THE PHARMACOLOGY, PHARMACOKINETICS AND METABOLISM OF A NOVEL NONSTEROIDAL SELECTIVE ANDROGEN RECEPTOR MODULATOR. DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Minoli A. Perera, Pharm.D. * * * * * * The Ohio State University 2003 Dissertation Committee: Professor Kenneth Chan, Adviser Approved by Professor William Hayton Professor Robert Lee _______________________ Professor Duxin Sun Adviser Professor Fredika Robertson College of Pharmacy ABSTRACT Testosterone is an important endogenous male hormone and used exogenously to treat a wide variety of disorders. However, there are many disadvantages to testosterone therapy, namely nonselective action, low oral bioavailability, and an unfavorable side effects profile. This has led to the investigation for compounds that bypass these obstacles. These compounds are known as selective androgen receptor modulators (SARMs). During in vitro studies in our laboratory, we discovered modifications to the structures of the known nonsteroidal antiandrogens, bicalutamide, produced androgen agonist activity. We used these preliminary finding to synthesize a series of bicalutamide analogs to determine if they possessed in vitro agonist activity. These studies identified the chemical modifications that increase binding to the androgen receptor (AR) and AR- mediated transcriptional activation. In addition, in vivo pharmacologic studies were conducted on compounds showing the most promising in vitro results. From this study we discovered the first nonsteroidal anabolic SARM. These findings established the structure-activity relationships (SARs), which lead to the development the lead compound, S-4. ii Pharmacokinetic studies of oral and intravenous doses were conducted in beagle dogs to determine the oral bioavailability and pharmacokinetic parameters associated with S-4. These studies showed that S-4 had a shorter half-life than bicalutamide (4 hours versus to 7 days), with a moderate to large volume of distribution (1.4 L/kg). In addition, S-4 showed dose dependent oral bioavailability, with the highest bioavailability of 91% seen following 0.1 mg/kg dose. Earlier investigations of compounds in this series showed that the AR agonist, R- 1 was metabolically converted to an AR antagonist through oxidation of the sulfur atom. In order to preserve the agonistic effects of these compounds, an oxygen atom was substituted for the sulfur atom on the B ring of the molecule. The metabolism and disposition of S-4 was studied to determine what metabolites were produced. A number of phase I and phase II metabolites were also identified in the urine and feces of dogs and rats on the basis of mass spectrometry. A mass balance and disposition study was conducted using [C14 ] S-4. The metabolites were quantified urine and feces. Total recovery of radioactivity was 97% and 83% after the 24 and 48-hours studies respectively. These studies showed that S-4 was extensively metabolized and excreted in both the urine and feces. The major metabolite of S-4 was the hydrolysis product found in the urine, which accounted for 25% of injected doses. In addition a large portion of the dose, approximately 32% was seen as parent compound in the feces. This may be due to biliary excretion of the glucuronide conjugates of S-4 or the parent drug itself. iii Dedicated to my parents and family, friends, and Eric iv ACKNOWLEDGMENTS I wish to thank my adviser, Dr. Kenneth Chan, for all his encouragement, and intellectual support. He allowed the scientific and scholarly freedom that pushed this project to its fruition. I also wish to thank Drs. Duane Miller and James Dalton, for their unwavering support both intellectually and scientifically. Your enthusiasm for “small victories” and overall vision has been inspiring. This work was supported by grants from the following organizations: The University of Tennessee, College of Pharmacy, and the National Institutes of Health (1 RO1 DK59800), American Foundation for Pharmaceutical Education (AFPE), and GTx., Inc. v VITA January 26, 1976 ............................................Born - Columbo, Sri Lanka May 1993 ................................................B.S. Biology, Christian Brothers University. June 2001 ................................................Pharm.D., University of Tennessee 2001- present ................................................Research Associate, The Ohio State University PUBLICATION 1. Perera MA, Yin D, Chung K, Chan KK, Miller DD and Dalton JT. Metabolism of a Novel Androgen Receptor Modulator. American Association of Pharmaceutical Scientists, Salt Lake City, Utah. October 2003. 2. Yin D, He Y, Perera MA, Hong SS, Marhefka C, Stourman N, Kirkovsky L, Miller DD, and Dalton JT. Key Structural Features of Nonsteroidal Ligands for Binding and Activation of the Androgen Receptor. Molecular Pharmacology 63 (2003) 211-223. 3. He Y, Yin D, Perera MA, Kirkovsky L, Stourman N, Dalton JT and Miller DD. Novel Nonsteroidal Ligands with High Affinity and Potent Functional Activity for the Human Androgen Receptor. European Journal of Medicinal Chemistry 37 (2002) 619-637. 4. Perera MA, Yin D, Chung K, Miller DD, and Dalton JT. Pharmacokinetics and Allometric Scaling of Andarine. American Association of Pharmaceutical Scientists, Toronto, Canada November 2002 . vi 5. Perera MA, Yin D, Chung K, Miller DD, and Dalton JT. Pharmacokinetics of Androxolutamide (GTx-007) in beagle dogs. The Endocrine Society, San Francisco, June 2002 6. Veverka K, Perera MA, Yin D, Gao W, Xu H, Kearbey JD, Chung K, Steiner MS, Miller DD, and Dalton JT. Preclinical Pharmacology and Pharmacokinetics of GTx-007, A Novel Selective Androgen Receptor Modulator. Society for Basic Urological Research, Tucson, AZ, December 2001 7. Dalton JT, Yin D, Perera MA, Gao W, Xu H, Kearbey JD, Chung K, and Miller DD. Preclinical Pharmacology and Pharmacokinetics of a Selective Androgen Receptor Modulator. International Society for Study of Xenobiotics. Drug Metabolism Reviews, 33(supplement 1): #222, 2001. 8. Reiner A, Perera MA, Paullus R, Medina L. Immunohistochemical localization of DARPP32 in striatal projection neurons and striatal interneurons in pigeons. Journal of Chemistry and Neuroanatomy 16 (1998) 17-33. FIELD OF STUDY Major Field: Pharmacy vii TABLE OF CONTENTS Page Abstract ......................................................................................................................ii Dedication..................................................................................................................iv Acknowledgments......................................................................................................v Vita ........................................................................................................................vi List of Tables .............................................................................................................xi List of Figures ............................................................................................................xiv List of Abbreviations .................................................................................................xvii Chapters: 1 BACKGROUND INFORMATION ............................................... 1 1.1. Testosterone ............................................................................................................ 1 1.2. Androgen Receptor................................................................................................. 2 1.3. Physiological Role of Testosterone ........................................................................ 7 1.4. Therapeutic Uses of Testosterone ........................................................................... 9 1.5. Ligands for the Androgen Receptor...................................................................... 10 1.6. Structure Activity Relationships for the Androgen Receptor ............................... 13 1.7. Selective Androgen Receptor Modulators ............................................................ 15 1.8. Andropause ........................................................................................................... 16 1.9. Overview of Dissertation...................................................................................... 18 2 IN VITRO AND IN VIVO PHARMACOLOGY OF NONSTEROIDAL ANDROGEN RECEPTOR LIGANDS ............................................ 22 2.1. Introduction........................................................................................................... 22 2.2. Materials and Methods.......................................................................................... 25 viii 2.2.1 Chemicals...................................................................................... 25 2.2.2 Buffers........................................................................................... 26 2.2.3 Preparation of Cytosolic AR................................................. 262.2.4 Competitive AR Binding Assay.................................................... 27 2.2.5 Determination of AR Binding Affinity......................................... 28 2.2.6 AR Mediated Transcriptional Activation Assay Method ............. 29 2.2.7 Animals ......................................................................................... 30 2.2.8 In vivo Pharmacologic Study Method..................................... 302.3. Results..........................................................................................