UNIVERSITY OF CINCINNATI

Date:______

I, ______, hereby submit this work as part of the requirements for the degree of: in:

It is entitled:

This work and its defense approved by:

Chair: ______

AN ASSESSMENT OF THE POTENTIAL INFLUENCE OF BEXAROTENE, A NOVEL X RECEPTOR AGONIST, ON THE HEPATIC METABOLISM OF

A thesis submitted to the

Division of Research and Advanced Studies of the University of Cincinnati

in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

in the Division of Pharmaceutical Sciences of the College of Pharmacy

2005

by

Purvi B. Shroff

MBBS, University of Mumbai, India, 2000.

Committee Chair: Dr. Pankaj B. Desai, Ph.D

i ABSTRACT

Background: Bexarotene, a novel RXR agonist, is used in the treatment of cutaneous T

cell lymphoma and is under investigation for the treatment of solid tumors in

combination with other chemotherapeutic agents including docetaxel. Both docetaxel and

bexarotene are metabolized by CYP3A4 and in animal studies bexarotene has been

shown to enhance the activity of CYP3A enzymes and increase its content. Thus, there

exists a potential for interactions between these two agents, which was evaluated in this

study.

Method: Bexarotene from commercial capsules was dissolved in DMSO. Employing pooled human liver microsomes, we assessed the influence of bexarotene on the metabolism of docetaxel. Microsomal fractions (1mg protein) were incubated with bexarotene (0.1 – 10µM) and docetaxel (0.5 – 5µM) in the presence of NADPH (1 mM).

Using liquid-liquid extraction, unreacted docetaxel was extracted and levels were measured using a validated HPLC method. Next, we employed primary human hepatocytes to assess the inductive effects of bexarotene. Hepatocytes were treated with bexarotene (1 – 50µM) for 72 hours. Docetaxel metabolism by microsomal fractions was then evaluated. Testosterone 6β-hydroxylation and CYP3A4- specific protein and mRNA levels were also measured to evaluate whether bexarotene acts as an inducer of CYP3A4.

Results: Bexarotene, at clinically relevant concentrations (1-10µM) did not inhibit

docetaxel metabolism. Also, incubation of primary human hepatocytes with bexarotene

did not enhance docetaxel metabolism, which was consistent with the observation that the

ii testosterone 6β-hydroxylation and CYP3A4 expression in bexarotene – treated cells were not statistically different than those from untreated cells.

Conclusions: Our findings suggest that bexarotene is unlikely to alter hepatic metabolism of docetaxel. Bexarotene appears to exhibit species – specific differences in the induction of CYP3A and further studies are required to understand the mechanistic basis of these differences.

iii

iv ACKNOWLEDGEMENTS

I take this opportunity to deeply thank my advisor, Dr. Pankaj B. Desai, who not only initiated this project but also gave me valuable guidance till its completion. I remain indebted to him for his support, encouragement, advice, affection and critique of my work. On a personal note, he has been a source of inspiration to me. His wife, Sangita and his two daughters, Shivani and Kiran, are an extended family to me!

I would also like to thank Dr. Jane Pruemer and Dr. Sadanand Patil for serving on my Masters Thesis Committee and giving me valuable inputs on this project. I appreciate them for reviewing my thesis and for their support and critique of my work.

None of this work would have been possible without my parents and my in-laws who were supportive throughout my stay here in Cincinnati. I express my sincere gratitude to my parents once again for giving me the strength to believe in myself and get through life with pride. I cannot thank my husband, Manoj, enough for being so cooperative and patient with me. I hope to make up for all the sacrifices that he made for me to be here and pursue graduate studies at the University of Cincinnati, USA. He has made me the person that I am today with his love and support.

I would like to appreciate Rucha, my senior in the laboratory, for not only teaching me laboratory skills but also being a great friend. I wish to thank her for all the wonderful moments that we shared while working together. I also thank Murad for his help in guiding me with HPLC techniques. I have enjoyed working with Niresh and Fang in the laboratory and I thank all of them for the healthy working environment that we have in the laboratory.

v I wish to thank the faculty at UC for being such wonderful teachers and for all the guidance, encouragement and challenges they provided us during the classes. I appreciate that they participated in so many extracurricular activities and gave us a chance to interact with them outside the academic settings.

I thank the Department of Pharmaceutical Sciences at the College of Pharmacy for their academic, fiscal and administrative support and for making our stay here in

Cincinnati so comfortable.

I deeply appreciate my friend, Hitesh Deshmukh, for all his patience, help, affection and encouragement. I cannot thank him enough for being such a kind human being that he is.

I would also like to acknowledge all my friends and family here in the US and back in India for their love and support.

Lastly, I would like to acknowledge my darling son, Taaraksh, who makes everything worthwhile in my life!

With this I seek Lord Ganesha’s blessings and thank Him for all that He has showered upon me!

vi

TABLE OF CONTENTS

ABSTRACT……………………………………………………………………………....ii

ACKNOWLEDGEMENTS……………………………………………………………....iv

TABLE OF CONTENTS…………………………………………………………………vi

LIST OF FIGURES……………………………………………………………………….x

LIST OF TABLES……………………………………………………………………...xiii

1. INTRODUCTION……………………………………………………………….....1

1.1. ADVERSE DRUG REACTIONS (ADRs)………………………………………1

1.2. DRUG – DRUG INTERACTIONS……………………………………………...2

1.3. DRUG INTERACTIONS IN ONCOLOGY…………………………………….2

1.4. TYPES OF DRUG-DRUG INTERACTIONS………………………………...... 3

1.5. NON-SMALL CELL LUNG CARCINOMA (NSCLC)………………………...5

1.6. BEXAROTENE………………………………………………………………….8

1.6.1. Chemistry

1.6.2. Mechanism of action of

1.6.3. Clinical Indications

1.6.4. of bexarotene

1.6.5. Metabolism of bexarotene: role of CYP3A4

1.7. DOCETAXEL…………………………………………………………………..14

1.7.1. Chemistry

1.7.2. Mechanism of action of docetaxel

1.7.2.1. Background on structure

vii

1.7.2.2. Mechanism of action of docetaxel

1.7.3. Clinical Indications

1.7.4. Pharmacokinetics of docetaxel

1.7.5. Metabolism of docetaxel: role of CYP3A4

1.8. CYTOCHROME P450 FAMILY…………………………………………….21

1.8.1. The CYP3A subfamily

1.8.2. CYP3A4 Inhibition

1.8.3. CYP3A4 Induction

1.8.4. Regulation of CYP3A4

2. RATIONAL AND HYPOTHESIS ………………………………………………28

2.1 SPECIFIC AIMS

2.1.1. Aim I

2.1.2. Aim II

3. MATERIALS AND METHODS…………………………………………………30

3.1. Experimental Strategy ……………………………………………...... 30

3.2. Human liver microsomal metabolism of docetaxel in the presence and in the absence of bexarotene…………………………………………………33

3.2.1. Chemicals

3.2.2. Reaction optimization

3.2.3. Assessment of the potential influence of bexarotene on docetaxel metabolism in human liver microsomal reactions

3.2.4. HPLC analysis of docetaxel

viii

3.3. Assessment of the potential induction of docetaxel metabolism by bexarotene in primary human hepatocytes…………………………...... 38

3.3.1. Hepatocyte culture

3.3.2. Drug treatments

3.3.3. Assay of CYP3A4 in human hepatocytes

3.3.4. Docetaxel metabolism by human hepatocytes

3.3.5. Western blot analysis of immunoreactive CYP3A4 protein levels in human hepatocytes

3.3.6. Real-time PCR to assess the levels of CYP3A4 mRNA in human hepatocytes

3.4. Statistical Analysis………………………………………………………..42

4. RESULTS……………………………………………………………………...... 43

4.1. Human liver microsomal metabolism of docetaxel in the presence / absence of bexarotene………..…………………………………………..43

4.1.1. Michaelis-Menten plot of docetaxel metabolism kinetics

4.1.2. – 4.1.5. Human liver microsomal metabolism of docetaxel (0.5 – 5 µM) in the presence and the absence of bexarotene (0.1 – 10 µM).

4.2. Assessment of the potential induction of docetaxel metabolism by bexarotene in primary human hepatocytes……………………………….53

4.2.1. Typical chromatogram showing effect of rifampicin and bexarotene treatment of hepatocytes on CYP3A4 activity measured by testosterone 6β-hydroxylation as compared to untreated controls.

4.2.2. Effect of rifampicin and bexarotene treatment on the CYP3A4 activity as compared to untreated controls.

4.2.3. Effect of rifampicin and bexarotene treatment on docetaxel metabolism by human hepatocytes as compared to untreated controls.

ix 4.2.4. Western blot analysis of rifampicin and bexarotene treatment on immunoreactive CYP3A4 protein levels as compared to untreated controls.

4.2.5. RT – PCR analysis of the effect of rifampicin and bexarotene treatment on the CYP3A4 specific mRNA levels as compared to untreated controls.

5 DISCUSSION……………………………………………………………………63

6 REFERENCES…………………………………………………………………..70

x LIST OF FIGURES

1.6.1. Chemical structure of bexarotene ……………………………………………….9

1.6.2. Mechanism of action of retinoids ..………………………………………………11

1.7.1. Chemical structure of docetaxel…..………………………………………………15

1.7.2. Biotransformation of docetaxel. The structural modifications of docetaxel are

highlighted……………………………………………………………………….20

3.1.1. Diagrammatic representation of the experimental strategy to assess the inductive

effects of bexarotene on human hepatocytes…….……………………………….32

4.1.1. Michaelis – Menten plot demonstrating the docetaxel human liver metabolism

kinetics…..………………………………………………………………………..45

4.1.2. Human liver microsomal metabolism of docetaxel (0.5 µM) in the presence and

the absence of the indicated concentration of bexarotene…….…………………47

4.1.3. Human liver microsomal metabolism of docetaxel (1 µM) in the presence and the

absence of the indicated concentration of bexarotene.…………………………..48

4.1.4. Human liver microsomal metabolism of docetaxel (2 µM) in the presence and the

absence of the indicated concentration of bexarotene………….………………..50

xi 4.1.5. Human liver microsomal metabolism of docetaxel (5 µM) in the presence and the

absence of the indicated concentration of bexarotene…………………………...51

4.2.1. Typical chromatogram showing 6β-hydroxytestosterone, 11α-hydroxyprogesterone

and testosterone from methanolic extracts of cell culture media obtained after

treatment of human hepatocytes with vehicle (control), rifampicin (10 µM) and

bexarotene (1, 10, 25 and 50 µM)…………………………………………………54

4.2.2. Fold change in CYP3A4 activity in rifampicin (10 µM) and bexarotene (1, 10, 25

and 50 µM) treated human hepatocytes measured as 6β-hydroxylation of

testosterone as compared to untreated controls…………………………………..55

4.2.3. Fold change in docetaxel metabolism by S9 fractions obtained from rifampicin (10

µM) and bexarotene (1, 10, 25 and 50 µM) treated human hepatocytes as

compared to untreated controls………………….………………………………..57

4.2.4. Effect of rifampicin (10 µM) and bexarotene (1, 10, 25 and 50 µM) treatment of

human hepatocytes on the CYP3A4 protein levels as measured by western blot

analysis and expressed as band intensity. Western blot showing the effect of

rifampicin (10 µM) and bexarotene (1, 10, 25 and 50 µM) treatment of human

hepatocytes on the CYP3A4 protein levels……………………………...……….59

4.2.5. Fold change in the CYP3A4-specific mRNA levels after treatment of human

xii hepatocytes with rifampicin (10 µM) and bexarotene (1, 10, 25 and 50 µM) as

compared to untreated controls and measured by RT-PCR………………………61

.

LIST OF TABLES

Table 1.8.1. CYP3A inducers in different species…………………………………..…27

xiii

Table 4.1.1. Summary of the data for human liver microsomal metabolism of docetaxel

(0.5 – 5 µM) in the presence and the absence of bexarotene…..………….52

Table 4.2.1. Summary of the fold change in CYP3A4 activity, CYP3A4-specific mRNA

levels and immunoreactive CYP3A4 protein levels (band intensity) in

human hepatocytes following treatment with vehicle (control), rifampicin

(10 µM) and bexarotene (1, 10, 25 and 50 µM) for 72 hours……………...62

xiv 1 INTRODUCTION

1.1. Adverse Drug Reactions (ADRs)

Adverse Drug Reactions (ADRs) represent a major cause of morbidity and mortality and as such an enormous global health issue. Adverse reactions to drugs are very common in everyday medical practice. Absolute statistical figures on the incidence of ADRs are limited due to incomplete reporting and methodological constraints; nevertheless they illustrate the severity of the problem. Studies conducted in the United

States estimated that 6.7% of hospitalized patients have serious ADRs with a fatality rate of 0.32%, causing over 106,000 deaths annually (Lazarou et al, 1998). In addition, 20% of injuries or deaths per year of hospitalized patients may be a result of systemic adverse reactions (Leappe et al, 1991). The magnitude of the problems arising with adverse drug reactions has only been escalating.

The major causes of ADRs include:

a) Inappropriate dosing regimens that lead to drug levels that either exceed minimum toxic concentration (MTC) leading to drug-dose related toxicities or that lead to sub- therapeutic drug levels that result in clinical inefficacy and/or development of drug reactions. b) Idiosyncratic side effects occur in certain individuals who may be more susceptible as compared to most other individuals probably due to genetically determined enzyme deficiencies or mutations causing increased enzyme effects. c) Drug-drug interactions (DDIs) occur when administration of one drug affects the pharmacokinetic or pharmacological/toxicological action of another drug co-

1 administered. This is particularly a major issue in the treatment of complex diseases that require use of multiple drugs, such as cancer and HIV.

1.2. Drug-Drug Interactions (DDIs)

The occurrence of DDIs represents 3-5% of all systemic adverse drug reactions.

DDIs are well known to have significant clinical importance. This is reflected in recently reported fatal drug interactions (Kudo et al., 1997; Ferslew et al., 1998), and withdrawal of prominent drugs from the US market such as thalidomide (Lazarou et al., 1998;

Duchateau, 1998). Due to the clinical significance, DDIs are extensively investigated throughout the drug development continuum as well as during post-marketing surveillance. Whenever possible, pharmaceutical companies strive to predict potential interactions of new drug candidates as early in drug development efforts as possible in an attempt to minimize economic losses and to more effectively safeguard the welfare of patients.

1.3. Drug Interactions in Oncology

The problem of DDIs in cancer treatment is particularly vexing due to several reasons. Firstly, most cytotoxic agents target not only tumor cells but they also affect numerous other cells in the body. Secondly, most anticancer drugs typically have a steep dose-response curve. Thus, most cytotoxic agents have an extremely narrow therapeutic index. Thirdly, a large number of these compounds undergo metabolic activation (i.e. and ) or inactivation (i.e. taxol, ). These metabolic reactions are mediated by phase I and II enzymes, which exhibit marked inter- and intra-

2 subject variability. Such variability makes it extremely difficult to optimize therapy for

an individual patient. Fourthly, cancer regimens usually contain many

structurally and mechanistically diverse antineoplastic agents as well as supportive care

medication for the management of the toxicity of anticancer compounds. Over-the-

counter products, alternative medicines, herbs, and nutritional supplements can also

adversely interact with anticancer drugs. The use of such combinations and the number of

drugs involved increases the potential for drug-drug interactions.

Taken together, these factors result in increased incidence of DDI-associated

adverse outcomes in cancer patients.

1.4. Types of Drug-Drug Interactions

Drug interactions may be pharmaceutical, pharmacokinetic or pharmacodynamic

in nature (Lin et al, 2000). However, in many cases, the interactions have a

pharmacokinetic, rather than a pharmacodynamic basis. The pharmacokinetic interactions

may be due to alterations in absorption, distribution, metabolism and processes

that influence the drug levels in the body.

Absorption: Development of oral anticancer drugs has been hampered because of the limited and variable bioavailability of the drug after oral administration. Drug transporters and drug metabolizing enzymes (DMEs) in the intestinal epithelium represent a major obstacle for efficient drug absorption. Multidrug transporters belonging to the ATP-binding cassette containing family of proteins, such as P-glycoprotein (P-gp),

Multidrug Resistance-associated Protein (MRP) or Resistance Protein

(BCRP) are located at several sites in the body but also in the intestinal epithelium and

3 can actively extrude substrates out of the cell. Studies with knockout mice that do not have Mdr1a/1b and their wild type littermates with functional P-glycoprotein have shown that P-glycoprotein substantially impedes oral uptake of several anticancer drugs, including : the AUC of paclitaxel after oral administration was six times higher in the knockouts than in the mdr1a/1b-null mice compared to that in the wild type mice

(Sparreboom et al, 1997; Bardelmeijer et al, 2000). Docetaxel is a weak P-glycoprotein substrate and its limited oral bioavailability is due to CYP3A4-mediated first-pass metabolism in the gut wall and liver. Plasma concentrations of docetaxel are 50 times higher in mice when oral docetaxel is combined with the potent CYP3A4 inhibitor, but weak P-glycoprotein inhibitor, ritonavir (Bardelmeijer et al, 2000).

Distribution: The unbound drug is regarded the biologically active fraction because it can extravasate to reach target tissues. Thus, drug displacement from blood components or tissue-binding sites increases the apparent distribution volume. The net pharmacodynamic effect, however, is difficult to predict because drug displacement with an increase of the free fraction not only makes the drug more available to its target, but also assists metabolic and renal elimination. Cytotoxic drugs such as paclitaxel and etoposide that are highly protein-bound, have the potential to interact with other protein- bound drugs such as warfarin. However, the therapeutic implications of displacement of many anticancer drugs from their protein binding have yet to be defined (Stewart et al,

1998).

Metabolism: The principle cause of DDIs, involves hepatic and extra-hepatic biotransformation of drugs. Of most importance is the cytochrome P450 (CYP) superfamily of enzymes. Drugs that are substrates for CYP1A, 2B, 2C, 2D and 3A

4 families commonly exhibit DDIs related to modulation of CYP activity or expression.

The role of CYP enzymes in DDIs is described in the section 1.8..

Excretion: While most anticancer drugs are eliminated via systemic biotransformation, many others are cleared by the renal or biliary excretion pathways. For example, platinum compounds and are eliminated mainly by the kidneys through glomerular filtration and active tubular secretion. ADRs resulting from the use of such drugs may involve DDIs resulting from changes in active secretion or reabsorption of filtered drugs. For example, probenecid, salicylates, and trimethoprim may increase plasma concentrations of methotrexate to toxic levels (Bannwarth et al, 1996).

1.5. Non-Small Cell Lung Carcinoma (NSCLC)

Lung cancer is one of the leading causes of cancer related deaths in the US,

accounting for an estimated 32% of cancer deaths in men versus 25% of such deaths in

women (Jemal et al, 2004). Approximately 173,770 new cases of were

predicted for 2004, while 160,440 people were estimated to die from lung cancer related

morbidity (Ginsberg et al, 2001). Non-small cell type lung carcinoma (NSCLC) accounts for approximately 85% of all lung cancer cases. It is an aggressive tumor with high rates of almost 30% with regional and 40% with distant metastases (Schiller JH et al, 2001) and is associated with poor prognosis and a 5-year survival rate of <5%.

Briefly, chemotherapy is aimed at providing patients with palliative relief and extending survival in advanced NSCLC. Despite multiple drugs available for treatment of

NSCLC, there exist no standard chemotherapy protocols. However, platinum-based

5 regimens have gained importance in the treatment paradigms in advanced NSCLC.

Cisplatin-based therapy in advanced NSCLC improves overall survival, relieves 68-78%

of symptoms and enhances quality of life (NSCLC Collaborative Group, 1995).

However, severe nephrotoxicity and neurotoxicity associated with , led to the

development of less toxic drugs, more or less targeted in their action. The other classes of

drugs used routinely in advanced NSCLC are such as

(Shepherd et al, 1997), (paclitaxel and docetaxel) (Cerny et al, 1998),

topoisomerase I inhibitors (irinotecan or CPT-11 and ) (Fukuoka et al, 1992;

Lynch et al, 1994) and vinka alkaloids such as (Depierre et al, 1991). These

have been evaluated in the treatment of NSCLC as single agents and in combination with

platinum-based drugs and have been shown to offer better results than the best supportive

care possible.

Various new agents offering a unique mechanism of action and an improved edge

over the toxicity profile of older drugs have been either approved for clinical usage or are

under investigation. These include the epithelial growth factor receptor (EGFR) tyrosine

kinase inhibitors gefitinib (Iressa®; AstraZeneca, Wilmington, DE;

http://www.astrazenecaus.com) and erlotinib (Tarceva®; Genentech, Inc.; South San

Francisco, CA; http://www.genentech.com), antisense oligonucleotide Affinitak or

LY900003/ISIS 3521 (Lynch et al, 2003), inhibitors of kinase signaling pathways,

antiangiogenesis agents, antivascular endothelial growth factor monoclonal antibody such as bevacizumab (Avastin®; Genentech, Inc.; South San Francisco, CA; http://www.genentech.com) (Sandler et al, 2004), matrix metalloproteinase inhibitors,

and (RXR)-specific agonists.

6

Retinoids are a promising class of agents in the treatment (Toma et al, 1999) and

prevention of cancer (Dragnev et al, 2000; Niles et al, 2000). The early retinoids were nonspecific for receptor binding, and were primarily ligands to receptors or

RARs. However, RAR-specific agonists were associated with side effects such as dry

skin, cheilitis, headache, mucosal dryness, hypercalcemia and hypertriglyceridemia

(Dragnev et al, 2000). The occurrence of these side effects often emphasized the need to

develop drugs acting via different pathways and with lower toxicity profiles. It was soon

realized that synthetic retinoids specific for RXR were less toxic and as such were better

candidates for clinical use than the RAR agonists. RXRs serve as key factors in nuclear

receptor signaling since they heterodimerize with numerous nuclear receptors such as the

RAR, the thyroid hormone receptor, the peroxisome proliferator-activated receptor or

PPAR and the vitamin D receptor (Leid et al, 1992). Thus RXR ligands are likely to have

multiple target genes including those that are involved in regulating cell proliferation.

Bexarotene (Targretin®; Ligand Pharmaceuticals; San Diego, CA) is the first

FDA-approved retinoid, selective for RXR and currently approved for the treatment of

cutaneous manifestations of cutaneous T-cell lymphoma. Bexarotene modulates gene

expression which in turn controls cell differentiation and apoptosis. Clinical studies have

demonstrated prolonged survival of patients with NSCLC on rexinoids as single agents or

as part of combination regimens. There is encouraging evidence from the study of

combination regimens of RXR agonists with other agents such as vinorelbine, paclitaxel,

cisplatin and (Rizvi et al, 2001; Khuri et al, 2001; Rizvi et al, 2002).

Currently, a phase I study of docetaxel and bexarotene in patients with advanced resistant

7 NSCLC is underway to define the maximum tolerated dose of bexarotene that could be

administered daily in combination with weekly docetaxel (ASCO meeting abstract,

2004).

1.6. Bexarotene (Targretin-Ligand Pharmaceuticals Incorporation, San Diego,

CA.)

1.6.1. Chemistry

Targretin or bexarotene, 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-

naphthalenyl)-1-ethenyl]-benzoic acid (Figure 1.6.1.), is a specific RXR agonist (Shirley et al, 1997). Retinoids are biological derivatives of or and exert pleiotropic effects. They play a vital role in vertebrate development, differentiation and homeostasis (Chambon P, 1996). Two classes of nuclear retinoid receptors, the retinoic acid receptors (RARs) and retinoic X receptors (RXRs) have been identified, each with three isotypes (α, β and γ) (Mangelsdorf et al, 1990; Mangelsdorf et al, 1995; Petkovich et al, 1987). The RARs are activated by both all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA), whereas the RXRs are activated only by 9-cis retinoic acid

(Mangelsdorf et al, 1994). Compounds that bind selectively to the RXRs are termed

“rexinoids” (Mukherjee et al., 1997). RXR is a key member of the nuclear receptor superfamily that is capable of forming homodimers or heterodimers with other ligand- bound nuclear receptors such as RAR, PPAR, vitamin D receptor, and thyroid hormone receptor and exert transcriptional modification (Chambon P, 1996). Although bexarotene is not a retinoic acid derivative, it has one carboxylate moiety and is structurally similar to other RAR specific agonists. The C-3 position has a methyl substitution on its

8 tetrahydro-naphthalenyl moiety which is believed to contribute to its RXR affinity

(Shirley et al, 1997).

The chemical structure of bexarotene is in the following figure.

C-3

Figure 1.6.1. Chemical structure of bexarotene. The C-3 position is highlighted in

the box.

9

1.6.2. Mechanism of Action of Retinoids and Bexarotene

The retinoid dependent signals are induced through nuclear retinoid receptors and

their co-regulators in a ligand-dependent manner leading to transcriptional activation of

target genes that ultimately signal retinoid growth and differentiation effects (Dragnev et

al, 2000). Once the ligand binds to the ligand-binding domain (LBD) of the nuclear

, the co-repressor/s is/are released followed by homodimerization or

heterodimerization and subsequent recruitment of co-activators. This sequence of events

is required for initiating gene transcription. Various ligands, retinoid receptor dimer,

repressor, and co-activator complexes that are formed determine which genes are

activated and which are repressed, resulting in diverse gene expression and pleiotropic effects (Leid et al, 1992; Glass et al, 1996). As a result of these ligand-receptor and

receptor-DNA interactions, direct retinoid target genes that contain retinoid response

elements in their promoter regions become transcriptionally activated or repressed. These

ultimately lead to changes in gene expression that mediate biological effects and is

demonstrated in Figure 1.6.2. These biological effects include modifying genes implicated in inhibition of cell growth, the induction of cell differentiation, and the induction of apoptosis in a variety of tumor cell lines. ATRA and 9-cis-RA both play an important role in the regulation of gene transcription that ultimately results in the

regulation of cell division, growth, differentiation, and proliferation (Mangelsdorf and

Evans, 1994).

10

9-cis-RA

RXR NR RXR/NR dimer Coactivator

Corepressor hormone RXR NR

RARE

DNA Transcription

Figure 1.6.2. Mechanism of action of retinoids. The sequence of events required for gene transcription after ligand binding to the NR is given in the diagram. (As adapted from Rigas et al, 2005). (RXR – Retinoid X Receptor, NR – Nuclear

Receptor, RA – Retinoic Acid, RARE – Retinoic Acid Response Element).

1.6.3. Clinical Indications

The US Food and Drug Administration (FDA) approved bexarotene capsules in

December 1999 for the treatment of cutaneous manifestations of cutaneous T-cell lymphoma in patients who are refractory to at least one prior systemic therapy.

Bexarotene gel was approved in 2000 for the topical treatment of cutaneous manifestations of cutaneous T-cell lymphoma in patients who are refractory to at least one prior systemic therapy (US FDA and CDER, Approved claims for lymphoma, 2005).

11

1.6.4. Pharmacokinetics of Bexarotene

Bexarotene is available as oral capsules in the strength of 75mg and the recommended dose is 300mg/m2/day due to dose-limiting toxicities seen in phase I studies (Miller et al, 1997). Micronization of the formulation increases the AUC0-6 from

1.4(ng.h/ml)/(mg/m2) to 19.8(ng.h/ml)/(mg/m2), suggesting there is an approximate 14- fold enhancement of LGD1069 absorption. Absorption is enhanced by 40% when bexarotene is taken with a fat containing dietary supplement in diabetic patients.

Bexarotene is highly protein bound (99.8-99.9%) (Rizvi et al, 1999). Pharmacokinetic parameters are linear up to 600mg/m2 indicating a saturation of the absorption processes at that dose. The time taken to achieve maximum concentration (Tmax) is 1.4 – 3.6hrs

(Rizvi et al, 1999). The elimination half-life or t1/2 is found to be 6-7hours. The repeat dose AUC values are consistent up to 140mg/m2/day and the discrepancies after that dose level are attributed to interindividual patient variability in pharmacokinetic parameters.

Elimination of bexarotene and its metabolites is mainly hepatobiliary by metabolism while their excretion via the renal pathway is minimal. Metabolism of bexarotene is discussed in the section 1.6.5.

1.6.5 Metabolism of Bexarotene

Howell SR et al in 2001 conducted a study to reveal the nature of bexarotene metabolism in rats, dogs and humans. CYP3A is demonstrated to be involved in the oxidation of bexarotene in rats and humans. However, CYP2B is also implicated in the rat while CYP2C9 and CYP2C19 in the human (Hein et al, 1996). The major metabolites

12 found in the rat are 6/7-hydroxybexarotene and 6/7-oxobexarotene and a number of phase

II metabolites, primarily glucuronides. Liver slices from dogs treated with bexarotene show the predominant metabolite as acyl glucuronide of bexarotene followed by 6/7- hydroxy and 6/7-oxobexarotene. The metabolite profile found in human liver microsomes is similar to that of the rat. The significant pathways for bexarotene metabolism in the dog and human include oxidation at C-6 and C-7 positions and glucuronidation.

Bexarotene glucuronidation has not been well studied and the identity of the member of uridine diphosphoglucuronyl transferase (UGT) family involved remains unknown. In all three species investigated, 6/7-hydroxybexarotene predominates over 6/7-oxobexarotene, while the C-6 isomer predominates over the C-7 isomer. It is noteworthy that at the highest metabolite concentration achieved in patients, the contribution of metabolites to the clinical retinoid receptor activity of bexarotene is probably insignificant.

A study conducted by Howell et al in 1998, using rat hepatic microsomes, shed light on the effect of bexarotene on the total CYP content, the individual CYP isozyme profiles and its effect on the rate of metabolism by CYP enzymes as well as UGT. The experiment was conducted by injecting male Sprague-Dawley rats with a fixed dose

(100mg/kg) of bexarotene once daily for 4 consecutive days. After preparation of rat hepatic microsomes, the microsomal reactions clearly indicated that bexarotene pretreatment increased bexarotene metabolism by both, CYP and UGT enzymes. The increase in the rates of metabolism corresponded well with the increase in the total cytochrome P450 enzyme content in the hepatic microsomes. Clearly, these studies employing animal models document the inductive effects of bexarotene. It is interesting

13 to note that various enzymes known to be PXR target genes, such as CYP3A, CYP2B

and UGT, were induced in animals treated with bexarotene.

1.7. Docetaxel (Taxotere - Sanofi–Aventis Pharmaceuticals, France)

1.7.1. Chemistry

Docetaxel (Taxotere developed by Aventis Pharmaceuticals, formerly Rhône-

Poulenc Rorer Pharmaceuticals Inc. and Hoechst Marion Roussel, Inc.) is chemically

(2R,3S)-N-carboxy-3-phenylisoserine, N-tert-butyl ester, 13-ester with 5β-20-epoxy-

12α,4,7β,10β,13α-hexahydroxytax- 11-en-9-one 4-acetate 2-benzoate, trihydrate. It

belongs to the family of taxoid compounds. Docetaxel is derived semi-synthetically via

direct acylation of its inactive precursor 10-deacetyl baccatin III at the C13 position,

which is extracted from the needles of the European yew Taxus baccata (Lavelle F et al,

1995). The structure-activity relationship studies have stated that the phenylisoserine

side chain at the C-13 position of the baccatin ring is essential for the anticancer activity

of taxanes. Newer taxoids, with a better cytotoxicity profile than paclitaxel and docetaxel

in vitro are being synthesized with various functional groups at C-10 position such as ethers, esters, carbamates and carbonates (Ojima et al, 1996). The structure of docetaxel

is given in Figure 1.7.1.

14

CH3

O

O O O O OH R2 OH H C R 3 3 13 O

H3C H C HO 3 O O HN O CH3 R4 R1

R1 R2 R3 R4

H3C O

H3C H H OH CH3 Docetaxel O H H O H3C

Figure 1.7.1. Structure of docetaxel. The C-13 side chain of the taxanes is indicated in the rectangular box.

1.7.2 Clinical Indications

Docetaxel received accelerated approval by the US FDA in 1996 for the treatment of patients with locally advanced or metastatic breast cancer who progressed during -based therapy or relapsed during anthracycline-based adjuvant therapy. In

15 1998, docetaxel or taxotere was approved for the treatment of locally advanced or

metastatic breast cancer, which progressed during anthracycline-based treatment or

relapsed during anthracycline-based adjuvant therapy. It was approved in December

1999 for locally advanced or metastatic non-small cell lung cancer after failure of prior

platinum-based chemotherapy. In 2002, docetaxel was approved for its indication in

combination with cisplatin for the treatment of patients with unresectable, locally

advanced or metastatic non-small cell lung cancer who did not previously receive

chemotherapy for this condition. In May 2004, the US FDA approved docetaxel for

treatment in combination with prednisone (a steroid), for the treatment of patients with

advanced metastatic prostate cancer (US FDA and CDER).

1.7.3. Mechanism of Action of Docetaxel

1.7.3.1. Background on Microtubular Structure

Microtubules are long, hollow cylinders assembled from a heterodimeric globular protein called “tubulin”. They consist of 13 aligned protofilaments within which the tubulin subunits interact through longitudinal and lateral bonds. Tubulin assembly derives energy from the guanosine triphosphate hydrolysis that takes place on tubulin polymerization. A steady state is maintained between assembled tubulin and the free tubulin called “critical concentration” and the individual in the steady state undergo considerable changes in length consisting of rapid transitions in between periods of lengthening and shortening. This so-called “dynamic instability” is essential for microtubule-dependent cellular functions. Microtubules are involved in a variety of cell functions along with actin microfilaments and intermediate filaments. When a cell

16 begins to divide, the interphasic microtubules disappear and the mitotic spindle

assembles. The depolymerization of the mitotic spindle is critical for movement of the

chromosomes to the metaphase plate and their optimum segregation during anaphase

(Lavelle F et al, 1995).

1.7.3.2. Docetaxel mechanism of action

Docetaxel belongs to the class of taxanes, which are microtubule-stabilizing

agents. It promotes tubulin assembly in microtubules and inhibits their depolymerization

(Schiff et al, 1979; Ringel and Horwitz, 1991). It induces a mitotic block in proliferating

cells by acting as a mitotic and causes arrest in the S phase

(Hennequin et al, 1995). Docetaxel induces apoptosis in cancer cells. Although

controversial, it is found to cause phosphorylation of the bcl-2 protein which leads to

apoptosis (Suzuki A et al, 1998; Schimming R et al, 1999). Other indirect antitumor activities of docetaxel include induction of cell differentiation of certain leukemic cell types (Lavelle et al, 1995). At the ultrastructural level, docetaxel seems to strengthen the lateral bonds between tubulin subunits and thus the “critical concentration” or the free tubulin concentration decreases.

Although docetaxel binds to the microtubules at the same site as paclitaxel, it has twice the binding affinity as paclitaxel. Thus, docetaxel is more potent than paclitaxel as a tubulin assembly promoter and as a microtubule stabilizer (in vitro and in vivo) in

various human tumor models of breast, lung, ovarian, colorectal cancer, melanoma and

leukemia (Bissery et al., 1995).

17 1.7.4. Pharmacokinetics of Docetaxel

Since docetaxel is poorly soluble in water, it is administered employing Tween 80

and ethanol to improve its solubility. The commercial formulation of docetaxel is first

diluted in saline or 5% glucose and then administered intravenously over 1-2 hours at a

dose of 70-115mg/m2. Docetaxel is highly protein bound (>95%) and has a very high

volume of distribution (72-126L/m2). Its clearance is 267-369ml/min/m2 and the elimination half life is approximately 11 hours (Clarke and Rivory, 1999). Docetaxel is primarily eliminated via hepatic metabolism followed by biliary excretion, with more than 75% of the docetaxel and its metabolites being eliminated in feces and less than 10% in urine (Marre et al., 1996). Docetaxel is converted to its inactive metabolites via

CYP3A4-mediated metabolism in liver as described below.

1.7.5. Docetaxel Metabolism

Docetaxel elimination is primarily via hepatic metabolism while the renal clearance is minimal. Hepatic CYP3A4 contributes almost exclusively to the metabolism of docetaxel. The biotransformation of docetaxel to hydroxydocetaxel involves initial oxidation on the tert-butyl of the lateral side chain followed by cyclization, both reactions

catalyzed by CYP3A4 (Bruno et al, 1993, Shou et al, 1998). Hydroxydocetaxel is the main metabolite formed by oxidation at the C-13 side chain of docetaxel. Two other metabolites formed are oxazolidine-type of compounds resulting in cyclization of an unstable intermediate aldehyde. Another carboxylic acid derivate metabolite formed may undergo further cyclization to form an oxazolidinedione derivative, the major docetaxel

18 metabolite in human feces (De Valeriola et al, 1993). The metabolism in the human and animal models is found to be similar.

CH 3 O

O O O O OH

OH H C H 3 O

H 3C H C HO 3 O N O O CH H C 3 Y P OH 3A O 4 CH -m 3 e di CH at 3 ed H3C CH 3 Docetaxel O O O O O OH

OH H C H 3 O

H 3C H C HO 3 O HN O O CH n 3 io OH at liz O yc c HO CH g 3 in R H3C O CH3

O O O O Hydroxydocetaxel - M2 OH

OH H C H 3 O

H 3 C H C HO 3 O N O O CH 3 C Y OH P O 3A 4 HO -m ed H3C ia CH3 t ed

Metabolites M1 + M3 CH 3 O

O O O O OH

OH H C H 3 O

H 3C H C HO 3 O O N O CH 3 OH O O

H3C CH3

Metabolite M4

Figure 1.7.2. Biotransformation of docetaxel by hepatic CYP3A4 in humans. M2 or hydroxydocetaxel is the major metabolite formed.

19 1.8. Cytochrome P450 Family

Cytochrome P450 (CYP) enzymes are undoubtedly the most important class of enzymes responsible for biotransformation of xenobiotics and endogenous substances in the body. These membrane bound heme-containing enzymes are present in the ribosomes on the endoplasmic reticulum (ER) or the inner mitochondrial membrane.

Ubiquitously expressed in the body, the CYP enzymes are abundant in the hepatic and small intestinal tissues. Of the fifty-seven human P450s known, at least fifteen are known to participate in phase I xenobiotic metabolism. While metabolizing drugs, carcinogens, food additives, pollutants, pesticides, and environmental chemicals, they are also responsible for oxidative, peroxidative, and reductive metabolism of endogenous compounds including steroids, bile acids, fatty acids, prostaglandins, leukotrienes, biogenic amines, and retinoids. Biotransformation of a substance by the CYP enzymes may result in one of the following: a) formation of a pharmacologically inactive metabolite which is subsequently excreted, b) activation of prodrugs to pharmacologically active metabolites, c) activation of non-toxic procarcinogens into potent carcinogens by the DMEs, d) metabolism of active drugs resulting in formation of toxic metabolites. These enzymes cause primary modifications in lipophilic compounds and catalyze mono-oxygenase reactions of lipophilic compounds allowing subsequent use of the attached hydroxyl group as a negative group that may be further modified by phase

II enzymes (Handschin et al, 2003).

20 1.8.1. The CYP3A subfamily

The CYP3A subfamily includes CYP3A4, CYP3A5, CYP3A7 and CYP3A43.

CYP3A5 is expressed in 2% of the adult population and contributes 10% to the total

CYP3A level. CYP3A7, a primary fetal isoform, is rarely detected in adults. The latest

CYP3A enzyme to be identified is CYP3A43, constituting only 0.2-5% of CYP3A and found in the prostate and testis. Members of the CYP3A subfamily contribute significantly to metabolism of clinically used drugs and other substances. The major sites of expression of CYP3A4 in the humans include the liver and gastrointestinal tract and it makes up 30-50% of the total cytochrome P450 content in these tissues (Watkins et al,

1994). It is responsible for the metabolism of more than 50% of the cytochrome P450 mediated catalysis of clinically used drugs (Lehmann et al, 1998). The CYP3A4 substrates include a range of chemically diverse compounds such as naturally occurring and synthetic glucocorticoids (for example, cortisol, dexamethasone), antiglucocorticoids

(for example, pregnenolone 16α-carbonitrile, PCN), antitubercular agent rifampicin

(RIF), macrolide antibiotics (for example, triacetyloleandomycin, erythromycin), anticonvulsants (for example, phenobarbital), antiestrogens (for example, tamoxifen) and many more classes of xenobiotics (Kocarek et al., 1995; Lu and Li, 2001). Given that

CYP3A4/5 is abundantly expressed in hepatic and extrahepatic tissues, and that numerous xenobiotics are metabolized by CYP3A4, there exists a significant potential for

DDIs. Furthermore, a number of compounds, many of which are substrates, alter

CYP3A4 function either by inhibiting its activity or modulating its expression. Indeed, numerous clinical DDIs involve CYP3A4/5 inhibition or induction.

21 1.8.2. CYP3A4 Inhibition

Enzyme inhibition involves several mechanisms that may be reversible or

irreversible. Changes in binding affinity (as apparent from changes in the Michaelis-

Menten constant of reaction, Km) or the rate of reaction (as apparent by the changes in the

maximal rate/velocity of reaction or Vmax), generally provide a basis for classification of

inhibition as being competitive, non-competitive or uncompetitive. In competitive

enzyme inhibition, the inhibitor and the drug-substrate may share chemical structure

similarity and compete for the same active site on the enzyme. This may be partly or

completely reversible with an increase in the drug or substrate concentration which

displaces the inhibitor from the enzyme. Whereas in noncompetitive enzyme inhibition,

the inhibitor may inhibit the enzyme by combining at a site on the enzyme that is

different from the active site, called the allosteric site. This type of inhibition is

completely dependent on the inhibitor concentration and is irreversible. In uncompetitive

enzyme inhibition, the inhibitor binds to the enzyme substrate complex after the binding

site becomes available only when the substrate is bound. Cyclosporin A is a competitive

inhibitor of CYP3A4, whereas quercetin is an uncompetitive and is a non- competitive inhibitor of CYP3A4. Some compounds bind irreversibly to the enzyme and are called “suicide inhibitors”. An example of this kind is ethinyl estradiol binding to

CYP3A4 (Wrighton and Thummel, 2000). Clinically, these interactions are important to know since they often result in considerable changes in drug safety and efficacy. For example, ketoconazole, a prototype inhibitor of CYP3A4 is known to increase oral midazolam AUC by 1490% which is a dramatic change in extent of drug exposure (von

22 Moltke et al., 1996b; Greenblatt et al., 1998b; Tsunoda et al., 1999; Yuan et al.,1999;

Venkatakrishnan et al., 2000).

1.8.3. CYP3A4 Induction

Numerous compounds, natural and synthetic, from diverse families have been known to induce various cytochrome P450s. CYP3A4 is one of the most inducible enzymes, and as such, its induction underlies clinically relevant drug-drug interactions.

The increased expression of both, hepatic and intestinal CYP3A4 has been shown to accelerate the rate of , including enhanced first-pass metabolism

(Pichard et al, 1990). The overall effect is reduced systemic availability of the drug, but an increased availability of the metabolite. Many clinically useful compounds such as rifampicin, phenobarbital and St. John’s wort are well known to cause serious DDIs resulting from CYP3A4 induction. The clinical outcomes have included increased rate of organ rejection (due to increased cyclosporin A metabolism by rifampicin), increased rate of clearance of aromatase inhibitors by tamoxifen and increased clearance of oral contraceptives in patients taking drugs such as rifampicin, phenobarbital and topiramate.

Usually, the CYP3A4 activity is measured by the metabolism of CYP3A4 marker substrate and the CYP3A4 protein levels. The induction potential of a compound is conventionally assessed in vitro by treating primary cultures of human hepatocytes with the test compound, preparing sub-cellular fractions from the treated hepatocytes and measuring the CYP3A4 activity as the rate of 6β-hydroxylation of testosterone (Li et al,

1992). It is interesting to note that the effect of the inducer drugs is not restricted to the regulation of the CYP enzymes, other enzymes and drug transporters but incorporates a

23 more pleitropic response including the up- or down- regulation of many other genes and

physiological systems.

1.8.4. Regulation of CYP3A4

CYP3A4 induction in humans remained an enigma to researchers for decades

before the discovery of PXR for a variety of reasons. Firstly, there was a marked

interspecies variability seen in the induction of CYP3A enzyme. Table 1.8.1. gives a few

examples of drugs showing interspecies differences in induction. Secondly, the role of

glucorticoid receptor in CYP3A induction was not clear before Schuetz et al reported in

2000 that glucorticoid receptor was required for CYP2B induction by steroids and not for

CYP3A in rodents. Lastly, compounds that induce CYP3A vary considerably in their

structure, molecular weight and lipophilicity.

Pregnane-activated receptor (PXR), also called steroid and xenobiotics receptor

has been implicated in CYP3A4 expression regulation since its discovery earlier in

mouse (Kliewer et al., 1998) and then in human (Bertilsson G et al, 1998; Lehmann et

al, 1998, Blumberg et al, 1998). It was discovered as an orphan nuclear receptor and was found to be activated by naturally occurring steroids such as pregnenolone and progesterone, and synthetic glucocorticoids and antiglucocorticoids (Kliewer et al.,

1998). It is primarily expressed in the liver and the small intestine. Like most other

nuclear receptors, it consists of an N-terminal transactivating domain, followed by a

DNA-binding domain (DBD) and a C-terminal ligand-binding domain (LBD). Three

transcripts of PXR have been characterized in the human liver while two distinct ones in

rat liver and three in the rabbit liver (Jones et al., 2000). The human, rat, mouse and

24 rabbit PXRs share an approximate 95% sequence homology in the DBD while only 75%

homology in the LBD (Lehmann et al, 1998). This disparity may account for the aforementioned difference in the activation profile of CYP3A4 by xenobiotics. The differences observed in the species-species induction by a variety of compounds may be explained on the basis of the structural differences in the LBD of the PXR since the DBD seems to be well conserved across species (Jones et al, 2000).

CYP3A gene expression occurs due to interaction of PXR with a response element well-conserved in the CYP3A gene promoter region. PXR binds to the response element in the 5’-flanking region of the CYP3A gene as a heterodimer with RXRα. When this interaction occurs with high affinity, CYP3A gene transcription enhances. The proximal promoter region of the CYP3A gene containing the response element is not well conserved across species. In humans, hPXR-RXRα heterodimer binds to the response element on the CYP3A4 gene promoter which is an ER6 motif and activates transcription through that. A similar ER6 motif exists in rabbits as the binding site for PXR-RXRα

heterodimer while DR3 PXR response element (PXRE) is the binding site for the PXR-

RXRα heterodimer in rat and mouse (Jones et al, 2000).

There is a remarkable interspecies variation in the activation of CYP3A enzyme.

For example, rifampicin is a potent inducer of CYP3A in rabbit and human livers but is a poor inducer in rats (Kocarek et al, 1995). However, pregnenolone 16α-carbonitrile

(PCN) is a potent inducer of CYP3A in rats and mice but not in human liver. The following table gives a few examples of drugs showing distinct differences in CYP3A activation across species.

25 Table 1.8.1. CYP3A Inducers in Different Species

Species CYP3A Inducers

Weak Potent

Mouse Rifampicin PCN

Clotrimazole Dexamethasone

Rat Rifampicin PCN

Dexamethasone

Clotrimazole

Rabbit PCN Rifampicin

Clotrimazole Dexamethasone

Phenobarbital

Human PCN Rifampicin

Dexamethasone Clotrimazole

Phenobarbital

26 2 RATIONALE AND HYPOTHESIS

As indicated earlier, docetaxel metabolism involves extensive hepatic biotransformation and CYP3A4 is exclusively involved in the conversion of docetaxel to its metabolite, hydroxydocetaxel. This metabolite is further converted into other species but neither of the metabolites show any pharmacological activity or toxicity (Marre et al,

1996). As such, docetaxel metabolism by CYP3A4 represents a detoxification pathway.

Modulation of hydroxydocetaxel formation by CYP3A4 due to either inhibition or induction of the enzyme is likely to profoundly affect the systemic exposure/area under plasma concentration versus time curve (AUC) of docetaxel, which may subsequently cause changes in therapeutic efficacy or toxicity.

Hepatic CYP3A4 is also predominantly involved in the metabolism of bexarotene. Pre-clinical data have shown that repeated administration of bexarotene in rats caused enhanced clearance of the drug, a phenomenon called as “autoinduction”.

This was accompanied by a marked increase in the level of hepatic CYP3A and UGT in rats. To the best of our knowledge, studies that specifically address potential CYP3A4 induction by bexarotene have not been reported. In contrast to the observation in animal studies, in a small phase I study, autoinduction of bexarotene was not noted. Thus, the potential that bexarotene may have inductive effects on the human CYP3A4 remains unresolved.

Given that bexarotene and docetaxel are likely to be used in combination chemotherapy (2004 ASCO Meeting Abstracts), and that CYP3A4 is exclusively

27 involved in the metabolism of both drugs, we hypothesize that bexarotene alters the hepatic metabolism of docetaxel.

2.1. Specific Aims

To address the overall hypothesis we undertook the following two specific aims:

2.1.1. AIM I

To investigate whether bexarotene inhibits the CYP3A4 mediated metabolism of docetaxel by human liver microsomes.

Working Hypothesis:

Since both compounds are CYP3A4 substrates we postulate that bexarotene may competitively inhibit docetaxel metabolism.

2.1.2. AIM II

To determine the mechanism of bexarotene induced change in CYP3A4 mediated metabolism of docetaxel in primary cultures of human hepatocytes.

Working Hypothesis:

Given that bexarotene increases CYP3A in animals, we tested the hypothesis that bexarotene acts as a CYP3A4 inducer in human hepatocytes and that results in increased docetaxel metabolism.

28

3 MATERIALS AND METHODS

3.1. Experimental Strategy

The effect of bexarotene on the hepatic CYP3A4-driven docetaxel metabolism was assessed in vitro.

Studies addressing Aim I: To determine whether bexarotene inhibited docetaxel metabolism, microsomal reactions using commercially pooled human liver microsomes were carried out. Our initial efforts attempted to optimize the microsomal incubation conditions. This involved varying reaction conditions with respect to microsomal protein, incubation period and docetaxel concentration. Using these optimum conditions, we carried out the docetaxel metabolism reactions by human liver microsomes. We tested a range of docetaxel concentrations (0.5-5µM) in the absence and the presence of bexarotene (0.1-10µM). Ketoconazole, a known inhibitor of CYP3A4 was used as the positive control. Docetaxel reactions were analyzed using a well-established high performance liquid chromatography (HPLC) detection method. The substrate depletion was monitored and was taken as a measure of the rate of reaction.

Studies addressing Aim II: To determine whether bexarotene increased docetaxel metabolism by inducing hepatic CYP3A4, primary human hepatocytes obtained from liver transplant surgeries, were cultured. The experimental strategy employed for the induction assessment studies is outlined in figure 3.1.1. below. Several known inducers of CYP3A4 (for example, rifampicin) require 48 hours to cause an increase in the enzyme activity. This information was valuable in designing our induction assessment studies. The primary hepatocytes were isolated, plated and then treated with various

29

drugs for 72 hours. Rifampicin, a potent inducer of CYP3A4 in humans, was used as the positive control. After completing the drug treatments, the cells were exposed to drug- free medium to help release of the drug from intact hepatocytes. CYP3A4 activity was measured as the rate of 6β-hydroxylation of testosterone, while CYP3A4 protein levels and CYP3A4-specific mRNA levels were analyzed with western blot assay and real-time polymerase chain reaction (PCR) respectively. Sub-cellular fractions or S9 fractions were used to determine if bexarotene-treated human hepatocytes had an effect on docetaxel metabolism. These metabolic reactions using S9 fractions were carried out using the method above outlined.

30 Experiments with Primary Human Hepatocytes

CYP3A4 Primary Activity Human (Testosterone / Hepatocytes Docetaxel)

Protein Detection – 48 hours Western Blot Analysis

mRNA Detection – Real-time PCR Drug-free medium (1-3 hours) S9 fractions after differential centrifugation Drug Treatment (72 hours)

Figure 3.1.1. Diagrammatic representation of the experimental strategy to assess the inductive effects of bexarotene on CYP3A4.

31

3.2. Human Liver Microsomal Metabolism of Docetaxel in the Absence and in the

Presence of Bexarotene

3.2.1. Chemicals

Paclitaxel, rifampicin, testosterone, 6β-hydroxytestosterone and NADPH were obtained from Sigma Chemicals Co. (St. Louis, MO). Docetaxel was a gift from Sanofi-

Aventis (Bridgewater, NJ). Bexarotene capsules were obtained from the UC Hospital pharmacy and bexarotene was dissolved in dimethylsulfoxide (DMSO) from the clinical formulation. Ketoconazole was a gift from Jensen Pharmaceuticals. Pooled human liver microsomes (HLMs) were purchased from Cellz Direct, (Tucson, AZ). William’s E culture medium and medium supplements were obtained from BioWhittaker (San Diego,

CA). HPLC solvents were ordered from Fisher Scientific. Monoclonal antibody for human CYP3A4 was obtained from Gentest Corporation (Woburn, MA). The 780- basepair CYP3A4 cDNA probe and the horseradish-peroxidase conjugated anti-mouse and anti-goat secondary antibodies were obtained from Oxford Biomedical Research Inc.,

(Oxford, MI).

3.2.2. Reaction Optimization

Initial efforts were focused on optimizing the conditions for microsomal metabolism of docetaxel by liver microsomes. We employed sheep liver microsomes for this purpose. It was estimated that for optimizing the protocols, at least 10 experiments each employing about 50mg microsomal protein would be required, which would be cost-prohibitive if human liver microsomes were to be used. Microsomes were prepared

32 from liver excised from untreated sheep in unrelated experiments conducted at the UC

College of Medicine. Typically, such tissues are discarded after sacrificing the animals at

the end of each experiment. Differential centrifugation was employed for the preparation

of microsomal fractions and docetaxel metabolism was carried in the presence of

NADPH. The rate of decrease in the docetaxel levels was employed to assess the overall

reaction rate.

In these initial experiments, we optimized the following three variables:

a) Microsomal Protein Needed for the Reaction

Incubations were carried out in triplicate with increasing amount (500µg-2mg) of

microsomal protein to ascertain the optimum amount of protein required to carry out

the reactions. The reaction (rate of substrate depletion) was found to be linear in this

range of concentrations and with due considerations of the assay sensitivity, we

determined that the optimum quantity of microsomal protein was 1mg.

b) Range of Docetaxel Concentrations

Incubations were carried out in triplicate with increasing concentrations (0.1-100µM)

of docetaxel, to obtain the linear range of docetaxel concentrations to be used in our

study. The reaction rate increased linearly with docetaxel concentration in the range

of 0.1-2µM, and the reaction reached near saturation at approximately 5µM

concentration. For further experimentation the concentration range of 0.5-5µM was

employed. c) Microsomal Incubation Period

To determine the optimum incubation period, experiments were designed with

incubations periods varying from 5-90minutes. The data obtained from these

33 reactions indicated that 30-45minutes was the optimal period for incubation. We

employed 30minute incubations for all our later experiments.

3.2.3. Assessment of the Potential Influence of Bexarotene on Docetaxel Metabolism

in Human Liver Microsomal Reactions

Based on the initial experiments conducted for establishing experimental conditions, we employed 1mg of microsomal protein, 30minute incubation period and docetaxel concentration range of 0.5-5µM. Under these conditions, the reaction rate at

1mg HLM was observed to be more or less similar to that with 1mg sheep liver microsomes (also confirmed by assessing the rate of testosterone 6β-hydroxylation, a reaction attributed to CYP3A activity). Docetaxel (0.5-5µM) was incubated with liver microsomes. The reaction was carried out in phosphate buffer (pH 7.4) and was initiated by the addition of NADPH (1mM). The total reaction volume was 1ml. The reaction was allowed to proceed in a shaking water bath at 37˚C. At the end of the incubation period

(30minutes), the reaction was terminated by the addition of the extraction solvent, ethyl acetate (2ml), followed by the addition of the internal standard, paclitaxel (1µM). Thus liquid-liquid extraction was employed to concentrate the drug levels in the sample. The extracted organic layer was dried under nitrogen and reconstituted in 250µl of acetonitrile:water (43:57). The reconstituted sample was analyzed by the HPLC method described in section 3.2.4.. This method is a modified version of a published method for docetaxel analysis by HPLC. To determine the effects of bexarotene on the metabolism of docetaxel, microsomal reactions with docetaxel were carried out in the absence or the

34 presence of bexarotene (0.1-10µM) and in the presence of ketoconazole (10µM), which is a known inhibitor of CYP3A4.

Bexarotene Concentrations – Microsomal reactions were carried out in the absence or the presence of increasing concentrations of bexarotene (0.1-10µM), a clinically relevant concentration range.

3.2.4. HPLC Analysis of Docetaxel

Docetaxel levels in reaction media were measured following liquid-liquid extraction of samples, using reverse-phase HPLC. Extracts were chromatographed isocratically with acetonitrile:water (43:57) at a flow rate of 1ml/min. Separation was accomplished on a Symmetry C18, 5µm, 3.9×150mm column. Pre-column C18 inserts or the guard column was replaced after approximately 80 samples to maintain optimum resolution. The internal standard used was paclitaxel (1µM). Docetaxel and paclitaxel were quantitated at 227nm using the dual wavelength ultraviolet tunable absorbance detector from Waters Corporation Inc. The docetaxel concentration in the microsomal extract was quantitated using Waters Millenium software utilizing linear regression analysis of peak area ratio (docetaxel/paclitaxel) versus docetaxel and paclitaxel standards in reaction media. The linear range of quantitation was 0.1-100µM. Retention times were 18minutes for docetaxel and 23minutes for paclitaxel.

3.3. Assessment of the Potential Induction of Docetaxel Metabolism by Bexarotene

in Primary Human Hepatocytes

3.3.1. Hepatocyte Culture

35 Human hepatocytes, isolated from surplus liver tissue unsuitable for

transplantation, were provided for research purposes, by Dr. Stephen Storm (Department

of Pathology, University of Pittsburgh, PA) under the auspices of NIH funded Liver

Tissue Procurement and Distribution System (LTPADS). Depending on the intended use

of the primary human hepatocytes, they were plated in different formats. Cell plating was

done within 48 hours of cell isolation. Cell viability was determined with trypan blue dye

exclusion test and found to be at least 80%. Usually, hepatocytes were plated in collagen

coated 6-well plates (1X106cells/well) for CYP3A activity and protein levels. In parallel,

collagen containing T25cm2 flasks were plated for total RNA extraction for real-time

PCR analysis. The cells were maintained in William’s E medium upon arrival and

incubated at 37ºC and 5% CO2 for at least 1-2hours. William’s E medium was supplemented with insulin (10-7M), dexamethasone (10-7M) and gentamicin (0.5µg/ml).

3.3.2. Drug Treatments

Hepatocytes were exposed to the different drug treatments for 72hours. The drug- free medium was substituted by the drug-containing medium and replaced every 24 hours. The primary human hepatocytes were exposed to the following concentrations of bexarotene: 1, 10, 25 and 50µM. Rifampicin (10µM), which is a known inducer of

CYP3A4 in human hepatocytes was used as the positive control in the experiment. The controls used were cells treated with vehicle and DMSO alone. Drug stock solutions

(1000x) were prepared in DMSO and were diluted directly in the medium. The final concentration of DMSO was 0.1% and equal in all treatments including the control

36 group. This concentration of DMSO was non-toxic to the hepatocytes as found in previous experiments.

On completion of the drug treatment after 72 hours, the CYP3A4 activity was measured and hepatocytes were scraped and collected for the microsomal metabolism of docetaxel, analysis of CYP3A4 protein content and CYP3A4 mRNA levels.

3.3.3. Assay of CYP3A4 in Human Hepatocytes

The rate of 6β-hydroxylation of testosterone, a reaction known to be catalyzed by

CYP3A4, is thus, substantially indicative of CYP3A4 activity in intact hepatocytes in vitro (Waxman et al., 1983). Following 72 hours of drug exposure, the drug-containing medium was replaced by drug-free medium facilitating the drug release from the cells.

The cells were incubated with the drug-free medium for a period of 30minutes.

Following this, the testosterone-containing medium was incubated for 30minutes and the supernatant collected for analysis with HPLC. The internal standard added to the supernatant was 11α-hydroxyprogesterone (final concentration being 1µg/100µl of reconstitution volume). The media was extracted using 3ml of the methylene chloride, after adequate vortexing. Liquid-liquid extraction was employed in order to concentrate the probable low levels of 6β-hydroxytestosterone. The extracted organic layer was then evaporated under a gentle nitrogen stream and reconstituted using the mobile phase (1:1

v/v methanol:water). This was injected into a µ-Bondapak C18 column (3.9×300mm,

10µm) with a µ-Bondapak C18 guard column (10µm) using Waters 717 WISP auto sampler. A modified and published method of 6β-hydroxytestosterone HPLC analysis

37 (Waxman et al., 1983) was employed. HPLC analysis entailed an isocratic flow of

mobile phase (1:1 v/v methanol: water) at the rate of 1ml/minute. The detector used was

Waters 486 UV/VIS and was set at a wavelength of 242nm. The total run lasted for 40

minutes, with 6β-hydroxytestosterone, 11α-hydroxyprogesterone and testosterone eluting

at 9, 15 and 23 minutes, respectively. The software used for the above HPLC system was

the Millennium version by Waters Corporation, Inc.

3.3.4. Docetaxel metabolism by human hepatocytes

Docetaxel metabolism was carried out employing S9 fractions prepared from

treated versus untreated human hepatocytes. Hepatocytes were subjected to differential

centrifugation (4ºC) at the speed of 9000xg for 20minutes in the Marathon 21K/BR

centrifuge (Fisher Scientific, Inc) for S9 preparation. The protein content was determined

using Lowry’s protein assay (Bio-rad DC assay) (Schuetz and Schinkel, 1999). The

metabolic reactions were carried out using bexarotene-treated S9 fractions with the

method described earlier. Docetaxel 1µM was incubated under the same optimum

conditions described in section 3.2.3., with the S9 fractions. These reactions were

compared to those done using vehicle and rifampicin-treated (10µM) S9 fractions.

Paclitaxel 1µM was used as internal standard and docetaxel analysis by HPLC was

carried out after extraction of the samples. The HPLC method of docetaxel analysis has

also been described in section 3.2.4.. The observed change in the docetaxel peak or substrate depletion was used as a measure of the rate of docetaxel metabolism.

3.3.5. Western blot analysis of immunoreactive CYP3A4 protein levels in human

38 hepatocytes

The CYP3A4 protein levels in untreated cells and bexarotene-treated cells were determined using western blot analysis. The human hepatocytes were harvested using a cell scraper after 72 hours of drug treatment and resuspended in 0.1M potassium phosphate (pH 7.6) buffer, and homogenized using a tissue homogenizer. S9 fractions from these homogenates were collected after centrifugation at 9000xg for at least 20 minutes. The protein content was determined using Lowry’s protein assay (Bio-rad DC assay) (Schuetz and Schinkel, 1999). Equal amount of protein (3µg) was loaded into each well for controls and various treatments. The proteins were separated using 12% sodium dodecyl sulfate-polyacrylamide electrophoresis and were electrophoretically blotted onto nitrocellulose membranes (Lecureur et al, 2000). The nitrocellulose membranes were blocked with 3% bovine serum albumin in PBS-T (0.1M sodium phosphate buffer, pH 7.4, containing 0.1% Tween 20, 10% fetal bovine serum) for 45 minutes and incubated with anti-CYP3A4 monoclonal antibody (1:500 dilution) in 5% milk and 0.1% PBS-T. The blots were then incubated with a horseradish-peroxidase conjugated anti-mouse secondary antibody (1:500), and visualized by enhanced chemiluminescence (ECL, Amersham). The membranes were placed on a photographic film for 5 minutes. After film development, the integrated densities of the resultant bands were determined using a densitometer. The immunoblots were quantitated employing

NucleoVision image analyzer with Gel Expert photodensitometry software (Nucleotech,

CA).

3.3.6. Real-time PCR to assess the levels of CYP3A4 mRNA in human hepatocytes

39 The total cellular RNA was isolated using TRIzol (GIBCO BRL, Rockville,

MD) and quantified spectrophotometrically at 260 nm. We utilized 2µg RNA for reverse transcription. The cDNA generated was amplified by real-time PCR on ABI 7000 instrument using SYBR green detection method. We used the ABI reagents and supplies for the same. CYP3A4-specific primers were designed using Primer Express software from Applied Biosystems, Inc. The Ct (Cycle number when amplification signal crosses the threshold value set in the exponential phase of amplification) values of CYP3A4 were normalized to the Ct values of GAPDH amplification. Fold induction over untreated controls were determined as per real-time PCR guidelines.

3.4. Statistical Analysis

The extent of metabolism or the rate of reaction of docetaxel metabolism was calculated as the measure of substrate or docetaxel depletion in presence of NADPH and absence of bexarotene. Various treatment groups were analyzed by Student’s t test

(Sigma, version 2.03). A p < 0.05 was interpreted as the level of statistical significance.

Fold change in the CYP3A4 activity, measured as the rate of 6β-hydroxylation of testosterone, immunoreactive CYP3A4 and CYP3A4 mRNA levels in various treatment groups was analyzed by Student’s t test (Sigma version 2.03). A p < 0.05 was interpreted as the level of statistical significance.

40 4 RESULTS

4.1. Human Liver Microsomal Metabolism of Docetaxel in the Presence / Absence

of Bexarotene

We employed human liver microsomes as an in vitro model to assess the potential

influence of bexarotene on the hepatic metabolism of docetaxel. To determine if

bexarotene inhibits docetaxel metabolism, human liver microsomal reactions were carried

out in triplicate with 1mg of microsomal protein for 30minutes (37˚C) with docetaxel

(0.5-5µM) concentrations in the absence of bexarotene and in the presence of bexarotene

(0.1-10µM) concentrations. Unchanged docetaxel was extracted using liquid-liquid

extraction and analyzed using HPLC/UV detection. Docetaxel levels extracted from

microsomal incubations in the absence of the reaction (accounted for recovery compared

to a pure standard) were compared to the unreacted docetaxel amount in the microsomal

standard extract. The difference between the two was considered as the loss due to

metabolism. Independently, it was seen that in the presence of ketoconazole (10µM), the

peaks that could be clearly attributed to CYP3A4 metabolites, were completely abolished

and docetaxel levels remain unchanged in the presence of ketoconazole (indicating nearly

complete inhibition of docetaxel metabolism). Thus, the extent of metabolism or the rate

of reaction was calculated as substrate depletion in the presence of NADPH and the

absence of bexarotene. To ascertain that docetaxel metabolism indeed occurred, the

difference between docetaxel recovered from the microsomal incubations in the absence

of NADPH or the microsomal standards and that recovered from microsomal reactions

41 was statistically analyzed by Student t’s test. The depletion was considered significant at p≤0.05, n=3. Likewise, statistical comparisons were also made for docetaxel depletion in human liver microsomal incubations (with NADPH present) in the absence and the presence of bexarotene (0.1-10µM).

From the human liver microsomal metabolism data, it was clear that with increasing concentrations of docetaxel, the rate of reaction approached saturation at concentrations nearing 2µM and was completely saturated at docetaxel 5µM. The reaction rate was linear from 0.5-1µM, which clearly represented the range that would yield the most reliable data for determining the influence of bexarotene on docetaxel metabolism. A plot of the rate of docetaxel metabolism versus docetaxel (substrate) concentration could be suitably described employing Michaelis-Menten kinetics (Figure

4.1.1.). This plot was used to calculate the Vmax and Km values, which were consistent with previously reported values (Monsarrat et al, 1997, Shou et al, 1998).

42

Figure 4.1.1. The Michaelis – Menten plot of substrate (docetaxel) concentration versus the rate of docetaxel metabolism. It indicates that the rate of reaction is reaching saturation near docetaxel concentration of 2µM and is completely saturated at docetaxel concentration of 5µM.

43 Figure 4.1.2. demonstrates the rate of metabolism of docetaxel (0.5µM) in the absence and presence of bexarotene (0.1-10µM). There was a statistically significant difference between the docetaxel peak in the absence of bexarotene and that obtained in the absence of reaction. However there was no statistical difference between the docetaxel peak in the absence of bexarotene and in the presence of bexarotene (0.1-10µM) (p ≤ 0.05).

The microsomal reaction of docetaxel (1µM) in the absence and presence of bexarotene

(0.1-10µM) is shown in Figure 4.1.3. A statistical significant difference was noted between docetaxel in the absence of reaction and docetaxel after reaction, but in the absence of bexarotene. There was no significant difference seen in the substrate reduction in the presence of bexarotene at any of the concentrations from (0.1-10µM) (p ≤ 0.05).

44

Figure 4.1.2. Human liver microsomal metabolism of docetaxel (0.5µM) in the absence and the presence of increasing concentrations of bexarotene (0.1-10µM).

Human liver microsomal protein (1mg) was incubated (37°C, 30minutes) with docetaxel (1µM) and bexarotene (0-10µM) in the presence of NADPH (1mM).

Results are expressed as the extent of docetaxel metabolism in the presence of bexarotene as compared to the control (no bexarotene, no NADPH). (Mean ± SEM, n = 3, p ≤ 0.05, * statistically significant from control).

45 Docetaxel (1µM) Metabolism

Figure 4.1.3. Human liver microsomal metabolism of docetaxel (1µM) in the absence and the presence of increasing concentrations of bexarotene (0.1-10µM). Human liver microsomal protein (1mg) was incubated (37°C, 30 minutes) with docetaxel

(1µM) and bexarotene (0-10µM) in the presence of NADPH (1mM). Results are expressed as the extent of docetaxel metabolism in the presence of bexarotene as compared to the control (no bexarotene, no NADPH). (Mean ± SEM, n = 3, p ≤ 0.05,

* statistically significant from control).

46 In Figure 4.1.4., which showed the microsomal reaction of docetaxel (2µM), there was evidence of metabolism reaction due to a significant difference between the substrate depletion in the reaction and the microsomal standard. However, there was no significant difference seen between the docetaxel depletion in the absence of bexarotene and the presence of bexarotene (0.1-10µM). The microsomal reactions of docetaxel 2µM in the presence of bexarotene 2.5µM showed a statistically significant difference. This finding seemed more like an outlier than a clinically significant result.

Similarly, for the microsomal reaction with docetaxel (5µM), Figure 4.1.5. showed a statistically significant difference between docetaxel depletion in the reaction without bexarotene and the docetaxel microsomal standard. However, the difference between docetaxel metabolism in the absence of bexarotene and in the presence of bexarotene (0.1-10µM) was not statistically significant (p ≤ 0.05).

Table 4.1.1. summarizes the rate of docetaxel metabolism observed while studying human liver microsomal incubations in the absence and the presence of bexarotene (0.1-10µM). The rate of reaction was measured as substrate depletion in the presence of NADPH.

47

Figure 4.1.4. Human liver microsomal metabolism of docetaxel (2µM) in the absence or the presence of increasing concentrations of bexarotene (0.1-10µM). Human liver microsomal protein (1mg) was incubated (37°C, 30 minutes) with docetaxel (1µM) and bexarotene (0-10µM) in the presence of NADPH (1mM). Results are expressed as the extent of docetaxel metabolism in the presence of bexarotene as compared to the control (no bexarotene, no NADPH). (Mean ± SEM, n = 3, p ≤ 0.05, * statistically significant from control, † statistically significant from bexarotene 0 µM).

48

Docetaxel (5µM) Metabolism

Figure 4.1.5. Human liver microsomal metabolism of docetaxel (5µM) in the absence

or the presence of increasing concentrations of bexarotene (0.1-10µM). Human liver

microsomal protein (1mg) was incubated (37°C, 30 minutes) with docetaxel (1µM)

and bexarotene (0-10µM) in the presence of NADPH (1mM). Results are expressed

as extent of docetaxel metabolism in presence of bexarotene as compared to the

control (no bexarotene, no NADPH). (Mean ± SEM, n = 3, p ≤ 0.05, * statistically significant from control).

49 Table 4.1.1. summarizes the rate of CYP3A4 driven human docetaxel metabolism as measured by substrate depletion. Docetaxel (0.5 – 5 µM) was incubated with human liver microsomes in the presence/absence of bexarotene (0 – 10 µM). a. docetaxel recovered from microsomal metabolism in the absence of NADPH (essentially, no reaction), b. docetaxel recovered from microsomal metabolism in the presence of NADPH but no bexarotene. Difference (a – b) provides the rate of docetaxel depletion in the presence and absence of indicated concentration of bexarotene.

* Statistically significant as compared to control (n = 3, p ≤ 0.05). ┼ Statistically significant from reaction in absence of bexarotene.

Docetaxel levels from microsomal incubations in the absence or presence of bexarotene

Microsomal Standard Bexarotene Bexarotene Bexarotene Bexarotene Bexarotene Bexarotene (Docetaxel without reaction) (0µM) (0.1µM) (0.5µM) (1µM) (2.5µM) (10µM) a. b. Docetaxel 0.5 µM 0.3215 0.0780 0.0457 0.0302 0.0749 0.0447 0.0301

±0.0015 ±0.012* ±0.0003 ±0.0007 ±0.012 ±0.0005 ±0.0002

Docetaxel 1 µM 0.6716 0.2750 0.2522 0.3301 0.3905 0.4823 0.5748

±0.0002 ±0.025* ±0.0444 ±0.0937 ±0.0440 ±0.0722 ±0.1120

Docetaxel 2 µM 1.1 0.7941 1.0707 0.9485 1.3484 2.1704 1.3667

±0.0001 ±0.0052* ±0.1076 ±0.1806 ±0.2452 ±0.4558† ±0.3411

Docetaxel 5 µM 2.9246 2.3044 2.1158 2.0963 1.7659 1.6812 1.602

±0.000 ±0.2059* ±0.0801 ±0.0869 ±0.1120 ±0.1439 ±0.2171

50

4.2. Assessment of the Potential Induction of Docetaxel Metabolism by Bexarotene

in Primary Human Hepatocytes

Primary cultures of hepatocytes were treated with vehicle (control), rifampicin

(10µM) and bexarotene (1, 10, 25 and 50µM) for 72 hours. At the end of 72 hours, the cells were incubated with drug-free medium. Following this, CYP3A4 activity was determined as the rate of testosterone 6β-hydroxylation by incubating the cells with testosterone-containing (250µM) medium. The cells were frozen for sub-cellular fractionation for docetaxel metabolism, western blot analysis for the CYP3A4 protein levels and RT-PCR for CYP3A4-specific mRNA levels. A typical HPLC profile of testosterone, 6β-hydroxytestosterone and 11α-hydroxyprogesterone (internal standard) peaks obtained from the methanolic extracts of cell culture media from untreated control hepatocytes, rifampicin (10µM) and bexarotene (1, 10, 25 and 50µM), treated hepatocytes is shown in Figure 4.2.1. In general, rifampicin, a known inducer of

CYP3A4 activity caused a 4- to 5- fold induction as compared to untreated controls, while bexarotene (1, 10, 25 and 50µM) showed no difference in the CYP3A4 activity as compared to the controls. The fold change of CYP3A4 activity in drug-treated cells over untreated control cells is graphically shown in Figure 4.2.2.

51 0.50 Testosterone

0.40 its

0.30 6β-OH testosterone AU

0.20 Bexarotene 1µM Absorbance Un Bexarotene 10µM 11α-OH progesterone Rifampicin 10µM (Internal Standard) Bexarotene 25µM 0.10 Bexarotene 50µM Control

0.00

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 MinutesMinutes

Figure 4.2.1.Typical chromatogram of testosterone, 6β-hydroxytestosterone and 11α-hydroxyprogesterone (internal standard) in the methanolic extract prepared from the cell culture media of bexarotene (1, 10, 25 and 50µM) and rifampicin (10µM) treated primary human hepatocytes and untreated control hepatocytes incubated with testosterone (250µM) for 30 minutes.

52 Fold Change in CYP3A4 Activity over Untreated Controls Controls Untreated over Activity CYP3A4 in Change Fold

Figure 4.2.2. Fold induction in CYP3A4 activity comparing untreated cells (vehicle

alone) with treated human hepatocytes (rifampicin 10µM and bexarotene - 1, 10, 25,

50µM). Primary cultures of human hepatocytes were treated with vehicle (control),

rifampicin (10µM) and bexarotene (1, 10, 25 and 50µM) for 72 hours. Following

drug treatment, CYP3A4 activity was evaluated by incubating cells with

testosterone (250µM) medium and measuring the rate of testosterone 6β-

hydroxylation.

53

Docetaxel (1µM) metabolism reactions were carried out using S9 fractions prepared from the control and drug-treated primary human hepatocytes by differential centrifugation. The protein content was evaluated using Lowry’s protein assay (Bio-rad

DC assay) (Schuetz and Schinkel, 1999). The optimum conditions for the metabolic reactions are outlined in section 3.3.4. Using liquid-liquid extraction, docetaxel was extracted and analyzed using HPLC / UV detection. Substrate or docetaxel depletion was taken as a measure of the rate of CYP3A4 driven metabolism.

Figure 4.2.3. shows the effect of vehicle (control), rifampicin (10µM) and bexarotene (1, 10, 25 and 50µM) pretreated human hepatocytes on docetaxel metabolism by hepatic CYP3A4. Rifampicin treated cells demonstrated 1.5-2 fold increase in docetaxel metabolism. The graph also makes it clear that the difference in docetaxel

(1µM) metabolism reactions between the untreated controls and bexarotene (1, 10, 25 and 50µM) treated cells showed no significant change.

54

Figure 4.2.3. Primary human hepatocytes were treated with vehicle (control), rifampicin (10µM) and bexarotene (1, 10, 25 and 50µM) for 72 hours. Sub-cellular or S9 fractions were obtained by differential centrifugation and docetaxel (1µM) metabolism was carried out using optimum conditions (37°C, ~ 1mg protein,

NADPH 1mM) for 30minutes. Rifampicin showed a marginal increase in CYP3A4 activity (1.5- 2fold) while bexarotene (1, 10, 25 and 50µM) failed to show any increase in docetaxel metabolism as compared to control.

55

We also determined the immunoreactive CYP3A4 protein levels in the bexarotene-treated cells, using the S9 fractions by western blot analysis. We used monoclonal anti-CYP3A4 antibody for detecting the CYP3A4 immunoreactive protein levels. Figure 4.2.4. displays western blots showing the immunoreactive CYP3A4 levels in bexarotene (1, 10 and 25µM) and rifampicin (10µM) treated cells and untreated controls. The immunoreactive CYP3A4 protein levels expressed as band intensity are graphically shown in Figure 4.2.4. Rifampicin-treated cells caused an increase of 2 to 3 fold as compared to the untreated controls. There was a statistically significant (71.29 ±

8.55, p ≤ 0.05) increase in the CYP3A4 protein levels. In contrast, bexarotene (1, 10 and

25µM) treated cells did not show any significant increase in the CYP3A4 levels. This was found to be consistent with the data on CYP3A4 activity and also its effect on docetaxel 1µM metabolism.

56

Figure 4.2.4. Western blot analysis data of bexarotene (1, 10 and 25µM) and rifampicin (10µM) treated human hepatocytes as compared to untreated controls.

Rifampicin shows a statistically significant increase in the CYP3A4 protein levels.

Bexarotene (1, 10 and 25µM) show no difference in the levels as compared to controls. (* statistically significant, p ≤ 0.05).

57

The CYP3A4 specific mRNA levels were evaluated using RT-PCR after RNA isolation from the bexarotene (1, 10, 25 and 50µM) and rifampicin (10µM) treated human hepatocytes and the untreated cells. RNA was subjected to reverse transcription and the cDNA thus formed was amplified by real-time PCR. CYP3A4 specific primers were used and Ct values were normalized to that of GAPDH.

Figure 4.2.5. demonstrates a fold change in the CYP3A4 mRNA levels following treatment with bexarotene (1, 10, 25 and 50µM) and rifampicin (10µM). Rifampicin shows a significant increase in the CYP3A4 mRNA levels (5.02 ± 1.1301, p ≤ 0.025) while bexarotene (1, 10, 25 and 50µM) showed a significant decrease in the CYP3A4 mRNA levels (1 µM = 0.0320 ± 0.0009, 10 µM = 0.0241 ± 0.0007, 25 µM = 0.0707 ±

0.0215, 50 µM = 0.0404 ± 0.0009 at p≤0.05).

Table 4.2.1. gives a summary of the fold change in CYP3A4 activity as measured by rate of testosterone 6β-hydroxylation, immunoreactive CYP3A4 levels and CYP3A4 mRNA levels in human hepatocytes treated with bexarotene (1, 10, 25 and 50µM) as compared with untreated controls (Mean ± SEM, p≤0.05 ).

58

Figure 4.2.5. CYP3A4 mRNA levels from vehicle, rifampicin (10µM) and

bexarotene (1, 10, 25 and 50µM) treated human hepatocytes (72hours) expressed as

fold change over untreated controls. RNA was isolated and subjected to reverse

transcription. The cDNA formed was amplified using CYP3A4 specific primers by

real-time PCR. Rifampicin showed a significant increase (5.02 ± 1.1301, p ≤ 0.025)

whereas bexarotene (1, 10, 25 and 50µM) treated cells showed a significant decrease

in CYP3A4 mRNA as compared to untreated controls. (* statistically significant as compared to controls, p ≤ 0.05).

59

Table 4.2.1. summarizes fold change in CYP3A activity, immunoreactive protein levels as band intensity and fold change in mRNA levels in human hepatocytes treated for 72hours with rifampicin (10µM), bexarotene (1, 10, 25 and 50µM) compared to untreated controls (Mean ± SEM).

* Statistically significant decrease as compared to untreated control (p ≤ 0.05).

Treatment Primary Human Hepatocytes

(72 hours) CYP3A4 CYP3A4 CYP3A4 Enzyme Activity Protein levels mRNA levels Fold change (Band Intensity) Fold change Rifampicin 10 µM 5.0940 (SD = 0.2991) 71.29 ± 8.55* 5.0000 ± 1.1301*

Bexarotene 1 µM 1.0322 (SD = 0.3409) 30.82 ± 5.45 0.0320 ± 0.0009*

Bexarotene 10 µM 1.1268 (SD = 0.0916) 33.95 ± 15.45 0.0241 ± 0.0007*

Bexarotene 25 µM 0.4003 (SD = 0.2306) 53.24 ± 12.51 0.0707 ± 0.0215*

Bexarotene 50 µM 0.0544 (SD = 0.0005) nd 0.0404 ± 0.0009*

SD= Standard Deviation; nd = not determined

60

5 DISCUSSION

We investigated the potential influence of bexarotene, a novel retinoid X receptor agonist on the hepatic metabolism of docetaxel, which belongs to the taxoid family of drugs. Docetaxel and bexarotene have shown promising results in the treatment of non- small cell lung cancer and their combination is being studied in phase I trials (Meeting

Abstracts, ASCO, 2004). Docetaxel biotransformation is as such, a detoxification pathway and is extensively and exclusively mediated by hepatic CYP3A4 (Shou et al,

1998). It is converted to its major metabolite-hydroxydocetaxel, which is further converted into various species. Neither of the metabolites formed show any pharmacological activity or toxicity (Bruno et al, 1993). The conversion of docetaxel in the liver by CYP3A4 to hydroxydocetaxel is a rate-limiting process. As indicated earlier,

CYP3A4 metabolizes a wide spectrum of drugs and xenobiotics which raises the possibility of drug interactions that may result in inhibition of CYP3A4 activity.

Likewise, PXR is also fairly accommodative and is activated by a wide array of compounds which lead to the induction of CYP3A4. Again, this underscores the possibility of DDIs due to CYP3A4 induction. Compounds with a potential to influence the activity of CYP3A4, either by inhibition or induction may affect the metabolism of other concurrently administered drugs.

Bexarotene, a novel retinoid X receptor agonist is also metabolized by hepatic

CYP3A4. Animal studies conducted during the development of bexarotene, clearly indicated that repeated administration of bexarotene in rats caused an increase in the

61

activity of hepatic CYP3A and UGT, while increasing the total hepatic CYP content

(Howell et al, 1998). Bexarotene clearance was also found to increase on repeated dosing in rats, a phenomenon called as “autoinduction” (Howell et al, 1998). However, the phase I/II studies conducted in humans did not show any autoinduction of bexarotene metabolism on repeat dosing (Miller et al, 1997; Rizvi et al, 1999). To the best of our knowledge, studies that specifically address potential CYP3A4 induction by bexarotene have not been reported and bexarotene effect on CYP3A4-mediated metabolism remains unresolved. We conducted this study to investigate if bexarotene potentially influences

CYP3A4-driven hepatic metabolism of docetaxel.

To determine if bexarotene inhibited the docetaxel metabolism by CYP3A4, pooled human liver microsomal reactions were carried out in the presence of a range of docetaxel concentrations (0.5-5µM) with NADPH, in the absence or presence of bexarotene (0.1-10µM). As a single agent, bexarotene is used at a dose of 300mg/m2, which yields peak and trough concentrations of 4 and 0.001µM, respectively (Rizvi et al,

1999; Khuri et al, 2001). From a drug-drug interaction perspective, the most relevant concentration range for bexarotene is 0.1 to 1µM. We measured the rate of reaction or docetaxel metabolism as substrate depletion. Figure 4.1.1. which is a plot of the

Michaelis-Menten kinetics of docetaxel metabolism, indicates clearly that the reaction reaches saturation at around 2µM but completely saturates by 5µM of docetaxel. This plot was used to calculate Vmax and Km, which were found to be consistent with those reported in literature (Monsarrat et al, 1997, Shou et al, 1998). There was a statistically significant difference found between microsomal standards of docetaxel (0.5-5µM) in the

62

absence of NADPH, bexarotene and docetaxel (0.5-5µM) in the presence of NADPH but no bexarotene (p≤0.05), assessed using pair-wise comparisons by Student t’s test. To assess whether bexarotene had an inhibitory effect on docetaxel, we compared substrate depletion in the absence of bexarotene with that obtained in the presence of increasing concentrations of bexarotene from 0.1-10µM. These differences were not found to be statistically significant. To strengthen our experimental approach, we carried out the metabolic reactions in the presence of ketoconazole (1 and 10µM), a prototype CYP3A4 inhibitor. In the presence of ketoconazole, the peaks clearly attributed to those of docetaxel metabolites were clearly abolished while they were prominent in the absence of ketoconazole. This finding validated our underlying assumption that CYP3A4 was predominantly involved in docetaxel metabolism. Thus our results indicated that bexarotene ranging from 0.1-10µM did not significantly affect docetaxel metabolism

(0.5-5µM).

The CYP3A4 inductive potential of bexarotene in humans was investigated using primary human hepatocyte cultures which were treated with bexarotene (1, 10, 25,

50µM) for 72hours. Rifampicin (10µM), a known inducer of CYP3A4 was used as the positive control. At the end of 72 hours, the cells were incubated with drug-free medium and were analyzed for CYP3A4 activity. Sub-cellular fractions were prepared after differential centrifugation and were used for assessing docetaxel (1µM) metabolism,

CYP3A4 protein levels by western blot analysis and CYP3A4-specific mRNA levels by

RT-PCR. We found that bexarotene (1-50µM) treatment of hepatocytes, produced no significant change in CYP3A4 activity, measured as fold difference in 6β-hydroxylation

63

of testosterone in vehicle-treated versus drug-treated hepatocytes. Rifampicin showed a

4-5 fold increase in CYP3A4 activity; whereas there was no change in the activity level of CYP3A4 at bexarotene 1 and 10µM. A marginal decrease in the activity was however noted at bexarotene 25µM and 50µM which may be related to the observed cytotoxicity of bexarotene at high concentrations.

We carried out the docetaxel 1µM metabolism reactions using S9 fractions of bexarotene (1, 10, 25 and 50µM) treated cells. Rifampicin treated cells showed a 1.5-2 fold increase in the rate of metabolism whereas there was no significant difference in the docetaxel metabolism of the control or untreated cells and bexarotene (1, 10, 25 and

50µM). This is illustrated in Figures 4.2.3. .

Rifampicin, a known inducer of CYP3A4, was found to induce docetaxel metabolism in human liver microsomes (Marre et al, 1996). Our experiments showed almost 2 fold induction of docetaxel metabolism by rifampicin (10µM). The consistency of findings in our experiments with those from previous studies and the information in the packaging insert of docetaxel, that states rifampicin as a drug known to induce docetaxel metabolism, validated our experimental approach for assessing induction of docetaxel metabolism.

The immunoreactive protein levels specific for CYP3A4 were assessed by western blot assays and they showed no increase in the protein levels in the bexarotene treated human hepatocytes.

CYP3A4-specific mRNA levels were evaluated by RT-PCR. The results of RT-

PCR expressed as fold change over untreated controls, demonstrate a 5 fold increase in

64

the mRNA levels of rifampicin (10µM) treated cells. Conversely, cells treated with bexarotene (1, 10, 25 and 50µM) showed a significant decrease in CYP3A4 mRNA levels. However, the reasons for the observed decrease in the CYP3A4 mRNA levels are not clear.

The major conclusion of this part of the study is that bexarotene does not induce human CYP3A4, which is in contrast to the observed induction in CYP3A4 in animal models. Thus, it appears that inductive effects of bexarotene are species-specific. There is no reported data explaining the species difference in the influence of bexarotene on the hepatic CYP3A enzyme.

Molecular mechanisms of species-specificity of bexarotene inductive effects are currently not clear. One of the plausible explanations is the difference in the amino acid sequence of LBD of PXR from different species. Cloning and characterization of PXR from humans, rabbits, rats and mice have revealed a ~95% sequence homology in the

DBD regions whereas the LBDs are not so well conserved across species. The LBDs of the PXR in various species are distinct from each other and the amino acid share only 75

– 80% sequence homology in these species. Thus, PXR displays markedly different activation profiles in response to xenobiotics (LeCluyse, 2001; Lehmann et al, 1998).

The CYP3A gene promoter region is also known to be well-conserved across species

(Jones et al, 2000). CYP3A is induced differentially by xenobiotics in various species as highlighted in Table 1.8.1. It is probable that the differences in the LBD among species are responsible for the selectivity in ligand binding and hence the striking differences in the induction profiles among species. Xie et al (2000a) reported a study where trans-

65

species transfection assays clearly demonstrated that transfection of human PXR was sufficient to convert the induction response characteristics of hepatocyte from rat to human. However the ability of a single transcription factor to determine differences in the response to xenobiotics in various species is without precedence. The assessment of bexarotene potential for hPXR activation has not been reported so far. It would be worthwhile studying the effects of bexarotene on hPXR by cell-based reporter assays to help predict its behaviour in vivo.

Screening of a new drug candidate early on in its development phase for its potential to cause drug-drug interactions is important. Withdrawing a developed drug from the market due to significant drug-drug interactions is cost-prohibitive for pharmaceutical companies. Several in vitro tools have been developed to screen compounds for such interactions. The question remains, can in vivo drug interactions be predicted accurately from in vitro metabolism studies? The enzyme inductive potential of drugs requires the use of primary human hepatocytes. The availability of human tissues and their use involve numerous complexities which limit their wide use in such screening procedures. The inter-individual variability in CYP3A4 expression also underscores the need for an appropriate model for drug screening and development.

Given the differences in the xenobiotic response in various species, the extrapolation of animal data to predicting in vivo drug interactions in humans is not always accurate.

Considering the possibility of inter-species variability in bexarotene-mediated CYP3A induction, choosing a good model to predict clearance and pharmacodynamics of this agent or coadministered drugs in humans is essential.

66

We report that bexarotene does not alter the hepatic metabolism of docetaxel by

CYP3A4 in vitro and the possibility of pharmacokinetic interactions between them seems minimal. However the pharmacodynamic interactions need to be investigated. Studies specifically addressing cellular uptake of bexarotene in various species need to be conducted. The transport mechanisms underlying the uptake of bexarotene by the cells are not clear. The possibility of limited uptake of bexarotene by the cells, rather than inability to activate PXR in humans, cannot be ruled out at this stage.

67

6 REFERENCES

1. Bannwarth B, Pehourcq F, Schaeverbeke T, Dehais J. Clinical pharmacokinetics of low-dose pulse methotrexate in rheumatoid arthritis. Clin Pharmacokinet. 1996; 30: p. 194–210.

2. Bardelmeijer HA, Beijnen JH, Brouwer KR, Rosing H, Nooijen WJ, Schellens JH, van Tellingen O. Increased oral bioavailability of paclitaxel by GF120918 in mice through selective modulation of P-glycoprotein. Clin Cancer Res. 2000. 6(11): p. 4416- 21.

3. Beijnen, J.H. and J.H. Schellens, Drug interactions in oncology. Lancet Oncol, 2004. 5(8): p. 489-96.

4. Bertilsson G, Heidrich J, Svensson K, Asman M, Jendeberg L, Sydow-Backman M, Ohlsson R, Postlind H, Blomquist P and Berkenstam A. Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proc Natl Acad Sci U S A. 1998; 95: p. 12208-12213.

5. Bissery MC, Nohynek G, Sanderink GJ and Lavelle F. Docetaxel (Taxotere): a review of preclinical and clinical experience. Part I: Preclinical experience. Anticancer Drugs. 1995; 6: p. 339-8.

6. Blackhall, F.H., F.A. Shepherd, and K.S. Albain, Improving survival and reducing toxicity with chemotherapy in advanced non-small cell lung cancer: a realistic goal? Treat Respir Med, 2005. 4(2): p. 71-84.

68 7. Blumberg B, Sabbagh W, Jr., Juguilon H, Bolado J, Jr., van Meter CM, Ong ES and Evans RM. SXR, a novel steroid and xenobiotic-sensing nuclear receptor. Genes Dev 12: 3195-3205.

8. Brabender, J., et al., The role of retinoid X receptor messenger RNA expression in curatively resected non-small cell lung cancer. Clin Cancer Res, 2002. 8(2): p. 438-43.

9. Braud, A.C., et al., Retinoids, cisplatin and interferon-alpha in recurrent or metastatic cervical squamous cell carcinoma: clinical results of 2 phase II trials. Eur Cytokine Netw, 2002. 13(1): p. 115-20.

10. Bruno R, Sanderink GJ. Pharmacokinetics and metabolism of Taxotere (docetaxel). Cancer Surv. 1993; 17: p. 305-13.

11. Cerny T, Calderoni A. Phase II results of taxanes in lung cancer. In: Johnson DH, Klatersky J, eds. Taxanes in Lung Cancer Therapy. New York: Marcel Dekker. 1998; p. 39-56.

12. Chambon, P., A decade of molecular biology of retinoic acid receptors. Faseb J, 1996. 10(9): p. 940-54.

13. Clarke SJ and Rivory LP. Clinical pharmacokinetics of docetaxel. Clin Pharmacokinet. 1999; 36: p. 99-114.

14. Classen, D.C., et al., Adverse drug events in hospitalized patients. Excess length of stay, extra costs, and attributable mortality. Jama, 1997. 277(4): p. 301-6.

15. Cresteil, T., et al., Regioselective metabolism of taxoids by human CYP3A4 and 2C8: structure-activity relationship. Drug Metab Dispos, 2002. 30(4): p. 438-45.

69 16. Depierre A, Lemarie E, Dabouis G. A phase II study of Navelbine (Vinorelbine) in the treatment of non-small cell lung cancer. Am J Clin Oncol. 1991; 14: p. 115-9.

17. De Valeriola D, Brassine C, Gaillard C, Ketler JP, Tomiak E, Van Vreckam A. et al. Study of excretion balance, metabolism and protein binding of C14-radiolabelled Taxotere in cancer patients. Proc Am Assoc Cancer Res. 1993; 34: p. 373.

18. Dimitroulis, J. and G.P. Stathopoulos, Evolution of non-small cell lung cancer chemotherapy (Review). Oncol Rep, 2005. 13(5): p. 923-30.

19. DiPaola, R.S., et al., Effect of 13-cis-retinoic acid and alpha-interferon on transforming growth factor beta1 in patients with rising prostate-specific antigen. Clin Cancer Res, 1997. 3(11): p. 1999-2004.

20. Dragnev, K.H., J.R. Rigas, and E. Dmitrovsky, The retinoids and cancer prevention mechanisms. Oncologist, 2000. 5(5): p. 361-8.

21. Duchateau A. Posicor: Veni, vidi and gone. Pharm. Weekblad. 1998; 133: p. 1294-1295.

22. Edwards, I.R. and J.K. Aronson, Adverse drug reactions: definitions, diagnosis, and management. Lancet, 2000. 356(9237): p. 1255-9.

23. Fenaux, P., et al., Long-term follow-up confirms the benefit of all-trans retinoic acid in acute promyelocytic leukemia. European APL group. Leukemia, 2000. 14(8): p. 1371-7.

24. Ferslew KE.; Hagardorn AN.; Harlan GC. McCormick WF. A fatal drug interaction between clozapine and fluoxetine. J. Forensic Sci. 1998; 43. p. 1082-1085.

70 25. Friedman, M.A., et al., The safety of newly approved medicines: do recent market removals mean there is a problem? Jama, 1999. 281(18): p. 1728-34.

26. Fukuoka M, Niitani H, Suzuki A, et al. A phase II study of CPT-11, a new derivative of , for previously untreated non-small cell lung cancer. For the CPT-11 Lung Cancer Study Group. J Clin Oncol. 1992; 10: p. 16 – 20.

27. Ginsberg RJ, Vokes EE, Rosenzweig K. Non-small cell lung cancer. In: DeVita VT Jr, Hellman S, Rosenberg SA, eds. Cancer: Principles and Practice of Oncology, 6th Edition. Philadelphia: Lippincott Williams and Wilkins, 2001: p. 925–983.

28. Glass CK. Some new twists in the regulation of gene expression by thyroid hormone and retinoic acid receptors. J Endocrinol 1996; 150: p. 349–357.

29. Glimelius, B., et al., The Swedish Council on Technology Assessment in Health Care (SBU) systematic overview of chemotherapy effects in some major tumour types-- summary and conclusions. Acta Oncol, 2001. 40(2-3): p. 135-54.

30. Gurwitz, J.H., et al., Incidence and preventability of adverse drug events in nursing homes. Am J Med, 2000. 109(2): p. 87-94.

31. Handschin, C. and U.A. Meyer, Induction of drug metabolism: the role of nuclear receptors. Pharmacol Rev, 2003. 55(4): p. 649-73.

32. Hein NI, Howell SR, Ulm EH. Evaluation of the cytochrome P450 (P450) isozymes involved in the metabolism of Targretin, an RXR-selective retinoid. ISSX Proc; 1996; 10; p. 364.

33. Hennequin, C., N. Giocanti, and V. Favaudon, S-phase specificity of cell killing by docetaxel (Taxotere) in synchronised HeLa cells. Br J Cancer, 1995. 71(6): p. 1194-8.

71 34. Howell SR, Shirley MA, Ulm EH. Effects of retinoid treatment of rats on hepatic microsomal metabolism and cytochromes P450. Correlation between /retinoid x receptor selectivity and effects on metabolic enzymes. Drug Metab Dispos. 1998; 26(3): p. 234-9.

35. Howell, S.R., et al., Bexarotene metabolism in rat, dog, and human, synthesis of oxidative metabolites, and in vitro activity at retinoid receptors. Drug Metab Dispos, 2001. 29(7): p. 990-8.

36. Jacubeit, T., D. Drisch, and E. Weber, Risk factors as reflected by an intensive drug monitoring system. Agents Actions Suppl, 1990. 29: p. 117-25.

37. Jemal A, Tiwari RC, Murray T et al. Cancer statistics, 2004. CA Cancer J Clin. 2004; 54: p. 8–29.

38. Johnson, J.A. and J.L. Bootman, Drug-related morbidity and mortality. A cost-of- illness model. Arch Intern Med, 1995. 155(18): p. 1949-56.

39. Khuri, F.R., et al., Multi-institutional phase I/II trial of oral bexarotene in combination with cisplatin and vinorelbine in previously untreated patients with advanced non-small-cell lung cancer. J Clin Oncol, 2001. 19(10): p. 2626-37.

40. Kliewer, S.A., et al., An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell, 1998. 92(1): p. 73-82.

41. Kocarek TA, Schuetz EG, Strom SC, Fisher RA and Guzelian PS. Comparative analysis of cytochrome P4503A induction in primary cultures of rat, rabbit, and human hepatocytes. Drug Metab Dispos. 1995; 23: p. 415-421.

72 42. Kudo K., Imamura T., Jitsufuchi N., Zhang XX., Tokunaga H. and Nagata T. Death attributed to the toxic interaction of triazolam, amitriptyline and other psychotropic drugs. Forensic. Sci. Int. 1997. 86; p. 35-41.

43. Lavelle, F., et al., Preclinical evaluation of docetaxel (Taxotere). Semin Oncol, 1995. 22(2 Suppl 4): p. 3-16.

44. Lazarou, J., B.H. Pomeranz, and P.N. Corey, Incidence of adverse drug reactions in hospitalized patients: a meta-analysis of prospective studies. Jama, 1998. 279(15): p. 1200-5.

45. Leape, L.L., et al., Systems analysis of adverse drug events. ADE Prevention Study Group. Jama, 1995. 274(1): p. 35-43.

46. Leape, L.L., et al., The nature of adverse events in hospitalized patients. Results of the Harvard Medical Practice Study II. N Engl J Med, 1991. 324(6): p. 377-84.

47. LeCluyse, E.L., Pregnane X receptor: molecular basis for species differences in CYP3A induction by xenobiotics. Chem Biol Interact, 2001. 134(3): p. 283-9.

48. Lehmann, J.M., et al., The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. J Clin Invest, 1998. 102(5): p. 1016-23.

49. Leid M, Kastner P, Chambon P. Multiplicity generates diversity in the retinoic acid signalling pathways. Trends Biochem Sci. 1992; 17: p. 427–433.

50. Li, A.P., et al., Primary human hepatocytes as an in vitro toxicology system, in In Advances in Animal Alternatives for Safety and Efficacy Testing, H. Salem and S.A. Katz, Editors. 1998, Taylor and Francis: Washington, D.C. p. 359–368.

73 51. Lin J.H.. Sense and Nonsense in the Prediction of Drug-Drug Interactions. Current Drug Metabolism. 2000; 1. p. 305-331.

52. Lu C and Li AP. Species comparison in P450 induction: effects of dexamethasone, omeprazole, and rifampin on P450 isoforms 1A and 3A in primary cultured hepatocytes from man, Sprague-Dawley rat, minipig, and beagle dog. Chem Biol Interact 2001; 134: p. 271-281.

53. Lucey MR, et al., Cyclosporin toxicity at therapeutic blood levels and cytochrome P-450 IIIA. Lancet, 1990. 335(8680): p. 11-5.

54. Lynch TJ Jr, Kalish L, Strauss G, et al. Phase II study of topotecan in metastatic non-small-cell lung cancer. J Clin Oncol. 1994; 12: p. 347-52.

55. Lynch TJ, Raju R, Lind M et al. Randomized phase III trial of chemotherapy and antisense oligonucleotide LY900003 (ISIS 3521) in patients with advanced NSCLC: initial report. Proc Am Soc Clin Oncol. 2003; 22: p. 623.

56. Mangelsdorf DJ, Ong ES, Dyck JA et al. Nuclear receptorthat identifies a novel retinoic acid response pathway. Nature 1990; 345: p. 224–229.

57. Mangelsdorf, D.J., et al., Retinoid receptors. Recent Prog Horm Res, 1993. 48: p. 99-121.

58. Mangelsdorf DJ, Umesono K, Evans RM. The retinoid receptors.In: Sporn MB, Roberts AB, Goodman DS, eds. The Retinoids: Biology, Chemistry, and Medicine, 2nd Edition. New York: Raven Press, 1994: p. 319–350.

59. Mangelsdorf DJ, Evans RM. The RXR heterodimers and orphan receptors. Cell. 1995; 83: p. 841–850.

74

60. Marre, F., et al., Hepatic biotransformation of docetaxel (Taxotere) in vitro: involvement of the CYP3A subfamily in humans. Cancer Res, 1996. 56(6): p. 1296-302. 34. 61. Medicine., C.o.Q.o.H.C.i.A.I.o., ed. To err is human: building a safer health system. ed. I.o. Medicine. 2000, National Academy Press: Washington, D.C.

62. Miller VA, Benedetti FM, Rigas JR, Verret AL, Pfister DG, Straus D, Kris MG, Crisp M, Heyman R, Loewen GR, Truglia JA, Warrell RP Jr. Initial of a selective retinoid X receptor ligand, LGD1069. J Clin Oncol. 1997; 15(2): p. 790-5.

63. Monsarrat B, Chatelut E, Royer I, Alvinerie P, Dubois J, Dezeuse A, Roche H, Cros S, Wright M and Canal P. Modification of paclitaxel metabolism in a cancer patient by induction of cytochrome P450 3A4. Drug Metab Dispos. 1998; 26: p. 229-233.

64. Mukherjee, R., et al., Sensitization of diabetic and obese mice to insulin by retinoid X receptor agonists. Nature, 1997. 386(6623): p. 407-10.

65. Non-small Cell Lung Cancer Collaborative Group. Chemotherapy in non-small cell lung cancer: A meta-analysis using updated data on individual patients from 52 randomized clinical trials. BMJ. 1995. 311: p. 899-909.

66. Niles RM. Recent Advances in the use of Vitamin A (retinoids) in the prevention and treatment of cancer. Nutrition. 2000; 16: p. 1084-1089.

67. Ojima I, Slater JC, Michaud E, Kuduk SD, Bounaud PY, Vrignaud P, Bissery MC, Veith JM, Pera P and Bernacki RJ. Syntheses and structure-activity relationships of the second-generation antitumor taxoids: exceptional activity against drug-resistant cancer cells. J Med Chem. 1996; 39: p. 3889-3896.

75 68. Pearson, T.F., et al., Factors associated with preventable adverse drug reactions. Am J Hosp Pharm, 1994. 51(18): p. 2268-72.

69. Petkovich M, Brand NJ, Krust A et al. A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature 1987; 330: p. 444–450.

70. Picard, E., et al., Expression of retinoid receptor genes and proteins in non-small- cell lung cancer. J Natl Cancer Inst, 1999. 91(12): p. 1059-66.

71. Pichard, L., et al., Cyclosporin A drug interactions. Screening for inducers and inhibitors of cytochrome P-450 (cyclosporin A oxidase) in primary cultures of human hepatocytes and in liver microsomes. Drug Metab Dispos, 1990. 18(5): p. 595-606.

72. Rapp, E., et al., Chemotherapy can prolong survival in patients with advanced non-small-cell lung cancer--report of a Canadian multicenter randomized trial. J Clin Oncol, 1988. 6(4): p. 633-41.

73. Recchia, F., et al., , cisplatin, and 13-Cis retinoic acid for patients with advanced or recurrent squamous cell carcinoma of the head and neck: a phase I-II study. Cancer, 2001. 92(4): p. 814-21.

74. Rigas, J.R. and K.H. Dragnev, Emerging role of rexinoids in non-small cell lung cancer: focus on bexarotene. Oncologist, 2005. 10(1): p. 22-33.

75. Ringel I and Horwitz SB. Studies with RP 56976 (taxotere): a semisynthetic analogue of taxol. J Natl Cancer Inst. 1991. 83: p. 288-291.

76. Rizvi, N.A., et al., A Phase I study of LGD1069 in adults with advanced cancer. Clin Cancer Res, 1999. 5(7): p. 1658-64.

76 77. Rizvi N, Hawkins MJ, Eisenberg PD et al. Placebo-controlled trial of bexarotene, a retinoid X receptor agonist, as maintenance therapy for patients treated with chemotherapy for advanced non-small cell lung cancer. Clin Lung Cancer. 2001; 2: p. 210–215.

78. Rizvi NA, Miller W Jr, Frank H et al. Phase I/II safety evaluation of bexarotene in combination with carboplatin and paclitaxel in patients with chemotherapy-naïve advanced non-small cell lung cancer (NSCLC). Proc Am Soc Clin. Oncol 2002; 21: p. 334a.

79. Rusch, V., et al., Differential expression of the epidermal growth factor receptor and its ligands in primary non-small cell lung cancers and adjacent benign lung. Cancer Res, 1993. 53(10 Suppl): p. 2379-85.

80. Sandler AB, Blumenschein GR, Henderson T et al. Phase I/II trial evaluating the anti-VEGF MAb bevacizumab in combination with erlotinib, a HER1/EGFR-TK inhibitor, for patients with recurrent non-small cell lung cancer. Proc Am Soc Clin Oncol 2004;23:127.

81. Schappert, S.M., Ambulatory care visits of physician offices, hospital outpatient departments, and emergency departments: United States, 1995. Vital Health Stat 13, 1997(129): p. 1-38.

82. Schiff, P.B., J. Fant, and S.B. Horwitz, Promotion of microtubule assembly in vitro by taxol. Nature, 1979. 277(5698): p. 665-7.

83. Schiller, J.H., Current standards of care in small-cell and non-small-cell lung cancer. Oncology, 2001. 61 Suppl 1: p. 3-13.

84. Schimming, R., et al., Lack of correlation between mitotic arrest or apoptosis and antitumor effect of docetaxel. Cancer Chemother Pharmacol, 1999. 43(2): p. 165-72.

77

85. Schuetz E, Schmid W, Schutz G et al. The glucocorticoids receptor is essential for induction of Cytochrome P4502B by steroids and not be drug or steroid induction of CYP3A or P450 reductase in mouse liver. Drug Metab. Dispos. 2000; 28: p. 268-278.

86. Schuetz EG and Schinkel AH. Drug disposition as determined by the interplay between drug- transporting and drug-metabolizing systems. J Biochem Mol Toxicol. 1999; 13: p. 219-222.

87. Shepherd FA. Single agent gemcitabine activity in non-small cell lung cancer. Lung Cancer. 1997; 18 (2): 114.

88. Shin, D.M., et al., Phase II study of paclitaxel, ifosfamide, and carboplatin in patients with recurrent or metastatic head and neck squamous cell carcinoma. Cancer, 2001. 91(7): p. 1316-23.

89. Shirley, M.A., et al., Oxidative metabolism of a rexinoid and rapid phase II metabolite identification by mass spectrometry. Drug Metab Dispos, 1997. 25(10): p. 1144-9.

90. Shou M, Martinet M, Korzekwa K, Krauz W, Gonzalez F, Gelboin H. Role of human cytochrome P450 3A4 and 3A5 in the metabolism of Taxotere and its derivatives: enzyme specificity, interindividual distribution and metabolic contribution in the human liver. Pharmacogenetics. 1998; 8: p. 391-401.

91. Song, J.I., et al., Abrogation of transforming growth factor-alpha/epidermal growth factor receptor autocrine signaling by an RXR-selective retinoid (LGD1069, Targretin) in head and neck cancer cell lines. Cancer Res, 2001. 61(15): p. 5919-25.

78 92. Sparreboom A, van Asperen J, Mayer U, Schinkel AH, Smit JW, Meijer DK, Borst P, Nooijen WJ, Beijnen JH, van Tellingen O. Limited oral bioavailability and active epithelial excretion of paclitaxel (Taxol) caused by P-glycoprotein in the intestine. Proc Natl Acad Sci U S A. 1997. 4; 94(5): p. 2031-5.

93. R. Suresh, W. Read, P. M. Fracasso and R. Govindan. A phase I study of docetaxel and bexarotene. Journal of Clinical Oncology, ASCO Annual Meeting Proceedings(Post-MeetingEdition).2004;22(S14);p.3200

94. Suzuki, A., T. Kawabata, and M. Kato, Necessity of interleukin-1beta converting enzyme cascade in taxotere-initiated death signaling. Eur J Pharmacol, 1998. 343(1): p. 87-92.

95. Toma S, Raffo P, Isnardi L et al. Retinoids in lung cancer chemoprevention and treatment. Ann Oncol 1999;10(S5): p. S95–S102.

96. Vaishampayan, U., et al., Phase II evaluation of paclitaxel, alpha-interferon, and cis-retinoic acid in advanced renal cell carcinoma. Cancer, 2001. 92(3): p. 519-23.

97. Watkins, P.B., Noninvasive tests of CYP3A enzymes. Pharmacogenetics, 1994. 4(4): p. 171-84.

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

99. Wrighton, S.A., et al., The human CYP3A subfamily: practical considerations. Drug Metab Rev, 2000. 32(3-4): p. 339-61.

100. Xie, W., et al., Humanized xenobiotic response in mice expressing nuclear receptor SXR. Nature, 2000. 406(6794): p. 435-9.

79

101. Xu, X.C., et al., Suppression of retinoic acid receptor beta in non-small-cell lung cancer in vivo: implications for lung cancer development. J Natl Cancer Inst, 1997. 89(9): p. 624-9.

102. Zhang, C., et al., Induction of apoptosis by bexarotene in cutaneous T-cell lymphoma cells: relevance to mechanism of therapeutic action. Clin Cancer Res, 2002. 8(5): p. 1234-40.

80