Genz-644282, a Novel Non-Camptothecin Topoisomerase I

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Genz-644282, a Novel Non-Camptothecin Topoisomerase I Inhibitor for Cancer

Treatment

Leslie S. Kurtzberg1, Stephanie Roth2, Roy Krumbholz2, Jennifer Crawford2, Christy

Bormann2, Sarah Dunham2, Min Yao1, Cecile Rouleau1, Rebecca G. Bagley1, Xian-Jie

Yu1, Fei Wang1, Steven M. Schmid2, Edmond J. LaVoie3 and Beverly A. Teicher1

1Genzyme Corporation, Framingham, MA 01701

2Genzyme Corporation, San Antonio, TX 78229

3Rutgers University, Piscataway, NJ 08855

Running Title: Genz-644282 Topoisomerase I Inhibitor Key Words: Topoisomerase I, Genz-644282, Bone Marrow CFU-GM, tumor CFU,

tumor xenografts

Corresponding Author: Leslie S. Kurtzberg

Genzyme Corporation

49 New York Avenue

Framingham, MA 01701

Phone: 508-271-3717

FAX: 508-872-4091

Email: [email protected]

Research. on January 22, 2019. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 17, 2011; DOI: 10.1158/1078-0432.CCR-10-0542

http://clincancerres.aacrjournals.org/

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STATEMENT OF RELEVANCE

Genz-644282 is a novel, non-camptothecin Topoisomerase I (Top1) inhibitor.

The value of Top1 inhibitors in cancer treatment is well-documented. The currently

approved Top1 inhibitors are the camptothecins, topotecan and irinotecan. We

demonstrate a potentially improved therapeutic index for Genz-644282 versus topotecan

and SN-38 (active metabolite of irinotecan) by comparing toxicity in bone marrow

colony formation assays (CFU) performed with both murine and human cells, and

efficacy against growth of a variety of human cancer lines. Unlike the camptothecins,

Genz-644282 is not a substrate for the MDR1 and BCRP efflux pumps. Further, we

tested Genz-644282 vs. standard drugs in a variety of human tumor xenograft models,

and found superior or equal efficacy in the models. Based on these and other results,

Genz-644282 has entered in Phase I clinical trial.




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ABSTRACT

Purpose: Genz-644282, 8, 9-dimethoxy-5-(2-N-methylaminoethyl)-2,3-methylenedioxy-

5H-dibenzo[c,h][1,6]naphthyridin-6-one, emerged as a promising candidate. This report

describes the bone marrow CFU-GM and tumor cell CFU activity of Top1 inhibitors,

Genz-644282, topotecan, irinotecan/SN-38 and ARC-111, and examines their activity in

several human tumor xenograft models.

Experimental Design: Colony-forming assays were performed with mouse and human

bone marrow and eight human tumor cell lines. In addition, 29 human tumor cell lines

representing a range of histology and potential resistance mechanisms were assayed for

sensitivity to Genz-644282 in a 72-hr exposure assay. The efficacy of Genz-644282 was

compared with standard anticancer drugs (irinotecan, docetaxel, and dacarbazine) in

human tumor xenografts of colon cancer, renal cell carcinoma, non-small cell lung cancer

and melanoma.

Results: Human bone marrow CFU-GM was more sensitive to the Top1 inhibitors than

was mouse bone marrow CFU-GM. The ratio of mouse to human IC90s was >10 for the

camptothecins and <10 for Genz-644282. Genz-644282 was a more potent cytotoxic

agent toward human tumor cells in culture than the camptothecins in the colony

formation assay and the 72-hr proliferation assay. Genz-644282 has superior or equal

antitumor activity in the human tumor xenografts than the standard drug comparators.

Conclusions: Based upon preclinical activity and safety, Genz-644282 was selected for

development and is currently undergoing Phase 1 clinical trial.




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INTRODUCTION

Topoisomerase 1 (Top1) is an essential enzyme in mammalian cells (1-4).

Topoisomerase activity is crucial for initiation and elongation during DNA synthesis, for

the proper separation of sister chromatids during mitosis, for RNA transcription and for

illegitimate recombination events (5-8). Top1 associates preferentially with

transcriptionally active genes and is thought to be involved in relaxing supercoils

introduced by RNA polymerase during RNA transcription (9, 10).

Top1 produces a single strand break in DNA allowing relaxation of DNA for

replication. The single strand break is then religated restoring the DNA double strands.

The Top1 reaction intermediate consists of the enzyme covalently linked to a nicked

DNA molecule. This assembly is known as a “cleavable complex” (12, 13). The Top1-

targeted drugs topotecan and irinotecan and the Top2-targeted drugs doxorubicin,

amsacrine, etoposide and teniposide, stabilize the covalent enzyme-DNA complex

preventing religation (11). The cytotoxicity of Top1 inhibitors is due to trapping Top1-

DNA cleavage complex as opposed to inhibition of Top1 catalytic activity. The Top1-

DNA cleavage complex causes DNA damage during DNA replication and transcription.

Repair of Top1-mediated DNA damage has been reviewed (2, 14).

Camptothecin analogs topotecan and irinotecan are FDA-approved Top1-targeted

drugs. In vivo, there is a chemical equilibrium between the lactone form and the E ring-

opened form of the camptothecins. The E ring-opened carboxylate form has less than

10% the potency of the lactone as a Top1 inhibitor and is inactive in cell culture, perhaps

due to inability to cross the cell membrane (15). Both topotecan and irinotecan are




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substrates for P-gp efflux pumps and for the pump known as breast cancer resistant

protein (BCRP) (16, 17).

Bone marrow is critically sensitive to many antineoplastic agents and contributes

to toxicity. Top1 inhibitors kill rapidly dividing bone marrow progenitor cells as well as

tumor cells, resulting in acute reversible neutropenia and thrombocytopenia 4 to 20 days

after administration (27). Bone marrow granulocyte-macrophage-colony forming unit

(CFU-GM) assays comparing the sensitivity of bone marrow cells across species are

useful in predicting the blood levels of an agent that might be achieved with acceptable

toxicity in patients (27-30). Generally, murine bone marrow has a lower sensitivity to

Top I inhibitors than does human bone marrow. The difference in sensitivity between

murine and human bone marrow progenitor cells to Top1 inhibitors may explain, in part,

why curative doses/blood levels of topotecan and 9-amino-camptothecin in mice with

human tumor xenografts are not achievable in patients (30). A compound with similar

bone marrow progenitor sensitivity across species may have better potential for reaching

the same blood level in patients as in mice. Pessina et al (27) suggested that through use

of the ratio of mouse/human CFU-GM IC90 values and the mouse maximum tolerated

dose that the human maximum tolerated dose of a compound could be predicted and the

potential for achieving a therapeutic blood level in patients estimated.

Nitidine and fagaronine are benzo[c]phenanthridine alkaloids with good antitumor

potency and are active Top1 inhibitors (31, 32). Synthetic compounds of this class are as

potent as camptothecin in stimulating Top1-mediated DNA cleavage using purified

human Top1 and are more potent than irinotecan in many human tumor xenografts (23,

33). An extensive structure-activity relationship was conducted around the




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dibenzo[c,h][1,6]naphthyridin-6-one compound family to identify structural features

associated with potent Top1-targeting and suitable pharmaceutical properties (34). Genz-

644282, 8, 9-dimethoxy-5-(2-N-methylaminoethyl)-2,3-methylenedioxy-5H-

dibenzo[c,h][1,6]naphthyridin-6-one, emerged as a promising candidate from the

structure-activity relationship. The current report describes the bone marrow CFU-GM

and tumor cell CFU activity of Top1 inhibitors, Genz-644282, topotecan, irinotecan/SN-

38 and ARC-111, and examines their activity in several human tumor xenograft models.

Genz-644282 is currently in Phase I clinical trial.




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MATERIAL AND METHODS

Materials

The purity and chemical identity of Genz-644282 (8, 9-dimethoxy-5-(2-N-

methylaminoethyl)-2,3-methylenedioxy-5H-dibenzo[c,h][1,6]naphthyridin-6-one) and

ARC-111 (8,9-dimethoxy-5-(2-N,Ndimethylaminoethyl)-2,3-methylenedioxy-5H-

dibenzo[c,h][1,6]naphthyridin-6-one) was confirmed (FIGURE 1A) (23, 32-35).

Topotecan, irinotecan and SN-38 were used as purchased from LKT Laboratories (St.

Paul, MN). Genz-644282 and ARC-111 were prepared as a 100 µM stock solution in

D5W (5% Dextrose, aqueous, Injection USP, B. Braun Medical Inc., Irvin, CA). For cell

culture, topotecan and SN-38 were prepared as 10 mM stock solutions in DMSO from

Sigma (#D-5879) and dilutions for experiments prepared in RPMI/5% FBS.

Mouse Bone Marrow

Male Balb/c mice were purchased from Charles River Laboratories (Wilmington,

MA) at 6-8 weeks of age and used for studies at 7-10 weeks of age. Procedures were

carried out according to a protocol approved by the Institutional Animal Care and Use

Committee in accordance with the Federal Animal Welfare Act (9 CFR, 1992) and were

conducted in an AAALAC accredited facility.

For sterile bone marrow collection, mouse femoral and tibial shafts were flushed

with sterile RPMI 1460/5% FBS with a syringe and blunt 27G needle. The resulting

cellular suspension was collected into a 50 ml conical bottom tube and kept on wet ice.

Approximately 25 million cells were obtained from each mouse.




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Human Bone Marrow

Viable human bone marrow cells were purchased as frozen vials from AllCells,

LLC via StemCell Technologies, (#ABM009). They were prepared with a DNase

treatment (#07900, StemCell Technologies), and washed in a protocol described by the

vendor in preparation for culture.

Bone Marrow CFU-GM Assay

For mouse CFU-GM assays, freshly isolated mouse bone marrow cells were

cultured in MethoCult, containing the cytokines rmSCF, rmIL-3, rhIL-6 (StemCell

Technologies, Vancouver, BC). For human CFU-GM assays, the culture medium used

was MethoCult GF, containing the cytokines hSCF, hGM-CSF, hIL-3 (StemCell

Technologies).

For experiments, final cell concentrations of 4 x 104 cells/ plate for mouse cells and 8 x

104 cells/ plate for human cells were set up in duplicate 100 cm2

culture dishes for each

compound concentration, and then placed in a 37°C incubator for 13 days for mouse bone

marrow and 15 days for human bone marrow. Human and mouse bone marrow

progenitor cells were continuously exposed to a concentration range of Genz-644282,

ARC-111, topotecan or SN-38 in three independent CFU-GM colony formation

experiments. The compound concentrations were at half-log intervals covering 5 logs (10

concentrations). Colonies were defined as clusters containing 30 or more cells. The IC50

and IC90 values were expressed as the mean with 95% confidence intervals in nanomolar

concentrations (27).




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Tumor Cell Survival CFU Assay

The MDA-MB-231, HCT-116, HT-29, NCI-H460, and RPMI 8226 cell lines

were purchased from ATCC (Manassas, VA) (36-39). The KB3-1 and KB-V1 cell lines

were a gift from Dr. Edmond LaVoie. The MDA-MB-231 is an ER, PR and Her2/neu

negative breast cancer cell line. HCT-116 and HT-29 are colorectal carcinoma cell lines.

The RPMI 8226 line is a B lymphocyte plasmacytoma, myeloma cell line, and NCI-H460

is a large cell lung carcinoma (39). The KB human carcinoma cell line was originally

thought to be derived from an epidermal carcinoma of the mouth but was subsequently

found to have been established via HeLa cell contamination and is now designated a

cervical carcinoma (41). The KB3-1 line was developed by subcloning the KB cell line.

The KB-V1 subline was established from the KB3-1 line by continuous exposure to

vinblastine (1 µg/ml) resulting in gene amplification of mdr1 and mdr2 (42, 43). The KB-

V1 cells have more than 300-fold higher expression of mdr1 mRNA than do KB3-1 cells

and are more than 100-fold less sensitive to colchicines, doxorubicin and vinblastine than

are KB3-1 cells.

For human tumor cell colony formation assays, the cell lines which grow as

monolayers, MDA-MB-231, HCT116, HT29, NCI-H460, KB3-1 and KBV-1, were

grown in RPMI medium (Invitrogen/Gibco, Grand Island, NY) supplemented with 5%

FBS (Invitrogen/Gibco). RPMI-8226 cells, which are non-adherent, were grown in 0.35%

agar in DMEM-F12 medium supplemented with 10% FBS over a base layer of 0.5% agar

in DMEM-F12 medium supplemented with 10% FBS. Human tumor cells were plated at

1 x 103 in 6-well plates in medium supplemented with 5% or 10% FBS. The compounds

were tested over a concentration range from 0.01 to 100 nM in half-log intervals covering




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5 logs along with vehicle controls. Each compound concentration was tested in duplicate

wells. Cultures were exposed to the compounds continuously for 7 days at 37oC in a

humidified atmosphere of 5% CO2. Each experiment was performed three independent

times. Colonies were defined as clusters containing 30 or more cells. For the monolayer

cultures, colonies were stained with crystal violet solution containing 0.41% crystal

violet, 12% ethanol and DI water (Becton Dickenson, Sparks, MD). The IC50 and IC90

values were expressed as the mean with 95% confidence intervals in nM concentrations.

Growth Inhibition Assay

Twenty-nine established human tumor cell lines were exposed to a concentration

range of Genz-644282 in two-four independent experiments. Human tumor cell lines

representing a range of histology and potential resistance mechanisms were purchased

from ATCC (Manassas, VA), including MIA PaCa-2, AsPC-1, BxPC-3, CFPAC-1,

Hs766T and Capan-1 pancreatic cancers, MEL624, C32, Hs695T and SK-MEL-3

melanomas, NCI-H1299, NCI-H292, NCI-H1915 and SW900 non-small cell lung

cancers, HCC1395, HCC1937, HCC202, Hs578T, T-47D and ZR-75-1 breast cancer,

ACHN, 769-P, A-498, A-704, SW156, Caki-2 and TK-10 renal cancers and OVCAR-4

and OVCAR-5 ovarian cancers. Cells were plated at 4 × 103/well in 96-well tissue

culture plates in 100 µl RPMI medium supplemented with 5% FBS and 12 concentrations

of Genz-644282 from 0.1 nM to 10 µM , with each concentration tested in triplicate.

Plates were incubated overnight at 37ºC in humidified air with 5% CO2. Plates were

incubated with Genz-644282 at 37ºC with humidified air/5% CO2 for 72 hrs. After the




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incubation period, the test plates were read utilizing Promega’s Cell Titer-Glo

Luminescent Cell Viability Assay according to the manufacturer’s instructions.

Luminescence was measured with a Bio-Tek (Highland Park, Winooski, VT) Synergy

HT plate reader utilizing the associated Kineticalc software, Version #3.4. Luminescence

data were converted to growth fraction by comparison to the luminescence for the

untreated control for each cell line and IC50 and IC90 values determined from the

graphical data. Each cell line was tested in at least two independent experiments.

Human Tumor Xenografts

The efficacy of Genz-644282 was compared with standard anticancer drug

therapy in human tumor xenograft studies. All procedures were carried out according to a

protocol approved by the Institutional Animal Care and Use Committee in accordance

with the Federal Animal Welfare Act (9 CFR, 1992) and were conducted in an AAALAC

accredited facility. Nu/nu mice from Harlan were implanted subcutaneously with a 4

mm3 tumor fragment, and treatments were initiated when tumors reached 200 mm3.

Animals were assigned to treatment (n = 8-10) or control groups (n = 8-10). Compounds

were prepared freshly prior to injection, with Genz-644282 was formulated in M/6

lactate, irinotecan in D5W, gemcitabine in saline, and docetaxel in ethanol, Cremophor

EL and saline. The doses, schedules and routes for each compound were determined in

earlier MTD studies. Genz-644282 was tested on three schedules and the most effective,

non-toxic regimen was selected for further study.

Genz-644282 was compared with irinotecan in experiments with the human HCT-

116, HT-29, HCT-15 and DLD-1 colon carcinoma and 786-O renal cell carcinoma

xenografts. Irinotecan was administered at 60 mg/kg/day by IV injection every fourth




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day for three injections. Genz-644282 was compared with docetaxel in the human NCI-

H460 non-small cell lung carcinoma xenograft. Docetaxel was administered at 12, 16 or

20 mg/kg/day by IV injection on alternate days for three injections. Genz-644282 was

compared with dacarbazine in the human LOX-IMVI melanoma xenograft. Dacarbazine

was administered at 90 mg/kg/day by IP injection once daily for 5 days. Genz-644282

was administered at 1, 1.36, 1.7, 2.7 or 4.1 mg/kg/day by IV on alternate days 3-times per

week for 2 weeks in all in vivo experiments.

Pharmacokinetic Studies

Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to

quantitate Genz-644282 in blood, plasma and tumor tissue of nude (nu/nu) mice both

naïve and bearing NCI-H460 human tumor xenografts. The pharmacokinetics of Genz-

644282 were evaluated in male nude/nude mice following a single IV dose of 2.0 mg/kg

administered as the free base prepared in a lactic acid-sodium lactate-water formulation.

Non-compartmental pharmacokinetic parameters were determined. Pharmacokinetic-

phamracodynamic modeling was carried out by the method of Simeoni et al (44).

Statistical Analysis

Concentration response data for bone marrow CFU-GM and human tumor cell

line CFU were analyzed using a nonlinear curve fit based upon the equation y =

a*exp(b*x). The IC50 and IC90 values and the 95% confidence interval for each

compound on human and mouse bone marrow were determined by nonlinear regression

analysis using SAS version 8.2. Tumor volumes were calculated using the formula (w2 x

l) x 0.52. Mouse weight and tumor dimensions were measured twice weekly. The data are

presented as mean tumor volume +/- SD. Antitumor activity of the compounds was




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determined by calculating tumor growth delay (T-C) in days at a tumor volume of 1500

mm3. Increase-in-lifespan was determined as a secondary endpoint with removal from

study due to tumor size. Fold increase-in-lifespan was calculated from median survival in

days for the treated versus control groups. Kaplan-Meier survival curves were prepared

using GraphPad Prism software and used to determine the median survival times for each

treatment group of mice.




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RESULTS

The chemical structures of the non-camptothecin Top1 inhibitors, Genz-644282,

ARC-111, are shown in Figure 1A (33-35). The compounds Genz-644282, ARC-111,

topotecan and SN-38 are potent cytotoxic agents toward both mouse and human bone

marrow CFU-GM. Exposure to the compounds produced killing of cells in an

exponential manner. The concentration response curves are shown in FIGURE 1B.

Coefficient of determination R2 values provides a measure of the goodness of fit of the

data to the model ranged between 0.75 and 0.97. For all compounds tested,

concentrations killing 50% and 90% of the cells were readily achieved. Human and

mouse bone marrow CFU-GM IC50’s were 3- to 4-fold lower than the corresponding

IC90’s, thus the concentration response curves for these compounds are steep (FIGURE

1B). Human bone marrow CFU-GM was more sensitive to the cytotoxicity of the

compounds than was mouse bone marrow CFU-GM. Genz-644282 and ARC-111 were

more potent cytotoxic agents than the camptothecins, topotecan and SN-38 (irinotecan).

The ratios of the mouse and human bone marrow CFU-GM at the IC50 and IC90

concentrations were calculated. For the camptothecins the ratios were greater than or

equal to 10 and for Genz-644282 and ARC-111 the ratios were 4 to 7.

Genz-644282, ARC-111, topotecan and SN-38 were potent cytotoxic agents

toward the eight human tumor cell lines included in the colony formation assay. Exposure

to the compounds produced exponential killing of human tumor cells as in the bone

marrow experiments (FIGURE 2). Concentrations killing 50% and 90% of the cells were




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readily achieved for all compounds. Genz-644282 and ARC-111 were found to be more

potent cytotoxic agents than the camptothecins, topotecan and SN-38 (active metabolite

of irinotecan).

KBV-1 cells are engineered to express much higher levels of MDR than parental

control KB3-1 cells, and H460, HT-29, and RPMI- 8226 cells are very high natural

expressers of BCRP (26). Cells with high expression of either MDR or BCRP were

markedly resistant to killing by topotecan and SN-38 but not to killing by Genz-644282

and ARC-111.

Twenty-nine human tumor cell lines of six histological types were tested for

sensitivity to growth inhibition by Genz-644282 in a 72-hr exposure study using an ATP-

content readout (FIGURE 1C). Exposure to the compound produced exponential killing

of cells in a manner consistent with potent inhibition of a critical molecular target. The

IC90 concentrations for the 29 human tumor cell lines spread over a 1000-fold

concentration range from 100 nM to >10 μM. Four of the six pancreatic cancer cell lines

tested were among the lines most sensitive to Genz-644282. Three of the four melanoma

cell lines tested were among the least sensitive to Genz-644282 while most of the six

breast cancer cell lines tended to have moderate response to the compound. The most

disparate results were obtained with the seven renal cell cancer cell lines which ranged

from the most sensitive to the least sensitive responders to the compound. Two ovarian

cancer lines were tested; the OVCAR-4 cell line which was from a previously untreated

patient was more sensitive to the compound than was the OVCAR-5 cell line which was

from a patient who had failed multiple chemotherapies. Ten of the 29 tumor cell lines

were documented to have originated from metastatic disease. There is no strong trend for




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these cell lines to be more or less sensitive to the compound; however, half of the

metastatic cell lines had IC50s >50 nM. The median IC50 value for the 29 human tumor

cell lines was 33 nM and the mean IC50 value for the 29 cell lines was 164 nM.

Following intravenous administration of Genz-644282 (2 mg/kg) to nude/nude

mice, the mean blood clearance, volume of distribution, and terminal half-life were 21.8

ml/min/kg, 5.44 L/kg and 5.14 hours. An intravenous injection of Genz-644282 (2

mg/kg) to nude/nude mice bearing NCI-H460 human tumor xenografts resulted in plasma

pharmacokinetic parameters of 104 ml/min/kg mean clearance, a 49.3 L/kg volume of

distribution and terminal half-life of 8.14 hours. The mean area-under-the curve (AUC) in

tumor tissues (9690 ng*h/ml) was approximately 30-fold higher than that observed in

plasma (319 ng*h/ml), demonstrating Genz-644282 has a high partition into tumor tissue

in the xenograft model. The Genz-644282 IC90 concentration for human tumor NCI-H460

cell line in the colony formation assay was 5 nanomolar (FIGURE 2). Analysis of the

PK/PD relationship of Genz-644282 compared the exponential growth of tumors in

control mice with tumor growth in the Genz-644282 treated mice. The growth decreased

by a factor proportional to drug concentration (44). The estimated average tumor

concentration of Genz-644282 at steady state ranged from 130-519 nM or 24- to 96-fold

higher than the in vitro IC90 (5.4 nM), thus predicting that the tumor would be responsive

to treatment.

Many schedules have been described in the literature for the administration of

irinotecan in mice (45). Both Genz-644282 and irinotecan were tested on several

schedules and the optimal ones for each compound were selected for doing comparisons

in several human tumor xenograft models. In vivo, Genz-644282 was tolerated at doses




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up to 4 mg/kg when administered intravenously on alternate days and the compound was

active at doses from 1 mg/kg to 4 mg/kg. The efficacy of Genz-644282 was compared

with irinotecan in four human colon carcinoma xenograft models. In the human HCT-116

colon cancer xenograft, tumor growth delay (TGD) values were 14 days for irinotecan

(60 mg/kg) and 34 days for Genz-644282 (2.7 mg/kg) giving maximum T/Cs of 23.7%

and 16.8 %, respectively (FIGURE 3A). A similar experiment was performed with the

human HT-29 colon carcinoma xenograft, and the TGD values were 11 days for

irinotecan (60 mg/kg) and 27 days for Genz-644282 with maximum T/Cs of 52% and

16.6%, respectively (FIGURE 3B). In the human HCT-15 colon carcinoma xenograft

the TGD, Genz-644282 was administered at 2 mg/kg resulting in a TGD of 33 days

compared with a TGD of 27 days with irinotecan (60 mg/kg) giving maximum T/Cs of

16% and 8.6 %, respectively (FIGURE 3C). Mice bearing the DLD-1 human colon

carcinoma xenograft were treated with Genz-644282 (1 mg/kg) resulting in a TGD of 14

days compared with a TGD of 11 days with irinotecan (60 mg/kg) with maximum T/Cs

of 50.2% and 51.3%, respectively (FIGURE 3D).

The antitumor efficacy of Genz-644282 was compared with the antitumor activity

of dacarbazine in the human LOX-IMVI melanoma xenograft (FIGURE 4A).

Administration of Genz-644282 (2 mg/kg) produced a TGD of 28 days in mice bearing

LOX-IMVI melanoma compared with a TGD of 14 days after administration of

dacarbazine (90 mg/kg) giving maximum T/Cs of 66.2% and 80.6 %, respectively. The

antitumor efficacy of Genz-644282 was compared with the antitumor activity of

irinotecan in the human 786-O renal cell carcinoma xenograft (FIGURE 4B).

Administration of Genz-644282 (1.7 mg/kg) produced a TGD of 23 days in mice bearing




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786-O tumors compared with a TGD of 16 days after administration of irinotecan (60

mg/kg) with maximum T/Cs of 8.7% and 20.7%, respectively.

The antitumor activity of Genz-644282 was compared with the antitumor activity

of docetaxel in NCI-H460 and NCI-H1299 human non-small cell lung carcinoma

xenografts. Administration of Genz-644282 (2.7 mg/kg) to mice bearing the NCI-H460

human non-small cell lung carcinoma produced at TGD of 27 days compared with a TGD

of 21 days from administration of docetaxel (20 mg/kg) giving maximum T/Cs of 4.6%

and 19.5%, respectively (FIGURE 4C). In the NCI-H1299 human non-small cell lung

carcinoma xenograft, treatment with Genz-644282 (1.7 mg/kg) resulted in a TGD of 33

days while treatment with docetaxel (20 mg/kg) produced a TGD of 15 days with

maximum T/Cs of 8.7% and 20.7%, respectively (FIGURE 4D). As an initial

combination chemotherapy regimen, administration of Genz-644282 (1.36 mg/kg) along

with docetaxel (12 mg/kg) was tested in mice bearing NCI-H460 human non-small cell

lung carcinoma xenografts (FIGURE 5). The TGD with docetaxel (12 mg/kg) alone was

12 days and the TGD obtained following administration of Genz-644282 (1.36 mg/kg)

was 24 days and the TGD achieved with the combination regimen was 27 days.




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DISCUSSION

Topoisomerase 1 is a fully validated target in cancer with two drugs specifically

targeted to the enzyme in clinical use. Top1 inhibition became an important target in

cancer with the discovery that camptothecin, a very potent anticancer plant alkaloid,

specifically targets Top1 (46). Top1 remains a target of active interest in the development

new anticancer agents because Top1 inhibitors are clearly active and effective anticancer

drugs and because the current Top1 inhibitors are molecules that can be improved upon.

There are camptothecin-derivatives and non-camptothecin Top1 inhibitors in preclinical

and clinical development. Each of these investigational molecules may have properties

that lead to improved therapeutic benefit to patients. There are also very active preclinical

efforts based upon protein phosphorylation levels and mRNA levels in tumor and blood

samples to define biomarkers that can select patients most likely to benefit from

treatment with specific Top1 inhibitors and to guide clinical investigators toward

definition of the lowest effective dose and optimal schedule for administration of these

agents (21).

Bone marrow granulocyte-macrophage-colony forming (CFU-GM) assays

comparing the toxic effects on bone marrow by investigational agents across species have

been useful in predicting the blood levels that might be reached in patients compared with

blood levels in preclinical efficacy and safety species (27). Frequently, mouse bone

marrow is less sensitive than human bone marrow to investigational agents allowing

blood levels to be achieved in mouse preclinical efficacy testing that can not be reached

in patients. The bone marrow toxicity of four Top1 inhibitors was examined (FIGURE

1B) (26-30). Murine bone marrow is 4- to 28-fold less sensitive to the Top1 inhibitors




20

than human bone marrow. The differential sensitivity between mouse and human bone

marrow to Top1 inhibitors may explain, in part, why curative doses/blood levels of

topotecan and irinotecan/SN-38 in mice with human tumor xenografts are not achievable

in patients (29, 30). Compounds with little differential in bone marrow sensitivity across

species may have greater potential for reaching similar blood levels in patients as in mice.

From these data, Genz-644282 and ARC-111 may have promise as development

candidates.

A hallmark of malignant cells is the capacity for unlimited renewal or

proliferation. The cancer stem cell hypothesis postulates that malignant disease is

maintained by a rare subset of cells with stem cell properties (47, 48). The colony

formation assay is the gold-standard for the determination of the cytotoxicity of

compounds toward malignant cells because it measures the survival/killing of cancer cell

capable of proliferating to form colonies or clones. Each colony originates from a single

cell capable of self-renew with the ability to generate malignant disease. Genz-644282

cytotoxicity was assessed in eight human tumor cell lines of varied histology and

resistance mechanisms by colony formation. Compared with topotecan and SN38, Genz-

644282 and ARC-111 were more potent cytotoxic agents. Genz-644282 and ARC-111

remained highly effective in cells expressing efflux pumps. Meng et al. (49) reported that,

like other Top1 inhibitors, the antiproliferative effects of ARC-111 were oxygen-

independent, which is distinguishable from inhibition of hypoxia-inducible factor-

1α accumulation by ARC-111, only observed under hypoxia and supporting the potential

anticancer efficacy of ARC-111 and similar compounds toward hypoxic tumors.




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In earlier reports, ARC-111 was shown to be an effective antitumor agent against

several standard human tumor xenografts. ARC-111 was as active as irinotecan in the

HCT-8 colon carcinoma and as active as topotecan or irinotecan in the SKNEP anaplastic

Wilm’s xenograft (23). ARC-111 was also a very effective antitumor agent in animals

bearing the SJ-BT45 medulloblastoma (34). In subsequent studies, ARC-111 was as or

more effective as an anticancer agent compared with irinotecan in the HCT116 and HT29

human colon carcinoma xenografts, docetaxel in the NCI-H460 human non-small lung

carcinoma xenograft and gemcitabine in the MiaPaCa2 human pancreatic carcinoma

xenograft (26). In the current study, Genz-644282 demonstrated greater or equal

antitumor efficacy as irinotecan in four human colon carcinoma xenografts, greater

antitumor efficacy than dacarbazine in the human LOX IMVI melanoma xenograft,

greater antitumor efficacy than irinotecan in the human 786-O renal cell carcinoma

xenograft, and greater than or equal antitumor efficacy than docetaxel in two human non-

small lung carcinoma xenografts. In an initial combination chemotherapy regimen,

Genz-644282 and docetaxel together showed a modest increase in tumor response in the

NCI-H460 non-small cell lung carcinoma xenograft compared with Genz-644282 as a

single agent.

Top1 inhibitors are active and effective anticancer drugs. The FDA approved

Top1 inhibitors are molecules that have flaws which newer compounds can improve

upon. For this reason, Top1 remains a target of active interest in the development of new

anticancer agents. Investigational molecules, both camptothecin and non-camptothecin

Top1 inhibitors in preclinical and clinical development, have properties that lead to

improved therapeutic benefit to patients. There is an active search in the Top1 inhibitor




22

field for biomarkers such as gene signatures or markers based upon protein

phosphorylation levels and mRNA levels in tumor or blood samples that can identify

patients most likely to benefit from treatment with a specific Top1 inhibitor.

Genz-644282 is a promising non-camptothecin Top1 inhibitor that based upon

preclinical activity and safety was selected for development and is currently undergoing a

Phase 1 clinical trial.




23

FIGURE LEGENDS FIGURE 1. A. Chemical structures of the non-camptothecin Top1 inhibitors, Genz-

644282 and ARC-111 are shown. B. Concentration response curves for each of the four

Top1 inhibitors in mouse (Δ) and human (▲) bone marrow CFU-GM showing an

exponential curve fit (dotted lines). C. Tumor cell IC90 values for 29 human tumor cell

lines upon exposure to Genz-644282 in a 72 hr growth inhibition assay in micromolar

concentrations are shown. The IC50 and IC90 concentrations dertermined from the

concentration response curves for the 29 tumor lines are shown in Supplemental

TABLE 1.

FIGURE 2. Concentration response curves in a colony formation assay for each of the

four Top1 inhibitors, Genz-644282 (♦), ARC-111 (◊), SN-38 (■) and topotecan (□), in

human HCT116 and HT29 colon carcinoma cells, NCI-H460 non-small cell lung

carcinoma cells, MDA-MB-231 breast carcinoma cells, RMPI8226 multiple myeloma

cells and KB3-1, KBV-1 and KBH5.0 cervical carcinoma cells as representative human

tumor cell lines. The red dot curves fit the nonlinear exponential model for the Genz-

644282 data.

FIGURE 3. Growth of subcutaneously implanted human tumor xenografts in nude mice

controls, treated with Genz-644282 or irinotecan. A. HCT-116 colon carcinoma untreated

control (♦), Genz-644282 vehicle treated (■), irinotecan vehicle treated (▲), Genz-

644282 (1.36 mg/kg, iv, alternate days for 2 wks) (), Genz-644282 (2.7 mg/kg, iv,

alternate days for 2 wks) (Δ), and irinotecan (60 mg/kg, iv each fourth day for 3




24

injections) (●). B. HT-29 colon carcinoma untreated controls (♦), Genz-644282 vehicle

treated (■), irinotecan vehicle treated (▲), Genz-644282 (1.36 mg/kg, iv, alternate days

for 2 wks) (), Genz-644282 (2.7 mg/kg, iv, alternate days for 2 wks) (Δ), Genz-644282

(4.1 mg/kg, iv, alternate days for 2 wks) (◊), and irinotecan (60 mg/kg, iv each fourth day

for 3 injections) (●). C. HCT-15 colon carcinoma untreated controls (♦), Genz-644282

vehicle treated (■), irinotecan vehicle treated (▲), Genz-644282 (1 mg/kg, iv, alternate

days for 2 wks (○), Genz-644282 (2 mg/kg, iv, alternate days for 2 wks) (), and

irinotecan (60 mg/kg, iv each fourth day for 3 injections) (●). D. DLD-1 colon carcinoma

untreated controls (♦), Genz-644282 vehicle treated (■), irinotecan vehicle treated (▲),

Genz-644282 (1 mg/kg, iv, alternate days for 2 wks) (○), and irinotecan (60 mg/kg, iv

each fourth day for 3 injections) (●). The data are the means +/- SD for groups of 8-10

mice.


controls, treated with Genz-644282 or dacarbazine or irinotecan or docetaxel. A. LOX-

IMVI melanoma untreated control (♦), Genz-644282 vehicle treated (■), Genz-644282 (1

mg/kg, iv, alternate days for 2 wks) (○), Genz-644282 (2 mg/kg, iv, alternate days for 2

wks) (), dacarbazine (90 mg/kg, ip, daily for 5 days) (●). B. 786-O renal carcinoma

untreated controls (♦), Genz-644282 vehicle treated (■), Genz-644282 (1 mg/kg, iv,

alternate days for 2 wks) (○), Genz-644282 (1.36 mg/kg, iv, alternate days for 2 wks) (◊),

Genz-644282 (1.7 mg/kg, iv, alternate days for 2 wks) (), and irinotecan (60 mg/kg, iv

each fourth day for 3 injections) (●). The data are the means +/- SD for groups of 8-10

mice. C. NCI-H460 non-small cell lung carcinoma untreated control (♦), Genz-644282

vehicle treated (■), docetaxel vehicle treated (▲), Genz-644282 (1 mg/kg, iv, alternate




25

days for 2 wks) (○), Genz-644282 (1.36 mg/kg, iv, alternate days for 2 wks) (), Genz-

644282 (2.7 mg/kg, iv, alternate days for 2 wks) (Δ) and docetaxel (20 mg/kg, iv,

alternate days for 3 injections. D. NCI-H1299 non-small lung carcinoma untreated

controls (♦),vehicle treated (■), Genz-644282 (1 mg/kg, iv, alternate days for 2 wks) (○),

Genz-644282 (1.36 mg/kg, iv, alternate days for 2 wks) (), Genz-644282 (1.7 mg/kg,

iv, alternate days for 2 wks) (Δ), docetaxel (16 mg/kg, iv, alternate days for 3 injections)

(●) and docetaxel (20 mg/kg, iv (▲).


controls, treated with Genz-644282 in combination with docetaxel. NCI-H460 non-small

cell lung carcinoma untreated controls (♦), Genz-644282 vehicle treated (■), Genz-

644282 (1.36 mg/kg, iv, alternate days for 2 wks (○), Genz-644282 (1.36 mg/kg, iv,

alternate days for 2 wks) + docetaxel (12 mg/kg, iv, alternate days for 3 injections) () ,

and docetaxel (12 mg/kg, iv, alternate days for 3 injections) (●). The data are the means

+/- SD for groups of 8-10 mice.




26

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33

Supplemental TABLE 1. Twenty-nine human tumor cell lines of six histological types

were tested for sensitivity to growth inhibition by Genz-644282 in a 72-hr exposure study

using an ATP-content readout. The IC90 concentrations for the 29 human tumor cell lines

spread over a 1000-fold concentration range from 100 nM to >10 μM. This data is

represented in FIGURE 1C.

DISEASE CELL LINE IC50, NANOMOLAR

IC90, MICROMOLAR

RATIO IC90/IC50

Pancreatic Ca MIA PaCa-2 3.5 0.11 31 Renal Cell Ca ACHN 1.9 0.12 63 Pancreatic Ca BxPC-3 15 0.14 9 Lung Ca NCI-H292 1.8 0.2 111 Pancreatic Ca Capan-1 4.8 0.3 63 Renal Cell Ca A-498 70 0.3 4 Ovarian Ca OVCAR-5 22 0.39 18 Pancreatic Ca CFPAC-1 1.9 0.5 263 Lung Ca SW900 9.5 0.65 68 Renal Cell Ca A-704 33 1.0 30 Breast ca T-47D 27 1.1 41 Ovarian Ca OVCAR-4 53 1.1 21 Melanoma C32 21 1.2 57 Lung Ca NCI-H1915 30 1.7 57 Breast Ca HCC1395 13 1.9 146 Breast Ca ZR-75-1 18 2.0 111 Breast ca HCC1937 200 2.1 10.5 Renal Cell Ca SW156 23 2.3 100 Pancreatic Ca Hs766T 280 2.5 89 Pancreatic Ca AsPC-1 300 3.0 10 Breast Ca HCC202 220 3.1 14 Renal Cell Ca 769-P 50 3.2 64 Breast Ca Hs578T 130 3.3 25 Renal Cell Ca CAKI-2 170 3.3 19 Melanoma Hs695T 86 3.9 45 Lung Ca NCI-H1299 80 4.5 56 Melanoma SK-MEL-3 100 6.0 60 Melanoma MEL624 1000 >10 10 Renal Cell Ca TK-10 1800 >10 5.6




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0.1

Hu: IC50 1.9nM; IC90 6.2nM

0.1

H IC50 10 M IC90 26 M

0.1

0.010.001 0.01 0.1 1 10 100 1000 10000

;

M: IC50 2.3nM; IC90 8.4nM0.01

0.001 0.01 0.1 1 10 100 1000 10000

M: IC50 8.0nM; IC90 27.9nM

0.010.1 1 10 100 1000 10000 100000

Hu: IC50 10nM; IC90 26nM

M: IC50 108nM; IC90 331nM0.01

0.1 1 10 100 1000 10000 100000 1E+06 1E+07 1E+08

Hu: IC50 6.5nM; IC90 18.8nM

M: IC50 166nM; IC90 518.8nM

Compound Concentration, nMFigure 1

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actio

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HCT116 Colon Carcinoma HT29 Colon Carcinoma NCI-H460 Non-small Cell Lung Cancerur

vivi

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ARC111: IC50 1.7; IC90 5.6nM

SN-38: IC50 2.7; IC90 8.4nM

0.1

Genz: IC50 0.15; IC90 0.9nM

ARC111: IC50 1.3; IC90 4.3nM

SN-38: IC50 0.5; IC90 1.8nM

0.1

Genz: IC50 1.2; IC90 5.0nMARC111: IC50 2.3; IC90 13.3nM

SN-38: IC50 4.7; IC90 6.2nM

MDA-MB-231 Breast Carcinoma RPMI8226 Multiple Myeloma

S 0.010.01 0.1 1 10 100

Topo: IC50 8.5; IC90 26.3nM0.01

0.01 0.1 1 10 100

Topo: IC50 2.9; IC90 11.2nM0.01

0.01 0.1 1 10 100

Topo: IC50 18.2; IC90 52.5nM

1 1

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Genz: IC50 0.2; IC90 0.7nM

ARC111: IC50 0 3; IC90 1 2nM

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SN-38: IC50 0.7; IC90 2.0nM

Topo: IC50 5.6; IC90 19.5nM 0.010.01 0.1 1 10 100

SN-38: IC50 0.9; IC90 3.0nMTopo: IC50 12.7; IC90 43.2nM

1 1 1

KBH5.0 Cervical Carcinoma

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0.1

Genz: IC50 1; IC90 3nMving

Fra

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Compound Concentration, nM Figure 20.01

0.01 0.1 1 10 100

ARC111: IC50 1.5; IC90 5.2nM

SN-38: IC50 5.3; IC90 19.1nM

Topo: IC50 32.7; IC90 107.2nM0.01

0.01 0.1 1 10 100

ARC111: IC50 1.8; IC90 5.7nM

SN-38: IC50 15; IC90 55nM

Topo: IC50 75; IC90 257nM0.01

0.01 0.1 1 10 100

ARC111: IC50 1.8; IC90 5.8nM

SN-38: IC50 6.1; IC90 18.8nM

Topo: IC50 32; IC90 114.8nMSur

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Untreated Controls

Genz Vehicle

Irinotecan Vehicle

Genz644282 1.36 mg/kg/day

Human HCT116 Colon Carcinoma 2000

Untreated Control

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Irinotecan Vehicle



Genz644282 4.1 mg/kg/dayHuman HT29 Colon Carcinoma

A B

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Human HCT-15 Colon Carcinoma

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12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 63

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Genz VehicleGenz644282 1 mg/kg

Genz644282 2 mg/kg

Human LOX-IMVI Melanoma

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Untreated ControlsGenz VehicleGenz644282 1 mg/kg/dayGenz644282 1.36 mg/kg/dayGenz644282 1.7 mg/kg/dayIrinotecan 60 mg/kg/day

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Genz644282 1 mg/kg

Genz644282 1.36 mg/kg

Genz644282 1.7 mg/kg

Docetaxel 16 mg/kg

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2200

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Genz Vehicle

Genz644282 1.36 mg/kg3

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Genz644282 1.36 mg/kg +Docetaxel 12 mg/kgDocetaxel 12 mg/kg

lum

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Published OnlineFirst March 17, 2011.Clin Cancer Res Leslie S. Kurtzberg, Stephanie D. Roth, Roy D. Krumbholz, et al. Inhibitor for Cancer TreatmentGenz-644282, a Novel Non-Camptothecin Topoisomerase I

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Genz-644282, a Novel Non-Camptothecin Topoisomerase I