side chain (table 1). The loss of 56 amu units can be explained by the loss of an isopropyl group, the only group which leads to -56 m/z in the side chain. Therefore, the suggested structure of the metabolite is aplidine C-dealkylated at the N(Me)-leucine group (apli-da). Figure 7. Product ion CAD spectra of apli-da as [M+H]+ (A) and [M+Na]+ (B) generated by ESI+ on the mass spectrometer. In the [M+H]+ spectrum the 1027 m/z is [M+H]+ with loss of C2H5 from isostatine and the 804 m/z is the ring with loss of CH.

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150m /z0

100

%

1055

1027

375307

239138 210

164 349479 804

749958

930817

A

200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250m /z0

100

%

885.30

518.91

431.41362.25

502.51

788.00

520.35

885.68

1076.95

1076.13

887.44

1077.64

1078.08

B

Chapter 4

76

Metabolite apli-da/h. The product ion spectra for apli-da/h as [M+H]+ and [M+Na]+ are illustrated in figure 8A and B, respectively. The product ions of the [M+H]+ show a loss of 56 amu in the side chain (table 1) and from the product ions of the [M+Na]+ a gain of 16 amu in the ring structure (table 2) can be seen. This indicates that an isopropyl group has been lost and an OH-group is gained and thus a combination of the two metabolic conversions previously mentioned. Thus, aplidine is hydroxylated at the isopropyl group and C-dealkylated at the N(Me)-leucine group (apli-da/h). Figure 8. Product ion CAD spectra of apli-da/h as [M+H]+ (A) and [M+Na]+ (B) generated by ESI+ on the mass spectrometer. In the [M+H]+ spectrum the 1043 m/z is [M+H]+ with loss of C2H5 from isostatine, the 985 m/z is [M+H]+ with loss of C4H9 from leucine, and the 803 m/z is the ring with loss of CH2O. In the [M+Na]+ spectrum the 1035 m/z and 844 m/z are losses of C4H9 from leucine.

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150m /z0

100

%

1072

375307

239210

138

113

192

164 268

347

1043

985

479

461

803

706497

594 688741

786 885858 946 1013

A

200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200m /z0

100

%

843.5

519.3

288.4 457.4

615.3589.4

520.3746.4

700.4673.4 747.4

1034.6

844.5

901.6

845.5

902.5

903.6

1092.6

1035.6

1036.6

1091.8

1093.7

1094.7

B


77

Metabolite apli-dm. The [M+H]+ and [M+Na]+ product ion spectra of metabolite apli-dm are shown in figure 9A and B. This metabolite shows a loss of 14 amu in the ring structure (table 1), indicating the loss of a methyl-group. From the comparison of the [M+Na]+ product ions of aplidine and this metabolite (table 2), it can be concluded that the demethylation has occured in the last group sequentially deleted from the ring. Therefore, aplidine is C-demethylated at the threonine group (apli-dm). Figure 9. Product ion CAD spectra of apli-dm as [M+H]+ (A) and [M+Na]+ (B) generated by ESI+ on the mass spectrometer. In the [M+H]+ spectrum the 1069 m/z is [M+H]+ with loss of C2H5 from isostatine and the 785 m/z is the ring with loss of H2O.

250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100m /z0

100

%

1097

295

268

1069

785

307

375349 522

440394 413 496

773683575547 612587 637 749715

803

845

827999912860 889 972940

1034

A

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200m /z0

100

%

1118.97

927.45

561.56

830.91

1119.84

B

Chapter 4

78

The loss of the isopropyl group from the side chain as in apli-da and apli-da/h may result in the loss of the cis-trans isomerism. The metabolite apli-dm with an intact isopropyl group will most likely show cis-trans isomerism, but both isomers may not show separation or possibly one elutes around aplidine A or aplidine B. The attachment of the OH to the hydroxyisovalerylpropionyl group leads to a more unstable structure in the [M+Na]+. Furthermore, the structure variations of the metabolites all correspond with their chromatographic elution order and thus supports the suggested structures of the metabolites. The gain of an OH-group and the loss of an alkyl group will give a more polar compound, which will elude before aplidine.

Table 1. [M+H]+ mass spectrometric results of aplidine and its metabolites. product ions [M+H]+

compound parent ion side chain ring aplidine A 1111 295 817 aplidine B 1111 295 817

apli-h 1 1127 295 833* apli-h 2 1127 295 833* apli-da 1055 239** 817

apli-da/h 1072 239** 803*** apli-dm 1097 295 803****

* gain of OH-group in 2S,4S-hydroxyisovalerylpropionyl ** loss of C4H8-group from (R)-N(Me)-leucine *** gain of OH-group in 2S,4S-hydroxyisovalerylpropionyl and loss of CH2O-group from

(S)-Me2-tyrosine **** loss of CH2-group from 1S,2R-threonine

Table 2. [M+Na]+ mass spectrometric results of aplidine and its metabolites. product ions [M+Na]+ loss of

compound parent

ion (S)-Me2-Tyr1 (S)-Pro2 (S)-Leu3 and 2S,4S-Hip4 3S,4R,5S-Ist5

aplidine A 1132.8 941.7 844.6 575.4 418.3 aplidine B 1132.8 941.7 844.6 575.4 418.3

apli-h 1 1148.8 957.7 861.0 575.4 418.3 apli-h 2 1148.8 957.7 861.0 575.4 418.3 apli-da 1077.0 885.3 788.0 518.9 362.3

apli-da/h 1092.6 901.6 804.9 519.3 362.3 apli-dm 1119.0 927.5 830.9 561.6 404.4 1 (S)-Me2-tyrosine 2 (S)-proline 3 (S)-leucine 4 2S,4S-hydroxyisovalerylpropionyl 5 3S,4R,5S-istostatine


79

Conclusions

The chemical structures of four metabolites of aplidine generated in vitro have been identified by mass spectrometric methods. Aplidine hydroxylated at the isopropyl group (figure 10 A), aplidine C-dealkylated at the N(Me)-leucine group (figure 10 B), aplidine with both these converted amino acid residues (figure 10 C), and aplidine C-demethylated at the threonine group (figure 10 D) have been found. Investigation of their presence in clinical samples can now be started, preferably using LC-MS/MS.

N

O

OH

NH

OO

NH

NH

NO

O

NO

O

O

O

N

OO

OOO

OH

N

O

OH

NH

O

NH

NH

NO

O

NO

O

O

O

N

OO

OOO

O

OH

N

O

OH

NH

OO

NH

NH

NO

O

NO

O

O

O

N

OO

OOO

N

O

OH

NH

OO

NH

NH

NO

O

NO

O

O

O

N

OO

OOO

(A)

(C)

(B)

(D)

Figure 10. Proposal for the structures of discovered metabolites of aplidine: apli-h1 and 2 (A), apli-da (B), apli-da/h (C), and apli-dm (D).

Chapter 4

80

References 1. Sakai R., Rinehart K.L., Kishore V., Kundu B., Faircloth G., Gloer J.B., Carney J.R.,

Namikoshi M., Sun F., Hughes Jr, R.G., Garcia Gravalos D., de Quesada T.G., Wilson G.R., and Heid R.M. (1996). Structure - activity relationships of the didemnins. J. Med. Chem. 39: 2819-2834.

2. Faircloth G., Hanauske A., Depenbrock H., Peter R., Crews C.M., Manzanares I., Meely K., Grant W., and Jimeno J.M. (1997). Pre-clinical characterization of aplidine, a new marine anticancer depsipeptide (abstract). Proc. Am. Assoc. Cancer Res. 38: 692

3. Depenbrock H., Peter R., Faircloth G.T., Manzanares I., Jimeno J., and Hanauske A.R. (1998). In vitro activity of aplidine, a new marine-derived anti-cancer compound, on freshly explanted clonogenic human tumour cells and haematopoietic precursor cells. Br. J. Cancer 78: 739-744.

4. Geldof A.A., Mastbergen S.C., Henrar R.E., and Faircloth G.T. (1999). Cytotoxicity and neurocytotoxicity of new marine anticancer agents evaluated using in vitro assays. Cancer Chemother. Pharmacol. 44: 312-318.

5. Jimeno J.M. (2002). A clinical armamentarium of marine-derived anti-cancer compounds. Anti-Cancer Drugs 13 (suppl 1): S15-S19.

6. Erba E., Ronzoni S., Bergamaschi D., Bassano L., Desiderio M.A., Faircloth G., Jimeno J., and D’Incalci M. (1996). Mechanism of antileukemic activity of aplidine (abstract). Proc. Am. Assoc. Cancer Res. 40: 3.


8. Urdiales J.L., Morata P., Nunez De Castro I., and Sanchez-Jimenez F. (1996). Antiproloferative effect of dehydrodidemnin B (DDB), a depsipeptide isolated from Mediterranean tunicates. Cancer Lett. 102: 31.

9. Ngoka L.C.M., Gross M.L., and Toogood P.L. (1999). Sodium-directed selective cleavage of lactones: a method for structure determination of cyclodepsipetides. Int. J. Mass Spectrom. 182/183: 289-298.

10. Sparidans R.W., Rosing H., Hillebrand M.J.X., Lopez-Lazaro L., Jimeno J.M., Manzanares I., van Kesteren Ch., Cvitkovic E., van Oosterom A.T., Schellens J.H.M., and Beijnen J.H. (2001). Search for metabolites of ecteinascidin 743, a novel, marine-derived, anti-cancer agent, in man. Anti-Cancer Drugs 12: 653-666.

11. Waterval J.C.M., Bloks J.C., Sparidans R.W., Beijnen J.H., Rodriguez-Campos I.M., Bult A., Lingeman H., and Underberg W.J.M. (2001). Degradation kinetics of aplidine, a new marine antitumoural cyclic peptide, in aqueous solution. J. Chromatogr. B 754: 161-168.

12. Ma S., Kálmán F., Kálmán A., Thunecke F., and Horváth C. (1995). Capillary zone electrophoresis at subzero temperatures I. Separation of the cis and trans conformers of small peptides. J. Chromatogr. A 716: 167-182.

13. Kálmán A., Thunecke F., Schmidt R., Schiller P.W., and Horváth C. (1996). Isolation and identification of peptide conformers by reversed-phase high-performance liquid chromatography and NMR at low temperature. J. Chromatogr. A 729: 155-171.

14. Sparidans R.W., Kettenes-van den Bosch J.J., van Tellingen O., Nuyen B., Henrar R.E.C., Jimeno J.M., Faircloth G., Floriano P., Rinehart K.L., and Beijnen J.H. (1999). Bioanalysis of aplidine, a new marine antitumoral depsipeptide, in plasma by high-performance liquid chromatography after derivatization with trans-4’-hydrazino-2-stilbazole. J. Chromatogr. B 729: 43-53.

81

CHAPTER In vitro characterization of the human biotransformation and CYP reaction phenotype of ET-743 (Trabectidin®, Yondelis®), a novel marine anti-cancer drug. Esther F.A. Brandon, Rolf W. Sparidans, Kees-Jan Guijt, Sjoerd Löwenthal, Irma Meijerman, Luis López Lázaro, Jos H. Beijnen, and Jan H.M. Schellens. Abstract

ET-743 (Yondelis®, Trabectedin®) is a potent marine anti-cancer drug and is currently being investigated in phase I and II clinical trials. To assess the biotransformation and CYP reaction phenotype and their potential implications for human pharmacology and toxicology, the in vitro metabolism of ET-743 was characterized using incubations with human liver preparations, cytochrome P450 (CYP) and uridine diphosphoglucuronosyl transferase (UGT) supersomes.

CYP supersomes and human liver microsomes showed that ET-743 was metabolized mainly by CYP3A4, but also by CYP2C9, 2C19, 2D6, and 2E1. The Km value of ET-743 in female human liver microsomes was significantly lower compared to male microsomes, while the Vmax values did not differ. ET-743 showed the highest affinity for CYP3A4 and the highest maximal metabolic rate for CYP2D6 among the CYPs shown to metabolize ET-743. Furthermore, ET-743 glucuronidation, catalyzed by UGT2B15, was observed in human liver microsomes and S9 fraction. In addition, glucosidation was observed for ET-743 in human liver cytosol and S9 fraction. No sulphation was observed for ET-743 in cytosol or S9 fraction. ET-743 was more extensively metabolized when CYP activity was combined with phase II enzymes UGT and glutathione-S-transferase (GST).

These results provide evidence that CYP3A4 has a major role in the metabolism of ET-743 in vitro with additional involvement of CYP2C9, 2C19, 2D6, and 2E1. Furthermore, ET-743 is conjugated in vitro by UGT and GST.

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82

Introduction

Ecteinascidin-743 (ET-743, Yondelis®, Trabectedin®) (figure 1) is a tetrahydro-isoquinoline isolated from the Caribbean tunicate Ecteinascidia turbinata [1]. The compound exhibited in vitro activity at nanomolar concentrations against various solid tumor cell lines, including melanoma and ovarian, renal, prostate, breast, and non-small cell lung cancer cell lines [2]. In addition, ET-743 appears effective against human xenografts of non-small cell lung, melanoma and breast tumors in vivo [2-3]. The mode of action of ET-743 has not been completely elucidated, but several mechanisms have been proposed. It is believed to involve binding to the minor groove of the DNA, interactions with transcription factors and DNA binding proteins, disorganization of the microtubule network, inhibition of topoisomerase I, pertubation of the cell cycle, and interference with DNA repair mechanisms [4, 5].

Multiple infusion schedules were investigated in phase I clinical trials and studies investigating the effects of ET-743 combined with cisplatin, carboplatin, and doxorubicin are currently in progress or preparation [4]. From phase I trials, a treatment schedule was chosen for phase II clinical trials. In these studies, ET-743 was administered as 3 or 24 h continuous i.v. infusion [6]. Phase II clinical trials are still ongoing, but activities against soft-tissue sarcomas, breast tumors, endometrial cancer, and ovarian cancer have already been shown [1, 6-8].

Reid et al. (2002) investigated the biotransformation of ET-743 and showed that ET-743 was metabolized by microsomes from cytochrome P40 (CYP) 2C9, 2D6, 2E1, and 3A4 transfected human B-lymphocyte cell lines [11]. Further, studies by Sparidans et al. (2001) showed that ET-743 was metabolized by human liver microsomes and was conjugated by rabbit UGT [12]. However, the enzyme kinetics of ET-743 and the relative contribution (%) of each CYP (CYP reaction phenotype) have not yet been identified. Knowledge about enzyme kinetics and CYP reaction phenotype is important in order to interpret the pharmacological properties found in clinical trials and to predict possible drug-drug interactions with other (anti-cancer) drugs. Furthermore, the biotransformation of ET-743 by human phase II enzymes and phase I in combination with phase II enzymes has not yet been reported. The elucidation of the biotransformation products of ET-743 may be complicated because of the presence of several potential sites for phase I and II reactions and the formation of degradation products (figure 1) [11, 12].

Different in vitro methods were therefore used in this explorative investigation, including pooled human liver microsomes, cytosol, and S9 fraction in combination with HPLC-UV analysis. The contribution of various isoforms of CYP and uridine diphosphoglucuronosyl transferase (UGT) to the biotransformation was investigated using pooled human liver microsomes in combination with specific CYP inhibitors and CYP and UGT supersomes.

In vitro biotransformation and CYP reaction phenotype of ET-743

83

N+

OOH

O O

O

ONH

O

O

NH

S

N

N

O

O

O

O

OH

O

O

OH

OH

O

O

a

b

c

a

ETM-204

b

ETM-305

(B)

C39H43N3O11SM.W. = 762 g/mol

= conjugation

= N-dealkylation

= hydroxylation

(A)

Figure 1. Chemical structure of ET-743 (A) and its degradation products ETM-204 and ETM-305 (B) [10]. The different squares and ovals indicate potential sites for biotransformation [11, 12]. In addition, all the ester bonds are potential sites for hydrolysis. Materials and methods

Materials. ET-743 was kindly donated by PharmaMar (Tres Cantos, Madrid, Spain). Methanol (HPLC grade) and acetonitrile (gradient grade) were purchased from Biosolve (Valkenswaard, The Netherlands) and formic acid (p.a.), ammonium acetate (p.a.), MgCl2

.6H2O (p.a.), and dimethyl sulfoxide (DMSO, synthesis grade) from Merck (Darmstadt, Germany). Water was purified on a multi-laboratory scale by reversed osmosis. Pooled human liver microsomes (mixed gender, male, and female), pooled human liver cytosol, pooled human liver S9 fraction, and human CYP and UGT supersomes (Baculovirus-insect-cell expressed) were provided by Gentest (Becton Dickinson, Woburn, MA, USA). Ritonavir was provided by Abbott (Chicago, IL, USA) and all other chemicals were purchased from Sigma Chemical Company (St. Louis, MO, USA) and were of analytical grade.

Chapter 5

84

ET-743 incubations with pooled human liver microsomes (mixed gender, male, and female). The incubation procedure of ET-743 with human liver microsomes was a modification of the method described by Sparidans et al. (2001) [10]. Twenty-five µl of 0.5 M potassium phosphate buffer (pH 7.4) were pipetted into a polypropylene micro tube on ice and 50 µl NADP regenerating system (NRS: 1.5 U/ml glucose-6-phosphate dehydrogenase, 0.5 mg/ml β-NADP, 4.0 mg/ml D-glucose-6-phosphate in 0.6 % NaHCO3), 7.5 µl of 20 mg/ml MgCl2

.6H2O solution, and 50 µl of an aqueous ET-743 solution (1% DMSO, final concentration of 50 µg/ml in the microsomes suspension) were added. After vortex-mixing briefly, the tubes were incubated for 2 min at 37°C in a shaking water bath. Next, 5 µl of mixed gender (lot number 18), male (lot number 2), or female (lot number 1) pooled human liver microsomes were added. The tube was vortex-mixed briefly again and the mixture was incubated for 4 h at 37°C in a shaking water bath. The reaction was terminated by adding 125 µl ice-cold methanol and vortex-mixing. The sample was centrifuged at approximately 15,000 g and 4°C for 1 min to remove proteins and the supernatant was injected for gradient chromatographic analysis. Control experiments were performed without ET-743 and without liver microsomes, respectively.

ET-743 incubated with pooled human liver microsomes in the absence and presence of CYP inhibitors. ET-743 incubations with liver microsomes in the absence and presence of CYP inhibitors were performed according to the method described for liver microsomes with slight modifications. Twelve and a half µl of 1 M potassium phosphate buffer (pH 7.4) were pipetted into a polypropylene micro tube on ice and 50 µl NRS, 7.5 µl of 20 mg/ml MgCl2

.6H2O solution, and 10 µl of an aqueous CYP inhibitor solution (1% (v/v) DMSO) were added. ET-743 was incubated with microsomes and the following inhibitors: 50 µM sulfaphenazole (CYP2C9), 200 µM (S)-(+)-mephenytoin (CYP2C19), 50 µM quinidine (CYP2D6), 200 µM chlorzoxazone (CYP2E1), and 100 µM ritonavir (CYP3A4). After vortex-mixing briefly, the tubes were incubated at 37°C in a shaking water bath for 2 min. Next, 5 µl of pooled human liver microsomes (mixed gender, lot number 21) were added. The tube was vortex-mixed briefly again and the mixture was then incubated at 37°C in a shaking water bath for 5 min. Fifty µl of an aqueous ET-743 solution (1% (v/v) DMSO, final concentration of 50 µg/ml) were added and vortex-mixed briefly. The tube was incubated further at 37°C in a shaking water bath for 4 h. The reaction was terminated and proteins removed as previously described. The supernatant was injected for gradient chromatographic analysis. Control experiments were performed without ET-743 and without liver microsomes, respectively.

ET-743 incubated with human CYP supersomes. Incubations with human CYP supersomes were performed as with the liver microsomes. Instead of liver microsomes, 5 µl of the CYP supersomes suspension were added. The following human CYP supersomes were tested: CYP1A1 (lot number 15), CYP1A2 (lot number 20), CYP2A6 (lot number 6), CYP2B6 (lot number 8), CYP2C8 (lot number 11), CYP2C9*1(Arg144) (lot number 17), CYP2C19 (lot number 12), CYP2D6*1 (lot number 27), CYP2E1 (lot number 9), CYP3A4 (lot number 40), and CYP4A11 (lot number 7). All CYPs were co-expressed with P450 reductase and CYP2A6, 2B6, 2C8, 2C9, 2C19, 2E1, and 3A4 were also co-expressed with cytochrome b5 in the insect cells. A concentration of 50 µg/ml ET-743 was incubated with the human CYP supersomes. The incubation was terminated as described previously after 4 h. The proteins were removed as previously described and the supernatant was injected for gradient chromatographic analysis. Control experiments were performed without substrate or with insect cell control supersomes (lot number 22).


85

Glucuronidation of ET-743 with rabbit UGT. The glucuronidation of ET-743 with rabbit UGT was a modification of the method described by Sparidans et al. (2001) [10]. Fourty µl of 0.1 M MgCl2

.6H2O solution, 10 µl of 0.5 mg/ml alamethicin, 50 µl ET-743 in water (1% (v/v) DMSO, final concentration of 50 µg/ml), 50 µl of 20 mM uridine diphosphoglucuronic acid (UDPGA), and 50 µl of 15 mg/ml rabbit UGT in 0.5 M potassium phosphate buffer (pH 7.4) (lot number 39H7848) were pipetted into a polypropylene micro tube on ice. The tube was vortex-mixed and the mixture was incubated for 5 h at 37°C in a shaking water bath. The reaction was terminated by adding 200 µl ice-cold methanol and vortex-mixing briefly. Proteins were removed and the supernatant was injected for gradient chromatographic analysis. Individual control experiments were performed without ET-743 and without rabbit UGT, respectively.

Glucuronidation of ET-743 by pooled human liver microsomes. The glucuronidation of ET-743 with human UGT was a modification of the method described for rabbit UGT. Thirty µl of 0.1 M MgCl2

.6H2O solution, 10 µl of 0.5 mg/ml alamethicin, 50 µl ET-743 in water (1% (v/v) DMSO, final concentration of 50 µg/ml), 25 µl of 1 M potassium phosphate buffer (pH 7.4), 50 µl of 15 mg/ml uridine diphosphoglucuronic acid (UDPGA), and 25 µl water were pipetted into a polypropylene micro tube on ice. After vortex-mixing briefly, the tube was incubated at 37°C in a shaking water bath for 2 min. Next, 10 µl of pooled human liver microsomes (lot number 21) were added. The tube was vortex-mixed briefly again and the mixture was then incubated at 37°C in a shaking water bath for 5 h. The reaction was terminated and proteins removed as previously described. The supernatant was injected for gradient chromatographic analysis. Individual control experiments were performed without ET-743 and without pooled human liver microsomes, respectively.

ET-743 incubated with human UGT supersomes. The incubation of ET-743 with human UGT supersomes was a modification of the method described by Gentest [13]. Twenty µl of 1 M potassium phosphate buffer (pH 7.4) were pipetted into a polypropylene micro tube on ice and 10 µl of 0.5 mg/ml alamethicin, 20 µl of 0.1 M MgCl2

.6H2O, 20 µl of 20 mM UDPGA, 50 µl ET-743 in water with 1% (v/v) DMSO (final concentration of 50 µg/ml in the supersomes suspension), 70 µl H2O, and 10 µl of the supersomes suspension were added. The following human UGT supersomes were tested: UGT1A1 (lot number 8), UGT1A3 (lot number 8), UGT1A9 (lot number 6), and UGT2B15 (lot number 5). After vortex-mixing briefly, the mixture incubated for 5 h at 37°C in a shaking water bath. The reaction was terminated and proteins removed as previously described. The supernatant was injected for gradient chromatographic analysis. Control experiments were performed without substrate and without UDPGA or with UGT insect cell control supersomes (lot number 5).

Conjugation of ET-743 by N-acetyl transferase, sulfotransferase and glutathione-S-transferase in pooled human liver cytosol. The incubation of ET-743 with pooled human liver cytosol was a modification of the method described by Gentest [13]. Equal volumes (20 µl) of 1 M potassium phosphate buffer (pH 7.4), 10 mM dithiotreitol (DTT), 1 mM acetyl-coenzyme A (acetyl-CoA), 45 mM acetyl-DL-carnitine, 80 units/ml carnitine acetyl transferase (from pigeon breast muscle), 1 mM adenosine 3’-phosphate 5’-phosphosulfate (PAPS), and 10 mM glutathione were pipetted into a polypropylene micro tube on ice. Six µl H2O and 50 µl of an aqueous dilution of ET-743 (1% (v/v) DMSO, final concentration of 50 µg/ml in the cytosol suspension) were added and vortex-mixed briefly. Next, 4 µl human liver cytosol (lot number 2) were added and, after vortex-mixing briefly, the mixture was incubated at 37°C in a shaking water bath for 5 h. The reaction was terminated by adding 200 µl acetonitrile and vortex-mixing. The proteins were removed as previously described and the supernatant was injected for gradient chromatographic analysis. Individual control experiments were performed without ET-743, only DTT, acetyl-CoA, acetyl-DL-carnitine, and carnitine acetyl transferase (only N-acetyltransferase (NAT) activity), only PAPS (only

Chapter 5

86

sulfotransferase (SULT) activity), only glutathione (only glutathione-S-transferase (GST) activity), and without all co-factors for enzyme activity. In addition, all four substrates of NAT were individually tested as controls.

ET-743 incubations with pooled human liver S9 fraction. The incubation of ET-743 with pooled human liver S9 fraction was a modification of the method described by Gentest [13]. Equal volumes (10 µl) of 75 mg/ml UDPGA, 10 mM DTT, 1 mM acetyl-CoA, 45 mM acetyl-DL-carnitine, 80 units/ml carnitine acetyl transferase, 1 mM PAPS, and 10 mM glutathione were pipetted into a polypropylene micro tube on ice. Twenty-four µl NRS (5 U/ml glucose-6-phosphate dehydrogenase, 1.67 mg/ml β-NADP, and 13.33 mg/ml D-glucose-6-phosphate in 2% (w/v) NaHCO3), 50 µl of an aqueous dilution of ET-743 (1% (v/v) DMSO, final concentration of 50 µg/ml in the S9 suspension), 12 µl of 20 mg/ml MgCl2

.6H2O, 20 µl of 1 M potassium phosphate buffer (pH 7.4), and 14 µl H2O were added and vortex-mixed briefly. Subsequently, the tubes were incubated at 37°C in a shaking water bath for 2 min. Next, 10 µl pooled human liver S9 fraction (lot number 5) were added and vortex-mixed. The mixture was incubated for 4 h at 37°C in a shaking water bath and the reaction was terminated by adding 200 µl ice-cold methanol and vortex-mixing. The sample was centrifuged at approximately 15,000 g and 4°C for 1 min and the supernatant was then injected for gradient chromatographic analysis. Individual control experiments were performed without substrate, without all co-factors for enzyme activity and with co-factors present for only one or two enzymes (only one enzyme or a combination of CYP with a phase II enzyme were active), respectively.

Lineweaver-Burke plot of ET-743 in mixed gender, male, and female pooled human liver microsomes. The incubation procedure of ET-743 with pooled human liver microsomes to obtain a Lineweaver-Burke plot was a modification of the method described previously for microsomes. Seven different concentrations of ET-743 (concentration range of 0.33-10 µg/ml) were incubated with human liver microsomes to generate one Lineweaver-Burke plot. The following human liver microsomes were tested: mixed gender (lot number 21), male (lot number 3), and female (lot number 2). Each ET-743 concentration was incubated for 7 different time points at 37°C in a shaking water bath and the length of the incubation depended on the substrate concentration (table 1). The incubation was terminated by adding 125 µl acetonitrile and vortex-mixing. The supernatant was injected for isocratic chromatographic analysis.

Table 1. ET-743 incubation times with pooled human liver microsomes. A concentration range of 0.33-10 µg/ml ET-743 were incubated with mixed gender, female, and male human liver microsomes.

ET-743 conc. (µg/ml) in incubation mixture incubation times 0.33 0.50 0.67 1.0 2.0 10

0-10-20-30-40-50-60 s X 0-0.5-1-1.5-2-2.5-3 min X X X X 0-5-10-15-20-25-30 min X


87

Lineweaver-Burke plot of ET-743 in CYP supersomes. Incubations with human CYP supersomes were performed according to the incubation method as described in the previous paragraph. The following CYP supersomes were tested: CYP2C9*1 (lot number 22), CYP2C19 (lot number 17), CYP2D6*1 (lot number 35), CYP2E1 (lot number 12), and CYP3A4 (lot number 50). Each ET-743 concentration was incubated for 7 different time points at 37°C in a shaking water bath (table 2). The supernatant was injected for isocratic liquid chromatographic analysis.

Table 2. ET-743 incubation times with supersomes. incubation time ET-743 conc. (µg/ml) in incubation mixture

CYP2C9*1 0.20 0.80 1.33 2.0 4.0 10

0-2.5-5-7.5-10-12.5-15 min X 0-5-10-15-20-25-30 min X X X X X

CYP2C19 1.0 1.6 2.0 3.0 4.0 10 20 40

0-5-10-15-20-25-30 min X X X 0-10-20-30-40-50-60 min X X X X X

CYP2D6*1 1.33 5 7.5 10 12.5 25 50

0-2.5-5-7.5-10-12.5-15 min X X 0-2-4-8-12-16-20 min X

0-5-10-15-20-25-30 min X X X X

CYP2E1 1.33 2.0 4.0 10 0-5-10-15-20-25-30 min X X X 0-10-20-30-40-50-60 min X

CYP3A4 0.33 0.40 0.50 0.67 1.0 2.0 4.0 10

0-05-1-1.5-2-2.5-3 min X X X X X X 0-2-4-5-6-8-10 min X

0-5-10-15-20-25-30 min X

Determination of the protein-binding of ET-743 in pooled human liver microsomes. ET-743 (final concentration range of 2-50 µg/ml) was pre-incubated with human microsomes for 15 min on ice. The reaction was terminated by removing proteins using ultra-centrifugation with Micronon YM-10 ultra-centrifuge tubes (cut-off filter of 10 kDa) (Millipore, Bedford, MA, USA) for 120 min at 14,000 g and 4°C. The protein binding was estimated by quantification of ET-743 in the ultra-filtrate. The samples were diluted 1:1 (v/v) with methanol and analyzed with gradient liquid chromatographic assay. Calibration was performed using the same concentration range of ET-743 incubated in phosphate buffered saline.

Analysis of ET-743 and metabolites by gradient HPLC. The chromatographic assay was a modification of the method described by Sparidans et al. (2001) [10]. The supernatants of the incubated mixtures were analyzed on an HPLC system consisting of two LC-10ATVP pumps, a SIL-10ADVP autoinjector (equipped with a 500 µl sample loop), a SCL-10AVP system controller, and a SPD-M10AVP photodiode array detector (all from Shimadzu, Kyoto, Japan). The column was thermostated by a Waters temperature control module and a Waters column heater module (Milford, MA, USA). Data were recorded on a Hermac Pentium 440,

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122 MB personal computer (Scherpenzeel, The Netherlands) equipped with the Class-VP 5.032 software (Shimadzu). Injections (50 µl) were made on a Symmetry C18 column (4.6 x 100 mm, dp=3.5 µm, Waters Chromatography, Milford, MA, USA) with a Sentry Guard Symmetry C18 pre-column (3.9 x 20 mm, dp=5 µm, Waters). The column temperature was maintained at 40°C. A gradient program was used with eluent A comprising 10 mM formic acid in water and eluent B comprising 10 mM formic acid in acetonitrile. After injection, elution started with 45% B and the eluent composition was raised linearly to 75% B during 20 min. This percentage was maintained for 2 min before conditioning with 45% B for 8 min. The eluent flow rate was 1.0 ml/min, the UV detection array was used between 190 and 300 nm and the peak areas were determined at 225 nm.

Analysis of ET-743 by isocratic HPLC. The system consisted of a P100 pump, an AS300 autoinjector (equipped with a 100 µl sample loop), and a UV100 detector (all from ThermoSeparation Products, Fremont, CA, USA). The column was thermostated by a Waters temperature control module and a Waters column heater module. Data analysis, column temperature and injections were performed as described previously. The eluent comprised of 65% (v/v) 25 mM phosphate buffer and 35% (v/v) acetonitrile. The eluent flow rate was 1.0 ml/min and the UV detection wavelength was set at 225 nm.

The CYP reaction phenotype. The involvement of each individual CYP with the biotransformation of ET-743 (CYP reaction phenotype) can be calculated by dividing the individual CYP contribution by the total contribution of all CYPs and multiplying it by 100 [14-16]. This contribution, which is an estimation of the relative rate of metabolism attributed to the CYP isozymes, can be calculated by multiplying the metabolism rate of ET-743 in CYP supersomes with the relative activity factor (RAF) value. The relative activity value is a factor for the calculation of the relative activity of the respective isoforms in human liver microsomes [14-16]:

RAF = formation rate in microsomes / formation rate in supersomes Both formation rates are in nmol/(mg protein * min) and are based on the activity data provided by Gentest with each lot number [13]. The RAF, contribution and % metabolism were calculated for female (lot number 2) and male (lot number 3) human liver microsomes for 50 µg/ml ET-743 (concentration used for microsomal incubations) and 1 ng/ml ET-743 (plasma concentration near Cmax in patients after 24 h infusion [17, 18])

Intrinsic metabolic clearance of ET-743 from microsomes and CYP supersomes. The intrinsic metabolic clearance (CLint) is the elimination of a compound by biotransformation at concentrations well below Km and can be calculated using the following equation [19]:

CLint = Vmax / Km The CLint was calculated for mixed gender, female, and male human liver microsomes and for CYP supersomes.

Data analysis. The results are expressed as mean ± standard deviation (SD). Differences between the results were analyzed by the student t-test for unpaired observations.


89

Results

Comparison of the ET-743 biotransformation in male, female, and mixed gender pooled human liver microsomes. Incubation of ET-743 with mixed gender human liver microsomes at 37°C for 4 h reduced the amount of ET-743 with 68.1 ± 6.0 %. Incubations of ET-743 with female human liver microsomes did not show a difference in percentage ET-743 remaining (61.8 ± 4.0 %). However, male human liver microsomes resulted in a significant decrease (p < 0.05) of the percentage ET-743 metabolized compared to mixed gender (50.3 ± 4.9 %). Some possible ET-743 metabolites were observed, but could not be identified due to the impurity of the supplied ET-743 (the impurity was mild (< 1%), but resulted in intervening peaks in the chromatogram (results not shown)). Further, two degradation products of ET-743 were observed, ETM-204 and ETM-305 (figure 1B, results not shown).

ET-743 biotransformation by human CYP supersomes. ET-743 is significantly metabolized by CYP2C9, 2C19, 2D6, 2E1, and 3A4 supersomes during 4 h at 37°C; 91.0 ± 2.5 %, 93.1 ± 4.3 %, 95.6 ± 2.7 %, 95.6 ± 3.0 %, and 90.9 ± 0.8 % of the initial amount of ET-743 was metabolized, respectively. The other CYP supersomes did not significantly metabolize ET-743.

ET-743 biotransformation by pooled human liver microsomes in the absence and presence of CYP inhibitors. To confirm the results found with CYP supersomes, ET-743 was incubated with human liver microsomes (mixed gender) in the presence of CYP inhibitors. Figure 2 shows that the CYP2D6, 2E1, and 3A4 inhibitors could significantly decrease the ET-743 biotransformation by pooled human liver microsomes. CYP3A4 is the main CYP isozyme responsible for the conversion of ET-743 in pooled human liver microsomes; the percentage ET-743 metabolized decreased in the presence of the CYP3A4 inhibitor ritonavir from 66.6 ± 1.3% to 17.8 ± 5.8%. Quinidine (CYP2D6) and chlorzoxazone (CYP2E1) reduced the ET-743 percentage metabolized to 55.9 ± 7.2 % and 54.1 ± 10.5 % respectively. The CYP2C9 and 2C19 inhibitors, sulfaphenazole and (S)-(+)-mephenytoin, had no influence on the biotransformation of ET-743 by human liver microsomes. Figure 2. Percentage ET-743 remaining after incubation of 50 µg/ml ET-743 with pooled mixed gender human liver microsomes in the presence of the CYP inhibitors sulfaphenazole (CYP2C9) (50 µM), (S)-(+)-mephenytoin (CYP2C19) (200 µM), quinidine (CYP2D6) (50 µM), chlorzoxazone (CYP2E1) (200 µM), and ritonavir (CYP3A4) (100 µM). The percentage remaining was determined using an ET-743 incubation without pooled human liver microsomes as control. Each column is the mean of 3 replicates; bars indicate the SD. * significantly different (p < 0.05) compared to no inhibitor.

0

10

20

30

40

50

60

70

80

90

100

no inhibitor sulfaphenazole mephenytoin quinidine chlorzoxazone ritonavir

% E

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3 re

mai

ning

*

**

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Glucuronidation of ET-743 by pooled human liver microsomes, isolated rabbit UGT and UGT supersomes. Significant glucuronidation was observed for ET-743 by pooled human liver microsomes (HLM) and rabbit UGT. After 5 h, 80.1 ± 2.2 % and 46.7 ± 6.3 % of the ET-743 was recovered, respectively. UGT2B15 supersomes significantly glucuronidated ET-743 (24.9 ± 6.0 % decrease in ET-743). UGT1A1, 1A3, and 1A9 did not metabolize ET-743.

ET-743 conjugation by pooled human liver cytosol. After 5 h, ET-743 was significantly conjugated by the phase II enzyme GST present in pooled human liver cytosol (81.4 ± 2.2 % of the ET-743 was recovered). SULT did not conjugate ET-743. The metabolism of ET-743 by NAT could not be studied due to degradation of ET-743 in the presence of the NAT substrates (results not shown).

Biotransformation of ET-743 by pooled human liver S9 fraction. Cytochrome P450, UGT, and GST in pooled human liver S9 fraction significantly metabolized ET-743 (figure 3). The CYPs present in the S9 fraction metabolized 25.0 ± 7.9 % of the ET-743 during 5 h at 37°C and UGT and GST conjugation resulted in 31.2 ± 10.1 % and 29.1 ± 7.7 % ET-743 metabolized, respectively. CYP activity in combination with the individual phase II enzymes UGT and GST resulted in a further reduction of ET-743 compared to CYP, UGT, or GST alone (46.5 ± 0.4 % and 47.3 ± 7.5 %, respectively). When all the enzyme substrates were present, 64.2 ± 11.2 % of the ET-743 is converted by pooled human liver S9 fraction. The phase II enzyme SULT did not metabolize ET-743. Figure 3. Comparison of the biotransformation of ET-743 in human liver S9 fraction by CYP and by CYP and phase II enzymes (UGT, SULT, and GST). The percentage ET-743 remaining was determined using an ET-743 incubation without S9 fraction as control. Each column is the mean of 3 replicates; bars indicate SD. * significantly different (p < 0.05) compared to control and ! significantly different (p < 0.05) compared to CYP.

Protein binding of ET-743 in human liver microsomes. ET-743 has a protein binding of 38.4 ± 7.4 % in human liver microsomes in the concentration range of 2-50 ng/ml (results not shown). The free fraction (fu) value was used to calculate the Km value from the Km(app) determined from the Lineweaver-Burke plot.

0

20

40

60

80

100

120

control CYP UGT SULT GST CYP+UGT CYP+SULT CYP+GST all

% E

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3 re

mai

ning

* * *

*!

**!

*!


91

Enzyme kinetics of ET-743 in mixed gender, female, and male human liver microsomes. The Vmax and Km values of ET-743 in human liver microsomes were calculated from Lineweaver-Burke plots (not shown) using weighed regression (1/x). The Vmax values are not significantly different in mixed gender, female, and male microsomes (table 3). However, the Km value of ET-743 in female liver microsomes is significantly lower compared to male microsomes, the Km decreased from 0.366 ± 0.067 µM to 0.118 ± 0.046 µM (table 3).

Table 3. The Km and Vmax values of ET-743 in mixed gender, female, and male human liver microsomes calculated from the Lineweaver-Burke plots.

human liver microsomes

Km ± SD [µM]

Vmax ± SD [nmol/(mg protein * min)]

mixed gender 0.304 ± 0.038 0.400 ± 0.014 female 0.118 ± 0.046* 0.455 ± 0.017 male 0.366 ± 0.067 0.462 ± 0.027

* significantly different (p < 0.05) compared to mixed gender and male human liver microsomes.

ET-743 enzyme kinetics in CYP2C9, 2C19, 2D6, 2E1, and 3A4 supersomes. The Vmax and Km values of ET-743 in CYP supersomes were also calculated from Lineweaver-Burke plots (not shown). The Vmax and Km values are shown in table 4. CYP3A4 has the highest Km value (2.27 ± 0.67 µM) and CYP2D6 has the highest Vmax value for ET-743 (2.34 ± 3.07 nmol/(mg protein * min)).

Table 4. The Km and Vmax values of ET-743 in human CYP supersomes calculated from the Lineweaver-Burke plots.

supersomes Km ± SD [µM]

Vmax ± SD [nmol/(mg protein * min)]

CYP2C9*1 7.8 ± 3.3 1.8 ± 0.77 CYP2C19 38 ± 17 6.6 ± 2.9

CYP2D6*1 31 ± 8.3 12 ± 3.1 CYP2E1 38 ± 17 11 ± 4.7 CYP3A4 2.3 ± 0.67 2.9 ± 0.77

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CYP reaction phenotype of ET-743. According to the calculations, CYP3A4 is the major isozyme involved in the biotransformation of 50 µg/ml ET-743 (>70%) and 1 ng/ml ET-743 (>94%) (figure 4 and 5 respectively). The contribution of the other CYPs involved in the biotransformation of 50 µg/ml ET-743 in human liver microsomes are in the order 2E1 > 2C9 > 2D6 > 2C19. One ng/ml ET-743 showed a slightly different order in the contribution of the other CYPs, namely 2C9 > 2E1 > 2D6 > 2C19. Figure 4. CYP reaction phenotype of 50 µg/ml ET-743 in female (A) and male (B) human liver microsomes, lot number 2 and 3 respectively. Figure 5. CYP reaction phenotype of 1 ng/ml ET-743 in female (A) and male (B) human liver microsomes, lot number 2 and 3 respectively.

(A)2C9

5.84%

2C190.38% 2D6

2.13%

2E19.77

3A481.9%

(B)

2C96.50%

2C190.91%

2D62.67%

2E118.1

3A471.8%

(A)2C9

2.26%

2C190.05% 2D6

0.31%

2E11.26

3A496.1%

(B)

3A494.0%

2E12.60%

2D60.43%

2C190.13%2C9

2.81%


93

The intrinsic metabolic clearance of ET-743 from human liver microsomes and CYP supersomes. The intrinsic metabolic clearance is shown in table 5. The intrinsic clearance was higher in female human liver microsomes (3.86 ± 1.50 ml/(mg protein * min)) compared to male microsomes (1.26 ± 0.23 ml / (mg protein * min)). CYP3A4 has the highest intrinsic clearance for ET-743 for all CYP supersomes tested (table 5).

Table 5. The intrinsic clearance (CLint) of ET-743 from human liver microsomes and CYP supersomes.

fraction CL [ml/(mg protein * min)]

human liver microsomes mixed gender 1.32 ± 0.16

female 3.86 ± 1.50* male 1.26 ± 0.23

CYP supersomes 2C9 0.23 ± 0.10 2C19 0.17 ± 0.08 2D6 0.39 ± 0.10 2E1 0.29 ± 0.13 3A4 1.26 ± 0.37

* significantly different (p < 0.05) compared to mixed gender and male microsomes. Discussion and conclusions

ET-743 is a promising new anti-cancer agent in clinical trials [3]. The biotransformation and CYP reaction phenotype of ET-743 in humans was investigated to support the pharmacokinetic findings of clinical studies and make predictions on drug-drug interactions for future co-treatment with other anti-cancer drugs.

Results from the CYP supersomes and inhibition experiments with human liver microsomes, indicate that CYP2C9, 2C19, 2D6, 2E1, and 3A4 may be involved in the biotransformation of ET-743 in the liver. The Km values of ET-743 in the different CYP supersomes and the effects of the different CYP inhibitors on the biotransformation in human liver microsomes, reveal that CYP3A4 was the main CYP responsible for the biotransformation of ET-743 in vitro.

The validity of the method using CYP inhibitors combined with microsomes was already proved by others [20-24]. The CYP inhibitors (sulfaphenazole, (S)-(+)-mephenytoin, quinidine, chlorzoxazone, and ritonavir) tested were selected using the human cytochrome P450 database from Gentest [20]. Sulfaphenazole, (S)-(+)-mephenytoin, and quinidine concentrations previously used were 5 µM, 200 µM, and 5 µM respectively [21]. Chlorzoxazone is a selective substrate for CYP2E1. However, it can also be used as a selective inhibitor for CYP2E1 and is used at concentrations between 10 and 500 µM [22, 23]. Furthermore, ritonavir is a potent CYP3A inhibitor at concentrations as low as 0.1 µM [24]. The inhibitor concentrations used in this study were within the range or above the concentrations used to inhibit CYPs by others. The results showed that quinidine, chlorzoxazone, and ritonavir were able to inhibit the metabolism of ET-743 by human liver microsomes, which indicates that CYP2D6, 2E1, and 3A4 are involved in the biotransformation of ET-743. The lack of effect of the CYP2C9 and 2C19 inhibitors sulfaphenazole and (S)-(+)-mephenytoin, indicates that both CYPs are hardly involved in the biotransformation of ET-743.

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The pooled mixed gender human liver microsomes were formulated from material derived from at least 21 individuals and the single gender pools were derived from 5 or more male or female donors [13]. The pooled microsomes have been designed for a profile of catalytic activities representative for many individuals and for minimal lot-to-lot variability. However, variation between the different lot numbers may occur, but the pools are large enough to examine gender-related differences in metabolism [13]. The biotransformation of ET-743 was not significantly different for mixed gender and female microsomes, but male microsomes showed a significant lower ET-743 biotransformation rate. This is probably caused by the significantly lower amount of CYP3A4 in the male liver microsomes, which was almost equal in mixed gender and female microsomes. As these lot numbers are representative for the whole male population, this indicates that on average the amount of CYP3A4 in the male liver is lower compared to female liver. Furthermore, the Km of CYP3A4 has an influence on the biotransformation rate when the unbound concentration is lower then the Km, probably resulting in in vitro gender differences. The Vmax and Km of ET-743 in female and male human liver microsomes were calculated for other lot numbers than those used to determine the percentage biotransformation after 4 h and thus the results of both experiments can not be compared without taking into account the differences in CYP activity levels (especially CYP3A4 differs between the lot numbers). The differences in biotransformation of ET-743 between female and male microsomes and the influence of the Km of CYP3A4 on the biotransformation may result in gender-related differences in metabolism of ET-743, the clinical relevance will be discussed later.

The CYP reaction phenotyping allows the assessment of the relative contribution of the CYP forms to metabolic pathways [25, 26]. The data retrieved from the human liver fractions microsomes and CYP supersomes, widely used to characterize the metabolic profile of drugs, were used to identify CYP reaction phenotypes [27]. Useful predictions on the in vivo pharmacokinetics can be made by assessment of RAF from the results obtained with subcellular fractions [28, 29]. However, the interpretation of microsomal data is difficult because of the complex factors involved, like phase II reactions following phase I metabolism, and the number of different hepatic enzymes involved in the biotransformation of ET-743 [28, 29]. Furthermore, biotransformation is not influenced by drug transporters as these are lacking in microsomes and supersomes [28]. The lack of drug transporters could result in higher biotransformation rates in subcellular fractions compared to the human in vivo situation [27]. Despite this, the information obtained with CYP reaction phenotyping can be indicative for which in vivo drug interaction studies are required and can alert clinicians for the need of genotyped patients, when polymorphically expressed enzymes are involved in the biotransformation [25, 26, 30, 31]. The CYP reaction phenotype of 1 ng/ml ET-743 showed that CYP3A4 was the major isozyme involved in the biotransformation of ET-743 (~95%). The contribution of the other CYPs involved in the biotransformation were in the order 2C9 > 2E1 > 2D6 > 2C19. However, the contribution of CYP2D6 and 2C19 to the CYP reaction phenotype was less than 0.5%, thus in vivo, it is most likely that both isozymes are not significantly involved in the biotransformation of ET-743. According to the CYP reaction phenotype, it is expected that CYP2C9 and 2E1 will only slightly be involved in the biotransformation of ET-743 in vivo.

RAF can also be used for calculating the in vitro intrinsic clearance into in vivo pharmacokinetic clearance. The in vitro intrinsic clearance showed to be comparable to the in vivo hepatic clearance when scaling factors were applied [19, 28]. The in vitro intrinsic clearance by human liver microsomes was higher in female than in male microsomes and this may also be the case in vivo in patients treated with ET-743, the clinical relevance will discussed later.


95

The results indicate that CYP3A4 has an important role in the metabolism of ET-743. Therefore, the risk of in vivo drug-drug interactions, when ET-743 is combined with other CYP3A4 substrates, is present [32, 33]. Consideration is warranted when ET-743 treatment is given in combination with other anti-cancer drugs that are metabolized by CYP3A4 or drugs that influence its activity, e.g. doxorubicin [18, 34, 35]. Furthermore, the intrinsic clearance and data obtained with the microsomes indicated that gender can play a role in the biotransformation and metabolic clearance in patients. However, gender differences are not always of clinical importance, due to high within-gender differences existing in CYP3A4 activity [36, 37]. Thus far, no gender differences in pharmacokinetics have been described for patients treated with ET-743. This emphasizes the influence of the high inter-individual variance in CYP3A4 activity on the pharmacokinetics of ET-743. The individual CYP isozyme activity is also influenced by food components, aging, disease, and genetic polymorphisms [36, 37]. The genetic component in the inter-individual variability in CYP3A4 activity has been estimated to be between 60 and 90%, but the underlying genetic factors are largely unknown [38]. Furthermore, it is most likely that CYP3A5 (same substrates as CYP3A4) is capable of metabolizing ET-743. In less than 9% of the Caucasians, CYP3A5 is functional [39]. Patients with functional CYP3A5, may show a higher metabolic clearance of ET-743. It is of interest to explore whether genotyping the patients for CYP3A5 may contribute to the safety of the patients treated with ET-743 [25, 26].

ET-743 was glucuronidated in vitro by UGT2B15 in human UGT supersomes and by the UGT isozymes present in pooled human liver microsomes and S9 fraction. Further, isolated rabbit UGT glucuronidated ET-743. In addition, GST conjugated ET-743 in pooled human liver cytosol and S9 fraction. The other phase II enzyme studied, SULT, did not metabolize ET-743 in human cytosol and S9 fraction. Gender differences have been observed in humans with glucuronidation activity being higher in men than in women [36]. Inter-individual variability in GST activity in patients has also been observed. Furthermore, the individual UGT and GST activity is also influenced by aging, disease, food or drug intake, and genetic polymorphisms [37, 40]. The pharmacokinetics and toxicity of ET-743 in cancer patients caused by UGT and GST should be taken into account. However, depending on the rate limiting step in the ET-743 metabolism (CYP, UGT, or GST mediated), the inter-individual variance in activity of the enzyme of the rate limiting step is of clinical importance [36].

In conclusion, ET-743 metabolism in human liver microsomes, cytosol and S9 fraction was catalyzed by cytochrome P450 and the phase II enzymes UGT and GST. CYP3A4 was the predominant CYP metabolizing ET-743 and CYP2C9, 2C19, 2D6, and 2E1 play a minor role in the applied test systems. These findings can help to interpret the pharmacokinetic data obtained from the clinical trials with ET-743 in patients.

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References 1. Jimeno J.M. (2002). A clinical armamentarium of marine-derived anti-cancer compounds.

Anti-Cancer Drugs 13 (suppl. 1): S15-S19. 2. Rinehart K.L., Gravalos L.G., Faircloth G., and Jimeno J. (1995). Ecteinascidin (ET-743):

Preclinical antitumor development of a marine derived natural product (abstract). Proc. Am. Assoc. Cancer Res. 36: 2322.

3. Jimeno J.M., Faircloth G., Cameron L., Meely K., Vega E., A. Gómez, Fernández Sousa-Faro J.M., and Rinehart K. (1996). Progress in the acquisition of new marine-derived anticancer compounds: development of Ecteinascidin-743 (ET-743). Drugs of the Future 21:1155-1165.




7. Zelek L., Yovine A., Brain E., Turpin F., Taamma A., Riofrio M., Spielmann M., Jimeno J., and Cvitkovic E. (2000). Preliminary results of phase II study of Ecteinascidin-743 with the 24 hour continuous infusion Q3 weeks schedule in pretreated advanced/metastatic breast cancer patients (abstract). Proc. 11th NCI-EORTC-AACR Symposium on New Drugs in Cancer Therapy, Amsterdam, The Netherlands: 85.


9. Reid J.M., Kuffel M.J., Ruben S.L., Morales J.J., Rinehart K.L., Squillace D.P., and Ames M.M. (2002). Rat and human liver cytochrome P-450 isoform metabolism of Ecteinascidin 743 does not predict gender-dependent toxicity in humans. Clin. Cancer Res. 8: 2952-2962.

10. Sparidans R.W., Rosing H., Hillebrand M.J.X., López-Lázaro L., Jimeno J.M., Manzanares I., van Kesteren Ch., Cvitkovic E., van Oosterom A.T., Schellens J.H.M., and Beijnen J.H. (2001). Search for metabolites of ecteinascidin 743, a novel, marine-derived, anti-cancer agent, in man. Anti-Cancer Drugs 12: 653-666.

11. Curry S.H. (1974). Drug disposition and pharmaconkinetics with a consideration of pharmacological and clinical relationships. Blackwell Scientific Publications (Oxford, UK): 42-48.

12. Gibson G.G. and Skett P. (1994). Introduction to drug metabolism. Blackie Academic and Professional (London, UK): 1-34.

13. Gentest, a Becton and Dickinson company. http://www.gentest.com (accessed July 2003). 14. Crespi C.L. and Miller V.P. (1999). The use of heterolously expressed drug

metabolizeing enzymes – state of the art and prospects for the future. Pharmacol. Ther. 84: 121-131.


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15. Venkatakrishnan K., von Moltke L.L., Court M.H., Harmatz J.S., Crespi C.L., and Greenblatt D.J. (2000). Comparison between cytochrome P450 (CYP) content and relative activity approaches to scaling from cDNA-expressed CYPs to human liver microsomes: ratios of accessory proteins as sources of discrepancies between the approaches. Drug Metab. Dispos. 28: 1493-1504.

16. Störmer W., von Moltke L.L., and Greenblatt D.J. (2000). Scaling drug biotransformation data from cDNA-expressed cytochrome P-450 to human liver: a copmparison of relative activity factors and human liver abundance in studies of mirtazapine metabolism. J. Pharmacol. Exp. Ther. 295: 793-801.

17. van Kesteren Ch., Cvitkovic E., Taamma A., López-Lázaro L., Jimeno J.M., Guzman C., Mathôt R.A.A., Schellens J.H.M., Misset J.-L., Brian E., Hillebrand M.J.X., Rosing H., and Beijnen J.H. (2000). Pharmacokinetics and pharmacodynamics of the novel marine-derived anticancer agent ecteinascidin 743 in a phase I dose-finding study. Clin. Cancer Res. 6: 4725-4732.

18. Puchalski T.A., Ryan D.P., Garcia-Carbonero R., Demetri G.D., Butkiewicz L., Harmon D., Seiden M.V., Maki R.G., López-Lázaro L., Jimeno J., Guzman C., and Supko J.G. (2002). Pharmacokinetics of ecteinascidin 743 administered as a 24-h continuous intravenous infusion to adult patients with soft tissue sarcomas: associations with clinical characteristics, pathophysiological variables and toxicity. Cancer Chemother. Pharmacol. 50: 309-319.

19. Obach R.S., Baxter J.G., Liston T.E., Silber B.M., Jones B.C., MacIntyre F., Rance D.J., and Wastall P. (1997). The prediction of human pharmacokinetic parameters from preclinical and in vitro metabolism data. J. Pharamcol. Exp. Ther. 283: 46-58.

20. Gentest Cytochrome P450 database. http://www.gentest.com/human_p450_database (accessed January 2003).

21. Wienkers L.C. and Wynalda M.A. (2002). Multiple cytochrome P450 enzymes responsible for the oxidative metabolism of the substituted (S)-3-phenylpiperidine, (S, S)-3-[3-(methylsulfonyl)phenyl]-1-propylpiperidine hydrochloride, in human liver microsomes. Drug Metab. Dispos. 30: 1372-1377.

22. Zhou S., Paxton J.W., Tingle M.D., and Kestell P. (2000). Identification of the human liver cytochrome P450 isoenzyme responsible for the 6-methylhydroxylation of the novel anticancer drug 5,6-dimethylxanthenone-4-acetic acid. Drug Metab. Dispos. 28: 1449-1456.

23. Ko J.W., Desta Z., Soukhova N.V., Tracy T., and Flockhart D.A. (2000). In vitro inhibition of the cytochrome P450 (CYP450) system by the antiplatelet drug ticlopidine: potent effect on CYP2C19 and CYP2D6. Br. J. Clin. Pharmacol. 49: 343-351.

24. Kumar G.N., Dykstra J., Roberts E.M., Jayanti V.K., Hickman D., Uchic J., Yao Y., Surber B., Thomas S., and Granneman G.R. (1999). Potent inhibition of the cytochrome P-450 3A-mediated human liver microsomal metabolism of a novel HIV protease inhibitor by ritonavir: a positive drug-drug interaction. Drug Metab. Dispos. 27: 902-908.

25. Lu A.Y.H., Wang R.W., and Lin J.H. (2003). Cytochrome P450 in vitro reaction phenotyping: a re-evaluattion of approaches used for P450 isofrom identification. Drug Metab. Dispos. 31: 345-350.

26. Rodrigues A.D. (1999). Integrated cytochrome P450 reaction phenotyping. Attempting to bridge the gap between cDNA-expressed cytochromes P450 and native human liver microsomes. Biochem. Pharmacol. 57: 465-480.

27. Brandon E.F.A., Raap C.D., Meijerman I., Beijnen J.H., and Schellens J.H.M. (2003). An update on in vitro test methods in human hepatic drug biotransformation research: pros and cons. Toxicol. Appl. Pharmacol. 189: 233-246.

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28. Bachman K.A. and Ghosh R. (2001). The use of in vitro methods to predict in vivo pharmacokinetics and drug interactions. Curr. Drug Metab. 2: 299-314.

29. Lin J.H. (2000). Sense and nonsense in the prediction of drug-drug interactions. Curr. Drug Metab. 1: 305-331.

30. Lin J.H. and Lu A.Y.H. (1998). Inhibition and induction of cytochrome P450 and the clinical implications. Clin. Pharmacokinet. 35: 361-390.

31. Dahl M.L. (2002). Cytochrome P450 phenotyping/genotyping in patients receiving antipsychotics: useful aid to prescribing? Clin. Pharmacokinet. 41: 453-470.

32. Levy R.H., Thummel K.E., Trager W.F., Hansten P.D., and Eichelbaum M. (2000). Metabolic drug interactions. Lippincott Williams and Wilkins (Philidelphia, USA).

33. Tucker G.T. (1992). The rational selection of drug interaction studies: implication of recent advantages in drug metabolism. Int. J. Clin. Pharmacol. Ther. Toxicol. 30: 550-553.

34. Laverdiere C., Kolb E.A., Supko G.J., Gorlick R., Meyers P.A., Maki R.G., Wexler L., Demetri G.D., Healey J.H., Huvor A.G., Goorin A.M., Bagatell R., Ruiz-Casado A., Guzman C., Jimeno J., and Harmon D. (2003). Phase II study of Ecteinascidin 743 in heavily pretreated patients with recurrent osteosarcoma. Cancer 98: 832-840.

35. Desai P.B., Duan J.Z., Zhu Y.W., Kouzi S. (1998). Human liver microsomal metabolism of paclitaxel and drug interactions. Eur. J. Drug Metab. Pharmacokinet. 23: 417-424.




39. van Schaik R.H., van der Heiden I.P., van den Anker J.N., and Lindemans J. (2001). CYP3A5 variant allele frequencies in Dutch Caucasians. Clin. Chem. 48: 1668-1671.

40. MacKenzie P.I., Miners J.O., and McKinnon R.A. (2000). Polymorphisms in UDP glucuronosyltransferase genes: functional consequences and clincial relevance. Clin. Chem. Lab. Med. 38: 889-892.

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CHAPTER In vitro cytotoxicity of ET-743 (Trabectedin®, Yondelis®), a marine anti-cancer drug, in the Hep G2 cell line; influence of cytochrome P450 and phase II inhibition and cytochrome P450 induction. Esther F.A. Brandon, Irma Meijerman, Joyce S. Klijn, Rianne Levink, Rolf W. Sparidans, Luis López Lázaro, Jos H. Beijnen, and Jan H.M. Schellens. Abstract

ET-743 is a marine anti-cancer drug and is currently in phase I clinical trials in which the effect of combination therapies will be investigated. In vitro studies have shown that ET-743 is mainly metabolized by cytochrome P450 (CYP) 3A4, but also by 2C9, 2C19, 2D6, and 2E1 and the phase II conjugating enzymes uridine diphosphoglucuronosyl transferase and glutathione-S-transferase. Based on this metabolic profile, there is a risk for drug-drug interactions possibly influencing the toxicity of ET-743. Therefore, the effect of CYP and phase II activity on the cytotoxicity of ET-743 was investigated in vitro.

The effect of different CYP and phase II enzyme inhibitors and CYP inducers on ET-743 cytotoxicity was studied after 48 and 120 h of treatment in Hep G2 cells using different assays. In addition, the toxicity of ET-743 metabolites was investigated.

A potent cytotoxic activity of ET-743 after 120 h treatment was observed in Hep G2, which could be increased in combination with the CYP inhibitors metyrapone (3A4), phenanthrene (substrate for 2E1 and 3A4), piperonyl butoxide (3A), proadifen (2C9, 2E1, and 3A4), ritonavir (3A4), and warfarin (2C9 and 2C19). No effect on the cytotoxicity of ET-743 was observed in combination with phase II enzyme inhibition and CYP induction. CYP metabolites of ET-743 were less toxic compared to ET-743.

These findings indicate that combination therapy of ET-743 with CYP inhibitors, e.g. other anti-cancer drugs, could lead to changes in the toxicity of ET-743.

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Introduction

The Ecteinascidins are a group of tetrahydroisoquinolines isolated from the Caribbean tunicate Ecteinascidia turbinata [1]. The potent cytotoxicity of extracts of this tunicate were first discovered in the late 1960s and were identified 12 years ago. Ecteinascidin-743 (ET-743, Trabectedin®, Yondelis®) (figure 1) was selected for further development, based on its promising cytotoxic activity and relative abundance in the tunicate [2].

The mode of action of ET-743 has not been completely elucidated, but several mechanisms have been proposed. They are believed to involve binding to the minor groove of the DNA, interactions with transcription factors and DNA binding proteins, disorganization of the microtubule network, inhibition of topoisomerase I, pertubation of the cell cycle, and interference with DNA repair mechanisms [3, 4]. Furthermore, ET-743 is a transcription interfering agent and could therefore be useful to treat multi-drug resistant tumors, which have induced transcription of drug transporters [5-10]. ET-743 is also an antagonist of the steroid and xenobiotic receptor (SXR) and thus inhibits the induction of several cytochrome P450s (CYP), phase II enzymes, and drug transporters [11, 12]. However, ET-743 inhibits only SXR-activated transcription of these enzymes and transporters and not the constitutive transcription [8].

NH

S

N

N

O

O

O

O

OH

O

O

OH

OH

O

O

C39H43N3O11SM.W. = 761.9 g/mol

Figure 1. Chemical structure and molecular weight of ET-743, a novel anti-cancer drug [20].

In vitro studies with ET-743 in cell lines of human origin exhibited activity at nanomolar concentrations against various solid tumor cell lines, including melanoma and ovarian, renal, prostate, breast, and non-small cell lung cancer cell lines, with potencies ranging from 1 pM to 10 nM [13]. In addition, in vivo ET-743 appears effective against human xenografts of non-small cell lung cancer, melanoma, and breast tumors [2, 13]. Activity against soft-tissue sarcomas and breast, endometrial, and ovarian cancer was shown in phase II trials [1, 14-18].

Reid et al. (2002) investigated the biotransformation of ET-743 and showed that ET-743 was metabolized by CYP2C9, 2D6, 2E1, and 3A4 microsomes from transfected human B-lymphocyte cell lines [19]. Enzyme kinetic studies with human liver microsomes and CYP supersomes at our laboratory showed that ET-743 is metabolized mainly by CYP3A4, but also by CYP2C9, 2C19, 2D6, and 2E1 (Chapter 5). Sparidans et al. (2001) showed that ET-743 is conjugated by rabbit uridine diphosphoglucuronosyl transferase (UGT) [20]. In

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other studies at our laboratory, we showed that in vitro human UGT2B15 is responsible for the glucuronidation of ET-743 (Chapter 5). Furthermore, in vitro ET-743 is also conjugated by glutathione-S-transferase (GST) (Chapter 5).

ET-743 has also been evaluated in vitro for the effect of combination therapy with other anti-cancer drugs, e.g. doxorubicin, paclitaxel, and cisplatin [11, 21-24]. Cisplatin, doxorubicin, and paclitaxel showed sequence-dependent synergistic effects in combination with ET-743 in human breast, ovarian, and soft tissue cancer cell lines and a human rhabdomyosarcoma cell line. Furthermore, in female rats and in patients co-administration of dexamethasone, a preventative anti-emetic, resulted in significantly lower hepatotoxicity of ET-743 [25, 26]. Dexamethasone, cisplatin, paclitaxel, and doxorubicin are CYP3A4 inhibitors which could lead to drug-drug interactions with ET-743, thereby decreasing its metabolic clearance and increasing hepatotoxicity [27]. However, dexamethasone and paclitaxel are also CYP3A4 inducers and this could lead to increased metabolic clearance and decreased hepatotoxicity [27]. Combination therapy is common in the treatment of cancer, but resulting drug-drug interactions are unpredictable because some drugs used can both act as CYP inhibitors and inducers. Therefore, in this study the effect of CYP and phase II enzyme activities on the cytotoxicity of ET-743 in vitro was investigated in order enable prediction of drug-drug interactions in patients.

Human cell lines can be used to study the cytotoxicity of a parent drug and its metabolites. The human hepatoma cell line (Hep G2) is the most frequently used and best characterized human hepatoma cell line [28]. It is known to express CYP1A, 2B1, 2B2, 2B6, 3A, monooxygenase, GST, and UGT and was therefore chosen as a model for this study [28, 29]. Materials and Methods

Materials. ET-743 was kindly donated by PharmaMar (Tres Cantos, Madrid, Spain). Methanol (HPLC grade) and dichloromethane were purchased from Biosolve (Valkenswaard, The Netherlands) and formic acid (p.a.), MgCl2

.6H2O (p.a.), and dimethyl sulfoxide (DMSO, synthesis grade) from Merck (Darmstadt, Germany). Water was purified on a multi-laboratory scale by reversed osmosis. Mixed gender pooled human liver microsomes were provided by Gentest (Becton Dickinson, Woburn, MA, USA). RPMI-1640 medium (with L-glutamine and 25 mM HEPES), MEM (with Earle’s salt, without L-glutamine and phenol red), heat-inactivated fetal calf serum, penicillin/streptomycin, L-glutamine, and Hanks’ Balanced Salt Solution (pH 7.4) were all obtained from Gibco BRL (Breda, The Netherlands). Trizol was purchased from Invitrogen Life Technologies (Paisley, UK) and the RevertAidTM first strand cDNA synthesis kit from Fermentas (St. Leon-Rot, Germany). Ritonavir was provided by Abbott (Chicago, IL, USA) and the Lactate dehydrogenase and WST-1 assay kit were obtained from Roche (Basel, Switzerland). The forward and reverse primer, Taqman®-MGB probe for CYP3A4, and HS99999905_m1 reagent were all purchased from Applied Biosystems (Foster City, CA, USA). All other chemicals were purchased from Sigma Chemical Company (St. Louis, MO, USA) and were of analytical grade.

Cell culture growth. The human hepatic carcinoma cell line (Hep G2) was obtained form the ATCC (Manassas, VA, USA). Routine cultivation of the monolayer cells was performed in RPMI-1640 medium (with L-glutamine and 25 mM HEPES) supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were sub-cultured weekly (ratio of 1:5 (v:v)) and the medium was refreshed after 3 days.

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Cytotoxicity of ET-743 in the absence and presence of cytochrome P450 inhibitors. The cytotoxicity of the CYP inhibitors for the Hep G2 cell line was determined by testing a concentration range and of the inhibitors concentrations below the IC5 were used in further experiments. For the determination of the cytotoxicity of ET-743 in the absence and presence of inhibitors, cells (passage 89-110) were seeded onto 96-well microtitre plates at a concentration of 8,000 cells/well. The cells were cultured at 37°C, 5% CO2, and 95% humidity. After 48 h, the cells were exposed to inhibitor. The following CYP inhibitors were examined for their effect on the cytotoxicity of ET-743: 50 µM chlorzoxazone (CYP2E1), 5 µM ketoconazole (CYP1A1, 2A6, 2C8, 2C19, 2D6, and 3A4), 200 µM metyrapone (CYP2A6 and 3A4), 50 µM phenanthrene (CYP2B6), 10 µM piperonyl butoxide (CYP3A), 10 µM proadifen (CYP2A6, 2B6, 2C9, 2E1, and 3A4), 10 µM ritonavir (CYP3A4), 100 µM sulfaphenazole (CYP2C9), and 50 µM warfarin (CYP2C9 and 2C19). As control, 0.1% DMSO was used. Twenty µl medium per well was replaced by 20 µl medium with inhibitor (10x the final concentration in the wells). The cells were incubated for 1 h at 37°C, 5% CO2, and 95% humidity. Next, the cells were exposed to ET-743 at concentrations of 0.001-500 ng/ml. Therefore, a concentration range of ET-743 with inhibitor (same concentration as in the wells) in medium was made and 20 µl was added to the wells, giving a final dilution of 1:10. Cell growth was determined after 48 h using the lactate dehydrogenase (LDH), WST-1, and the sulforhodamine B (SRB) assay and after 120 h using the LDH and SRB assay. Cell survival (%) was calculated relatively to control cells and 100% killed cells (killed with 1% tributyltinchloride 1 h prior to the assay). Concentration-viability curves were constructed with this data and the IC50 (concentration of compound giving 50 % cell death) was calculated by the Softmax®Pro 3.1 software (Molecular Devices, Sunnyvale, CA, USA). Control experiments were performed without ET-743 and without inhibitor.

ET-743 cytotoxicity in the absence and presence of dexamethasone as inhibitor. The induction effect was studied by pre-incubating the cells for 2 days with the inducers before exposure to with ET-743. The direct effect of dexamethasone was studied in Hep G2 cells (passage 107-110) by adding dexamethasone together with ET-743 as described for the CYP inhibitors. After 48 h, the cells were exposed to 50 µM dexamethasone (concentration below the IC5 value). The cells were treated with ET-743 and dexamethasone as previously described for CYP inhibitors. The IC50 values of ET-743 were determined after 48 and 120 h of treatment using the assays previously described for CYP inhibitors. Control experiments were performed without ET-743 and without dexamethasone.

The effect of phase II inhibitors on the cytotoxicity of ET-743. The Hep G2 cells (passage 86-86) were seeded as described for CYP inhibitors and after 48 h the cells were incubated with the phase II inhibitors: 200 µM acetaminophen (UGT), 1 µM berberine (N-acetyltransferase (NAT)), 20 µM of 2,6-dichloro-4-nitrophenol (DNP) (sulfotransferase (SULT)), 200 µM S-hexylglutathion (GST), or 0.1% DMSO (control). After addition of ET-743, the cytotoxicity was determined after 48 and 120 h of treatment with ET-743 as previously described. Control experiments were performed without ET-743 and without phase II inhibitor.

ET-743 cytotoxicity after CYP induction with rifampicin or dexamethasone. The cells (passage 87-91) were seeded onto 96-well microtitre plates at a concentration of 8,000 cells/well in the presence of 10 µM rifampicin (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2E1, and 3A4), 10 µM dexamethasone (CYP2A6, 2B6, 3A4, and 4A11), or 0.1% DMSO (control) (concentrations were below the IC5 value). After 2 days, the cells were exposed to ET-743 at concentrations of 0.001-500 ng/ml by replacing the medium with medium containing ET-743 and inducer. The IC50 values were determined after 48 and 120 h


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of treatment with ET-743 as previously described. Control experiments were performed without ET-743.

Cytotoxicity of ET-743 and metabolites formed by pooled human liver microsomes. The incubation procedure of ET-743 with human liver microsomes was a modification of the method described by Sparidans et al. (2001) [20]. Twenty-five µl of 0.5 M potassium phosphate buffer (pH 7.4) were pipetted into a polypropylene micro tube on ice and 50 µl NADP regenerating system (NRS: 1.5 U/ml glucose-6-phosphate dehydrogenase, 0.5 mg/ml β-NADP, 4.0 mg/ml D-glucose-6-phosphate in 0.6 % (w/v) NaHCO3), 7.5 µl of 20 mg/ml MgCl2

.6H2O solution, and 50 µl of an aqueous ET-743 solution (1% (v/v) DMSO, final concentration of 50 µg/ml in the microsomes suspension) were added. After vortex-mixing briefly, the tubes were incubated for 2 min at 37°C in a shaking water bath. Next, 5 µl of pooled human liver microsomes (mixed gender, lot number 21) were added. The tube was vortex-mixed briefly again and the mixture was incubated at 37°C in a shaking water bath for 3 h (~50% of the ET-743 was metabolized). The reaction was terminated by removing proteins using ultra-centrifugation with Micronon YM-10 ultra-centrifuge tubes (cut-off filter of 10 kDa) (Millipore, Bedford, MA, USA) for 90 min at 14,000 g. The polypropylene micro tubes used to collect the filtrate were sterile and the filtrate was handled aseptically. The ultra-filtrate was injected for liquid chromatographic analysis to determine the ET-743 concentration. Control experiments were performed incubating for 3 h at 4°C, without liver microsomes, and without ET-743. The ultra-filtrate was diluted in RPMI-1640 medium to an ET-743 concentration range of 0.001-500 ng/ml. The Hep G2 cells (passage 96-98) were seeded and treated as previously described for CYP inhibitors. The IC50 values were determined as previously described.

Analysis of ET-743 by gradient HPLC. The chromatographic assay was a modification of the method described by Sparidans et al. (2001) [20]. The supernatants of the incubated mixtures were analyzed on an HPLC system consisting of two LC-10ATVP pumps, a SIL-10ADVP autoinjector (equipped with a 500 µl sample loop), a SCL-10AVP system controller, and a SPD-M10AVP photodiode array detector (all from Shimadzu, Kyoto, Japan). Data were recorded on a Hermac Pentium 440, 122 MB personal computer (Scherpenzeel, The Netherlands) equipped with the Class-VP 5.032 software (Shimadzu). Injections (50 µl) were made on a Symmetry C18 column (4.6 x 100 mm, dp=3.5 µm, Waters Chromatography, Milford, MA, USA) with a Sentry Guard Symmetry C18 pre-column (3.9 x 20 mm, dp=5 µm, Waters). The column temperature was maintained at 40°C. A gradient program was used with eluent A comprising 10 mM formic acid in water and eluent B comprising 10 mM formic acid in acetonitrile. After injection, elution started with 45% B and the eluent composition was raised linearly to 75% B during 20 min. This percentage was maintained for 2 min before conditioning with 45% B for 8 min. The eluent flow rate was 1.0 ml/min, the peak areas were determined at 225 nm.

Lactate dehydrogenase assay (LDH assay). The LDH assay was performed as described in the kit protocol. A volume of 100 µl supernatant was removed from the original plate and pipetted into a new 96 wells plate. A volume of 100 µl reaction solution was added and the plates were incubated for 30 min at room temperature in the dark. The absorption at 492 nm, using a reference wavelength of 600 nm, was determined using a Versamax microtitre plate reader (Molecular Devices, Sunnyvale, CA, USA). Data were recorded and analyzed on a Hermac Pentium 440, 122 MB personal computer (Scherpenzeel, The Netherlands) equipped with the Softmax®Pro 3.1 software (Molecular Devices).

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WST-1 assay. The WST-1 assay was performed according to the protocol provided with the kit. Medium was removed, leaving 100 µl medium, and 10 µl dye solution was added. The plates were incubated at 37°C for 2.5 h. The absorbance was measured at 450 nm, with a reference wavelength of 620 nm, using a Versamax microtitre plate reader. Data were recorded and analyzed as previously described for the LDH assay.

Sulforhodamine B assay (SRB assay). The SRB assay was a modification of the method described by Higgins et al. (1993) [30]. The cell culture medium was removed and the cells were fixed in 100 µl of 10% (w/v) trichloroacetic acid for 60 min at 4°C. The wells were rinsed three times with tap water to remove solutes and cells were stained with 50 µl of 0.4% (w/v) sulforhodamine B (SRB) in 1% (v/v) acetic acid for 15 min. The cells were washed three times with 1% (v/v) acetic acid and air-dried. After drying, 120 µl of 10 M Tris in Hanks’ Balanced Salt Solution (pH 7.4) were added to solubilize the protein bound SRB. After mixing, the absorbance was measured at 540 nm using a Versamax microtitre plate reader. Data were recorded and analyzed as described for the LDH assay.

CYP3A4 mRNA levels before and after induction with rifampicin and dexamethasone analyzed with real-time PCR. The cells (passage 95-98) were seeded onto 6-well microtitre plates at a concentration of 1 * 105 cells/well in the presence of 10 µM rifampicin, 10 µM dexamethasone, or 0.1% DMSO (control). After 4 days, the total RNA was extracted from the Hep G2 cells using Trizol according to the protocol described by the manufacturer (Invitrogen Life Technologies). cDNAs were prepared from total RNA using RevertAidTM first strand cDNA synthesis kit. The primers and probe used for CYP3A4 were designed using Primer Express software (Applied Biosystems, Foster City, CA, USA): forward primer 5'-TCA ATA ACA GTC TT CCA TTC CTC AT-3', reverse primer 5'-CTT CGA GGC GAC TTT CTT TCA-3', and Taqman®-MGB (major groove binder) probe 5'-TGT TTC CAA GAG AAG TTA CAA A-3' labeled with phosphoramidite (FAM). As internal control the mRNA level of glyceraldehyde-3’-phosphatedehydrogenase (GAPDH) was measured using the assay-on-demand Hs99999905_m1 reagents for human GAPDH. The amplification and detection was performed using the ABI Prism® 7000 sequence detection system. The relative quantification of the CYP3A4 mRNA expression was achieved using the comparative CT method (Applied Biosystems).

CYP3A4 activity levels after induction with rifampicin for 4 days. The cells were cultured in T75 culture bottles as described above for the routine cultivation of the cells. After 2 days the medium was replaced by medium without FCS to which 50 µM rifampicin, or 0.1% DMSO was added. The medium was refreshed daily. After 4 days of induction, the CYP3A4 activity was determined by measuring the 6β-hydroxylation of the model substrate testosterone, as described by Wortelboer et al. (1990) [31]. For the activity measurement the cell were incubated for 3 h with 5 ml MEM supplemented with 2 mM L-glutamine and 250 µM testosterone. 11β-Hydroxytestosterone was added to the medium as internal standard. Testosterone, its metabolites, and the internal standard were extracted from the medium using 6 ml dichloromethane. The samples were vortex-mixed thoroughly and were centrifuged at 2200 g for 30 min. The aqueous phase was removed and the organic phase was evaporated. The residue was re-dissolved in 130 µl of 50% (v/v) methanol/water. The testosterone and 6β-hydroxytestosterone were analyzed using an HPLC system equipped with two consecutive reversed phase Chromsep C18 columns (3 x 100 mm, dp=5 µm, Varian Inc., Palo Alto, CA, USA) with a Safe Guard C18 pre-column (3 x 10 mm, dp=5 µm, Varian Inc.). The column temperature was maintained at 60°C. A gradient program was used with eluent A comprising 25% (v/v) methanol and eluent B comprising 40% (v/v) methanol and 3.5% (v/v) acetonitrile. After injection (20 µl), elution started with 100% A and the eluent composition was raised linearly from 10 to 45 min to 100% B. The eluent flow rate was 0.8 ml/min and


105

the peak areas were determined at 254 nm. The protein contents of each flask was determined according to the method described by Lowry et al. (1951) [32] and data was expressed as pmol/(mg protein * min).

Data analysis. The results are expressed as mean ± standard deviation (SD). Differences between the results were analyzed by the student t-test for unpaired observations. Results

IC5 values of the inhibitors and inducers. In table 1, the IC5 values of the CYP and phase II enzyme inhibitors and the CYP inducers after 120 hours using the SRB assay are shown. Berberine showed to be the most cytotoxic in Hep G2 cells, with an IC5 value of 1.2 µM, while acetaminophen was the least toxic (IC5 value >500 µM). Most of the inhibitors and inducers were used below their IC5 value, with the exception of ketoconazole, proadifen, sulfaphenazole, and 2,6-dichloro-4-nitrophenol, which were used at their IC5 concentration.

Table 1. IC5 values of CYP and Phase II inhibitors and CYP inducers in the Hep G2 cell line.

compound IC5-value (µM) CYP inhibitors chlorzoxazone 61 ketoconazole 5 metyrapone >200

phenanthrene 60 piperonyl butoxide 27

proadifen 10 ritonavir 18

sulfaphenazole 100 warfarin 87

CYP inducers dexamethasone 80

rifampicin 50 phase II inhibitors

acetaminophen >500 berberine 1.2

2,6-dichloro-4-nitrophenol 20 S-hexylglutathion >200

Cytotoxicity of ET-743 in combination with CYP inhibitors and dexamethasone. Using the SRB assay, it was shown that ritonavir (CYP3A4 inhibitor) significantly increased the cytotoxicity of ET-743 after 48 and 120 h (figure 2). Also the CYP inhibitors metyrapone (2A6 and 3A4), phenanthrene (2B6), piperonyl butoxide (3A), proadifen (2A6, 2B6, 2C9, 2E1, and 3A4), and warfarin (2C9 and 2C19) significantly decreased the IC50 value of ET-743 (figure 2 and table 2). The same pattern was observed after 48 h with the LDH and WST-1 assay and after 120 h with the LDH assay, however, not all decreases were significant (table 2). The direct effect of dexamethasone when co-administered with ET-743 on the cytotoxicity of ET-743 was not statistically significant.

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Figure 2. ET-743 cytotoxicity in Hep G2 cells (passage 89-110) after 48 h (A) and 120 h (B) in the absence and presence of CYP inhibitors or dexamethasone as CYP inhibitor determined with the SRB assay. ET – ET-743, CZ - chlorzoxazone, KC - ketoconazole, MR - metyrapone, PT - phenanthrene, PB - piperonyl butoxide, PA - proadifen, RN - ritonavir, SP - sulfaphenazole, WF – warfarin, DM - dexamethasone. Each column is the mean of 12 replicates for ET-743 (12 different passages) and 3 replicates for ET-743 in combination with the inhibitor (3 different passages); bars indicate SD. * significantly different (p < 0.05) compared to ET-743 without inhibitor. Table 2. ET-743 cytotoxicity (IC50 value) in Hep G2 cells (passage 89-110) after 48 and 120 h

in the absence and presence of CYP inhibitors or dexamethasone in [ng/ml]. 48 h 120 h

inhibitor LDH WST SRB LDH SRB none 3.34 ± 1.45 1.07 ± 0.72 1.41 ± 0.79 0.91 ± 0.22 0.65 ± 0.13

chlorzoxazone 3.25 ± 1.09 1.36 ± 1.37 1.79 ± 1.76 0.92 ± 0.12 0.85 ± 0.67 ketoconazole 1.61 ± 1.02 1.01 ± 0.95 0.64 ± 0.40 0.80 ± 0.21 0.35 ± 0.25 metyrapone 3.13 ± 0.43 0.53 ± 0.16* 0.74 ± 0.60 0.75 ± 0.66 0.30 ± 0.16*

phenanthrene 1.69 ± 1.21 0.49 ± 0.59 1.11 ± 0.68 1.20 ± 0.27 0.30 ± 0.04* piperonyl butoxide 0.62 ± 0.20* 0.17 ± 0.05* 0.60 ± 0.47 0.49 ± 0.11* 0.19 ± 0.07*

proadifen 0.73 ± 0.28* 0.26 ± 0.11* 0.85 ± 0.57 1.26 ± 0.69 0.21 ± 0.04* ritonavir 0.99 ± 0.51* 0.40 ± 0.50 0.24 ± 0.09* 0.49 ± 0.11* 0.11 ± 0.06*

sulfaphenazole 1.88 ± 0.30* 1.19 ± 0.72 1.55 ± 1.00 1.22 ± 0.33 0.80 ± 0.45 warfarin 3.24 ±0.77 0.25 ± 0.16* 1.17 ± 0.35 0.83 ± 0.06 0.49 ± 0.05*

dexamethasone 5.50 ±1.63 1.25 ± 0.39 2.11 ± 0.85 1.20 ± 0.55 0.76 ± 0.52 * significantly different (p < 0.05) compared to no inhibitor.

A

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

ET + CZ + KC + MR + PT + PB + PA + RN + SP + WF + DM

mea

n IC

50 v

alue

*

B

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

ET + CZ + KC + MR + PT + PB + PA + RN + SP + WF + DM

mea

n IC

50 v

alue

*

*

* **

*


107

ET-743 cytotoxicity in the presence of phase II inhibitors. The IC50 values of ET-743 in Hep G2 cells after 48 and 120 hours using the SRB assay were respectively 0.64 ± 0.33 and 0.41 ± 0.17 µg/ml (table 3). No significant effect on the IC50 values could be observed in the presence of phase II inhibitors.

Table 3. ET-743 cytotoxicity (IC50 value) in Hep G2 cells (passage 86-86) after 48 and 120 h in the absence and presence of phase II inhibitors in [ng/ml].

48 h 120 h inhibitor LDH WST SRB LDH SRB

none 2.20 ± 0.59 0.36 ± 0.32 0.64 ± 0.33 1.03 ± 0.90 0.41 ± 0.17 acetaminophen 1.56 ± 0.45 0.32 ± 0.27 0.82 ± 0.21 1.27 ± 0.68 0.22 ± 0.09

berberine 2.07 ± 0.64 0.33 ± 0.08 0.67 ± 0.25 0.99 ± 0.42 0.32 ± 0.11 DNP 2.00 ± 0.69 0.34 ± 0.27 1.34 ± 0.54 0.74 ± 0.34 0.39 ± 0.12

S-hexylglutathion 2.32 ± 1.52 0.47 ± 0.08 0.80 ± 0.29 0.84 ± 0.56 0.41 ± 0.17

IC50 values of ET-743 after CYP induction. CYP3A4 in Hep G2 cells was induced for 2 days. In the presence of the inducers rifampicin and dexamethasone, no significant effect on the cytotoxicity of ET-743 could be observed (table 4). However, RT-PCR experiments showed that rifampicin induced CYP3A4 after 4 days in three different experiments by a factor 6.9, 1.2, and 3.1, resulting in a median up regulation of a factor of 3.7. The CYP3A4 activity levels measured by testosterone 6β-hydroxylation before and after induction with rifampicin were increased by a factor of 12 after 4 days. Furthermore, dexamethasone induced CYP3A4 mRNA levels after 4 days in four separate experiments by a factor of 7.6, 3.6, 6.6, and 1.7, resulting in a median up regulation of 4.9.

Table 4. ET-743 cytotoxicity (IC50 value) in Hep G2 cells (passage 87-91) after 48 and 120 h in the absence and presence of CYP inducers in [ng/ml].

48 h 120 h inducer LDH WST SRB LDH SRB

none 1.26 ± 0.59 0.52 ± 0.41 1.33 ± 0.72 0.80 ± 0.29 0.27 ± 0.18 rifampicin 1.03 ± 0.37 0.54 ± 0.46 1.30 ± 0.98 0.85 ± 0.72 0.18 ± 0.11

dexamethasone 1.72 ± 0.79 0.53 ± 0.39 1.34 ± 0.85 1.21 ± 0.46 0.27 ± 0.13 Cytotoxicity of ET-743 and its metabolites formed by cytochrome P450 in human

liver microsomes. The cytotoxicity of CYP metabolites of ET-743, formed by human liver microsomes, were less toxic compared to ET-743 (table 5). A slight decrease in the IC50 value could be observed for ET-743 with its CYP metabolites after 48 h compared to ET-743 alone, but this was not significant (p > 0.05) and not observed after 120 h. Table 5. Cytotoxicity of ET-743 and its metabolites formed in human liver microsomes (IC50 value) in Hep G2 cells (passage 96-98) after 48 and 120 h in [ng/ml].

48 h 120 h ET-743

incubation LDH WST SRB LDH SRB

no HLM at 37°C 4.28 ± 0.58 2.18 ± 0.77 3.55 ± 1.19 3.22 ± 0.51 1.94 ± 0.17 HLM at 37°C 3.66 ± 0.57 1.71 ± 0.93 3.20 ± 0.71 2.21 ± 0.50 2.05 ± 0.07 HLM at 4°C 4.19 ± 1.08 2.05 ± 0.12 3.42 ± 0.91 2.38 ± 0.41 2.04 ± 0.31

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Discussion and conclusions

ET-743 has shown activity as single agent in clinical trials in patients with advanced cancer. The clinical efficacy of ET-743 in combination with other anti-cancer drugs is now under investigation. However, the metabolic profile of ET-743 indicates that in the application of combination therapy drug-drug interactions could be a risk factor [19] (Chapter 5). Therefore, the effects of CYP and phase II inhibition and CYP induction on the cytotoxicity of ET-743 were investigated.

An increase of the cytotoxicity of ET-743 (IC50 decrease) could be observed in the Hep G2 cell line after incubation with the CYP inhibitors metyrapone (CYP2A6 and 3A4), phenanthrene (CYP2B6), piperonyl butoxide (CYP3A), proadifen (CYP2A6, 2B6, 2C9, 2E1, and 3A4), ritonavir (CYP3A4), and warfarin (CYP2C9 and 2C19). This indicates that the inhibited CYPs are involved in the biotransformation and detoxicification of ET-743 in humans. For CYP2C9, 2C19, 2E1, and 3A4, these results were confirmed by previous in vitro enzyme kinetic studies, which showed that ET-743 is metabolized by these enzymes and CYP2D6, but not by other CYPs [19] (Chapter 5). Phenanthrene is an inhibitor of CYP2B6, but it is also a substrate for CYP2E1 and 3A4 [33]. As it was previously shown in our laboratory that ET-743 is not metabolized by CYP2B6, most likely competitive inhibition of either CYP2E1, 3A4, or both is involved in the inhibiting effect of phenanthrene. Dexamethasone is an inhibitor and inducer of CYP3A4, but under the inhibition conditions tested (no pre-incubation) dexamethasone had no significant direct effect on the cytotoxicity of ET-743 [27]. The lack of a significant effect of chlorzoxazone (CYP2E1), ketoconazole (CYP1A1, 2A6, 2C8, 2C19, 2D6, and 3A4), sulfaphenazole (CYP2C9), and dexamethasone (CYP3A4) may be caused by incomplete inhibition of the isozyme(s) due to the practical limitation that the concentration had to be below the IC5 value to prevent a direct cytotoxic effect of the inhibitors on the viability of the cells.

Inhibition of phase II enzymes by acetaminophen (UGT), berberine (NAT), 2,6-dichloro-4-nitrophenol (SULT), and S-hexylglutathion (GST) showed no significant influence on the cytotoxicity of ET-743. Previous in vitro studies showed that ET-743 can be conjugated by UGT and GST and phase II metabolism was successfully studied by others in the Hep G2 cell line [34-38] (Chapter 5). The lack of effect in this study indicates that in Hep G2 cells, phase II enzymes are not significantly involved in the biotransformation and detoxicification of ET-743 and that CYP metabolism may be the rate limiting step. This was confirmed in in vitro studies in pooled human liver S9 fractions, where it was shown that ET-743 was preferably metabolized by CYPs compared to UGTs or GSTs (Chapter 5).

Based on the human CYP database, different CYP inhibitors were selected [33]. The applied concentrations were selected from results obtained in other studies in which the effect was investigated of CYP inhibitors on the biotransformation in human primary cells or cell lines. The phase II enzyme inhibitors tested were also selected based on other studies [39-42]. Acetaminophen is normally used as substrate for UGT, mainly UGT1A1, 1A6, and 2B7, but in this study it was used as a competitive inhibitor for UGT [39]. As with the CYP inhibitors, the applied concentrations were limited by their IC5 value in Hep G2 cells. This could possibly lead to a lack of effect of some inhibitors due to concentrations below a minimal level needed for complete inhibition.

The metabolites of ET-743 formed by cytochrome P450 in human liver microsomes were found to be less toxic than ET-743 itself. Together with the decrease in IC50 value of ET-743 in combination with specific CYP inhibitors, this indicates that ET-743 is detoxified in the liver.


109

This study showed that up-regulation of CYP3A4, as measured by mRNA levels after induction by rifampicin and dexamethasone, did not result in a significant change in the IC50 value of ET-743 in the Hep G2 cell line. In vitro studies by Reid et al. (2002) and at our laboratory with human liver microsomes showed a high metabolic conversion rate of ET-743, depending on the CYP3A4 activity levels [19] (Chapter 5). Based upon these data, it is expected that the half-life of ET-743 in Hep G2 cells is very short compared to the incubation time in the present experiments, leading to a short exposure compared to its less toxic metabolites. Shortening this exposure time to the parent compound even further by induction of CYP3A4 may therefore not result in a measurable decrease of the total toxicity during the whole duration of the experiment. However, adding inhibitors of metabolism will most likely increase the exposure time to ET-743 by a significant factor, increasing its contribution to the cytotoxicity. This was confirmed in the CYP inhibition experiments.

In a clinical trial, it was shown that combination therapy of ET-743 with dexamethasone increased the hepatic clearance and reduced the hepatotoxicity [27]. The hepatotoxic potential of ET-743 in female rats was also decreased by pretreatment with dexamethasone and other modulators of drug metabolism [43]. However, comparable with our experiments, Donald et al. (2003) showed that the cytotoxicity of ET-743 in vitro did not significantly decrease in primary rat hepatocytes isolated from animals treated with dexamethasone [47]. This discrepancy of in vitro and in vivo result warrants further studies both in vitro and in vivo to determine the influence of cytochrome P450 induction on the cytotoxicity and therapeutic efficacy of ET-743 in patients. To investigate the in vitro effect of CYP induction on the toxicity of ET-743, another experimental set-up should be used, where cytotoxicity is measured after a shorter time period of incubation, with other more sensitive assays and maybe a different in vitro cell model.

The Hep G2 cell line is the most frequently used and best-characterized human hepatoma cell line [29]. This cell line has a variety of liver specific metabolic functions. Under standard culturing conditions, the cells show stable, but relatively low levels of CYP and phase II enzymes, inducible by pretreatment with inducing agents [29, 44]. Still, compared to freshly isolated human hepatocytes, the overall CYP activity remains low [45]. However, freshly isolated human hepatocytes are difficult to obtain and furthermore a disadvantage is the high inter-individual variability and a gradual loss of liver specific functions during cultivation, with special reference to a decreased CYP expression [29]. The Hep G2 cell line is therefore a suitable model to study the effect of CYP inhibitors and inducers and phase II enzyme inhibitors on the cytotoxicity of ET-743, although, the low expression levels might result in a lack of effect of some inhibitors.

Furthermore, a study performed by Wilkening and Bader (2003) showed that the expression levels of several CYPs and phase II enzymes are highly variable between different passages of Hep G2 cells [46]. This could explain the high standard deviations in ET-743 cytotoxicity between different passages and also the lack of a significant effect of some of the applied inhibitors on the cytotoxicity of ET-743. The high variation in expression levels could be caused by the variation of the CYP3A4 mRNA levels between the different passages after induction with rifampicin and dexamethasone and the lack of a significant effect of these inducers on the cytotoxicity of ET-743.

Three different cytotoxicity assays were used, each having their own specific endpoint: measurement for the mitochondrial activity (cellular metabolic function) of the cell (WST-1), detection of the release of the cytosolic component lactate dehydrogenase into the culture medium (LDH), and measurement of cellular protein material attached to the plate (SRB) indicative for the number of living cells [30, 47, 48]. Luber-Narod et al. (2001) showed that combining different cytotoxicity assays provides the best information about the cytotoxicity of chemotherapeutic drugs as ET-743 and, therefore, all three assays were used [49]. In our

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experimental set-up the most sensitive and reproducible method was shown to be the SRB assay.

Based on the in vitro cytotoxicity results, there is a potential risk for drug-drug interactions between ET-743 and CYP3A4 inhibitors or inducers, but also with CYP2C9, 2C19, 2D6, and 2E1 substrates [50, 51]. They warrant for consideration when ET-743 treatment is given in combination with other anti-cancer drugs, e.g. cisplatin, paclitaxel, and doxorubicin, which are capable of CYP modulation [27, 52, 53]. Co-treatment with CYP3A4 inhibitors could lead to reduced metabolism and thus reduced hepatic clearance. In this study, it was shown that metabolites of ET-743 are less toxic than ET-743, therefore, reduced clearance could lead to increased hepatotoxicity. The difficulty with some of the applied compounds is that they are inhibitors (direct effect) and inducers (long term effect), like paclitaxel and dexamethasone. Therefore, in clinical trials with combination therapies, extra attention should be paid to the final effect: hepatic toxicity when plasma levels increase, or decreased plasma levels, possibly reducing the efficacy of the therapy.

In conclusion, ET-743 showed high cytotoxicity in Hep G2 cells, which could be increased by CYP2C9, 2C19, 2D6, 2E1, and 3A4 inhibitors, while phase II enzyme inhibitors had no influence on the toxicity. Most likely, CYP metabolism is the rate limiting step in the detoxification of ET-743. Furthermore, no effect could be observed for CYP inducers with the current experimental set-up. These findings can be used for the interpretation of the data obtained from the clinical trials with ET-743 in combination with other drugs in patients. Acknowledgment

We thank Yolanda Simarro Doorten of the department of Veterinary Pharmacology, Pharmacy and Toxicology of the faculty of Veterinary Medicine from Utrecht University for performing the CYP3A4 activity determination. This work was supported with a grant from the Nijbakker-Morra Foundation, The Netherlands.


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References 1. Jimeno J.M. (2002). A clinical armamentarium of marine-derived anti-cancer compounds.

Anti-Cancer Drugs 13 (suppl. 1): S15-S19. 2. Jimeno J.M., Faircloth G., Cameron L., Meely K., Vega E., Gómez A.,

Fernández Sousa-Faro J.M., and Rinehart K. (1996). Progress in the acquisition of new marine-derived anticancer compounds: development of Ecteinascidin-743 (ET-743). Drugs of the Future 21: 1155-1165.





7. Takebayashi Y., Pourquier P., Zimonjic D.B., Nakayama K., Emmert S., Ueda T., Urasaki Y., Kanzaki A., Akiyama S.-L., Popescu N., Kreamer K.H., and Pommier Y. (2001). Antiproliferative activity of ecteinascidin 743 is dependent upon transcription-coupled nucleotide-excision repair. Nature Med. 7: 961-966.


9. Donald S., Verschoyle R.D., Edwards R., Judah D.J., Davies R., Riley J., Dinsdale D., Lopez-Lazaro L., Smith A.G., Gant T.W., Greaves P., and Gescher A.J. (2002). Hepatobiliary damage and changes in hepatic gene expression caused by the antitumor drug ecteinascidin-743 (ET-743) in the female rat. Cancer Res. 62: 4256-4262.


11. Synold T.W., Dussault L., and Forman B.M. (2001). The orphan nuclear receptor SXR coordinately regulates drug metabolism and efflux. Nature Med. 7: 584-590.

12. Sparfel L., Payen L., Gilot D., Sidaway J., Morel F., Guillouzo A., and Fardel O. (2003). Pregnane X receptor-dependent and –independent effects of 2-acetylaminofluorene on cytochrome P450 3A23 expression and liver cell proliferation. Biochem. Biophys. Res. Commun. 300: 278-284.

13. Rinehart K.L., Gravalos L.G., Faircloth G., and Jimeno J.M. (1995). Ecteinascidin (ET-743): Preclinical antitumor development of a marine derived natural product (abstract). Proc. Am. Assoc. Cancer Res. 36: 2322.


Chapter 6

112

15. Yovine A., Riofrio M., Brain E., Blay J.Y., Kahatt C., Delaloge S., Bautier L., Coffu P., Jimeno J. , Cvitkovic E., and Misset J.L. (2001). Ecteinascidin (ET-743) given as a 24 hour (H) intravenous continuous infusion (IVCI) every 3 weeks: results of a Phase II trial in patients (pts) with pretreated soft tissue sarcomas (PSTS) (abstract). Proc. Am. Soc. Clin. Oncol. 20: 36.

16. Le Cesne A., Blay J., Judson I., van Oosterom A., Verweij J., Radford J., Lorigan P., Rodenhuis S., Di Paola E.D., van Glabbeke M., Jimeno J., and Nielsen O. (2001). ET-743 is an active drug in adult soft-tissue sarcoma (STS): a STBSG-EORTC phase II trial (abstract). Proc. Am. Soc. Clin. Oncol. 20: 1407.

17. Zelek L., Yovine A., Brain E., Turpin F., Taamma A., Riofrio M., Spielmann M., Jimeno J., and Cvitkovic E. (2000). Preliminary results of phase II study of Ecteinascidin-743 with the 24 hour continuous infusion Q3 weeks schedule in pretreated advanced/metastatic breast cancer patients (abstract). Proc. 11th NCI-EORTC-AACR Symposium on New Drugs in Cancer Therapy, Amsterdam, The Netherlands: 85.


19. Reid J.M., Kuffel M.J., Ruben S.L., Morales J.J., Rinehart K.L., Squillace D.P., and Ames M.M. (2002). Rat and human liver cytochrome P-450 isoform metabolism of Ecteinascidin 743 does not predict gender-dependent toxicity in humans. Clin. Cancer Res. 8: 2952-2962.

20. Sparidans R.W., Rosing H., Hillebrand M.J.X., López-Lázaro L., Jimeno J.M., Manzanares I. , van Kesteren Ch., Cvitkovic E., van Oosterom A.T., Schellens J.H.M., and Beijnen J.H. (2001). Search for metabolites of ecteinascidin 743, a novel, marine-derived, anti-cancer agent, in man. Anti-Cancer Drugs 12: 653-666.

21. D’Incalci M., Colombo T., Ubezio P., Nicoletti I., Giavazzi R., Erba E., Ferrarese L., Meco D. , Riccardi R., Sessa C., Cavallini E., Jimeno J., and Faircloth G.T. (2003). The combination of yondelis and cisplatin is synergistic against human tumor xenografts. Eur. J. Cancer 39: 1920-1926.

22. Meco D., Colombo T., Ubezio P., Zucchetti M., Zaffaroni M., Riccardi A., Faircloth G., Jimeno J., D’Incalci M., and Riccardi R. (2003). Effective combination of ET-743 and doxorubicin in sarcoma: preclinical studies. Cancer Chemother. Pharmacol. 52: 131-138.

23. Takahashi N., Li W.W., Banerjee D., Scotto K.W., and Bertino J.R. (2001). Sequence-dependent enhancement of cytotoxicity produced by Ecteinascidin 743 (ET-743) with doxorubicin or paclitaxel in soft tissue sarcoma cells. Clin. Cancer Res. 7: 3251-3257.

24. Takahashi N., Li W.W., Banerjee D., Guan Y., Wada-Takahashi Y., Brennan M.F., Chou T.-C. , Scotto K.W., and Bertino J.R. (2002). Sequence-dependent synergistic cytotoxicity of Ecteinascidin-743 and paclitaxel in human breast cancer cell lines in vitro and in vivo. Cancer Res. 62: 6909-6915.

25. Donald S., Verschoyle R.D., Greaves P., Gant T.W., Colombo T., Zaffaroni M., Frapolli R. , Zucchetti M., D’Incalci M., Meco D., Riccardi R., Lopez-Lazaro L., Jimeno J., and Gescher A.J. (2003). Complete protection by high-dose dexamethasone against the hepatotoxicity of the novel antitumor drug yondelis (ET-743) in the rat. Cancer Res. 63: 5902-5908.

26. Puchalski, T.A., Ryan D.P., Garcia-Carbonero R., Demetri G.D., Butkiewicz L., Harmon D., Seiden M.V., Maki R.G., Lopez-Lazaro L., Jimeno J., Guzman C., and Supko J.G. (2002). Pharmacokinetics of Ecteinascidin 743 administrated as a 24-h continuous intravenous infusion to adult patients with soft tissue sarcomas: associations with clinical characteristics, pathophysiological variables and toxicity. Cancer Chemother. Pharmacol. 50: 309-319.


113


28. Knasmuller S., Parzefall W., Sanyal R., Ecker S., Schwab C., Uhl M., Mersch-Sundermann V., Williamson G., Hietsch G., Langer T., Darroudi F., and Natarajan A.T. (1998). Use of metabolically competent human hepatoma cells for the detection of mutagens and antimutagens. Mutat. Res. 402: 185-202.


30. Higgins III J.D., Neely L., and Fricker S. (1993). Synthesis and cytotoxicity of some cyclometallated palladium complexes. J. Inorg. Biochem. 49: 149-156.

31. Wortelboer H.M., de Kruif C.A., van Iersel A.A., Falke H.E., Noordhoek J., and Blaauboer B.J. (1990). The isoenzyme pattern of cytochrome P450 in rat hepatocytes in primary culture, comparing different enzyme activities in microsomal incubations and in intact monolayers. Biochem. Pharmacol. 40: 2525-2534.

32. Lowry O.H., Rosebrough N.J., Farr A.L., and Randall R.J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275.

33. Gentest Cytochrome P450 database. http://www.gentest.com/human_p450_database (accessed July 2004).

34. Berhane K., Widersten M., Engstrom A., Kozarich J.W., and Mannervik B. (1994). Detoxication of base propenals and other alpha, beta-unsaturated aldehyde products of radical reactions and lipid peroxidation by human glutathione transferases. Proc. Natl. Acad. Sci. USA 91: 1480-1484.

35. Wu L.T., Chung J.G., Chen J.C., and Tsauer W. (2001). Effect of norcantharidin on N-acetyltransferase activity in HepG2 cells. Am. J. Chin. Med. 29: 161-172.

36. Barbier O., Duran-Sandoval D., Pineda-Torra I., Kosykh V., Fruchart J.-C., and Staels B. (2003). Peroxisome proliferator-activated receptor α induces hepatic expression of the human bile acid glucuronidation UDP-glucuronosyltransferase 2B4 enzyme. J. Biol. Chem. 278: 32852-32860.

37. O’Leary K.A., Day A.J., Needs P.W., Mellon F.A., O’Brien N.M., and Williamson G. (2003). Metabolism of quercitin-7- and quercitin-3-glucuronides by in vitro hepatic model: the role of human β-glucuronidase, sulfotransferase, catechol-O-methyltransferase and multi-resistant protein 2 (MRP2) in flavonoid metabolism. Biochem. Pharamacol. 65: 479-491.

38. Zhang K., Chew M., Yang E.B., Wong K.P., and Mack P. (2001). Modulation of cisplatin cytotoxicity and cisplatin-induced DNA cross-links in HepG2 cells by regulation of glutathione-related mechanisms. Molec. Pharmacol. 59: 837-843.

39. Ferguson S.S., LeCluyse E.L., Negishi M., and Goldstein J.A. (2002). Regulation of human CYP2C9 by the constitutive androstane receptor: discovery of a new distal binding site. Mol. Pharmacol. 62: 737-746.

40. Court M.H., Duan S.X., von Moltke L.L., Greenblatt D.J., Patten C.J., Miners J.O., and MacKenzie P.I. (2001). Interindividual variability in acetaminophen glucuronidation by human liver microsomes: identification of relevant acetaminophen UDP-glucuronosyltransferase isoforms. J. Pharmacol. Exp. Ther. 299: 998-1006.

41. Wang D.Y., Yeh C.C., Lee J.H., Hung C.F., and Chung J.G. (2002). Berberine inhibited arylamine N-acetyltransferase activity and gene expression and DNA adduct formation in human malignant astrocytoma (G9T/VGH) and brain glioblastoma multiforms (GBM 8401) cells. Neurochem. Res. 27: 883-889.

Chapter 6

114

42. Nakagawa Y., Suzuki T., and Tayama S. (2000). Metabolism and toxicity of benzophenone in isolated rat hepatocytes and estrogenic activity of its metabolites in MCF-7 cells. Toxicology 156: 27-36.

43. Donald S., Verschoyle R.D., Greaves P., Orr S., Jimeno J., and Gescher A.J. (2003). Comparison of four modulators of drug metabolism as protectants against the hepatotoxicity of the novel antitumor drug yondelis (ET-743) in the female rat and in hepatocytes in vitro. Cancer Chemother. Pharmacol. Epub ahead of print.


45. Fardel O., Morel F., Ratanasanh D., Fautrel A., Beaune P., and Guillouzo A. (1992). Expression of drug metabolizing enzymes in human HepG2 hepatoma cells. Cell. Molec. Aspects Cirrhosis 216: 327-330.

46. Wilkening S. and Bader A. (2003). Influence of culture time on the expression of drug-metabolizing enzymes in primary human hepatocytes and hepatoma cell line Hep G2. J. Biochem. Molec. Toxicol. 17: 207-213.

47. Roche product information. http://www.roche-applied-science.com/ibuybiochem/ (accessed July 2003).

48. Slater K. (2001). Cytotoxicity tests for high-throughput drug discovery. Curr. Opin. Biotechnol. 12: 70-74.

49. Luber-Narod J., Smith B., Grant W., Jimeno J.M., Lopez-Lazaro L., and Faircloth G.T. (2001). Evaluation of the use of in vitro methodologies as tools for screening new compounds for potential in vivo toxicity. Toxicol. In Vitro 15: 571-577.

50. Levy R.H., Thummel K.E., Trager W.F., Hansten P.D., and Eichelbaum M. (2000). Metabolic drug interactions. Lippincott Williams and Wilkins (Philidelphia, USA).

51. Tucker G.T. (1992). The rational selection of drug interaction studies: implication of recent advantages in drug metabolism. Int. J. Clin. Pharmacol. Ther. Toxicol. 30: 550-553.

52. Laverdiere C., Kolb E.A., Supko G.J., Gorlick R., Meyers P.A., Maki R.G., Wexler L., Demetri G.D., Healey J.H., Huvor A.G., Goorin A.M., Bagatell R., Ruiz-Casado A., Guzman C., Jimeno J., and Harmon D. (2003). Phase II study of Ecteinascidin 743 in heavily pretreated patients with recurrent osteosarcoma. Cancer 98: 832-840.


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CHAPTER Validation of in vitro cell models used in drug metabolism studies; genotyping of cytochrome P450 and phase II enzyme polymorphisms in the human hepatoma (Hep G2) and ovarian carcinoma (IGROV-1) cell lines. Esther F.A. Brandon, Tessa Bosch, Rianne Levink, Everdina van der Wal, Joyce B.M. van Meerveld, Monique Bijl, Jos H. Beijnen, Jan H.M. Schellens, and Irma Meijerman. Abstract

Several in vitro methods are available to study biotransformation of new drugs, including human cell lines. In vivo genetic polymorphisms have been identified, leading to altered enzyme activity or a change in the inducibility of the enzyme. Therefore, the aim of our study was to pharmacogenotype two cell lines used in drug metabolism studies, the Hep G2 and IGROV-1 cell lines, for genetic polymorphisms in biotransformation enzymes using allele-specific polymerase chain reaction (PCR), PCR with restriction fragment length polymorphism or PCR-sequencing.

The Hep G2 and IGROV-1 cell line were found to have the CYP3A5*3 polymorphism, which is known to have no enzymatic activity. The Hep G2 cell line was shown to be *4 (wild type) for N-acetyltransferase (NAT) 1 and heterozygous for the NAT2*6 polymorphism, both associated with a slow acetylation phenotype. The cell line was also heterozygous for the sulfotransferase (SULT) enzyme 1A1*2 and homozygous mutant for SULT1A2*2, probably leading to a reduction in the enzymatic activity of these enzymes. The IGROV-1 cell line most likely has limited glutathione-S-transferase (GST) activity, because of a deletion of the GSTM1 gene and the *B polymorphism in GSTP1, which decreases enzymatic activity. UGT1A1*28 polymorphism was also found to be heterozygous, which is associated with a decreased activity. The cell line was also shown to be wild type (*4) for NAT1 and 2, which is associated with a slow and rapid acetylation phenotype, respectively.

This characterization will allow the interpretation of the results of biotransformation studies using these in vitro cell models.

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Introduction

An important aspect in the development of new drugs is the elucidation of drug metabolism pathways, assessment of the pharmacological and toxicological activity of formed metabolites compared to the parent drug and identification of the metabolic enzymes involved in the biotransformation. Several in vitro methods are available to study these processes, including human cell lines.

Biotransformation pathways can be divided into two categories; phase I (oxidation, reduction, and hydrolysis) and phase II (conjugation) reactions [1, 2]. The cytochrome P450 (CYP) superfamily is the largest and most important group of the phase I enzymes and the CYP3A, CYP2D, and CYP2C subfamilies are responsible for respectively 50%, 25%, and 20% of the biotransformations of all drugs [3-5]. Phase II reactions are conjugation reactions to increase the water solubility of a drug, thereby making the drug more readily excretable. Major enzymes involved in phase II reactions are uridine diphosphoglucuronosyl transferase (UGT), sulfotransferase (SULT), N-acetyltransferase (NAT), and glutathione-S-transferase (GST) [3, 6, 7]. The majority of the metabolites formed by biotransformation are inactive, but sometimes bioactivation by CYP occurs. Drug metabolism plays an important role in human drug toxicology and therapeutic efficacy [8].

In vivo, CYP and phase II enzyme activity shows pronounced inter-individual variability, which is determined by gender, age, disease, food or drug intake, and genetic factors [8-13]. The genetic component in the inter-individual variability in enzyme activity has been estimated to be high [8]. A polymorphism in a biotransformation enzyme may have a direct, indirect, or no effect on the enzyme activity [14]. The polymorphism can lead to a non-active enzyme when the whole gene or part of the coding gene is deleted, when it leads to a slicing defect or altered transcriptional initiation site, or when it leads to an amino acid change or introduction of a stop codon. Polymorphisms can also lead to a decreased activity by causing a change in the active site or protein folding leading to a change in substrate specificity. An increased enzyme activity can occur when there is a multiplication of the gene. A polymorphism can also have an effect on the inducibility of the enzyme, by changes in the promoter region. In clinical studies, it was already shown that genetic polymorphisms in biotransformation enzymes can have a large impact on adverse drug reactions and the therapeutic efficacy of drugs like warfarin and several anti-cancer drugs, e.g. irinotecan [15-17].

The human hepatoma cell line (Hep G2) and human ovarian carcinoma cell line (IGROV-1) were established from Caucasian cancer patients in 1979 and 1985, respectively. The Hep G2 cell line is the most frequently used and best-characterized human hepatoma cell line and expresses a variety of liver specific metabolic enzymes, with varying activities between different passages [18-20]. It is known to express CYP1A, 2B1, 2B2, 2B6, and 3A and the phase II enzymes GST, NAT, SULT, and UGT in low amounts [21, 22]. Furthermore, biotransformation experiments at our laboratory have indicated that CYP2A6, 2C9, 2C19, 2E1, and 4A11 are also present. Thus far, mRNA levels and activity levels of some CYP and phase II enzymes in these cells have been determined. The IGROV-1 cell line is less extensively investigated and used for biotransformation research purposes, but is an important cell line in the National Cancer Institute anti-cancer screening program and for generating drug resistant cell lines. Only GST was reported to be present in these cells and there is no literature describing the presence of CYPs, UGT, NAT, or SULT [23, 24]. However, biotransformation experiments at our laboratory have indicated that the enzymes CYP2A6, 2E1, 3A4, and 4A11 are active in IGROV-1 cells. Both cell lines have been used at our laboratory to investigate the biotransformation pathways and bio(in)activation of several novel anti-cancer drugs.

Metabolic polymorphisms in Hep G2 and IGROV-1

117

Thus far, no studies have been performed to screen these frequently used cell models for genetic polymorphisms. These polymorphisms could influence the outcome of pre-clinical biotransformation studies of drugs performed with the Hep G2 and IGROV-1 cell lines. The aim of our study was therefore to screen Hep G2 and IGROV-1 cells for polymorphisms in the main CYPs and phase II enzymes involved in the biotransformation of drugs. Materials and Methods

Materials. RPMI-1640 medium (with L-glutamine and 25 mM HEPES), heat-inactivated fetal calf serum, penicillin/streptomycin, and Hanks’ Balanced Salt Solution (pH 7.4) were all obtained from Gibco BRL (Breda, The Netherlands). Primers were purchased from Invitrogen Life Technologies (Paisley, UK) and the restriction enzymes BcgI, BsaI, BstEII, BstUI, DdeI, NciI, NlaIII, Tsp509 I, and XcmI were provided by New England BioLabs (Hitchin, UK). The other restriction enzymes, Taq polymerise, and dNTPs were obtained from Fermentas (St. Leon-Rot, Germany). PCR buffers and adjuvants were all provided by Stratagene (Cedar Creek, TX, USA), except for PCR buffer II, which was purchased from Applied Biosystems (Foster City, CA, USA). ExoSAP-IT was obtained from Amersham Biosciences (Uppsala, Sweden) and the Big Dye Terminator Ready Reaction mix, Terminator buffer, and Amplitaq Gold were purchased from Applied Biosystems. Water was purified on a multi-laboratory scale by reversed osmosis and autoclaved before usage. All other chemicals were purchased from Sigma (St. Louis, MO, USA) and were of analytical grade.

Cell culture growth. The human hepatic carcinoma cell line (Hep G2) was obtained from the ATCC (Manassas, VA, USA) and the human ovarian adenocarcinoma cell line (IGROV-1) was kindly donated by the Netherlands Cancer Institute (Amsterdam, The Netherlands). Routine cultivation of the monolayer cells was performed in RPMI-1640 medium (with L-glutamine and 25 mM HEPES) supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were sub-cultured weekly (ratio of 1:5 (v/v) for Hep G2 and 1:25 (v/v) for IGROV-1 cells) and medium was refreshed after 3 days.

DNA isolation. The Hep G2 or IGROV-1 cells were detached from the flask at 75% confluency using trypsinization. The cells were centrifuged for 5 min at 1000 rpm (4°C) and resuspended in 1 ml of RPMI-1640 medium. The genomic DNA was purified from the cell suspension by a Si-GuSCN procedure modified from the procedure described by Boom et al. (1991) [27]. Nine hundred µl of lysis buffer (965 mg/ml guanidine isothiocyanate, 80% (v/v) of 0.1 M Tris-HCl, 18% (v/v) of 0.2 M EDTA, 2% (v/v) Triton-X 100) and 40 µl Celite® suspension (20% (v/v) Celite® in 0.37% HCl) was vortex mixed in a polypropylene micro tube. Hundred µl cell suspension was added and vortex mixed briefly. Next, the samples were incubated at room temperature for 15 min, vortex mixed, and centrifuged for 1 min at 14,000 rpm. The DNA pellets were washed twice with wash buffer (1.20 g/ml guanidine isothiocyanate in 0.1 M Tris-HCl (pH 6.4)), twice with 70% (v/v) ethanol, and once with acetone. After drying (20 min at 56°C), DNA was dissolved in 120 µl of 10 mM Tris-HCl (pH 8.3) by incubating the samples for 15 min at 37°C. The samples were centrifuged for 3 min at 14,000 rpm and 70 µl of the supernatant was used for PCR and stored at -20°C until use.

PCR amplification. All PCR reactions (unless mentioned otherwise) were performed in a total volume of 25 µl with ~100 ng of genomic DNA, 200 µM dNTPs, 1x PCR buffer, 0.5 U Taq polymerase and 200 nM of forward and reverse primer. Adjuvant was added if necessary, to improve the specificity of the PCR. Amplitaq Gold (0.625 U in 25 µl) was used instead of Taq polymerase for the PCR sequence reactions. The PCR buffer conditions were

Chapter 7

118

optimized using the OptiPrime PCR optimization kit (Stratagene, La Jolla, CA, USA). Amplification was carried out on a PTC-100 or PTC-200 Thermal Cycler (MJ Research Inc., Waltham, MA, USA). Details of the used PCR buffers, additives, and amplification programs are mentioned further on. Aliquots of each PCR product were subjected to agarose gel electrophoresis to ensure proper amplification. All PCR methods were validated with sequencing.

β-Globulin was used as internal control in allele-specific PCR (CYP2A6*9) or in case of a deletion of the gene (GSTM1 and GSTT1). The following primers were used to generate a 268 bp product: 5’-CAA CTT CAT CCA CGT TCA CC-3’ (forward primer) and 5’-GAA GAG CCA AGG ACA GGT AC-3’ (reverse primer).

CYP polymorphisms. Identification of the most common Caucasian polymorphisms in CYP isozymes were performed using allele-specific PCR, PCR with restriction fragment length polymorphism (PCR-RFLP), or PCR-sequencing (table 1). Using PCR-RFLP, the polymorphisms were detected by digestion with a restriction enzyme (protocol and buffer supplied by manufacturer). The resulting fragments were analyzed on a agarose gel (percentage agarose based on the expected fragment sizes) stained with ethidium bromide. Product and fragment sizes are mentioned in table 1.

Uridine diphosphoglucuronosyl transferase. PCR followed by sequencing was used to determine the *28 polymorphism (A(TA)6TAA to A(TA)7TAA), *33 (A(TA)6TAA to A(TA)5TAA) and *34 (A(TA)6TAA to A(TA)8TAA) in the UGT1A1 gene which are all polymorphisms in the promotor region. The forward primer sequence was 5’-TCC CTG CTA CCT TTG TGG AC-3’ and the reverse primer sequence 5’-CCA TCC ACT GGG ATG CAA CA-3’. The amplification was carried out in the presence of 3 mM MgCl2 and PCR buffer II (Perkin and Elmer). After initial denaturation, the samples were subjected to 40 cycles of 95°C for 30 s, 55°C for 45 s, and 76°C for 45 s, not followed by a final extension step.

The UGT2B15 polymorphisms *2 (D85Y) and *3 (L86S) were identified using the primers and PCR method described by MacLeod et al. (2000) [45]. Briefly, a 326 bp fragment was amplified with the forward primer 5’-GAC TGT GTT GAC ATC TTC GGC TTC T-3’ and the reverse primer 5’-CCA GTA GCT CAC CAC AGG GAT TAA G-3’. The final buffer conditions were 1.5 mM MgCl2, 25 mM KCl and 10 mM Tris-HCl (pH 9.2). Amplification was performed using the following protocol: 5 min at 95°C, 35 cycles of: 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s, and a final extension step at 72°C for 7 min. The PCR product was sequenced to determine the *2 (G253→T) and *3 (T256→C) mutation, as described in the sequencing section.

Sulfotransferase polymorphisms. The *2 and *3 polymorphisms in SULT1A1 and 1A2 were genotyped according to Peng et al. (2003) [46]. A 281 bp fragment was amplified for SULT1A1*2 and *3 using the following buffer conditions: 1.5 mM MgCl2, 25 mM KCl, 10 mM Tris-HCl (pH 9.2), and the primer 5’-GGT TGA GGA GTT GGC TCT GC-3’ (forward primer) and 5’-ATG AAC TCC TGG GGG ACG GT-3’ (reverse primer). The PCR-product was amplified by 35 cycles of 1 min at 95°C, 1 min at 60°C, and 2 min 72°C. The product was digested with HhaI to determine the *2 polymorphism; wild type was digested to 176 and 105 bp. NlaII was used to determine the *3 polymorphism, digesting the wild type to 207 and 74 bp.


119

ref.

[26]

[27]

[28]

[29]

[30]

[31]

prod

uct

size

(bp)

wt:

324

mt:

209+

133

wt a

nd m

t: 32

2

wt:

139+

65

mt:

204

wt:

314+

83+5

2+34

m

t: 39

6+52

+34

wt:

597

mt:

489+

108

wt:

123+

120

mt:

242

anal

ysis

RFL

P w

ith

Msp

I

sequ

enci

ng

RFL

P w

ith

Bsa

I

RFL

P w

ith

Tsp5

09I

RFL

P w

ith

Dde

I

RFL

P w

ith

Apa

I

buff

er

cond

ition

s

1.5

mM

MgC

l 2 25

mM

KC

l 10

mM

Tris

-HC

l pH

8.8

1.5

mM

MgC

l 2 25

mM

KC

l 10

mM

Tris

-HC

l pH

9.2

1.5

mM

MgC

l 2 25

mM

KC

l 10

mM

Tris

-HC

l pH

9.2

1.5

mM

MgC

l 2 25

mM

KC

l 10

mM

Tris

-HC

l pH

9.2

1.5

mM

MgC

l 2 75

mM

KC

l 10

mM

Tris

-HC

l pH

9.2

3.5

mM

MgC

l 2 25

mM

KC

l 10

mM

Tris

-HC

l pH

9.2

PCR

pr

ogra

m

30’’

94°

C

30’’

56°

C

30’’

72°

C

(35

cycl

es)

1’ 9

4°C

1’

63°

C

1.5’

72°

C

(35

cycl

es)

30’’

94°

C

30’’

63°

C

30’’

72°

C

(35

cycl

es)

30’’

94°

C

10’’

58°

C

1’ 7

2°C

(4

0 cy

cles

)

1.5’

94°

C

2’ 5

8°C

2’

72°

C

(35

cycl

es)

30’’

94°

C

30’’

60°

C

30’’

72°

C

(40

cycl

es)

5’-3

’ seq

uenc

e

CA

G T

GA

AG

A G

GT

GTA

GC

C G

CTF

TAG

GA

G T

CT

TGT

CTC

ATG

CC

TR

GA

A C

TG C

CA

CTT

CA

G C

TG T

CTF

AA

G A

CC

TC

C C

AG

CG

G G

CA

ATR

CTG

TC

T C

CC

TC

T G

GT

TAC

AG

G A

AG

CF

TTC

CA

C C

CG

TTG

CA

G C

AG

GA

T A

GC

CR

AG

C C

CT

TGA

GTG

AG

A A

GA

TG

F G

GT

CTT

GC

T C

TG T

CA

CTC

AR

GC

T A

CA

CA

T G

AT

CG

A G

CT

ATA

CF

CA

G G

TC T

CT

TCA

CTG

TA

A A

GT

TAR

CC

C A

GA

AG

T G

GA

AA

C T

GA

GA

F G

GG

TTG

AG

A T

GG

AG

A C

AT

TCR

SNP

T 380

1C

A24

55G

*4

*1B

*1C

*1F

Tab

le 1

. PC

R m

etho

ds fo

r CY

P po

lym

orph

ism

s

CY

P is

ozym

e

1A1

1A2

Chapter 7

120

ref.

[32]

[32]

[33]

[33]

[34]

[35]

prod

uct

size

(bp)

wt:

119+

49

mt:

168

wt:

107+

61

mt:

168

wt:

213

mt:

151+

62

wt:

213

mt:

143+

70

wt o

r mt:

368

wt:

385+

338

mt:

723

anal

ysis

RFL

P w

ith

Eco4

7I

RFL

P w

ith

Nci

I

RFL

P w

ith

Xcm

I

RFL

P w

ith

Dde

I

alle

le

spec

ific

RFL

P w

ith

Bsp

143I

I

buff

er

cond

ition

s

3.5

mM

MgC

l 2 75

mM

KC

l 10

mM

Tris

-HC

l pH

9.2

sam

e as

for *

1J

1.5

mM

MgC

l 2 75

mM

KC

l 10

mM

Tris

-HC

l pH

9.2

sam

e as

for *

2

3.5

mM

MgC

l 2 75

mM

KC

l 10

mM

Tris

-HC

l pH

9.2

3.5

mM

MgC

l 2 25

mM

KC

l 10

mM

Tris

-HC

l pH

8.3

10

0 µm

g/m

l BSA

PCR

pr

ogra

m

15’’

95°

C

20’’

54°

C

1’ 7

2°C

(4

5 cy

cles

)

sam

e as

for

*1J

1’ 9

5°C

1’

50°

C

1’ 7

2°C

(3

5 cy

cles

)

sam

e as

for

*2

1’ 9

5°C

1’

54°

C

1’ 7

2°C

(4

0 cy

cles

)

30’’

95°

C

30’’

50°

C

1’ 7

2°C

(4

0 cy

cles

)

5’-3

’ seq

uenc

e

TGG

AA

G C

TA G

TG G

GG

AC

AF

TTG

TG

C T

AA

GG

G G

GA

AG

CR

sam

e as

for *

1J

AC

C T

CC

CC

A G

GC

GTG

GTA

F TC

G T

CC

TG

G G

TG T

TT T

CC

TTC

R

sam

e as

for *

2

GA

T TC

C T

CT

CC

C C

TG G

AA

CF

GG

C T

GG

GG

T G

GT

TTG

CC

T TT

AW

R

GG

C T

GG

GG

T G

GT

TTG

CC

T TT

CM

R

AC

A T

TC A

CT

TGC

TC

A C

CTF

GTA

AA

T A

CC

AC

T TG

A C

CA

R

SNP

*1J

*1K

*2

*3

*9

C64

T

Tab

le 1

con

tinue

d. P

CR

met

hods

for C

YP

poly

mor

phis

ms

CY

P is

ozym

e

1A2

2A6

2B6


121

ref.

[35]

[35]

[35]

[35]

[36]

[37]

prod

uct

size

(bp)

wt:

267+

236+

23

mt:

503+

23

wt:

299+

196+

145

mt:

341+

299

wt:

296+

171+

117+

56

mt:

467+

117+

56

wt:

1400

m

t: 11

86+2

14

wt:

89+3

5 m

t: 12

5

wt:

521+

169

mt:

690

anal

ysis

RFL

P w

ith

Bse

NI

RFL

P w

ith

Bsp

143I

I

RFL

P w

ith

Eco1

30I

RFL

P w

ith

Bgl

II

RFL

P w

ith

TaqI

RFL

P w

ith

Ava

II

buff

er

cond

ition

s

1.5

mM

MgC

l 2 25

mM

KC

l 10

mM

Tris

-HC

l pH

8.8

1.5

mM

MgC

l 2 75

mM

KC

l 10

mM

Tris

-HC

l pH

8.8

1.5

mM

MgC

l 2 75

mM

KC

l 10

mM

Tris

-HC

l pH

8.8

3.5

mM

MgC

l 2 25

mM

KC

l 10

mM

Tris

-HC

l pH

9.2

1.5

mM

MgC

l 2 25

mM

KC

l 10

mM

Tris

-HC

l pH

9.2

3.5

mM

MgC

l 2 25

mM

KC

l 10

mM

Tris

-HC

l pH

9.2

PCR

pr

ogra

m

30’’

95°

C

30’’

56°

C

40’’

72°

C

(35

cycl

es)

30’’

95°

C

30’’

60°

C

1’ 7

2°C

(4

0 cy

cles

)

30’’

95°

C

30’’

60°

C

1’ 7

2°C

(4

0 cy

cles

)

30’’

95°

C

30’’

60°

C

1.5’

72°

C

(35

cycl

es)

1’ 9

4°C

2’

55°

C

2’ 7

2°C

(4

0 cy

cles

)

1’ 9

4°C

1.

5’ 6

0°C

30

’’ 7

2°C

(3

5 cy

cles

)

5’-3

’ seq

uenc

e

GG

T C

TG C

CC

ATC

TA

T A

AA

CF

CTG

ATT

CTT

CA

C A

TG T

CT

GC

GR

GA

C A

GA

AG

G A

TG A

GG

GA

G G

AA

F C

TC C

CT

CTG

TC

T TT

C A

TT C

TG T

R

GA

C A

GA

AG

G A

TG A

GG

GA

G G

AA

F C

TC C

CT

CTG

TC

T TT

C A

TT C

TG T

R

TGA

GA

A T

CA

GTG

GA

A G

CC

ATA

GA

F TA

A T

TT T

CG

ATA

ATC

TC

A C

TC C

TG C

R

AA

A A

AT

GTT

TC

T C

TT A

CA

CG

F A

TT T

TA C

CT

GC

T C

CA

TTT

TG

R

TAC

AA

A T

AC

AA

T G

AA

AA

T A

TC A

TGF

CTA

AC

A A

CC

AG

A C

TC A

TA A

TGR

SNP

G51

6T

C77

7A

A78

5G

C14

59T

*4

*2

Tab

le 1

com

tinue

d. P

CR

met

hods

for C

YP

poly

mor

phis

ms

CY

P is

ozym

e

2B6

2C8

2C9

Chapter 7

122

ref. -

[38]

[39]

[39]

[40]

[41]

prod

uct

size

(bp)

wt a

nd m

t: 20

8

wt:

212+

109

mt:

321

wt:

188+

84

mt:

175+

84+1

2

wt:

250+

105

mt:

355

wt:

351+

62

mt:

413

wt:

572+

305+

121

mt:

877+

121

anal

ysis

sequ

enci

ng

RFL

P w

ith

SmaI

RFL

P w

ith

Msp

I

RFL

P w

ith

Mva

I

RFL

P w

ith

Rsa

I

RFL

P w

ith

Dra

I

buff

er

cond

ition

s

1.5

mM

MgC

l 2 25

mM

KC

l 10

mM

Tris

-HC

l pH

9.2

3.5

mM

MgC

l 2 25

mM

KC

l 10

mM

Tris

-HC

l pH

8.3

1.5

mM

MgC

l 2 25

mM

KC

l 10

mM

Tris

-HC

l pH

9.2

5%

DM

SO

1.5

mM

MgC

l 2 25

mM

KC

l 10

mM

Tris

-HC

l pH

9.2

10

0 µg

/ml B

SA

1.5

mM

MgC

l 2 25

mM

KC

l 10

mM

Tris

-HC

l pH

8.8

3.5

mM

MgC

l 2 25

mM

KC

l 10

mM

Tris

-HC

l pH

8.8

PCR

pr

ogra

m

1’ 9

4°C

1’

57°

C

1’ 7

2°C

(3

5 cy

cles

)

20’’

94°

C

10’’

53°

C

10’’

72°

C

(37

cycl

es)

1’ 9

4°C

1’

60°

C

2’ 7

2°C

(4

5 cy

cles

)

1’ 9

4°C

1’

60°

C

1.5’

72°

C

(38

cycl

es)

1’ 9

4°C

1’

55°

C

1’ 7

2°C

(3

5 cy

cles

)

1’ 9

4°C

1’

60°

C

4’ 7

2°C

(3

5 cy

cles

)

5’-3

’ seq

uenc

e

GA

A C

GT

GTG

ATT

GG

C A

GA

AA

F TC

G A

AA

AC

A T

GG

AG

T TG

C A

GR

CA

G A

GC

TTG

GC

A T

AT

TGT

ATC

F G

TA A

AC

AC

A A

AA

CTA

GTC

AA

T G

R

GA

T G

AG

CTG

CTA

AC

T G

AG

CC

CF

CC

G A

GA

GC

A T

AC

TC

G G

GA

R

GC

C T

TC G

CC

AA

C C

AC

TC

C G

F A

AA

TC

C T

GC

TC

T TC

C G

AG

GC

R

CC

A G

TC G

AG

TC

T A

CA

TTG

TC

AF

TTC

ATT

CTG

TC

T TC

T A

AC

TG

GR

TCG

TC

A G

TT C

CT

GA

A A

GC

AG

GF

GA

G C

TC T

GA

TG

C A

AG

TA

T C

GC

AR

SNP

*3

*2

*3

*4

*5

*6

Tab

le 1

con

tinue

d. P

CR

met

hods

for C

YP

poly

mor

phis

ms

CY

P is

ozym

e

2C9

2C19

2D6

2E1


123

ref. -

[42]

[43]

[42]

[44]

[44]

prod

uct

size

(bp)

wt a

nd m

t: 20

1

wt a

nd m

t: 36

9

wt:

226+

23

mt:

249

wt a

nd m

t: 42

1

wt:

269

mt:

183+

86

wt:

168+

125

mt:

293

anal

ysis

sequ

enci

ng

sequ

enci

ng

RFL

P w

ith

Mph

1103

I

sequ

enci

ng

RFL

P w

ith

TasI

RFL

P w

ith

SspI

buff

er

cond

ition

s

1x P

CR

buf

fer I

I 2.

5 m

M M

gCl 2

0.5x

PC

R b

uffe

r II

1.5

mM

MgC

l 2 0.

5% D

MSO

3.5

mM

MgC

l 2 75

mM

KC

l 10

mM

Tris

-HC

l pH

8.3

sam

e as

*2

1.5

mM

MgC

l 2 75

mM

KC

l 10

mM

Tris

-HC

l pH

8.8

3.5

mM

MgC

l 2 75

mM

KC

l 10

mM

Tris

-HC

l pH

8.3

PCR

pr

ogra

m

30’’

95°

C

45’’

56°

C

45’’

72°

C

(40

cycl

es)

30’’

95°

C

30’’

56°

C

1’ 7

0°C

(3

5 cy

cles

)

1’ 9

4°C

1.

5’ 5

5°C

1’

72°

C

(35

cycl

es)

sam

e as

*2

1’ 9

4°C

1’

55°

C

1’ 7

2°C

(4

0 cy

cles

)

1’ 9

4°C

1’

55°

C

1’ 7

2°C

(3

5 cy

cles

)

5’-3

’ seq

uenc

e

TGC

CA

A C

AG

AA

T C

AC

AG

A G

GF

GTC

CC

T A

CC

AG

G G

TG C

TG T

AR

CC

T G

TT G

CA

TG

C A

TA G

AG

GF

GA

T G

AT

GG

T C

AC

AC

A T

AT

CR

TGG

AC

C C

AG

AA

A C

TG C

AT

ATG

CF

GA

T C

AC

AG

A T

GG

GC

C T

AA

TTG

R

GA

G T

TA G

TC T

CT

GG

A G

CT

CC

F C

AA

CC

A C

AT

GA

C T

GT

CC

T G

TA G

R

CTG

TTT

CTT

TC

C T

TC C

AG

GC

F C

TC C

AT

TTC

CC

T G

GA

GA

C T

TGR

CA

T C

AG

TTA

GTA

GA

C A

GA

TG

AF

GG

T C

CA

AA

C A

GG

GA

A G

AA

ATA

R

SNP

*1B

*2

*3

*12

*2

*3

Tab

le 1

con

tinue

d. P

CR

met

hods

for C

YP

poly

mor

phis

ms

CY

P is

ozym

e

3A4

3A5

F fo

rwar

d pr

imer

R

reve

rse

prim

er

WR

wild

type

reve

rse

prim

er

MR

mut

ant r

ever

se p

rimer

re

f. re

fere

nce

Chapter 7

124

Genetic polymorphism SULT1A2*2 was analyzed using forward primer 5’-GAA CAT GGA GCT GAT CCA GGT C-3’ and reverse primer 5’-CTG AGG TGA GCA TGA CCT CG-3’ and the following buffer conditions: 1.5 mM MgCl2, 25 mM KCl, and 10 mM Tris-HCl (pH 8.3). Thirty-five cycles of 1 min 94°C, 1 min 58°C, and 2 min 72°C were used to amplify a 227 bp fragment. BstEII digested the mutant to 208 and 19 bp. For the genotyping of SULT1A2*3, amplification with forward primer 5’-GGA ACC ACC ACA TTA GAG C-3’ and reverse primer 5’-GGC TCT GCA AAG TAC TTG ATG CG and digestion with BstUI was performed. The same buffer conditions were used as described for SULT1A1*2 and *3 and the PCR program was the same as for SULT1A2*2. The wild type was digested to 245 and 23 bp bands.

Glutathione-S-transferase polymorphisms. GSTM1 and T1 deletions were analyzed using a PCR method with β-globulin as internal control as described by Sreelekha et al. (2001) [47]. Forward primer 5’-GAA CTC CCT GAA AAG CTA AAG C-3’ and reverse primer 5’-GTT GGG CTC AAA TAT ACG GTG G-3’ were used for GSTM1 genotyping. The PCR amplification was performed in the presence of 3.5 mM MgCl2, 75 mM KCl, and 10 mM Tris-HCl (pH 8.3) and 35 cycles at 94°C for 1 min, 61°C for 1 min, and 72°C for 1 min. A homozygous deletion of the GSTM1 gene was shown by absence of the 219 bp band.

To determine the GSTT1 gene deletion, forward primer 5’-TTC CTT ACT GGT CCT CAC ATC TC-3’ and reverse primer 5’-TCA CCG GAT CAT GGC CAG CA-3’ were used. The PCR reaction was performed in 3.5 mM MgCl2, 25 mM KCl and 10 mM Tris-HCl (pH 8.8) and 35 cycles of 30 s at 94°C, 1 min at 64°C, and 1 min at 72°C. The homozygous deletion of the gene was shown by the absence of the 459 bp product.

GSTP1*B was genotyped with PCR-RFLP according to Jerónimo et al. (2002) [48]. The presence of the polymorphism I105V (*B) was determined with the forward primer 5’-ACC CCA GGG CTC TAT GGG AA-3’ and reverse primer 5’-TGA GGG CAC AAG AAG CCC CT-3’. The same buffer conditions as for GSTM1 were used with 5% DMSO as adjuvant. A fragment of 176 bp was amplified using the following protocol: 45 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. Using restriction enzyme Alw26I, the mutant was digested to 93 and 83 bp.

N-acetyl transferase polymorphisms. The NAT1 mutations were analyzed using PCR-RFLP and sequencing according to the methods described by Loktionov et al. (2002), while the NAT2 mutations were analyzed using a method described by van Duijnhoven et al. (2002) [49, 50]. For NAT1 and NAT2, two fragments were amplified to identify the different polymorphisms; 62 and 112 bp for NAT1 and 230 and 524 bp for NAT2. The PCR method and product sizes for NAT1 and NAT2 are described in table 2.


125

prod

uct

size

(bp)

wt:

99+1

3 bp

m

t: 11

2 bp

wt a

nd m

t: 11

2 bp

wt:

112

bp

mt:

103

bp

wt:

31+1

6+15

bp

mt:

31 b

p

wt:

34+1

7+5

bp

mt:

62 b

p

wt:

54+8

bp

mt:

62 b

p

wt:

34+1

7+5

bp

mt:

62 b

p

wt:

112

bp

mt:

115

bp

wt:

112

bp

mt:

109

bp

wt:

112

bp

mt:

115

bp

anal

ysis

RFL

P w

ith

Mbo

II

sequ

enci

ng

-

RFL

P w

ith

Mbo

II

and

Bcg

I

RFL

P w

ith

Eco5

7I

and

Bcg

I

sequ

enci

ng

-

sequ

enci

ng

buff

er

cond

ition

s

3.5

mM

MgC

l 2 75

mM

KC

l 10

mM

Tris

-HC

l pH

9.2

sam

e as

for C

1095

A

sam

e as

for C

1095

A

sam

e as

for C

1095

A

sam

e as

for C

1095

A

sam

e as

for C

1095

A

sam

e as

for C

1095

A

sam

e as

for C

1095

A

PCR

pr

ogra

m

1’ 9

4°C

1’

48°

C

1’ 7

2°C

(3

5 cy

cles

)

sam

e as

for

C10

95A

sam

e as

for

C10

95A

1’ 9

4°C

1’

59°

C

1’ 7

2°C

(3

5 cy

cles

)

sam

e as

for

*14

sam

e as

for

C10

95A

sam

e as

for

C10

95A

sam

e as

for

C10

95A

5’-3

’ seq

uenc

e

ATG

TC

A T

CA

TA

T A

TA A

TT A

AA

CA

GF

GA

T A

AC

CA

C A

GG

CC

A T

CT

TTA

GA

AR

sam

e as

for C

1095

A

sam

e as

for C

1095

A

CTT

CA

T TC

T G

AT

CTC

CTA

GC

A G

AC

AG

F G

GC

TTA

AG

A G

TA A

AG

GA

G C

AG

ATT

CTT

R

sam

e as

for *

14

sam

e as

for C

1095

A

sam

e as

for C

1095

A

sam

e as

for C

1095

A

SNP

C10

95A

*10

*11

*14

*15

*16

*18

*26

Tab

le 2

. PC

R m

etho

ds fo

r NA

T1 a

nd N

AT2

pol

ymor

phis

ms

NA

T

1

Chapter 7

126

prod

uct

size

(bp)

wt:

112

bp

mt:

106

bp

wt:

138+

92 b

p m

t: 23

0 bp

wt:

220+

10 b

p m

t: 12

2+10

8+10

bp

wt:

202+

170+

152

bp

mt:

372+

152

bp

wt:

430+

94 b

p m

t: 52

4 bp

wt:

345+

111+

45

+23

bp

mt:

345+

134+

45 b

p

wt:

470+

54 b

p m

t: 52

4 bp

anal

ysis

-

RFL

P w

ith

Bse

GI

RFL

P w

ith

Dde

I

RFL

P w

ith

TaqI

RFL

P w

ith

Kpn

I

RFL

P w

ith

Dde

I

RFL

P w

ith

Bam

HI

buff

er

cond

ition

s

sam

e as

for

C10

95A

3.5

mM

MgC

l 2 25

mM

KC

l 10

mM

Tris

-HC

l pH

9.2

75

mM

(N

H4)

2SO

4

sam

e as

for

C28

2T

sam

e as

for

C28

2T

sam

e as

for

C28

2T

sam

e as

for

C28

2T

sam

e as

for

C28

2T

PCR

pr

ogra

m

sam

e as

for

C10

95A

30’’

95°

C

30’’

62°

C

1.5’

72°

C

(40

cycl

es)

sam

e as

for

C28

2T

30’’

95°

C

30’’

60°

C

1’ 7

2°C

(3

5 cy

cles

)

sam

e as

for

C48

1T

sam

e as

for

C48

1T

sam

e as

for

C48

1T

5’-3

’ seq

uenc

e

sam

e as

for C

1095

A

GG

A G

GT

GG

G C

TT A

GA

GG

CF

AC

C C

AG

CA

T C

GA

CA

A T

GT

AA

T TC

C T

GC

CC

T C

AR

sam

e as

for C

282T

CC

A G

AT

GTG

GC

A G

CC

TC

T A

F G

GT

GA

T A

CA

TA

C A

CA

AG

G G

TT T

R

sam

e as

for C

481T

sam

e as

for C

481T

sam

e as

for C

481T

SNP

*28

C28

2T

T 341

C

C48

1T

G59

0A

A80

3G

G85

7A

Tab

le 2

con

tinue

d. P

CR

met

hods

for N

AT1

and

NA

T2 p

olym

orph

ism

s

NA

T

1 2

F fo

rwar

d pr

imer

R

reve

rse

prim

er


127

Sequencing of polymorphism. Sequencing was used to determine the polymorphisms CYP1A1 A2455G, CYP2C9*3, CYP3A4*1B, *2, and *12, and UGT1A1*28, *33, and *34. The PCR product was purified by adding 2 µl ExoSAP-IT to 10 µl PCR product. This was incubated for 15 min at 37°C and the ExoSAP-IT was inactivated by heating the mixture to 80°C for 15 min. The primers used for sequencing were the same as used for the polymerase chain reactions. The sequence reaction was performed in a total volume of 20 µl with 4 µl purified PCR product, 1µl Big Dye Terminator Ready Reaction mix, 7 µl of 2.5x Big Dye Terminator buffer, 6.5 µl H2O and 1.5 µl of forward or reverse primer (5µM). The PCR mixtures were subjected to 25 cycles of 10 s at 96°C, 5 s at 50°C, and 4 min at 60°C. Residual dideoxy terminators were removed by ethanol/sodium acetate precipitation according to the protocol supplied by the manufacturer. The sequencing was performed on an Applied Biosystems 3100-Avant Genetic Analyzer and analysis of the data was performed using SeqScape® v2.0 (Applied Biosystems).

Nomenclature. Base numbering and allele designation were based on literature and the general guidelines for naming human gene mutations in the different CYP and phase II enzymes [51-53]. The polymorphisms were assigned based on the general nomenclature and the different SNPs observed. Results

Genotyping of cytochrome P450 enzymes. The results of the CYP genotyping for both cell lines are shown in table 3. To compare the observed polymorphisms with in vivo data, the occurrence in Caucasians is also mentioned as is the effect of the polymorphism on the activity of the concerning enzyme. The Hep G2 and IGROV-1 cell line were found to have the CYP3A5*3 polymorphism. The CYP3A5*3 polymorphism is a common genetic polymorphism in Caucasians (around 90%) and is known to have no enzymatic activity [46]. The IGROV-1 cell line was shown to be heterozygous for the CYP1A2*1B polymorphism and homozygous for the CYP2C9*3 polymorphism. Thus far, no effect of the CYP1A2*1B polymorphism on enzyme activity has been identified. The CYP2C9*3 polymorphism is associated with a decreased CYP2C9 activity [54]. No other polymorphisms were detected in both cell lines for the other measured CYPs.

Chapter 7

128

Table 3. CYP polymorphisms

CYP polymorphism % in

Caucasiansenzyme activity Hep G2 IGROV-1

1A1 *2A 5-8 ↑inducibility W W *2B 3 ↑inducibility W W *2C 5 normal W W *4 3 ? W W

1A2 *1B 33-38 ? W H *1C 2 decreased W W *1D 5 ? W W *1F 33-68 ↑inducibility W W *1J 1 Normal W W

2A6 *2 1-3 none W W *3 0-1 none W W *9 5 decreased W W

2B6 *2 5 ? W W *4 4 ? W W *5 10-14 decreased W W *6 9-25 increased W W *7 3 decreased W W

2C8 *4 8 decreased W W 2C9 *2 8-22 decreased W W

*3 5-15 decreased W M 2C19 *2 13-15 none W W 2D6 *3 1-3 none W W

*4 12-23 none W W 2E1 *5 1-5 increased W W

*6 10 ? W W 3A4 *1B 5-9 normal W W

*2 3 decreased W W *3 2-4 normal W W

3A5 *2 1-5 none W W *3 90 none M M

? unknown, W wild type, H heterozygous, and M mutant type

Genotyping of Phase II enzymes. In table 4, the results are shown for the genetic polymorphisms in phase II enzymes together with occurrence in Caucasians and corresponding enzyme activities. The Hep G2 cell line was shown to have polymorphisms in the sulfotransferase enzymes, heterozygous for SULT1A1*2 and homozygous mutant for SULT1A2*2. Both polymorphisms have been associated with reduced enzyme activities [55]. The GSTM1 gene was deleted in the IGROV-1 cell line and also the homozygous GSTP1*B polymorphism, associated with a reduced GSTP1 activity, was observed [48]. The heterozygous UGT1A1 *28 polymorphism was found in this cell line and has been connected with decreased UGT1A1 activity [56]. Both Hep G2 and IGROV-1 were found to be wild type (*4) for NAT1. The IGROV-1 cell line was also wild type (*4) for NAT2, while the Hep G2 cells showed to be heterozygous mutant for the *6 polymorphism in this gene. NAT1*4 and NAT2*4 have been designated as the wild type human NAT1 and NAT2 alleles,


129

respectively. They are the most common occurring alleles in some, but not all ethnic groups [52]. The NAT1*4 allele is present in around 70 to 75% of the Caucasian population, while NAT2*4 is only present in 20 to 25% of the Caucasians [49, 57]. The NAT1 wild type has been associated with a slow acetylation phenotype, while NAT2 results in a rapid acetylation phenotype [49]. Furthermore, NAT2*6 polymorphism leads to a decreased acetylation activity compared to the wild type [49].

Table 4. Phase II enzyme polymorphisms

enzyme polymorphism % in

Caucasiansenzyme activity Hep G2 IGROV-1

SULT1A1 *2 30-38 decreased H W *3 1 ? W W

SULT1A2 *2 20-39 decreased M W *3 10-18 ? W W

GSTM1 *0 40-60 none W M GSTP1 *B 28-34 decreased W M GSTT1 *0 10-24 none W W

UGT1A1 *28 32 decreased W H UGT2B15 *2 56 decreased W W

*3 ? ? W W NAT1 *3 3 decreased W W

*10 16-20 increased W W *11 3-5 increased W W *14 3 decreased W W *15 0.3 none W W *16 ? ? W W *18A ? ? W W *18B ? ? W W *26A ? ? W W *26B ? ? W W *28 ? ? W W

NAT2 *5 43-49 decreased W W *6 27-36 decreased H W *7 2-5 decreased W W *11 ? ? W W *12 1 increased W W *13 1 increased W W

? unknown, W wild type, H heterozygous, and M mutant type

Validation. Sequencing showed that the PCR methods for PCR-RFLP and allele-specific PCR were valid (results not shown). Discussion and conclusion

In vitro cell models are frequently used in preclinical research of novel compounds and can be used to elucidate the biotransformation route of new drugs. The aim of our study was to control the validity of two different cell lines, a hepatoma cell line (Hep G2) and ovarian carcinoma (IGROV-1) cell line, in drug metabolism studies by pharmacogenotyping the cells for polymorphisms in the main CYP and phase II enzymes.

Chapter 7

130

Based on the genetic polymorphisms, the Hep G2 cell line is expected to have low SULT and NAT activity levels. This is confirmed by Fardel et al. (1992), who showed that Hep G2 cells possess low levels of N-acetyltransferase and sulfotransferase activity [21], but still high enough to study the biotransformation of drugs by these enzymes [20, 58-62]. No other polymorphisms in CYP and phase II enzymes were detected and this was confirmed by metabolic studies at our laboratory and by others showing that this cell line is a suitable model to study biotransformation and the role of the most common CYP, GST, and UGT enzymes [20, 21, 63-66].

The IGROV-1 cell line will probably have reduced CYP2C9, UGT1A1, and GSTP1 activity, due to polymorphisms in these genes. It will also have no GSTM1 activity, because of the deletion of the whole gene. Besides metabolic studies performed in our laboratory with this cell line, no further information is known about the activity of non-polymorphic cytochrome P450 and phase II enzymes in this cell line. Therefore, for further validation of the IGROV-1 cell line, activity levels of CYP and phase II enzymes, should be measured.

The overall therapeutic and toxic profile of a drug in patients is to a large extent affected by the biotransformation of a drug. Therefore, biotransformation research is a pivotal part in the early developmental stage of new drugs and in the prediction of drug-drug interactions at the metabolic level. There are several useful in vitro model system with a strong predictive power for human biotransformation, ranging from (recombinant) isolated enzymes to primary human hepatocytes and cell lines [19]. Supersomes (insect cells transfected with human cytochrome P450 DNA) and human liver microsomes (CYP and UGT activity) are often used models to study cytochrome P450 mediated biotransformation and to investigate the inter-individual variability in drug metabolism. However, a disadvantage of these two in vitro models is that only CYP and UGT mediated biotransformation can be studied and they are a poor representation of the in vivo human liver. Primary human hepatocytes better resemble the human liver and are a popular in vitro system for drug biotransformation research [19]. However, a problem encountered with primary hepatocytes is the considerable inter-individual variation, mainly caused by genetic polymorphisms in the CYP and phase II enzymes, and the gradual loss of liver specific functions during cultivation, with special reference to a decreased CYP expression [19]. Due to the disadvantages of primary human hepatocytes and their poor availability, human cell lines are also used to study biotransformation. They are a reliable in vitro model, because of their stable enzyme expressing levels and the lack of inter-individual variability. Furthermore, they are a useful tool to study drug-drug interactions and the cytotoxicity of the parent compound and its metabolites. Validation of the cell lines used in biotransformation research is important, because polymorphisms in these cell lines could influence the enzyme activity or inducibility and therefore the outcome of the studies. Based on the data retrieved about the genetic polymorphisms, the investigated cell line models seem to be suitable to study general biotransformation. The Hep G2 and IGROV-1 cell lines complement one another in the investigation of the metabolic route of a drug, because the observed polymorphisms in the two cell lines are present in different metabolic enzymes. However, the effects of the genetic polymorphisms should be kept in mind when interpreting the data obtained from biotransformation studies in the Hep G2 and IGROV-1 cell lines, because of the lack of or reduced activity of some metabolic routes. To investigate the effect of different polymorphisms on the biotransformation of a drug, a panel of genetically characterized and validated cell lines should be used.


131

In conclusion, the results indicate that the Hep G2 cell line is a good model to study biotransformation, because no common Caucasian CYP polymorphisms that will influence its capacity to metabolize compounds were found. However, the Hep G2 cell line is a less suitable model for SULT conjugation reactions, because of the *2 polymorphisms in SULT1A1 and 1A2. The IGROV-1 cell line is not an appropriate model to study biotransformation by the CYP2C9, UGT1A1, GSTM1, and GSTP1 enzymes, but is probably an useful model to study metabolism by other enzymes. Depending on the metabolic route of a compound, the polymorphisms in the enzymes should be kept in mind when interpreting the data obtained from biotransformation studies in the Hep G2 and IGROV-1 cell lines. Acknowledgment This work was supported with a grant from the Nijbakker-Morra Foundation, The Netherlands.

Chapter 7

132

References 1. Derelanko M.J. and Hollinger M.A. (1995). Handbook of toxicology. CRC press

(West Palm Beach, USA): 539-579. 2. Crommentuyn K.M.L., Schellens J.H.M., van den Berg J.D., and Beijnen J.H. (1998).

In-vitro metabolism of anti-cancer drugs, methods and applications: Paclitaxel, docetaxel, tamoxifen and ifosfamide. Cancer Treat. Rev. 24: 345-366.

3. Lu F.C. (1996). Basic Toxicology – Fundamentals, Target Organs and Risk Assessment. Taylor and Francis (Washington DC, USA), 3rd editition: 27-39.

4. Wrighton S.A. and Stevens J.C. (1992). The human hepatic cytochromes P450 involved in drug metabolism. Crit. Rev. Toxicol. 22: 1-21.


6. Range H.P., Dale M.M., and Ritter J.M. (1996). Pharmacology. Churchill Livingstone (Edinburgh, UK), 3rd edition: 83.

7. Ritter J.K. (2000). Roles of glucuronidation and UDP-glucuronosyltransferase in xenobiotic bioactivation reactions. Chem. Biol. Interact. 129: 171-193.



10. Tukey R.H. and Strassburg C.P. (2000). Human UDP-glucuronosyltransferases: metabolism, expression, and disease. Annu. Rev. Pharmacol. Toxicol. 40: 581-616.

11. MacKenzie P.I., Miners J.O., and McKinnon R.A. (2000). Polymorphisms in UDP glucuronosyltransferase genes: functional consequences and clinical relvance. Clin. Chem. Lab. Med. 38: 889-892.

12. Glatt H., Boeing H., Engelke C.E.H., Ma L., Kuhlow A., Pabel U., Pomplun D., Teubner W., Meinl W. (2001). Human cytosolic sulphotransferases: genetics, characterization, toxicological aspects. Mutat. Res. 482: 27-40.

13. Coughtrie M.W.H. (2002). Sulfation through the looking glass – recent advances in sulfotransferase research for the curious. Pharmacogenomics J. 2: 297-308.

14. Pirmohamed M. and Park B.K. (2003). Cytochrome P450 enzyme polymorphisms and adverse drug reaction. Toxicology 192: 23-32.

15. Scordo M.G., Pengo V., Spina E., Dahl M.L., Gusella M., and Padrini R. (2002). Influence of CYP2C9 and CYP2C19 genetic polymorphisms on warfarin maintenance dose and metabolic clearance. Clin. Pharmacol. Ther. 72: 702-710.

16. Toffoli G., Cecchin E., Corona G., and Boiocchi M. (2003). Pharmacogenetics of irinotecan. Curr. Med. Chem. Anti-Canc. Agents 3: 225-237.

17. Sekine I. and Saijo N. (2001). Polymorphisms of metabolizing enzymes and transporter proteins involved in the clearance of anticancer agents. Ann. Oncol. 12: 1515-1525.

18. Knasmuller S., Parzefall W., Sanyal R., Ecker S., Schwab C., Uhl M., Mersch-Sundermann V., Williamson G., Hietsch G., Langer T., Darroudi F., and Natarajan A.T. (1998). Use of metabolically competent human hepatoma cells for the detection of mutagens and antimutagens. Mutat. Res. 402: 185-202.



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20. Wilkening S. and Bader A. (2003). Influence of culture time on the expression of drug-metabolizing enzymes in primary human hepatocytes and hepatoma cell line HepG2. J. Biochem. Molec. Toxicol. 17: 207-213.

21. Fardel O., Morel F., Ratanasanh D., Fautrel A., Beaune P., Guillouzo A. (1992). Expression of drug metabolizing enzymes in human HepG2 hepatoma cells. Cell. Molec Aspects Cirrhosis 1992; 216: 327-330.

22. Grant H., Duthie S.J., Gray, A.G. and Burke D. (1988). Mixed function oxidase and UDP-glucuronyltransferase activities in the human Hep G2 hepatoma cell line. Biochem. Pharmacol. 37: 4111-4116.

23. Ferretti A., D’Ascenzo S., Knijn A., Iorio E., Dolo V., Pavan A., and Podo F. (2002). Detection of polyol accumulation in a new ovarium carcinoma cell line, CABA I: a 1H NMR study. Br. J. Cancer 86: 1180-1187.

24. Perego P., Paolicchi A., Tongiani R., Pompella A., Tonarelli P., Carenni N., Romanelli S., and Zunino F. (1997). The cell-specific anti-proliferative effect of reduced glutathione is mediated by gamma-glutamyl transpeptidase-dependent extracellular pro-oxidant reactions. Int. J. Cancer 71: 246-250.

25. Boom R., Sol C.J.A., Salimans M.M., Jansen C.L., Wertheim-van Dillen P.M.E., and van der Noordaa J. (1991). Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28: 495-503.

26. Hayashi S.-I., Watanabe J., Nakachi K., and Kawajiri K. (1991). Genetic linkage of lung cancer-assocciated MSPI polymorphisms with amino acid replacement in the heme binding region of the human cytochrome P450IA1 gene. J. Biochem. 110: 407-411.

27. Silvestri L., Sonzogni L., De Silvestri A., Gritti C., Foti L., Zavaglia C., Leveri M., Cividini A., Mondelli M.U., Civardi E., and Silini E.M. (2003). CYP enzyme polymorphisms and susceptibility to HCV-related chronic liver disease and liver cancer. Int. J. Cancer 104: 310-317.

28. Cascorbi I., Brockmöller J., and Roots I. (1996). A C4887A polymorphism in exon 7 of human CYP1A1: population frequency, mutation linkages, and impact on lung cancer susceptibility. Cancer Res. 56: 4965-4969.

29. Sachse C., Bhambra U., Smith G., Lightfoot T.J., Barrett J.H., Scollay J., Garner C., Boobis A.R., Wolf C.R., Gooderham N.J. and the colorectal cancer study group (2003). Polymorphisms in the cytochrome P450 CYP1A2 gene (CYP1A2) in colorectal cancer patients and controls: allele frequencies, linkage disequilibrium and influence on caffeine metabolism. Br. J. Clin. Pharmacol. 55: 68-76.

30. Nakajima M., Yokoi T., Mizutani M., Kinoshita M., Funayama M., and Kamataki T. (1999). Genetic polymorphism in the 5’-flanking region of human CYP1A2 gene: effect on the CYP1A2 inducibility in humans. J. Biochem. 125: 803-808.

31. Chida M., Yokoi T., Fukui T., Kinoshita M., Yokota J., and Kamataki T. (1999). Detection of three genetic polymorphisms in the 5’-flanking region and intron 1 of human CYP1A2 in the Japanese population. Jpn. J. Cancer Res. 90: 899-902.

32. Aklillu E., Carrillo J.A., Makonnen E., Hellman K., Pitarque M., Bertilsson L., and Ingelman-Sunberg M. (2003). Genetic polymorphism of CYP1A2 in Ethiopians affecting induction and expression: characterization of novel haplotypes with single-nucleotide polymorphisms in intron 1. Mol. Pharmacol. 64: 659-669.

33. Paschke T., Riefler M., Schuler-Metz A., Wolz L., Schere G., McBride C.M., Bepler G. (2001). Comparison of cytochrome P450 2A6 polymorphism frequencies in Caucasians and African-Americans using a new one-step PCR-RFLP genotyping method. Toxicology 168: 259-268.

Chapter 7

134

34. Yoshida R., Nakajima M., Nishimura K., Tokudome S., Kwon J.-T., and Yokoi T. (2003). Effect of polymorphism in promoter region of human CYP2A6 gene (CYP2A6*9) on expression level of messenger ribonucleic acid and enzymatic activity in vivo and in vitro. Clin. Pharmacol. Ther. 74: 69-76.

35. Lang T., Klein K., Fisher J., Nüssler A.K., Neuhaus P., Hofmann U., Eichelbaum M., Schwab M., and Zanger U.M. (2001). Extensive genetic polymorphism in the human CYP2B6 gene with impact on expression and function in human liver. Pharmacogenetics 11: 399-415.

36. Bahadur N., Leathart J.B.S., Mutch E., Steimel-Crespi D., Dunn S.A., Gilissen R., van Houdt J., Hendrickx J., Mannens G., Bohets H., Williams F.M., Armstrong M., Crespi C.L., and Daly A.K. (2002). CYP2C8 polymorphisms in Caucasians and their relationship with paclitaxel 6α-hydroxylase activity in human liver microsomes. Biochem. Pharmacol. 64: 1579-1589.

37. Sullivan-Klose T.H., Ghanayem B.I., Bell D.A., Zhang Z.Y., Kaminsky L.S., Shenfield G.M., Miners J.O., Birket D.J., and Goldstein J.A. (1996). The role of the CYP2C9-Leu359 allelic variant in the tolbutamide polymorphism. Pharmacogenetics 6: 341-349.

38. Goldstein J.A. and Blaisdell J. (1996). Genetic tests which identify the principal defects in CYP2C19 responsible for the polymorphism in mephenytoin metabolism. Methods Enzymol. 272: 210-218.

39. Schur B.C., Bjerke J., Nuwayhid N., and Wong S.H. (2001). Genotyping of cytochrome P450 2D6*3 and *4 mutations using conventional PCR. Clin. Chim. Acta 308: 25-31.

40. Wu X., Shi H., Jiang H., Kemp B., Hong W.K., Delclos G.L., and Spitz M.R. (1997). Associations between cytochrome P4502E1 genotype, mutagen sensitivity, cigarette smoking and susceptibility to lung cancer. Carcinogenesis 18: 967-973.

41. Kato S., Shields P.G., Caporaso N.E., Sugimura H., Trivers G.E., Tucker M.A., Trump B.F., Weston A., and Harris C.C. (1994). Analysis of cytochrome P450 2E1 genetic polymorphisms in relation to human lung cancer. Cancer Epidemiol. Biomark. Prev. 3: 515-518.

42. Sata F., Sapone A., Elizondo G., Stocker P., Miller V.P., Zheng W., Raunio H., Crespi C.L., and Gonzalez F.J. (2000). CYP3A4 allelic variants with amino acid substitutions in exons 7 and 12: Evidence for an allelic variant with altered catalytic activity. Clin. Pharmacol. Ther. 67: 48-56.

43. van Schaik R.H., de Wildt S.N., Brosens R., van Fessem H., van den Anker J.N., and Lindemans J. (2001). The CYP3A4*3 allele: is it really rare? Clin. Chem. 47: 1104-1106.

44. van Schaik R.H.N., van der Heiden I.P., van den Anker J.N., and Lindemans J. (2002). CYP3A5 variant allele frequencies in Dutch Caucacians. Clin. Chem. 48: 1668-1671.

45. MacLeod S.L., Nowell S., Plaxco J., and Lang N.P. (2000). An allele-specific polymerase chain reaction method of the D85Y polymorphism in the human UDP-glucuronosyltransferase 2B15 gene in a case-control study of prostate cancer. Ann. Surg. Oncol. 7: 777-782.

46. Peng C. T., Chen J.C., Yeh K.T., Wang Y.F., Hou M.F., Lee T.P., Shih M.C., Chang J.Y., and Chang J.G. (2003). The relationship among the polymorphisms of SULT1A1, 1A2, and different types of cancer in Taiwanese. Int. J. Molec. Med. 11: 85-89.

47. Sreelekha T.T., Ramadas K., Pandey M., Thomas G., Nalinakumari K.R., and Pillai M.R. (2001). Genetic polymorphism of CYP1A1, GSTM1 and GSTT1 genes in Indian oral cancer. Oral Oncol. 37: 593-598.

48. Jerónimo C., Vazim G., Henrique R., Oliveira J., Bento M.J., Silva C., Lopes C., and Sidransky D. (2002). I105V polymorphism and promoter methylation of the GSTP1 gene in prostate adenocarcinoma. Cancer Epidemiol. Biomark. Prev. 11: 445-450.


135

49. Loktionov A., Moore W., Spencer S.P., Vorster H., Nell T., O’Neill I.K., Bingham S.A., and Cummings J.H. (2002). Differences in N-acetylation genotypes between Caucasians and Black South Africans: implications for cancer prevention. Cancer Detec. Prev. 26: 15-22.

50. Duijnhoven F.J.B., van der Hei O.L., van der Luijt R.B., Bueno de Mesquita B., van Noord P.A.H., and Peeters P.H.M. (2002). Quality of NAT2 genotyping with restriction fragment length polymorphism using DNA isolated from frozen urine. Cancer Epidemiol. Biomark. Prev. 11: 771-776.

51. Human Cytochrome P450 (CYP) Allele Nomenclature Committee homepage. http://www.imm.ki.se/CYPalleles (accessed January 2004).

52. Arylamine N-Acetyltransferase (NAT) Nomenclature homepage. http://www.louisville.edu/medschool/pharmacology/NAT.html (accessed January 2004).

53. UDP Glucuronosyltransferase homepage http://som.flinders.edu.au/FUSA/ClinPharm/UGT (accessed January 2004).

54. Takahashi H., Wilkinson G.R., Caraco Y., Muszkat M., Kim R.B., Kashima T., Kimura S., and Echizen H. (2003). Population differences in S-warfarin metabolism between CYP2C9 genotype-matched Caucasian and Japanese patients. Clin. Pharmacol. Ther. 73: 253-263.

55. Glatt H. and Meinl W. (2004). Pharmacogenetics of soluble sulfotransferases (SULTs). Naunyn Schmiedebergs Arch. Pharmacol. 369: 55-68.

56. Bosma P.J., Roy Chowdhury J., Bakker C., Gantla S., de Boer A., Oostra B.A., Lindhout D., Tytgat G.N.J., Jansen P.L.M., Oude Elferink R.P.J., and Roy Chowdhury N. (1995). The genetic basis of the reduced expression of bilirubin UDP-glucuronosyltransferase 1 in Gilbert's syndrome. N. Engl. J. Med. 333: 1171-1175.

57. Cascorbi I., Brockmoller J., Mrozikiewicz P.M., Muller A., and Roots I. (1999). Arylamine N-acetyltransferase activity in man. Drug Metab. Rev. 31: 489-502.

58. Rueff J., Chiapella C., Chipman J.K., Darroudi F., Silva I.D., Duverger-van Bogaert M., Fonti E., Glatt H.R., Isern P., Laires A., Leonard A., Llagostera M., Mossesso P., Natarajan A.T. , Palitti F., Rodrigues A.S., Schinoppi A., Turchi G., and Werle-Schneider G. (1996). Development and validation of alternative metabolic systems for mutagenicity testing in short-term assays. Mutat. Res. 353: 151-76.

59. Coroneos E., Gordon J.W., Kelly S.L., Wang P.D., and Sim E. (1991). Drug metabolising N-acetyltransferase activity in human cell lines. Biochim. Biophys. Acta 1073: 593-599.

60. Coroneos E. and Sim E. (1993). Arylamine N-acetyltransferase activity in human cultured cell lines. Biochem. J. 294 (Pt 2): 481-486.

61. Honma W., Shimada M., Sasano H., Ozawa S., Miyata M., Nagata K., Ikeda T., and Yamazoe Y. (2002). Phenol sulfotransferase, ST1A3, as the main enzyme catalyzing sulfation of troglitazone in human liver. Drug Metab. Dispos. 30: 944-949.

62. Suiko M., Sakakibara Y., and Liu M.C. (2000). Sulfation of environmental estrogen-like chemicals by human cytosolic sulfotransferases. Biochem. Biophys. Res. Commun. 267: 80-84.

63. Chun H.S., Kim H.J., and Choi E.H. (2001). Modulation of cytochrome P4501-mediated bioactivation of benzo[a]pyrene by volatile allyl sulfides in human hepatoma cells. Biosci. Biotechnol. Biochem. 65: 2205-2212.

64. Parker R.S., Sontag T.J., and Swanson J.E. (2000). Cytochrome P4503A-dependent metabolism of tocopherols and inhibition by sesamin. Biochem. Biophys. Res. Commun. 277: 531-534.

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General discussion and conclusions

Chances for patients with advanced cancer are still poor and most anti-cancer drugs show serious side effects when applied at clinically relevant dose-levels. Therefore, new anti-cancer drugs are still desired to improve the treatment of cancer patients and reduce the side effects of the current therapies. Many of the registered anti-cancer drugs originate from natural resources and 50 years ago investigation of marine organisms for the discovery of novel anti-cancer drugs was initiated. In this thesis, three novel marine anti-cancer drugs were investigated, namely thiocoraline, aplidine, and ET-743. At this moment these agents are used in preclinical, phase II clinical, and phase II clinical studies, respectively. The aim was to elucidate the human biotransformation pathways involved in the metabolism of these anti-cancer drugs using in vitro techniques. Biotransformation is important for a better excretion of a lipophilic drug from the body and, therefore, plays an important role in the pharmacokinetics of the drug. Determination of the enzymes involved in the biotransformation of drugs may help to improve individual therapies and a prediction of side effects may be possible when anti-cancer drugs are combined with other drugs or food components that influence the biotransformation of the drugs. Furthermore, it helps to interpret pharmacokinetic and toxicity data already obtained from clinical trials.

The phase I and II enzymes involved in the biotransformation pathways of thiocoraline, aplidine, and ET-743 were identified using different in vitro techniques. The involvement of non-specific esterases and amidases (phase I enzymes), which may be involved in hydrolysis of the anti-cancer drugs, was studied in human plasma by determining the half-life of a drug in human plasma and comparing it with the half-life in phosphate buffered saline [1]. The influence of carboxyl and cholesterol esterases on the biotransformation was determined by investigating the effect of specific esterase inhibitors on the half-lifes of drugs in human plasma. Pooled human liver fractions (microsomes, cytosol and S9 fraction) are widely used to characterize the metabolic profiles of novel pharmaceutical compounds [2]. Microsomes were used to obtain information on the biotransformation of the marine compounds by cytochrome P450 (CYP) enzymes and the phase II enzyme uridine diphosphoglucuronosyl transferase (UGT). Furthermore, gender specific microsomes were used to examine gender-related differences in CYP mediated metabolism of the compounds [3]. The involvement of the soluble human phase II enzymes (glutathione-S-transferase (GST) and sulfotransferase (SULT)) was studied in human liver cytosol by adding specific cofactors needed for an enzyme to be active. Human liver S9 fraction was used to study the combination of phase I and II biotransformation. However, in cytosol and S9 fraction the metabolism of the anti-cancer drugs by NAT could not be studied because of the degradation of these compounds in the presence of NAT cofactors. The involvement of various isoforms of CYP and UGT on the metabolism was investigated using CYP and UGT supersomes, which are also valuable tools to identify the corresponding metabolites. Only the role of UGT isozymes 1A1, 1A3, 1A9, and 2B15 on the glucuronidation was investigated, because these UGT isozymes are involved in the conjugation of large molecules. To elucidate the involvement of different CYP isozymes to the total biotransformation in microsomes, the effect of different CYP inhibitors on the biotransformation percentages of the anti-cancer drugs was studied in human liver microsomes. Furthermore, CYP supersomes were used to determine the enzyme kinetics of ET-743, from which the CYP reaction phenotype was calculated. The CYP reaction phenotype allows the assessment of the relative contribution of the CYP isoforms to the metabolic pathways [4, 5].


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Finally, the biotransformation of thiocoraline, aplidine, and ET-743, was investigated in the human cancer cell lines Hep G2 and IGROV-1 by determining the effects of different enzyme inhibitors on the cytotoxicity of these compounds. Furthermore, the cytotoxicities of metabolites could also be studied with these cell lines and compared to the cytotoxicities of their parent compounds. Freshly isolated human hepatocytes are also used in biotransformation studies. However, they are difficult to obtain and, furthermore, the high inter-individual variability and a gradual loss of liver specific functions during cultivation, with special reference to a decreased CYP expression, are great disadvantages of this in vitro model [2]. The human hepatoma cell line Hep G2 is the most frequently used and best-characterized human hepatoma cell line [2]. This cell line has a variety of liver specific metabolic functions and under standard culturing conditions, the cells have low levels of CYP and phase II enzymes, inducible by pretreatment with inducing agents [2, 6, 7]. Furthermore, the activities of drug-metabolizing enzymes were highly variable between the different passages, which can result in different metabolic rates between the different passages [8]. Genotyping the genes coding for CYP and phase II enzymes in the Hep G2, showed that the Hep G2 cell line has no common Caucasian CYP polymorphisms that may influence its capacity to metabolize drugs except for the common Caucasian CYP3A5*3 polymorphism. The incidence of this polymorphism is more than 90% in the Caucasian population and results in absence of CYP3A5 activity. Furthermore, SULT1A1*2 and 1A2*2 polymorphisms were identified, which lead to a decreased activity compared to the wild type, indicating that the cell line is not a suitable model to study SULT conjugation reactions. The Hep G2 cell line was a suitable model to study the effect of phase I and II enzyme inhibitors and inducers on the cytotoxicity of the marine compounds, although, the low expression levels might result in the lack of effect of some inhibitors, because these enzymes are not significantly involved in the (de)toxification of the compounds.

The IGROV-1 cell line was used in this thesis as a model to study the effect of different phase I enzyme inhibitors on the cytotoxicity of thiocoraline, aplidine, and ET-743. However, the IGROV-1 cell line has been less extensively investigated for biotransformation research purposes. It is used by the National Cancer Institute in its anti-cancer screening program and is used by other research groups to generate drug resistant cell lines [9]. Genotyping of the IGROV-1 cell line revealed that the cells have the CYP1A2*1B, 2C9*3, and 3A5*3 polymorphisms, leading to decreased CYP2C9 activity and no CYP3A5 activity. Thus far, the influence of the CYP1A2*1B polymorphism on the activity of the enzyme is unknown. Furthermore, the cells were shown to have the GSTM1 gene deleted, resulting in no GSTM1 activity, and the *B polymorphism in the GSTP1 gene, leading to a decreased activity. Furthermore, the *28 polymorphism was identified in the UGT1A1 gene, resulting in a decreased activity. No other common Caucasian CYP and phase II enzyme polymorphisms were observed. The polymorphisms identified indicate that the IGROV-1 cell line is a less suitable model to study glutathione conjugation because of the reduced GST activity. It is also a less suitable model to study CYP2C9 metabolism. These results indicate that the IGROV-1 cell line may be used as a model to study the biotransformation of a drugs, but further validation by activity measurements should be conducted.


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Thiocoraline

The enzymes in human plasma significantly decreased the half-life of thiocoraline, which could not be influenced by esterase inhibitors. Thiocoraline was metabolized by the CYPs present in human liver microsomes, but not directly by phase II enzymes. The metabolites formed by cytochrome P450 could be conjugated by the phase II enzymes UGT, SULT, and GST in the human liver S9 fraction. CYP2C8 and 3A4 supersomes did show significant metabolization of thiocoraline, but the inhibitors of these CYPs had no effect on the cytotoxicity of thiocoraline in the Hep G2 and the IGROV-1 cell lines. The lack of effect of the different CYP3A4 inhibitors may be caused by incomplete inhibition of the isozyme(s) due to the practical limitation that the inhibitor concentration used had to be below the IC5 value to prevent a direct effect of the inhibitors on the viability of the cells. However, the most likely cause is the high variation in CYP3A4 expression levels resulting in high standard deviations in the IC50 value between the different passages. Aplidine

Enzymes present in human plasma significantly metabolized aplidine and its half-life could be significantly increased in the presence of a carboxyl esterase inhibitor. This indicates that carboxyl esterases are partly responsible for the clearance of aplidine from human plasma. CYP2A6, 2E1, 3A4, and 4A11 were also involved in the biotransformation of aplidine. A significantly lower (p < 0.05) percentage of biotransformation was observed for aplidine by male human liver microsomes compared to mixed gender and female microsomes, indicating a gender-related difference in biotransformation of aplidine. Aplidine was significantly conjugated by UGT1A3 and 1A9, but not by the other phase II enzymes. However, the phase II enzymes UGT, SULT and GST significantly conjugated the aplidine metabolites formed by CYPs in human liver S9 fraction. After microsomal and supersomal incubations four metabolites were identified for aplidine, one was eluted chromatographically as two conformers of one another, analogous to aplidine. The aplidine metabolites were identified by off-line MSMS as: apli-da (aplidine dealkylated at the (R)-N(methyl)-leucine group), apli-h (aplidine hydroxylated at the isopropyl-group), and apli-da/h (aplidine dealkylated at the (R)-N(methyl)-leucine and hydroxylated at the isopropyl group), which all three were specifically formed by CYP3A4 supersomes, and apli-dm (aplidine demethylated at the C-atom in the threonine group), formed only after incubation of aplidine with CYP2A6 supersomes. No metabolites were observed after incubation with CYP2E1 and 4A11 supersomes. Inhibitor studies in human liver microsomes showed that CYP3A4 was the main CYP isozyme responsible for the biotransformation of aplidine, but the other inhibitors tested also significantly decreased the aplidine biotransformation by pooled human liver microsomes. A significant decrease in IC50 value in the presence of the CYP2A6, 2E1, 3A4, and 4A11 inhibitors was observed in the Hep G2 and IGROV-1 cell lines. Ritonavir (CYP3A4) showed the highest decrease in IC50 value of aplidine. Furthermore, in the IGROV-1 cell line also ketoconazole (CYP1A1, 2A6, 2C8, 2C19, 2D6, and 3A4) significantly decreased the IC50 value of aplidine. The lack of effect of the carboxyl esterase inhibitor indicates that these enzymes are not significantly involved in the biotransformation of aplidine in Hep G2 and IGROV-1 cells.


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ET-743

CYP2C9, 2C19, 2D6, 2E1, and 3A4 showed to be involved in the in vitro metabolism of ET-743. Male human liver microsomes showed a significantly lower (p < 0.05) percentage of ET-743 biotransformation compared to mixed gender and female microsomes, indicating that there is a gender-related difference in biotransformation of ET-743. Significant conjugation was observed for ET-743 mediated by UGT2B15 and GST. ET-743 was significantly metabolized by CYP, UGT, and GST in the human liver S9 fraction and CYP activity in combination with the individual phase II enzymes UGT and GST resulted in a further reduction of ET-743. ET-743 biotransformation by pooled human liver microsomes could be significantly decreased with CYP2D6, 2E1, and 3A4 inhibitors, but not with CYP2C9 and 2C19 inhibitors. The CYP reaction phenotype was used to determine the contribution of each CYP involved in the biotransformation of ET-743 at therapeutic concentrations, which lead to the following order 3A4 >> 2C9 > 2E1 > 2D6 > 2C19. Furthermore, some CYP2A6, 2C9, 2C19, 2E1, and 3A4 inhibitors significantly increased the cytotoxicity of ET-743 in the Hep G2 cell line. No effect on the cytotoxicity of phase II enzyme inhibitors could be observed in the Hep G2 cell line, indicating that CYP metabolism may be the rate limiting step in the detoxicification of ET-743. In addition, up-regulation of CYP3A4 did not result in a significant change in the IC50 value of ET-743 in the Hep G2 cell line. Based upon the enzyme kinetic data, it is expected that the half-life of ET-743 in Hep G2 cells is very short compared to the incubation time in the present experiments, leading to a short exposure time compared to its less toxic metabolites. Shortening the exposure time to the parent compound even further by induction of CYP3A4 may therefore not result in a measurable decrease of the total toxicity during the whole duration of the experiment. Adding inhibitors of metabolism will most likely increase the exposure time to ET-743 by a significant factor, increasing its contribution to the cytotoxicity. This was confirmed in the CYP inhibition experiments. The IGROV-1 cell line was not used to study the biotransformation of ET-743 because significant information on the metabolism already could be obtained with the Hep G2 cell line. Discussion

All the results indicate that CYP3A4 is the main enzyme involved in the biotransformation of thiocoraline, aplidine, and ET-743. CYP3A4 is, together with CYP2C9, the pre-dominant cytochrome P450 isozyme in the human liver and is responsible for approximately 50% of all biotransformation reactions in the liver [15-17]. The individual CYP3A4 enzyme activity is influenced by gender, genetic polymorphisms, food components, drugs, aging, and disease, leading to high inter-individual variances in activity [18-20]. Gender specific human liver microsomes showed for aplidine and ET-743 that gender could play a role in the biotransformation in patients. However, these gender differences are not always of clinical importance due to the high within-gender differences [18, 19]. The influence of this high inter-individual variance in CYP3A4 activity on the biotransformation is supported by the fact that thus far no gender differences have been described for patients treated with aplidine and ET-743. The genetic factors that can cause the inter-individual variability in CYP3A4 activity are unknown, but are estimated to be high [21]. Several polymorphisms known for CYP3A4, but thus far a decrease in CYP3A4 activity was observed only for the *2 polymorphism.


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CYP3A5 was not tested for its influence on the biotransformation of thiocoraline, aplidine, and ET-743, because it is only functional in about 10% of the Caucasian population. However, CYP3A5 has the same substrate specificity as CYP3A4 and is present in higher amounts than 3A4 [22]. Therefore, it is most likely that thiocoraline, aplidine, and ET-743 are also metabolized by CYP3A5 when it is functional, leading to increased metabolic rates and most likely also increased clearance. However, clinical consequences are currently unknown. Genotyping patients for CYP3A4 may contribute to the safety of patients treated with thiocoraline, aplidine, and ET-743 and genotyping for CYP3A5 to the therapeutic efficacy of the patients treated with these compounds.

Food components are known to decrease or increase CYP3A4 activity, e.g. grapefruit juice inhibits CYP3A and St. John’s wort induces CYP3A [23]. Thus, consumption of CYP3A4 significant inhibiting and inducing food components may needed to be monitored during the therapy with thiocoraline, aplidine, and ET-743. There is also a risk of in vivo drug-drug interactions when thiocoraline, aplidine, and ET-743 are combined with other drugs that influence the CYP3A4 activity [24, 25]. Most of the applied drugs in anti-cancer combination therapy are CYP3A4 inhibitors (e.g. tamoxifen), inducers (e.g. cisplatin, doxorubicin, and vinblastine), or both (e.g. paclitaxel, docetaxel, and dexamethasone). Therefore, in clinical trials with combination therapies with thiocoraline, aplidine, or ET-743, attention should be paid to the final effect: hepatic toxicity may result from increased plasma levels or a possible reduced efficacy of the therapy when plasma levels decrease. Conclusions

Thiocoraline, aplidine, and ET-743 metabolisms in vitro are mainly catalyzed by CYP3A4. For aplidine and ET-743 some other CYPs were found to play a minor role in the applied test systems. These findings can help to interpret the pharmacokinetic data obtained from the clinical trials with these anti-cancer drugs in patients and predictions may be made from these results regarding side effects when these anti-cancer drugs are combined with other drugs or food components that influence CYP3A4 activity. Overall, results of studies obtained in this thesis may help to optimize clinical development of the studied novel and promising anti-cancer drugs thiocoraline, aplidine, and ET-743. Future recommendations

Information has been obtained regarding the biotransformation of thiocoraline, aplidine, and ET-743, but still several issues remain to be elucidated in future studies. First, the effect of CYP3A5 on the cytotoxicity and clearance of thiocoraline, aplidine, and ET-743 is not yet known. CYP3A5*3 is a common genetic polymorphisms, but in patients without this polymorphism CYP3A5 could influence the therapeutic efficacy. Thus, it is important to study the effect of wild type CYP3A5 with, for example, CYP3A5 supersomes and other cell lines than the Hep G2 and IGROV-1 cell lines, both lacking CYP3A5 activity.

Second, no effect of CYP3A4 induction on the cytotoxicity of ET-743 was observed. However, in vivo experiments indicate that hepatotoxicity of ET-743 is decreased and clearance is increased after pretreatment with dexamethasone, a CYP3A4 inducer [26, 27]. Therefore, it is important to investigate the underlying mechanisms in vitro. Another experimental set-up should be used, where cytotoxicity can be measured after a shorter time and with other, possibly more sensitive, assays. The effect of CYP induction on the cytotoxicity of thiocoraline and aplidine remains also to be studied.


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Third, drug transporters are known to influence the internal cell concentration of a drug and the transport from the blood to the tumor. The metabolic rate of a drug in the liver can be influenced by drug transporters. Thus far, no studies were performed to find the drug transporters involved in the transport of thiocoraline and aplidine. ET-743 is not transported by P-glycoprotein and multidrug resistance protein 1, but thus far no transporters have been identified being involved with the transport of ET-743 [28]. Therefore, discovery of the drug transporters involved in the transport of thiocoraline, aplidine, and ET-743 could lead to a better understanding of the pharmacokinetic data obtained in trials.

Fourth, to obtain more information on drug interactions that may occur with thiocoraline, aplidine, and ET-743 in combination therapy, future in vitro studies should be performed with human liver fractions, CYP supersomes, and human cell lines. The CYP reaction phenotype of thiocoraline and aplidine should be determined to assess the contribution of each CYP isozyme to the biotransformation. Furthermore, by co-incubation of thiocoraline, aplidine, and ET-743 with other drugs used in cancer therapy in human liver fractions and CYP supersomes, the effect on the biotransformation can be studied. The inhibition and induction effects on the cytotoxicity of the combination of drugs can be studied in cell lines. Most of the current drugs in anti-cancer therapy are CYP3A4 inhibitors, inducers, or both and in combination therapy this could lead to drug-drug interactions. ET-743 was shown to be a transcription interfering agent and an antagonist of the steroid and xenobiotic receptor (SXR), inhibiting the induction of several cytochrome P450s, phase II enzymes, and drug transporters [29-34]. However, only activated transcription is inhibited and not the constitutive transcription [32, 35, 36]. For thiocoraline and aplidine no studies have been performed to examine their inhibiting or inducing effect on CYP, phase II enzymes, and drug transporter activity via SXR or other mechanisms. Therefore, it is important to study the effect of thiocoraline and aplidine on the CYP3A4 activity before combination therapy is started.

Fifth, in addition to the four in vitro metabolites discovered for aplidine formed by CYP3A4 and CYP2A6, no metabolites have been observed for CYP2E1 and 4A11 so far. Therefore, identification of the metabolites formed by these CYP isozymes may be of interest for future research. In clinical trials, patients could be screened for the formation of these metabolites. Furthermore, possible metabolites of thiocoraline and ET-743 were observed, which could not be further identified. The thiocoraline metabolite concentrations were too low for correct isolation for off-line MS analysis using the current chromatographic assay. For ET-743 this was due to the impurity of the supplied raw material (the impurity was mild (< 1%), but resulted in intervening peaks in the chromatogram). In future studies, HPLC with online MS analysis should be used for the identification of the in vitro thiocoraline and ET-743 metabolites. The identification of the in vitro metabolites may help to identify metabolites in patients.

Finally, the different in vitro techniques used in this thesis were found to be useful tools for the identification of the enzymes involved in the biotransformation of novel compounds. Each in vitro technique has its own advantages and disadvantages, but together they are powerful tools in the prediction of the enzymes involved in the in vivo biotransformation of a drug. Furthermore, the contribution of each isozyme to the biotransformation at different drug concentrations can be determined and also drug-drug interactions can be predicted. Therefore, these in vitro techniques can be used in the future to elucidate the biotransformation pathways of other novel compounds.


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References 1. Lu F.C. (1996). Toxicology - Fundamentals, Target Organs and Risk Assessment. Taylor

and Francis (Washington DC, USA), 3rd edition: 3-39. 2. Brandon E.F.A., Raap C.D., Meijerman I., Beijnen J.H., and Schellens J.H.M. (2003). An

update on in vitro test methods in human hepatic drug biotransformation research: pros and cons. Toxicol. Appl. Pharmacol. 189: 233-246.

3. Gentest, a BD Biosciences Company. http://www.gentest.com/ (accessed March 2003). 4. Lu A.Y.H., Wang R.W., and Lin J.H. (2003). Cytochrome P450 in vitro reaction

phenotyping: a re-evaluattion of approaches used for P450 isofrom identification. Drug Metab. Dispos. 31: 345-350.

5. Rodrigues A.D. (1999). Integrated cytochrome P450 reaction phenotyping. Attempting to bridge the gap between cDNA-expressed cytochromes P450 and native human liver microsomes. Biochem. Pharmacol. 57: 465-480.


7. Fardel O., Morel F., Ratanasanh D., Fautrel A., Beaune P., and Guillouzo A. (1992). Expression of drug metabolizing enzymes in human HepG2 hepatoma cells. Cell. Molec. Aspects Cirrhosis 216: 327-330.

8. Wilkening S. and Bader A. (2003). Influence of culture time on the expression of drug-metabolizing enzymes in primary human hepatocytes and hepatoma cell line HepG2. J Biochem Molec Toxicol 17: 207-213.

9. Righetti S.C., Perego P., Corna E., Pierotti M.A., and Zunino F. (1999). Emergence of p53 mutant cisplatin-resistant ovarian carcinoma cells following drug exposure: spontaneously mutant selection. Cell Growth Differ. 10: 473-478.


11. Wu L.T., Chung J.G., Chen J.C., and Tsauer W. (2001). Effect of norcantharidin on N-acetyltransferase activity in HepG2 cells. Am. J. Chin. Med. 29: 161-172.


13. O’Leary K.A., Day A.J., Needs P.W., Mellon F.A., O’Brien N.M., and Williamson G. (2003). Metabolism of quercitin-7- and quercitin-3-glucuronides by in vitro hepatic model: the role of human β-glucuronidase, sulfotransferase, catechol-O-methyltransferase and multi-resistant protein 2 (MRP2) in flavonoid metabolism. Biochem. Pharamacol. 65: 479-491.

14. Zhang K., Chew M., Yang E.B., Wong K.P., and Mack P. (2001). Modulation of cisplatin cytotoxicity and cisplatin-induced DNA cross-links in HepG2 cells by regulation of glutathione-related mechanisms. Molec. Pharmacol. 59: 837-843.

15. Whrighton S.A. and Stevens J.C. (1992). The human hepatic cytochromes P450 involved in drug metabolism. Crit. Rev. Toxicol. 22: 1-21.


17. Wilkinson G.R. (1996). Cytochrome P4503A (CYP3A) metabolism: prediction of in vivo activity in humans. J. Pharmacokinet. Biopharm. 24: 475-490.


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20. MacLeod S.L., Nowell S., Massengill J., Jazieh A., McClure G., Plaxco J., Kadlubar F.F., and Lang N.P. (2000). Cancer therapy and polymorphisms of cytochrome P450. Clin. Chem. Lab. Med. 38: 883-887.


22. Kuehl P., Zhang J., Lin Y., Lamba J., Assem M., Schuetz J., Watkins P.B., Daly A., Wrighton S.A., Hall S.D., Maurel P., Relling M., Brimer C., Yashuda K., Venkataramanan R., Strom S., Thummel K., Boguski M.S., and Schuetz E. (2001). Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression. Nat. Genet. 27: 383-391.

23. Drug interaction table. http://medicine.iupui.edu/flockhart/table.htm (last accessed January 2004).



26. Donald S., Verschoyle R.D., Greaves P., Gant T.W., Colombo T., Zaffaroni M., Frapolli R., Zucchetti M., D’Incalci M., Meco D., Riccardi R., Lopez-Lazaro L., Jimeno J., and Gescher A.J. (2003). Complete protection by high-dose dexamethasone against the hepatotoxicity of the novel antitumor drug yondelis (ET-743) in the rat. Cancer Res. 63: 5902-5908.

27. Puchalski T.A., Ryan D.P., Garcia-Carbonero R., Demetri G.D., Butkiewicz L., Harmon D., Seiden M.V., Maki R.G., López-Lázaro L., Jimeno J., Guzman C., and Supko J.G. (2002). Pharmacokinetics of ecteinascidin 743 administered as a 24-h continuous intravenous infusion to adult patients with soft tissue sarcomas: associations with clinical characteristics, pathophysiological variables and toxicity. Cancer Chemother. Pharmacol. 50: 309-319.

28. Poindessous V., Koeppel F., Raymond E., Comisso M., Waters S.J., Larsen A.K. (2003). Marked activity of irofulven toward human carcinoma cells: comparison with cisplatin and ecteinascidin. Clin. Cancer Res. 9: 2817-25.



31. Takebayashi Y., Pourquier P., Zimonjic D.B., Nakayama K., Emmert S., Ueda T., Urasaki Y., Kanzaki A., Akiyama S.-L., Popescu N., Kreamer K.H., and Pommier Y. (2001). Antiproliferative activity of ecteinascidin 743 is dependent upon transcription-coupled nucleotide-excision repair. Nat. Med. 7: 961-966.


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33. Donald S., Verschoyle R.D., Edwards R., Judah D.J., Davies R., Riley J., Dinsdale D., Lopez-Lazaro L., Smith A.G., Gant T.W., Greaves P., and Gescher A.J. (2002). Hepatobiliary damage and changes in hepatic gene expression caused by the antitumor drug ecteinascidin-743 (ET-743) in the female rat. Cancer Res. 62: 4256-4262.


35. Synold T.W., Dussault L., and Forman B.M. (2001). The orphan nuclear receptor SXR coordinately regulates drug metabolism and efflux. Nat. Med. 7: 584-590.

36. Sparfel L., Payen L., Gilot D., Sidaway J., Morel F., Guillouzo A., and Fardel O. (2003). Pregnane X receptor-dependent and –independent effects of 2-acetylaminofluorene on cytochrome P450 3A23 expression and liver cell proliferation. Biochem. Biophys. Res. Commun. 300: 278-284.


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Summary

Cancer is the second cause of death in The Netherlands. Although the treatment options over the past few decades have substantially improved, the cure rate for patients with advanced cancer remains low. In addition, hopefully new therapies will induce less severe side effects compared to the present therapies. Overall, new anti-cancer drugs are still very much needed to improve treatment outcome of patients. Many active cytotoxic agents originate from natural resources, mainly plants (e.g. paclitaxel originates from the Taxus tree). The search for marine compounds started 50 years ago and new compounds isolated from various marine sources have been described as anti-cancer agent and some have already entered clinical studies.

In this thesis, three novel marine derived anti-cancer drugs were investigated, i.e. thiocoraline, aplidine, and ET-743. Thiocoraline is derived form the Micromonospora marina, which grows in the Mozambique strait. Aplidine originates from the Mediterranean tunicate Aplidium albicans (figures on thesis cover) and ET-743 from the Ecteinascidia turbinata from the Caribbean. All three anti-cancer drugs showed promising activity against various tumor types in preclinical studies. Aplidine and ET-743 have already entered clinical trials and thiocoraline will be investigated in patients in the near future.

Drugs are often converted in the body for better excretion mainly via the urine, feces, or both. The conversion process is called biotransformation or metabolism and its product a metabolite. The main organ involved is the liver. Biotransformation can be divided into two categories: phase I reactions and phase II reactions. An enzyme superfamily called cytochrome P450 (CYP) is the main enzyme system catalyzing phase I reactions. During phase II reactions (conjugation) a water-soluble group is attached to the compound or to its reaction product from a phase I reaction. The main phase II enzyme families are glutathion-S-transferase (GST), N-acetyltransferase (NAT), uridine diphosphoglucuronosyl transferase (UGT), and sulfotransferase (SULT). CYP and UGT enzymes show large inter-individual variability that is determined by gender, age, disease, food or drug intake, and genetic factors.

Little is known about the biotransformation of thiocoraline, aplidine, and ET-743 in the human body. Information on the enzymes involved and the knowledge of the patient enzyme activity may help to improve the treatment of the individual patient and reduction of side effects. Therefore, it is important to elucidate the involvement of the different enzymes with the biotransformation of the anti-cancer drugs thiocoraline, aplidine, and ET-743. The research, as described in this thesis, is focused on the elucidation of the human biotransformation pathways of these drugs. Furthermore, the toxicity of the metabolites was investigated to elucidate if bioactivation (metabolite more toxic than parent compound) or bioinactivation (metabolite less toxic than parent compound) occurred.

It is possible to study the biotransformation pathways and the toxicity of a compound and its metabolites using in vitro techniques. An overview of all the different in vitro techniques is described in Chapter 1 with their advantages and disadvantages. In this research project, several different in vitro techniques were used, including human liver fractions (different parts of grinded human liver) and special fractions of transfected insect cells containing a specific human enzyme, also called supersomes. Furthermore, human cancer cell lines were used. A cancer cell lines is derived from one cell, which keeps dividing continuously and the cells have the same characteristics. In this study, the cell lines Hep G2, a human liver cancer cell line, and IGROV-1, an ovarian cancer cell line, were used.

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The cytochrome P450 enzyme 3A4 was found to be very important in the biotransformation of thiocoraline (Chapter 2) and CYP2C8 was involved to a lesser extent. UGT1A1 and 1A9 were involved in the conjugation of thiocoraline. No involvement of other phase II enzymes was found. Furthermore, the metabolites formed from the CYP reaction were further conjugated by the phase II enzymes UGT, SULT, and GST.

Aplidine (Chapter 3 and 4) was mainly metabolized by CYP3A4, but also by CYP2A6, 2E1, and 4A11. Four metabolites were identified from which three were specifically formed by CYP3A4 and one by CYP2A6. Only the UGTs 1A3 and 1A9 were involved in the direct conjugation of aplidine. The metabolites formed by CYP were further conjugated by UGT, SULT, and GST. Cell culture experiments showed that the aplidine metabolites were less toxic compared to aplidine, thus aplidine was bioinactivated by these enzymes.

ET-743 (Chapter 5 and 6) was also mainly metabolized by CYP3A4 and a number of other CYPs played a minor role, namely CYP2C9, 2C19, 2D6, and 2E1. In addition, ET-743 was conjugated by GST and UGT. The metabolites formed by cytochrome P450 were less toxic compared to ET-743 itself in cell culture experiments, thus ET-743 was bioinactivated by the CYPs involved in its biotransformation.

Based on these results, the pharmacological data obtained from patients in clinical trials may be analyzed better and individual therapies may be improved. Furthermore, based on these results possible drug-drug interactions can be predicted For instance grapefruit juice decreases CYP3A4 activity, which may lead to lower metabolic rates and increased drug levels in blood. This could result in more pronounced side effects. However, a component like Saint John’s wort increases CYP3A4 activity and thiocoraline, aplidine, and ET-743 may be metabolized more rapidly during consumption of these roots. This could lead to a reduced therapeutic efficacy. Furthermore, a number of anti-cancer drugs are known to have a positive or negative influence on the CYP3A4 activity. A patient’s therapy could be improved based on these results and the risk of side effects may be decreased.

Finally, the genetic variations (polymorphisms) in the genes coding for CYPs and phase II enzymes were determined in both cell lines used in this research, the human Hep G2 and IGROV-1 cell lines (Chapter 7). These polymorphisms can lead to loss, decrease or increase in activity of these enzymes. The cell lines can be characterized using the genetic data and thus it can be determined if they are a useful in vitro tool to study biotransformation in these cell lines. The results showed that the Hep G2 cell line was a suitable in vitro model to study CYP biotransformation. However, the SULT activity may be lowered due to polymorphisms, thus making the Hep G2 cell line a less suitable model to study conjugation by SULT. Based on the genetic data, the IGROV-1 was also found to be a suitable in vitro model to study biotransformation, but not for CYP2C9 and GST biotransformation. However, to be certain if the cell lines are suitable in vitro models, the enzyme activities should be determined in both cell lines.

The studies performed to unravel the biotransformation of the here investigated marine derived anti-cancer agents may help to improve clinical evaluation of anti-cancer activity and safety of these drugs.

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Samenvatting

Kanker is doodsoorzaak nummer 2 in Nederland. De behandeling van kanker is de laatste jaren sterk verbeterd. Patiënten in een vergevorderd stadium van kanker, bijvoorbeeld met uitzaaiingen, hebben echter nog meestal een kleine overlevingskans. Nieuwe therapieën zullen tevens de vaak voorkomende bijwerkingen bij de huidige therapie sterk kunnen verminderen. Daarom is het belangrijk dat er onderzoek plaatsvindt naar nieuwe antikankergeneesmiddelen. Veel antikankergeneesmiddelen hebben een natuurlijke oorsprong, zoals planten (bijvoorbeeld paclitaxel dat uit de taxusboom geïsoleerd is). Sinds een jaar of 50 wordt er ook onderzocht of er nieuwe antikankermiddelen uit zeeorganismen verkregen kunnen worden. Enkele nieuwe middelen worden al getest in patiënten (klinische studie).

In dit onderzoek zijn drie nieuwe antikankermiddelen, afkomstig uit zeeorganismen, onderzocht, namelijk thiocoraline, aplidine en ET-743. Thiocoraline werd uit de Micromonospora marina, welke in de straat van Mozambique voorkomt, geïsoleerd. Aplidine werd oorspronkelijk geïsoleerd uit de Aplidium albicans (figuur op de omslag), welke leeft in de Middellandse Zee, en ET-743 uit de Ecteinascidia turbinata uit het Caribische gebied. Alle drie de antikankermiddelen hebben in preklinische (niet in een patiënt) studies (bijvoorbeeld in een geïsoleerde tumor) grote werkzaamheid getoond ten aanzien van verschillende soorten kanker. Intussen worden aplidine en ET-743 al in patiënten (klinische studies) onderzocht en binnenkort zal thiocoraline ook bij patiënten gebruikt gaan worden.

Veel geneesmiddelen worden in het lichaam afgebroken, zodat ze het lichaam beter kunnen verlaten meestal via de urine, ontlasting of beiden. Dit omzettingsproces wordt biotransformatie of metabolisme genoemd en het product een metaboliet. Het belangrijkste orgaan waar dit plaatsvindt is de lever. Biotransformatie verloopt over het algemeen in twee reactiestappen, namelijk een zogenoemde fase-I-reactie, meestal gevolgd door een fase-II-reactie. Een product, welke gevormd wordt uit de uitgangsstof door een fase-I- of II-reactie, heet een metaboliet. Een enzymfamilie, genaamd cytochroom P450 (CYP), is het voornaamste enzymsysteem, welke de fase-I-reactie katalyseert. Tijdens een fase-II-reactie wordt er een wateroplosbare groep aan de stof of het reactieproduct van de fase-I-reactie gekoppeld, dit heet conjugatie. De belangrijkste fase-II-enzymen zijn glutathion-S-transferase (GST), N-acetyltransferase (NAT), uridine difosfoglucuronosyl transferase (UGT) en sulfotransferase (SULT). Deze enzymen zijn eigenlijk een grote familie waaronder verschillende subvormen gerangschikt zijn. Tevens vertonen de CYP- en UGT-enzymen een grote interindividuele variatie in activiteit, welke bepaald wordt door geslacht, leeftijd, ziekte, voedsel- en medicijninname en genetische factoren.

Er is weinig tot niets bekend over hoe thiocoraline, aplidine en ET-743 in het lichaam omgezet worden en hoe ze het lichaam verlaten via de urine of ontlasting. Kennis van de enzymen die betrokken zijn bij de biotransformatie en informatie over de enzymactiviteit van een patiënt kunnen leiden tot een verbetering van de therapie en vermindering van de bijwerkingen. Het is dus erg belangrijk om de betrokkenheid van de verschillende enzymen bij de biotransformatie van thiocoraline, aplidine en ET-743 te identificeren. Het onderzoek zoals beschreven in dit proefschrift is dan ook gericht op de opheldering van de biotransformatieroutes van deze middelen. Ook is onderzocht of de reactieproducten (metabolieten) giftiger zijn dan hun uitgangsstof (bioactivatie) of juist onschadelijk gemaakt zijn (bioinactivatie) door de betrokken enzymen.

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Met behulp van in vitro onderzoek (in vitro is Latijn voor in glas, dus in testbuis en niet in een dier) is het mogelijk om de biotransformatieroute te bestuderen en tevens de giftigheid (toxiciteit) van een stof en zijn metabolieten te bepalen. Een overzicht van alle in vitro technieken is in Hoofdstuk 1 weergegeven met alle voor- en nadelen van iedere techniek. In dit onderzoeksproject is er gebruik gemaakt van een aantal in vitro modellen, onder andere menselijke leverfracties (verschillende soorten gepureerde lever, die elk bepaalde enzymen bevatten) en speciale fracties van insectencellen, waarin bepaalde menselijke enzymen tot expressie gebracht zijn, ook wel supersomen genoemd. Verder is er gekozen voor speciale kankercellijnen. Een kankercellijn bestaat uit cellen die door deling ontstaan zijn uit een moedercel en welke continu blijven delen en dezelfde eigenschappen hebben. In dit promotieonderzoek zijn de cellijnen Hep G2, een leverkanker-cellijn, en IGROV-1, een eierstokkanker-cellijn, gebruikt.

Bij de biotransformatie route van thiocoraline (Hoofdstuk 2) bleek dat het cytochroom P450 enzym 3A4 een belangrijke rol speelde in de omzetting en dat CYP2C8 in mindere mate betrokken was bij de biotransformatie van thiocoraline. Bij de conjugatie waren UGT1A1 en 1A9 betrokken, maar geen van de andere fase II enzymen. Tevens bleken de metabolieten uit de CYP-reactie verder omgezet te worden door de fase II enzymen UGT, GST en SULT.

Aplidine (Hoofdstuk 3 en 4) werd eveneens voornamelijk omgezet door CYP3A4, maar CYP2A6, 2E1 en 4A11 speelden ook een rol. Er konden vier in vitro metabolieten geïdentificeerd worden, waarvan er drie door CYP3A4 gevormd werden en één door CYP2A6. Alleen de UGTs 1A3 en 1A9 waren betrokken bij de directe conjugatie van aplidine. De metabolieten gevormd na de CYP-reactie werden verder omgezet door UGT, SULT en GST. Uit de celkweekexperimenten bleek dat de metabolieten minder toxisch waren dan aplidine zelf, dus aplidine werd geïnactiveerd door deze enzymen.

Ook ET-743 (Hoofdstuk 5 en 6) werd voornamelijk door CYP3A4 omgezet. Tevens speelden er een aantal CYPs een kleine rol bij de biotransformatie, namelijk CYP2C9, 2C19, 2D6 en 2E1. Verder werd ET-743 door GST en UGT geconjugeerd. De metabolieten die gevormd werden door cytochroom P450 bleken uit celkweekexperimenten minder toxisch te zijn dan ET-743 zelf, ET-743 werd dus geïnactiveerd door de CYPs.

Op basis van deze resultaten kunnen de gegevens die verkregen worden uit patiëntenstudies beter geanalyseerd worden en zou de individuele therapie verbeterd kunnen worden. Tevens zouden mogelijke geneesmiddelen-interacties, welke tot bijwerkingen kunnen leiden, voorspeld kunnen worden als deze antikankermiddelen gecombineerd worden met medicijnen of voedselcomponenten, die invloed hebben op de CYP3A4-activiteit. Grapefruitsap zorgt er bijvoorbeeld voor dat de CYP3A4-activiteit minder wordt, waardoor er minder van de antikankermiddelen omgezet zal worden, wat een hogere concentratie in het bloed kan opleveren en daardoor kan leiden tot meer geprononceerde bijwerkingen. Sint Jan’s kruid daarentegen heeft een verhogende werking op de CYP3A4-activiteit, waardoor thiocoraline, aplidine of ET-743 sneller worden omgezet, wat tot een verminderd effect van de therapie kan leiden. Verder is er van een aantal andere antikankermiddelen bekend dat zij zowel een positieve als negatieve invloed kunnen hebben op de CYP3A4-activiteit. De therapie voor een individuele patiënt zou met behulp van de resultaten verbeterd kunnen worden, waardoor het aantal en de kans op bijwerkingen af zou kunnen nemen.

Tenslotte zijn ook nog de variaties in de genen (polymorfismen), coderend voor de CYPs en fase II enzymen, bepaald in beide cellijnen welke gebruikt zijn in dit promotieonderzoek, de menselijke Hep G2 en IGROV-1 cellijn (Hoofdstuk 7). Deze polymorfismen kunnen leiden tot een verlies, verlaging of verhoging in activiteit van deze enzymen. Met behulp van deze polymorfismen konden beide cellijnen gevalideerd worden voor hun gebruik als in vitro model om de biotransformatie te bestuderen. Het bleek dat op basis van de afwezigheid van

Samenvatting

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polymorfismen in CYP-genen, de Hep G2-cellijn een geschikt in vitro biotransformatiemodel is. De SULT activiteit is echter verlaagd in deze cellijn, waardoor het een minder geschikt model is om de conjugatie door SULT te bestuderen. De IGROV-1 cellijn is ook een geschikt in vitro model voor biotransformatie op basis van de genetische gegevens, echter niet als men de CYP2C9 of GST biotransformatie wil bestuderen. Om echter definitief met zekerheid vast te stellen of deze cellijnen geschikte in vitro biotransformatiemodellen zijn, zou tevens de activiteit van de enzymen bepaald moeten worden.

De studies uitgevoerd om de biotransformatie van de hier onderzochte, uit zeeorganismen verkregen antikankergeneesmiddelen, op te helderen kunnen helpen om de klinische evaluatie van antikankeractiviteit en de veiligheid van deze geneesmiddelen in patiënten te verbeteren.

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List of publications Journal articles Esther F.A. Brandon, Christiaan D. Raap, Irma Meijerman, Jos H. Beijnen, and Jan H.M. Schellens. An update on in vitro test methods in human hepatic drug biotransformation research: pros and cons. Toxicology and Applied Pharmacology 189: 233-246, 2003. Esther F.A. Brandon, Rolf W. Sparidans, Irma Meijerman, Ignasio Manzanares, Jos H. Beijnen, and Jan H.M. Schellens. In vitro characterization of the biotransformation pathway of thiocoraline, a novel marine anti-cancer drug. Investigational New Drugs 22: 241-251, 2004. Esther F.A. Brandon, Rolf W. Sparidans, Ronald D. van Ooijen, Irma Meijerman, Luis López Lázaro, Jos H. Beijnen, and Jan H.M. Schellens. In vitro characterization of the human biotransformation pathways of aplidine, a novel marine anti-cancer drug. (submitted). Esther F.A. Brandon, Ronald D. van Ooijen, Rolf W. Sparidans, Albert J.R. Heck, Jos H. Beijnen, and Jan H.M. Schellens. Structure elucidation of aplidine metabolites formed in vitro by human liver microsomes using triple quadrupole mass spectrometry. (submitted). Esther F.A. Brandon, Rolf W. Sparidans, Kees-Jan Guijt, Sjoerd Löwenthal, Irma Meijerman, Luis López Lázaro, Ignacio Manzanares, Jos H. Beijnen, and Jan H.M. Schellens. In vitro characterization of the human biotransformation and CYP reaction phenotype of ET-743 (Yondelis®, Trabectidin®), a novel marine anti-cancer drug (submitted). Esther F.A. Brandon, Irma Meijerman, Joyce S. Klijn, Rianne Levink, Rolf W. Sparidans, Luis López Lázaro, Jos H. Beijnen, and Jan H.M. Schellens. In vitro cytotoxicity of ET-743 (Trabectedin®, Yondelis®), a marine anti-cancer drug, in the Hep G2 cell line; influence of cytochrome P450 and phase II inhibition and cytochrome P450 induction. (submitted). Esther F.A. Brandon, Tessa Bosch, Rianne Levink, Everdina van der Wal, Joyce B.M. van Meerveld, Monique Bijl, Jos H. Beijnen, Jan H.M. Schellens, and Irma Meijerman. Validation of in vitro cell models used in drug metabolism studies; genotyping of cytochrome P450 and phase II enzyme polymorphisms in the human hepatoma (Hep G2) and ovarian carcinoma (IGROV-1) cell lines. (submitted). Conference proceedings Esther F.A. Brandon, Rolf W. Sparidans, Irma Meijerman, Jos H. Beijnen and Jan H.M. Schellens. In vitro characterization of the biotransformation pathway of thiocoraline. AIO/OIO-days of the Netherlands Society of Toxicology, De Bilt, The Netherlands, 14-15 January 2003. Proceedings of the scientific meeting of the Netherlands Society of Toxicology: 34, 2003 (poster).

List of publications

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Esther F.A. Brandon, Rolf W. Sparidans, Ronald D. van Ooijen, Irma Meijerman, Luis López Lázaro, Ignacio Manzanares, Jos H. Beijnen, and Jan H.M. Schellens. In vitro characterization of the human biotransformation pathways of aplidine, a novel marine anti-cancer drug. The 12th North American meeting of the International Society for the Study of Xenobiotics, Providence, RI, USA, 12-16 October 2003. Drug Metabolism Reviews 35 (suppl. 2): 182, 2003 (poster). Esther F.A. Brandon, Rianne Levink, Joyce B.M. van Meerveld, Tessa Bosch, Monique Bijl, Everdina van der Wal, Jos H. Beijnen, Jan H.M. Schellens, and Irma Meijerman. Validation of in vitro methods for drug metabolism in oncology; genotyping of cytochrome P450 and phase II enzyme polymorphisms in the human hepatoma (Hep G2) and ovarian carcinoma (IGROV-1) cell lines. The 1st international conference on cancer therapeutics: molecular targets, pharmacology and clinical applications of the International Society of Chemotherapy, Florence, Italy, 19-21 February 2004. Journal of Chemotherapy 16 (suppl. 1): 201, 2004 (poster). Esther F.A. Brandon, Rolf W. Sparidans, Ronald D. van Ooijen, Irma Meijerman, Ignacio Manzanares, Jos H. Beijnen, and Jan H.M. Schellens. In vitro characterization of the human biotransformation pathways of aplidine, a novel marine anti-cancer drug. AIO/OIO-days of the Netherlands Society of Toxicology, Veldhoven, The Netherlands, 10-11 June 2004. Proceedings of the scientific meeting of the Netherlands Society of Toxicology: ?, 2004 (oral presentation).

155

Curriculum Vitae

Esther Fleur Annette Brandon was born on July 3rd 1977 in Leiderdorp. After finishing high school (VWO) at the Twents Carmel Lyceum in Oldenzaal in 1995, she started the study of Biopharmaceutical Sciences at Leiden University. During her study she did two traineeships, one internal and one external. In the internal traineeship, the accumulation of Protoporphyrin IX after topical application of 5-aminolevulinic acid esters on pig and human skin was investigated for 9½ months at the Department of Medicinal Photochemistry of the Leiden Amsterdam Center for Drug Research (LACDR), Leiden University, under the supervision of Dr. G.J.M. Beijersbergen van Henegouwen. An external traineeship was performed for 6 month at the Toxicology department of Novartis Crop Protection in Basel, Switzerland. The route by which a novel class of insecticides induces cytotoxicity and identification of possible modulators of this cytotoxicity were investigated using primary rat hepatocytes and animal cell lines under the supervision of Dr. P. Bouis (Novartis) and Professor Dr. G.J. Mulder (LACDR). The masters degree in Biopharmaceutical Sciences was obtained in May 2000. The research described in this thesis was performed from June 2000 to May 2004 at the Division of Clinical and Analytical Drug Toxicology, Department of Biomedical Analysis, Faculty of Pharmaceutical Sciences, Utrecht University. This research was conducted under supervision of Professor Dr. J.H.M. Schellens, Professor Dr. J.H. Beijnen, Dr. Ir. I. Meijerman and Dr. Ir. R.W. Sparidans. During the same period, the author followed some modules of the Postdoctoral Education in Toxicology for registration as a Toxicologist.

Curriculum Vitae

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157

Dankwoord

Het promotieonderzoek van de afgelopen vier jaar heb ik niet alleen gedaan, veel mensen hebben direct of indirect geholpen bij de totstandkoming van dit proefschrift. Zonder hun hulp was het niet gelukt, daarom wil ik hen via deze weg bedanken.

Mijn promotoren Jan Schellens en Jos Beijnen voor de gelegenheid die zij mij hebben geboden om aan de in dit proefschrift beschreven onderzoek te werken en dit af te ronden met een promotie. Natuurlijk mijn co-promotor Irma Meijerman, die de afgelopen 2½ jaar mij heeft begeleid op praktisch gebied met de celkweek-experimenten, met het genotyperingswerk en het nakijken van de artikelen.

Rolf Sparidans dank je wel voor al je inzet de afgelopen vier jaar bij het doen van de vele biotransformatie-experimenten en het bediscussiëren van de resultaten. Zonder jou was het niet gelukt om het biotransformatie onderzoek op te zetten. Dianne van der Wal, ook al ben je er nog niet zo lang, je kennis zorgde ervoor dat het genotyperingswerk toch tot een goed einde gebracht is, ondanks alle problemen met de PCR-apparatuur. Natuurlijk ook Ronald van Ooijen voor de hulp bij de identificatie van de aplidine-metabolieten met behulp van de MS en voor de gezelligheid die je altijd meebracht als je langskwam. Verder nog bedankt voor de vakanties de je mij aanraadde om te gaan maken, misschien ga ik nog wel een keer een paardrijdtochtje op IJsland maken. Frits Dost, je was af en toe de rots in de branding en ik kon altijd bij je terecht voor hulp, ook al had je niks met mijn onderzoek te maken. Vivienne Verweij, we zijn ongeveer tegelijkertijd begonnen en ook al werkte je bij een andere groep, er is een hechte vriendschap ontstaan en je was altijd bereid om mij te helpen met de experimenten. Ed Volkerts, je kwam altijd gezellig koffie halen en was altijd geïnteresseerd in mijn onderzoek. Ook was je er altijd als vraagbaak voor allerlei zaken, dank hiervoor.

Prof. Dr. Gerard M. Mulder hartelijk dank dat u mijn opleider voor de postdoctorale opleiding toxicologie wilde zijn en voor alle hulp die u mij al tijdens mijn studie biofarmaceutische wetenschappen gaf.

Sjoerd Löwenthal en Kees-Jan Guijt hebben heel wat tijd van hun stageperiode doorgebracht met het incuberen van ET-743 samples met microsomen en supersomen en het letten op de stopwatch om de reactie stop te zetten. Zonder jullie was het enzymkinetiek gedeelte in hoofdstuk 5 nooit zo snel afgerond en was ik nu waarschijnlijk nog bezig met de experimenten. Joyce Klijn heeft lange tijd achter de downflowunit doorgebracht met het behandelen van cellen om de cytotoxiciteit van ET-743 in combinatie met verschillende inhibitoren te testen. Je kon je energie, die je opbouwde met het steriel werken, in ieder geval kwijt met de SRB assay, want je labjas zat altijd onder de roze kleurstof. Het was af en toe saai werk, maar je hebt veel werk verzet voor het ET-743 onderzoek. Het genotyperen van de cellijnen was niet zo snel afgerond als Rianne Levink mij niet in mijn laatste jaar had geholpen met het opzetten van alle nieuwe methoden en het screenen van de Hep G2 en IGROV-1 cellijn. Het was erg gezellig om met jou samen te werken. Veel succes nog met het afronden van je opleiding Medisch en Biologisch Laboratoriumonderzoek en met het paardrijden. Christiaan Raap heeft geholpen met het tot stand komen van hoofdstuk 1 door tijdens zijn studie een scriptie te schrijven over de verschillende in vitro technieken, die gebruikt worden bij het biotransformatie onderzoek. Je hebt veel artikelen doorgespit en een mooi overzicht geschreven. Joyce van Meerveld en Monique Bijl hebben een aantal genotyperingsmethoden opgezet voordat ik zelf met dit werk bezig was. Dankzij jullie waren een aantal polymorphismen kant-en-klaar te bepalen voor beide cellijnen. De vele andere studenten die er waren gedurende mijn aio-schap hebben voor veel gezelligheid op het lab gezorgd, vooral op vrijdag.

Dankwoord

158

Zonder de Instrumentele Dienst, met name Christina van Ek en Micha Kanters, was er de afgelopen vier jaar weinig van onderzoek terecht gekomen. Jullie stonden altijd klaar om mij te helpen de HPLC weer aan de praat te krijgen of de pipetten te ijken. De Financiële Administratie, Dan van Regteren en Sander Stekelenburg, was altijd weer bereid om een spoed-fax de deur uit te doen, als er met spoed iets besteld moest worden. Zonder de hulp van de Grafische Dienst waren de posters en de omslag van dit proefschrift niet tot stand gekomen.

Alle familieleden en vrienden voor hun belangstelling en steun gedurende de afgelopen vier jaar. Maar toch vooral Patrick en mijn ouders, jullie stonden altijd voor mij klaar. Mijn oma was altijd geïnteresseerd in mijn onderzoek, maar heeft helaas de afronding niet meer mogen meemaken.

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