Antonia Tolson Baaqee, Ph.D. 11 South Eutaw Street #1602 Baltimore, MD 21201 Phone: 301·602·9083 Email: [email protected]

Antonia Tolson Baaqee, Ph.D. 11 South Eutaw Street #1602 Baltimore, MD 21201 Phone: 301·602·9083 Email: Atols001@Umaryland.Edu

Identification of Drugs of Abuse as Modulators of Drug-Metabolizing Enzymes through Nuclear Receptor-Mediated Mechanisms

Item Type dissertation

Authors Baaqee, Antonia Tolson

Publication Date 2012

Abstract To date, the majority of reports discussing opioid-drug interactions focus intensively on characterizing how other drugs affect the metabolic and pharmacokinetic (MPK) profile of opioids, however little has been published regarding the potential for ...

Keywords constitutive androstane receptor; CYP; drugs of abuse; pregnane x receptor; Opioids; Methadone

Download date 02/10/2021 11:24:43

Link to Item http://hdl.handle.net/10713/2306 Antonia Tolson Baaqee, Ph.D. 11 South Eutaw Street #1602 Baltimore, MD 21201 Phone: 301·602·9083 Email: [email protected]

EDUCATION

Ph.D. in Pharmaceutical Sciences 2012 University of Maryland, School of Pharmacy, Baltimore, Maryland

B.S. in Chemical Engineering 2004 University of Virginia, Charlottesville, Virginia

PROFESSIONAL EXPERIENCE

Graduate Research Assistant University of Maryland, School of Pharmacy, Department of Pharmaceutical Sciences Advisors: Hongbing Wang, Ph.D., and Dean Natalie Eddington, Ph.D., FAAPS, FCP (2006-2012), Dissertation Title: Identification of Drugs of Abuse as Modulators of Drug-Metabolizing Enzymes Through Nuclear Receptor- Mediated Mechanisms

Purpose  To elucidate how nuclear receptors Pregnane X Receptor and Constitutive Androstane Receptor modulate enzymes implicated in metabolic- or pharmacokinetics-based drug-drug interactions which occur upon treatment with commonly abused drugs such as opioids methadone and buprenorphine Technical Experience  In vitro techniques Human primary hepatocyte culture Real-time polymerase chain reaction (TaqMan and SYBR Green) ABI Prism 7000 sequence detection Western Immunobloting Rat primary hepatocyte culture Cell-based reporter assay methods: cell culture (HepG2, Caco-2), seeding, counting (hemacytometer), transfection, treatments, dual- luciferase analyses (Promega) Confocal laser scanning microscopy (Nikon C1-LU3, inverted Nikon Eclipse TE2000 microscope)  In vivo techniques Animal handling (rats and mice), cardiac puncture Animal injections (i.p.) Euthanasia (rats and mice) Rat tissue dissection (liver, brain, kidney, small intestine) Additional responsibilities  Supervised high school and rotational graduate students  Managed and coordinated laboratory meeting/journal club scheduling  Reviewed journal articles, literature  Reviewed laboratory grant applications and manuscripts  Wrote, and received funding for, Ruth L. Kirschstein National Research Service Award (NRSA) Pre-doctoral (F31) grant application  PROMISE Peer Mentor, Maryland’s Alliance for Graduate Education & Professorate (AGEP)  Pharmacy Graduate Student Association Big Sibling

Science, Technology and Weapons Analyst, United States Government, Office of Transnational Issues, Langley, Virginia (April 2005-2006)  Successfully graduated from rigorous Career Analyst Training program  Assessed challenging national security issues, such as foreign weapons proliferation, and emerging technologies  Served as professional intelligence officer, by applying scientific and technical knowledge to solving complex intelligence problems  Presented assessments to senior policymakers as needed, through use of creativity, innovative analytical skills and technical expertise  Selected as a mentee to participate in mentorship program with Associate Deputy Director for Intelligence

Technical Operations Intern, Merck & Company, Incorporated, Merck Manufacturing Division, Stonewall Plant, Elkton, Virginia (2003)  Responsible for three bulk chemical process optimization projects to reduce costs and improve cycle times while ensuring product quality, one of which required designing laboratory simulation model of factory dryer in order to reduce cycle time of drying operations in factory  Worked as part of a team to help prepare and implement Process Change Request and Analytical Change Request

Capitol Hill Congressional Intern, United States House of Representatives, Washington, DC (2002)  Implemented daily office mission of the Assistant to the Democratic Leader, Hon. Rosa L. DeLauro, by accelerating and delivering party message through caucus-wide scheduling and House floor one-minute calls  Attended Press Conferences, Subcommittee Hearings, Bill Mark-ups, House/Senate Message Meetings, historical House trial  Met with other personal and leadership congressional members  Produced large-scale project for use by Prescription Drugs Message Teams  Became certified through Congressional Research Service, researched in Library of Congress, and participated in Intern Lecture Series with other technology policy interns from UVA & MIT

Mathematics Tutor, Center for Diversity in Engineering at the University of Virginia, Charlottesville, Virginia (2000-2004) Responsible for providing comprehensive tutoring services Courses • APMA 1110 – Single Variable Calculus II • APMA 2120 – Multivariable Calculus • APMA 2130 – Ordinary Differential Equations • APMA 3080 – Linear Algebra

PUBLICATIONS

Manuscripts  Antonia H. Tolson, Li H, Eddington ND, and Wang H. Methadone induces the expression of hepatic drug-metabolizing enzymes through the activation of pregnane X receptor and constitutive androstane receptor. Drug Metab Dispos. 2009 Sep; 37(9):1887-94  Antonia H. Tolson and Hongbing Wang. Regulation of Drug-Metabolizing Enzymes by Xenobiotic Receptors: PXR and CAR. Adv Drug Deliv Rev. 2010 Oct 30; 62(13):1238-49  Li L, Stanton JD, Antonia H. Tolson, Luo Y, and Wang H. Bioactive terpenoids and flavonoids from Ginkgo biloba extract induce the expression of hepatic drug-metabolizing enzymes through pregnane X receptor, constitutive androstane receptor, and aryl hydrocarbon receptor-mediated pathways. Pharm Res. 2009 Apr; 26(4):872-82  Li L, Hassan HE, Antonia H. Tolson, Ferguson SS, Eddington ND, and Wang H. Differential Activation of Pregnane X Receptor and Constitutive Androstane Receptor by Buprenorphine in Primary Human Hepatocytes and HepG2 cells. J Pharmacol Exp Ther. 2010 Dec; 335(3):562-71

Abstracts and Presentations  Antonia H. Tolson, Tao Chen, Natalie D. Eddington and Hongbing Wang (2007). “Synthetic Opioid Methadone Activates PXR and Induces the Expression of Related DMEs in Human Primary Hepatocyte Cultures.” Nuclear Receptors in Liver and Digestive Diseases Research Workshop Poster Presentation, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), Bethesda, Maryland.  Antonia H. Tolson (2008). “Synthetic Opioid Methadone Activates PXR and Induces the Expression of Related DMEs in Human Primary Hepatocyte Cultures." Pharmacy Research Day Poster Presentation, University of Maryland at Baltimore, Baltimore, Maryland.  Antonia H. Tolson, Tao Chen, Natalie D. Eddington and Hongbing Wang (2008). "Synthetic Opioid Methadone Activates PXR and Induces the Expression of Related DMEs in Human Primary Hepatocyte Cultures." AAPS 22nd Annual Meeting and Exposition, Atlanta, Georgia.

SCHOLARSHIPS & FUNDING

 Pharmaceutical Sciences Departmental Merit Award, UMB (2010)  Ruth L. Kirschstein National Research Service Award (NRSA), UMB (2009- 2011) F31 DA026684-01  School of Engineering and Applied Science Scholarship, UVA (2000-2004)

HONORS & AWARDS

 Graduate Student of the Month for September (2009)  Inducted into The Rho Chi Honor Society, Omicron Chapter, UMB, School of Pharmacy, Omicron Chapter (2008)  Dean's List, School of Engineering & Applied Science, UVA (2004)  National Society of Black Engineers Torchbearer (2003)  Selected to Grad Preview at Cal Tech Program (2003)  University of Virginia Intermediate Honors (2002)  University of Virginia "Z" Society Recognition (2001)  AP Scholar with Honor, Highest Achievement in Mathematics & Science (2000)

PROFESSIONAL ASSOCIATIONS

 American Association of Pharmaceutical Scientists, UMB  National Society of Collegiate Scholars, UVA  American Institute of Chemical Engineers, UVA  National Society of Black Engineers, UVA  Society of Women Engineers, UVA

LEADERSHIP & SERVICE

 Graduate Student Peer Mentor, PROMISE: Maryland’s Alliance for Graduate Education & Professorate (AGEP) (2006-2012)  Treasurer and Social Chair, AAPS Student Chapter, UMB, School of Pharmacy (2006-2008)  First Year Representative for School of Pharmacy, Graduate Student Association, UMB, School of Pharmacy (2006-2007)  BRIDGE Counselor, University of Virginia Center for Diversity in Engineering, UVA (2004)  Historian and Parliamentarian, Delta Sigma Theta Sorority, Incorporated, Kappa Rho Chapter, UVA (2003-2004)  Peer Advisor, Office of African American Affairs, UVA (2003-2004)  Volunteer, Madison House Community Service (2000–2002)

HOBBIES & INTERESTS

 Basketball, UMB & UVA, UVA University Intramural Champions 3-on-3 Fall 2001, 2002; 5-on-5 Fall 2001, Spring 2000, 2001  Softball, UMB League Champions 2009; Arlington Athletic Social League Champions 2006; Central Intelligence Agency League 2005-2007

Abstract

Title of the Dissertation: Identification of Drugs of Abuse as Modulators of Drug-

Metabolizing Enzymes through Nuclear Receptor-Mediated

Mechanisms

Antonia Tolson Baaqee Doctor of Philosophy, 2012

Dissertation Directed by: Natalie D. Eddington, Ph.D., FAAPS, FCP

Dean and Professor

School of Pharmacy

University of Maryland, Baltimore, MD

To date, the majority of reports discussing opioid-drug interactions focus intensively on characterizing how other drugs affect the metabolic and pharmacokinetic

(MPK) profile of opioids, however little has been published regarding the potential for opioids to modulate MPK-based drug-drug interactions (DDIs) involving other commonly co-administered or co-abused drugs. Moreover, virtually no mechanistic evidence has been explored. Thus, the objective of this work was to elucidate how opioids affect the MPK of other drugs, thereby undertaking research from a perspective that has been historically overlooked. Accordingly, the specific aims of this study were to: 1) Screen several different drugs of abuse for nuclear receptor (NR) activation potential, 2) Determine the expression profiles of key drug-metabolizing enzymes

(DMEs) or drug transporters for selected drugs in human primary hepatocytes (HPHs), and 3) Characterize the mechanistic roles played by xenoreceptors Pregnane X Receptor (PXR) and Constitutive Androstane Receptor (CAR) underlying observed DME modulation. Results: Here we show that several opioids were identified as potential NR activators, and selected drugs of abuse exhibited differential induction profiles at the mRNA level for target genes CYP2B6 and CYP3A4. Overall, for opioid therapies MD and BUP: 1) MD induced the hepatic expression of multiple key DMEs by activating

PXR- and CAR-mediated pathways; 2) More specifically, MD treatment resulted in significant nuclear accumulation of adenovirus/enhanced yellow fluorescent protein tagged-hCAR in HPHs, which has been regarded as the initial step of CAR activation, and additional analysis of the two enantiomers of racemic MD, R-(–)-MD (active) and S-

(+)-MD (inactive), indicated a lack of stereoselectivity pertaining to MD-mediated DME induction; 3) For BUP, although hPXR-mediated CYP2B6 and CYP3A4 reporter activities were significantly increased in HepG2 cells, treatment with identical concentrations of buprenorphine in HPHs resulted in literally no induction of target gene expression. Taken together, these results provide much-needed mechanistic evidence which demonstrates that MD may be more likely than BUP to modulate CAR- and PXR- mediated DME perturbation during opioid-drug interactions. This research is of great importance to the overall public health industry, particularly to those clinicians and research scientists whom administer MD or BUP as part of opioid maintenance pharmacotherapy.

Identification of Drugs of Abuse as Modulators of Drug-Metabolizing Enzymes through Nuclear Receptor-Mediated Mechanisms

Antonia Tolson Baaqee

Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, Baltimore in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2012

Dedicated wholeheartedly to the Best Daughter a Mom could ask for, my Kaedynn-Bear, and to my Wonderful Husband, Mikal… Thank you both for your unconditional love and support… I will always be your Biggest Fan; Thanks for being mine…

“Let all who run to you for protection always sing joyful songs… Our Lord, you bless those who live right, and you shield them with your kindness”

Psalms 5:11-12

iii

Acknowledgments

Thank You, Dear Heavenly Lord, for giving me the resilience to finish; it is only through You that I achieve. Thank you for always providing me with all that I need, and for showing me the way, especially at times when I felt all alone.

I would like to express my sincere gratitude to my advisors, Dr. Hongbing Wang and Dean Natalie D. Eddington. Most people call her “Dean” but we call her “Dr. E.” I have enjoyed learning from each of them. I thank Dr. E for offering continuous assistance, trying to instill confidence in me, and sharing her wisdom. I thank her also for taking me as a student at a time when most others would have turned one down; and moreover, for keeping me as a student thereafter. To say that I have learned so much from her as a leader and investigator is an understatement. So I instead simply want to thank her for everything, especially the many spoken and unspoken lessons well learned that I know will stay with me as I move forward in my career. I thank Dr. Wang for taking a chance on me, and for turning a chemical engineer into a molecular biologist.

His guidance was absolutely essential, not just for my work, but for me as a person and scientist as well. I enjoyed coming to understand Dr. Wang’s vision, perspective and overall approach to things. I appreciate his precision and way of thinking; and I am so glad to have been able to learn these things from him first-hand. I am proud to consider myself to be his “first UMB student.”

Hardly anyone is crazy enough to have two primary co-mentors for their dissertation work. But for me, it worked; I received twice the mentorship, twice the expertise and twice the benefits of having each of them shape my career. Each of my mentors has positively molded me as an investigator, and each has inspired me to excel,

mainly by challenging me as much as I challenge myself. Specifically, my mentors practiced patience but firmness in guiding me; showed me how to employ shrewdness and creativity in navigating the scientific atmosphere of academia; and each has taught me above all that there are many ways succeed. I have learned so much from watching them lead, teach, and run their labs. I sincerely appreciate all they have done, both individually and together, to promote my development as a scientist. I jokingly referred to them as being just like my “real parents” when I started working for them; that analogy proved to be very accurate throughout the course of my career. Similar to how one may come to understand and appreciate more about their parents over time or as they prepare to “leave the nest,” I too understand and deeply appreciate more about my mentors today than I did yesterday, or last month, or last year, and so forth. The good has far outweighed anything else, and it has been a pleasure working for them. So thank you,

Drs. E and Wang, I am so fortunate to have called you both “boss” for my Ph.D. career and I could not have done it without you. I will always be grateful to each of you.

I wish to thank my Ph.D. Committee Members, Dr. Kenneth Bauer, Dr. Yan Shu, and Dr. Donna Volpe, for their significant assistance, valuable advice and professional attention. I had absolutely tremendous labmates, and having formally been in two labs, I have a lot of them to thank! Therefore I want to recognize and complement my wonderful co-workers for creating such a helpful and cordial work environment. In the Eddington

Lab, thank you to Ahmed, Vijay, Lisa, Christina, and, most of all, Dr. Hazem Hassan.

Hazem, you are such a major influence on me as a co-worker and investigator. Thank you for always offering your professional input and help. It was beyond valuable, and I am going to take your guidance along with me throughout the rest of my career. Thank you

also to the many previous postdoctoral fellows and lab managers. In the Wang Lab, thank you to Tao, Haishan, Leslie, Hui, Brandy, Duan; and a special thanks to Caitlin and “Dr.

Linhao Li!” Caitlin and Linhao, the Wang Lab would not be the Wang Lab without both of you; it is truly a unique atmosphere that we have enjoyed while working together in our lab these last several years. Overall, the congeniality and camaraderie that I have enjoyed amongst such great groups of people is special and I will miss it. Lastly, I want to thank Tao, Leslie, Caitlin, Hazem, and Linhao again – for being fantastic friends in addition to being great co-workers.

I thank my treasured friends and colleagues at UMB; and very special warm thanks go to my PROMISE family – especially Drs. Warnick, Tull and Pollack. The

PROMISE mentors, friends and colleagues that I’ve made across the campuses have been an integral part of my success. Namely, I want to thank Dr. Trudy Smith, Dr. Marishka

K. Brown, and Dr. Stacey Williams. Thank you for listening to me, encouraging me, and helping me through some really challenging times. I am more than glad to have met you all, proud to call you all colleagues, and even more proud to call you all friends.

Lastly, from the bottom of my heart I would like to thank my precious daughter,

Kaeds, just for being her, and my other half, Mikal, for everything. They have been instrumental in helping and supporting me in this journey; and helping me become Dr.

“Antonia Mommy.” I really appreciate their love, patience, perseverance, encouragement, sacrifice and unconditional support. KK, we are a “Number One Family”… and I look forward to us all continuing to take the world by storm -- together -- at whatever we set out to do in life! I love you… Let’s go!

Table of Contents

Dedication ……………………………………………………………………………….iii

Acknowledgements …………….……………………………………………………….iv

List of Tables ………...…………………………………………………………………..x

List of Figures ………………………………………………………………………...…xi

List of Abbreviations ………………………………………………………………….xiv

Chapter 1: Introduction ...... 1

1.1 The Magnitude of Drug Abuse ...... 2

1.2 Commonly Abused or Misused Drugs ...... 4

1.2.1 Opioids ...... 5

1.2.2 Metabolism and Pharmacokinetics (PK) of Opioids ...... 7

1.3 Investigating the Problem: Cytochrome P450s (CYPs) and Nuclear Receptor

(NR) Involvement ...... 12

1.4 Rationale, Hypothesis and Specific Aims ...... 19

1.5 References ...... 22

Chapter 2: Opioid-Mediated Activation of Xenobiotic Receptors Pregnane X

Receptor (PXR) and Constitutive Androstane Receptor (CAR) ...... 32

2.1 Introduction ...... 33

2.2 Materials and Methods ...... 41

vii

2.3 Results ……...... 44

2.4 Discussion ...... 51

2.5 References ...... 56

Chapter 3: Induction of Hepatic Drug-Metabolizing Enzymes (DMEs) by

Selected Drugs of Abuse in Human Primary Hepatocytes (HPHs) ...... 59

3.1 Introduction ...... 60

3.2 Materials and Methods ...... 61

3.3 Results ……...... 65

3.4 Discussion ...... 77

3.5 References ...... 84

Chapter 4: Characterization of the Mechanistic Roles of Xenoreceptors

Pregnane X Receptor and Constitutive Androstane Receptor Underlying Drug-

Metabolizing Enzyme Modulation ...... 87

4.1 Introduction ...... 88

4.2 Materials and Methods ...... 90

4.3 Results ……...... 95

4.4 Discussion ...... 107

4.5 References ...... 116

viii

Chapter 5: Future Directions: Exploring the Potential for Methadone to Modulate

Corresponding Rodent Drug Metabolizing Enzymes ...... 122

5.1 Preliminary Induction Profiles in Rat ...... 123

5.2 Future Directions and Conclusions ...... 128

5.3 References ...... 130

List of Tables

Chapter 2

Table 2.1. Drugs of abuse, including opioids, used in cell-based reporter assays ...... 35

Chapter 3

Table 3.1. Primer and probe sequences for real-time PCR assays ...... 67

List of Figures

Chapter 2

Figure 2.1. Chemical structures of: cocaine (COC), buprenorphine (BUP), and

foxymethoxy (FOXY) ...... 36

Figure 2.2. Chemical structures of: methadone (MD), and diazepam (DZP) ...... 37

Figure 2.3. Chemical structures of: flavone (FLV), and quercitin (QUE) ...... 38

Figure 2.4. Chemical structures of: genistein (GEN), indole-3-carbinol (I3C), oltipraz

(OLT), and brassinin (BRS) ...... 39

Figure 2.5. Activation of hPXR-3A4 by drugs of abuse in cell-based reporter assays .... 46

Figure 2.6. Activation of hPXR-3A4 by opioids in cell-based reporter assays ...... 47

Figure 2.7. Activation of hPXR-2B6 by opioids in cell-based reporter assays ...... 48

Figure 2.8. Activation of hPXR-3A4 by anticancer agents in cell-based assays ...... 50

Figure 2.9. Concentration-related activation of hPXR-3A4 by anticancer agent ...... 52

Figure 2.10. Concentration-related activation of hPXR-3A4 by drugs of abuse ...... 53

Figure 2.11. Concentration-related activation of hPXR-3A4 by opioids ...... 54

Chapter 3

Figure 3.1. Induction of CYP2B6 and CYP3A4 after treatment with FOXY, FLV, or DZP

in HPHs...... 68

Figure 3.2. Induction of CYP2B6 and CYP3A4 after treatment with MD or METH in

HPHs…...... 69

Figure 3.3. Induction of CYP2B6 and CYP3A4 after treatment with MD in HPHs ...... 71

Figure 3.4. Induction of UGT1A1(UDP glucuronosyltransferase 1A1) and MDR1

(Multidrug resistance protein 1) after treatment with MD in HPHs ...... 72

Figure 3.5. Protein expression of CYP3A4 and CYP2B6 after treatment with MD in

HPHs…...... 74

Figure 3.6. Induction of CYP3A4 and CYP2B6 after treatment with BUP in HPHs ...... 75

Figure 3.7. Protein expression of CYP3A4 and CYP2B6 after treatment with BUP in

HPHs...... 76

Figure 3.8. Metabolic stability of BUP and DIP in HepG2 cells and HPHs ...... 78

Figure 3.9. DIP fails to induce CYP2B6 and CYP3A4 expression in HPHs ...... 79

Chapter 4

Figure 4.1. Schematic illustration of the activation mechanisms and target genes of CAR

and PXR ...... 91

Figure 4.2. Activation of hCAR3 and hCAR1+A by prototypical hCAR activators ...... 94

Figure 4.3. Effects of MD on hPXR-, hCAR3-, and hCAR-mediated CYP2B6 reporter

gene activation ...... 96

Figure 4.4. MD enantiomers increase the activities of hPXR in HepG2 cells ...... 98

Figure 4.5. Effects of racemic MD and enantiomers on the activation of hCAR ...... 99

Figure 4.6. Effects of BUP on hPXR-mediated reporter gene activation in cell-based

reporter assays ...... 101

Figure 4.7. Effects of BUP on the activation of hCAR ...... 102

Figure 4.8. Localization of Ad/EYFP-hCAR in HepG2 cells and HPHs ...... 105

xii

Figure 4.9. Known hCAR activators promote nuclear translocation of Ad/EYPF-hCAR in

HPHs…...... 106

Figure 4.10. Methadone translocates Ad/EYFP-hCAR in HPHs ...... 108

Figure 4.11. MD and constituent isomers promote nuclear translocation of Ad/EYPF-

hCAR in human hepatocytes...... 109

Figure 4.12. Localization of Ad-EYFP-hCAR upon BUP Treatment in HPHs ...... 111

Chapter 5

Figure 4.1. MD increases the expression of rodent cyp2b2 and cyp3a1 in rat

hepatocytes...... 125

Figure 5.2. MD does not increase the expression of rodent ugt1a1 and mdr1 in rat

hepatocytes...... 126

Figure 5.3. PB induction profiles for rodent cyp2b2 and cyp3a1 in rat hepatocytes ...... 127

xiii

List of Abbreviations

Ad/EYFP Adenovirus Yellow Fluorescence-tagged Protein

ADRs Adverse Drug Reactions

AMPK AMP-activating Protein Kinase

BUP Buprenorphine

CAR Constitutive Androstane Receptor

CNS Central Nervous System

COC Cocaine

CAM Complementary and Alternative Medicine

CYPs Cytochrome P450s

CCRP Cytoplasmic CAR Retention Protein

DAWN Drug Abuse Warning Network

DDIs Drug-Drug Interactions

DMEs Drug-Metabolizing Enzymes

ED Emergency Department

FLAV/FLV Flavone

FOXY Foxymethoxy

GSTs Glutathione S-transferases

Hsp90 Heat shock protein 90

HPHs Human Primary Hepatocytes

LBDs Ligand Binding Domains

MD Methadone

µOR/MOR µ-Opioid Receptor

xiv

MDR1 Multidrug Resistance 1

MRPs Multidrug Resistance associated Proteins

NIDA National Institute on Drug Abuse

NRs Nuclear Receptors

OATPs Organic Anion-transporting Polypeptides

PK Pharmacokinetics/Pharmacokinetic

PB Phenobarbital

PXR Pregnane X Receptor

PP2A Protein Phosphatase 2, Protein Phosphatase 2A

Q-PCR Quantitative Polymerase Chain Reaction

RXR Retinoid X Receptor

SULTs Sulfotransferases

UGT Uridine diphosphate Glucuronosyltransferase

WT Wild Type

Chapter 1

Introduction

1.1 The Magnitude of Drug Abuse

Drug abuse is a rapidly escalating problem: for example, currently an estimated 1 million people in the United States are addicted to heroin or other opiates.1 Moreover, drug abuse is one of the most challenging public health problems facing America today, and the devastating effects from drug abuse extend far beyond detriment for just the individuals abusing drugs. Rather, drug abuse negatively impacts our society on multiple levels: the individual, families, communities, children, our nation’s economy, and even its workplace infrastructure. Each year, approximately 40 million debilitating illnesses or injuries occur among Americans as a result of their usage of tobacco, alcohol, or other addictive drugs;2 and substance abuse costs our nation approximately more than $484 billion per year.3 Major problems such as drugged driving, violence, stress, missing work, child abuse, mental illness, crimes, and of course death can all be attributed to drug abuse.

The Drug Abuse Warning Network (DAWN) is a public health surveillance system that monitors drug-related emergency department (ED) visits for the nation, and for selected metropolitan areas located therein. Although the overall number of ED visits attributable to drug misuse or abuse was stable from 2004 to 2009, increases were seen in

ED visits involving nonmedical use of pharmaceuticals, both alone (117% increase), with illicit drugs (97%), with alcohol (63%), and combined with both illicit drugs and alcohol

(76%). In 2009, DAWN estimated that approximately 2.1 million ED visits resulted from medical emergencies involving drug misuse or abuse, the equivalent of 674.4 ED visits per year per 100,000 people. Equivalent rates for individuals aged 20 or younger numbered 473.3 visits, and for those aged 21 or older, the rate was 754.8 visits.4 Of the

2.1 million visits associated with drug misuse or abuse in 2009, the following statistics were determined: 35.3 percent of ED visits involved pharmaceuticals alone, 23.0 percent involved illicit drugs alone, 10.2 percent involved illicit drugs plus alcohol, 11.0 percent involved pharmaceuticals plus alcohol, 10.0 percent involved pharmaceuticals plus illicit drugs, 6.7 percent involved alcohol alone in patients aged 20 or younger, and 3.9 percent involved pharmaceuticals and illicit drugs plus alcohol.4

Slightly over 120 million total visits were made to EDs in general-purpose hospitals in the United States in 2009, and DAWN estimates that at least 4.5 million of these visits were drug related. Drug-related ED visits have increased by over 80 percent since 2004.5 It is quite interesting that this increase in drug-related ED visits likely reflects greater numbers of medical emergencies associated with some of the same major risks observed clinically due to polypharmacy, such as adverse drug reactions (ADRs), drug-drug interactions (DDIs), and misuse or abuse of prescription drugs or over-the- counter medications. The term “polypharmacy,” meaning “many drugs,” commonly refers to the concomitant use of three or more drugs, or individuals receiving a drug regimen involving the use of more drugs than are clinically indicated. Inherent risks associated with DDIs and ADRs arise for several reasons in addition to the increasingly standard practice of using polypharmacy approaches to therapy, such as the pharmacological complexity of modern drugs and the ageing population. Overall, both rampant drug abuse and the increasing instances of polypharmacy pose major problems to optimizing pharmacotherapy in today’s clinical setting. For example, accurately prescribing medication is becoming an increasingly complex and challenging process given the frequency of patients who might be abusing drugs, taking multiple therapies at

3 once, or both. Another problem is excessive cost: health expenditures in the United States alone approached nearly $2.6 trillion in 2010.6 Lastly, a particularly confounding problem resulting from polypharmacy scenarios (a problem which can also be easily exacerbated if drug abuse is involved beyond a polypharmacy regimen) is that the side effects or symptoms seen in these patient situations can easily be confused with those of a different disease state other than the original health problem being treated. This can lead to a dangerous cycle of more prescriptions and declining health rather than less prescriptions and improved outcomes.

1.2 Commonly Abused or Misused Drugs

Topping DAWN’s list of most commonly abused or misused drugs are drugs such as stimulants, dissociative drugs, “club drugs,” hallucinogens, and opioids. Examples of popularly abused stimulants are cocaine and amphetamines; examples of dissociative drugs are ketamine, phencyclidine, otherwise known as PCP, and its analogs. The category “club drugs” commonly refers to some of the drugs already named in the aforementioned drug classes (e.g. ketamine and methamphetamine), as well other drugs such as flunitrazepam (also called Rohypnol, or “roofies”) and methylenedioxymethamphetamine (MDMA, ecstasy). As the name of this group so aptly suggests, club drugs tend to be used most often by teenagers and young adults in social settings such as concerts, nightclubs and parties. Hallucinogens include psilocybin and lysergic acid diethylamide (LSD); meanwhile opioids, which are also one of the most commonly abused prescription drug types today, include heroin, oxycodone and

4 methadone. Research groups at the National Institute on Drug Abuse (NIDA) like the

Community Epidemiology Work Group comprise an early warning network of investigators that regularly publish issues of a Research Report Series about the nature and patterns of drug abuse in major areas of the U.S. Publications like this help depict what types of data have been collected about drugs of abuse and provide useful tracking statistics; however, aside from overall trends or surveillance data, other research information regarding drugs of abuse, such as mechanistic evidence, or specific data describing DDIs or ADRs with other drugs is deficient. Take for example opioids: opioid analgesics are by far the most appropriate medication used for management of moderate to severe pain, especially cancer and post-operative-related pain. Yet, despite the long history of opioid use for therapeutic purposes, and the frequent prevalence of opioid-drug interactions, there is a very little mechanistic information available from scientific literature reports to characterize opioid-drug interactions.

1.2.1 Opioids

Opioids are compounds that are both structurally related to morphine (a phenanthrene derivative) and its derivatives, such as diacetylmorphine (heroin) and codeine; and structurally unrelated to morphine such as methadone and methadone- related compounds (e.g., methadone, dextropropoxyphene), phenylpiperidine analogs

(e.g., meperidine, fentanyl, loperamide), benzomorphan analogs (e.g., pentazocine) and semisynthetic thebaine derivatives (e.g., oxycodone, buprenorphine and etorphine). Both types of compounds exert their actions by binding to specific opioid receptors, namely

5 the mu-opioid receptor (μOR or MOR), δ-opioid receptor, and κ -opioid receptor [δ, κ], where they decrease pain impulses and sensations after binding to nerve cells in the spinal cord and brain. Some opioids are mainly prescribed for mild to moderate pain

(e.g., codeine, meperidine); meanwhile others are primarily prescribed for moderate to severe pain (e.g., morphine, oxycodone, methadone, fentanyl).

Overall, opioid analgesics are well known for their ability to reduce the perception of pain without loss of consciousness. However, they are also known for being associated with problems of physical dependence and tolerance upon repeated administration. The development of tolerance to opioid analgesics is quite common, and presents a practical challenge, specifically for the use of opioids in the clinical settings.7

Nonetheless, opioids constitute the major class of analgesics used in the management of moderate to severe pain because of their effectiveness, ease of titration and favorable risk-to-benefit ratio. Opioids produce analgesia by binding to μ-, δ- and κ-opioid receptors both within and outside the central nervous system. These opioid receptors are glycoproteins that exist in many organs including brain, gastrointestinal tract, cardiovascular system, bladder and lungs.

Opioids can be classified into naturally occurring (e.g., morphine, codeine), semisynthetic (e.g., oxycodone, diamorphine) or synthetic (e.g., levorphanol, methadone), according to their origin. Thereafter, opioids can also be classified as full agonists, partial agonists or antagonists. Full agonists (e.g., morphine, oxycodone) are strong opioids that occupy small percentage of the available opioid receptors (10-20 %) to produce maximum analgesic effects. On the other hand, partial agonists (e.g., buprenorphine, nalorphine) are opioids that need to occupy a high percentage of the

6 available receptors (75-100 %) while still producing less than the maximum analgesic effects. Partial agonists in some cases can be considered as antagonist, meaning they compete with the full agonists for the available receptors, thereby decreasing the potency of the full agonists. In addition, some opioids can exhibit agonist properties at one opioid receptor but antagonist properties at another opioid receptor. Such opioids are known as mixed agonist-antagonist opioids (e.g., buprenorphine). So called antagonists (e.g., naloxone, naltrexone, diprenorphine) are compounds that bind to the opioid receptors but are unable to activate these receptors and inhibit the action of the full antagonists.

1.2.2 Metabolism and Pharmacokinetics of Opioids

Metabolism refers to the biotransformation process by which drugs are broken down into something that can get eliminated from the body. Pharmacokinetics (PK), which is often described as what the body does to a drug, commonly refers to the time- course of movement of a drug throughout the body. Pharmacokinetics is often discussed in terms of not just metabolism, but also in terms of other parameters that characterize the absorption, bioavailability, distribution, and excretion of drugs. Drug PK determines the onset, duration, and intensity of a drug's effect; yet the PK of a drug depends on patient- related factors as well as on the drug's chemical properties. Some patient-related factors such as age, sex, genetic makeup, etc., can be used to predict pharmacologic response of populations, while other factors are related to individual physiology, such as body weight or disease state. The effects of some individual factors (e.g., renal failure, obesity, hepatic failure, dehydration) can be reasonably predicted, but other factors are idiosyncratic and

7 thus have unpredictable effects.8 Opioid metabolism and PK vary greatly among different opioids, across different patient populations, and even within an individual. There is a narrow therapeutic index associated with opioid administration; and careful consideration of pharmacokinetic concerns about dosing, route of administration and clearance become particularly imperative to optimizing clinical outcomes associated with opioid therapy.

Because of individual differences, drug administration must be based on each patient's needs, and knowledge of pharmacokinetic principles helps prescribers adjust dosage more accurately and rapidly.

Although some drugs are able to perform their functions before being excreted from the body intact, other xenobiotics depend on metabolism first to enable them to reach the appropriate target sites of action and remain there long enough to exert their effects efficiently prior to elimination from the body. Altered metabolism and/or the PK of opioids within an individual patient or within a particular patient population can result in several suboptimal therapeutic outcomes. For example, the opioid may leave the body too quickly, therefore not having enough time to exert an effect. It may not be able to reach its therapeutic target, or it may remain in systemic circulation too long, therefore eliciting toxic effects.

As with a myriad of other types of drugs, the main site of metabolic transformation of opioids within the body is the liver, where opioids undergo varying degrees of both phase 1 metabolism (modification reactions) and phase 2 metabolism

(conjugation reactions). The cytochrome P450 (CYP) enzymatic system, which is the major superfamily of drug-metabolizing enzymes (DMEs), is likely the dominant system implicated in biotransformation of opioids during phase 1 metabolism. During phase 2

8 metabolism, uridine diphosphate glucuronosyltransferase (UGT) enzymes likely catalyze glucuronidation reactions which may further transform opioids. The CYP3A4 isozyme is responsible for metabolizing more than 50% of all drugs; and CYP3A4, along with

CYP2D6, are the main isozymes responsible for metabolizing opioids. Most opioids undergo extensive first-pass metabolism (ex. morphine, diazepam, buprenorphine), which reduces the bioavailability of the opioid prior to entering systemic circulation. Opioids are typically lipophilic, which allows them to cross cell membranes in order to reach target tissues, meanwhile, drug metabolism is ultimately intended to make a drug hydrophilic to facilitate its excretion in the urine.9 Of note regarding opioid metabolism is the fact that it often results in the production of both inactive and active metabolites, the latter of which may be more potent than the parent compound. Therefore, although metabolism is ultimately a process of detoxification, particularly concerning opioid metabolism, the production of intermediate products should render caution. These intermediates may either possess useful clinical activity, or be associated with toxicity, or both.

Several of the abovementioned points regarding the metabolism and PK of opioids can be illustrated in discussing opioids morphine, oxycodone, buprenorphine and methadone, for example. Morphine is a phenanthrene derivative that activates the µ- opioid receptors and is recommended by the World Health Organization as the first line therapy for management of moderate to severe pain. It is considered as the standard opioid that is used to determine the relative potency of all other opioids, and, interestingly, it undergoes phase 2 metabolism primarily via glucuronidation catalyzed by

UGT2B7, not phase 1 metabolism. It is usually administered via intramuscular,

9 intravenous or subcutaneous injection; and has a half-life of 2-3 h. Morphine can also be administered orally but it undergoes extensive first-pass metabolism in the liver prior to being metabolized into its two major metabolites morphine-3- and morphine-6- glucuronide. Oxycodone (4,5 δ-epoxy-14-hydroxy-3-methoxy-17-methyl-morphinan-6- one) is a semi-synthetic derivative of thebaine. It is an opioid agonist that is used in the management of moderate to severe pain; and it is primarily metabolized by CYP3A4 and

CYP2D6. Oxycodone does not undergo subsequent phase 2 metabolism; however, relatively small quantities of oxymorphone are produced during this process, which will be transformed via the same pathway as morphine.

Buprenorphine is unique in that it is a partial agonist at the opioid μ receptor. It undergoes extensive first-pass metabolism and therefore has very low oral bioavailability; however, its bioavailability sublingually is extensive enough to make this a feasible route of administration for the treatment of opioid dependence.10 Buprenorphine is extensively metabolized by N-dealkylation to norbuprenorphine primarily through cytochrome

CYP3A4, and the mean time to maximum plasma concentration following sublingual administration is variable, ranging from 40 minutes to 3.5 hours. As could be said for many other opioids, although there is limited evidence in the literature to date, drugs that are known to inhibit, induce, or be a substrate of CYP3A4 could feasibly likely have the potential to diminish or enhance buprenorphine metabolism. The relationship between buprenorphine plasma concentration and response in the treatment of opioid dependence has not been well studied.10

Methadone is the most established substance abuse pharmacotherapy of choice for the treatment of heroin dependence. Within the United States alone, approximately 20%

10 of an estimated 1 million heroin addicts receive long term methadone maintenance treatment.11 Methadone is a long-lasting synthetic opioid, commonly used not only to treat addiction but also as a narcotic analgesic for treating chronic pain, and has been prescribed by healthcare professionals for decades. Methadone is primarily metabolized by CYP3A4 and CYP2B6; however, CYP2C8, CYP2C19, CYP2D6 and CYP2C9 also contribute in varying degrees.9 The primary metabolic route of MD is hepatic N- demethylation and cyclization to its stable metabolite, 2-ethyl-1,5-dimethyl-3,3- diphenylpyrrolidine, or EDDP, which is pharmacologically inactive. Both clearance and metabolism rates for methadone vary considerably among patients.12 Such high interindividual variability is thought to be accredited to genetic inconsistency in the production of associated enzymes CYP3A4, CYP2B6 and CYP2D6.13 Additionally, factors such as urine pH, sex and auto-induction of enzymes influence methadone metabolism and clearance rates. Metabolism rates for methadone vary by sex (women metabolize methadone faster than men); elimination rates can fluctuate based upon urine pH (enhanced by acidic urine and decreased by alkaline urine), or due to auto-induction of CYP3A4, which can be enhanced upon chronic administration.14 Negative effects resulting from auto-induction include a time-dependent increase in clearance, and greater occurrence of first-pass metabolism. Overall, variations in CYP expression levels and enzymatic activity account for the large individual variations associated with methadone pharmacokinetics.

Ultimately, drugs that induce the CYP3A4 or CYP2B6 system lower the levels of methadone circulating in plasma; conversely, drugs that inhibit these systems increase the levels of methadone. This example of the complex interplay of methadone with as many

11 as six different players in the CYP system illustrates how easily opioid therapy can be accompanied by considerable interaction potential in many clinical scenarios. Over the last several decades, methadone has been administered universally to some millions of persons in recovery from opioid addiction. Yet, the problem is that the particular function of methadone, the potential for it to affect other drugs, and the mechanisms through which these effects may result in clinically significant methadone-drug interactions, are all phenomena that remain not fully understood. The same can be said regarding other opioids in general. Overall, there is a major research gap in existing literature; and filling this gap is vital to improving current methadone and opioid pharmacotherapy when opioids are administered as part of a polypharmacy approach. Studies such as those outlined in this dissertation are essential to establish the role that opioids play in pharmacotherapy, and to begin to evaluate the impact of opioid-mediated alterations in the metabolism and PK of other commonly co-administered or co-abused drugs.

1.3 Investigating the Problem: CYPs and Nuclear Receptor (NR) Involvement

First, in order to begin filling this research gap, it is important to consider a major area of research related to the CYPs: nuclear receptors (NRs), which are a relatively recently characterized family of proteins that act as transcriptional regulators of gene expression in response to extracellular stimuli such as hormones, xenobiotics or other ligands. When trying to understand how nuclear receptors may be modulating a process or interaction, it is important to pay close attention to the major players involved in

12 xenobiotic metabolism, Pregnane X Receptor (PXR) and Constitutive Androstane

Receptor (CAR).

The pregnane X receptor (PXR, NR1I2) is an approximately 434-amino acid, 50- kDa protein, primarily expressed in the liver and intestine.15 In 1998, three research groups independently isolated cDNAs encoding a novel orphan receptor, PXR, which was subsequently shown to play a central role in the transcriptional regulation of CYP3A genes across multiple species.16 Prior to being designated NR1I2, this receptor was also named SXR (steroid and xenobiotic receptor) and PAR (pregnane-activated receptor) in humans, which proved to be reflective of its subsequently identified versatility in recognizing a broad array of both synthetic steroids and xenobiotics.17

Compared with other NRs, PXR possesses a bulky and flexible ligand-binding cavity, which enables it to accommodate a more structurally promiscuous library of ligands.18 Multiple structural studies have facilitated the rationalization of the ligand- binding characteristics of PXR, such as the determination of the structure of human

(h)PXR, in both the SR-12813-bound and unbound forms,19 followed by similar characterization of hPXR bound to hyperforin,20 or rifampicin.21 Among the many key insights that have been gained from these studies are the realization that the ligand- binding cavity of PXR can be, for example, differentially induced to change shape, expand in volume, or adopt unique conformational structures. This suggests that the promiscuity of PXR with respect to ligand binding can be attributed to the tremendously unique binding capabilities of this receptor.19, 22 Additionally, although most human and rodent NR orthologs share greater than 90% amino acid identity, the LBDs of PXR are less conserved among species; for instance, hPXR and rat PXR share 76% amino acid

13 identity.23 The sequence divergence in the LBD of PXR among species is believed to be responsible for the species-specific PXR activation and target gene induction.

Initially PXR was thought to be the more “conventional” NR, as it appears to exert its effects through a similar mechanism of action as the other steroid hormone receptors. However, its apparently ever-evolving library of structurally diverse ligands has come to distinguish PXR as a unique, promiscuous, but integral mediator of inductive expression of many DMEs and transporters. Using transgenic PXR mouse model, approximately 150 gene tags expressed in a PXR-dependent manner were identified, which include a spectrum of biologically important phase I and II DMEs, as well as uptake and efflux drug transporters.24 The extremely flexible nature of PXR in ligand and target gene recognition has set it apart as a special xenobiotic sensor in a class of its own.

Like PXR, CAR also has distinguishing characteristics that make it too a major xenosensor involved in metabolic processes. Because the two NRs are closely related, extensive cross-talk exists between them, however, many key differences between the two receptors are important.

In the nuclear receptor superfamily tree, CAR (NR1I3) is the closest relative to the abovementioned PXR and is expressed primarily in the liver and intestine. Initially named MB67 in 1994, this receptor was designated as constitutive activated receptor

(CAR), because it forms a heterodimer with retinoid X receptor (RXR) that binds to retinoic acid response elements (RAREs) and transactivates target genes in the absence of ligand stimulation.25 In 1998, the first class of CAR ligands including androstanol and androstenol was identified.26 Interestingly, these compounds were characterized as inverse agonists because instead of activation they repressed the constitutive activity of

CAR in vitro.26 Accordingly, this receptor is also referred to as constitutive androstane receptor. Major progress in our understanding of the physiological roles of CAR, however, came with the observation that activation of CAR was linked to the induction of the CYP2B gene family by phenobarbital (PB) and PB-like inducers.27 This finding has triggered a wealth of subsequent studies exploring the role of CAR on xenobiotic detoxification and excretion;28 and the definite role of CAR on CYP2B induction was established eventually by using CAR knockout mice.29

During the last decade, mounting evidence suggests that CAR induces a broad spectrum of hepatic and intestinal genes involving xenobiotic metabolism and transport.28b, 30 Notably, CAR and PXR share significant cross-talk in both target gene recognition by binding to the similar xenobiotic responsive elements in their target gene promoters, and in accommodating a diverse array of xenobiotic activators.31

Coordinately, CAR and PXR regulate a largely overlapping set of xenobiotic metabolizing genes. These target genes include several CYPs (i.e. CYP3A4, CYP2B6,

CYP2Cs, and CYP2A6),32 UGTs (i.e. UGT1A1, UGT1A6, and UGT1A9),33 GSTs, and

SULTs; as well as drug transporters such as MRPs, MDR1, and OATPs.34 On the other hand, CAR displays unique activation mechanisms compared with PXR and other orphan receptors, involving both direct ligand binding and indirect ligand-independent pathways

(Fig. 1).35 In addition, CAR may also respond to stress and energy crisis in a way distinct from PXR.36 As will be discussed further below, our focus is given to the differential rather than the redundant roles of CAR from PXR in xenobiotic metabolizing gene regulation and the mechanisms underlying CAR activation.

Compared with the highly overlapping target genes between CAR and PXR, these two receptors share much less similarity in their activation mechanisms upon chemical stimulation. Different from PXR, where activation is dependent exclusively on ligand- binding, CAR can be activated by either direct ligand binding or ligand-independent

(indirect) pathways. As a matter of fact, the majority of CAR activators identified to date actually activate this receptor through rather mutedly defined indirect mechanisms.37

Interestingly, CAR exhibits a unique subcellular distribution and activation pattern between immortalized cell lines and physiologically relevant primary cells.

In primary hepatocytes and intact liver in vivo, CAR is primarily located in the cytoplasm prior to activation and translocates to the nucleus in an activator-dependent manner.35a, 38 Nevertheless, consistent with its designated name, CAR is spontaneously accumulated in the nucleus and constitutively activated in all known immortalized cell lines without xenobiotic stimulation.39 A CAR protein complex seems to be required to retain this receptor in the cytoplasm, and components of this complex identified thus far include heat shock protein 90 (Hsp90), cytoplasmic CAR retention protein (CCRP),

PPP1R16A, and possibly PP2A.40 In HepG2 cells, over-expression of CCRP retained mouse (m) CAR in the cytoplasm of transfected cells; the cytoplasmic retained CAR, however, seems to have lost its nuclear translocation upon PB-stimulation, indicating that

CCRP may not be a xenobiotic responsive component of the CAR complex.40a Notably,

PB, the prototypical activator of CAR and inducer of CYP2B in multiple species, does not bind to either mCAR or human (h) CAR, but triggers nuclear accumulation of CAR in hepatocytes under primary culture or in vivo conditions.

Ligand-independent activation of CAR distinguishes it from typical NRs, but also poses major difficulties for evaluating drug-mediated CAR activation, particularly, in high throughput manners in vitro. Due to the multi-mechanistic activation of CAR, in vitro ligand-binding assays offer only limited value in identifying CAR activators.

Moreover, cell-based luciferase reporter assays in immortalized cell lines are not sensitive to chemical activation. In mouse primary hepatocyte cultures, PB-mediated

CAR translocation and Cyp2b10 expression were efficiently repressed by the pretreatment of okadaic acid (OA), the prototypical inhibitor of PP2A.35a, 41 Further analysis revealed that PB treatment of mouse hepatocytes led to the recruitment of PP2A to the CAR-Hsp90 cytoplasmic complex, suggesting a possible mechanism in PB- triggered CAR translocation.42 To this end, although the definitive mechanisms behind

PB-mediated CAR translocation have yet to be elucidated, primarily cultured hepatocytes seem to be an attractive in vitro model for the investigation of CAR localization/translocation and the identification of xenobiotics as CAR activators.

Utilizing human primary hepatocytes infected with adenovirus expressing EYFP- tagged hCAR (Ad/EYFP-hCAR), Li et al.,38 evaluated CAR nuclear translocation- triggered by 22 compounds including known CAR activators, non-activators, and selective activators of other receptors. Results obtained from this study indicate a close correlation between chemically-mediated CAR translocation and activation in this system, where nuclear accumulation of Ad/EYFP-hCAR was clearly increased upon the treatment of known human CAR activators; whereas no changes were observed after treatment with selective activators of other receptors such as RIF for PXR, 3MC for AhR,

Wy-14643 for PPARα, or TCPOBOP, as a selective activator, of mCAR.

In addition to the unique feature of indirect activation, CAR can be activated also by a prototypical ligand-dependent mechanism. TCPOBOP is the first potent and selective mCAR ligand identified through a combination of in vivo and in vitro approaches.35c Unlike the universal CAR activator PB, TCPOBOP induces the expression of CYP2B in mice but not in humans.35c Comparatively, identifying selective hCAR activators has been proven to be difficult because: 1) known mCAR invert agonists androstanol and androstenol are not effective repressors of hCAR, 2) hPXR appears to have evolved into a more promiscuous xenobiotic sensor than its rodent counterparts; and

3) significant overlap in the pharmacology of hCAR and hPXR exists. For instance, PB activates rodent CAR but not PXR, but is an activator of both hCAR and hPXR.32a

Recently identified hCAR deactivators such as clotrimazole (CLZ), and PK11195 are also potent activators of hPXR.43

In 2003, CITCO was reported as the first selective hCAR activator by directly binding and activating this receptor. Indeed, preferential induction of CYP2B6 over

CYP3A4 has been observed in CITCO treated human hepatocytes.35b, 44 Notably,

PK11195 repressed constitutive activity of hCAR could be efficiently reactivated by

CITCO but not PB in luciferase reporter assay in HepG2 cells (Fig. 3), thus PK11195 may represent a chemical tool for distinguishing direct vs. indirect activators of hCAR.43a

Recently, several reports have discussed a novel role of AMP-activating protein kinase signaling in PB- but not CITCO-mediated induction of CYP2B6, indicating that AMPK may exert differential effects in direct vs. indirect activation of CAR.45 Nevertheless,

CITCO also activates hPXR at slightly higher concentrations with an EC50 of approximately 3 μM in cell-based reporter assays;35b as such, more selective hCAR

18 activators and deactivators are still necessary for delineating the specific function of CAR in humans.

1.4 Rationale, Hypothesis and Specific Aims

Taken altogether, based on the widespread use and abuse opioids, it is surprising that investigations examining the impact of opioid-drug interactions have not been more thoroughly examined. Therefore, the aim of this work is to examine how opioids and other drugs of abuse may affect co-administered or co-abused drugs in opioid-drug interaction scenarios. We were also interested in including anticancer agents in these investigations, since interest in complementary and alternative medicine (CAM) has grown rapidly in the industrialized world in recent years,46 and CAM use is more common among patients with cancer than in the general population.47 In fact, it has been estimated that up to one third of the entire population in the United States has used CAM, and further, that approximately 15 million adults have combined herbal remedies with prescription medications.46 Thus, the risk for herb-drug interactions is a growing concern not unlike the risks associated with opioid-drug interactions: a major cause of concern is the potential for herbs, opioids, drugs of abuse, etc., to interact with prescription drugs, and possibly alter their pharmacokinetic characteristics, which could lead to clinically significant drug-drug interactions (DDIs). As such, the hypothesis of this work is that:

“Drugs of abuse such as synthetic opioids are involved in the nuclear receptor-mediated perturbation of clinically important drug-metabolizing enzymes (DMEs) and drug transporters.”

More specifically, we hypothesize that 1) opioids such as methadone play a key role in the perturbation of one or more clinically important drug metabolizing enzymes

(DMEs) or drug transporters that are responsible for the metabolism of a myriad of xenobiotics; and 2) that the opioid-mediated induction of DMEs and drug transporters is governed predominantly by the activation of nuclear receptors (NR) pregnane X receptor

(PXR), and constitutive androstane receptor (CAR). Our goals for investigating our hypothesis were to screen several different drugs of abuse for the potential to activate

PXR and CAR; to obtain induction profiles for selected opioids for clinically relevant

DMEs and drug transporters; and to demonstrate some mechanistic evidence underlying the observed induction. Therefore, in order to address our hypothesis and achieve these goals, the following specific aims were pursued:

Specific Aim 1: To screen several different compounds, including drugs of abuse and opioids, for nuclear receptor (NR) activation. Popular drugs of abuse such as modafinil, oxycodone, nicotine, cocaine, foxymethoxy, ecstasy and diazepam; as well as opioids such as methadone, buprenorphine, and diprenorphine, were screened using cell- based reporter assays to determine the potential for nuclear receptor activation. Specific

Aim 2: To determine the expression profiles of key DMEs or drug transporters for drugs of abuse in human primary hepatocytes (HPH). Since polypharmacy is commonplace in current pharmacotherapy, and opioids (e.g., oxycodone) are often concomitantly administered with other therapeutic agents that are substrates for transporters and/or metabolizing enzymes, monitoring the changes in the level of expression of these transporters and/or metabolizing enzymes may yield valuable insight into how opioids are exerting their influence in common clinical drug-drug interactions scenarios. Specific

Aim 3: To characterize the mechanistic roles of xenoreceptors PXR & CAR underlying observed DME modulation. The majority of NR-modulated processes occurs through xenosensors PXR and CAR. Therefore, demonstrating the way in which these two xenobiotic receptors are involved in any observed induction is paramount to beginning to investigate the mechanisms underlying induction.

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Nuclear Receptor CAR. Molecular and Cellular Biology 2000, 20 (9), 2951-2958.

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Medicine Use Among Women With Breast Cancer. Journal of Clinical Oncology

2002, 20 (suppl 1), 34-38.

Chapter 2

Opioid-Mediated Activation of Xenobiotic Receptors Pregnane X

Receptor (PXR) and Constitutive Androstane Receptor (CAR)

2.1 Introduction

Cell-based reporter assays are a popular tool utilized currently in multiple sectors of scientific research. One of the most attractive features of this technique is that any capacity ranging from just a small number of molecules synthesized in the laboratory, up to a high volume of lead compounds identified from virtual screening of databases containing hundreds or thousands of molecules alike, can be biologically screened. In the pharmaceutical industry for example, over the past few decades especially, high throughput screening of compounds has become a mainstay in the drug discovery process both in lead discovery and during subsequent stages of lead optimization. Via this technique, compounds are likely tested for their ability to activate a receptor and its associated target gene expression by measuring an easily detectable marker as the endpoint of measurement.

Notably, activation of PXR and CAR, two xenobiotic sensors, has been associated with increased expression of major drug-metabolizing enzymes (DMEs), and enhanced potential for drug-drug interactions (DDIs). Accordingly, in the following studies, we aimed to screen compounds belonging to two main classes -- drugs of abuse and anticancer agents -- for their potential to achieve nuclear receptor activation in cell-based

PXR/CAR reporter assays. Of particular interest in our screening of the drugs of abuse were twelve opioids, as opiate addiction is now recognized by the World Health

Organization as a disorder of the central nervous system. Additionally, many anticancer agents are natural components of our diet, and, due to a burgeoning interest in utilizing these types of agents in alternative medicine for their numerous beneficial anti- inflammatory, antioxidant and chemoprotective properties, they are increasingly being

consumed by the general population. We were also interested in including anticancer agents in these investigations since the risk for herb-drug interactions to result in clinically significant DDIs is a growing concern not unlike the concerns associated with opioid-drug interactions.

The eighteen drugs of abuse investigated, including twelve frequently clinically prescribed opioids, were: modafinil (MOD), oxycodone (OXY), nicotine (NIC), cocaine

(COC), buprenorphine (BUP), 5-methoxy-diisopropyltryptamine (5-MeO-DiPT), or foxymethoxy (FOXY), methadone (MD), (±)-3,4-Methylenedioxymethamphetamine hydrochloride or ecstasy (MDMA), diazepam (DZP), 3-methylmorphine, or codeine

(CDN), morphine, meperidine hydrochloride, 6-desoxycodeine, naloxone, diprenorphine

(DIP), N-phenyl butyl normeperidine, 6-desoxymorphine, and normeperidine. Table 2.1 summarizes the drugs of abuse tested in the PXR/CAR reporter assay studies, and figures

2.1-2.2 illustrate the chemical structures and formulas of selected opioids for which dose- response curves were subsequently obtained after initial screening (Figs 2.1-2.2).

In an additional effort, we also examined six anticancer agents, including flavone

(FLV), quercitin dihydrate (QUE), 3-Indomethanol, or indole-3-carbinol (I3C), genistein

(GEN), oltipraz (OLT), and brassinin (BRS). Figures 2.3-2.4 depict the chemical structures and formulas for the anticancer agents investigated. Also, along with the drugs of abuse and anticancer agents listed above, bilobalide was included in initial cell-based reporter assay screening, and further testing was performed later to demonstrate the role that it plays in nuclear receptor activation.1 Bilobalide is just one of many active constituents comprising popular herbal supplement EGb 761, or gingko biloba; and recent studies have shown that EGb 761 improves cognitive function, neuropsychiatric

Table 2.1. Drugs of abuse, including opioids, used in cell-based reporter assays

No. Name Drug Type Schedule No. Opioid? Derivative of 1 modafinil analeptic IV - adrafinil 2 oxycodone analgesic II + thebaine 3 nicotine rec./smoking cessation - - tobacco 4 cocaine rec./surgery/local anesthetic II - erythroxylon coca 5 buprenorphine opioid addiction/pain III + oripavine/thebaine 6 foxymethoxy psychedelic/hallucinogen I - tryptamine 7 methadone opioid addiction/pain II + morphine 8 MDMA psychoactive, entactogen I - phenethylamine 9 diazepam anticonvulsant IV - benzodiazepine 10 codeine pain/cough suppression II (III-V) + morphine 11 morphine analgesic II + N/A 12 meperidine analgesic II + piperidine 13 6-desoxycodeine analgesic with reduced - + codeine tolerance 14 naloxone opioid antagonist - - thebaine 15 diprenorphine opioid antagonist/veterinary IIb + oripavine 16 N-phenyl butyl analgesic with reduced - + meperidine normeperidine tolerance 17 6-desoxymorphine analgesic with reduced - + morphine tolerance 18 normeperidine analgesic with reduced II + meperidine tolerance a “rec” – recreational b classified as Schedule II narcotic, but requires additional storage/ordering requirements

A B

Buprenorphine

Cocaine

Foxymethoxy

Figure 2.1. Chemical structures of: cocaine, C17H21NO4 (A, top left), buprenorphine,

C29H41NO4 · HCl (B, top right), and 5-methoxy-diisopropyltryptamin or foxymethoxy,

C17H26N2O · xHCl · yH2O (C, bottom).

Methadone

Diazepam

Figure 2.2. Chemical structures of: methadone, C21H27NO · HCl (A, top), and diazepam,

C16H13ClN2O (B, bottom).

Flavone

Quercitin

Figure 2.3. Chemical structures of: flavone, C15H10O2 (A, top) and quercitin, C15H10O7 ·

2H2O (B, bottom).

A B

Indole-3-carbinol Genistein

C D

Brassinin Oltipraz

Figure 2.4. Chemical structures of: genistein, C15H10O5 (A, top left), indole-3-carbinol

(I3C), C9H9NO (B, top right), oltipraz, C8H6N2S3 (C, bottom left), and brassinin,

C11H12N2S2 (D, bottom right).

symptoms, activities of daily living and quality of life in patients with mild to moderate dementia.2 In total, a battery of approximately twenty-five compounds was screened.

Briefly, flavone is a crystalline compound that is also the parent substance of a number of important white or yellow pigments naturally occurring in plants. Quercitin is also a type of plant-based chemical called a phytochemical, or flavonoid, known for a growing list of properties that make it an effective treatment against an increasing variety of disease states. Quercitin represents the most abundant dietary flavonoid found in a broad range of fruits, vegetables and beverages; and both its antioxidant and anti- inflammatory properties have been associated with the prevention and therapy of cardiovascular diseases and cancer.3 Indole-3-carbinol (I3C) is produced endogenously from naturally occurring glucosinolates contained in a wide variety of plant food substances. Notably, in some studies, I3C has been shown to inhibit the growth of human cancer cells.4 Genistein is a plant phenolic that is one of the most commonly consumed phytoestrogens, and is the predominant isoflavone found in soy products. It has been extensively studied because of both its pharmacological effects and beneficial effects;5 and has been shown to inhibit carcinogenesis in animal models.6 Oltipraz is an antischistosomiasis drug found to inhibit carcinogenesis, and is quite effective against several chemically induced tumor models. It is believed that its mechanism of action is through the induction of phase II detoxifying enzymes, which results in diminished carcinogen-DNA binding. Brassinin is a phytoalexin found in Chinese cabbage, and is known for being an effective inhibitor against two stage skin carcinogenesis. Like OLT,

BRS is also an inducer of Phase II enzymes and an inhibitor of chemically induced carcinogenesis.

2.2 Materials and Methods

Chemicals and Biological Reagents. The eighteen drugs of abuse investigated were: (±)-2-(benzhydrylsulfinyl) acetamide, or modafinil (MOD), (5R,9R,13S,14S)-4,5α- epoxy-14-hydroxy-3-methoxy-17-methylmorphinan-6-one, or oxycodone (OXY), 3-

[(2S)-1-methylpyrrolidin-2-yl]pyridine, or nicotine (NIC), methyl (1R,2R,3S,5S)-3-

(benzoyloxy)-8-methyl-8-azabicyclo[3.2.1] octane-2-carboxylate, or cocaine (COC),

[5α,7α(S)]-17-(Cyclopropylmethyl)-α-(1,1-dimethylethyl)4,5-epoxy-18,19-dihydo-3- hydroxy-6-methoxy-α-methyl-6,14-ethenomorphinan-7-methanol hydrochloride, or buprenorphine (BUP), 5-methoxy-diisopropyltryptamine (5-MeO-DiPT); 3-[2-

(Diisopropylamino)ethyl]-5-methoxyindole, or foxymethoxy (FOXY), 6-Dimethylamino-

4,4-diphenylheptan-3-one hydrochloride, or methadone (MD), (±)-3,4-

Methylenedioxymethamphetamine hydrochloride or ecstasy; XTC hydrochloride, or

(MDMA), 7-Chloro-1-methyl-5-phenyl-3H-1,4-benzodiazepin-2(1H)-one, Ro 5-2807, or diazepam (DZP), (5α,6α)-7, 8-Didehydro-4,5-epoxy-3-methoxy-17-methylmorphinan-6- ol; 3-methylmorphine; morphine monomethyl ether, or codeine, (5α,6α)-7,8-didehydro-

4,5-epoxy-17-methylmorphinan-3,6-diol, or morphine, 1-Methyl-4-phenylpiperidine-4- carboxylic acid ethyl ester hydrochloride; pethidine hydrochloride, or meperidine hydrochloride, 6-desoxycodeine, naloxone, (5α,7α)-17-(Cyclopropylmethyl)- 4,5-epoxy-

18,19-dihydro- 3-hydroxy- 6-methoxy- α,α-dimethyl- 6,14-ethenomorphinan- 7- methanol, or diprenorphine (DIP), N-phenyl butyl normeperidine, 6-desoxymorphine, and normeperidine. BUP, RIF, MD, diprenorphine (DIP), PK11195, sulforaphane (SFN), and phenobarbital (PB) were purchased from Sigma-Aldrich (St. Louis, MO). 6-(4-

Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl) oxime

(CITCO) was obtained from Enzo Life Sciences Research Laboratories (Plymouth

Meeting, PA). Morphine, codeine, and naloxone were supplied from Mallinckrodt, Inc.

(St. Louis, MO). Meperidine, 6-desoxycodeine, N-phenyl butyl normeperidine, 6- desoxymorphine, and normeperidine were kindly provided by Andrew Coop (University of Maryland School of Pharmacy, Baltimore, MD).

Additionally, the six anticancer agents were 2-Phenyl-4H-1-benzopyran-4-one, 2-

Phenylchromone, or flavone (FLV), 2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-4H-1- benzopyran-4-one dihydrate, 3,3′,4′,5,7-Pentahydroxyflavone dihydrate, or quercitin dihydrate (QUE), 3-Hydroxymethylindole; 3-Indolyl- carbinol; 3-Indomethanol, or indole-3-carbinol (I3C), 4′,5,7-Trihydroxyisoflavone, 5,7-Dihydroxy-3-(4- hydroxyphenyl)-4H-1-benzopyran-4-one, or genistein (GEN), 4-Methyl-5-pyrazinyl-3H-

1,2-dithiole-3-thione, or oltipraz (OLT), and methyl (1H-indol-3-ylmethylamino) methanedithioate; methyl N-(1H-indol-3-ylmethyl)-carbamodithioate, or brassinin

(BRS). Anticancer agents were purchased from either Sigma Aldrich or LKT Sciences.

The dual-luciferase reporter assay system was purchased through Promega (Madison,

WI). Matrigel, insulin, and insulin/transferrin/selenium were obtained from BD

Biosciences Discovery Labware (Bedford, MA). Other cell culture reagents were purchased from Invitrogen (Carlsbad, CA) or Sigma-Aldrich.

Plasmid Constructs. The pSG5-hPXR expression vector was obtained from Steven

Kliewer (University of Texas Southwestern Medical Center, Dallas, TX). The CYP2B6 reporter construct, containing both phenobarbital-responsive enhancer module and the distal XREM (CYP2B6–2.2kb), were generated as described previously.7 The CYP3A4-

PXRE/XREM reporter vector was obtained from Bryan Goodwin (GlaxoSmithKline,

Research Triangle Park, NC). The pRL-TK Renilla reniformis luciferase plasmid used to normalize firefly luciferase activities was obtained from Promega.

Transient Transfection in HepG2 Cells and Human Primary Hepatocytes. HepG2 cells in 24-well plates were transfected with CYP3A4-PXRE/XREM or CYP2B6–2.2kb reporter construct in the presence of an hPXR, hCAR, hCAR3, or hCAR1+A expression vector using a FuGENE 6 transfection kit according to the manufacturer's instructions

(Roche Applied Science, Indianapolis, IN). Twenty-four hours after transfection, cells were treated for another 24 h with solvent (0.1% DMSO) or test compounds RIF (10

μM), CITCO (1 μM), morphine (10 μM), MD (10 μM), oxycodone (10 μM), codeine (10

μM), meperidine (10 μM), BUP (1, 5, 10, 25, 50, and 100 μM), 6-desoxycodeine (10

μM), naloxone (10 μM), DIP (10 μM), N-phenyl butyl normeperidine (10 μM), 6- desoxymorphine (10 μM), or normeperidine (10 μM). Cell lysates were subsequently assayed for firefly activities normalized against the activities of cotransfected R. reniformis luciferase using the Dual-Luciferase Kit (Promega). Data were represented as mean ± S.D. of three individual transfections. To analyze PXR- and CAR-mediated induction of CYP3A4 in hepatoma cells, HepG2 cells seeded in 12-well plates were transfected with a hCAR or hPXR expression vector; 24 h after transfection, cells were treated for another 24 h with RIF (10 μM), CITCO (1 μM), PK11195 (10 μM), SFN (10

μM), or BUP (10 and 50 μM) or were cotreated for 24 h with SFN plus RIF or BUP, and

PK11195 plus CITCO or BUP, respectively. Total RNA was isolated and subjected to real-time RT-PCR analysis as described above. In separate experiments, HPHs seeded in

24-well BioCoat plates were transfected with CYP3A4-PXRE/XREM or CYP2B6–2.2kb construct in the presence of the pGL-TK vector using Effectene reagent (QIAGEN) as

described previously.8 Transfected HPHs were treated with DMSO [0.1% (v/v)], RIF (10

μM), CITCO (1 μM), BUP (10 and 50 μM), or DIP (10 μM) for 24 h, respectively. Cell lysates were subjected to dual-luciferase analysis as described above.

Statistical Analysis. All data represent at least three independent measurements and are expressed as the mean ± S.D. Statistical comparisons were made using either one-way analysis of variance followed by a post hoc Dunnett's test or Student's t test where appropriate. Statistical significance was set at P < 0.05, and assessed at 3 levels (***, p ≤

0.001; **, p ≤ 0.01, and *, p ≤ 0.05).

2.3 Results

Effects of Drugs of Abuse on the Activation of hPXR in HepG2 Cells. To assess whether selected drugs of abuse could have an effect on the activation of xenobiotic receptor hPXR, all eighteen drugs of abuse, including twelve popular clinically prescribed opioids, were screened in HepG2 cells transfected with an hPXR expression vector in the presence of the CYP3A4-PXRE/XREM luciferase reporter construct. The test compounds were MOD, OXY, NIC, COC, BUP, FOXY, MD, MDMA, DZP, CDN, morphine, meperidine, 6-desoxycodeine, naloxone, DIP, N-phenyl butyl normeperidine,

6-desoxymorphine, and normeperidine. Natural product biloba (BIL) was also included.

All compounds were tested at 10 µM concentration; RIF (10 µM) and DMSO (0.1%, v/v) were the positive and vehicle controls, respectively. Figures 2.5-2.7 demonstrate the influence of drugs of abuse and opioids on CYP3A4 (Figs. 2.5-2.6) reporter activities, and figure 2.7 demonstrates the influence of opioids on CYP2B6 reporter activities (Fig

2.7), in comparison with vehicle control as a percentage of the increase achieved by the

hPXR positive control RIF (10 µM). Opioids methadone (MD), oxycodone (OXY), codeine (CDN), and buprenorphine (BUP) appear in all figures (Fig 2.5-2.7). Notably, several compounds achieved 40% of RIF induction, including COC, BUP, FOXY, MD,

DZP, and DIP; whereas the remaining drugs of abuse were classified as non-activators, due to lack of major differences between compound-mediated induction levels as compared to vehicle. It is also worth noting that BUP and DIP, which share high structural similarity, both display strong activation of hPXR, reaching approximately

80% and 70% of RIF response, respectively (Fig. 2.6-2.7), whereas all of the other opioids except for MD exhibited negligible PXR activation (Fig. 2.6-2.7).

7 hPXR-3A4

6 ***

4 *** 3 *** *** 2 ** ** 1 * Fold To Control To Fold 0

Figure 2.5. Activation of hPXR by different drugs of abuse in cell-based reporter assays.

HepG2 cells were transfected with an hPXR expression vector in the presence of

CYP3A4-PXRE/XREM reporter construct as described under Materials and Methods.

Transfected cells were then treated with indicated compounds at 10 μM for 24 h. RIF (10

μM) was used as a positive control for hPXR activation. Relative luciferase (luc) activities were determined and expressed as fold to vehicle control (0.1% DMSO). All data are expressed as mean ± S.D. (n = 3). *, P < 0.05; **, P < 0.01; ***, P <0.001.

hPXR-3A4

** ** **

** Relative Luc Activity Relative

Figure 2.6. Activation of hPXR by different opioids in cell-based reporter assays. HepG2 cells were transfected with an hPXR expression vector in the presence of CYP3A4-

PXRE/XREM reporter construct as described under Materials and Methods. Transfected cells were then treated with different opioids (morphine, MD, oxycodone, codeine, meperidine, BUP, 6-desoxycodeine, naloxone, DIP, N-phenyl butyl normeperidine, 6- desoxy-morphine, or normeperidine) at 10 μM for 24 h. RIF (10 μM) was used as a positive control for hPXR activation. Luciferase activities were determined and expressed as a percentage of RIF activation. All data are expressed as mean ± S.D. (n = 3). *, P <

0.05; **, P < 0.01; ***, P <0.001.

hPXR-2B6 (2.2kb)

** ** **

** Relative Luc Activity Relative

Figure 2.7. Activation of hPXR by different opioids in cell-based reporter assays. HepG2 cells were transfected with an hPXR expression vector in the presence of CYP2B6-2.2kb reporter construct as described under Materials and Methods. Transfected cells were then treated with different opioids (morphine, MD, oxycodone, codeine, meperidine, BUP, 6- desoxycodeine, naloxone, DIP, N-phenyl butyl normeperidine, 6-desoxy-morphine, or normeperidine) at 10 μM for 24 h. RIF (10 μM) was used as a positive control for hPXR activation. Luciferase activities were determined and expressed as a percentage of RIF activation. All data are expressed as mean ± S.D. (n = 3). *, P < 0.05; **, P < 0.01; ***, P

<0.001.

Effects of Anticancer Agents on the Activation of hPXR in HepG2 Cells. To assess whether the anticancer agents we selected could have an effect on the activation of xenobiotic receptor hPXR, we screened all six anticancer agents in HepG2 cells transfected with an hPXR expression vector in the presence of the CYP3A4-

PXRE/XREM luciferase reporter construct. The test anticancer agents were FLV, QUE,

I3C, GEN, OLT and BRS. All compounds were tested at 10 µM concentration; RIF (10

µM) and DMSO (0.1%, v/v) were the positive and vehicle controls, respectively. Figure 7 demonstrates the influence of anticancer agents on CYP3A4 (Fig. 7) reporter activities in comparison with vehicle control as a percentage of the increase achieved by the hPXR positive control RIF (10 µM). The criteria adopted from previous publications 9 classify compounds that achieve more than 40% of RIF-normalized hPXR activation in reporter assays as strong activators of hPXR, and those exhibiting between 15 and 40% of RIF response as moderate activators, respectively. Compounds exhibiting less than 15% of

RIF response are recognized as non-activators. Notably, FLV rivaled RIF induction, achieving 7.1-fold induction, which is approximately 90% of that reached by the RIF positive control. The remaining five anticancer agents were classified as non-activators, due to lack of major discernible differences between compound-mediated and vehicle- treated induction levels.

Concentration-Related Activation of hPXR in HepG2 Cells. For selected compounds which reached approximately 40% of RIF control, subsequent initial concentration-related hPXR reporter experiments were performed in HepG2 cells over a broader concentration range. Concentration-related responses for compound-induced

PXR activation of CYP3A4 reporter activities for FLV (Fig. 2.9), COC (Fig. 2.10 A),

10 hPXR-3A4 9 **

*** 8 7 6 5 4 3 Fold To Control To Fold 2 * 1 0

Figure 2.8. Activation of hPXR by different anticancer agents in cell-based reporter assays. HepG2 cells were transfected with an hPXR expression vector in the presence of

CYP3A4-PXRE/XREM reporter construct as described under Materials and Methods.

Transfected cells were then treated with indicated compounds at 10 μM for 24 h. RIF (10

μM) was used as a positive control for hPXR activation. Luciferase activities were determined and expressed as fold to vehicle control (0.1% DMSO). All data are expressed as mean ± S.D. (n = 3). *, P < 0.05; **, P < 0.01; ***, P <0.001.

DZP (Fig. 2.10 B), FOXY (Fig. 2.10 C), BUP (Fig. 2.11 A), and MD (Fig. 2.11 B), are shown in figures 2.9-2.11 (Figs. 2.9-2.11).

In total, six of the twenty-five compounds screened were classified as hPXR activators, namely FLV, COC, DZP, FOXY, MD, and BUP. Interestingly, only one anticancer agent potently induced hPXR-mediated reporter activities; meanwhile several opioids or drugs of abuse achieved potent hPXR activation.

2.4 Discussion

Metabolism induction-related DDIs are one of the critical concerns in the overall safety profiles of clinical medication and drug development today; and transcriptional up- regulation of drug-metabolizing genes by activation of NRs represents the principle mechanism by which induction-related DDIs occur.10 Due to their ability to transactivate the expression of multiple DMEs and transporters, as well as the promiscuous selectivity of ligands and activators, both PXR and CAR have been widely accepted as xenobiotic sensors that can mediate the major inductive responses in hepatic metabolism and transport.11 Cell-based PXR reporter assays in particular have been extensively used in the prediction of xenobiotic-mediated induction of PXR target genes such as CYP3A4 and CYP2B6.9b, 12 Therefore, we sought to investigate the potential for several different drugs of abuse and anticancer agents to activate one of these major xenosensors, PXR, by performing some screening in cell-based reporter assays. Our current results revealed that several test compounds that were screened may be activators of hPXR in vitro. In our cell-based reporter assay systems, six compounds were able to transactivate hPXR-3A4 to an extent rivaling the threshold of 40% of the RIF positive control: FLV, COC, FOXY,

7 *** hPXR-3A4

6 5 *** 4 *** 3 *** 2 **

1 Fold To Control To Fold 0

Figure 2.9. Concentration-related activation of hPXR by anticancer agent in cell-based reporter assays. HepG2 cells were transfected as described previously under Materials and Methods, then treated with FLV at increasing concentrations for 24 h. RIF (10 μM) was used as a positive control for hPXR activation. Luciferase activities were determined and expressed relative to vehicle control (0.1% DMSO). All data are expressed as mean ±

S.D. (n = 3). *, P < 0.05; **, P < 0.01; ***, P <0.001.

6 hPXR-3A4 A ** *** 4 ** ** 2 *** *

0 Fold To Control To Fold

8 B hPXR-3A4 6 *** 4 ** ** ** *** ** 2 **

0 Fold To Control To Fold

6 C *** hPXR-3A4 4 *** *** * ***

2 **

0 Fold To Control To Fold

Figure 2.10. Concentration-related activation of hPXR by drugs of abuse in cell-based reporter assays. HepG2 cells were transfected as described previously under Materials and Methods, then treated with COC (A), DZP (B), and FOXY (C) at increasing concentrations for 24 h. RIF (10 μM) was used as a positive control. Luciferase activities were determined and expressed as fold to vehicle control (0.1% DMSO). All data are expressed as mean ± S.D. (n = 3). *, P < 0.05; **, P < 0.01; ***, P <0.001.

hPXR-3A4 6

5 *** *** *** ** *** 4 * 3 2 ***

Fold To Control To Fold 1 0

6 hPXR-3A4 5

*** 4 *** ** ** 3 ** ** 2 **

1 Fold To Control To Fold 0

Figure 2.11. Concentration-related activation of hPXR by opioids in cell-based reporter assays. HepG2 cells were transfected as described previously under Materials and

Methods, then treated with BUP (A) or MD (B) at increasing concentrations for 24 h. RIF

(10 μM) was used as a positive control for hPXR activation. Luciferase activities were determined and expressed relative to vehicle control (0.1% DMSO). All data are expressed as mean ± S.D. (n = 3). *, P < 0.05; **, P < 0.01; ***, P <0.001.

DZP, MD, and BUP. Therefore, these compounds became candidates for further concentration-related hPXR assays and for subsequent experimental investigation in a more sophisticated system of human primary hepatocyte cultures.

Sandwich cultures of human primary hepatocytes have been used extensively for evaluating the ability of drugs to induce the expression and activity of DMEs and transporters in humans.13 Enzyme induction in human primary hepatocytes possesses the distinct advantage of better mimicking physiological in vivo conditions and exhibiting species-specific induction patterns than that which is possible using a cell-based reporter assay system. Because significant drawbacks associated with the PXR reporter assay can affect the proper interpretation of data obtained from this broadly used in vitro system, it is important not to over-interpret the screening data. Rather, the compounds identified as hPXR activators should next undergo further testing in human primary hepatocytes, which are known as the “gold standard” since they are the more physiologically relevant system. Lastly, the current in vitro observations warrant further investigations overall, which would ultimately better characterize how activators such as FLV, COC, FOXY,

DXP, MD, and BUP, can mediate changes in the metabolic and pharmacokinetic profiles of other drugs.

2.5 References

1. Li, L.; Stanton, J.; Tolson, A.; Luo, Y.; Wang, H., Bioactive Terpenoids and

Flavonoids from Ginkgo Biloba Extract Induce the Expression of Hepatic Drug-

Metabolizing Enzymes Through Pregnane X Receptor, Constitutive Androstane

Receptor, and Aryl hydrocarbon Receptor-Mediated Pathways. Pharmaceutical

Research 2009, 26 (4), 872-882.

2. Ihl, R., Gingko biloba extract EGb 761®: clinical data in dementia. International

Psychogeriatrics 2012, 24 (SupplementS1), S35-S40.

3. Russo, M.; Spagnuolo, C.; Tedesco, I.; Bilotto, S.; Russo, G. L., The flavonoid

quercetin in disease prevention and therapy: Facts and fancies. Biochemical

Pharmacology 2012, 83 (1), 6-15.

4. Brandi, G.; Paiardini, M.; Cervasi, B.; Fiorucci, C.; Filippone, P.; De Marco, C.;

Zaffaroni, N.; Magnani, M., A New Indole-3-Carbinol Tetrameric Derivative

Inhibits Cyclin-dependent Kinase 6 Expression, and Induces G1 Cell Cycle Arrest

in Both Estrogen-dependent and Estrogen-independent Breast Cancer Cell Lines.

Cancer Research 2003, 63 (14), 4028-4036.

5. Yang, Z.; Zhu, W.; Gao, S.; Yin, T.; Jiang, W.; Hu, M., Breast Cancer Resistance

Protein (ABCG2) Determines Distribution of Genistein Phase II Metabolites:

Reevaluation of the Roles of ABCG2 in the Disposition of Genistein. Drug

Metabolism and Disposition 2012.

6. Banerjee, S.; Li, Y.; Wang, Z.; Sarkar, F. H., Multi-targeted therapy of cancer by

genistein. Cancer Letters 2008, 269 (2), 226-242.

7. (a) Wang, H.; Faucette, S.; Sueyoshi, T.; Moore, R.; Ferguson, S.; Negishi, M.;

LeCluyse, E. L., A Novel Distal Enhancer Module Regulated by Pregnane X

Receptor/Constitutive Androstane Receptor Is Essential for the Maximal

Induction of CYP2B6 Gene Expression. Journal of Biological Chemistry 2003,

278 (16), 14146-14152; (b) Chen, T.; Tompkins, L. M.; Li, L.; Li, H.; Kim, G.;

Zheng, Y.; Wang, H., A Single Amino Acid Controls the Functional Switch of

Human Constitutive Androstane Receptor (CAR) 1 to the Xenobiotic-Sensitive

Splicing Variant CAR3. Journal of Pharmacology and Experimental

Therapeutics 2010, 332 (1), 106-115.

8. Wang, H.; Faucette, S.; Moore, R.; Sueyoshi, T.; Negishi, M.; LeCluyse, E.,

Human Constitutive Androstane Receptor Mediates Induction of CYP2B6 Gene

Expression by Phenytoin. Journal of Biological Chemistry 2004, 279 (28), 29295-

29301.

9. (a) Faucette, S. R.; Zhang, T.-C.; Moore, R.; Sueyoshi, T.; Omiecinski, C. J.;

LeCluyse, E. L.; Negishi, M.; Wang, H., Relative Activation of Human Pregnane

X Receptor versus Constitutive Androstane Receptor Defines Distinct Classes of

CYP2B6 and CYP3A4 Inducers. Journal of Pharmacology and Experimental

Therapeutics 2007, 320 (1), 72-80; (b) Chu, V.; Einolf, H. J.; Evers, R.; Kumar,

G.; Moore, D.; Ripp, S.; Silva, J.; Sinha, V.; Sinz, M.; Skerjanec, A., In Vitro and

in Vivo Induction of Cytochrome P450: A Survey of the Current Practices and

Recommendations: A Pharmaceutical Research and Manufacturers of America

Perspective. Drug Metabolism and Disposition 2009, 37 (7), 1339-1354; (c) Sinz,

M.; Kim, S.; Zhu, Z.; Chen, T.; Anthony, M.; Dickinson, K.; Rodrigues, A. D.,

Evaluation of 170 Xenobiotics as Transactivators of Human Pregnane X Receptor

(hPXR) and Correlation to Known CYP3A4 Drug Interactions Current Drug

Metabolism 2006, 7 (4), 375-388.

10. Li, L.; Hassan, H. E.; Tolson, A. H.; Ferguson, S. S.; Eddington, N. D.; Wang, H.,

Differential Activation of Pregnane X Receptor and Constitutive Androstane

Receptor by Buprenorphine in Primary Human Hepatocytes and HepG2 Cells.

Journal of Pharmacology and Experimental Therapeutics 2010, 335 (3), 562-571.

11. (a) Honkakoski, P.; Sueyoshi, T.; Negishi, M., Drug-activated nuclear receptors

CAR and PXR. Annals of Medicine 2003, 35 (3), 172-182; (b) Willson, T. M.;

Kliewer, S. A., Pxr, car and drug metabolism. Nat Rev Drug Discov 2002, 1 (4),

259-266.

12. Kliewer, S. A.; Goodwin, B.; Willson, T. M., The Nuclear Pregnane X Receptor:

A Key Regulator of Xenobiotic Metabolism. Endocr Rev 2002, 23 (5), 687-702.

13. LeCluyse, E. L., Human hepatocyte culture systems for the in vitro evaluation of

cytochrome P450 expression and regulation. European Journal of

Pharmaceutical Sciences 2001, 13 (4), 343-368.

Chapter 3

Induction of Hepatic Drug-Metabolizing Enzymes by Selected

Drugs of Abuse in Human Primary Hepatocytes

3.1 Introduction

Pharmaceutical scientists regularly utilize several immortalized cell lines such as

HepG2 cells, or liver-derived model systems, in order to investigate drug disposition in vitro. However, due to both the lack of phenotypic gene expression in almost all immortalized cell lines and other limitations associated with the use of liver slices, cultures of human primary hepatocytes (HPHs) have become recognized as the “gold standard” for the in vitro testing of drugs.1 Primary cultures of human hepatocytes, which have been utilized for toxicological and pharmacological studies for decades, are particularly useful for studying drug metabolism and the induction of liver CYP enzymes.

Primary hepatocytes appear as the closest model for the liver in vivo. Moreover, in vitro hepatocyte models represent very useful systems in both fundamental research and various application areas.2 Especially as our understanding of the species-specific differences in the regulation and substrate specificity of the mammalian CYP enzymes has broadened over the last several decades, the need for an in vitro hepatic model system that is specific to humans is evident.

This can best be exemplified by the CYP3A4 isozyme, which represents the basis for a number of common DDIs. Many xenobiotic examined for their effects on CYP3A expression reveal marked species-specific differences.3 Pregnenolone 16α-carbonitrile and dexamethasone are known efficacious CYP3A inducers in rodents, but not in humans; conversely, rifampicin is a known efficacious inducer of CYP3A in humans but not rodents. Therefore, when investigating CYP induction, especially for the purposes of predicting the potential for drug interactions in humans, it is advantageous to use the more physiologically relevant system, i.e. human primary hepatocyte cultures.

Primary cultures of hepatocytes provide a sensitive method for evaluating the ability of drugs to induce microsomal CYP450 enzymes in humans. The response of human hepatocyte cultures to treatment with various compounds (both positive and negative modulators) reflects that observed in vivo both in regards to enzyme specificity and potency of enzyme induction. Due to species differences in liver response, substrate specificity, and inducibility, the use of human hepatocytes in culture remains arguably the best system making it possible to predict enzyme induction in humans.4

3.2 Materials and Methods

Chemicals and Biological Reagents. BUP, RIF, MD, DZP, Methamphetamine

(Meth), 5-methoxy-diisopropyltryptamine (5-MeO-DiPT), FOXY, 2-Phenyl-4H-1- benzopyran-4-one, 2-Phenylchromone, FLAV, and phenobarbital (PB) were purchased from Sigma-Aldrich (St. Louis, MO). 6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5- carbaldehyde-O-(3,4-dichlorobenzyl) oxime (CITCO) was obtained from Enzo Life

Sciences Research Laboratories (Plymouth Meeting, PA). Oligonucleotide primers were synthesized by either Sigma Genosys (The Woodlands, TX) or by Integrated DNA

Technologies, Inc. (Coralville, IA); and TaqMan fluorescent probes were synthesized by

Applied Biosystems (Foster, CA). CYP2B6 and CYP3A4 antibodies were obtained from

Millipore Corporation (Billerica, MA). β-Actin antibody was obtained from Sigma-

Aldrich. Matrigel, insulin, and insulin/transferrin/selenium were obtained from BD

Biosciences Discovery Labware (Bedford, MA). Horseradish peroxidase-labeled antirabbit antibody was purchased from Amersham Biosciences (Pittsburgh, PA). HPLC-

61 grade acetonitrile was purchased from Thermo Fisher Scientific (Waltham, MA). Other cell culture reagents were purchased from Invitrogen or Sigma-Aldrich.

Induction Studies in Human Primary Hepatocyte Cultures. Human liver tissues were obtained following surgical resection by qualified pathology staff after diagnostic criteria were met and prior approval was obtained from the Institutional Review Board at the University of Maryland at Baltimore. Hepatocytes were isolated by a modification of the two-step collagenase digestion method as described previously.5 Hepatocytes were seeded at 1.5 × 10^6 cells/well in six-well BioCoat plates in Dulbecco's modified Eagle's medium supplemented with 5% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 4

μg/ml insulin, and 1 μM dexamethasone. After attachment at 37°C in a humidified atmosphere of 5% CO2, cells were overlaid with Matrigel (0.25 mg/ml) in Williams' E medium supplemented with insulin, transferrin, and selenium; 0.1 μM dexamethasone;

100 U/ml penicillin; and 100 μg/ml streptomycin. Hepatocytes were maintained for 36 h before treatment with RIF (10 μM), CITCO (1 μM), PB (1 mM), BUP (10 and 50 μM),

MD (10, 25, and 50 μM ), or DIP (10 μM for another 24 h or 72 h for detection of mRNA and protein expression, respectively.

Real-Time PCR Analysis. Total RNA was isolated from treated hepatocytes using the RNeasy Mini Kit (QIAGEN, Valencia, CA) and reverse-transcribed using the High-

Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) following the manufacturers' instructions. Primers and probes for CYP2B6, CYP3A4, UGT1A1, and

MDR1 genes (Table 3.1) were designed using Primer Express Version 2.0; and entered into the NCBI Blast to ensure specificity as described previously.6 For MD-treated HPHs, the mRNA expression of CYP2B6, CYP3A4, UGT1A1, and MDR1, was normalized

62 against that of human β-actin, which was detected using a pre developed primer/probe mixture (Applied Biosystems, Foster, CA), and TaqMan PCR assays were performed in

96-well optical plates on an ABI Prism 7000 Sequence Detection System (Applied

Biosystems). For BUP-treated HPHs, CYP2B6 and CYP3A4 mRNA expression was normalized against that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and real-time PCR assays were performed in 96-well optical plates on an ABI Prism 7000 sequence detection system with SYBR Green PCR Master Mix (Applied Biosystems).

Induction values (fold over control) were calculated according to the 2ΔΔCt method, where ΔCt represents the differences in cycle threshold numbers between the target gene and housekeeping gene; and ΔΔCt represents the relative change in these differences between the control and treatment groups.

Western Immunoblot Analysis. Whole-cell homogenate proteins (either 40 or 20 μg each) from treated HPHs were separated on either a NuPAGE® 4-12 % Bis-Tris Gel

(Invitrogen, Carlsbad, CA) and transferred onto PVDF Transfer Membrane (Pierce,

Rockford, IL), or resolved on SDS-polyacrylamide gels and electrophoretically transferred onto Immobilon-P polyvinylidene difluoride membranes (Millipore

Corporation). Subsequently, membranes were incubated with specific antibodies against

CYP2B6 or CYP3A4 (Millipore Chemicon, CA) diluted 1:4000 and 1:5000, respectively.

β-actin (Sigma-Aldrich. St. Louis, MO) was used as internal control. Blots were washed and incubated with horseradish peroxidase goat antirabbit IgG antibody diluted 1:4000.

Films were developed using ECL Western blotting detection reagent (GE Healthcare,

Chalfont St. Giles, UK).

Metabolic Stability Studies. Metabolism studies were conducted in cryopreserved hepatocytes and HepG2 cells. Cell suspensions consisting of 0.5 × 106 viable cells per incubation were diluted in serum-free Dulbecco's modified Eagle's medium supplemented with ITS+ (BD Biosciences), 0.1 μM dexamethasone, and penicillin-streptomycin. Cells were incubated in triplicate under standard culture conditions with BUP and DIP at the final concentration of 5 μM. Time points used were 0, 30, 60, 90, 120, 150, and 180 min for BUP, and 0, 60, 120, and 180 min for DIP, respectively. The reactions were terminated using acetonitrile.

HPLC Quantitation of Buprenorphine and Diprenorphine. BUP and DIP samples were analyzed according to a validated HPLC method.7 The assay was performed using a mobile phase of 5 mM sodium acetate buffer, pH 3.75, in acetonitrile [2:8 (v/v)] and a

Waters 474 fluorescence detector (excitation at 210 nm, emission at 352 nm; Waters,

Milford, MA). A flow rate of 1.2 ml/min at 25°C was used, and the injection volume was

150 μl. BUP and DIP were eluted at 3.83 and 4.57 min, respectively. The assay was linear (r2 ≥ 0.987) over the tested concentrations (0.05–4 μg/ml). The chromatographic

HPLC system was composed of 1) a Waters 1525 Binary HPLC pump (Waters), 2) a 717

Waters autosampler, 3) a 3390A Hewlett Packard integrator/plotter (Hewlett Packard,

Avondale, PA), and 4) Waters Symmetry C18 (4.6 × 250 mm) column (Waters).

Accuracy and precision were determined by replicate injection of quality control samples.

Both precision and accuracy were of satisfactory results below 11% CV.

Statistical Analysis. All data represent three independent measurements and are expressed as the mean ± S.D. Statistical comparisons were made using one-way analysis of variance followed by a post hoc Dunnett's test and Student's t test where appropriate.

Statistical significance was set at P < 0.05., and statistical significance was assessed at 3 levels (***, p ≤ 0.001; **, p ≤ 0.01, and *, p ≤ 0.05).

3.3 Results

Induction of CYP2B6 and CYP3AA4 After Treatment with Drugs of Abuse, and

FLAV (Anticancer Agent). To assess whether or not FOXY, FLAV, DZP, MD and

METH (methamphetamine) could achieve induction of CYP2B6 and CYP3A4 target genes, two shared common target genes of PXR and CAR in HPHs, cultures were treated with the aforementioned compounds at the indicated concentrations (Figs. 3.1-3.2). Total

RNA was collected, reverse-transcribed, and subjected to real-time RT-PCR for detecting the CYP2B6 (A) and CYP3A4 (B) expression levels as outlined under Materials and

Methods. For FOXY and DZP (Fig. 3.1 A), over the concentration range investigated, a

“U”-shaped pattern was observed for CYP2B6; meanwhile the opposite pattern, a bell- shaped curve, was observed for CYP2B6 for the anticancer agent FLAV (Fig. 3.1 A). For

CYP3A4 (Fig. 3.1 B), none of the compounds appeared to mimic similar patterns to those which they displayed for CYP2B6, rather the compounds illustrated alternative profiles.

FOXY displayed obvious increases over the concentration range from 10-50 µM, achieving well beyond the threshold of 40% RIF induction at its peak of 50 µM. FLV demonstrated virtually no induction at all. Rather, at the highest concentration examined,

50 µM, the severity of the decrease in fold induction instead might suggest a possible sign that repression could occur, although the avenue of repression was not pursued in any investigations during these studies.

For DZP, due to the limited amount of human primary hepatocytes for this culture, two concentrations were investigated instead of the three concentrations investigated for CYP2B6. Based on the CYPP3A4 fold induction achieved at 10 and 25

M, it appears that DZP might display a profile similar to that which it displayed for

CYP2B6, but to a less potent extent. However, since the 50 µM concentration was unable to be assessed, a direct comparison of the extent of CYP2B6 induction to the extent of

CYP3A4 induction over the entire 10-50 µM concentration range cannot be made for

DZP. For both METH and MD (Fig. 3.2), two concentrations were also assessed. For

METH, moderate-to-potent induction was observed for CYP2B6, whereas weak-to- moderate induction was observed for CYP3A4. However, most notably of all the drugs of abuse examined, methadone not only displayed a potent and concentration-related increase in CYP2B6 induction from 10-25 µM, but methadone also achieved induction which was more potent than the RIF positive control at the highest concentration tested,

50 µM (Fig. 3.2). For CYP3A4, methadone displayed a similar profile of induction whereby induction increased with increasing concentration, however the induction pattern appeared to occur only moderately, to a much lesser extent than the CYP2B6 induction profile.

Influence of Methadone on mRNA Expression for DMEs and Drug Transporter in

Human Primary Hepatocytes. In the current study, the effects of methadone on the expression of CYP2B6, CYP3A4, UGT1A1, and MDR1 genes were evaluated in HPH cultures using real-time reverse transcription-PCR analysis. In HPHs prepared from two donors (HL3, HL6), the mRNA expression of CYP2B6, CYP3A4, UGT1A1, and MDR1 was increased significantly after treatment with methadone at 10, 25, and 50 μM (Figs.

Table 3.1 Primer and probe sequences for real-time PCR assays.

Gene Sequence Reference

CYP2B6 Forward primer 5-AAGCGGATTTGTCTTGGTGAA-3 (Faucette et al. 2007) Reverse primer 5-TGGAGGATGGTGGTGAAGAAG-3 Probe 6-FAM-CATCGCCCGTGCGGAATTGTTC-TAMRA

CYP3A4 Forward primer 5-TCAGCCTGGTGCTCCTCTATCTAT-3 (Faucette et al. 2007) Reverse primer 5-AAGCCCTTATGGTAGGACAAAATATTT-3 Probe 6-FAM-TCCAGGGCCCACACCTCTGCCT-TAMRA

UGT1A1 Forward primer 5-GGCCCATCATGCCCAATAT-3 (Smith et al. 2005) Reverse primer 5-TTCAAATTCCTGGGATAGTGGATT-3 Probe 6-FAM-TTTTTGTTGGTGGAATCAACTGCCTTCAC-TAMRA

MDR1 Forward primer 5-GTCCCAGGAGCCCATCCT-3 (Maglich et al. 2002) Reverse primer 5-CCCGGCTGTTGTCTCCAT-3 Probe 6-FAM-TGACTGCAGCATTGCTGAGAACATTGC-TAMRA

A 20

18 * 16 ** 14 12 *** ** 10 8 6 *** ** 4 *** *

CYP 2B6 FoldInduction 2 0

50 * 40 30 *** 20 *** 10 * *** *** CYP 3A4 FoldInduction *** 0

Figure 3.1. Induction of CYP2B6 (A) or CYP3A4 (B) after treatment with FOXY, FLV, or DZP at indicated concentrations in human primary hepatocytes as described under

Materials and Methods. RIF (10 μM) was used as a positive control. All data are expressed as mean ± S.D. (n = 3). *** and ###, p ≤ 0.001; ** and ##, p ≤ 0.01; * and #, p

≤ 0.05.

90 ### 80 70 CYP2B6 60 CYP3A4 50 *** 40 *** 30 FoldInduction ** ## ## *** 20 ## 10 0

Figure 3.2. Induction of CYP2B6 or CYP3A4 after treatment with methadone (MD) or methamphetamine (METH) at indicated concentrations in human primary hepatocytes as described under Materials and Methods. RIF (10 μM) was used as a positive control. All data are expressed as mean ± S.D. (n = 3). *** and ###, p ≤ 0.001; ** and ##, p ≤ 0.01; * and #, p ≤ 0.05.

3.3-3.4). It is noteworthy that maximal induction of CYP2B6 at higher concentrations of methadone treatment again equals or exceeds that induced by the positive control RIF (10

μM).

Potent induction of CYP3A4 mRNA was also observed in HL3, where 6-, 25-, and 33-fold CYP3A4 mRNA increases resulted from 10, 25, and 50 μM methadone exposures, respectively. Compared with the highly inducible CYP2B6 and CYP3A4 genes, the induction of UGT1A1 and MDR1 was relatively moderate (Fig. 3.4, A and B).

As expected, the positive control RIF efficiently induced CYP2B6 and CYP3A4; and achieved moderate UGT1A1 and MDR1 induction in both hepatocyte preparations, despite obvious interindividual variations.

Induction of CYP2B6 and CYP3A4 Protein in Human Primary Hepatocytes Upon

Methadone Treatment. To assess whether methadone could induce CYP2B6 and

CYP3A4 expression at the protein level, whole-cell homogenate isolated from two HPH preparations (HL14, HL16) treated with different concentrations of MD was analyzed for

CYP2B6 and CYP3A4 protein content by Western blot analysis. As shown in Fig. 3.5,

MD robustly induced the protein expression of CYP3A4 in a dose-dependent manner, whereas the CYP2B6 protein was only induced at higher concentrations of methadone

(25 and 50 μM) to a relatively minor extent (Fig. 3.5, A and B). In both hepatocyte preparations, RIF displayed the greatest extent of CYP2B6 and CYP3A4 protein induction.

Influence of Buprenorphine on CYP3A4 and CYP2B6 Expression in Human

Primary Hepatocytes. Extensive literature and systematic reviews show that maintenance treatment for opioid addiction with either methadone or buprenorphine is associated with

50 HL #3 *** 40 HL #6 ***

30 FoldInduction 20 ### ***### *** ###

10 ### CYP 2B6 0

40 ### ***

35 HL #3 ** 30 HL #6 25 ***

FoldInduction

15 ## 10 ## ### *** CYP 3A4 5 ## 0

Fig 3.3. Induction of CYP2B6 (A) or CYP3A4 (B) after treatment with methadone (MD) at indicated concentrations in human primary hepatocytes as described under Materials and Methods. RIF (10 μM) was used as a positive control. All data are expressed as mean

± S.D. (n = 3). *** and ###, p ≤ 0.001; ** and ##, p ≤ 0.01; * and #, p ≤ 0.05. Adapted from Tolson AH, et.al., Drug Metabolism and Disposition, 2009.

A 8

7 *** HL #3 6 HL #6 5 4 # 3 *** *** 2 ***

UGT 1A1 FoldInduction 1 0

B 5 *** HL #3 4 HL #6

3 ** 2

1 MDR1 FoldInduction

Fig 3.4. Induction of UGT1A1 (A) or MDR1 (B) after treatment with methadone (MD) at indicated concentrations in human primary hepatocytes as described under Materials and

Methods. RIF (10 μM) was used as a positive control. All data are expressed as mean ±

S.D. (n = 3). *** and ###, p ≤ 0.001; ** and ##, p ≤ 0.01; * and #, p ≤ 0.05. Adapted from Tolson AH, et.al., Drug Metabolism and Disposition, 2009.

72 many benefits, such as retention in treatment, reduction in illicit opiate use, decreased craving, and improved social function.8 Accordingly, we also evaluated the effect of buprenorphine on the expression of CYP3A4 and CYP2B6 at both the mRNA and protein levels in HPHs.

Cultures of HPHs from four different donors were treated with CITCO, RIF, PB, and BUP as described under Materials and Methods. As expected, all prototypical

PXR/CAR activators exhibited potent induction of CYP2B6 and/or CYP3A4 at both the mRNA and protein levels (Fig. 3.6-3.7). Nevertheless, surprisingly the expression of

CYP2B6 and CYP3A4 was literally not increased after the treatment of buprenorphine at concentrations of 10 and 50 μM. These unexpected results indicate that buprenorphine may not be an efficient activator of either PXR or CAR in the more physiologically relevant cell system of HPHs as opposed to in derived immortal cell lines.

Metabolic Stability of Buprenorphine and Diprenorphine in HepG2 and Human

Hepatocytes. To investigate the surprising lack of induction observed in HPHs after buprenorphine treatment, the metabolic stability of buprenorphine in these two cell systems was investigated. HepG2 cells and suspension of cryopreserved human hepatocytes were used to compare the metabolic stability of buprenorphine as described under Materials and Methods. Because diprenorphine also demonstrated potent activation of PXR in HepG2 cells (Fig. 2.9, A and B), and has high structural similarity with buprenorphine (Fig. 3.8), the metabolic stability of diprenorphine was also investigated.

As demonstrated in Fig. 3.8 B, buprenorphine was quickly metabolized in HPHs with a half-life (t1/2) of approximately 2 h, whereas its concentration remains virtually unchanged up to the maximal incubation time (3 h) in HepG2 cells. It is noteworthy that

A HL #14 B HL #16

CYP3A4

CYP2B6

β-Actin

5 80 4 60 3 CYP3A4 40 2 1 20

0 0

CT RIF10 MD10 MD25 MD50 CT RIF10 MD10 MD25 MD50

2.5 2.0 2 1.5 1.5 1.0 CYP2B6 1 0.5 0.5 0.0 0 CT RIF10 MD10 MD25 MD50 CT RIF10 MD10 MD25 MD50

Fig 3.5. Effects of methadone (MD) treatment on the expression of CYP2B6 and

CYP3A4 immunoreactive proteins. Human primary hepatocytes from HL-14 (A) and

HL-16 (B) were treated for 72 h with vehicle control, RIF (10 μM), or MD (10, 25, and

50 μM). After harvesting, whole-cell homogenates (40 μg of each) were subjected to

CYP2B6 and CYP3A4 Western blot and densitometric analyses as described under

Materials and Methods. Adapted from Tolson AH, et.al., Drug Metabolism and

Disposition, 2009.

CYP 3A4 Fold 3A4 Induction CYP

6 Fold 6 Induction

B CYP 2 CYP

Fig 3.6. Induction of CYP3A4 (A) or CYP2B6 (B) after treatment with buprenorphine

(BUP). Human primary hepatocytes from HL-17, HL-18, and HL-23 were treated for 72 h with vehicle control, CITCO (1 μM), RIF (10 μM), PB (1 mM), or BUP (10, 25, and 50

μM). After harvesting, whole-cell homogenates (20 μg of each) were subjected to

CYP2B6 and CYP3A4 Western blot analyses as described under Materials and Methods.

Adapted from Li L, Hassan HE, Tolson AH, et. al., J Pharmacol Exp Ther, 2010.

BUP BUP A CTL CITCO RIF PB 10 µM 50 µM ~57kDa CYP3A4 ~56kDA HL #19 CYP2B6 ~42kDa β-Actin

B ~57kDa CYP3A4

~56kDa HL #21 CYP2B6 ~42kDa β-Actin

CYP3A4 ~57kDa

HL #23 CYP2B6 ~56kDa

β-Actin ~42kDa

Fig 3.7. Effects of buprenorphine (BUP) treatment on the expression of CYP3A4 and

CYP2B6 immunoreactive proteins. Human primary hepatocytes from HL-19 (A), HL-21

(B) and HL-23 (C) were treated for 72 h with vehicle control, CITCO (1 μM), RIF (10

μM), PB (1 mM), or BUP (10 or 50 μM). After harvesting, whole-cell homogenates (40

μg of each) were subjected to CYP2B6 and CYP3A4 Western blot analyses as described

under Materials and Methods. Adapted from Li L, Hassan HE, Tolson AH, et. al., J

Pharmacol Exp Ther, 2010.

76 diprenorphine also displayed quick metabolism in HPHs versus HepG2 cells, where the estimated t1/2 was approximately 50 min, and less than 10% diprenorphine was detected only in HPHs at the end of incubation (3 h) (Fig. 3.8 C). Subsequent experiments in

HPHs demonstrated that diprenorphine, similarly to buprenorphine, also failed to induce the expression of CYP2B6 and CYP3A4 (Fig. 3.9). Taken together, these dramatic differences in the metabolic stability of buprenorphine and diprenorphine in HepG2 versus HPHs may significantly contribute to the observed discrepancy in BUP- and DIP- mediated target gene induction, and in turn BUP- and DIP-mediated NR activation in these two cell systems.

3.4 Discussion

It is well known that CYP3A4 has been estimated to account on average for approximately 40% of the total CYP content in human liver. Additionally, regarding

CYP2B6, the importance of this isozyme in drug metabolism and detoxification has been firmly established through a plethora of studies within the past 10 years. Although it was regarded historically as a minor drug-metabolizing enzyme with insignificant roles in pharmacology and toxicology, increasing evidence has demonstrated that CYP2B6 is expressed in all human livers with variable amounts and at much higher levels than previously estimated (Tompkins and Wang, 2008). Further, major xenosensors PXR and

CAR exhibit a high degree of cross-talk, thereby sharing a growing spectrum of overlapping target genes such as CYP2B6 and CYP3A4.

Thus, we wanted to assess whether or not FOXY, FLAV, DZP, METH, MD and

BUP could achieve induction of CYP2B6 and CYP3A4 target genes. We also

B C

Fig 3.8. Metabolic stability of BUP and DIP in HepG2 cells and human primary hepatocytes. Chemical structures of BUP and DIP are depicted (A). The metabolic stability of BUP and DIP was determined in suspensions of HepG2 cells and cryopreserved hepatocytes via HPLC analysis as described under Materials and Methods.

The percentages of remaining BUP (B) and DIP (C) over time in HepG2 and HPHs are shown. All data represent the average of three measurements ± S.D. Adapted from Li L,

Hassan HE, Tolson AH, et. al., J Pharmacol Exp Ther, 2010.

25 HL-HL 023 #23 CYP3A4 CYP2B6

InductionFold FoldInduction

PB 1mM 0.1% DMSORIF 10uMCITCO 1uM DIP 10uM

B 25 HL-HL 024 #24 CYP3A4 20 CYP2B6

Fold Induction

FoldInduction

0 RIF 10uM PB 1mMDIP 10uM 0.1% DMSO CITCO 1uM

Fig 3.9. DIP fails to induce CYP2B6 and CYP3A4 expression in human primary hepatocytes. Human hepatocytes from HL-023 (A) and HL-024 (B) were treated for 24 h with CITCO (1 μM), RIF (10 μM), PB (1 mM), or DIP (10 μM). Real-time RT-PCR analysis of CYP2B6 and CYP3A4 expression was carried out as outlined under Materials and Methods. All data are expressed as mean ± S.D. (n = 3). **, P < 0.01. Adapted from

Li L, Hassan HE, Tolson AH, et. al., J Pharmacol Exp Ther, 2010.

79 investigated diprenorphine, due to its structural similarity to buprenorphine. Overall, multiple DME expression profiles were obtained, yet, most notably, the most potent

DME induction achieved was that reached by methadone, which acted as a potent

CYP2B6 inducer and moderate CYP3A4 inducer.

Methadone is widely prescribed for the management of heroin dependence and different types of chronic pain. Because of frequent co-administration with other therapeutics, DDIs involving methadone resulting from a polypharmacy approach to therapy are common, as are DDIs involving other opioids. Nevertheless, mounting evidence shows thus far that methadone-drug interactions have been characterized asymmetrically among existing literature, where focus has been placed heavily on describing how other drugs affect the metabolic or pharmacokinetic profiles of methadone. However, limited data exist regarding the potential for methadone to influence similar fates of co-administered drugs, and little to no mechanistic evidence has been provided. Although methadone is not a new medication, to our knowledge, the current study is the first to show that methadone can induce the expression of multiple hepatic DMEs. Enzyme induction in human primary hepatocytes possesses the distinct advantage of mimicking physiological in vivo conditions and exhibiting species-specific induction patterns. Using human primary hepatocytes as a model, our data revealed that the expression of CYP2B6 and CYP3A4 was potently and dose-dependently induced after treatment with methadone, whereas the expression of phase II enzyme UGT1A1 and efflux transporter MDR1 was only moderately induced.

Given that the clinical use or abuse of methadone is usually chronic, and prescribed dosages could range from approximately 30 to 180 mg/day up to as much as

1300 mg/day under certain circumstances,9 exposure levels could vary dramatically. The current in vitro studies have tested methadone over a concentration range of 10 to 50 μM.

Although it is difficult to quantitatively correlate in vitro data with in vivo conditions, our findings lead to the speculation that methadone may also induce CYP2B6 and CYP3A4 expression to a clinically significant level in vivo. Because the occurrence of HIV disease is common among injection drug users and opioid dependents that often require methadone maintenance therapy, DDIs occurring between methadone and anti-HIV agents have been investigated previously. However, the particular DDI between methadone and antiretroviral drugs has been commonly examined in a unilateral manner, where anti-HIV agents have been reported to affect methadone disposition,10 but little has been written about how methadone can affect antiretroviral drugs. A number of anti-HIV agents such as efavirenz and nevirapine are primarily metabolized in the liver by

CYP3A4 and CYP2B6.10b Thus, methadone induction of CYP2B6 and CYP3A4 may result in altered pharmacokinetics of such agents and may contribute to the frequently observed efavirenz adaptation in clinical settings. Likewise, methadone itself is metabolized predominantly by CYP2B6, CYP3A4, and CYP2D6, with CYP2B6 exhibiting the highest affinity and efficacy.11 Therefore, methadone-mediated autoinduction of P450s may enhance the clearance of methadone itself. Overall, the current in vitro observations warrant further in vivo and clinical investigations, with a focus on how the long-term administration of methadone may affect the efficacy and toxicity of concomitantly administered medications.

Because buprenorphine was among the most potent PXR activators identified in previous studies (Fig. 2.9) in the cell-based reporter assays, and because of the frequency

81 with which buprenorphine and methadone are prescribed for the management of opioid addiction, we were highly interested in comparing the influences of buprenorphine and methadone on CYP2B6 and CYP3A4 induction. In doing so, an unexpected profile of

CYP2B6/CYP3A4 induction was uncovered in cultured HPHs treated with buprenorphine, whereby buprenorphine treatments resulted in virtually no induction of target genes CYP2B6 and CYP3A4; despite indications from screening assays that buprenorphine potently activates hPXR.

It is well established that buprenorphine was predominantly metabolized in the liver by CYP3A4, CYP2C8, and UGTs before eliminating in the bile.12 Thus the metabolic stability of buprenorphine in HPHs versus HepG2 cells was compared.

Substantial differences in the rate of buprenorphine elimination were observed between these two cell systems (Fig. 3.8), which strongly support the speculation that rapid clearance of buprenorphine in HPHs contributes significantly to the non-induction of

CYP2B6 and CYP3A4. In the meantime, these results imply that metabolites of buprenorphine are not able to activate PXR and CAR or induce their target genes; therefore, in separate observations, the metabolic stability of diprenorphine, an opioid antagonist structurally parallel to buprenorphine, was also dramatically decreased in

HPHs versus HepG2 cells. Moreover, a similarly differential induction of CYP2B6 and

CYP3A4 in HPHs, was noticed with the treatment of diprenorphine (Fig. 8), suggesting that this phenomenon represents a shared class effect rather than a unique compound- specific role of buprenorphine.

In conclusion, evidence unearthed in these studies revealed that buprenorphine lost its effects in the more physiologically relevant cell system, the HPHs. Further, the

82 loss of inductive effects for buprenorphine in HPHs is mainly due to the rapid metabolism and clearance of buprenorphine. In addition, diprenorphine displayed similar differential responses as that which buprenorphine displayed in HepG2 versus HPHs, indicating that a class of structurally similar opioids and their derivatives may behave alike in this regard. This example raises sincere overall concerns regarding the use and interpretation of data obtained from cell-based nuclear receptor assays; in particular, the widely used PXR reporter assays can be misleading, especially as compared to data obtained from the more physiologically relevant HPH system. Meanwhile, the current studies show that methadone can induce the expression of multiple hepatic DMEs and drug transporters, including CYP2B6, CYP3A4, UGT1A1, and MDR1. Given that opioid drug abuse is a rapidly escalating problem, which has the potential to lead to clinically significant DDIs and adverse effects, the in vitro discoveries from the current studies warrant more systematic future in vivo and clinical studies that focus on how methadone may mediate changes in the metabolic and pharmacokinetic profiles of other drugs.

3.5 References

1. LeCluyse, E. L., Human hepatocyte culture systems for the in vitro evaluation of

cytochrome P450 expression and regulation. European Journal of

Pharmaceutical Sciences 2001, 13 (4), 343-368.

2. Guguen-Guillouzo, C.; Guillouzo, A., General Review on In Vitro Hepatocyte

Models and Their Applications. Hepatocytes. Maurel, P., Ed. Humana Press:

2010; Vol. 640, pp 1-40.

3. (a) Kocarek, T. A.; Schuetz, E. G.; Strom, S. C.; Fisher, R. A.; Guzelian, P. S.,

Comparative analysis of cytochrome P4503A induction in primary cultures of rat,

rabbit, and human hepatocytes. Drug Metabolism and Disposition 1995, 23 (3),

415-421; (b) Barwick, J. L.; Quattrochi, L. C.; Mills, A. S.; Potenza, C.; Tukey,

R. H.; Guzelian, P. S., Trans-species gene transfer for analysis of glucocorticoid-

inducible transcriptional activation of transiently expressed human CYP3A4 and

rabbit CYP3A6 in primary cultures of adult rat and rabbit hepatocytes. Molecular

Pharmacology 1996, 50 (1), 10-16.

4. Kern, A.; Bader, A.; Pichlmayr, R.; Sewing, K. F., Drug metabolism in

hepatocyte sandwich cultures of rats and humans. Biochemical Pharmacology

1997, 54 (7), 761-772.

5. LeCluyse EL; Alexandre E; Hamilton GA; Viollon-Abadie C; Coon DJ; Jolley S;

Richert L., Isolation and culture of primary human hepatocytes. . Methods Mol

Biol 2005, 290, 207–229.

6. (a) Faucette, S. R.; Zhang, T.-C.; Moore, R.; Sueyoshi, T.; Omiecinski, C. J.;

LeCluyse, E. L.; Negishi, M.; Wang, H., Relative Activation of Human Pregnane

X Receptor versus Constitutive Androstane Receptor Defines Distinct Classes of

CYP2B6 and CYP3A4 Inducers. Journal of Pharmacology and Experimental

Therapeutics 2007, 320 (1), 72-80; (b) Li, L.; Chen, T.; Stanton, J. D.; Sueyoshi,

T.; Negishi, M.; Wang, H., The Peripheral Benzodiazepine Receptor Ligand 1-(2-

Chlorophenyl-methylpropyl)-3-isoquinoline-carboxamide Is a Novel Antagonist

of Human Constitutive Androstane Receptor. Mol Pharmacol 2008, 74 (2), 443-

453; (c) Maglich, J. M.; Stoltz, C. M.; Goodwin, B.; Hawkins-Brown, D.; Moore,

J. T.; Kliewer, S. A., Nuclear Pregnane X Receptor and Constitutive Androstane

Receptor Regulate Overlapping but Distinct Sets of Genes Involved in Xenobiotic

Detoxification. Mol Pharmacol 2002, 62 (3), 638-646; (d) Smith, C. M.; Graham,

R. A.; Krol, W. L.; Silver, I. S.; Negishi, M.; Wang, H.; Lecluyse, E. L.,

Differential UGT1A1 Induction by Chrysin in Primary Human Hepatocytes and

HepG2 Cells. J Pharmacol Exp Ther 2005, 315 (3), 1256-1264.

7. Hassan, H. E.; Myers, A. L.; Coop, A.; Eddington, N. D., Differential

involvement of P-glycoprotein (ABCB1) in permeability, tissue distribution, and

antinociceptive activity of methadone, buprenorphine, and diprenorphine: In vitro

and in vivo evaluation. Journal of Pharmaceutical Sciences 2009, 98 (12), 4928-

4940.

8. Bart, G., Maintenance Medication for Opiate Addiction: The Foundation of

Recovery. Journal of Addictive Diseases 2012, 31 (3), 207-225.

9. Ali, J.; Woods, D., 475850 - SEIZURE IN A CANCER PATIENT ON

METHADONE. Can J Anesth 2008, 55 (suppl_1), 475850-.

10. (a) Clarke, S. M.; Mulcahy, F. M.; Tjia, J.; Reynolds, H. E.; Gibbons, S. E.;

Barry, M. G.; Back, D. J., The pharmacokinetics of methadone in HIV-positive

patients receiving the non-nucleoside reverse transcriptase inhibitor efavirenz. Br

J Clin Pharmacol 2001, 51(3), 213-217; (b) Desta, Z.; Saussele, T.; Ward, B.;

Blievernicht, J.; Li, L.; Klein, K.; Flockhart, D. A.; Zanger, U. M., Impact of

CYP2B6 polymorphism on hepatic efavirenz metabolism in vitro.

Pharmacogenomics 2007, 8 (6), 547-58; (c) Kharasch, E. D.; Walker, A.;

Whittington, D.; Hoffer, C.; Bedynek, P. S., Methadone metabolism and clearance

are induced by nelfinavir despite inhibition of cytochrome P4503A (CYP3A)

activity. Drug and Alcohol Dependence 2009, 101 (3), 158.

11. (a) Gerber, J. G.; Rhodes, R. J.; Gal, J., Stereoselective metabolism of methadone

N-demethylation by cytochrome P4502B6 and 2C19. Chirality 2004, 16 (1), 36-

44; (b) Kharasch, E. D.; Hoffer, C.; Whittington, D.; Sheffels, P., Role of hepatic

and intestinal cytochrome P450 3A and 2B6 in the metabolism, disposition, and

miotic effects of methadone[ast]. Clin Pharmacol Ther 2004, 76 (3), 250.

12. (a) Cone, E. J.; Gorodetzky, C. W.; Yousefnejad, D.; Buchwald, W. F.; Johnson,

R. E., The metabolism and excretion of buprenorphine in humans. Drug

Metabolism and Disposition 1984, 12 (5), 577-581; (b) Orman, J. S.; Keating, G.

M., Buprenorphine/Naloxone: A Review of its Use in the Treatment of Opioid

Dependence. Drugs 2009, 69 (5), 577-607 10.2165/00003495-200969050-00006.

Chapter 4

Characterization of the Mechanistic Roles of Xenoreceptors

Pregnane X Receptor and Constitutive Androstane Receptor

Underlying Drug-Metabolizing Enzyme Modulation

4.1 Introduction

In the past few years, novel mouse, rat, rabbit and human orphan members of the nuclear hormone receptor superfamily have been cloned and shown to be activated by a variety of known inducers of CYP3A expression.1 These orphan nuclear receptors have been named pregnane X receptor (PXR) by Kliewer and colleagues based upon their efficacious activation by natural C21 steroids (pregnanes). Although activation of nuclear receptors by ligands involves a complex series of events, such as dissociation of corepressors and recruitment of coactivators, there is now substantial evidence that PXR is a major determinant of CYP3A gene regulation by drugs and other xenobiotics;2 and moreover, that the species origin of PXR determines the induction profile of CYP3A.3

First, the PXR gene is predominantly expressed in liver and intestine; and, to a lesser extent, in kidney and lung.1b, 4 The tissue distribution and the relative abundance of

PXR mRNA among these tissues resemble those of CYP3A, suggesting that PXR is important not only for induction but possibly for constitutive expression of these enzymes also. Second, PXR binds to xenobiotic response elements previously identified in the

CYP3A promoters from several species and activates expression of the CYP3A4 promoter in transfection assays.1, 4b PXR forms a heterodimer with the retinoid X receptor

(RXR) and binds to the ER6 and DR3 response elements identified in CYP3A gene promoters from different species. Third, structurally diverse inducers of CYP3A gene expression activate PXR, including antibiotics, antimycotics, protease inhibitors,

NNRTIs, glitazones and statins.4b Fourth, PXR activation correlates with CYP3A gene induction in primary hepatocytes from different species.4b Based upon these data, the

PXRs have been suggested to serve not only as one of the key regulators of CYP3A

88 expression, but also as a major determinant of the observed species differences in CYP3A regulation by xenobiotics.

Recently, a number of studies have reported that PXR might not be the sole regulator of CYP3A gene expression by drugs.5 In experiments performed with PXR knockout mice, no induction of CYP3A11 was observed after treatment with PCN and dexamethasone, but the induction of CYP3A11 by phenobarbital was not affected.

Induction of mouse CYP2B10 was similar in wild type and PXR knockout mice, indicating that mPXR does not mediate mouse CYP2B induction, which is in agreement with reports that have shown rodent CYP2B to be predominantly regulated by CAR.6

Importantly, dexamethasone-induced TAT activity was the same in wild type and mPXR knockout mice indicating that responses mediated via the glucocorticoid receptor were maintained.

Therefore, these intriguing results suggest that induction of mouse CYP3A is not mediated solely by PXR, and the most likely candidate for the PB-mediated increase in

CYP3A expression is CAR.5c, 6a, b It has been shown that CAR binds to response elements in both the CYP2B6 and CYP3A4 promoter region and its activation by PB increases the expression of reporter genes driven by elements from both genes.5c, 6 Overall, the emerging story suggests that there is significant cross-talk between the various receptor signaling pathways (see Figure 4.1) and that the regulation of CYP3A4 in vivo is much more complex than initially assumed.7 Figure 4.1 illustrates a schematic of the activation mechanisms and overlapping target genes of CAR and PXR.

Accordingly, in the following experiments, we aimed to uncover mechanistic evidence that may elucidate the roles that both PXR and CAR play in underlying the

89 observed DME modulation. To do so, cell-based reporter assays utilizing HepG2 cells transfected with hPXR, hCAR, and hCAR3, a splicing variant of the wild-type human

CAR that contains an insertion of five amino acids (APYLT), were performed. Also used in the reporter assays was an additional hCAR construct, hCAR1+A, which is a unique chimeric construct shown previously to be both significantly activated by a series of known hCAR activators (Fig. 4.2 A), and to display activation superior to that of hCAR3

(Fig. 4.2 B). Moreover, intracellular localization assays revealed that hCAR1+A exhibits nuclear accumulation upon 6-(4-chlorophenyl) imidazo[2,1-b][1,3]thiazole-5- carbaldehyde-O-(3,4-dichlorobenzyl) oxime (CITCO) treatment in COS1 cells, which differs from the spontaneous nuclear distribution of hCAR1 and the nontranslocatable hCAR3.

4.2 Materials and Methods

Transient Transfection in HepG2 Cells. HepG2 cells seeded in 24-well plates were transfected with CYP2B6-2.2kb reporter construct in the presence of hPXR, hCAR1 or hCAR3 expression vector using Fugene 6 Transfection Kit according to the manufacturer’s protocol. Twenty-four hours post-transfection, cells were treated with solvent (0.1% DMSO) or test compounds at the concentrations of RIF (10 μM), CITCO

(1 μM), or MD (10 μM, 25 μM, or 50 μM) for another 24 hrs. Subsequently, cell lysates were assayed for firefly activities normalized against the activities of Renilla luciferase using the Dual-Luciferase Kit (Promega, WI). Data are represented as mean ± S.D. of three individual transfections.

Fig 4.1. Schematic illustration of the activation mechanisms and target genes of

CAR and PXR. CAR can be activated by either direct (ligand binding) or indirect mechanisms, while activation of PXR is purely ligand dependent. CAR and PXR shared target genes are grouped in a red box, CAR-specific targets in a blue box, and PXR- specific targets in a purple box (modified from Qatanani and Moore, Curr. Drug Metab.

2005). Adapted from Tolson AH and Wang, H, Advanced Drug Delivery Reviews, 2010

(source).

Translocation of Ad/EYFP-hCAR in Human Primary Hepatocyte Cultures. Human hepatocytes were seeded at 3.75 × 105 cells/well in 24-well BioCoat plates and cultured as described previously (Wang et al., 2003). Twenty four hours later, hepatocyte cultures were infected as described previously 8 with Ad/EYFP-hCAR for 12 hrs before treatment with vehicle control (0.1% DMSO) or test compounds for another 12 hrs. Confocal laser scanning microscopy was performed with a Nikon C1-LU3 instrument based on an inverted Nikon Eclipse TE2000 microscope. The subcellular localization of Ad/EYFP- hCAR was visualized, and quantitatively characterized as nuclear (N), cytosolic (C), and mixed (N + C) by counting 100 Ad/EYFP-hCAR expressing hepatocytes from each group.

(Roche Applied Science, Indianapolis, IN). Twenty-four hours after transfection, cells were treated for another 24 h with solvent (0.1% DMSO) or test compounds RIF (10

μM), CITCO (1 μM), morphine (10 μM), MD (10 μM), oxycodone (10 μM), codeine (10

μM), meperidine (10 μM), BUP (1, 5, 10, 25, 50, and 100 μM), 6-desoxycodeine (10

92 induction of CYP3A4 in hepatoma cells, HepG2 cells seeded in 12-well plates were transfected with a hCAR or hPXR expression vector; 24 h after transfection, cells were treated for another 24 h with RIF (10 μM), CITCO (1 μM), PK11195 (10 μM), SFN (10

μM), or BUP (10 and 50 μM) or were cotreated for 24 h with SFN plus RIF or BUP, and

PK11195 plus CITCO or BUP, respectively. Total RNA was isolated and subjected to real-time RT-PCR analysis as described above. In separate experiments, HPHs seeded in

24-well BioCoat plates were transfected with CYP3A4-PXRE/XREM or CYP2B6–2.2kb construct in the presence of the pGL-TK vector using Effectene reagent (QIAGEN) as described previously (Wang et al., 2004). Transfected HPHs were treated with DMSO

[0.1% (v/v)], RIF (10 μM), CITCO (1 μM), BUP (10 and 50 μM), or DIP (10 μM) for 24 h, respectively. Cell lysates were subjected to dual-luciferase analysis as described above.

Translocation of Adenovirus Expressing Enhanced Yellow Fluorescent Protein-

Tagged hCAR in Human Primary Hepatocytes. The adenovirus expressing enhanced yellow fluorescent protein-tagged hCAR (Ad/EYFP-hCAR), which infects HPHs with high efficiency, was generated as described previously.8 Hepatocyte cultures in 24-well

BioCoat plates were infected with 5 μl of Ad/EYFP-hCAR for 12 h before treatment with the vehicle control (0.1% DMSO), PB (1 mM), BUP (10 and 50 μM), or DIP (10 μM).

After 24 h of incubation, cells were washed twice with phosphate-buffered saline and fixed for 30 min in 4% buffered paraformaldehyde. The cells were then stained with 4, 6- diamidine-2-phenylindole dihydrochloride for 30 min. Confocal microscopy analysis was performed with a Nikon C1-LU3 instrument based on an inverted Nikon Eclipse TE2000 microscope (Nikon, Melville, NY). The subcellular localization of hCAR was visualized

CYP2B6 Reporter

Relative Luc Activity Relative B

CYP2B6 Reporter

Relative Luc Activity Relative

Fig 4.2. Activation of hCAR3 and hCAR1+A by prototypical hCAR activators.

HepG2 cells were transfected with CYP2B6-PBREM reporter, and hCAR3 or hCAR1+A expression vectors as described previously,9 prior to subsequent treatment with vehicle control (0.1% DMSO) or CITCO at the concentration of 0.1, 1.0, and 5.0 μM (A); or with known hCAR activators, including PB, ART, PHN, EFV, and NVP, at indicated concentrations for 24 h (B). RIF (10 μM) was included as non-hCAR activator. Adapted from Chen T, et. al., Journal of Pharmacology and Experimental Therapeutics, 2010.

94 and quantitatively characterized as a nuclear, cytosolic, or mixed (nuclear + cytosolic) expression by counting 100 Ad/EYFP-hCAR-expressing hepatocytes from each group.

4.3 Results

Activation of hPXR and hCAR by Racemic MD. Drug-induced expression of

CYP2B6 and CYP3A4 is predominantly controlled at the transcriptional level by the two

NRs hCAR and hPXR. Here, we investigated the ability of racemic MD to activate these receptors in cell-based reporter assays conducted in HepG2 cells. As shown in Fig. 4.3 A,

MD showed significant increases of hPXR-mediated CYP2B6 reporter activities over the concentration range of 10 to 50 μM, where significant CYP2B6 and CYP3A4 gene induction was observed in human primary hepatocytes. In contrast to hPXR, in vitro assessment of hCAR activation has been problematic because of the constitutive activation nature of CAR in all the immortalized cell lines. More recently, however, several reports indicate that an hCAR splicing variant (hCAR3) displays low basal activity while still retaining chemical-mediated activation in several cell lines.10 Thus, in the current study, we also evaluated hCAR3 activation in HepG2 cells. As expected, the hCAR-selective agonist CITCO strongly enhanced hCAR3 activity, whereas MD resulted in moderate but statistically significant activation of hCAR3 (Fig. 4.3 B). In addition, earlier work conducted in this laboratory showed that the constitutive activity of the wild- type hCAR in HepG2 cells could be substantially repressed by PK11195, a typical ligand for peripheral benzodiazepine receptor, and this inhibitory effect was only recovered by cotreatment with direct hCAR activator CITCO but not by indirect activators such as

B C

Fig 4.3. Effects of MD on hPXR-, hCAR3-, and hCAR-mediated CYP2B6 reporter gene activation. HepG2 cells were transfected with hPXR (A), hCAR3 (B), or hCAR (C) expression vectors in the presence of CYP2B6-2.2 kb reporter construct. Transfected cells were then treated with vehicle or MD (10, 25, or 50 μM) for 24 h. RIF (10 μM) and

CITCO (1 μM) were used as positive controls for hPXR and hCAR, respectively.

Luciferase activities were determined and expressed relative to vehicle control. Data represent the mean ± S.D. (n = 3). ***, p ≤ 0.001; **, p ≤ 0.01. Adapted from Tolson

AH, et. al., Drug Metabolism and Disposition, 2009.

96 phenobarbital (PB).11 Using this system, our current results showed that MD has no effect on the PK11195-mediated deactivation of hCAR (Fig. 4.3 C).

Both R-(–)-MD and S-(+)-MD Contribute to the Activation of hPXR in HepG2

Cells. MD is clinically administered as a mixture of two stereoisomers, R-(–)-MD and S-

(+)-MD, with opioid activity residing in the R-enantiomer. To assess the contribution of each enantiomer to the observed increases of hPXR-mediated CYP2B6 reporter activities, similar reporter assays in HepG2 cells were conducted as described above using R-(–)-MD (active) and S-(+)-MD (inactive). As shown in Fig. 4.4, both the active

R-enantiomer and the inactive S-enantiomer achieved potent and concentration-related activation of hPXR, with maximal activity occurring at 50 μM. These results suggest that although the metabolism and clearance of racemic MD are highly variable, the potential for each enantiomer to achieve DME induction is most likely nonstereoselective.

Enantiomers R-(–)-MD and S-(+)-MD Equally Contribute to Weak Activation of hCAR1+A in HepG2 Cells. The relative contribution of each enantiomer to the activation of hCAR1+A was also assessed. As compared to one another, each enantiomer contributed similarly to the weak-to-moderate activation of hCAR1+A achieved by the racemic mixture (Fig. 4.5). These increases however can be considered weak compared to the potent and concentration-related activities of hPXR described above achieved by

R-(–)-MD (active) and S-(+)-MD (inactive). As was the case with assessing enantiomeric contributions for hPXR, again, no difference in the activation profile of the active versus inactive enantiomer was observed. This result may also provide further support for the suggestion that the potential for each enantiomer to achieve DME induction is most likely

Fig 4.4. MD enantiomers increase the activities of hPXR in HepG2 cells. HepG2 cells were transfected with hPXR expression vector in the presence of CYP2B6-2.2 kb reporter construct, then treated with vehicle control, R-(–)-MD (A) or S-(+)-MD (B) (10,

25, or 50 μM) for 24 h. RIF (10 μM) was used as positive control. Luciferase activities were determined and expressed relative to vehicle control. Data represent the mean ±

S.D. (n = 3). ***, p ≤ 0.001; **, p ≤ 0.01. Adapted from Tolson AH, et. al., Drug

Metabolism and Disposition, 2009.

9 hCAR1+A-2B6 8

7 6 5 4 3

2 Relative Luc ActivityRelative 1 0

Fig 4.5. Effects of racemic MD and enantiomers on the activation of hCAR. HepG2 cells were transfected with an hCAR1+A expression vector in the presence of CYP2B6–

2.2kb reporter construct as described under Materials and Methods. Transfected cells were subsequently treated with vehicle control (0.1% DMSO), racemic MD, R-(–)-MD, or S-(+)-MD at the indicated concentrations (10, 25, or 50 μM for each) for 24 h. RIF (10

μM) and CITCO (1 μM) were used as negative and positive controls for hCAR activation, respectively. Luciferase (Luc) activities were determined and expressed relative to vehicle control. Data represent the mean ± S.D. (n = 3). ***, p ≤ 0.001; **, p ≤

0.01.

99 nonstereoselective despite the metabolism and clearance of racemic MD being highly variable.

Effects of BUP on the Activation of hPXR in HepG2 Cells. To characterize the potential role of partial mu opioid agonist BUP on the activation of hPXR, HepG2 cells were transfected with an hPXR expression vector in the presence of either CYP3A4-

PXRE/XREM or CYP2B6–2.2kb reporter construct as described above, then treated with

BUP over a range of concentrations (1–100 μM). Our preliminary experiments showed that BUP at a concentration of 100 μM is associated with cytotoxicity in HepG2 cells

(data not shown). Obvious concentration-dependent responses (1–50 μM) for BUP- induced PXR activation of CYP2B6 and CYP3A4 reporter activities are shown in Fig.

4.6.

Effects of BUP on the Activation of CAR in HepG2 Cells. Because PXR and CAR share a number of overlapping target genes and many xenobiotics as co-activators, we further investigated whether BUP could activate human CAR in HepG2 cells. Consistent with previous reports, cell-based reporter assays using the reference hCAR (hCAR1) displayed constitutively high basal activity and moderate response to the known hCAR activator CITCO in HepG2 cells 8(Li et al., 2009a). There was no activation of hCAR1 after the exposure of BUP in this system (Fig. 4.7A). On the other hand, using a hCAR ligand-responsive variant (hCAR3), BUP caused a marginal but concentration-dependent enhancement of CYP2B6 reporter activity, with the highest activation at 50 μM (1.8-fold over vehicle control), whereas using the positive control CITCO resulted in 4.34-fold increase (Fig. 4.7B). Moreover, in separate reporter assays, we applied a recently developed hCAR1+A construct, which demonstrated robust response to chemical-

100

Fig 4.6. Effects of BUP on hPXR-mediated reporter gene activation in cell-based reporter assays. HepG2 cells were transfected with an hPXR expression vector in the presence of CYP3A4-PXRE/XREM (A) or CYP2B6–2.2kb (B) reporter construct as described under Materials and Methods. Transfected cells were then treated with BUP at the indicated concentrations for 24 h. RIF (10 μM) was used as a positive control for hPXR activation. Luciferase (Luc) activities were determined and expressed relative to vehicle control (0.1% DMSO). All data are expressed as mean ± S.D. (n = 3). *, p < 0.05;

**, p < 0.01. Adapted from Li L, Hassan HE, Tolson AH, et. al., J Pharmacol Exp Ther,

2010.

101

A B

Fig 4.7. Effects of BUP on the activation of hCAR. HepG2 cells were transfected with

an hCAR1 (A), hCAR3 (B), or hCAR1+A (C) expression vector in the presence of

CYP2B6–2.2kb reporter construct as described under Materials and Methods.

Transfected cells were subsequently treated with BUP at concentrations of 1 to 50 μM for

24 h. CITCO (1 μM) was used as a positive control for hCAR activation. Luciferase

(Luc) activities were determined and expressed as fold activation relative to vehicle

control (0.1% DMSO). All data are expressed as mean ± S.D. (n = 3). *, p < 0.05; **, p <

0.01. Adapted from Li L, Hassan HE, Tolson AH, et. al., J Pharmacol Exp Ther, 2010.

102 mediated activation of hCAR in immortalized cell lines.9 As shown in Fig. 4.7 C, BUP significantly enhanced the hCAR1+A-mediated expression of CYP2B6 reporter gene, with the highest response at 50 μM (6.43-fold over vehicle control) challenging that of the positive control CITCO (6.92-fold over vehicle control).

Translocation of Ad/EYFP-hCAR by MD in Human Primary Hepatocytes. The high constitutive activation of hCAR in immortalized cell lines can be attributed to the spontaneous nuclear accumulation of hCAR in these cells. In contrast, in primary hepatocytes and in vivo, CAR is sequestered predominantly in the cytoplasm and translocated to the nucleus only after exposure to xenobiotics. More recently, our laboratory has generated an Ad/EYFP-hCAR, which showed exceptional efficiency in transducing human primary hepatocytes 8. As shown in Fig. 4.8, our recombinant

Ad/EYFP-hCAR infected both HepG2 cells and HPHs with high efficiency (Fig. 4.8).

Further, a series of 22 compounds was chosen to evaluate the correlation between chemical-mediated hCAR nuclear accumulation and target gene induction in current studies. All of the eight known hCAR activators resulted in remarkable nuclear accumulation after 24 h of treatment, for which nucleus and mixed hCAR distribution accounts for approximately 90% of the infected hepatocytes (Fig. 4.9 C). Representative images of hCAR localization were demonstrated in Fig. 4.9, A and B, after treatment with the vehicle control (0.1% DMSO), the typical hCAR activator PB (1 mM), or

CITCO (1 μM). It is noteworthy that the selective hCAR agonist, CITCO, revealed a unique pattern of hCAR translocation in which up to 64.5% of infected cells exhibit mixed hCAR distribution (Fig. 4.9 B).

103

Using this system, we sought to determine whether MD can translocate hCAR as the initial step of hCAR activation. Cultured hepatocytes were infected with Ad/EYFP- hCAR and treated with vehicle control, known hCAR indirect activator PB, or 50 μM

MD. Fluorescence microscopy analysis showed that both PB and MD treatment result in abundant nuclear accumulation of hCAR (Fig. 4.10 A). Of the hepatocytes expressing hCAR prepared from two liver donors (HL8, HL9), 87 to 93% displayed cytoplasmic localization and 7 to 13% exhibited mixed (cytoplasm and nucleus) localization, whereas cells in the vehicle the control group showed pure nuclear localization (Fig. 4.10 B). The

Ad/EYFP-hCAR expression was predominantly accumulated inside the nucleus after the treatment with known indirect hCAR activator PB, where 63 to 80% exhibited nuclear localization, 7 to 38% exhibited a mixed distribution pattern, and only 0 to 13% displayed cytoplasmic localization. It is noteworthy that MD (50 μM) treatment also resulted in 62 to 81% nuclear, 11 to 13% mixed, and 8 to 25% cytoplasmic localizations (Fig. 4.10 B).

Subsequently, a parallel experiment was conducted to expand the scope of MD-mediated hCAR translocation by including both racemic MD and its constituent isomers at 10, 25, and 50 μM concentrations. As shown in Fig. 4.11, all the isomers of MD translocated hCAR efficiently from the inactive cytoplasmic localization to the nucleus at all three concentrations. The maximal translocation extent mimicked that of the positive control

PB. Overall, these results indicate that MD and its constituent isomers are capable of accumulating hCAR inside the nucleus of human hepatocytes in a nonstereoselective manner.

Nuclear Translocation of hCAR by BUP in Human Primary Hepatocytes. In contrast to the constitutive nuclear expression in immortalized cells, human CAR is

104

A B

Fig 4.8. Localization of Ad/EYFP-hCAR in HepG2 cells and HPHs. HepG2 cells and

HPHs (HL-#009) were infected with Ad/EYFP-hCAR as described previously (Li H, et., al. source). Confocal images depict the localization of Ah/EYFP-hCAR in HepG2 and

HPHs (A). Left panels, Ad/EYFP-hCAR expression (green); center panels, nuclear staining (red); right panels, merged images. One hundred hCAR-expressing cells from each group were counted and classified by cytosolic, nuclear, or mixed (cytosolic + nuclear) hCAR cellular localizations (B). Adapted from Li H, et. al., Drug Metabolism and Disposition, 2009.

105

A B

Fig 4.9. Known hCAR activators promote nuclear translocation of Ad/EYPF-hCAR in HPHs. HPHs (HL-#009 and HL-#014) were infected with Ad/EYFP-hCAR as described previously (source) and treated with vehicle (0.1% DMSO) or eight known hCAR activators at the indicated concentrations. After 24 h of treatment, hepatocytes were stained with 4,6-diamidine-2-phenylindole dihydrochloride and subjected to confocal microscopy. Representative Ad/EYFP-hCAR localizations from vehicle control,

PB (1 mM), or CITCO (1 μM) treatment in HPHs are depicted (A). Three panels are shown for each treatment: left, Ad/EYFP-hCAR (green); center, nuclear staining (red); and right, merged image. For each treatment, 100 hCAR-expressing cells were counted and classified based on cytosolic, nuclear, or mixed (cytosolic + nuclear) hCAR cellular localizations (B and C). Adapted from Li H, et. al., Drug Metabolism and Disposition,

2009.

106 predominantly sequestered in the cytoplasm of primary hepatocytes and translocated to the nucleus after exposure to prototypical CAR activators such as CITCO and PB.6b, 12

We have recently generated an Ad-EYFP-hCAR. With high infection efficiency in HPHs, the Ad-EYFP-hCAR also displays the unique feature of hCAR distribution and activation in HPHs.8 Because our results thus far demonstrated that BUP-mediated activation of hCAR in HepG2 cells is associated with enhanced expression of CYP3A4, we continue to test whether this opioid could translocate hCAR to the nucleus as the initial step of hCAR activation in HPHs. Cultured hepatocytes were infected with Ad-EYFP-hCAR and treated with vehicle control, positive control PB (1 mM), or BUP (10 and 50 μM) as outlined under Materials and Methods. Confocal microscopic analysis revealed that without activation, Ad-EYFP-hCAR was localized primarily in the cytoplasm (90%), and the prototypical CAR activator PB efficiently translocated hCAR to the nucleus (92%), whereas BUP at 10 and 50 μM showed no effect on hCAR nuclear accumulation with a cytoplasmic localization of 87 and 88%, respectively (Fig. 6, A and B). Overall, these results demonstrate that BUP is not an inducer of CYP2B6 and CYP3A4 because it fails to activate either PXR or CAR in HPHs.

4.4 Discussion

Prescription drug abuse and misuse has become a major public health concern worldwide. In particular, opioid dependence results in high socioeconomic costs to individuals, health care systems, and society 13. Given that they continue to be the mainstay for the treatment of chronic pain because of their stable pharmacokinetic and pharmacodynamic features, as well as for reasons of therapy compliance,14 opioids

107

Fig 4.10. Methadone translocates Ad/EYFP-hCAR in human primary hepatocytes.

Human hepatocytes (HL8 and HL9) were infected with Ad/EYFP-hCAR as described under Materials and Methods and treated with vehicle, PB (1 mM), or MD (50 μM).

After 24 h of treatment, hepatocytes were subjected to confocal microscopy. A, representative Ad/EYFP-hCAR localizations from vehicle control, PB- (1 mM), and MD-

(50 μM) treated hepatocytes. B, for each treatment, more than 60 hCAR-expressing cells were counted and classified based on cytosolic, nuclear, or mixed (cytosolic + nuclear) hCAR localizations.

108

Fig 4.11. MD and constituent isomers promote nuclear translocation of Ad/EYPF- hCAR in human hepatocytes. Hepatocytes were infected with Ad/EYFP-hCAR and treated with vehicle (0.1% DMSO), or racemic MD and its constituent isomers at 10, 25, and 50 μM for 24 h. For each treatment group, 100 hCAR-expressing cells were counted and classified based on cytosolic, nuclear, or mixed (cytosolic + nuclear) hCAR distributions. Adapted from Tolson AH, et. al., Drug Metabolism and Disposition, 2009.

109 constitute the highest prevalence of drug abuse and misuse. They are extensively used for the management of heroin dependence, and frequently, opioid abuse is often associated with a variety of other addictive behaviors and disease states, such as alcohol consumption, nicotine addiction, and HIV- or Hepatitis C-infected populations. For instance, cigarette smoking rates are higher in the methadone-maintained opiate- dependent population than in the general population. At least 80% of methadone patients smoke, and similarly, opioid substitution therapy with buprenorphine showed increased smoking rates 15. Yet, despite such associations, characterization of opioid interactions with other drugs has been heavily one-sided to date, focusing on how other drugs affect the metabolic and/or pharmacokinetic profiles of opioids. In contrast, the potential for opioids to influence the similar fates of coadministered drugs remains largely unexplored.

For example, although MD and BUP have both emerged as desirable options for maintenance treatment of opioid dependence, very little mechanistic evidence can be found among available literature reports for either therapy. MD is not a new medication, and is the pharmacotherapy of choice for the management of heroin addiction; yet, to our knowledge, the current investigation is the first to show that MD can induce the expression of multiple hepatic DMEs through the activation of PXR- and CAR- dependent pathways. Moreover, although BUP is a partial agonist that also makes an excellent candidate therapy for use during maintenance due to its enhanced safety profile for certain pharmacological effects (eg, respiratory depression and sedation) as compared with full opioid agonist profiles, little-to-no mechanistic evidence has been provided for buprenorphine thus far either.

110

A B

Fig 4.12. Localization of Ad-EYFP-hCAR upon BUP Treatment in human primary

hepatocytes. Human hepatocytes from donors (HL-018 and HL-020) in 24-well BioCoat

plates were infected with Ad-EYFP-hCAR for 12 h and then treated with vehicle control

(0.1% DMSO), PB (1 mM), and BUP (10 and 50 μM) for 24 h. Cells were subsequently

fixed for 30 min in 4% buffered paraformaldehyde and stained with 4,6-diamidine-2-

phenylindole dihydrochloride for 30 min before being examined by a confocal

microscopy. A, representative Ad-EYFP-hCAR localizations from vehicle control, PB-,

and BUP-treated hepatocytes. B, percentage of Ad/EYFP-hCAR cellular localization in

HPHs after treatment with vehicle control, PB, and BUP. One hundred Ad/EYFP-hCAR-

expressing HPHs were counted from each treatment group. Adapted from Li L, Hassan

HE, Tolson AH, et. al., J Pharmacol Exp Ther, 2010.

111

We report here that buprenorphine differentially transactivates PXR, CAR, and their target genes (CYP2B6 and CYP3A4) in HepG2 and HPHs. In the current studies, we showed that both BUP and MD were identified as potent PXR activators in cell-based reporter assays. Subsequent experiments in HepG2 cells confirmed that buprenorphine can activate both PXR and CAR; yet, on the other hand, an unexpected profile of

PXR/CAR activation and CYP2B6/CYP3A4 induction was uncovered in cultured HPHs treated with buprenorphine (Figs. 3.6 & 3.7), whereby buprenorphine treatments resulted in virtually no activation of PXR and CAR or induction of their target genes CYP2B6 and CYP3A4.

Metabolism induction-related DDIs are one of the critical concerns in the overall safety profiles of clinical medication as well as drug development; and transcriptional up- regulation of drug-metabolizing genes by activation of nuclear receptors represents the principle mechanism by which induction-related DDIs occur. Our current results illustrate that buprenorphine is not a physiologically relevant activator of PXR and CAR or inducer of associated P450s in HPHs (Figs. 3.6 & 3.7), despite its demonstrated potent activation of PXR and induction of CYP3A4 in HepG2 cells (Fig. 4.6). These findings trigger the speculation that the obvious lack of metabolic capability in an immortalized cell line compared with an HPH may contribute to the differential induction. Meanwhile, in delineating the molecular mechanisms underlying MD-mediated induction of CYP2B6 and CYP3A4, our cell-based reporter assays in HepG2 cells showed that treatment of methadone resulted in potent and concentration-related activation of hPXR-mediated luciferase reporter gene expression (Fig. 4.3 A). Given that activation of PXR coordinately induces the expression of a plethora of DMEs and drug transporters besides

112

CYP2B6 and CYP3A4, methadone treatment holds great potential for causing DDIs by interacting with a broader spectrum of DMEs and transporters.

Compared with PXR, in vitro evaluation of CAR activation was more challenging because of the nature of constitutive activation of CAR in immortalized cell lines and the fact that CAR can be activated by both direct ligand binding and indirect mechanisms.16

We have established several novel strategies to efficiently identify hCAR activators in vitro. A chimeric construct generated by inserting alanine to amino acid position 270 of hCAR (hCAR1+A) converts the constitutively activated hCAR to a xenobiotic-sensitive receptor; this construct was significantly activated by a series of known hCAR activators.9 Using this unique system, we showed that buprenorphine efficiently activated hCAR1+A to the extent that it is clearly superior to that of hCAR1 or hCAR3 in cell- based reporter assays. In addition, the Ad/EYFP-hCAR infection of HPHs was established as a valuable model to efficiently detect chemical-mediated nuclear translocation of hCAR as the initial step of CAR activation in HPHs.8 In agreement with the unstable feature of buprenorphine in HPHs, buprenorphine was unable to translocate hCAR to the nucleus in transfected HPHs.

Meanwhile, using the hCAR3 reporter and PK11195-based hCAR reactivation assays, our data showed that methadone mediated a moderate but concentration-related activation of hCAR3 while failing to reactivate PK11195-suppressed hCAR activity in

HepG2 cells, indicating that methadone may not function as a direct agonist of hCAR.

Nevertheless, it is noteworthy that the majority of known hCAR activators are PB-like compounds that activate hCAR through indirect mechanisms without direct ligand binding. As a matter of fact, CITCO and artemisinin are the only two hCAR agonists

113 identified thus far.17 Compared with the cell-based hCAR reporter assays, chemical- mediated hCAR nuclear translocation in hepatocytes of primary culture or in vivo appears to correlate well with hCAR activation and target gene induction, regardless of the distinction between direct or indirect mechanisms.12, 18 However, in vitro transfection of human primary hepatocytes has been extremely challenging mostly because of the quiescent nature of hepatocytes in cultures. Most recently, we have generated an

Ad/EYFP-hCAR that transduces human primary hepatocytes with high efficiency and exhibits a physiologically relevant hCAR distribution pattern.8 Further evaluation of methadone in Ad/EYFP-hCAR-infected human primary hepatocytes revealed that methadone treatment resulted in remarkable nuclear accumulation of hCAR at all the tested concentrations, which achieves an extent similar to the positive control PB.

Combined with the reporter assays, these results indicate that methadone activates hCAR most likely through indirect, PB-like mechanisms, and that methadone’s constituent isomers are capable of accumulating hCAR inside the nucleus of human hepatocytes in a nonstereoselective manner.

Also nonstereoselective was the intriguing finding that both methadone isomers robustly activated hPXR in cell-based reporter assays in HepG2 cells. Methadone is clinically administered as a racemic mixture of two stereoisomers with only the R- enantiomer possessing opioid activity. Several lines of evidence indicate that MD metabolism and clearance are stereoselective, where CYP2B6, but not CYP3A4, contributes significantly to the highly variable plasma R/S-MD ratios.19 Accordingly, to gain insight into the potential stereoselectivity between R-(–)-MD and S-(+)-MD in the induction of DMEs, the current study further evaluated the active R-(–)-MD and the

114 inactive S-(+)-MD isomers for their activation of hPXR and hCAR. The results that both isomers robustly activated hPXR in cell-based reporter assays in HepG2 cells and translocated Ad/EYFP-hCAR in human primary hepatocytes overall suggest that the two enantiomers of methadone may equally induce hepatic DMEs through similar molecular mechanisms, even though their own disposition is considerably stereoselective.

In conclusion, the current studies illustrate that both methadone and buprenorphine play a role in the activation of xenobiotic receptors PXR and CAR. Given that opioid drug abuse is a rapidly escalating problem, which has the potential to lead to clinically significant DDIs and adverse effects, the in vitro discoveries from the current studies warrant more systematic future in vivo and clinical studies that focus on how both methadone and buprenorphine can mediate changes in the metabolic and pharmacokinetic profiles of other drugs. Further, that buprenorphine was shown to function as a potent activator of PXR and CAR as well as an inducer of CYP3A4 in the immortalized cell line, yet such effects were totally lost in a more physiologically relevant cell system, the

HPHs illustrates a paradox which necessitates careful consideration. Further evidence revealed that the loss of inductive effects for buprenorphine in HPHs is mainly due to the rapid metabolism and clearance of buprenorphine. In addition, diprenorphine displayed similar responses in HepG2 and HPHs, indicating that a class of structurally similar opioids and their derivatives may behave alike in this regard. Overall, in the case of buprenorphine, these results raise sincere concerns in our use and interpretation of data obtained from cell-based nuclear receptor assays, and in particular, the widely used PXR reporter assays.

115

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16. (a) Honkakoski, P.; Sueyoshi, T.; Negishi, M., Drug-activated nuclear receptors

CAR and PXR. Annals of Medicine 2003, 35 (3), 172-182; (b) Qatanani, M.;

Zhang, J.; Moore, D. D., Role of the Constitutive Androstane Receptor in

Xenobiotic-Induced Thyroid Hormone Metabolism. Endocrinology 2005, 146 (3),

995-1002.

17. (a) Maglich, J. M.; Parks, D. J.; Moore, L. B.; Collins, J. L.; Goodwin, B.; Billin,

A. N.; Stoltz, C. A.; Kliewer, S. A.; Lambert, M. H.; Willson, T. M.; Moore, J. T.,

Identification of a Novel Human Constitutive Androstane Receptor (CAR)

Agonist and Its Use in the Identification of CAR Target Genes. Journal of

Biological Chemistry 2003, 278 (19), 17277-17283; (b) Simonsson, U. S. H.;

Lindell, M.; Raffalli-Mathieu, F.; Lannerbro, A.; Honkakoski, P.; Lang, M. A., In

vivo and mechanistic evidence of nuclear receptor CAR induction by artemisinin.

European Journal of Clinical Investigation 2006, 36 (9), 647-653.

18. Faucette, S. R.; Zhang, T.-C.; Moore, R.; Sueyoshi, T.; Omiecinski, C. J.;

LeCluyse, E. L.; Negishi, M.; Wang, H., Relative Activation of Human Pregnane

X Receptor versus Constitutive Androstane Receptor Defines Distinct Classes of

CYP2B6 and CYP3A4 Inducers. Journal of Pharmacology and Experimental

Therapeutics 2007, 320 (1), 72-80.

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19. Totah, R. A.; Allen, K. E.; Sheffels, P.; Whittington, D.; Kharasch, E. D.,

Enantiomeric metabolic interactions and stereoselective human methadone

metabolism. The Journal of pharmacology and experimental therapeutics 2007,

321 (1), 389-99.

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Chapter 5

Future Directions: Exploring the Potential for Methadone to

Modulate Corresponding Rodent Drug Metabolizing Enzymes

5.1 Preliminary Induction Profiles in Rat

Significant species-specific differences have been observed in both PXR and CAR activation profiles, whereby both PXR and CAR exhibit the ability to bind multiple ligands, and each receptor’s ligand specificity is species-dependent.1 A large number of xenobiotics may affect the efficacy and toxicity of certain drugs in animals but not in humans.2 For example, in the case of CAR, a variety of compounds bind to it in addition to the classic CYP2B inducer PB, including but not limited to: androstane steroidal compounds, 5β-pregnane-3,20-dione, retinoic acids, clotrimazole, chlorpromazine, o,p’-

DDT, methoxychlor, and hydrocarbons such as CITCO and 1,4-bis [2-(3,5- dichloropyridyloxy)] benzene (TCPOBOP) [need original sources 2, 16-18], constituting a structurally-diverse panel of compounds.1 However, their affinities for CAR vary across species and in some cases their direct binding to CAR remains a matter of debate;

TCPOBOP and CITCO are the only compounds shown to specifically bind to mouse

CAR (mCAR) and human CAR (hCAR), respectively.

Meanwhile, in the case of PXR, ligands include the naturally occurring steroids 5α- pregnane-3, 20-dione, progesterone, 17α-hydroxyprogesterone, 17α- hydroxypregnenolone and corticosterone, hyperforin (a component of St. John’s Wort), dexamethasone, the anti-glucocorticoids PCN and RU486, taxol, and the bisphosphonate ester SR12813. Selectivity for these compounds also differs across species. For instance, pregnenolone 16α-carbonitrile (PCN) binds to the rodent form of PXR, while rifampicin

(RIF) and SR12813 are both specific to human PXR. The structural nature of PXR’s ligand binding domain explains in part its broad substrate specificity;1 and generally speaking, the ligand binding domains of PXRs are more divergent across species,

123

whereas the DNA binding domains of mammalian PXRs are highly conserved, sharing more than 95% amino acid identity.

Thus, species-specific differences present a major obstacle to being able to extrapolate data regarding DME induction from one species across another species.

Rather, induction profiles need to be obtained for each individual, unique system for which DME induction and nuclear receptor-mediated effects are being studied.

Accordingly, in these investigations, we desired to examine whether or not corresponding rodent DME induction could be observed for cyp2b2, cyp3a1, ugt1a1 and mdr1, similar to that which was observed for human CYP2B6, CYP3A4, UGT1A1 and MDR1 upon

MD treatment. Representative induction profiles for cyp2b2, cyp 3a1, ugt1a1, and mdr1 are shown in figs. 5.1-5.2. Peak induction for cyp2b2 was observed around a MD concentration of 5 µM, whereas maximal induction for cyp3a1 was observed at 50 µM; virtually no induction was observed for either ugt1a1 or mdr1. Since it was observed that maximal induction was achieved closer to the middle of the MD concentration range investigated for cyp2b2 than at the higher end of the concentration range (like that observed for cyp3a1), a concentration range for PB was also investigated for comparison.

Interestingly, a similar trend of maximal induction occurring closer to the middle of the

PB concentration range for cyp2b2, while maximal induction did not occur until the higher end of the concentration range for cyp3a1, was also observed after PB treatment.

In human, it has been well accepted that maximal induction for corresponding target genes CYP2B6 and CYP3A4 occurs at differing PB concentrations; however, no such In human, it has been well accepted that maximal induction for corresponding target genes CYP2B6 and CYP3A4 occurs at differing PB concentrations; however, no such

124

A 70 ***

60 ***

40 Fold InductionFold

20 cyp 2b2 *** 10 *** * *** *** 0 ***

20 18 *** 16 *** 14 12

Fold InductionFold 10

8 6 cyp 2b2 4 *** *** *** *** 2 0

Fig 5.1. MD increases the expression of rodent cyp2b2 and cyp3a1 in rat hepatocytes. Primary rat hepatocytes were isolated and cultured as described previously

(source), prior to treatment with the following compounds at indicated concentrations: PB

(100 μM), DEX (10 μM), PCN (10 μM), MD (10, 25 or 50 μM) or 3MC (5 μM) for 24 h.

Cells were harvested, RNA was isolated, and real-time PCR was performed for (A) cyp2b2 and (B) cyp3a1 as described previously. All data are expressed as mean ± S.D. (n

= 3). ***, p ≤ 0.001; **, p ≤ 0.01; *, p ≤ 0.05.

125

A 1.4

1.2

0.8

0.6

Fold Induction

0.4

ugt1a1 0.2

B 2.5

2 *

1.5 *

Fold InductionFold

0.5 mdr1

Fig 5.2. MD does not increase the expression of rodent ugt1a1 and mdr1 in rat hepatocytes. Primary rat hepatocytes were isolated and cultured as described previously

(source), prior to treatment with the following compounds at indicated concentrations: PB

(100 μM), DEX (10 μM), PCN (10 μM), MD (10, 25 or 50 μM) or 3MC (5 μM) for 24 h.

Cells were harvested, RNA was isolated, and real-time PCR was performed for (A) ugt1a1 and (B) mdr1 as described previously. All data are expressed as mean ± S.D. (n =

3). ***, p ≤ 0.001; **, p ≤ 0.01; *, p ≤ 0.05.

126

A B 8 *** 60 ***

7 *** *** 50 6 *** 5 40 4 ***

** 30 Fold InductionFold

3 ** Fold Induction 20 2

1 10 *** cyp 2b2 cyp 3a1 0 0

C PB Dose-Response Curves 60

50 cyp3a1 cyp 2b2 40

Fig 5.3. PB Induction Profiles for Rodent cyp2b2 and cyp3a1 in rat hepatocytes.

Primary rat hepatocytes were isolated and cultured as described previously (source), prior to treatment with the following compounds at indicated concentrations: PB (100 μM),

DEX (10 μM), PCN (10 μM), MD (10, 25 or 50 μM) or 3MC (5 μM) for 24 h. Cells were harvested, RNA was isolated, and real-time PCR was performed for (A) cyp2b2 and (B) cyp3a1 as described previously. All data are expressed as mean ± S.D. (n = 3). ***, p ≤

0.001; **, p ≤ 0.01; *, p ≤ 0.05.

127

trend has been reported for cyp2b2 and cyp3a1 in rat. That MD could possibly mimic a pattern similar to PB in rat is a potentially novel finding that may provide support for MD functioning similar to known indirect activator PB, however this warrants further investigation.

5.2 Future Directions and Conclusions

Some future directions for this work include obtaining more MD induction profiles in additional primary rat hepatocyte cultures, as well as obtaining profiles in mouse hepatocytes, and possibly also in higher species. Panels of MD induction profiles across different species could be compared against PB concentration ranges, and subsequently, against both PXR and CAR activation profiles in those species. Close attention should be paid to the considerable species-specific differences for each target gene and each nuclear receptor to be investigated. Use of humanized rodent models may be desirable for further testing of compounds that have profiles already obtained in human primary hepatocytes, although the cost and maintenance of such models is expensive and time-consuming. Otherwise, after obtaining profiles in primary hepatocyte cultures across species, in vivo studies to find the relevant induction window for detecting DME induction within the corresponding applicable in vivo models would be appropriate.

Overall, the objectives of these studies were to: 1) screen several different compounds, including popular drugs of abuse, opioids, and anticancer agents for nuclear receptor activation potential, 2) to determine the expression profiles of key DMEs or drug transporters for drugs of abuse in human primary hepatocyte cultures, and 3) to

128

characterize the mechanistic roles of xenoreceptors PXR & CAR underlying observed

DME modulation. Cell-based reporter assay screening identified seven compounds as potential NR activators, and subsequently, studies in human primary hepatocytes were conducted to obtain DME induction profiles. Of the compounds for which induction profiles were obtained, for two opioids, MD and BUP, multiple mechanistic studies to investigate the roles played by xenosensors PXR and CAR were performed. In these studies, MD was shown for the first time to potently and concentration-dependently induce CYP2B6; as well as efficiently and non-stereoselectively activate hPXR.

Additionally, MD appears to mimic indirect CAR activator PB. For MD, the highest

DME induction was achieved for CYP2B6 and CYP3A4, as compared to only slight induction observed for phase II enzyme UGT1A1 or transporter MDR1. For BUP, treatment resulted in virtually no induction of target genes CYP2B6 and CYP3A4 despite results obtained in HeG2 cells. Taken together, MD acted as potent CYP2B6 inducer, as a moderate CYP3A4 inducer, and MD modulates DMEs much more strongly than BUP.

Interestingly, these findings may have impact on whether or not BUP then may be the more desirable choice for maintenance over MD in some instances, since it seems that

BUP may be less likely than MD to be involved in adverse interactions involving

CYP2B6, PXR or CAR.

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5.3 References

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2. Tolson, A. H.; Wang, H., Regulation of drug-metabolizing enzymes by xenobiotic

receptors: PXR and CAR. Advanced Drug Delivery Reviews 2010, 62 (13), 1238-

1249.

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