Studies on Drug Interactions between CYP3A4 Inhibitors and ...ethesis.helsinki.fi/julkaisut/laa/kliin/vk/varis/studieso.pdf · Department of Clinical Pharmacology University of Helsinki

Department of Clinical PharmacologyUniversity of Helsinki

Finland

Studies on Drug Interactions

between CYP3A4 Inhibitors and

Glucocorticoids

by

Tiina Varis

ACADEMIC DISSERTATIONTo be presented, with the permission of the Medical Faculty of the University ofHelsinki, for public examination in the small lecture hall of Haartman Institute,

Haartmaninkatu 3, on December 15th, 2000, at 12 noon.

Helsinki 2000

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Supervised by:

Professor Pertti Neuvonen, MDDepartment of Clinical PharmacologyUniversity of HelsinkiHelsinki, Finland

Docent Kari Kivistö, MDDepartment of Clinical PharmacologyUniversity of HelsinkiHelsinki, Finland

Reviewed by:

Docent Arja Rautio, MDDepartment of Pharmacology and ToxicologyUniversity of OuluOulu, Finland

Docent Kari Aranko, MDFocus Inhalation OYTurku, Finland

Official opponent:

Docent Risto Huupponen, MDDepartment of Pharmacology and Clinical PharmacologyInstitute of BiomedicineUniversity of TurkuTurku, Finland

ISBN 952-91-2933-5 (nid.)ISBN 952-91-2934-3 (PDF)Helsinki 2000Yliopistopaino

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To Juha, Antti, and Kristiina

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CONTENTS

ABBREVIATIONS..................................................................................................................6

LIST OF ORIGINAL PAPERS..............................................................................................7

ABSTRACT .............................................................................................................................8

INTRODUCTION ...................................................................................................................9

REVIEW OF THE LITERATURE......................................................................................10

1. Drug metabolism.............................................................................................................101.1. Principles of drug metabolism.................................................................................101.2. The CYP enzymes...................................................................................................101.3. CYP3A subfamily ...................................................................................................111.4. Drug interactions .....................................................................................................141.5. Drug interactions mediated by inhibition of CYP3A4 ............................................161.6. CYP3A4 inhibitors studied .....................................................................................171.6.1. Itraconazole..........................................................................................................171.6.2. Diltiazem..............................................................................................................181.6.3. Mibefradil.............................................................................................................201.6.4. Grapefruit juice ....................................................................................................211.7. CYP3A4 and P-glycoprotein...................................................................................24

2. Glucocorticoids...............................................................................................................242.1. General ....................................................................................................................242.2. Regulation of cortisol secretion...............................................................................272.3. Pharmacodynamics..................................................................................................272.4. Therapeutic use .......................................................................................................282.5. Adverse effects of glucocorticoid treatment............................................................302.6. Methylprednisolone.................................................................................................302.7. Prednisolone............................................................................................................332.8. Dexamethasone .......................................................................................................35

AIMS OF THE STUDY ........................................................................................................38

MATERIALS AND METHODS ..........................................................................................39

1. Ethical considerations .....................................................................................................392. Study subjects .................................................................................................................393. Study protocol.................................................................................................................39

3.1. Study design ............................................................................................................393.2. Determination of plasma drug and cortisol concentrations .....................................423.3. Pharmacokinetic calculations ..................................................................................433.4. Pharmacodynamic measurements............................................................................443.5. Statistical methods...................................................................................................453.6. Archiving.................................................................................................................45

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RESULTS .............................................................................................................................. 46

1. Interaction between itraconazole and glucocorticoids .................................................... 461.1. Oral methylprednisolone (Study I) ......................................................................... 461.2. Intravenous methylprednisolone (Study II) ............................................................ 471.3. Oral prednisolone (Study IV) ................................................................................. 481.4. Oral and intravenous dexamethasone (Study VI) ................................................... 49

2. Interaction between diltiazem or mibefradil and oral methylprednisolone (Study III) ... 533. Interaction between grapefruit juice and oral methylprednisolone (Study V) ................ 55

DISCUSSION ........................................................................................................................ 57

1. Methodological considerations....................................................................................... 572. Interaction between CYP3A4 inhibitors and glucocorticoids......................................... 58

2.1. Methylprednisolone ................................................................................................ 582.2. Prednisolone ........................................................................................................... 602.3. Dexamethasone....................................................................................................... 61

3. General discussion.......................................................................................................... 62

SUMMARY AND CONCLUSIONS.................................................................................... 65

ACKNOWLEDGEMENTS.................................................................................................. 67

REFERENCES...................................................................................................................... 69

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ABBREVIATIONS

ACTH adrenocorticotropin hormoneANOVA analysis of varianceAUC(t-ti) area under the plasma drug concentration-time curve from t to ti hoursCBG corticosteroid-binding globulinCl systemic clearanceCPMP committee for proprietary medicinal productsCmax peak concentrationCRH corticotropin-releasing hormoneCV coefficient of variationCYP cytochrome P450DDD defined daily doseECG electrocardiogramEDTA ethylendiaminetetraacetic acidGRE glucocorticoid regulatory elementHIV human immunodeficiency virusHMG-CoA 3-hydroxy-3-methylglutaryl-coenzyme AHPA-axis hypothalamic-pituitary-adrenal axisHPLC high-performance liquid chromatographyIBW ideal body weightkel elimination rate constantLC/MS/MS liquid chromatography-ionspray-tandem mass spectrometryMDR multiple drug resistanceMI-complex metabolic intermediate complexP-gp P-glycoproteinQT QT interval in electrocardiogramQTc heart rate-corrected QT intervalSD standard deviationSEM standard error of meantmax time of peak concentrationt½ elimination half-lifeVd volume of distribution

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LIST OF ORIGINAL PAPERS

This thesis is based on the following articles which will be referred to in the textby the Roman numerals I to VI.

I Varis T, Kaukonen K-M, Kivistö KT, Neuvonen PJ. Plasmaconcentrations and effects of oral methylprednisolone are considerablyincreased by itraconazole. Clin Pharmacol Ther 1998;64:363-368

II Varis T, Kivistö KT, Backman JT, Neuvonen PJ. Itraconazole decreasesthe clearance and enhances the effects of intravenously administeredmethylprednisolone in healthy volunteers. Pharmacol Toxicol1999;85:29-32

III Varis T, Backman JT, Kivistö KT, Neuvonen PJ. Diltiazem andmibefradil increase the plasma concentrations and greatly enhance theadrenal-suppressant effect of oral methylprednisolone. Clin PharmacolTher 2000;67:215-221

IV Varis T, Kivistö KT, Neuvonen PJ. The effect of itraconazole on thepharmacokinetics and pharmacodynamics of oral prednisolone. Eur J ClinPharmacol 2000;56:57-60

V Varis T, Kivistö KT, Neuvonen PJ. Grapefruit juice can increase theplasma concentrations of oral methylprednisolone. Eur J Clin Pharmacol2000;56:489-493

VI Varis T, Kivistö KT, Backman JT, Neuvonen PJ. The cytochrome P4503A4 inhibitor itraconazole markedly increases the plasma concentrationsof dexamethasone and enhances its adrenal-suppressant effect. ClinPharmacol Ther, in press

The original publications are reprinted with permission of the copyright holders.

ABSTRACT

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ABSTRACT

The aim of this thesis was to investigate the effect of CYP3A4 inhibitors on thepharmacokinetics and pharmacodynamics of systemic glucocorticoids; theeffect of itraconazole on oral and intravenous methylprednisolone, on oralprednisolone, and on oral and intravenous dexamethasone; and the effect ofdiltiazem, mibefradil, and grapefruit juice on oral methylprednisolone. Theeffect of route of administration of methylprednisolone and dexamethasone onthe possible interaction with itraconazole was also evaluated.

A total of 8 to 10 healthy subjects were enrolled in each study. Each was adouble-blind, randomised, placebo-controlled, cross-over study, except thegrapefruit juice-methylprednisolone interaction study, with its open, randomisedcross-over design. The pretreatment was followed by a single dose ofglucocorticoid, blood samples were collected and plasma drug and cortisolconcentrations determined.

The use of itraconazole enhanced the adrenal-suppressant effect of bothmethylprednisolone and dexamethasone. Itraconazole increased the AUC oforal and intravenous methylprednisolone and oral and intravenousdexamethasone 3.9-fold (p <0.001), 2.6-fold (p <0.001), 3.7-fold (p <0.001),and 3.3-fold (p <0.001), respectively. In contrast, itraconazole increased theAUC of oral prednisolone by only 24% (p <0.001), and the adrenal-suppressanteffect of prednisolone was slightly enhanced. Both diltiazem and mibefradilincreased the AUC of oral methylprednisolone (2.6-fold and 3.8-fold; p <0.001)and its adrenal-suppressant effect was greatly enhanced. Grapefruit juiceincreased the AUC of oral methylprednisolone by 75% (p <0.001), but itsadrenal-suppressant effect was not significantly affected. The route ofmethylprednisolone and dexamethasone administration may have only a minoreffect on the extent of itraconazole-methylprednisolone and itraconazole-dexamethasone interactions. Despite the differences seen in pharmacokineticinteractions, the extent of the adrenal-suppressant effect was similar after asingle oral or intravenous dose of these glucocorticoids.

In conclusion, these interactions probably result from inhibition of the CYP3A4enzyme both during the first-pass and the elimination phases. Inhibition of theP-glycoprotein may have contributed to the interactions of methylprednisoloneand dexamethasone. Care should be taken if methylprednisolone ordexamethasone is used concomitantly with a potent inhibitor of CYP3A4 over along period. However, the itraconazole-prednisolone and grapefruit juice-methylprednisolone interactions are probably of limited clinical significance.

INTRODUCTION

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INTRODUCTIONThe cytochrome P450 (CYP) enzymes catalyse the oxidative metabolism ofmany drugs (Pelkonen et al 1998, Dresser et al 2000). Among the human drug-metabolising CYP enzymes, CYP3A4 is the most abundant enzyme both in theliver and intestine (Shimada et al 1994, de Waziers et al 1990).

Because several drugs and also dietary factors can inhibit the catalytic activity ofCYP3A4 (Pelkonen et al 1998, Evans 2000), inhibitors of CYP3A4 can increaseexposure to drugs metabolised mainly via this pathway. Drug-drug interactionscan therefore significantly enhance both the therapeutic effects of the drug andalso the risk of adverse effects (Dresser et al 2000). For example, concomitantuse of itraconazole with commonly used drugs like terfenadine, midazolam, andsimvastatin is associated with potentially serious adverse effects (Pohjola-Sintonen et al 1993, Olkkola et al 1994, Segaert et al 1996). However, enzymeinhibition may also result in therapeutic failure if the drug is administered as aninactive prodrug and its metabolic activation is inhibited (Lin et Lu 1998).

Many important drug-drug interactions have been reported after the drug inquestion has received marketing approval. Some interactions have been seriousenough to cause limitations in the use of the drug or even its withdrawal from themarket. Concern for the safe use of drugs has increased, and the drug interactionpotential of a new investigational compound has to be evaluated beforemarketing approval (Note for guidance on the investigation of drug interactions,CPMP, 1997). In vitro studies are used, for example, to give information aboutspecific CYP enzymes involved in the metabolism of new compounds andpossible drug-drug interactions. However, an interaction observed in an in vitrostudy should be confirmed in relevant, controlled in vivo studies.

Intravenous and oral glucocorticoids have been widely used for many decades invarious clinical conditions for their anti-inflammatory and immunosuppressiveproperties (Hench et al 1949, Swartz and Dluhy 1978, Mies et al 1998). Systemicglucocorticoids are eliminated mainly by metabolism (Vree et al 1999, Begg et al1987, Tomlinson et al 1996), and inhibition of the catalytic activity of CYP3A4by other drugs or dietary constituents can affect their elimination. CYP3A4enzyme, however, probably has a dissimilar role in the metabolism of differentglucocorticoids.

The purpose of the present study was to investigate the potential of CYP3A4inhibitors to affect the pharmacokinetics and adrenal-suppressant effect ofsystemic methylprednisolone, prednisolone, and dexamethasone in controlledclinical studies in healthy volunteers. Furthermore, the effect of the route ofglucocorticoid administration on the interaction potential was evaluated.

REVIEW OF THE LITERATURE

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1. Drug metabolism1.1. Principles of drug metabolismMost pharmacologically active compounds are lipophilic because lipophilicityenables them to pass through biological membranes. In order to be excreted fromthe body, these lipophilic compounds must be biotransformed into more water-soluble metabolites (Meyer 1996). Metabolites generated are generallypharmacologically less active than the parent drug or even inactive, but themetabolites may also show enhanced pharmacological activity. Furthermore, themetabolism of drugs can produce harmful reactive and toxic products, which maybe responsible for various forms of toxicity, e.g., hepatotoxicity (Nelson et al1996, Mitchell et al 1974).

One major enzyme system involved in drug biotransformation is the cytochromeP450 (CYP) monooxygenase system. Other important enzymes include esterases,epoxide hydrolase, and conjugating enzymes such as glucuronosyltransferasesand sulfotransferases. These metabolic reactions are divided into functionalisation(phase I) and conjugation (phase II) reactions (Meyer 1996). The phase Ioxidative biotransformations catalysed by the hepatic and extrahepatic CYPenzymes include oxidation, reduction, and hydrolysis. Oxidative metabolismrepresents a major route of elimination for many drugs (Lin and Lu 1998).

During phase II reactions, the parent drug or its phase I metabolite is conjugatedwith an endogenous substrate. The phase II conjugation reactions involveglucuronidation, sulphation, acetylation, and conjugation with glutathione. Theconjugated metabolites are usually highly water-soluble and are readily excretedinto urine and bile, which are the main routes of elimination for most drugs(Meyer 1996).

1.2. The CYP enzymesCYP enzymes are a family of enzymes which are found not only in human beingsbut also in bacteria, fungi, plants, and animals (Nelson et al 1999). CYP enzymesare membrane-bound, localised to the endoplasmic reticulum (Meyer 1996). Theyare responsible for most of the oxidative metabolism of both xenobiotics andendogenous substrates (Dresser et al 2000, Lin and Lu 1998).

The nomenclature of all CYP enzymes is based on their amino acid sequence(Nelson et al 1996). The CYP enzymes within the same family are designated byan Arabic numeral (e.g., CYP3) and share more than 40% identity in the amino


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acid sequence. The families are further divided into subfamilies, with theenzymes within a subfamily designated by a capital letter (e.g., CYP3A) andsharing more than 55% identity in the amino acid sequence. Finally, an Arabicnumerical after the letter denotes each individual isoenzyme (e.g., CYP3A4).

Functionally, the CYP enzymes are divided into two major classes, those with aspecific role e.g, in steroid biosynthesis, bile and arachidonic acid metabolism(CYP4, CYP5, and higher) and those primarily involved in the metabolism ofxenobiotics such as drugs, anti-oxidants, and chemicals (CYP1, CYP2, CYP3)(Nelson et al 1999). Many of these enzymes are involved in metabolism of a widerange of different substrates, e.g., endogenous compounds, chemicals, and naturalplant products (Nelson et al 1996). Among the 17 CYP gene families reported inhumans by Nelson in 1999, three families: CYP1, CYP2, and CYP3, areimportant for hepatic drug metabolism (Wrighton et al 1996). The mostprominent CYP isoenzymes with regard to the number of substrate drugs areCYP3A4 and CYP2D6, with a smaller number of drugs metabolised by CYP2C9,CYP2C19, CYP1A2, and CYP2E1 (Wrighton and Stevens 1992, Meyer 1996).CYP enzymes are found mainly in the liver, which plays the major role in humandrug metabolism, but they are present in lower amounts in many extrahepatictissues (Krishna and Klotz 1994).

The expression and catalytic activity of CYP enzymes may vary greatly betweenindividuals, due to genetic, non-genetic endogenous (e.g. diseases), andenvironmental (drugs, diet) factors (Pelkonen et al 1998). Genetic polymorphismgreatly affects the activity of some CYP enzymes, e.g., CYP2C19 and CYP2D6(Wrighton et al 1996, Nakamura et al 1985). Furthermore, some CYP enzymesare inducible by environmental factors such as drugs (Wrighton et al 1996,Pelkonen et al 1998). In addition, catalytic activity of CYP enzymes can beinhibited, or the CYP enzymes can be inactivated. Induction and inhibition ofCYP enzymes increase the intraindividual and interindividual variability of drugmetabolism. Examples of the substrates, inhibitors, and inducers of CYP3A4,CYP2D6, CYP2C9, CYP2C19, CYP1A2, and CYP2E1 isoenzymes are presentedin Table I (page 12).

1.3. CYP3A subfamilyThe CYP3A subfamily is involved in the metabolism of a large number ofendogenous and exogenous compounds, e.g., cortisol, testosterone, cyclosporin,midazolam, nifedipine, and simvastatin (Table I). Because approximately 40-50%of the drugs used in man are metabolised at least partly by CYP3A-mediated


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Table I. Examples of substrates, inhibitors, and inducers of the main humanhepatic drug-metabolising CYP enzymes and their relative amounts of the totalhepatic CYP content. * indicates that the enzyme exhibits genetic polymorphism.Modified from Bertz and Granneman (1997) and Pelkonen et al (1998).

CYP enzyme Substrates Inhibitors Inducers

CYP2C19*< 5%

omeprazolemephenytoindiazepam

fluoxetinefluvoxamine

rifampicin

CYP2C8/9*∼ 20%

warfarinphenytoincerivastatin

sulphaphenazolefluconazole

rifampicin

CYP3A4∼ 30%

simvastatinmidazolamtriazolamnifedipinecyclosporincisapridetestosteroneethinylestradiolcortisolterfenadine

ketoconazoleitraconazolediltiazemverapamilgrapefruit juiceerythromycinchlarithromycin

rifampicinphenytoincarbamazepine

CYP1A2∼ 15%

caffeinetheophyllineclozapine

fluvoxamine smokingrifampicin

CYP2E1< 10%

ethanolhalothanechlorzoxazone

disulfiram ethanolisoniazide

CYP2D6*<5%

dextrometorphandebrisoquinecodeinemetoprolol

quinidinefluoxetine

not inducible


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oxidation, it appears to be the most important drug-metabolising CYP subfamily(Thummel and Wilkinson 1998).

CYP3A consist of three isoenzymes, CYP3A4, CYP3A5, and CYP3A7(Wrighton and Stevens 1992, de Wildt et al 1999). Among these, CYP3A4 is themost abundant isoenzyme in adult liver, accounting for about 30% of total CYPcontent (Shimada et al 1994). CYP3A4 is shown to be present also e.g, in smallintestine, colon, and stomach (de Waziers et al 1990). CYP3A5 is found in 25-30% of adult livers (Wrighton and Stevens 1992). More recently, however,CYP3A5 protein was detected in 74% of livers examined (Jounaidi et al 1996).CYP3A5 is the major isoenzyme in the lung tissue, in which CYP3A4 isexpressed in only the minority of cases (Kivistö et al 1996a, Anttila et al 1997).Furthermore, CYP3A5 is expressed in the small intestine in 70 to 100% ofindividuals (Lown et al 1994, Kivistö et al 1996b).

CYP3A5 displays 84% similarity in its amino acid sequence to CYP3A4(Aoyama et al 1989). Although the substrate specificity of CYP3A5 is similar tothat of CYP3A4, differences exist in these isoensymes’ catalytic properties(Wrighton and Stevens 1992). In contrast to CYP3A4, CYP3A5 does not appearto metabolise erythromycin, quinidine, or 17α-ethinylestradiol at significant rates(Wrighton et al 1990). Furthermore, testosterone, cortisol, and nifedipine aremetabolised by CYP3A5 at a slower rate than by CYP3A4 (Wrighton et al 1990).

CYP3A7 is the major CYP isoenzyme in the fetal liver, accounting for up to 50%of total CYP content (Kitada et al 1985, Wrighton and VandenBranden 1989). Inthe adult liver, CYP3A7 mRNA is found in about 55 to 85% of samples, althoughat lower levels than CYP3A4 (Schuetz et al 1994, Hakkola et al 1994).

In vitro, the expression and catalytic activity of hepatic CYP3A4 can vary asmuch as 6-fold and 30-fold, respectively (Shimada et al 1994, Yun et al 1993).Despite this large interindividual variation in CYP3A4 activity, CYP3A4 does notappear to be subject to genetic polymorphism, in contrast to CYP2C9, CYP2C19,and CYP2D6 (Aithal et al 1999, Gaedigk 2000). Recent studies have, however,identified a mutation in the CYP3A4 that has been linked to tumour stage andgrade at the time of diagnosis of prostatic cancer (Rebbeck et al 1998)Furthermore, this variation may influence the risk of treatment-related leukemias(Felix et al 1998). A recent study has found this promoter region polymorphismnot to affect the CYP3A4-mediated metabolism of erythromycin or thepharmacokinetics of nifedipine (Ball et al 1999). Sata et al (2000) have identifiedother variant alleles of CYP3A4 designated CYP3A4*2 and CYP3A4*3;CYP3A4*2, which is present at a frequency of 2.7% in a white Finnishpopulation, showed in vitro altered catalytic activity compared with that of wild-type P450 (Sata et al 2000). However, the impact of these mutations on CYP3A4


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activity and on the pharmacokinetics of CYP3A4 substrates needs furtherevaluation.

Erythromycin is metabolised about 25% more rapidly by microsomes fromfemale than from male human liver (Hunt et al 1992). Furthermore, midazolam iscleared 20-40% faster by women than by men (Harris et al 1995). Not all studies,however, have found a sex-related difference in CYP3A4 activity (Schmucker etal 1990, Shimada et al 1994). Neither menstrual-cycle phase, smoking, norethanol consumption appear to influence CYP3A4 activity (Kharasch et al 1999,Hunt et al 1992). Sotaniemi et al (1997) have suggested that liver CYP contentdeclines after age 40. However, two other studies have failed to find anyrelationship between age and liver CYP3A activity (Hunt et al 1992, Blanco et al2000). The CYP3A4 activity is decreased in severe liver disease (Pelkonen et al1998), and celiac disease can decrease CYP3A4 activity in gut (Lang et al 1996).

During the last few years research interest has increasingly focused on drugmetabolism in the intestinal wall. This location makes suitable to play animportant role in first-pass metabolism and in limiting the bioavailability of orallyadministered drugs (Hoppu et al 1991, Dresser et al 2000). Many CYP enzymesincluding CYP3A4, CYP3A5, and CYP2D6 appear to be expressed in theintestinal wall (de Waziers et al 1990, Kivistö et al 1996b). After the liver, thesmall intestine contains the highest amount of CYP3A4 (de Waziers et al 1990).Hepatic and intestinal CYP3A4 appear to be regulated independently (Lown et al1994), and the content of intestinal CYP3A4 varies 11-fold and its catalyticactivity 6-fold among individuals (Lown et al 1994). This finding may contributeto the interindividual variability in disposition of CYP3A4 substratesadministered orally. The small intestine is an important place for first-passmetabolism of oral drugs. Interestingly, the intestinal first-pass metabolism ofsome oral drugs, for example, midazolam and cyclosporin, may even exceed thatoccurring in the liver (Thummel et al 1996, Wu et al 1995).

1.4. Drug interactionsDrug interaction can occur by several different mechanisms, and these can beeither harmful or beneficial. Pharmacodynamic interactions cause changes in theeffects of drugs. For example, alcohol enhances the sedative effect ofbenzodiazepines (Seppälä et al 1982). Pharmacokinetic interactions can takeplace at the level of drug absorption, distribution, metabolism, and excretion. Forexample, antacids containing aluminium, calcium, or magnesium and ironproducts reduce the absorption of tetracyclines, resulting in reduced antibacterialeffects (Neuvonen et al 1970). An example of an interaction due to changes in


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excretion is reduced excretion of penicillin by probeniside (Kampmann et al1972).

Enzyme induction and inhibition affect drug metabolism. CYP-enzyme inductionusually results from an increased amount of enzyme protein, and a period of timeis needed before enzyme induction becomes clinically significant (Pelkonen et al1998). Enzyme induction increases the metabolism of drugs that are metabolisedby induced enzymes, leading to decreased plasma drug concentrations (Lin andLu 1998). For example, rifampicin can greatly decrease the plasma concentrationsof triazolam and abolishes the effect of triazolam (Villikka et al 1997).

CYP enzyme inhibition usually occurs rapidly, even after a single dose ofinhibitor (Pelkonen et al 1998). Enzyme inhibition can be competitive or non-competitive (Lin and Lu 1998). In case of competitive inhibition, enzymeinhibitor competes the active site of CYP enzyme. Competitive inhibition isprobably the most common mechanism responsible for the drug-drug-interactions. For example, itraconazole and indinavir, a potent humanimmunodeficiency virus (HIV) protease inhibitor, are competitive inhibitors ofCYP3A4 (Back and Tjia 1991, Fitzsimmons and Collins 1997). Competitiveinhibitors are often substrates of the CYP isoenzyme whose activity they inhibit.However, this is not always the case. Quinidine is a substrate for CYP3A4, but isa potent reversible inhibitor of CYP2D6 (Guengerich et al 1986, Nielsen et al1990).

Many drugs, including the macrolide antibiotics troleandomycin anderythromycin, undergo metabolic activation by CYP3A4 to form inhibitorymetabolites (Pessayere et al 1982, Danan et al 1981). These metabolites can formcomplexes with the enzyme called metabolic intermediate (MI) complexes,resulting in a functionally inactive CYP3A4 enzyme. Ethinylestradiol andgrapefruit juice, for example, are classified as mechanism-based inactivators orsuicide substrates, because they cause irreversible inactivation of the enzyme(Guengerich 1988, Lown et al 1997).

In addition to the hepatic, the intestinal CYP3A4 can be induced by such drugs asrifampicin (Kolars et al 1992) and inhibited by known CYP3A4 inhibitors such asketoconazole (Tsunoda et al 1999). The orally administered drug is exposed toCYP3A4-mediated first-pass metabolism, and inhibition of CYP-mediatedmetabolism during the first-pass can result in increased bioavailability. Afterintravenous administration, the drug bypasses the first-pass metabolism, and itssystemic bioavailability is complete. Thus, the effect of enzyme inhibitiondepends on whether the drug is administered orally or intravenously.


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Drugs can be classified by wheather their hepatic clearance is enzyme-limited(low; hepatic extraction ratio < 0.3) or flow-limited (high; hepatic extraction ratio> 0.7) (Lin and Lu 1998). If the drug is metabolised mainly in the liver, decreasein metabolic activity by enzyme inhibition results in an almost proportionalincrease in the area under the plasma drug concentration-time curve (AUC) of anorally administered drug, regadless whether the drug is a high- or low-clearancedrug (Lin and Lu 1998). However, after intravenous administration enzymeinhibition significantly affects the AUC of a low-clearance drug, whereas theAUC of high-clearance drug is only slightly affected (the hepatic clearance islimited by the hepatic blood flow) (Lin and Lu 1998).

1.5. Drug interactions mediated by inhibition of CYP3A4The drug-drug interactions mediated through CYP3A4 result either frominduction or from inhibition of this enzyme (Lin and Lu 1998, de Wildt et al1999). Both in vitro and in vivo studies have demonstrated that CYP3A4 isinhibited, for example, by the azole antimycotics ketoconazole and itraconazole(Back and Tjia 1991, Varhe et al 1994, Olkkola et al 1994), the macrolideantibiotics troleandomycin, erythromycin, roxithromycin, and clarithromycin(Gascon et al 1991, Fleming et al 1992, Olkkola et al 1993, Backman et al 1994a,Bailey et al 1996, Yeates et al 1996), the calcium-channel antagonists diltiazem,verapamil, and mibefradil (Backman et al 1994b, Kantola et al 1998a, Welker etal 1998), and by grapefruit juice (Bailey et al 1991, Hukkinen et al 1995, Lilja etal 1998a).

Many drugs from different therapeutic areas are substrates for CYP3A4, which ispresent in high amounts in both the liver and the intestine (Shimada et al 1994, deWaziers et al 1990). Use of a CYP3A4 inhibitor can decrease drug metabolismboth during the first-pass and the elimination phases. Decreased drug eliminationmay enhance a drug’s therapeutic and also its toxic effects. There are severalexamples of an interaction due to inhibition of CYP3A4 being clinicallyimportant. QT-interval prolongation or torsades de pointes ventricular arrhytmiahave been reported with concomitant use of a CYP3A4 inhibitor with terfenadineor cisapride (Pohjola-Sintonen et al 1993, van Haarst et al 1998, Piquette 1999).The risk of rhabdomyolysis appeared to increase with co-administration of aCYP3A4 inhibitor and lovastatin or simvastatin (Neuvonen and Jalava 1996,Neuvonen et al 1998, Lees and Lees 1995). Furthermore, concomitant use ofdrugs such as ketoconazole, itraconazole, or verapamil increases the sedativeeffects of midazolam and triazolam (Olkkola et al 1994, Backman et al 1994b,Varhe et al 1994).


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Drug-drug interactions can also prove useful. The reduction in cyclosporin dosagewould result in extensive cost savings (Jones et al 1997, Martin et al 1999).Concomitant use of ketoconazole with cyclosporin allows the reduction ofcyclosporin dosage by 70 to 85% in renal-, cardiac-, and liver-transplant patients.

1.6. CYP3A4 inhibitors studied

1.6.1. ItraconazoleItraconazole is a triazole antimycotic introduced onto the market in the early1990’s. Itraconazole inhibits the fungal CYP enzyme 14-demethylase, therebyinhibiting the conversion of lanosterol to ergosterol, the primary sterol in fungalmembranes. The azole antifungals are primarily fungistatic (Gupta et al 1994).

Itraconazole is used in treatment and prevention of systemic fungal infections andin oral therapy of superficial dermatomycoses such as onychomycosis (Gupta1994, Huijgens et al 1999, Harari 1999, Venkatakrishnan et al 2000). Therecommended daily dose is 100 to 400 mg for superficial infections, but doses upto 800 mg/day may be needed for treatment of systemic mycoses (Sanchez et al1995). Itraconazole is usually well tolerated; incidence of adverse effects is 7%,rising to 12.5% with therapy longer than one month. The most common adverseeffects are gastrointestinal symptoms, headache, dizziness, and rash. Furthermore,elevation of transaminases has occurred in 0.3 to 5% of patients (Gupta et al1994). Unlike ketoconazole, itraconazole has no significant effect on adrenalfunction and plasma cortisol concentration at doses up to 400 mg daily (Gupta etal 1994, Phillips et al 1987, Queiroz-Telles et al 1997).

Itraconazole is well absorbed when administered with a meal, but its absorption isreduced during fasting (Van Peer et al 1989). The time of the peak concentration(tmax) in plasma ranges from 1.5 to 4 h (Hardin et al 1988). It is highly bound toplasma proteins (Grant and Clissold 1989, Gupta et al 1994). About 95% is boundto plasma protein, mainly albumin, 5% to blood cells, and only 0.2 to 3% remainsfree in plasma (Arredondo at al 1995, Grant and Clissold 1989, Backman et al2000). The volume of distribution (Vd) of itraconazole is about 11 l/kg, whichindicates extensive tissue distribution (Heykants et al 1989). Due to itslipophilicity, concentrations of itraconazole are 3 to 10 times as high in skin, and10 to 20 times as high in liver as in plasma (Heykants et al 1989, Backman et al2000). Itraconazole exhibits non-linear pharmacokinetics at therapeutic doses.The mean AUC increases more than proportionally with dose, suggestingsaturable metabolism (Hardin et al 1988, Van Peer et al 1989). The eliminationhalf-life (t½) of itraconazole after a single dose ranges from 15 h to 25 h and atsteady state from 30 h to 50 h (Heykants et al 1989).


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Itraconazole has a significant first-pass metabolism and is eliminated bymetabolism. More than 30 metabolites have been found, but onlyhydroxyitraconazole possesses clinically significant activity (Heykants et al1989). CYP3A4 appears to have an important role in metabolism of itraconazole(Ducharme et al 1995a). About 54% of an oral dose is excreted in faeces and 35%in urine (Heycants et al 1989).

It had been suggested that itraconazole is a selective inhibitor of fungal CYPenzymes and will not inhibit drug metabolism in humans (Grant and Clissold1989). However, it is known today that itraconazole inhibits the metabolism ofmany CYP3A4 substrates and causes many clinically significant drug-druginteractions. Itraconazole has increased the AUC of midazolam and triazolam 11-and 27-fold, respectively and markedly enhanced their hypnotic effects (Olkkolaet al 1994, Varhe et al 1994). In addition, itraconazole has increased the AUC ofbuspirone about 20-fold and increased its pharmacodynamic effects (Kivistö et al1997). Plasma concentrations of lovastatin, lovastatin acid, simvastatin, andsimvastatin acid are greatly increased by itraconazole (Neuvonen and Jalava1996, Kivistö et al 1998, Neuvonen et al 1998). Importantly, concomitant use ofitraconazole and certain HMG-CoA reductase inhibitors may increase risk ofmyalgia, and cases of rhabdomyolysis have been reported (Lees and Lees 1995,Segaert et al 1996). Furthermore, itraconazole inhibits the metabolism ofterfenadine, resulting in increased risk for torsades de pointes ventriculartachycardia (Pohjola-Sintonen et al 1993).

Itraconazole potently inhibits P-glycoprotein (P-gp) in vitro and probably also invivo (Miyama et al 1998, Kurosawa et al 1996, Backman et al 2000). P-gp andCYP3A show substrate overlap (Wacher et al 1995), although the CYP3A4substrate midazolam, for instance, is not a substrate of P-gp (Kim et al 1999). Therelative role of P-gp- and CYP3A-inhibition to the effect of itraconazole on thepharmacokinetics of dual substrates of P-gp and CYP3A is unclear(Venkatakrishnan et al 2000).

A summary of selected characteristics of itraconazole, diltiazem, mibefradil, andgrapefruit juice is shown in Table II (page 23).

1.6.2. DiltiazemDiltiazem is a benzothiazepine calcium-channel blocker used in treatment ofangina pectoris, hypertension, and supraventricular arrhythmias (Dollery 1999a,Buckley et al 1990). Diltiazem inhibits the influx of calcium into vascular smoothmuscle and myocardial cells through ”slow” calcium channels and therebyreduces the tone of coronary and peripheral arterioles (Jones and Hall 2000). Therecommended oral daily dose of diltiazem ranges from 120 to 360 mg divided


19

into one to three doses (Buckley et al 1990). Diltiazem is usually well tolerated;the incidence of adverse effects is less than 2% (McGraw et al 1982). The mostcommon adverse effects are related to the vasodilator properties of diltiazem, likeflushing, headache, ankle oedema, and hypotension.

Diltiazem is almost completely absorbed after oral administration; it undergoes asignificant first-pass metabolism, resulting in a bioavailability of only about 40%(Kelly and O’Malley 1992). The peak plasma concentration (Cmax) is reachedwithin 1.5 h with fast-release formulation (Buckley et al 1990).

Diltiazem is about 80 to 90% bound to plasma proteins, mainly albumin (Buckleyet al 1990). The Vd of diltiazem is about 5.0 l/kg (Hermann et al 1983). It islargely eliminated through CYP3A4-mediated metabolism (Pichard et al 1990,Sutton et al 1997). About 70% of the dose is excreted into the urine and 17% intothe faeces (Höglund and Nilsson 1988). Metabolites of diltiazem possesspharmacologic activity, but their role in therapeutic effects is unclear (Jones andHall 2000). The t½ of orally administered diltiazem averages about 4.5 h (range 2-11 h) (Buckley et al 1990). Multiple oral dosing with diltiazem results inprolongation of its elimination, which may be due to inhibition of diltiazemmetabolism by diltiazem itself or by accumulated metabolites (Tsao et al 1990).

The ability of diltiazem to inhibit microsomal drug oxidation was first reported byRenton in 1985. More recently, in a study in human liver microsomes, bothdiltiazem and its metabolites inhibit CYP3A4 (Sutton et al 1997). However, theN-desmethyl and N,N-didesmethyl metabolites were much more potent inhibitorsof CYP3A4 than the parent drug itself (Sutton et al 1997, Jones and Hall 2000).Thus, the metabolites of diltiazem probably contribute to enzyme inhibition anddrug-drug interactions in vivo (Sutton et al 1997, Jones and Hall 2000).

In humans, the plasma concentrations of several CYP3A4 substrates such ascyclosporin, midazolam, triazolam, and simvastatin are increased by diltiazem(Pochet and Pirson 1986, Brockmöller at al 1990, Backman et al 1994b, Varhe etal 1996, Mousa et al 2000). Furthermore, diltiazem increases the concentrationsof carbamazepine, resulting in its enhanced neurotoxicity (Brodie and MacPhee1986, Eimer and Carter 1987). In addition, diltiazem has been shown to increasethe metabolic ratio of debrisoquine, indicating its potential to inhibit the CYP2D6enzyme (Sakai et al 1991). Because diltiazem does not significantly affect hepaticblood flow (Bauer et al 1986), the changes in drug metabolism are most probablydue to the inhibition of CYP enzymes by diltiazem and/or its metabolites.

Diltiazem is also an inhibitor of P-gp (Cornwell et al 1987), and may therebyaffect the pharmacokinetics of substrates of P-gp. In fact, diltiazem can prolongthe t½ and decrease the Cl of digoxin, which is actively secreted by P-gp in renaltubular cells (Rameis et al 1984, de Lannoy and Silverman 1992).


20

1.6.3. MibefradilMibefradil belongs to a new class of calcium blockers; unlike traditional calciumblockers, it selectively blocks T-type (transient, low-voltage-activated) calciumchannels (Welker et al 1998). Mibefradil can thus serve as an oral treatment forhypertension and stable angina pectoris. It dilates the peripheral and coronaryvessels, which results in a reduction in arterial pressure and peripheral vascularresistance, and an increase in coronary artery blood flow (Welker et al 1998).Mibefradil reduces heart rate dose-dependently and does not cause reflextachycardia, as is commonly seen with dihydropyridine calcium blockers (Welkeret al 1998). The recommended dosage is 50 to 100 mg once daily.

About 99.5% of mibefradil is bound to plasma proteins, mainly to α1-acidglycoprotein (Welker et al 1998). The Vd of mibefradil in the steady state rangesfrom 130 l to 200 l (Dollery 1999b, du Souich et al 2000). Mibefradil exhibitsdose- and time-dependent pharmacokinetics that can be explained by partialsaturation of the metabolism or by inhibition of mibefradil metabolism bymibefradil itself or by accumulated metabolites (du Souich et al 2000). Its t½

averages 15 h, with a range of about 10 to 38 h (Dollery 1999b, Welker et al1998, du Souich et al 2000).

Mibefradil is eliminated largely by metabolism, and excreted mainly in the faeces(75%), to a lesser extent in the urine (25%); less than 3% is excreted unchangedin the urine. The metabolism of mibefradil is mediated by two main pathways,CYP3A4-mediated oxidation and hydrolysis of the ester side-chain. Suchhydrolysis produces Ro 40-5966, which is, however, the major plasma metabolite,possessing about 10% of the pharmacodynamic activity of the parent drug(Welker et al 1998). A total of some 30 metabolites of mibefradil have beendetected (Wiltshire et al 1997).

In vitro studies on human liver microsomes have shown mibefradil to be a potentinhibitor of CYP3A4 and CYP2D6 (Welker et al 1998, Wang et al 1999, Bohetset al 2000). In vivo, mibefradil has increased the total AUC of CYP3A4 substratescyclosporin and triazolam 2.7-fold and 9-fold, respectively, and enhanced thesedative effects of triazolam (Welker et al 1998, Backman et al 1999). Inaddition, mibefradil raises the plasma concentration of terfenadine and the meanQTc interval, resulting in an increased risk for cardiac arrhythmias (Welker et al1998). Mibefradil also considerably increases plasma concentrations ofmetoprolol, a substrate of CYP2D6 (Welker et al 1998). Besides inhibitingCYP3A4, mibefradil also inhibits P-gp in vitro (Wandel et al 2000). However, inhumans mibefradil raises plasma concentrations of digoxin only atsupratherapeutic levels (Siepmann et al 1995, Welker et al 1998).


21

Mibefradil was withdrawn from the market in 1998, within its first year afterlaunch, because of severe interactions with other drugs (Ellison 1998, Mullins etal 1998, Schmassmann-Suhijar et al 1998, Krähenbühl et al 1998, Anonymous1998).

1.6.4. Grapefruit juiceA finding in 1989 in an ethanol-felodipine interaction study suggested thatgrapefruit juice may increase the bioavailability of felodipine (Bailey et al 1989).This observation was confirmed in a subsequent study, in which the mean AUCof felodipine was increased 284% by grapefruit juice (Bailey et al 1991).

Grapefruit juice has been shown also to raise plasma concentrations of otherdihydropyridine calcium-channel blockers such as nifedipine, nisoldipine, andnitrendipine (Bailey et al 1991, Bailey et al 1993a, Soons et al 1991). Grapefruitjuice has increased the AUC of orally administered midazolam by 52% andenhanced its effects (Kupferschmidt et al 1995). However, Vanakoski et al (1996)found no interaction between grapefruit juice and oral midazolam. In addition,grapefruit juice has increased the plasma concentrations of such drugs ascyclosporin, terfenadine, and cisapride (Ducharme et al 1995b, Benton et al 1996,Kivistö et al 1999). Grapefruit juice appears to affect particularly those drugs withan extensive CYP3A4-mediated first-pass metabolism. It has greatly increased theAUC of of simvastatin (16-fold) and simvastatin acid (7-fold), the AUC oflovastatin (15-fold) and lovastatin acid (5-fold), and the AUC of buspirone (9-fold) (Lilja et al 1998a, Kantola et al 1998b, Lilja et al 1998b).

A study by Lee et al (1996) suggests that grapefruit juice inhibits 11β-hydroxysteroid dehydrogenase that oxidises cortisol to cortisone. However,grapefruit juice does not interact with prednisone and it had no effect on themetabolism of prednisone to prednisolone, a reaction catalysed by 11β-hydroxysteroid dehydrogenase (Hollander et al 1995).

Graprefruit juice reduces intestinal CYP3A4 without affecting hepatic CYP3A4activity. Therefore, the most probable mechanism for grapefruit juice interactionsis inhibition of first-pass metabolism by reduced CYP3A4-mediated metabolismin the intestine (Lown et al 1997, Evans 2000). For example, although grapefruitjuice raises plasma concentrations of oral cyclosporin, felodipine, and midazolam,it has no effect on their clearance after intravenous administration (Ducharme etal 1995b, Lundahl et al 1997, Kupferschmidt et al 1995).

Because grapefruit juice does not reduce the amount of CYP3A4 mRNA, itappears to damage the CYP3A4 enzyme, resulting in accelerated degradation ofthe enzyme (mechanism-based inhibition) (Lown et al 1997, Bailey et al 1998a,


22

Evans 2000). This ihibition is consistent with the finding that grapefruit juiceaffects felodipine pharmacokinetics even when the juice is ingested 24 h beforethe drug (Lundahl et al 1995). Thus, the return of CYP3A4 activity would need denovo enzyme synthesis (Bailey et al 1998a).

Grapefruit juice-drug interactions can be greater after repeated consumption thanafter a single glass of grapefruit juice (Gross et al 1999, Kivistö et al 1999). TheAUC values of oral cisapride have been increased by about 40% and 145% after asingle glass of regular strength and after repeated doses of double-strengthgrapefruit juice, respectively (Gross et al 1999, Kivistö et al 1999). In addition,repeated consumption of double-strength grapefruit juice has increased the t½ oforal cisapride, buspirone, and atorvastatin (Kivistö et al 1999, Lilja et al 1998b,Lilja et al 1999). Increase in t½ indicates an effect on hepatic CYP3A4.Accordingly, high amounts of grapefruit juice have been reported to affect bothpresystemic and systemic metabolism of oral midazolam, a well-known marker ofCYP3A4 (Veronese et al 1999).

Because of the overlapping substrate specificity of CYP3A4 and P-gp (Wacher etal 1995), it is possible that grapefruit juice affects the intestinal P-gp. Lown et al(1997) found grapefruit juice to reduce the amount of intestinal CYP3A4 withoutaffecting intestinal P-gp expression. Recently grapefruit juice has beendemonstrated in vitro to inhibit vinblastine cellular efflux by P-gp (Takanaga et al1998). Eagling et al (1999) have reported grapefruit juice components to inhibitCYP3A4-mediated metabolism of saquinavir, and to modulate to a limited extentP-gp-mediated transport in vitro. Eagling et al (1999) suggest that the in vivoeffects of grapefruit juice on saquinavir pharmacokinetics most likely result frominhibition of CYP3A4 and only to a minor extent from modulation of P-gp.

It was originally thought that the flavonoid naringin, which is related to thetypical bitter taste of grapefruit juice, and is its most abundant flavonoid, isresponsible for the interaction of grapefruit juice with drugs (Bailey et al 1998a,Evans 2000). Both naringin and its aglucone naringenin inhibit the metabolism offelodipine and nifedipine in vitro (Bailey et al 1998b, Miniscalco et al 1992).Also, furanocumarins can cause a mechanism-based inhibition of CYP enzymesand a furanocumarin called 6’,7’-dihydroxybergamottin is present in grapefruitjuice (Evans 2000). However, in vivo studies show that neither naringin nor 6’,7’-dihydroxybergamottin is the component responsible for grapefruit juice-druginteractions in humans (Bailey et al 1993b and 1998b).

A summary of selected characteristics of itraconazole, diltiazem, mibefradil, andgrapefruit juice is shown in Table II.


23


24

1.7. CYP3A4 and P-glycoproteinThe P-gp in humans is a MDR-1 (multiple drug resistance) gene product, whichacts as a transmembrane efflux pump. It has an important role in the developmentof multidrug resistance by tumour cells exposed to cancer chemotherapeuticagents (Sikic et al 1997). P-gp is found in a number of human organs, includinggut, liver, adrenal gland, and kidney (Thiebaut et al 1987).

P-gp has an important role in elimination of drugs into bile and urine, and inlimiting the drug penetration into brain through the blood-brain barrier (Jalava etal 1997, Schinkel et al 1994 and 1995). P-gp and CYP3A4 are colocalised inintestine, where they act together to limit the drug absorption into portalcirculation and to decrease plasma drug concentrations. Both P-gp and CYP3A4are expressed and upregulated co-ordinately in human cancer cells (Schuetz et al1996). In addition, known CYP3A4 inducers like rifampicin also induce P-gp(Schuetz et al 1996) and many inhibitors of CYP3A4 e.g. itraconazole are alsoinhibitors of P-gp (Kurosawa et al 1996, Wacher et al 1995).

Many drugs metabolised by CYP3A4 are also substrates for P-gp (Wacher et al1995). Accordingly, the coadministration of two CYP3A4 substrates can result ininteractions that reflect inhibition of the CYP3A4-mediated metabolism, reducedefflux by P-glycoprotein or a combination of both effects (Kivistö et al 1995).

2. Glucocorticoids2.1. GeneralThe adrenal glands are endocrine organs essential for life. This was established in1849 by Addison, who described a fatal outcome in a patient with adrenaldestruction. The adrenal gland consists of cortex and medulla. The adrenal cortexsecretes corticosteroids, glucocorticoids that regulate carbohydrate and proteinmetabolism, the mineralocorticoid aldosterone that regulates fluid and electrolytebalance, and also precursors of the sex steroids. The main glucocorticoid inhumans is cortisol. (Schimmer and Parker 1996)

Chemical modifications of the cortisol molecule have generated syntheticglucocorticoid derivates. Because effects of natural and synthetic glucocorticoidsare mediated by the same receptor, the glucocorticoids do not differ in theireffects in humans. However, changes in chemical structure may cause changes inpotencies relative to glucocorticoid and mineralocorticoid activity, alterations inabsorption, protein binding, and metabolism (Schimmer and Parker 1996). Allglucocorticoids, cortisol and its synthetic analogues, are referred as to C21steroids (Figure 1). Some areas of the steroid molecule are critical (Figure 2), and


25

alteration in any of these areas will result in loss of glucocorticoid activity(Swartz and Dluhy 1978).

Figure 1. Structure of cortisone, hydrocortisone (cortisol), and selected syntheticglucocorticoids.


26

Figure 2. Groups/areas essential for anti-inflammatory activity of glucocorticoids(Adapted from Swartz and Dluhy 1978)

Synthetic corticosteroids are grouped according to their relative potencies in Na+

retention and anti-inflammatory effects in relation to cortisol (Table III). Basedon these potencies, synthetic corticosteroids are divided into glucocorticoids andmineralocorticoids, although some steroids traditionally classified asglucocorticoids, such as prednisolone, also possess some mineralocorticoidactivity. Synthetic glucocorticoids with long plasma half-lives also possess longbiological half-lives, reflecting the duration of the activity at tissue level (TableIII) (Swartz and Dluhy 1978, Schimmer and Parker 1996).

Table III. Relative potencies of some natural and synthetic glucocorticoids(Modified from Swartz and Dluhy 1978, Schimmer and Parker 1996)

Compound Equivalentpotency (mg)

Anti-inflammatory

potency

Na+-retainingpotency

Biologicalhalf-life (h)

Cortisol 20 1 1 8-12Cortisone 25 0.8 0.8 8-12

Prednisone 5 4 0.8 18-36

Prednisolone 5 4 0.8 18-36

Methylprednisolone 4 5 0.5 18-36

Dexamethasone 0.75 25 0 36-54


27

2.2. Regulation of cortisol secretionThe main glucocorticoid of human beings is cortisol (hydrocortisone). Thesecretion of cortisol follows a circadian diurnal variation with peakconcentrations in the morning and the lowest concentrations occurringapproximately at midnight (Jusko et al 1975, Meibohm et al 1999). Esteban et al(1991) suggest that cortisol secretion rather than metabolism is mainlyresponsible for the changes in plasma cortisol.

Hypothalamus, pituitary, and adrenal cortex are collectively called thehypothalamic-pituitary-adrenal (HPA) axis. This system is responsible for thediurnal rhythm in basal cortisol secretion and the increase in cortisol secretion instress, e.g., in infection or surgery (Naito et al 1992). The adrenocorticotropinhormone (ACTH), which is secreted by the anterior pituitary gland, stimulates thesecretion of cortisol. The secretion of ACTH is regulated by a hypothalamichormone, corticotropin-releasing hormone (CRH). Cortisol has negative feedbackeffects on the hypothalamus and anterior pituitary gland to reduce formation ofCRH and ACTH and thereby can control its own secretion.

2.3. PharmacodynamicsIntracellular action of glucocorticoids. Glucocorticoids enter the target cell andbind the glucocorticoid receptor in cytoplasm. This binding activates the receptorthat moves into the nucleus and binds to specific glucocorticoid regulatoryelements (GREs). Glucocorticoids mainly increase the expression of the targetgenes. They can, however, also suppress gene transcription. Glucocorticoids havewidespread actions affecting many cells and tissues (Schimmer and Parker 1996,Berne and Levy 1998).

Carbohydrate, protein, and lipid metabolism. In the liver, glucocorticoidsenhance the formation of glucose from amino acids and glycerol, and thedeposition of glucose as glycogen. In peripheral tissues, glucocorticoids increaseprotein breakdown in muscle, bone, connective tissue, and skin. Furthermore,glucocorticoids antagonise the effect of insulin on glucose metabolism and inhibitinsulin-stimulated glucose uptake in cells. The net effect of glucocorticoids is anincrease in blood glucose concentrations. Glucocorticoids facilitate the effect oflipolytic substances, such as growth hormone, resulting in an increase in free fattyacids. An excess of glucocorticoids results in the typical distribution of fat in theback and neck, face, and abdomen, with loss of fat in the extremities.

Effects on muscle, bone, and connective tissue. Glucocorticoids reduce musclemass and strength and inhibit bone formation by several mechanisms. Theyreduce synthesis of the type I collagen essential for bone matrix. Glucocorticoids


28

reduce the availability of calcium for bone mineralisation. In addition,glucocorticoids increase bone reabsorption. Finally, the net effect is a decrease inbone density (osteoporosis). Glucocorticoids inhibit the synthesis of collagen,resulting in thinning of the skin and capillary walls.

Effects on the vascular system. Glucocorticoids enhance vascular reactivity toother vasoactive substances, e.g., angiotensin II. Glucocorticoids, even thoselacking mineralocorticoid activity, may cause hypertension, the mechanism ofwhich is still unclear.

Effects on the central nervous system. Glucocorticoids modulate behaviour andmood. Lack of cortisol in Addison’s disease may cause apathy and depression. Anexcess of glucocorticoids may cause insomnia, decrease memory skills, lower thethreshold to seizure activity, or even cause psychoses.

Effects on inflammatory and immune responses. Glucocorticoids reduce thenumber of lymphocytes and reduce the mass of lymphoid tissue. In addition,glucocorticoids inhibit such processes as the synthesis and release of arachinodicacid, a precursor for many mediators of immune responses. The hormone reducesthe number and function of thymus-derived lymphocytes (T-cells), therebyinhibiting cell-mediated immunity. The production of e.g., interleukin-1, -2, -6,and tumour necrosis factor α is reduced. The suppression of T-cell activation alsosuppresses the activation of B-lymphocytes. The net effects of these complexactions are diminished inflammatory and immune responses.

2.4. Therapeutic useThe finding of Hench et al in 1949 that cortisone has beneficial effects intreatment of rheumatoid arthritis generated great interest in cortisol and itssynthetic analogues. In addition to being replacement therapy for adrenalinsufficiency, glucocorticoids are widely used because of their anti-inflammatoryand immunosuppressive properties in treatment of nonendocrine diseases.

Systemic glucocorticoids are included in the standard immunosuppressive drugregimen for organ-transplant patients (Mies et al 1998, Sarna et al 1997, Pericoand Remuzzi 1997). They are used in treatment of diseases of connective tissue,e.g., rheumatoid arthritis, polymyositis, and systemic lupus erythematosus(LaRochelle et al 1993, Laan et al 1999) and in treatment of acute exacerbation ofasthma (Rowe et al 2000). Many antineoplastic combinations used to treat acuteleukaemia, lymphomas, and myeloma include glucocorticoids (Gaynon andCarrel 1999, Cain et al 1998, Oken 1997). Furthermore, renal diseases, e.g.,nephrotic syndrome secondary to minimal change disease usually respond well tothe glucocorticoids (Friedman and Chesney 1982). In addition, glucocorticoidsare used for the management of nausea and vomiting induced by cancer


29

chemotherapy and for management of brain tumours (Ater et al 1997, Perez1998).

Usually the appropriate dose has to be determined case by case. The dosage ofglucocorticoid varies, depending on nature and severity of the disease. Prolongedtherapy with doses that exceed the normal physiological level of glucocorticoidrequire careful consideration. If possible, the total daily dose should be given as asingle morning dose, and an alternate day therapy has been recommended tominimise the catabolic actions and HPA-axis suppression of glucocorticoids(Melby 1974, Swartz and Dluhy 1978).

Interestingly, the use of systemic glucocorticoids in Finland is steadily increasing(Figure 3); for example by about 60% from the year 1988 to 1998 measured asdefined daily doses per 1000 inhabitants per day (DDD/1000 inh/day). In 1998,the use of systemic glucocorticoids was 12.42 DDD/1000 inh/day, which means62 100 DDD/5 million inhabitants every day.

02468

101214

1988

1990

1992

1994

1996

1998

Years

DD

D/1

000

inh

/day

Figure 3. Use of systemic glucocorticoids in Finland, 1988-1998, in defined dailydoses per 1000 inhabitants per day (DDD/1000 inh/day) (Finnish Statistics onMedicines 1988-1998; National Agency for Medicines and Social InsuranceInstitution)


30

2.5. Adverse effects of glucocorticoid treatmentGlucocorticoid adverse effects can be described as an extension of theirpharmacodynamic and physiological effects. Synthetic glucocorticoids cansuppress endogenous cortisol secretion and abolish the daily circadian rhythm ofcortisol (Salassa et al 1953, Melby 1974, Slayter et al 1996). The failure of theadrenal gland to response to stress (infection, surgery) is one major andunpredictable complication of glucocorticoid therapy (Fraser et al 1952, Salassaet al 1953, Melby 1974, Henzen et al 2000). Treatment with high doses ofglucocorticoids even for less than a week and daily doses as low as 5 mgprednisone equivalent or less can, in some sensitive individuals, impair theadrenal response (Streck and Lockwood 1979, Schlaghecke et al 1992, Henzen etal 2000). Neither the dose of glucocorticoid nor the duration of the treatmentappears to predict the adrenal insufficiency (Livanou et al 1967, Schlaghecke et al1992, Henzen et al 2000).

Glucocorticoids increase the susceptibility to bacterial, viral, and fungalinfections (Twycross 1994). Severe, even fatal varicella cases have beenassociated with corticosteroid use (Kasper and Howe 1990, Dowell and Bresee1993). Glucocorticoids can cause myopathy, which may be severe enough todisturb daily activities and even impair respiratory function (Batchelor et al1997). Osteoporosis and vertebral compression fractures are seriouscomplications of glucocorticoid therapy (Adachi et al 1993). It has been estimatedthat the incidence of fractures in patients receiving chronic glucocorticoid therapyis about 30% to 50% (Adachi et al 1993). In addition, glucocorticoids can causehyperglycaemia and increase insulin requirements of diabetes patients (David etal 1980, Twycross 1994). Furthermore, glucocorticoids can cause electrolyteabnormalities, hypertension, psychiatric disorders, cataract, a typical moon-face,striae, and truncal obesity (Melby 1974, Swartz and Dluhy 1978, Twycross 1994,Jenkins 1999). In children treatment with glucocorticoids can result in growthretardation (Sarna et al 1997).

2.6. MethylprednisolonePharmacokinetics. Methylprednisolone is rapidly absorbed following oraladministration with its Cmax reached within 1 to 3 h (Antal et al 1983, Al-Habetand Rogers 1989). The mean absolute bioavailability of methylprednisolone ishigh, ranging from 80% to 90% (Antal et al 1983, Al-Habet and Rogers 1989,Patel et al 1993). However, Patel et al (1993) reported large interindividualvariability in absolute bioavailability, ranging from 50% to completebioavailability.


31

Methylprednisolone is about 80% bound to plasma protein with only a littlebound to the corticosteroid-binding protein, transcortin (Szefler et al 1986, Konget al 1989). The mean Vd of methylprednisolone averages 1.2 to 1.5 l/kg (Al-Habet and Rogers 1989, Kong et al 1989, Szefler et al 1986). Ferry et al (1994)suggest that methylprednisolone exhibits dose- and time-dependentpharmacokinetics. Others have, however, shown the pharmacokinetics to belinear in the dose ranges 0 to 40 mg (Kong et al 1989), 20 to 80 mg (Al-Habet andRogers 1989), and 540 to 1026 mg (Assael et al 1982). The mean t½ ofmethylprednisolone averages 1.9 to 3.3 h (Szefler et al 1986, Ferry et al 1994, Al-Habet and Rogers 1989). The systemic plasma clearance (Cl) ofmethylprednisolone averages 350 to 450 ml/h/kg (Al-Habet and Rogers 1989,Patel et al 1993).

Due to its lipophilicity and low water-solubility, methylprednisolone itself can notbe administered intravenously. Methylprednisolone sodium succinate is a prodrugand is hydrolysed in the body rapidly and almost completely with a half-life ofabout 4 minutes to active methylprednisolone alcohol (Al-Habet and Rogers1989).

Methylprednisolone undergoes reduction of the ketone group at the C20 position,yielding the inactive metabolites 20α- and 20β-hydroxymethylprednisolone, andfurther oxidation of the C20,C21 side chain (Stjernholm and Katz 1975, Vree et al1999). The 6α-methyl group has been suggested to prevent the hydroxylation atC6 position. However, Vree et al (1999) found 6β-hydroxy-6α-methylprednisolone and a compound that might be 6α-hydroxy-6β-methylprednisolone in the urine of patients receiving methylprednisolone.Oxidation at C11 of methylprednisolone produces a minor metabolite,methylprednisone; this reaction is reversible. In addition, Rodchenkov et al(1987) found 6,7-dehydro metabolites in urine, indicating that formation of a 6,7-double band may be possible. The suggested metabolic pathways ofmethylprednisolone are presented in Figure 4. The specific CYP enzymesinvolved in the various metabolic pathways of methylprednisolone have not beendetermined.

After radio-labelled doses of methylprednisolone, a total of 75% of the label wasdetected in urine and 20% in bile (Slaunwhite and Sandberg 1961). Furthermore,during the first 24 h after administration of methylprednisolone sodium succinate,about 30% of the dose was excreted in the urine: 10% as unmetabolised ester,12% as methylprednisolone, 8% as 20α- and 1.0% as 20β-hydroxymethylprednisolone, and less than 1.0% as methylprednisone (Lawson etal 1992). In urine, glucuronides, sulphates and unconjugated metabolites werepresent (Slaunwhite and Sandberg 1961).


32

Figure 4. Metabolic pathways of methylprednisolone reported in the literature(Adapted from Vree et al 1999).

Women appear to eliminate methylprednisolone more rapidly than do men;however, the clinical importance of this observation is unclear (Lew et al 1993).The Cl of methylprednisolone is slower in elderly (range 69-82 years) than inyounger (range 24-37 years) men (Tornetore et al 1994). This pharmacokineticalteration may increase the risk for adverse effects among elderly patients(Tornetore et al 1994). Furthermore, the Cl of methylprednisolone was lower inthe obese than in non-obese subjects (Dunn et al 1991). The metabolism ofmethylprednisolone may be impaired in severe liver disease; however, patientswith renal impairment appears to need no dose adjustment of methylprednisolone(Dollery 1999c).

Pharmacokinetic drug interactions. Enzyme inducers phenobarbital, phenytoin,and carbamazepine induce metabolism of methylprednisolone. Phenobarbitalincreased the Cl of methylprednisolone by 208%, phenytoin by 478%, andcarbamazepine by 342%, suggesting clinically significant interactions(Stjernholm and Katz 1975, Bartoszek et al 1987).

The effect of some CYP3A4 inhibitors on intravenous methylprednisolone hasbeen evaluated. Troleandomycin decreased the Cl of intravenousmethylprednisolone by 64% and increased its mean t½ from 2.5 to 4.6 h (Szefleret al 1980). Erythromycin 250 mg 4 times daily for one week decreased the Cl ofmethylprednisolone by 46% and increased its t½ by 51%, from 2.4 to 3.5 h(LaForce et al 1983). In addition, ketoconazole increased the methylprednisoloneAUC by 135% and reduced its Cl by 66% after intravenous administration (Glynn


33

et al 1986). Kandrotas et al (1987) recommend a 50% lower dose ofmethylprednisolone during concomitant use of ketoconazole andmethylprednisolone.

Cimetidine did not affect methylprednisolone disposition (Green et al 1984).Furthermore, cyclosporin appears not to affect methylprednisolone dispositionfollowing intravenous administration of methylprednisolone (Tornatore et al1993). Oral contraceptives decreased the Cl of intravenous methylprednisolone,increased total AUC by 49%, and t½ from 1.7 to 2.2 h (Slayter et al 1996).

A summary of pharmacokinetic variables of methylprednisolone, prednisolone,and dexamethasone is shown in Table IV (page 37).

2.7. PrednisolonePharmacokinetics. Prednisolone is rapidly absorbed after oral administration andreaches the Cmax within approximately 1 to 2 h (Leclercq and Copinschi 1974,Pickup 1979). The mean absolute bioavailability of prednisolone ranged from80% to about 100% (Petereit and Meikle 1977, Al-Habet and Rogers 1980). Theprotein binding of prednisolone varies from 65% to 90% (Pickup 1979).Prednisolone is reversibly bound to albumin and to transcortin. The degree ofprotein binding of prednisolone is concentration-dependent, since binding totranscortin is saturable (Pickup 1979).

The Vd of prednisolone increases with dose from about 0.35 l/kg to 0.7 l/kg (Begget al 1987). In addition, the Cl of prednisolone is dose-dependent. After a 5 mgand 40 mg dose of prednisolone the Cl has been about 7 l/h and 12 l/h,respectively (Rose et al 1981a). These dose-dependent changes may result from aconcentration-dependent change in protein binding. Because both Vd and the Clincrease, there may be none (Tanner et al 1979, Bergrem et al 1983) or only aminor dose-dependent change in the t½ of prednisolone (Rose et al 1981a). Themean t½ of prednisolone has ranged from 2.6 h to 4.5 h (Al-Habet and Rogers1980, Rose et al 1981a, Bergrem et al 1983).

The oxidation of prednisolone at C11 position produces prednisone, a syntheticglucocorticoid in an inactive form. This metabolic reaction is interconversible andfavours the formation of prednisolone. The concentrations of prednisolone are 4to 10 times as high as those of prednisone after administration of eitherprednisone or prednisolone (Rose et al 1981a). Prednisolone can be hydroxylatedat the 6β position like cortisol, and the urinary 6β-hydroxyprednisolone makes up8% to 10% of the administered prednisolone dose and some 2% to 5% is excretedas prednisone in the urine (Rose et al 1981a, Frey et al 1987, Zürcher et al 1989).About 7% to 24% of the prednisolone dose is excreted in the urine as the parent


34

drug (Rose et al 1981a, Frey et al 1987, Zürcher et al 1989). Furthermore, bothprednisone and prednisolone can undergo reduction of the ketone group at C20(Gray et al 1956). After radiolabelled doses of prednisolone, more than 90% ofthe label is detected in urine and less than 5% in bile (Slaunwhite and Sandberg1961).

The specific CYP enzymes involved in the metabolic pathways of prednisolonehave not been determined. Based on the fact that 6β-hydroxylation of cortisol iscatalysed by CYP3A4 and CYP3A5 (Ged et al 1989, Wrighton et al 1990), theseenzymes have also been assumed to be responsible for the 6β-hydroxylation ofprednisolone, although this is a relatively minor metabolic pathway ofprednisolone.

The Cl of prednisolone appears to be slightly, about 20%, higher in females thanin males (Meffin et al 1984), the clinical significance of which remains unclear.The Cl of prednisolone is 35% higher in obese subjects than in non-obesesubjects, and dosage should reflect total body weight (Milsap et al 1984). Its Clmay be slightly decreased in chronic active hepatitis (Powell and Axelsen 1972);however, in a study by Kawai et al (1985), liver disease does not affect itspharmacokinetics. Smoking does not affect the pharmacokinetics of prednisolone(Rose et al 1981b). In addition, results concerning the autoinduction ofprednisolone metabolism during long-term therapy are contradictory (Begg et al1987).

Pharmacokinetic drug interactions. Rifampicin, phenobarbital, carbamazepine,and phenytoin induce the metabolism and increase the Cl of prednisolone(McAllistar et al 1983, Brooks et al 1976, Bartoszek et al 1987, Petereit andMeikle 1977). Concomitant use of rifampicin or phenobarbital with prednisolonecan worsen the symptoms of the disease being treated, e.g., nephrotic syndromeor rheumatoid arthritis (Hendrickse et al 1979, Brooks et al 1976).

Lanzoprazole and omeprazole, following prednisone administration, do not affectthe pharmacokinetics of prednisolone (Cavanaugh and Karol 1996). Thepharmacokinetics of prednisolone are altered only slightly after cimetidine andranitidine, and this interaction appears not to be clinically significant (Sirgo et al1985). Neither azathioprine nor interleukin-10 affect the pharmacokinetics ofprednisolone (Frey et al 1981, Chakraborty et al 1999). In addition, indomethacinand naproxen have increased free prednisolone concentration by 30 to 60%without affecting total prednisolone concentrations (Rae et al 1982). Oralcontraceptive steroids have increased the t½ of prednisolone about 1.5- to 2.5-fold, and careful monitoring is recommended when oral contraceptives andprednisolone are used concomitantly (Legler and Benet 1986).


35

The CYP3A4 inhibitor ketoconazole (200 mg/day) has increased plasmaconcentrations of prednisolone by about 50% following oral prednisone orintravenous prednisolone administration (Zürcher et al 1989). Two other studies,however, failed to find any effect with 200 mg/day ketoconazole on either thepharmacokinetics or pharmacodynamics of prednisolone (Ludwig et al 1989,Yamashita et al 1991). Furthermore, neither troleandomycin nor grapefruit juiceaffected its pharmacokinetics (Szefler et al 1982, Hollander et al 1995). However,administration of diltiazem increased the AUC of prednisolone by about 20%after ingestion of prednisone (Imani et al 1999). Whether cyclosporin affects theelimination of prednisolone is unclear. Two reports have suggested cyclosporininhibits the elimination of prednisolone (Öst 1984, Langhoff et al 1985). Incontrast, some investigators have found cyclosporin not to alter its metabolism(Frey et al 1987, Rocci et al 1988).

2.8. DexamethasonePharmacokinetics. Dexamethasone is rapibly absorbed after oral administration.The Cmax of oral dexamethasone is reached between 1.2 and 2.7 h (Rose et al1981b, Loew et al 1986, O’Sullivan et al 1997). Although mean absolutebioavailability of dexamethasone has ranged from 61 to 78% (Duggan et al 1975,Rose et al 1981b, O’Sullivan et al 1997), large interindividual variation in theabsolute bioavailability can exist ,e.g., from 51% to 89% in healthy volunteersand from 34% to 79% in depressed patients (O’Sullivan et al 1997).

Dexamethasone is about 77% bound to plasma protein, mainly albumin, and itappears neither to bind transcortin nor to compete with cortisol for its bindingprotein (Peets et al 1969). Dexamethasone bound to albumin is unaffected byincreasing dexamethasone concentrations (Peets et al 1969). The mean Vd ofdexamethasone averages 0.58 to 1.5 l/kg (Rose et al 1981b, Workman et al 1986,O’Sullivan et al 1997). Loew et al (1986) found the AUC of dexamethasone toincrease less than proportionally to dose after oral 0.5- to 1.5-mg doses. However,no dose-dependency was observed when dexamethasone was administered tohealthy subjects at a 15-mg dose and at high 1.5 mg/kg doses or to cancer patientsat 40- to 200-mg doses (Rohdewald et al 1987, Brady et al 1987). The t½ ofdexamethasone averages 3.1 to 6.1 h and its Cl 110 to 180 ml/h/kg (Rose et al1981b, O’Sullivan et al 1997).

Dexamethasone sodium phosphate is a prodrug. After intravenous administration,the hydrolysis of the phosphate ester to dexamethasone is rapid, with half-life of 5min, and the Cmax of dexamethasone is reached after 10 min (Rohdewald et al1987). Conversion of dexamethasone phosphate to dexamethasone alcoholappears to be about 90% (Hare et 1975).


36

The 6β-hydroxylation is the major metabolic pathway of dexamethasone(Minagawa et al 1986), and hydroxylation both at the 6α and at the 6β positionhave shown to be CYP3A4-mediated in human liver microsomes in vitro (Gentileet al 1996). About 9 to 10% of the dose was excreted as unchangeddexamethasone in the urine (Duggan et al 1975, Miyabo et al 1981), withglucuronides present in small amounts (Minagawa et al 1986, Tomlinson et al1996). After an intravenous dose of labelled dexamethasone, about 64% wasexcreted in the urine within 24 h, and unconjugated metabolites formed thelargest fraction, about 30% of the dose (Haque et al 1972).

The t½ of dexamethasone has been reported to be prolonged in liver disease andshortened in chronic renal failure (Kawai et al 1985). However, Workman et al(1986) found chronic renal failure not to affect the pharmacokinetics ofdexamethasone. The t½ of oral dexamethasone was longer in men than in women(6.4 h vs. 4.8 h), and the gender difference appears to be weight-related(O’Sullivan et al 1997). In addition, smoking did not affect the pharmacokineticsof dexamethasone (Rose et al 1981b).

Pharmacokinetic drug interactions. Phenobarbital has increased the Cl ofdexamethasone by 88% and decreased its t½ by 44% (Brooks et al 1972).Phenytoin increased the Cl and reduces the systemic bioavailability ofdexamethasone (Chalk 1984). Carbamazepine 800 mg daily raises the dose ofdexamethasone needed to suppress plasma cortisol 2- to 4-fold, when measuredas a part of the dexamethasone suppression test (Kobberling and v zur Muhlen1973).

Treatment with ephedrine has decreased the t½ of dexamethasone by 36% andincreased its Cl by 42%, but theophylline appears not to interact withdexamethasone (Brooks et al 1977). Cimetidine does not affect thepharmacokinetics of intravenous dexamethasone (Peden et al 1984). There appearto be no controlled studies concerning the pharmacokinetic interactions betweenCYP3A4 inhibitors and dexamethasone in humans.

A summary of pharmacokinetic variables of methylprednisolone, prednisolone,and dexamethasone is shown in Table IV.


37

AIMS OF THE STUDY

38

AIMS OF THE STUDY

Synthetic glucocorticoids are widely used because of their anti-inflammatory andimmunosuppressive properties in many different types of formulations. Thisstudy, however, will focus on systemic oral and/or intravenous glucocorticoidsmethylprednisolone, prednisolone, and dexamethasone. It has been shown thatthese glucocorticoids are eliminated mainly by metabolism, and CYP3A4 mayplay a different role in their biotransformation. Many drugs currently on themarket are known to inhibit CYP3A4-mediated metabolism in vitro and in vivo.CYP3A4 is also present in the gut, and inhibition of drug metabolism can occureven in the intestinal mucosa, a fact which may be important in regard to orallyadministered drugs.

Glucocorticoids have a very wide therapeutic margin, and high doses can begiven intravenously, e.g., in an emergency. However, for continuous use at dosesas low as possible, intermediate-acting glucocorticoids and alternate-day therapyhave been recommended to minimise the catabolic action and HPA-axissuppression of glucocorticoids. Thus, pharmacokinetic interaction ofglucocorticoids with other drugs, resulting in an increased and prolongedexposure to glucocorticoids, can be clinically important. Increased knowledge ofpotential drug-drug interactions of systemic glucocorticoids would result in moreproper and safe use of these drugs.

The specific aims of this thesis were to study:

1. The effect of itraconazole on the pharmacokinetics and adrenal-suppressanteffect of oral and intravenous methylprednisolone (Studies I and II), with specialattention to the route of methylprednisolone administration.

2. The effect of itraconazole on the pharmacokinetics and adrenal-suppressanteffect of oral prednisolone (Study IV)

3. The effect of itraconazole on the pharmacokinetics and adrenal-suppressanteffect of oral and intravenous dexamethasone (Study VI), with special attentionto the route of dexamethasone administration.

4. The effect of diltiazem and mibefradil on the pharmacokinetics and adrenal-suppressant effect of oral methylprednisolone (Study III)

5. The effect of grapefruit juice on the pharmacokinetics and adrenal-suppressanteffect of oral methylprednisolone (Study V).

MATERIALS AND METHODS

39


1. Ethical considerationsAll study protocols were approved by independent Ethics Committee of theDepartment of Clinical Pharmacology, University of Helsinki, and reviewed by theNational Agency of Medicines in Finland. Before entering the study all subjectsreceived both oral and written information and gave their written consent.

2. Study subjectsAll subjects participating in Studies I to VI were healthy volunteers (age range 19-42 years; weight 45-90 kg). Altogether 43 study subjects (25 females, 18 males)participated in Studies I to IV. A total of ten subjects participated in more than onestudy; seven subjects in two, two subjects in three and one subject in four times.None of the subjects were smokers or used continuous medication, except that inStudies I to III eight female subjects used oral contraceptive steroids. Use of otherdrugs was prohibited starting 2 weeks before each study day. Furthermore,participation in another trial and blood donation within one month before the studyperiod were exclusion criteria. Each subject underwent a medical screening 1 to 3weeks before participation in a study. This screening included clinicalexamination, medical history, and routine laboratory tests, and in Study III astandard electrocardiogram (ECG) to ascertain that each was in good health.

3. Study protocol3.1. Study designIn Studies I to IV and VI the subjects were pretreated with a potential enzymeinhibitor or placebo and in Study V with double-strength grapefruit juice or water.A randomised crossover design was used in two or three phases with a washoutperiod of 4 weeks. All studies were double-blind, except Study V, which was anopen study.

The pretreatment drugs and matched placebos were supplied, packed, and labelledaccording to a randomisation list for each subject by the Pharmacy of the HelsinkiUniversity Central Hospital. Pretreatment drugs and matched placebos wereidentical in order to maintain the the double-blind nature of the studies. The studydrugs (glucocorticoids) were also supplied by the Pharmacy of the HelsinkiUniversity Central Hospital. Glucocorticoids were given under the supervision ofthe investigator in the morning at 9:00 after an overnight fast. The oral study drugs


40

were ingested with 150 ml of water, except in Study V with 200 ml of water ordouble-strength grapefruit juice. Consumption of grapefruit juice was prohibited,except in Study V, and use of alcohol was not allowed during the study days and 2days beforehand. A light standard meal was served at 2 and 10 h and lunch 6 hafterwards. The structure of the studies is described below and in Table V.

Table V. Structure of Studies I-VI

Study Pretreatment Study drug/ GlucocorticoidNo. Subject Drug Daily dose Duratio

nDrug, dose androute

Timing

I n = 10 ItraconazolePlacebo

200 mg x 1 Days 1-4Days 1-4

Methylprednisolone16 mg orally

On day 4,1 h afterpretreatment

II n = 9 ItraconazolePlacebo


Methylprednisolone16 mg intravenouslyas sodium succinate


III n = 9 Diltiazem

MiberadilPlacebo

60 mg x 360 mg x 250 mg x 1

Days 1-2Day 3Days 1-3Days 1-3


On day 3,2 h aftermorningpretreatmentdose

IV n = 10 ItraconazolePlacebo


Prednisolone20 mg orally


V n = 10 GrapefruitjuiceWater

200 ml x 3 Days 1-3

Days 1-3


With 200 mlgrapefruit juicein morning ofday 3

VI n = 8 ItraconazolePlacebo


Dexamethasone4.5 mg orallyDexamethasonesodium phosphate5.0 mgintravenously



41

Study I. Ten subjects (7 females/3 males) each received 200 mg itraconazole orplacebo orally once daily for 4 days. On day 4, a dose of 16 mgmethylprednisolone was ingested at 9:00 a.m., one hour after theitraconazole/placebo. Blood samples were drawn before the methylprednisoloneadministration and ½, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, and 24 h later. Plasma wasseparated within 30 min and stored at -40° C until analysis. Methylprednisolone,cortisol, itraconazole, and hydroxyitraconazole concentrations were determined.

Study II. Nine subjects (4 females/5 males) each received 200 mg itraconazole orplacebo orally once daily for 4 days. On day 4, an intravenous dose of 16 mgmethylprednisolone as sodium succinate was given within 2 min at 9:00 a.m., onehour after itraconazole/placebo. Blood samples were drawn beforemethylprednisolone administration and 3 min and ½, 1, 2, 3, 4, 5, 6, 8, 10, 12, and24 h later. The sampling tubes were immediately placed into ice water, and plasmawas separated within 30 min by centrifugation at +4°C. The samples were stored at-80°C until analysis. Methylprednisolone, cortisol, itraconazole, andhydroxyitraconazole concentrations were determined.

Study III. Nine subjects (8 females/1 male) each received 60 mg diltiazem 3 timesa day (at 7:00 a.m., 3:00 p.m., and 9:00 p.m.), 50 mg mibefradil once a day (at 7:00a.m. and corresponding placebo at 3:00 p.m. and 9:00 p.m.), or placebo three timesa day, all of these orally and all for 2 days. On the next day (day 3), only twopretreatment doses were given (at 7:00 a.m. and 3:00 p.m), and a dose of 16 mgmethylprednisolone was ingested at 9:00 a.m., 2 h after diltiazem, mibefradil, orplacebo. Blood samples were drawn before methylprednisolone administration andat ½, 1, 1½, 2, 3, 4, 5, 6, 8, 10, 12, 23, and 47 h. Plasma was separated within 30min and stored at -80° C until analysis. Methylprednisolone, cortisol, diltiazem,and mibefradil concentrations were determined. Each subject underwent ascreening including ECG, blood pressure, and heart rate on the first and third dayof each study period.

Study IV. Ten subjects (5 females/5 males) each received 200 mg itraconazole orplacebo orally once daily for 4 days. On day 4, a dose of 20 mg prednisolone wasingested at 9:00 a.m., one h after itraconazole/placebo. Blood samples were drawnbefore prednisolone administration and at ½, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 23, and47 h. Plasma was separated within 30 min and stored at -40 °C until analysis.Prednisolone, cortisol, itraconazole, and hydroxyitraconazole concentrations weredetermined.

Study V. Ten subjects (2 females/8 males) each drank 200 ml double-strengthgrapefruit juice or 200 ml water 3 times a day for 2 days. On day 3, a dose of 16mg methylprednisolone was ingested at 9:00 a.m. with 200 ml double-strengthgrapefruit juice or water. In addition, 200 ml grapefruit juice or water was given ½


42

and 1½ h after methylprednisolone administration. Blood samples were drawnbefore methylprednisolone administration and at ½, 1, 1½, 2, 3, 4, 5, 6, 8, 10, 12,23, and 47 h. Plasma was separated within 30 min and stored at -40 °C untilanalysis. Methylprednisolone and cortisol concentrations were determined.

Study VI. Eight subjects (7 females/1 male) each ingested either 200 mgitraconazole (during 2 phases) or placebo (during 2 phases) once daily for 4 days.On the fourth day, at 9:00 a.m., one h after itraconazole/placebo, each subjectreceived 4.5 mg dexamethasone orally or a dose of 5.0 mg dexamethasone sodiumphosphate (corresponds to 3.85 mg dexamethasone) intravenously during both theitraconazole and placebo phases. Dexamethasone sodium phosphate was given asan intravenous injection over 2 min. Blood samples were drawn beforedexamethasone administration and at 1, 2, 3, 4, 6, 8, 10, 12, 23, 47, and 71 h. Afterintravenous administration, an additional sample was drawn 0.25 h afterdexamethasone injection. Plasma was separated within 30 min and stored at -70°Cuntil analysis. Dexamethasone, cortisol, itraconazole, and hydroxyitraconazoleconcentrations were determined.

3.2. Determination of plasma drug and cortisol concentrationsAll plasma drug and cortisol concentrations were determined in the AnalyticalLaboratory of the Department of Clinical Pharmacology, University of Helsinki.

Methylprednisolone. In Studies I, II, and III, plasma methylprednisoloneconcentrations were determined by high-performance liquid chromatography(HPLC) with ultraviolet detection (Ebling et al 1984, Kong et al 1988).Dexamethasone served as the internal standard. The quantification limit was 3ng/ml in Studies I and II and 2 ng/ml in Study III. The interday coefficient ofvariation (CV) for methylprednisolone ranged from 5.8% to 11.9% at < 20 ng/mlconcentrations and 1.2% to 4.9% at higher methylprednisolone concentrations.

In Study V, the plasma methylprednisolone concentrations were determined byliquid chromatography-ionspray-tandem mass spectrometry with use of the PESCIEX API 3000 LC/MS/MS system (Sciex Division of MDS Inc, Toronto,Canada) (Kong et al 1988, Dodds et al 1997). Dexamethasone served as theinternal standard. The quantification limit was 0.5 ng/ml and the interday CV 6.3%at 4.3 ng/ml, 2.3% at 25 ng/ml, and 3.6% at 118 ng/ml (n = 4 at all concentrations).

Prednisolone. Plasma prednisolone concentrations were determined by HPLCwith ultraviolet detection (McWhinney et al 1996). Dexamethasone served as theinternal standard. The quantification limit was 5 ng/ml and the interday CV 8.5%at 11 ng/ml, 2.8% at 49 ng/ml, and 3.8% at 227 ng/ml (n = 6 at all concentrations).


43

Dexamethasone. Plasma dexamethasone concentrations were determined byHPLC with ultraviolet detection (McWhinney et al 1996). Methylprednisoloneserved as the internal standard. The quantification limit was 2 ng/ml and theinterday CV 9.0% at 3.7 ng/ml (n = 11), 1.6% at 36 ng/ml (n = 14), and 2.2% at158 ng/ml (n = 14).

Itraconazole and hydroxyitraconazole. Itraconazole and hydroxyitraconazoleplasma concentrations were determined by HPLC with fluorescence detection(Allenmark et al 1990, Remmel et al 1988). R51012 (Janssen Pharma, Beerse,Belgium) served as the internal standard. The quantification limit was 10 ng/ml forboth compounds. The interday CV for itraconazole ranged from 0.5% to 5.6% andthat for hydroxyitraconazole from 0.7% to 9.3% at relevant itraconazoleconcentrations.

Diltiazem. Plasma diltiazem concentrations were determined by HPLC withultraviolet detection (Dube et al 1988, Hynning et al 1988). Verapamil served asthe internal standard. The quantification limit was 10 ng/ml, and the interday CVfor diltiazem was 4.0% at 24.7 ng/ml (n = 6) and 3.4% at 232 ng/ml (n = 6).

Mibefradil. Plasma mibefradil concentrations were determined by HPLC(Skerjanec and Tam 1995) with fluorescence detection. An external standardserved in this assay. The quantification limit was 10 ng/ml. The intraday CV formibefradil was 1.5% at 15.8 ng/ml (n = 10) and 2.0% at 50.9 ng/ml (n = 10).

Cortisol. In Studies I to IV and VI plasma cortisol concentrations were determinedby HPLC with ultraviolet detection (Ebling et al 1984, Kong et al 1988,McWhinney et al 1996). Dexamethasone served as the internal standard. Thequantification limit was 3 ng/ml in Studies I and II, 2 ng/ml in III and IV, and 5ng/ml in VI. The interday CV for cortisol ranged from 3.7% to 14.5% at < 20ng/ml concentrations and from 1.6% to 6.5% at higher cortisol concentrations.

In Study V, the plasma cortisol concentrations were determined by liquidchromatography-ionspray-tandem mass spectrometry with use of the PE SCIEXAPI 3000 LC/MS/MS system (Sciex) with dexamethasone as the internal standard.The quantification limit was 0.5 ng/ml. The interday CV was 1.2% at 4.1 ng/ml,6.1% at 25 ng/ml, and 4.3% at 123 ng/ml (n = 5 at all concentrations).

3.3. Pharmacokinetic calculationsThe peak concentration in plasma (Cmax) and time to Cmax (tmax) were obtaineddirectly from the data. The elimination rate constant (kel) was determined forcalculation of elimination half-life (t½) and total area under the plasmaconcentration-time curve [AUC(0-∞)]. The terminal log-linear phase of the plasmaconcentration-time curve was visually identified for each subject. The elimination


44

rate constant (kel) was calculated by regression analysis of this log-linear part of theplasma concentration-time curve. The t½ was calculated from the equation

t½ = ln2 / kel

The area under the plasma concentration-time curve (AUC) values were calculatedby use of the linear trapezoidal rule (Studies I, II and IV) or by log trapezoidal rule(Studies III, V, and VI). To determine the AUC to infinity, the extrapolation toinfinity was done by dividing the last measured concentration by the correspondingkel value. AUC values were calculated, referring to the time from 0 to 24 h after theadministration of the study drug in Study I. Furthermore, AUC values from 12 to24 h (Study II), from 12 to 23 h (Studies III and VI), and from 23 to 47 h (StudyVI) were calculated as a posthoc analysis of AUC. Plasma concentrations forcalculation of the AUC(12-24), AUC(12-23), and AUC(23-47) values wereestimated with use of kel values when the concentrations were below thequantification limit. Furthermore, for itraconazole, diltiazem, and mibefradil, theAUC values [AUC(0-24) or AUC(0-23)] after administration of the study drugwere calculated by linear trapezoidal rule.

In Studies II and VI, after intravenous administration of methylprednisolone anddexamethasone the systemic clearance (Cl) and volume of distribution (Vd) werecalculated from the equations

Cl = Dose / AUC(0-∞)

Vd = Cl / kel

In Study VI the bioavailability of oral dexamethasone (F) was calculated (Gibaldiand Perreir 1982) from the equation

F = [AUC(0-∞)po·Doseiv] / [AUC(0-∞)iv·Dosepo]

For the pharmacokinetic calculations, the conversion of intravenousdexamethasone sodium phosphate to dexamethasone alcohol was expected to be90% (Hare et al 1975). The pharmacokinetic program, MK model, version 5(Biosoft, Cambridge, UK) were used for calculation of the pharmacokineticparameters.

3.4. Pharmacodynamic measurementsIn all studies, endogenous cortisol secretion and the adrenal-suppressant effect ofglucocorticoids were evaluated by measuring morning plasma cortisolconcentrations before the administration of the glucocorticoid and in the morning


45

at 23 or 24, 47, and 71 h after glucocorticoid administration. In addition, in StudiesIII and V, the AUC of cortisol from 0 to 12 h after methylprednisoloneadministration [AUC(0-12)] was calculated by use of the linear trapezoidal rule.

3.5. Statistical methodsAll individual subject data, medical history, results from the clinical examination,chemical laboratory tests, ECG, and plasma drug and cortisol concentrations areconsidered as source data. The individual data were recorded into a database.However, plasma drug and cortisol concentrations were sent as an electronicdatabase.

Results are expressed as mean values with standard deviation (SD) and as meanvalues with standard error of mean (SEM) in figures. Each tmax value is presentedas median with range. The pharmacokinetic variables and the 0-, 23- or 24-, 47-,and 71-hour plasma cortisol concentrations between the phases were comparedwith Student’s 2-tailed t-test for paired data. In Study III, the statistical analysiswas done with two-way analysis of variance (ANOVA) with the Tukey test. In allstudies, the Wilcoxon test was used for analysis of tmax. The 95% confidenceintervals (CI) and ranges were presented for selected variables.

Pearson’s correlation coefficient was used to evaluate the linear correlations.Spearman rank-order correlation coefficient was used to investigate the possiblerelationship between the morning plasma cortisol concentration and the AUC(12-23) and AUC(0-∞) of methylprednisolone (Study III). In Study VI, nonlinearregression was used to evaluate the relationship between the AUC(23-47) ofdexamethasone and morning plasma cortisol concentrations at 47 and 71 h.

In all studies, the level of statistical significance was p <0.05. The number ofsubjects in each study was 8-10, which was considered to be sufficient in apharmacokinetic cross-over study to discover an about 30 to 50% change in theAUCs, sufficient large to be clinically significant. The statistical analyses weredone with the statistical program Systat for Windows, version 6.0.1 (SPSS Inc.,Chicago, Il, USA).

3.6. ArchivingAll study-related documents, e.g., protocols, original signed informed consents,source data are archived at the Department of Clinical Pharmacology, University ofHelsinki.

RESULTS

46

RESULTS

1. Interaction between itraconazole and glucocorticoids1.1. Oral methylprednisolone (Study I)Itraconazole 200 mg daily for 4 days markedly increased plasma concentrationsof oral methylprednisolone. Itraconazole increased the AUC(0-�) ofmethylprednisolone 3.9-fold (p <0.001) and the Cmax 1.9-fold: from 118 (25)ng/ml to 221 (49) ng/ml; p <0.001, and prolonged the t½ of methylprednisolonefrom 1.9 (0.3) h to 4.4 (0.7) h (p <0.001), compared with placebo (Figures 5 and11). The tmax of methylprednisolone was not significantly affected byitraconazole.

The mean morning plasma cortisol concentration, measured 24 h after theingestion of 16 mg methylprednisolone, was, during the itraconazole phase,about 13% of that during the placebo phase: 18 (23) ng/ml versus 139 (60)ng/ml; p <0.001; Figure 6. No significant difference existed in the morningplasma cortisol concentration between the itraconazole and placebo phases justbefore ingestion of methylprednisolone.

Figure 5. Pharmacokinetic changes of methylprednisolone. Mean relative changes (withranges) in pharmacokinetic variables of oral and intravenous methylprednisolone afteritraconazole compared with placebo pretreatment (= 100%; indicated with a dashed line).AUC, area under the plasma concentration-time curve; T½, elimination half-life; Cmax, peakplasma concentration; Cl, systemic clearance; Vd, volume of distribution** p <0.001 compared with placebo phase* p <0.01 compared with placebo phase

RESULTS

47

1.2. Intravenous methylprednisolone (Study II)Itraconazole 200 mg daily for 4 days increased the AUC(0-�) of intravenousmethylprednisolone 2.6-fold (p <0.001) and the AUC(12-24) 12.2-fold (range,6.7-fold to 29.3-fold; p <0.001). Itraconazole decreased the Cl ofmethylprednisolone by 60%: 167 (51) ml/h/kg versus 449 (237) ml/h/kg; p <0.01. The t½ of methylprednisolone was increased from 2.1 (0.3) h to 4.8 (0.8) h(p <0.001) by itraconazole (Figures 5 and 11). The Vd of methylprednisolone wasunaffected by itraconazole (Figure 5).

The mean morning plasma cortisol concentration measured 24 h after theingestion of 16 mg methylprednisolone was during the itraconazole phase 9% ofthat during the placebo phase: 11 (9.0) ng/ml versus 117 (50) ng/ml; p <0.001;Figure 6. No significant difference existed in morning plasma cortisolconcentration between the itraconazole and placebo phases just before ingestionof methylprednisolone.

Figure 6. Mean (with SD) plasma cortisol concentrations, measured 24 hours afteroral and intravenous (Iv) methylprednisolone during placebo and itraconazolephases.** p <0.001 compared with placebo phase

RESULTS

48

1.3. Oral prednisolone (Study IV)Itraconazole 200 mg daily for 4 days slightly increased the AUC(0-∞) ofprednisolone by 24% (p <0.001), and the t½ by 29%: 3.6 (0.4) h versus 2.8 (0.4)h; p <0.001, compared with placebo (Figures 7 and 11). The Cmax and tmax ofprednisolone were not significantly affected by itraconazole (Figure 7).

The mean morning plasma cortisol concentration, measured 23 h after theingestion of 20 mg prednisolone, was during the itraconazole phase about 73% ofthat during the placebo phase: 104 (39) ng/ml versus 141 (33) ng/ml; p <0.001;Figure 8. No significant difference existed in morning plasma cortisolconcentration between the itraconazole and placebo phases just before ingestionof prednisolone or 47 h after it.

Figure 7. Pharmacokinetic changes of prednisolone. Mean relative changes (withranges) in pharmacokinetic variables of oral prednisolone after itraconazolecompared with placebo pretreatment (= 100%; indicated with a dashed line).AUC, area under the plasma concentration-time curve; T½, elimination half-life; Cmax, peakplasma concentration** p <0.001 compared with placebo phase

RESULTS

49

Figure 8. Mean (with SD) plasma cortisol concentrations, measured 23 hours afteroral prednisolone during placebo and itraconazole phases.** p <0.001 compared with placebo phase

1.4. Oral and intravenous dexamethasone (Study VI)Itraconazole 200 mg daily for 4 days markedly increased the AUC(0-�),AUC(12-23), and AUC(23-47) of oral dexamethasone 3.7-fold (p <0.001), 8.2-fold (p <0.001), and 46-fold (p <0.001), respectively, compared with placebo(Figures 9 and 11). The Cmax of oral dexamethasone was increased 1.7-fold: 60(17) ng/ml versus 38 (16) ng/ml; p <0.001 and t½ 2.8-fold: 10.9 (2.0) h versus 4.0(0.9) h; p <0.001, by itraconazole (Figures 9 and 11). The absolute bioavailabilityof oral dexamethasone rose from 75 (9)% during the placebo phase to 86 (11)%during the itraconazole phase (p =0.13).

Itraconazole 200 mg daily for four days increased the AUC(0-�), AUC(12-23),and AUC(23-47) of intravenous dexamethasone 3.3-fold (p <0.001), 9.1-fold (p<0.001), and 74-fold (p <0.001), respectively, compared to placebo (Figures 9and 11). The Cl of dexamethasone was decreased by 68% (p <0.001), and the t½

was increased 3.2-fold: 12.2 (2.3) h versus 4.1 (1.4) h; p <0.001), by itraconazole(Figures 9 and 11). The Vd was unaffected by itraconazole (Figure 9).

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Mean morning plasma cortisol concentrations, measured 47 and 71 h afteradministration of dexamethasone, were lower after exposure to itraconazole thanto placebo: 47 h after oral dexamethasone, 6.4 (2.1) ng/ml versus 134 (39) ng/ml;p <0.001 and 71 h after oral dexamethasone, 42 (37) ng/ml versus 164 (50)ng/ml; p <0.001; Figure 10. Additionally, 47 h after intravenous dexamethasone,6.8 (2.9) ng/ml versus 130 (40) ng/ml; p <0.001 and 71 h after intravenousdexamethasone, 35 (27) ng/ml versus 174 (40) ng/ml; p <0.001; Figure 10.

Figure 9. Pharmacokinetic changes of dexamethasone. Mean relative changes (withranges) in pharmacokinetic variables of oral and intravenous dexamethasone afteritraconazole compared with placebo pretreatment (= 100%; indicated with a dashedline). AUC, area under the plasma concentration-time curve; T½, elimination half-life; Cmax, peak plasma concentration; Cl, systemic clearance; Vd, volume of distribution ** p <0.001 compared with placebo phase

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However, no significant difference appeared in morning plasma cortisolconcentration between the itraconazole and placebo phases just before or 23 hafter administration of either intravenous or oral dexamethasone. Very goodexponential relationships existed between the AUC(23-47) of dexamethasoneand the morning cortisol concentrations at 47 h (r =0.960) and 71 h (r =0.912)after intravenous and at 47 h (r =0.953) and 71 h (r =0.874) after oraldexamethasone.

Figure 10. Mean (with SD) plasma cortisol concentrations, measured 23, 47, and 71hours after oral and intravenous dexamethasone during placebo and itraconazolephases.** p <0.001 compared with placebo phase

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Figure 11. Mean relative changes (with ranges) in AUC (upper panel) and t½ values(lower panel) of different glucocorticoids after itraconazole compared with placebopretreatment (= 100%; indicated with a dashed line).

Mepre = methylprednisolone, Pred = prednisolone, Dexa = dexamethasone, iv = intravenousAUC, area under the plasma concentration-time curve; T½, elimination half-life** p <0.001 compared with placebo phase

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2. Interaction between diltiazem or mibefradil and oralmethylprednisolone (Study III)Compared with placebo, diltiazem and mibefradil increased the AUC(0-∞) oforal methylprednisolone 2.6-fold (p <0.001) and 3.8-fold (p <0.001), and theCmax 1.6-fold (p <0.001) and 1.8-fold (p <0.001), respectively (Figure 12).Furthermore, diltiazem and mibefradil increased the t½ of methylprednisolone1.9-fold (p <0.001) and 2.7-fold (p <0.001), respectively (Figure 12). Comparedwith placebo, the AUC(12-23) of methylprednisolone was increased 28.2-fold (p<0.01) by diltiazem and 72.1-fold (p <0.001) by mibefradil. The tmax ofmethylprednisolone was not significantly altered by diltiazem, but mibefradilincreased the tmax of methylprednisolone from 1.5 to 3.0 h (p <0.05) comparedwith placebo. Mibefradil increased the AUC(0-�) and AUC(12-23) ofmethylprednisolone more than did diltiazem (p <0.001).

Figure 12. Pharmacokinetic changes of methylprednisolone. Mean relative changes(with ranges) in pharmacokinetic variables of oral methylprednisolone afterdiltiazem (Dilt) and mibefradil (Mibe) compared with placebo pretreatment (=100%; indicated with a dashed line).AUC, area under the plasma concentration-time curve; T½, elimination half-life; Cmax, peakplasma concentration** p <0.001 compared with placebo phase

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The morning plasma cortisol concentration, measured 23 h after ingestion of 16mg methylprednisolone, was substantially lower after exposure to diltiazem andmibefradil than to placebo: 22 (16) ng/ml and 4.2 (1.6) ng/ml, respectively,versus 186 (80) ng/ml; p <0.001; Figure 13. Furthermore, a significant negativecorrelation appeared between the morning plasma cortisol concentration and theAUC(12-23) (r = -0.81; p <0.001) and the AUC(0-∞) (r = -0.76; p <0.001) ofmethylprednisolone. However, no significant differences existed in the AUC(0-12) of cortisol between the phases or in the morning plasma cortisolconcentrations just before ingestion of methylprednisolone or 47 h afterwards(Figure 13).

Figure 13. Mean (with SD) plasma cortisol concentrations, measured 23 and 47hours after oral methylprednisolone during placebo, diltiazem, and mibefradilphases.** p <0.001 compared with placebo phase

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3. Interaction between grapefruit juice and oralmethylprednisolone (Study V)Grapefruit juice increased the AUC(0-∞) of oral methylprednisolone by 75% (p<0.001), the Cmax by 27% (p <0.01), and the t½ by 35%: 2.8 (0.3) h versus 2.1(0.5) h; p <0.001, compared with water (Figure 14). The tmax ofmethylprednisolone was prolonged from 2.0 to 3.0 h (p < 0.05) by grapefruitjuice.

Figure 14. Pharmacokinetic changes of methylprednisolone.Mean relative changes (with ranges) in pharmacokinetic variables of orallyadministered methylprednisolone during grapefruit juice phase compared with waterphase (= 100%; indicated with a dashed line).

AUC, area under the plasma concentration-time curve; T½, elimination half-life; Cmax, peakplasma concentration** p <0.001 compared with placebo phase* p <0.01 compared with placebo phase

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The morning plasma cortisol, measured 23 h after the administration of 16 mgmethylprednisolone, was slightly lower during the grapefruit juice phase thanduring the water phase :133 (43) ng/ml versus 149 (39) ng/ml; p =0.09; Figure15]. The AUC(0-12) of cortisol was slightly greater during the grapefruit juicephase than during the water phase: 500 (165) ng � h/ml versus 453 (103) ng �h/ml; p =0.08. On the other hand, the plasma cortisol concentration, measured inthe morning before administration of methylprednisolone, was lower during thegrapefruit juice phase than during the water phase: 140 (47) ng/ml versus 169(37) ng/ml; p <0.05.

Figure 15. Mean (with SD) plasma cortisol concentrations, measured 23 hours afteroral methylprednisolone during grapefruit juice (GFJ) and water phases.# p =0.09 compared with water phase

DISCUSSION

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DISCUSSION

1. Methodological considerationsThe present study evaluated the effect of CYP3A4 inhibitors on syntheticglucocorticoids after their systemic administration. In addition tomethylprednisolone, prednisolone, and dexamethasone, also betamethasone andprednisone are in clinical use in Finland. Betamethasone and prednisone were notevaluated in the present study. Prednisone, however, is considered a prodrug andis further metabolised to prednisolone.

All studies were carried out in healthy subjects, and after their pretreatment withCYP3A4 inhibitor, a single dose of glucocorticoid was administered. This shouldbe considered in the extrapolation of the pharmacokinetic and pharmacodynamicresults to the patient using systemic glucocorticoids as a long-term treatment ofchronic disease. The doses of 20 mg prednisolone and of 16 mgmethylprednisolone used in the present study are equivalent regarding theirpotency to glucocorticoid activity (Table III, page 26). However, an equivalentdose of dexamethasone would be 3.0 mg. Thus, dexamethasone was used at higher(3.85 mg and 4.5 mg) doses regarding the potency than were methylprednisoloneand prednisolone.

The susceptibility of glucocorticoids to interact with CYP3A4 inhibitors wasstudied with potential CYP3A4 inhibitors currently on the market and in clinicaluse. Mibefradil was, however, withdrawn from the market in 1998 during theclinical phase of Study III. Each subject received oral and written information andgave additional written consent.

The CYP3A4-enzyme inhibitors of the present study possess different inhibitorymechanisms. Itraconazole, for example, is a competitive inhibitor of CYP3A4, andgrapefruit juice and maybe also mibefradil are non-competitive mechanism-basedinactivators of CYP3A4 (Back and Tjia 1991, Lown et al 1997, Prueksaritanont elal 1999). The pretreatment period ranged from 3 to 4 days, because even such ashort treatment is sufficient to reveal a clinically significant inhibition ofCYP3A4. The t½ of mibefradil ranges from 10 h to 38 h, and it may be suggestedthat the steady-state concentration was not reached during the 3-day treatment. Inaddition, diltiazem has been shown dose-dependently to increase the plasmaconcentrations of nifedipine (Tateishi et al 1989). Because the steady-stateconcentration of the CYP3A4 inhibitors was perhups not reached during the 3 to 4days, the interactions observed may become even more pronounced afterpretreatment for longer period or with a higher dose.

DISCUSSION

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The adrenal-suppressant effect of glucocorticoids was evaluated by measuringmorning plasma cortisol concentrations. The normal range for the morning plasmacortisol concentration taken between 8:00 a.m. and 10:00 a.m. varies between 50and 250 ng/ml according to the laboratory of Helsinki University Central Hospital.In the present study blood samples for plasma cortisol determination were taken at8:00 a.m. or at 9:00 a.m.. Cortisol secretion is pulsatile and exhibits a diurnalpattern with the highest concentration occurring early in the morning. Morningcortisol measurement has been used as an index of adrenal function, because itreflects the peak endogenous activation of the HPA-axis (Grinspoon and Biller1994). According to Grinspoon and Biller (1994), patients with a morning cortisolconcentration above 190 ng/ml do not need further testing for adrenalinsufficiency; although a low morning cortisol concentration may reflect the nadirbetween pulses of cortisol secretion, a cortisol concentration of less than 30 ng/mlis considered diagnostic for adrenal insufficiency. In the present series ofinteraction studies, cortisol concentrations were not measured during the night-time, which could have provided additional information.

2. Interaction between CYP3A4 inhibitors andglucocorticoids2.1. MethylprednisoloneItraconazole markedly affected the pharmacokinetics both of oral and ofintravenous methylprednisolone. Itraconazole increased the AUC of oralmethylprednisolone 3.9- fold, the AUC of intravenous methylprednisolone 2.6-fold, and decreased the Cl of methylprednisolone by 60%. The effect ofitraconazole on the AUC of methylprednisolone was about 50% greater after oralthan after intravenous administration. This finding can be explained by theinhibition of the first-pass metabolism after oral administration, resulting inincreased bioavailability of methylprednisolone. This absolute bioavailability,however, could not be calculated, because the effects of itraconazole on the oraland intravenous methylprednisolone were evaluated in separate studies.

Diltiazem and mibefradil significantly affected the pharmacokinetics of oralmethylprednisolone. The extent to which mibefradil affected the pharmacokineticsof oral methylprednisolone was as great as that seen after itraconazole in Study I.This finding is in good agreement with that of an in vitro study in whichmibefradil was as potent inhibitor of CYP3A4 as was itraconazole (Wang et al1999). Thus, these in vitro results predicted well the interactions observed in vivoin the present studies. Furthermore, the effect of diltiazem on thepharmacokinetics of methylprednisolone was smaller than that of mibefradil.

DISCUSSION

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However, in some subjects diltiazem affected the AUC of methylprednisolone asalmost as greatly as mibefradil.

Troleandomycin, a potent inhibitor of CYP3A4, has shown to decrease the Cl ofmethylprednisolone by 64% (Szefler et al 1980). Furthermore, ketoconazole,which is an even more potent inhibitor of CYP3A4 in vitro than is itraconazole,increased the AUC of methylprednisolone about 2.4-fold and decreased its Cl by66% (Glynn et al 1986). Erythromycin decreased the Cl of methylprednisolone by46%, and in a recent study, clarithromycin reduced the apparent oral Cl ofmethylprednisolone by 65% (LaForce et al 1983, Fost et al 1999). In summary, ourresults for itraconazole, diltiazem, and mibefradil are in concordance with those ofprevious studies on the effect of other potent CYP3A4 inhibitors on thepharmacokinetics of methylprednisolone. Furthermore, in a congress report,itraconazole for 4 days increased the AUC of oral methylprednisolone (48 mg)about 2.5-fold, confirming the results of the present study (Lebrun-Vignes et al1998).

In this study, high amounts of grapefruit juice increased the AUC of oralmethylprednisolone by 75%. Because grapefruit juice reduced intestinal CYP3A4without affecting hepatic CYP3A4 activity (Lown et al 1997), it may be suggestedthat the increase in AUC of oral methylprednisolone mainly resulted frominhibition of its first-pass metabolism. However, recent studies have shown highand repeated consumption of grapefruit juice to increase also the t½ of such drugsas oral buspirone and cisapride (Lilja et al 1998b, Kivistö et al 1999). In thepresent study, grapefruit juice slightly but significantly increased the t½ of oralmethylprednisolone, suggesting that it inhibited also systemic (hepatic) CYP3A4-mediated methylprednisolone metabolism. Grapefruit juice alters thepharmacokinetics of methylprednisolone considerably less than that of drugs suchas simvastatin, lovastatin, and buspirone (Lilja et al 1998a, Kantola et al 1998b,Lilja et al 1998b). This may be because of the rather small first-pass metabolismof methylprednisolone compared to that of simvastatin, lovastatin, and buspirone.

The increased and prolonged exposure to methylprednisolone during theitraconazole, the diltiazem, and the mibefradil phases was associated with muchlower morning plasma cortisol concentrations at 23/24 h than those seen duringthe placebo phase. Thus, concomitant use of methylprednisolone with itraconazoleor with these calcium-channel blockers enhances the adrenal-suppressant effect ofmethylprednisolone by completely suppressing the peak cortisol concentrationseen in the placebo phase.

The concentration-effect relationship is log-linear, and despite the fact thatitraconazole increased the AUC of methylprednisolone by some 50% more afteroral than after intravenous administration, the extent of adrenal-suppressant effectduring the itraconazole phases was about the same. Furthermore, a negative

DISCUSSION

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correlation appeared between the night-time exposure to methylprednisolone[AUC(12-23)] and the morning plasma cortisol concentrations (Study III). It isknown that endogenous cortisol is secreted almost immediately after synthesis(Berne and Levy 1998). Therefore, increases in t½ and the night-timeconcentrations of methylprednisolone may effectively suppress the peak cortisolconcentrations occurring early in the morning.

It can be concluded that these interactions between itraconazole, diltiazem, ormibefradil and methylprednisolone are clinically significant and enhance theadrenal-suppressant and perhups also other effects of methylprednisolone. In thepresent study, the adrenal-suppressant effect of methylprednisolone tended toincrease during the grapefruit juice phase compared with that of the water phase.Despite the statistically significant increase in the AUC, Cmax, and t½ of oralmethylprednisolone caused by grapefruit juice, the clinical significance of thisinteraction appears to be minor. However, it might enhance the effects ofmethylprednisolone in a few subjects.

2.2. PrednisoloneDespite the statistically significant increase in the AUC and t½ of oralprednisolone caused by itraconazole, the interaction was quantitatively minor andis probably of limited clinical significance. This observation is in agreement withearlier findings that prednisolone does not significantly interact withtroleandomycin, ketoconazole, or clarithromycin, all potent inhibitors of CYP3A4(Szefler et al 1982, Ludwig et al 1989, Yamashita et al 1991, Frost et al 1999). Inaddition, the pharmacodynamics of prednisolone, evaluated by its suppressiveeffects on serum cortisol, blood basophil, and helper T-lymphocyte values, werenot affected during the ketoconazole phase (Ludwig et al 1989, Yamashita et al1991). In the present study, the adrenal-suppressant effect slightly increasedduring the itraconazole phase compared with that of the placebo phase. Theclinical significance of this change is probably minor.

However, in contrast to other reports, Zürcher et al (1989) found ketoconazole toraised prednisolone plasma concentration by 50% after both oral prednisone andintravenous prednisolone, and Imani et al (1999) reported diltiazem to raise theAUC of prednisolone slightly, by about 20%, after prednisone administration.Imani et al (1999) suggested that diltiazem may in some patients enhance theeffects of prednisone. Furthermore, Zürcher et al (1989) suggested that the enzymeresponsible for the interconversion of prednisolone ↔ prednisone was unaffectedby ketoconazole. It may be assumed that the effect of itraconazole, another azoleantimycotic agent and potent inhibitor of CYP3A4, on plasma concentrations ofprednisolone is also similar after both prednisolone and prednisone administration.

DISCUSSION

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A congress report by Lebrun-Vignes et al (1998) confirms that itraconazole forfour days does not affect the pharmacokinetics prednisolone after oral prednisone(60 mg).

In conclusion, the results of the present study strongly suggest that CYP3A4 doesnot play a major role in the metabolic pathways of prednisolone. The concomitantuse of prednisolone or prednisone with itraconazole or with other potent CYP3A4inhibitors should not markedly alter the effects of these glucocorticoids.

2.3. DexamethasoneItraconazole greatly affected the pharmacokinetics both of oral and of intravenousdexamethasone. Itraconazole increased the AUC of oral dexamethasone 3.7-fold,the AUC of intravenous dexamethasone 3.3-fold, and decreased the Cl ofdexamethasone by 68%. The effect of itraconazole on the AUC of dexamethasonewas similar after oral and after intravenous administration. Furthermore, theabsolute bioavailability of oral dexamethasone was slightly elevated, from 75% to86%, by itraconazole. It is known that a significant increase in the AUC byenzyme inhibition both after oral and after intravenous administration togetherwith small change in the absolute bioavailability are typical of a drug with lowhepatic clearance (Lin and Lu 1998).

The extent of itraconazole-dexamethasone interaction was approximately as greatas the extent of itraconazole-methylprednisolone interaction. Thus, thesusceptibility of dexamethasone and of methylprednisolone to interact withitraconazole is similar. There seem to be no previous studies on the effects ofCYP3A4 inhibitors on the pharmacokinetics of oral and intravenousdexamethasone. However, CYP3A4 has been shown to be important inmetabolism of dexamethasone in vitro (Gentile et al 1996). The present resultsstrongly suggest that this is the case also in vivo in human beings.

The increased exposure to dexamethasone during the itraconazole phases wasassociated with much reduced morning plasma cortisol concentrations at 47 and71 h than those during the placebo phases. The extent of the adrenal-suppressanteffect of dexamethasone during the itraconazole phase was about the same,regardless of the route of dexamethasone administration.

In conclusion, concomitant use of itraconazole or probably also other potentCYP3A4 inhibitors woth dexamethasone greatly enhances the adrenal-suppressantand perhaps also other effects of dexamethasone.

DISCUSSION

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3. General discussion

Itraconazole, diltiazem, and mibefradil are known to inhibit CYP3A4-mediateddrug metabolism. Therefore, it can be assumed that inhibition of CYP3A4-mediated metabolism during first-pass and elimination phases is the most probablemechanism explaining the observed interactions in the present study. However,itraconazole, diltiazem, and mibefradil are also inhibitors of P-gp (Miyama et al1998, Cornwell et al 1987, Wandel et al 2000). In addition, grapefruit juice hasbeen demonstrated to inhibit the vinblastine cellular efflux by P-gp and tomodulate to a limited extent P-gp mediated transport of saquinavir in vitro(Takanaga et al 1998, Eagling et al 1999). Eagling et al (1997) suggest, however,that the in vivo effects of grapefruit juice most likely result from inhibition ofCYP3A4 and only to a minor extent from modulation of P-gp. In contrast toprednisolone (Saitoh et al 1998), methylprednisolone and dexamethasone havebeen shown to be substrates for P-gp (Saitoh et al 1998, Ueda et al 1992);therefore, inhibition of this transmembrane efflux pump may have contributed tothe observed interactions. However, present studies do not allow the separation ofthe role of CYP3A4 and P- gp in these interactions.

All glucocorticoids are readily eliminated by metabolism. Although the role ofindividual CYP enzymes in the metabolic pathways of various glucocorticoidsremains unclear, CYP3A4 probably has a dissimilar role in the metabolism ofeach glucocorticoid. Based on the studies presented here, itraconazole, a potentinhibitor of CYP3A4, greatly interacted with both methylprednisolone anddexamethasone, but only slightly affected the pharmacokinetics of prednisolone.This strongly suggests that the metabolic pathways of these glucocorticoids aredissimilar and that, in contrast to prednisolone, CYP3A4 has an important role inthe metabolism of methylprednisolone and dexamethasone.

Induction of CYP3A4 by glucocorticoids may counteract the effects of CYP3A4inhibitors on these glucocorticoids during long-term use. Dexamethasone has beenshown to induce CYP3A4 in vitro (Pichard et al 1992), and in cancer patients toincrease the Cl of cyclophosphamide, a substrate for CYP3A4 (Yule et al 1996,Busse et al 1997). However, in controlled studies, dexamethasone does not affectthe AUC of triazolam and midazolam, both known substrates for CYP3A4(Villikka et al 1998, Palmer et al 2000). Thus, dexamethasone seems not to be apotent inducer of CYP3A4-mediated metabolism in human beings.

The concomitant use of the CYP3A4 inhibitors itraconazole, diltiazem, ormibefradil with methylprednisolone, and of itraconazole with dexamethasonemarkedly enhanced the adrenal-suppressant effect of these glucocorticoids. Theprologned suppression was due to the increased effect of methylprednisolone ordexamethasone, because neither itraconazole, diltiazen nor mibefradil

DISCUSSION

63

significantly affected the plasma cortisol concentrations measured in the morningbefore glucocorticoid administration. The marked decrease in elimination ofmethylprednisolone and dexamethasone is particularly important, because itappears also to prolong the biological half-lives of these steroids. Thus, not onlythe exposure to methylprednisolone and dexamethasone increased, but also the”biological profile” of these steroids changed. Furthermore, the increasedexposure to methylprednisolone, especially during night-time, enhanced itsadrenal-suppressant effect by suppressing the peak cortisol concentration evidentduring the placebo phase. This may be important in patients receiving low dosesof methylprednisolone once daily.

Present studies were performed in healthy volunteers after administration of asingle dose of glucocorticoid. It can be assumed, however, that the great effect ofCYP3A4 inhibitors on the pharmacokinetics and pharmacodynamics ofmethylprednisolone or dexamethasone can be seen also in patients receiving long-term glucocorticoid treatment. During long-term treatment, the increased andprolonged exposure to glucocorticoids caused by other concomitant medicationmay alter the clinical response to glucocorticoid. Besides plasma cortisolconcentratios and the addrenal-suppressant effect of glucocorticoid, no otherpharmacodynamic parameters were assessed in the studies of this thesis. However,there are several lines of evidence in the literature to suggest that concomitant useof a potent inhibitor of CYP3A4 with glucocorticoid, or increased exposure to aglucocorticoid may enhance many different glucocorticoid-related effects.

In one case report, addition of itraconazole to a transplant patient´simmunosuppressive regimen which includied methylprednisolone led to myopathyand diabetes, suggesting a clinically important interaction between itraconazoleand methylprednisolone (Linthoudt et al 1996). Similar, or even more severeitraconazole-methylprednisolone interactions are known to have occurred duringtheir combined clinical use. Furthermore, combination of troleandomycin andmethylprednisolone had a beneficial ”steroid-sparing” effect on difficult asthma(Siracusa et al 1993). However, this combination led to increased risk ofglucocorticoid-related adverse effects (e.g., reduction in bone mineral content,increase in plasma glucose levels), and even one case of fatal varicella has beenreported, suggesting enhanced immunosuppression (Harris et al 1989, Siracusa etal 1993, Lantner et al 1990). Diltiazem and verapamil can improve the outcome oforgan-transplant patients treated with methylprednisone (Wagner et al 1987,Kunzendorf et al 1991, Dawidson et al 1989, Mies et al 1998), which may in partbe explained by the increase in the AUC of methylprednisolone caused bydiltiazem. Furthermore, methylprednisolone exposure rather than themethylprednisolone dose predicts adrenal suppression and growth inhibition inchildren with liver and renal transplants (Sarna et al 1997). In addition, the

DISCUSSION

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diabetogenic effect of dexamethasone appears to be dose-dependent (Matsumotoet al 1996).

The pharmacokinetic interaction between itraconazole and dexamethasone andbetween itraconazole and methylprednisolone was slightly greater after oral thanafter intravenous administration of these two steroids. This finding may favourintravenous rather than oral use of methylprednisolone and dexamethasone.However, the extent of the adrenal-suppressant effect was similar after their oraland their intravenous administration. The extent of interaction observed after oraladministration might be reduced by giving itraconazole some 12 h afterglucocorticoid, as shown in a recent study of ketoconazole with budesonide(Seidegård 2000).

SUMMARY AND CONCLUSIONS

65


One of the most important drug-metabolising enzymes in human beings isCYP3A4. Many drugs and also grapefruit juice can inhibit the catalytic activityof CYP3A4, which may result in a clinically significant increase in exposure todrugs metabolised mainly by this enzyme.

The systemic glucocorticoids methylprednisolone, prednisolone, anddexamethasone have been used for many years for various clinical indicationsand are eliminated mainly by metabolism. Although the role of individual CYPenzymes in the metabolic pathways of these glucocorticoids seems not to beclear, CYP3A4 probably plays a dissimilar role in their metabolism.

The aim of this series of studies was to investigate the effect of CYP3A4inhibitors on the pharmacokinetics and pharmacodynamics of systemicglucocorticoids: the effect of itraconazole on oral (Study I) and intravenous(Study II) methylprednisolone, on oral prednisolone (Study IV), and on oral andintravenous dexamethasone (Study VI). In addition, the effect of diltiazem ormibefradil (Study III), and grapefruit juice (Study V) on oral methylprednisolonewas investigated. The effect of route of administration of methylprednisolone anddexamethasone on their possible interaction with itraconazole was also evaluated(Studies I, II, and VI).

The studies were double-blind, randomised, placebo-controlled, and cross-over,except for the grapefruit juice-methylprednisolone interaction study, which hadan open, randomised, cross-over design. All studies were carried out in 8 to 10healthy subjects. A single dose of glucocorticoid was administered after or duringthe administration of a CYP3A4 inhibitor for 3 to 4 days. Blood samples werecollected, and plasma drug and cortisol concentrations determined by specifichigh-performance liquid chromatographic and mass spectrometric methods.

Itraconazole increased the AUC of oral and intravenous methylprednisolone 3.9-fold (p <0.001) and 2.6-fold (p <0.001). The AUC of oral and intravenousdexamethasone was increased 3.7-fold (p <0.001) and 3.3-fold (p <0.001) byitraconazole. The use of itraconazole enhanced the adrenal-suppressant effect ofboth methylprednisolone and dexamethasone. In contrast, itraconazole increasedthe AUC of oral prednisolone by only 24% (p <0.001) and had a minor effect onthe adrenal-suppressant effect of prednisolone. Both diltiazem and mibefradilincreased the AUC of oral methylprednisolone (2.6-fold and 3.8-fold; p <0.001)and greatly enhanced the adrenal-suppressant effect of methylprednisolone.Grapefruit juice increased the AUC of oral methylprednisolone by 75% (p<0.001); however, it did not significantly affect the adrenal-suppressant effect ofmethylprednisolone. The route of methylprednisolone or dexamethasone


66

administration may have a only minor effect on the extent of itraconazole-methylprednisolone and itraconazole-dexamethasone interactions.

The following conclusions can be drawn on the basis of Studies I-VI:

1. Interactions between the antifungal agent itraconazole and methylprednisoloneor dexamethasone are clinically important. Itraconazole and probably also otherpotent CYP3A4 inhibitors should be used with caution in patients receivingdexamethasone or methylprednisolone, particularly during long-termconcomitant use.

2. The interaction between itraconazole and prednisolone is unlikely to beclinically significant.

3. The interactions between the calcium-channel blockers diltiazem or mibefradiland methylprednisolone are clinically important. Diltiazem and mibefradilshould be used with caution in patients receiving methylprednisolone,particularly during long-term concomitant use.

4. The clinical significance of the interaction between grapefruit juice and oralmethylprednisolone is probably minor. However, high repeated doses ofgrapefruit juice may enhance the effects of oral methylprednisolone in somesensitive subjects.

5. The route of methylprednisolone or dexamethasone administration may have aminor effect on the extent of itraconazole-methylprednisolone and itraconazole-dexamethasone interactions. Despite the differences seen in pharmacokineticinteractions, the extent of the adrenal-suppressant effect was similar after oral orintravenous dose of these glucocorticoids.

6. All of these interactions are probably due to inhibition of the CYP3A4 enzymeduring both the first-pass and the elimination phases. Inhibition of the P-glycoprotein may have contributed to the observed interactions ofmethylprednisolone and of dexamethasone.

7. The metabolism and interaction potential of different glucocorticoids aredissimilar. In contrast to prednisolone and also prednisone, CYP3A4 appears tobe important in the metabolism of methylprednisolone and dexamethasone.Therefore, the pharmacokinetics of methylprednisolone and dexamethasone aresensitive to itraconazole and probably also to other potent CYP3A4 inhibitors.However, interactions of itraconazole and other CYP3A4 inhibitors withprednisolone and probably with prednisone are likely to be of limited clinicalsignificance.

ACKNOWLEDGEMENTS

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ACKNOWLEDGEMENTS

These studies were carried out at the Department of Clinical Pharmacology,University of Helsinki, during the years 1997 to 1999. I am grateful to all thosewho have contributed to my work during these years.

I express my deepest gratitude to my supervisers:

Professor Pertti Neuvonen, Head of the Department of ClinicalPharmacology, University of Helsinki, for introducing me to the field ofdrug-drug interactions, for his encouraging supervision, his time, and alsofor his providing excellent working facilities. His amazing energy combinedwith his wide knowledge in the field of clinical pharmacology creates astrong foundation for high quality research.

Docent Kari Kivistö, for his continuous interest, his time, and for hisscientific guidance during these years. His enthusiasm for scientific workand wide expertise in the field of clinical pharmacology has alwaysimpressed me.

Docent Arja Rautio and Docent Kari Aranko are gratefully acknowledged fortheir valuable review and comments on the present work.

I express my warm thanks to Kerttu Mårtensson, Lisbeth Partanen, EijaMäkinen-Pulli, Mikko Neuvonen, and Jouko Laitila for their guidance and skilfuldetermination of plasma drug and cortisol concentrations. Airi Järvinen is alsowarmly acknowledged.

I thank all my colleagues at the Department of Clinical Pharmacology, includingJanne Backman, Maija Kaukonen, Kirsti Villikka, Jari Lilja, Outi Lapatto-Reiniluoto, Kari Raaska, Harri Luurila, Jun-Sheng Wang, Xia Wen, MikkoNiemi, Elina Saarenmaa, Mika Jokinen, Carl Kyrklund, Matti Kivikko, KatiAaltonen, and Laura Juntti-Patinen, for their friendship and collaboration. I alsowant to thank all the people who join us in the ”first-pass group” meetings. Veryspecial thanks belong to Tuija Itkonen for her joyful company during these yearsand for her help in many practical matters.

Docent Kalle Hoppu and the staff of the Poison Information Centre are alsowarmly acknowledged.

Sincere thanks to Carol Norris for editing of the English language of this thesis.

Financial support by the Helsinki University Central Hospital Research Fund, theNational Technology Agency of Finland (TEKES), the Finnish Medical Society

ACKNOWLEDGEMENTS

68

Duodecim, and the Clinical Drug Research Graduate School are gratefullyacknowledged.

Haluan osoittaa lämpimät kiitokseni myös vanhemmilleni, jotka ovat tukeneetminua tutkimustyöni aikana ja osoittaneet jatkuvaa kiinnostusta sitä kohtaan.Lisäksi kiitos myös käytännön avusta, jota olen teiltä saanut erityisesti kuluneenkesän ja syksyn aikana.

Finally, I express my deepest and most loving thanks to my husband Juha for hisunselfish support and love during these years. My gratitude and love go to ourchildren Antti and Kristiina, who are the most important challenge of my life,more important even than science.

Karkkila, November 2000

Tiina Varis

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Studies on Drug Interactions between CYP3A4 Inhibitors and ...ethesis.helsinki.fi/julkaisut/laa/kliin/vk/varis/studieso.pdf · Department of Clinical Pharmacology University of Helsinki