Pharmacokinetics, Metabolism, and Pharmacologic Activities of Nonsteroidal Selective Androgen Receptor Modulators and Their Potential Application to Osteoporosis

Home , Fluoxymesterone, Hydroxyflutamide

PHARMACOKINETICS, METABOLISM, AND PHARMACOLOGIC ACTIVITIES OF NONSTEROIDAL SELECTIVE ANDROGEN RECEPTOR MODULATORS AND THEIR POTENTIAL APPLICATION TO OSTEOPOROSIS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the

Graduate School of The Ohio State University

Juhyun Kim, M.S.

*****

The Ohio State University 2006

Dissertation Committee:

Dr. James T. Dalton, Advisor Approved by

Dr. Robert W. Brueggemeier

Dr. William L. Hayton

Dr. Thomas D. Schmittgen Advisor Graduate Program in Pharmacy

ABSTRACT

The overall objective of this dissertation was to develop a safe, orally active, and affordable drug for the treatment of osteoporosis in men and women. Based on a report by the National Institutes of Health in 2000, 10 million individuals already have osteoporosis, and 18 million more have low bone mass, placing them at increased risk for this disorder. It is estimated that $ 10 to $15 billion are spent each year for treatment of osteoporotic fracture. Osteoporotic fractures, particularly vertebral fractures, can result in chronic disabling pain. Although abundant data suggest that androgens are useful for the treatment for osteoporosis, undesirable side effects and pharmacokinetic properties limit their clinical uses.

Nonsteroidal selective androgen receptor modulators (SARMs), which we were the first to develop, display higher anabolic activity than androgenic activity in vivo

without androgen-related side effects. These nonsteroidal SARMs are more amenable to

structural modification and demonstrate high oral bioavailability with long half-life. Due

to their favorable characteristics, SARMs are promising as a new treatment of

osteoporosis.

In the current study, in vitro structure-activity relationship of novel SARMs

modified in the aromatic B-ring was analyzed by determining in vitro AR binding affinity ii

and AR-mediated transcriptional activation (Chapter 2). In vivo pharmacologic activity

and pharmacokinetics of 4-halogen substituted SARMs were determined and based on

results of this study, we hypothesized that two simple criteria (i.e., Ki < 10 nM and lower

in vivo clearance) could be used to identify efficacious and potent SARMs. Using these

criteria, we identified S-22 as a compound with the most potent and tissue-selective in

vivo activity that we have observed to date and favorable pharmacokinetic properties

(Chapter 3). In vivo metabolism and plasma protein binding of a series of SARM in male

rats were determined. Halogen-substituted SARMs underwent three major phase I

metabolism pathways; 1) hydrolysis of the amide bond, 2) B-ring hydroxylation, 3) A-

ring nitro reduction to an aromatic amine. Some of these phase I metabolites underwent

phase II metabolisms including sulfation and/or glucuronidation. Moreover, plasma

protein binding study suggests that the total clearance of halogen-substituted SARMs was

significantly affected by the fraction of drug unbound (Chapter 4). The effects of a

SARM (S-22) alone or in combination with SERM (i.e., raloxifene) in bone, muscle,

uterus, and body composition were determined. S-22 and raloxifene additively increase

bone density and improve microarchitecture of bone with less stimulating effect on uterus

than steroidal counterpart. Moreover, S-22 exerted anabolic effect on soleus muscle

(Chapter 5). The effects of SARM (S-22) alone or in combination with SERM

(raloxifene) on bone cells in vitro were also studied (Chapter 6).

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Dedicated to my parents and Tai

ACKNOWLEDGMENTS

I would like to deeply thank my advisor, Dr. James T. Dalton, for his guide, support, and understanding. He has patiently advised me to be a better “re-searcher” and scientist. He is such a great person and teacher and I am so blessed to have spent this time with him.

I appreciate Dr. Duane D. Miller and his research group for syntheis of the compounds used in my studies. I would like to thank Dr. Peter J. Reiser for generously giving his time and expertise on muscle strength measurement studies. I also thank my other committee members, Dr. Robert W. Brueggemeier, Dr. William L. Hayton, and Dr.

Thomas D. Schmittgen for their valuable discussion. I sincerely appreciate all my lab mates for their friendship and help.

I express thanks to my Mom and Dad, who is now in haven, for their unconditional love, support, and sacrifice. I would like to thanks my husband, Tai Ahn, for his love, encouragement, and support.

VITA

August 21, 1976………………………….. Born – Gwangju, South Korea

1998……………………………………… B.S. Pharmacy Chosun University, Gwangju, South Korea

2000 ………………………………………M.S. Pharmaceutics Chosun University, Gwangju, South Korea

1998 - 2000……………………………… Graduate Research Associate Chosun University

2000 - 2001……………………………… Intern Researcher Korean Science and Engineering Foundation

2001 - 2003………………………..………Graduate Teaching Associate The Ohio State University

2004 ……………………………………... Summer Intern PDM department, Pfizer Inc., St. Louis, MO

2003 - present…………………………… Graduate Research Associate The Ohio State University

PUBLICATIONS

Research Publications

1. Gao W, Kim J , Dalton JT, Pharmacokinetics and Pharmacodynamics of Nonsteroidal Androgen Ligands, Pharm Res. 23(8): 1641-1658, 2006

2. Kim J , Wu D, Hwang DJ, Miller DD, Dalton JT, The Para Substituent of S-3- (Phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamides Is a Major Structural Determinant of in Vivo Disposition and Activity of Selective Androgen Receptor Modulators, J Pharmacol Exp Ther. 315(1): 230-9, 2005

3. Chen J, Kim J , Dalton JT. Discovery and Therapeutic Promise of Selective Androgen Receptor modulators, Molecular Interventions 5(3): 173-188, 2005

4. Marhefka CA, Gao W, Chung K, Kim J , He Y, Yin D, Casey B, Dalton JT, Miller DD, Design, Synthesis, and Biological Characterization of Metabolically Stable Selective Androgen Receptor Modulators. J Med. Chem. 47(4): 993-998, 2004

5. Kim J and Choi H-K, Effects of Additives on the Crystallization and the Permeation of Ketoprofen from Adhesive matrix. Int. J. Pharm. 236: 81-85, 2002

6. Kim J , Lee CH, Choi H-K, Transdermal Delivery of Physostigmine: Effect of Enhancers and Pressure Sensitive Adhesives, Drug Devel. Ind. Pharm., 28, 7: 833-9, 2002

7. Kim J , Lee CH, Choi H-K, A method to Measure the Amount of Drug Penetrated across the Nail Plate. Pharm Res. 18, 10: 1468-71, 2002

8. Kim J and Choi H-K, Complexation of Prioxicam and Tenoxicam with Hydroxypropyl-β-cyclodextrin, J. Kor. Pharm. Sci., 33-37, 2000

9. Kim J , Cho Y-J, Choi H-K, Effect of Vehicles and Pressure Sensitive Adhesives on the Permeation of Tacrine across Hairless Mouse Skin. Int. J. Pharm. 196: 105-113, 2000

FIELD OF STUDY

Major Field: Pharmacy

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TABLE OF CONTENTS

Page

Abstract...... ii

Dedication...... iv

Acknowledgments...... v

Vita...... vi

List of Tables ...... xiii

List of Figures...... xviii

Abbreviations...... xx

Chapters:

1. Introduction ...... 1

1.1 Androgens...... 1 1.2 Androgen Receptor...... 3 1.3 Steroidal Androgens...... 4 1.4 Development of Nonsteroidal Selective Androgen Receptor Modulators ...... 6

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1.5 Actions of Androgen...... 7 1.5.1 Bone ...... 8 1.5.2 Muscle...... 9 1.6 Therapeutic Promise of SARMs ...... 9 1.6.1 Osteoporosis...... 10 1.6.2 Muscle Wasting...... 13 1.7 Overview of Dissertation and Research...... 16

2. In Vitro Structure Activity Relationship of Nonsteroidal Selective Androgen Receptor Modulators ...... 27

2.1 Introduction...... 27 2.2 Materials and Methods...... 29 2.2.1 Chemical and Animals...... 29 2.2.2 Prepartionof Cytosolic ARs...... 30 2.2.3 AR Competitive Binding Assay...... 31 2.2.4 AR-Mediated Transcriptional Activation...... 32 2.3 Results and Discussion...... 33 2.3.1 B-ring Modificaiton...... 33 2.3.2 Linkage Modificaiton...... 37 2.4 Conclusion...... 38

3. The 4-para Substitueent of S-3-(phenoxy)-2-hydroxy-2-methyl-N-(4-mitro- 3-trifluoromethyl-phenyl)-propionamides is a Major Structural Determinant of In Vivo Disposition and Activity of Selective Androgen Receptor Modulators ...... 44

3.1 Introduction...... 44 3.2 Materials and Methods...... 47 3.2.1 Chemicals...... 47 3.2.2 In Vitro Pharmacologic Activity...... 47 3.2.3 Assay for In Vivo Pharmacological Activity in Rats...... 48 3.2.4 Pharmacokinetic studies...... 50 3.2.5 Bioanalytical Methods...... 50 3.3 Results ...... 52 3.3.1 Halogen Substituted SARMs...... 52

3.3.1.1 In Vivo Phamacological Activity of 4-Halogen Substituted SARMs ...... 52 3.3.1.2 Pharmacokinetics of 4-Halogen Substituted SARMs.....54 3.3.1.3 Exposure-Response Relationship for SARMs...... 55 3.3.2 Cyano/Nitro Group Substituted SARMs...... 55 3.3.2.1 Binding Affinity and In Vitro Functional Activity of Cyano/Nitro Group Substituted SARMs ...... 55 3.3.2.2 Pharmacokinetics of Cyano/Nitro Group Substituted SARMs...... 56 3.3.2.3 In Vivo Phamacological Activity of Cyano/Nitro Group Substituted SARMs ...... 57 3.4 Discussion...... 58

4. Effect of Para Halogen Modification of S-3-(phenoxy)-2-hydroxy-2-methyl- N-(4-nitro-3-trifluoromethyl-phenyl)-propionamides on Metabolism; Fraction Unbound vs Intrinsic Clearance ...... 75

4.1 Introduction...... 75 4.2 Materials and Methods...... 78 4.2.1 Chemicals...... 78 4.2.2 Animals...... 78 4.2.3 Metabolism Studies...... 78 4.2.4 LC-MS n Analysis...... 79 4.2.5 Plasma Protein Binding Study...... 80 4.2.6 LC-MS Analysis...... 82 4.3 Results ...... 82 4.3.1 Identification of Metabolites of S-9, S-10, and S-11 in Rats...... 83 4.3.1.1 Metabolites of S-9 in Rats ...... 83 4.3.1.2 Metabolites of S-10 in Rats...... 87 4.3.1.3 Metabolites of S-11 in Rats...... 88 4.3.2 Plasma Protein Binding Study of Various SARMs ...... 89 4.4 Discussion ...... 90

5. Anabolic Effects of Selective Androgen Receptor Modulator Alone and in Combination with Selective Estrogen Receptor Modulator on Bone and Muscle ...... 104

5.1 Introduction...... 104 5.2 Materials and Methods...... 108 5.2.1 Materials...... 108 5.2.2 Animals and Experimental design...... 108 5.2.3 Dexa Analysis...... 110 5.2.4 Muscle Strength Measurement...... 110 5.2.5 Micro CT Measurement ...... 112 5.3 Results ...... 112 5.3.1 Body Weight and Tissue Weights...... 112 5.3.2 BMD and Body Composition...... 115 5.3.3 Soleus Muscle Strength...... 117 5.3.4 Structural Parameters of Trabecular Bone...... 118 5.4 Discussion ...... 119

6. Effects of Selective Androgen Receptor Modulator Alone and in Combination with Selective Estrogen Receptor Modulator on Osteoblast and Osteoclast...... 134

6.1 Introduction...... 134 6.2 Materials and Methods...... 137 6.2.1 Experimetal Animals and Materials...... 137 6.2.2 Cell Cultures...... 138 6.2.3 Proliferation Studies...... 139 6.2.4 Alkaline Phosphatase Activity ...... 140 6.2.5 Alizarin Red-S Assay ...... 140 6.2.6 TRAP Assay ...... 141 6.2.7 Statistical Analysis ...... 141 6.3 Results ...... 142 6.3.1 Effect on Proliferation, Differentiation, and Mineralization of Osteoblast...... 142 6.3.2 Effect on Osteoclastogenesis...... 144 6.4 Discussion ...... 145

7. Summary...... 154

BIBLIOGRAPHY...... 157

Apendices:

Appendix A: Data Related to Chapter 2 ...... 172 Appendix B: Data Related to Chapter 3 ...... 182 Appendix C: Data Related to Chapter 4 ...... 208 Appendix D: Data Related to Chapter 5 ...... 211 Appendix E: Data Related to Chapter 6...... 245

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LIST OF TABLES

Table Page

1.1 Mode of application and dosage of various androgen preparations ...... 17

1.2 Effects of androgens on osteoblast proliferation ...... 18

1.3 Effect of androgens on osteoblast differentiation markers ...... 19

2.1 In vitro AR binding affinities of B-ring modified SARMs ...... 42

2.2 AR binding affinities and transcriptional activation of linkage group modified compounds...... 43

3.1 Chemical structures for nonsteroidal AR ligands ...... 63

3.2 Mean androgenic and anabolic activities of 4-halogen substituted SARMs to IP in castrated male rats ...... 64

3.3 Pharmacokinetic parameters of 4-halogen substituted SARMs in male rats after a single intravenous or oral administration at 10 mg/kg ...... 65

3.4 Chemical structure, AR relative binding affinity, in vitro transcriptional activities, and pharmacokinetic parameter of cyano/nitro group substituted SARMs...... 66

4.1 Structures and plasma protein binding of nonsteroidal AR ligands ...... 94

4.2 LC-MS analysis condition to measure drug concentration in plasma side and buffer side ...... 95

4.3 Proposed structure of S-9 and its urinary metabolites in rats ...... 97

4.4 Proposed structure of S-10 and its urinary metabolites in rats ...... 98

4.5 Proposed structure of S-11 and its urinary metabolites in rats ...... 99

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5.1 Animal status, treatment regimen, and animal number of treatment groups...... 125

5.2 Body weight, soleus muscle weight, and soleus muscle weight to body weight ratio in different treatment groups ...... 126

5.3 Body mineral density (BMD) and % of fat in different treatment groups. Statistical significance was determined within weight bearing or hindlimb unloading...... 127

5.4 Contractile properties of the soleus muscle in different treatment groups ...... 128

5.5 3D parameters of trabecular bone in femur analyzed by µCT...... 129

A.1 Competitive binding affinity of S-23 and S-25 to the AR...... 173

A.2 Competitive binding affinity of S-26 and S-27 to the AR...... 174

A.3 Competitive binding affinity of S-28 and S-29 to the AR...... 175

A.4 Competitive binding affinity of S-30 and S-31 to the AR...... 176

A.5 Competitive binding affinity of S-32 and S-33 to the AR...... 177

A.6 Competitive binding affinity of S-34 and S-35 to the AR...... 178

A.7 Competitive binding affinity of R-2 and R-3 to the AR ...... 179

A.8 Competitive binding affinity of R-5 and R-8 to the AR ...... 180

A.9 Competitive binding affinity of R-9 to the AR...... 181

B.1 Competitive binding affinity of S-9 and S-10 to the AR...... 183

B.2 Competitive binding affinity of S-11 to the AR ...... 184

B.3 Pharmacokinetic parameters of S-9 in rats after i.v. administration...... 185

B.4 Pharmacokinetic parameters of S-9 in rats after p.o. administration...... 186

B.5 Pharmacokinetic parameters of S-10 in rats after i.v. administration...... 187

xiv

B.6 Pharmacokinetic parameters of S-10 in rats after p.o. administration...... 188

B.7 Pharmacokinetic parameters of S-11 in rats after i.v. administration...... 189

B.8 Pharmacokinetic parameters of S-11 in rats after p.o. administration...... 190

B.9 Competitive binding affinity S-19 and S-20 to the AR ...... 191

B.10 Competitive binding affinity S-21 and S-22 to the AR ...... 192

B.11 Transcriptional activational activities of various SARMs...... 193

B.12 Pharmacokinetic parameters of S-19 after i.v. administration...... 194

B.13 Pharmacokinetic parameters of S-20 after i.v. administration...... 195

B.14 Pharmacokinetic parameters of S-21 after i.v. administration...... 196

B.15 Pharmacokinetic parameters of S-22 after i.v. administration...... 197

B.16 Normalized tissue weights of androgenic and anabolic organs in S-9 administered male rats ...... 198

B.17 Normalized tissue weights of androgenic and anabolic organs in S-10 administered male rats ...... 199

B.18 Normalized tissue weights of androgenic and anabolic organs in S-11 administered male rats ...... 200

B.19 Plasma concentration of S-9 after i.v. or p.o. administration ...... 201

B.20 Plasma concentration of S-10 after i.v. or p.o. administration ...... 202

B.21 Plasma concentration of S-11 after i.v. or p.o. administration ...... 203

B.22 Plasma concentration of S-19 and S-20 after i.v. administration ...... 204

B.23 Plasma concentration of S-21 and S-22 after i.v. administration ...... 205

B.24 Normalized tissue weights of anabolic and adrogenic organs in male rats administered with cyano/nitro substituted SARMs ...... 206

B.25 Normalized tissue weights of anabolic and adrogenic organs in S-22 administered male rats ...... 207

C.1 Concentration of drug bound (CB) and unbound (CU) in plasma protein binding study...... 209

C.2 Concentration of drug bound (CB) and unbound (CU) of S-22 in plasma protein binding study ...... 210

D.1 Body, soleus, and uterus weight of OVX rats administered with various compounds ...... 212

D.2 DEXA parameters of OVX rats administered with various compounds ...... 215

D.3 Parameters for muscle strength measurement...... 223

D.4 Micro CT parameters of group 1 ...... 225

D.5 Micro CT parameters of group 1 and 2...... 226

D.6 Micro CT parameters of group 2 ...... 227

D.7 Micro CT parameters of group 2 and 3...... 228

D.8 Micro CT parameters of group 3 ...... 229

D.9 Micro CT parameters of group 4 ...... 230

D.10 Micro CT parameters of group 4 and 5...... 231

D.11 Micro CT parameters of group 5 ...... 232

D.12 Micro CT parameters of group 6 ...... 233

D.13 Micro CT parameters of group 6 and 7...... 234

D.14 Micro CT parameters of group 7 ...... 235

xvi

D.15 Micro CT parameters of group 7 and 8...... 236

D.16 Micro CT parameters of group 8 ...... 237

D.17 Micro CT parameters of group 9 ...... 238

D.18 Micro CT parameters of group 9 and 10...... 239

D.19 Micro CT parameters of group 10 ...... 240

D.20 Micro CT parameters of group 10 and 11...... 241

D.21 Micro CT parameters of group 11 ...... 242

D.22 Micro CT parameters of group 11 and 12...... 243

D.23 Micro CT parameters of group 12 ...... 244

E.1 Proliferation of osteoblast...... 246

E.2 ALP activities in MC3T3-E1 cells...... 247

E.3 Alizarin Red-S activities in MC3T3-E1 cells...... 248

E.4 TRAP activities in MC3T3-E1 cells...... 249

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LIST OF FIGURES

Figure Page

1.1 Biosynthesis pathways of sex steroids in testis and peripheral tissue ...... 20

1.2 Hypothalamus-pituitary-testis axis of androgen regulation...... 21

1.3 Structural and fuctional organization of androgen receptor protein ...... 22

1.4 Various possibilities for metabolism of testosterone...... 23

1.5 The chemical structures of steoroidal androgens...... 24

1.6 The chemical structures of AR nonsteroidal ligands ...... 25

1.7 Bone remodeling cycle ...... 26

2.1 Chemical structure of nonsteroidal AR antagonists (flutamide and bicalutamide) and nonsteroidal AR agonists (S-1 and S-4)...... 39

2.2 AR-mediated transcriptional activation of S-25, S-27, S-36, and S-37...... 40

2.3 Antagonist activities of R-8 and R-9 on DHT-induced transcription...... 41

3.1 Dose-response relationships of S-9, S-10, and S-11 in androgenic and anabolic organs of castrated male rats ...... 68

3.2 Plasma concentrations of 4-halogen substituted SARMs after a single intravenous dose of 10 mg/kg or a single oral dose of 10 mg/kg to male rats...... 70

3.3 AUC-response relationship of S-4 and halogen substituted SARMs in the levator ani muscle ...... 71

3.4 Plasma concentration of S-19, S-20, S-21, and S-22 after a single intravenous dose of 10 mg/kg to male rats ...... 72

xviii

3.5 In vivo pharmacologic activity of S-19, S-20, S-21, and S-22 in androgenic and anabolic organs of castrated male rats ...... 73

3.6 Dose-response relationships of S-22 in androgenic and anabolic organs of castrated male rats ...... 74

4.1 spectra of M9-1 (A) and M10-1 (B) isolated from rat urine...... 100

4.2 Proposed major metabolism pathways of S-9, S-10, and S-11...... 101

4.3 Relationship between lipophilicity and plasma protein binding (50 µM) ...... 102

4.4 Unbound fraction of S-22 at various concentrations ...... 103

5.1 Effect of S-22, raloxifene (Ral), 5 α-dihydrotestosterone (DHT) and 17 β- estradiol (E2) in alone and/or combination on uterus in ovariectomized adult rats...... 130

5.2 The Body composition change in (A) hindlimb unloading groups (B) weight bearing groups...... 131

5.3 Pt/CSA (A) and P o/CSA (B) in different treatment groups...... 132

5.4 BMD (A) and percent bone volume (B) measured by µCT...... 133

6.1 Effect of DHT and S-22 on the proliferation of Saos-2 cells ...... 147

6.2 Effect of DHT and S-22 on the proliferation of hFOB-AR6 cells...... 148

6.3 Time course study of DHT (1 nM to 10 µM) in MC3T3-E1 cells ...... 149

6.4 ALP activities of MC3T3-E1 cells treated with S-22, raloxifene, estradiol, and DHT alone (A), and S-22 and raloxifene in combination (B)...... 151

6.5 Mineralization of MC3T3-E1 cells treated with S-22, raloxifene, estradiol, and DHT...... 152

6.6 Effect of S-22 and raloxifene alone or in combination on differentiation of bone morrow cell toward osteoclast ...... 153

xix

ABBREVIATIONS

ANOVA Analysis of variance BMC Bone mineral content AR Androgen Receptor ARE Androgen Response Elements AUC Area under the curve AUC ∞ Area under the plasma concentration-time curve from time zero to infinity BMD Bone Mineral Density BMA Bone mineral area BPH Benign Prostate Hyperplasia BS/TV Bone surface density BV/TV Percent trabecular bone volume CL Clearance CSA Cross sectional area DA Degree of anisotropy DBD DNA-binding domain DEXA Dual energy x-ray absorptiometry DHEA Dehydroepiandrosterone DHT Dihydrotestosterone DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethyl sulfoxide Emax Maximal pharmacologic effect ESI Electrospray ionization Fp.o. Oral bioavailability HAP Hydroxyapatite i.S Intersection surface i.v. Intravenous FBS Fetal bovine serum FSH Follicle-Stimulating Hormone GnRH Gonadotropin-Releasing Hormone MIB Mibolerone LH Luteinizing Hormone LBD Ligand Binding Domain PBS Phosphate buffer solution PEG 300 Polyethylene glycol 300 PMSF Phenylmethylsulfonly fluoride xxi

PTH Parathyroid Hormone Po Tetanus amplitude Pt Maximal twitch tension SAR Structure and Activity Relationships SARM Selective Androgen Receptor Modulator SERM Selective Estrogen Receptor Modulator SHBG Sex hormone binding globulin SIM Single ion monitoring SMI Structure model index TBP TATA-Box Binding Protein Tb.Pf Trabecular pattern factor Tb.Th Trabecular thickness Tb.N Trabecular number Tb.Sp Trabecular separation TPt Time to peak twitch tension T1/2R Time to one-half relaxation TP Testosterone propionate T1/2 The terminal half-life Vss Apparent volume of distribution at equilibrium

xxii

CHAPTER 1

INTRODUCTION

1.1 Androgens

Testosterone is the major circulating androgen in the human male and female [1].

In males, more than 95% of circulating testosterone (approximately 6-7 mg per day) is secreted by the testis and less than 5% of the testosterone is produced by peripheral tissues, mainly the adrenal cortex [2-4]. In females, 50% of circulating testosterone is secreted by the adrenal glands and ovaries and the rest is produced by the conversion of dehydroepiandrosterone (DHEA) and androstenedione in peripheral tissues [5]. Figure

1.1 shows the steroidogenic pathways in the human testis and peripheral tissues. The source material of steroid biosynthesis is cholesterol. This substrate seems to be supplied by the transport from lipoproteins or de novo synthesis from acetate [6]. The conversion

of cholesterol (C 27 ) to pregnenolone (C 21 ) in the mitochondria is the start of the steroidogenic cascade. Subsequently, pregnenolone undergoes a series of side-chain cleavage and hydroxylation reactions in the smooth endoplasmic reticulum to form

DHEA and androstenedione [7, 8]. DHEA and androstenedione are the major end products of steroid biosynthesis in the adrenal gland, and their androgenic activities are 1

less than 10% of that of testosterone. In the Leydig cells of the testes, androstenedione is converted into testosterone by 17 β-hydroxysteroid dehydrogenase. Testosterone

circulates in the blood and plays an important role in the hypothalamus-pituitary-testis

loop to control the proper secretion of sex steroids and the induction of virilization of

internal sex organs (i.e., testis, epididymis, vas deferens, and seminal vesicles). Certain

peripheral tissues, such as adult reproductive tissues and skin, convert testosterone to

dihydrotestosterone (DHT) via 5 α-reductase. The conversion of testosterone to DHT

amplifies the activity of androgen since DHT binds the androgen receptor (AR) with a

higher binding affinity than testosterone [1, 9]. Testosterone is alternatively converted to

estradiol by aromatase (CYP19) in adipose tissue, certain brain nuclei and other tissues

[10].

The concentration of androgen in the circulation is tightly regulated by the

hypothalamus-pituitary-testis axis (Figure 1.2) [11]. Gonadotropin-releasing hormone

(GnRH), which is released from the hypothalamus, binds to the cell surface receptor of

pituitary gonadotropes and stimulates the secretion of luteinizing hormone (LH) and

follicle-stimulating hormone (FSH) [12]. The released gonadotropins, LH and FSH,

increase the expression of steroidogenic genes in Leydig cells, resulting in higher levels

of testosterone secretion. When testosterone reaches high levels, it inhibits the secretion

of GnRH and gonadotropin as means of negative feedback. Besides Leydig cells, Sertoli

cells of the testis respond to FSH by releasing important factors for sperm maturation,

inhibin and activin, of which the latter two control gonadotropin secretion [13].

1.2 Androgen Receptor

Androgens, mediating a various physiological responses, act through AR. The AR belongs to the nuclear receptor superfamily. This family includes the receptors for endogenous hormones, such as androgens, estrogens, progestins, glucocorticoids, and mineralocorticoids, retinoids, thyroid hormones, vitamin D, fatty acids and other small hydrophobic molecules [14]. Based on inheritance patterns of AR-related dysfunction, the AR gene was theorized to exist in the X chromosome. The cloning of the AR cDNA revealed that this hypothesis was correct [15]. Due to a polymorphic variation in the length of a CAG repeat or GGN repeat ends, the sizes of AR cDNA vary. The AR gene has a length of more than 90 kb and consists of eight exons and seven introns [16]. Figure

1.3 shows the domain structure of AR protein. The AR protein can be divided into four functional domains; an N-terminal transactivation domain, a DNA-binding domain

(DBD), a hinge domain, and a ligand binding domain (LBD). Two transcriptional activation functions, AF-1 and AF-2, were also identified by deletion and mutational analyses [17]. AF-1 domain is located in the N-terminal region and functionally independent from ligand binding [17, 18]. On the other hand, mutation or deletion of the

AF-2 domain dramatically reduces AR-mediated transcriptional activation [18].

The unliganded AR (apoAR) is bound to a complex of molecular chaperones

(Hsp90, Hsp70, Hsp40, HOP/p60, p23 and immunophilins). The chaperone proteins help the receptor to fold properly for efficient ligand binding [19]. Upon ligand binding, the conformational changes in the AR trigger the dissociation of chaperone proteins, dimerization, and translocation to the nucleus. The translocated AR-ligand complex

(holoAR) binds to specific DNA sequences called androgen response elements (AREs),

which are located at the promoter or enhancer regions of AR target genes (e.g., probasin, prostate binding protein, and prostate specific antigen) [20-22]. Binding to DNA triggers interaction of the holoAR with components of the transcription machinery including

TFIIH, TFIIF, TATA-box binding protein (TBP), and other cofactor proteins, which subsequently leads to production of target mRNA and protein [23, 24].

1.3 Steroidal Androgens

The overall physiological effects of endogenous steroidal androgens are

contributed largely by testosterone and DHT. Circulating testosterone plays important

roles in the differentiation and growth of male accessory reproductive organs (e.g.,

prostate and seminal vesicles), control of male sexual behavior, and the development and

maintenance of male secondary characteristics including bone, muscle, and hair [1]. Low

endogenous testosterone concentrations are associated with sarcopenia and low bone

mineral density (BMD), lessened muscle strength, and diminished sexual drive and

fuction. Due to the essential actions of androgens in men, they have been used for

hormonal replacement in hypogonadal men. The potential benefits of testosterone

replacement therapy include increase in BMD, improvement in body composition and

strength, sexual function, cognitive function, and mood. However, the potential risks of

this treatment, including those in prostate and blood (e.g., hematocrit and hemoglobin

levels) are widely recognized [25-28].

In spite of the therapeutic benefits of androgens, their clinical use has been limited.

This is partly due to the limitations of current androgen preparations. Table 1.1 shows the

mode of application and dosage of various androgen preparations. Unmodified

testosterone is absorbed well from the gut but is impractical for clinical use due to its rapid hepatic elimination and high variability in absorption and elimination [29]. To overcome this limitation, three approaches have been used: 1) chemical modification of the molecule, 2) esterification at position 17, and 3) different routes of administration.

Introduction of alkyl group in the 17 α position (17 α-methyltestosterone and fluoxymesterone) protects testosterone from hepatic metabolism (Figure 1.4). However, these androgens are obsolete due to a risk of hepatotoxicity with long-term use [30].

Moreover, esterification of the 17 β-position improves absorption and prolongs half-life.

Currently, testosterone undecanoate (Figure 1.5) is available in the market for oral administration, but interindividual and intraindividual variability in serum testosterone levels during therapy is problematic [31, 32]. Intramuscular injection of testosterone esters is also a widely used approach. However, the intramuscular injection of testosterone propionate, testosterone enanthate, testosterone cypionate, and testosterone cyclohexanecarboxylate (Figure 1.5) results in wide fluctuation in testosterone serum concentrations [33, 34]. To maintain effective testosterone serum concentrations, unacceptably frequent doses need to be injected. Intramuscular testosterone undecanoate, recently approved for clinical use, demonstrates more favorable pharmacokinetic characteristics and may be more acceptable for routine clinical use [35]. Androgen administration via alternative routes (e.g., buccal administration, subcutaneous injection of microcapsules, transdermal application by scrotal and non-scrotal patch/gel, and pellet implantation) improves the pharmacokinetic profile but has limitations [30, 36-38].

Microcapsule injection and transdermal patches are associated with early burst release and skin irritation, respectively. Moreover, the disadvantages of subcutaneous pellet

implantation are pellet extrusion (about 10% per procedure), bruising, infection, and the inconvenience of removal.

1.4 Development of Nonsteroidal Selective Androgen Receptor Modulators

Nonsteroidal AR agonists potentially possess several advantages over steroidal androgens, such as favorable pharmacokinetic profile, oral bioavailability, better receptor selectivity, and greater flexibility in structural modification. Due to these potential advantages, several research groups, including ours, are interested in the discovery and development of nonsteroidal AR agonists [39-44]. Our laboratory was the first to report the discovery of nonsteroidal AR ligands having in vivo anabolic and androgenic activities [45]. In our early studies, we synthesized several novel electrophilic nonsteroidal AR ligands in an attempt to identify chemo affinity ligands for AR. The AR binding affinity and ability to stimulate AR-mediated transcriptional activation were determined. Among this initial series of compounds, R-1 (Figure 1.6) showed the most efficacious AR agonist activity. However, owing to concerns about its electrophilic properties that can potentially alkylate nucleophilic sites in cells, Yin et al. designed, synthesized and tested the pharmacologic activity of our first reversible nonsteroidal androgen (i.e., acetothiolutamide), an aryl propionamide derivative with a thioether linkage and a para-acetamido substituent in the aromatic B-ring (Figure 1.4) [46].

Although acetothiolutamide exerted high AR binding affinity and full agonist activity during in vitro studies, its in vivo activity was relatively low [46]. Subsequent pharmacokinetic and metabolism studies demonstrated that the discrepancy between in vitro and in vivo activities was due to extensive hepatic metabolism of acetothiolutamide,

mainly oxidation at the thioether linkage. Therefore, we designed and synthesized a series of compound incorporating an ether linkage instead of a thioether linkage in acetothiolutamide to improve to metabolic stability [45]. Among these compounds, S-1 and S-4 (Figure 1.4) were identified as selective androgen receptor modulators (SARMs) with potent and efficacious anabolic and androgenic activities in vivo . This second generation of SARMs showed full agonistic activity in anabolic tissue (i.e., levator ani muscle) but partial agonistic activity in androgenic tissues (i.e., prostate and seminal vesicles) without affecting the hypothalamus-pituitary-testis axis. These SARMs displayed better pharmacokinetic properties than those of their steroidal counterparts, including higher oral bioavailability, longer half-life, and linear kinetics [47, 48].

Animal models demonstrating that SARMs can be potentially used for benign prostate hyperplasia (BPH), male contraception, muscle wasting, and osteoporosis have been reported recently [48-53]. The great potential of SARMs has generated immense interest and research toward optimization and understanding of SARMs. Based on existing knowledge of AR crystallography, structure and activity relationships (SAR) for ligands, and pharmacokinetics of SARMs, structural modifications on A- and B-ring of

SARMs appear to be one of the most reasonable approaches for ongoing discovery of

SARMs [54, 55].

1.5 Actions of Androgen

Androgens promote growth, differentiation, and function of male reproductive

organs. In addition, androgens play an important role in development and maintenance of

extragenital aspects of the male phenotype including actions on hair growth, skeletal and

muscle growth, fat distribution, and deepening of the voice [1, 56]. The physiological roles for androgens in women have been more difficult to define, but studies suggest that androgens have effects on libido, BMD, and lean body mass in women [56, 57]. In this chapter, the action of androgen in only bone and muscle will be discussed.

1.5.1 Bone

Bone is a living tissue, which is continuously being broken down (i.e., bone resorption) and regenerated (i.e., bone formation). Cells responsible for bone resorption and subsequent bone formation are osteoclasts and osteoblasts, respectively. AR ligands affect BMD by changing osteoblastic activity and osteoclastic activity, which result from cell number and individual cell functional capacity (Figure 1.7) [58]. These actions of AR ligands are mediated directly via the AR and/or via paracrine and autocrine action. The underlying mechanisms for paracrine and autocrine action have been reviewed in detail elsewhere [58]. Androgens seem to have the ability to decelerate the bone remodeling cycle and tilt the focal balance of the cycle toward bone formation. The loss of androgens is thought to increase the rate of bone remodeling by removing restraining effects on osteoblastogenesis and osteoclastogenesis [58]. Also, androgens exert dual effects on the lifespan of mature bone cells, with anti-apoptotic effects on osteoblasts and osteoclasts and pro-apoptotic effects on osteoclasts [59, 60]. DHT also stimulates osteoblast proliferation under experimental conditions (Table 1.2) [61]. The effects on osteoblast differentiation are rather controversial, but most in vitro studies suggest that androgens have a stimulatory effect on alkaline phosphatase, type I collagen, osteocalcin, and

mineralization of the extracellular bone matrix (Table 1.3) [60]. Moreover, DHT has been demonstrated to have a suppressive effect on osteoclast differentiation [62].

1.5.2 Muscle

The mechanisms of androgen action on muscle remain largely unknown. The common hypothesis is that androgens promote muscle protein synthesis [63-65]. There is evidence to support the idea that testosterone supplementation increases muscle protein synthesis in elderly men [63, 64] and young hypogonadal men [65]. Also, androgen- induced increases in muscle mass seem to be due to muscle fiber hypertrophy, rather than hyperplasia [66]. The hypertropic action of androgen is caused by promoting myogenesis via increasing satellite cell numbers. The molecular mechanisms of androgen effect in satellite cell number are not well understood. Bhasin et al. reported that androgen stimulates the differentiation of mesenchymal pluripotent cells to the myogenic lineage

[67, 68]. However, other possible pathways, including increases in satellite cell proliferation and decreases in satellite cell apoptosis are possible but remain unknown.

1.6 Therapeutic Promise of SARMs

SARMs could have utility in the treatment for male hypogonadism, BPH, male contraception, erythropoiesis, osteoporosis, and muscle wasting disease [48-53]. In this chapter, the potential uses of SARMs for osteoporosis and muscle wasting will be discussed.

1.6.1 Osteoporosis

Numerous lines of evidence indicate that androgens are important in bone, and that SARMs may represent a novel approach to the treatment of osteoporosis. Reduction of androgen concentration correlates well with bone resorption markers in men admitted for hip fracture, suggesting that loss of androgen may contribute to bone loss and increased risk of fracture [69]. Loss of androgen by surgical or medical castration increases the cumulative incidence of fracture in prostate cancer patients [70]. Androgen replacement prevents bone loss and increases BMD in hypogonadal men [71]. However, the effect of androgen replacement on fracture risk remains unknown. Women with hirsutism and polycystic ovary syndrome, who have excess endogenous androgen, have increased BMD compared with normal young women [72]. Estrogen and androgen therapy increases BMD to a greater degree than does estrogen therapy alone [73].

An important issue from the perspective of developing SARMs as anabolic agents for bone is whether aromatization of androgen is necessary for anabolic action. In humans, androgen-insensitive males that have inactivating AR mutations are frequently osteopenic [74]. In experimental animals, the non-aromatizable androgens significantly prevent osteopenia that results from orchidectomy [75]. DHT also prevents bone loss after ovariectomy in female rats [76], and flutamide (an AR antagonist) induces osteopenia even in estrogen-replete female rats [77]. In addition to this indirect evidence from studies of steroidal androgens, several studies of SARMs have already demonstrated their potential as a treatment for osteoporosis. SARMs significantly increased BMD and bone strength in orchidectomized and ovariectomized rats [51, 52, 78]. Hanada et al. reported that a SARM (i.e., S-40503) induced a significant increase in femoral BMD in a 10

dose-dependent manner in a hypogonadal model, with lesser activities in androgenic organs. Rosen and Negro-Vilar also reported that a SARM (i.e., LGD2226) prevents bone loss observed in orchidectomized animals [78]. In ovariectomized rats, SARMs demonstrated a significant elevation in femoral BMD (S-40503) and mechanical strength of femoral bone (S-4 and S-40503) [51, 52].

The majority of clinically available therapies for osteoporosis are bone resorption inhibitors, such as bisphosphonates, estrogen, selective estrogen receptor modulators

(SERMs), and calcitonin, which are not sufficient to restore bone mass for patients who have already lost a significant amount of bone. Intermittent parathyroid hormone (PTH) treatment is the only clinically available option to promote bone formation [79, 80].

However, PTH treatment for osteoporosis is very limited due to concerns related to side effects and osteosarcoma [81]. The lack of satisfactory treatments has piqued interest in use of SARM as a treatment of osteoporosis. This interest appears rational given the accumulating evidence to support the anabolic activities of SARMs, especially at periosteal cortical bone. Androgen seems to be responsible for obvious sexual differences in skeletal morphologies after puberty. Skeletal size in men is larger than that in women, including radial width and cortical thickness of long bone, femoral neck cross-sectional area, and vertebral cross-sectional area [82]. In humans and rodents, genetic males with complete androgen insensitivity achieve a bone mass more typical of females, suggesting the anabolic action of androgen [82, 83]. Turner et al. showed that DHT increases bone formation in the periosteal surface of cortical bone, while estrogens depress it [84]. Even though volumetric BMD (i.e., the amount of mineral per volume of bone (g/cm 3)) is very similar in adult men and women, the larger diameter and cortical thickness of the long 11

bones in men offers great advantage to their mechanical strength, explaining the lower incidence of fracture of men compared with women [85]. Hanada et al. provided direct evidence for the anabolic action of SARMs in bone, which is the only published evidence to date [52]. In this study, a SARM (S-40503) remarkably elevated the periosteal mineral apposition rate in the cortical bone of ovariectomized rats, demonstrating the bone formation activity of SARM in the periosteal surface of cortical bone [52]. In orchidectomized rats, S-40503 treatment significantly increased cortical BMD in femoral and tibial bone compared with a vehicle control group, but did not affect the cancellous

BMD in both bones. This result suggests that SARMs significantly promote BMD mainly by anabolic action, rather than anti-resorptive action. Interestingly, S-40503 treatment enhanced cortical BMD to a similar extent as estrogen in an immobilized, orchidectomized model of osteoporosis [52]. In fact, S-40503 increased cortical BMD significantly higher than that observed in intact rats or orchidectomized rats treated with estrogen. Although this comparison was performed in different bones, it suggests that

SARMs might increase BMD by two mechanisms: (1) direct anabolic action on bone and

(2) indirect action via muscle stimulation.

In conclusion, SARMs are still in the early stages of drug development. As such, knowledge on their activity in the skeleton remains sparse. However, SARMs have great potential for treatment of osteoporosis. First, their unique tissue selectivity can remove the shackles restraining the clinical usage of AR ligands as a treatment of osteoporosis.

SARMs can minimize undesirable side effects, resulting from stimulation of androgenic organs and cross-reactivity of androgens and their metabolites. Moreover, the anabolic effect of SARMs in bone could likely be promoted by higher dosage regimen than 12

conventional dosage for steroidal androgen, since conventional steroidal androgen dosages are restricted by side effects. Second, SARMs promote bone formation, rather than reduce resorptive action, which suggest that SARMs can restore bone mass even for severe osteoporosis as well as preventive and early stage osteoporosis. Combination therapy with other anti-resorptive agents might synergistically increase bone mass and strength. Finally, the anabolic effects of androgen on muscle are beneficial for increasing bone mass and reducing fracture risk. The pharmacokinetic advantages, selectivity, and dual activity of SARMs in muscle and bone suggest that they may indeed become an important new addition to the armamentarium of drugs to treat osteoporosis.

1.6.2 Muscle Wasting

Testosterone replacement in young hypogonadal men at a physiologic dose is associated with changes in body composition (i.e., gain of lean and loss of fat mass) and increase in muscle protein synthesis [65, 86-88]. Moreover, administration of testosterone at a physiologic dose increases maximal voluntary strength in young hypogonadal men

[86, 88]. Administration of a supraphysiologic dose of testosterone (about 6 times the dose needed to achieve normal serum concentrations) to healthy normal men increases fat-free mass to a similar extent as resistance-exercise training [89]. The combination therapy of resistance exercise and a supraphysiologic dose of testosterone showed additive effects on fat-free mass and muscle size and strength [89]. Moreover, administration of a supraphysiologic dose of testosterone is associated with increase in maximal voluntary strength and quadriceps cross-sectional area and volume [89, 90].

The majority of studies to date examined the effects of testosterone replacement in older men [28, 91-95]. Generally, testosterone replacement in elderly men produced modest increases in muscle mass and strength. Some studies reported modest increases in lean mass [92, 94]. However, few studies have reported that androgen administration increased grip strength [28]. In fact, some have shown that it does not [93, 94]. Likewise, the effects of testosterone administration on lower body strength were not significant in several studies [28, 91-94]. While the effect of testosterone on muscle mass and strength in elderly men has not been consistent or impressive, these varying results do not suggest that the administration of SARMs will not be beneficial in terms of muscle mass and strength in elderly men. Only lower doses of testosterone were considered for studies in elderly men, due to the concerns about side effects at higher doses, especially accelerating the risk of prostate cancer. A study showed that administration of testosterone at 125, 300, and 600 mg/week significantly increased muscle mass and strength equally in older and younger men [66, 96]. These actions of androgen in muscle seem to have a dose (and concentration)-dependent relationship [66, 96]. In healthy young men, it was observed that testosterone dose and testosterone concentrations, including total, free, and steady-state concentration, were highly correlated. Also, there were correlations between testosterone dose and the gains in fat-free mass, leg press strength, and leg power [96]. Testosterone dose and change in fat mass were inversely correlated. Moreover, preoperative administration of supraphysiologic doses of testosterone tended to shorten hospital stay and improve walking and stair climbing in older men undergoing knee replacement surgery. At postoperative Day 3, there was a significant improvement in ability to stand in the testosterone treatment group [97].

SARMs demonstrate strong agonist activity and ability to promote growth of the levator ani muscle, maximally to a size significantly greater than that of intact control animals [98]. However, the anabolic activity of SARMs in the levator ani muscle does not directly support the contention that SARMs will improve muscle performance.

Recently, the effects of a SARM (i.e., S-4) on the mass and strength of skeletal muscle

(isolated soleus muscle) in orchidectomized rats were measured [99]. S-4 treatment (3 mg/kg and 10 mg/kg) significantly increased the skeletal muscle strength (measured as peak titanic tension) in orchidectomized animals, even though the effect of S-4 in muscle size was not significant. S-4 restored castration-induced losses in lean body mass. Similar changes in muscle size, muscle strength, and lean body mass were also observed in DHT

(3 mg/kg) treated animals. However, DHT (3 mg/kg) also fully restored the androgenic tissue weights, while S-4 (3 mg/kg) only returned the prostate and seminal vesicle to 16% and 17%, respectively, of the control levels.

In summary, administration of androgen significantly increases muscle mass and strength in young hypogonadal men (physiologic replacement dose) and eugonadal men

(supraphysiologic dose). In elderly men, testosterone effects on muscle mass and strength have not been consistent or impressive, possibly due to the low dosages used in clinical trials. The high correlation between dose (and concentration) and the anabolic actions of androgen in muscle suggest that androgen administration of higher doses in elderly men may significantly increase muscle mass and strength. In orchidectomized animals, a

SARM (S-4) showed strong anabolic effects in skeletal muscle without affecting the androgenic tissues. This evidence strongly supports the great potential of SARMs as

anabolic agents to treat muscle wasting, improve muscle performance in the frail, and/or shorten rehabilitation time after.

1.7 Overview of Dissertation and Research

We have identified a series of SARMs having favorable properties, such as tissue selectivity, long half-life, high oral bioavailability, and AR specificity. Since numerous lines of evidence indicate that androgens play an important role in bone and muscle, we hypothesized that SARMs, which mimic androgen in anabolic tissues (e.g., bone and muscle), could represent a novel approach to the treatment of osteoporosis with minimized side effects. To test this hypothesis, we employed rational experimental designs as followed.

The specific aims of this project were to: analyze in vitro structure-activity

relationship by determining in vitro AR binding affinity and AR-mediated transcriptional activation of novel SARMs modified in the aromatic B-ring (Chapter 2); determine the in vivo pharmacologic activity and pharmacokinetics of B-ring substituted SARMs with halogen atom, discover the major factor(s) determining in vivo pharmacologic activities of SARMs, and based on this information, select a potent SARM (S-22) for osteoporosis studies (Chapter 3); examine the in vivo metabolism and plasma protein binding of a series of SARM in male rats (Chapter 4); determine effects of a SARM (S-22) alone or in combination with SERM (i.e., raloxifene) in bone, muscle, uterus, and body composition in order to evaluate a SARM as a treatment of osteoporosis (Chapter 5); determine in vitro activities of SARM (S-22) alone or in combination with SERM (raloxifene) in bone cells (Chapter 6). A summary of our findings of current studies is presented in Chapter 7. 16

Preparation Route of application Full substitution dose

In clinical use 2-4 capsules (40mg) per Testosterone undecanoate Oral day Testosterone tablets Buccal 30mg / twice daily 200-250 mg every 2-3 Testosterone enanthate Intramuscular injection weeks Testosterone cypionate Intramuscular injection 200 mg every 2 weeks 1000 mg every 10-12 Testosterone undecanoate Intramuscular injection weeks 4 implants (200mg) every Testosterone implants Implantation under skin 5-6 months Transdermal testosterone Scrotal skin 1 patch per day patch Transdermal testosterone Non-scrotal skin 1or 2 systems per day patch Transdermal testosterone gel Non-scrotal skin 5 to 10 g gel per day

Under development

Testosterone cyclodextrin Sublingual Not yet determined

Testosterone buciclate Intramuscular injection Not yet determined

Testosterone microcapsules Subcutaneous injection Not yet determined

Obsolete

17 α-Methyltestosteone Oral

Fluoxymesterone Sublingual/oral

Table 1.1 Mode of application and dosage of various androgen preparations.

Cells Androgen(s) Effect

Primary human osteoblasts DHT ↑ 50%

Primary mouse calvarial cells DHT ↑ 100% DHT ↑ 200% Primary human osteoblasts DHEA ↑ 88% MC3T3-E1 cells DHT ↑ 15% DHT ↑ 57% MC3T3-E1 cells Testosterone ↑ 39% Primary rat diaphyseal osteoblasts Testosterone ↑ 100%

Primary rat epiphyseal cells Testosterone ↑ 57% DHT ↑ 150% Primary rat calvarial osteoblasts Testosterone ↑ 70% DHT ↓ 25% Osteosarcoma cell line TE-85 Testosterone ↓ 20% hFOB/AR6 cell line DHT ↓ 30%

Table 1.2 Effects of androgens on osteoblast proliferation.

↑ increase, ↓ decrease [100].

Marker of osteoblast differentiation Androgen(s) Effect

Alkaline phosphatase (AP) - Percentage of AP-positive cells DHT ↑ - AP activity DHT, DHEA ↑ DHT, Testosterone ↔ DHT, Testosterone ↓

Type I collagen - mRNA levels DHT, Testosterone ↑ DHT, Testosterone ↔ - Protein secretion DHT, Testosterone ↓ - [3H]proline incorporation DHT ↑ DHT ↔

Osteocalcin secretion DHT, Testosterone, DHEA ↑ DHT, Testosterone, DHEA ↔

Mineralization of intracellular bone matrix DHT, DHEA ↑

Table 1.3 Effect of androgens on osteoblast differentiation markers.

DHT, dihydrotestosterone; DHEA, dehydroepiandrosterone; ↑ increase, ↔ no change, ↓ decrease [100].

Cholesterol

CYP11A1 H3C H3C O O

3β-HSD

HO O Pregnenolone Progesterone

CYP17 CYP17 α (17 -hydroxylase) H C (17 α-hydroxylase) H3C 3 O O Adrenal β OH OH 3 -HSD and Testis HO O 17-OH Pregnenolone 17-OH Progesterone

CYP17 CYP17 (17,20 Lyase) (17,20 Lyase) O O

3β-HSD

HO O Dehydroepiandrosterone Androstenedione

17 β-HSD OH Testis and Peripheral Tissue 5α-reductase O Aromatase Testosterone

OH OH Peripheral Tissue O HO Dihydrotestosterone Estradiol

Figure 1.1 Biosynthesis pathways of sex steroids in testis and peripheral tissue. 3β-HSD,

3β-hydroxysteroid dehydrogenase; 17 β-HSD, 17 β-hydroxysteroid dehydrogenase.

Figure 1.2 Hypothalamus-pituitary-testis axis of androgen regulation. 21

1 537 587 626 722 771 814 867 918

NH 2 1 2 3 4 5 6 7 8 COOH

Transactivation AF-1 DBD NLS LBD/AF-2 Domain

Figure 1.3 Structural and functional organization of androgen receptor protein.

AF, activation function; DBD, DNA binding domain; LBD, ligand binding domain; NLS, nuclear localization signal.

Conjugation or Oxidation OH

CH3 17 Hydroxylation

CH3

Conjugation O

Hydroxylation Reduction

Figure 1.4 Various possibilities for metabolism of testosterone.

OH OH

CH3

O O Testosterone 17 α-Methyltestosterone

OH OCO(CH2)9CH3 HO CH3 F

O O Fluoxymesterone Testosterone undecanoate

OCOCH2CH3 OCO(CH2)5CH3

O O Testosterone propionate Testosterone enanthate

OCO(CH2)2 OCO (CH2)3CH3

O O Testosterone cypionate Testosterone butylcyclohexylcarcoxylate

Figure 1.5 The chemical structures of steroidal androgens.

H NC N CH2Cl O

O F3CN S H HO R-1

H NC N CH3 O

O F3CN S H HO Acetothiolutamide

O2N F O

F3CN O H HO S-1

H O2N N CH3 O

O F3CN O H HO S-4

Figure 1.6 The chemical structures of nonsteroidal AR ligands

Figure 1.7 Bone remodeling cycle

(at http://www.surgeongeneral.gov/library/bonehealth/chapter_2.html).

CHAPTER 2

IN VITRO STRUCTURE ACTIVITY RELATIONSHIP OF NONSTEROIDAL

SELECTIVE ANDROGEN RECEPTOR MODULATORS

2.1. INTRODUCTION

Androgen receptor (AR) belongs to the nuclear receptor superfamily. By binding to AR, androgen mediates various physiological processes including differentiation, homeostasis, and development of male secondary characteristics [101]. In the absense of a ligand, AR is bound to a complex of molecular chaperones, which facilitate proper folding of AR for efficient ligand binding [19]. Upon androgen binding, AR conformation changes and it sequentially leads to the dissociation of chaperone proteins, dimerization, and translocation to nucleus. The translocated AR-ligand complex binds to specific DNA sequences (i.e., AREs) and triggers the production of target mRNA and protein with the aid of transcription machinery [24].

Both agonist and antagonist ligands for AR are clinically valuable. For AR agonists, only steroidal compounds are available. Synthesized steroidal AR agonists are

mainly used for androgen-deficient disorders since those steroids are able to mimic the action of endogenous androgen [102]. However, unfavorable pharmacokinetic properties and steroid-related side effects of steroidal AR agonists hamper their common use in the clinic [36]. On the other hand, steroidal and nonsteroidal derivatives are used clinically as

AR antagonists. AR antagonists have been used for disorders that originate from excessive androgen, such as androgen-dependent prostatic hyperplasia, male-pattern baldness, and acne [103]. Steroidal AR antagonists (e.g., cyproterone acetate and megestrol acetate) display mixed androgenic and antiandrogenic activity and cross- reactivity to progesterone receptors, resulting in side effects. Nonsteroidal AR antagonists

(e.g., flutamide, nilutamide, and bicalutamide) are advantageous over their steroidal counterparts in terms of AR specificity, selectivity and pharmacokinetics [104, 105].

Although it is expected that nonsteroidal androgens would be advantageous over their steroidal counterparts, the discovery and development of nonsteroidal androgens was unsuccessful for decades. Recently, several laboratories, including ours, demonstrated that nonsteroidal AR agonists can be obtained by structural modification of existing nonsteroidal AR antagonists [40-43, 55, 106-108]. In our early study to search for electrophilic affinity ligands for the AR, we found that structural modification of flutamide and bicalutamide could produce compounds having in vitro AR agonistic

activity [40]. In further attempts, we developed nonsteroidal AR ligands (i.e., S-1 and S-

4) that possess in vivo agonistic activity [45]. These compounds are superior to steroidal agonists in terms of pharmacokinetic properties and AR specificity [47]. Moreover, S-1 and S-4 exerted potent and tissue-selective pharmacologic activity and represented the first members of a new class of selective androgen receptor modulators (SARMs). The

great therapeutic potential of SARMs motivated us to understand the structure-activity relationships (SARs) of a series of SARMs on in vitro AR binding and pharmacokinetics

in order to improve their in vivo pharmacologic activity and pharmacokinetics for clinical

use. In previous studies, we outlined the key SARs of a series of SARMs on AR binding

affinity and AR transcriptional activation [55, 107]. These studies demonstrated the

importance of structural modification of the A-ring and B-ring (figure 2.1) of the aryl

propionamide pharmacophore to enhance binding affinity, and demonstrated that the

linkage group plays an important role in transcriptional signaling toward either agonistic

or antagonistic action. In this study, we focused on further modification of the B-ring and

linkage group of aryl propionamide SARMs and we herein report AR binding affinities

and abilities to induce AR transcriptional activation of these derivatives. Moreover, we

speculate on the three-dimensional structure of the AR ligand-binding domain (LBD)

bound to these derivatives based on x-ray crystallography of the AR LBD bound to a

nonsteroidal AR ligand, which was recently solved in our lab [54].

2.2. MATERIALS AND METHODS

2.2.1. Chemicals and Animals

The nonsteroidal AR ligands were synthesized by Dr. Duane Miller’s research group at the University of Tennessee using similar procedures as previously described

[107, 109]. The purities of synthesized compounds were greater than 99%, as confirmed by elemental analysis and mass spectrometry. Unlabeled mibolerone (MIB) and 17 α- methyl-[3H] MIB ([ 3H]-MIB, 83.5 Ci/mmol) were purchased from PerkinElmer Life

Sciences (Boston, MA). Triamicinolone acetonide, phenylmethylsulfonly fluoride

(PMSF), sodium molybdate, and dihydrotestosterone (DHT) were purchased from

Sigma-Aldrich (St. Louis, MO). Hydroxyapatite (HAP) was purchased from Bio-Rad

Laboratories (Hercules, CA). EcoLite (+) scintillation cocktail was purchased from ICN

Pharmaceuticals (Costa Mesa, CA). Dulbecco’s modified Eagle’s medium (DMEM), penicillin-streptomycin, trypsin-EDTA, and LipofectAMINE reagent were purchased form Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was purchased form Atlanta

Biologicals (Norcross, GA).

The animal protocol was reviewed and approved by the Institutional Laboratory

Animal Care and Use Committee of The Ohio State University. Male Sprague-Dawley rats were purchased form Harlan (Indianapolis, IN). All animals were maintained on a

12-h light/dark cycle. Standard rat chow and water were provided ad libitum .

2.2.2. Preparation of Cytosolic ARs

Cytosolic AR was prepared from ventral prostates of castrated male Sprague-

Dawley rats as described previously [110]. Briefly, rats were castrated via a scrotal incision under anesthesia. One day after castration, the ventral prostates were excised and kept in ice-cold homogenization buffer, containing 10 mM Tris, 1.5 mM Disodium

EDTA, 0.25 M sucrose, 10 mM sodium molybdate, and 1 mM PMSF at pH 7.4. The prostates were minced with scissors in a homogenization buffer (a ratio of 0.5g prostate to 1 ml buffer) and homogenized with a mechanical homogenizer (Model PRO 200, Pro

Scientific, Monroe, CT). The homogenate was centrifuged at 114,000g for 1 hr. The cytosolic supernatant was collected and stored at - 80°C until it was used.

2.2.3. AR Competitive Binding Assay

The AR binding affinity of nonsteroidal AR ligands was determined using an in

3 vitro competitive radioligand-binding assay with [ H]-MIB as described previously [110].

3 Briefly, 50 µl of AR cytosol was incubated with [ H]-MIB (1 nM) and triamcinolone

acetonide (1 µM) at 4 °C for 18 h in the absence (total binding) or presence of increasing

3 concentrations (10 -2 nM to 5,000 nM) of each ligand. Nonspecific binding of [ H]-MIB

was determined by adding an excess amount (1 µM) of unlabeled MIB to separate

incubates. At the end of incubation, HAP slurry was added to the incubate in order to

3 collect the [ H]-MIB bound to AR. HAP pellet was centrifuged at 1,000g for 10 min and

then rinsed three times with washing buffer (50 mM Tris at pH 7.2). An aliquot (1 ml) of

ethanol was added to HAP pellet and kept at room temperature for 1 hr to extract the

3 [ H]-MIB bound to AR. After the extraction, the suspension was centrifuged at 1,000g

for 10 min and the supernatant was transferred to 5 ml of scintillation cocktail.

Radioactivity was counted in a Beckman LS6500 liquid scintillation counter (Beckman

Instruments Inc., Irvine, CA).

3 The specific binding of [ H]-MIB to AR was calculated by subtracting the

3 nonspecific binding of [ H]-MIB from total binding, and expressed as the percentage of

3 the total binding. The concentration of each ligand that inhibited [ H]-MIB binding by

50% (i.e., the IC 50 ) at each ligand concentration was determined by nonlinear regression

of the specific binding data for each ligand using WinNonlin software. WinNonlin was

generously provided by pharsight corporation (Mountain View, CA) through an academic

license to The Ohio State University, Division of Pharmaceutics. The apparent equilibrium dissociation constant of the inhibitor (i.e., K i) for each compound was

3 calculated as K i = K d × IC 50 / (K d + L), where K d was the dissociation constant of H-

MIB (0.19 ± 0.01 nM, previously determined by Mukherjee et al., 1999), and L was the concentration of 3H-MIB used in the experiment (1 nM). Binding affinities of the ligands

were then compared using the calculated K i values .

2.2.4. AR-Mediated Transcriptional Activation

The abilities of the compounds to modulate the transcriptional activity of AR in a cellular context were measured using a cotransfection assay in CV-1 cells, as described previously [55]. Briefly, CV-1 cells (American Type Culture Collection, Manassas, VA) were plated into DMEM with 10% FBS one day prior to transfection. When CV-1 cells reached 90% confluence, transient transfection of plated cells was performed in serum- free medium using LipofectAMINE according to the manufacturer’s instruction. Cells in each flask were cotransfected with a human AR expression vector (pCMVhAR; generously provided by Dr Donald Tindall, Mayo Clinic), an androgen-dependent luciferase reporter vector (pMMTV-Luc; generously provided by Dr. Ron Evans, Salk

Institute), and a control β-galactosidase expression vector (pSV- β-galactosidase;

Promega Corporation) for 10 hr. After washing with DMEM, cells were allowed to

recover in fresh DMEM supplemented with 0.2% FBS for 12 hr prior to drug treatment.

Cells were treated with the compound of interest in the presence or absence of DHT (1

nM) for 12 hr. At the end of drug treatment, cells were washed with PBS twice and lysed

with passive lysis buffer (100 µl/well) for 30 min at room temperature. For β-

galactosidase assays, an aliquot (50 µl) of cell lysate was incubated with β-galactosidase

assay buffer (50 µl) at 37 ˚C for 3 hr. The UV absorbance at the wavelength of 420 nm was measured. For luciferase assay, an aliquot (50 µl) of cell lysate was mixed with 50 µl of 1 mM beetle luciferin (Promega, Madison, WI) and the luminescence was read by an

AutoLumate LB953 luminometer (Wallace Inc., Gaithersberg, MD).

Transcriptional activation in each well was calculated as the ratio of luciferase activity to β-galactosidase activity to normalize the variance on cell number and transfection efficiency. Transcriptional activation induced by each ligand of interest, in the absence or presence of 1 nM DHT, was expressed as a percentage of that induced by

1 nM DHT.

2.3. RESULTS AND DISCUSSION

2.3.1. B-ring modification

Our previous studies demonstrated that the in vitro and in vivo pharmacologic

activities of aryl propianamide SARMs could be improved by structural modification(s)

of the A-ring, B-ring, and/or linkage of the pharmacophore. We designed and synthesized

several series of nonsteroidal compounds, based on the SARs for AR binding affinity

obtained from our previous studies [55, 109, 111]. The AR binding affinity was

determined using a radioligand competitive binding assay as described in section 2.2.3

and reported as K i values. As the K i value decreased, AR binding affinity increased. The

average K i of DHT was 0.64 ± 0.32 nM. The AR binding affinities of various B-ring modified SARMs are shown in Table 2.1. In a previous study, the SARM substituted

with an C(O)CH 2CH 3 group in the 4-position of the B-ring displayed high binding affinity (K i = 6.07 ± 0.14 nM) [109]. Therefore, we synthesized compounds having

similar structures, S-23 and S-24. S-23, the derivative with an -C(O)OCH 3 moiety in the

4-position of the B-ring, exerted an AR binding affinity of 25 ± 3. Unexpectedly, S-24,

the carboxylic acid derivative did not bind to AR. Another modification at the 4-position

of the B-ring that exerted high binding affinity was a fluorine atom (i.e., S-1), an

electron-withdrawing substituents. The K i of S-1 was 6.1 ± 0.2 nM [45]. To improve binding affinity, we attempted structural modification of S-1. We incorporated a variety of electron-withdrawing substituents at the para -position (S-25, S-26) or meta -position

(S-27) of the B-ring. Incorporation of a trifluoromethyl group at either the para or meta

position of the B-ring negatively affected AR binding affinity, with no significant

difference between the two sites. The substitution of trifluoromethoxy moiety slightly

increased AR binding affinity, compared to that of trifluoromethyl derivative (i.e., S-25)

but still exerted lower binding affinity than that of S-1 or S-4, suggesting that the fluorine

atom provided near optimal size or elcetonegativity at this position for interaction with

the AR.

Since the derivative incorporating an acetamido group at the para position of the

B-ring (i.e., S-4) exerted a high binding affinity in previous studies [45], derivatives with

similar hydrogen bonding and steric properties to the acetamido group in the B-ring were

synthesized and tested (Table 2.1). Crystallographic data of the AR LBD bound to S-4

(data not shown) showed that the -CH 3 of the acetamido moiety in S-4 was located close

to an electron acceptor group (i.e., ketone moiety) of the LBD and was surrounded by a

lipophilic LBD pocket that appeared to be able to accommodate several more atoms.

Therefore, we hypothesized that 1) replacement of the -CH 3 with a hydrogen bond donor

and/or 2) the addition of a lipophilic moiety to the acetamido group of S-4 would increase

AR binding affinity. We designed and synthesized compounds S-28 to S-31 to test these

hypotheses. Replacement of the oxygen atom in the ketone group with a sulfur atom

exerted minimal effect on AR binding affinity. The addition of a methyl group on the

NHC(O)NH 2 moieties also did not change binding affinity. These results indicate that the addition of a lipophilic moiety to the acetamido group of S-4 does not improve AR binding affinity. Moreover, the addition of a hydrogen bond donor moiety in S-28, S-29, and S-30 did not improve, or even caused drastic decreases, in AR binding affinity. These observations could be due to the fact that the -NH- and -NH 2 group might be more hydrophilic than -CH 2- and -CH 3 at physiological pH, resulting in less favorable interactions within the lipophilic surroundings of the LBD.

While the SAR of SARMs for AR binding was being studied, in vivo metabolism

and pharmacokinetic studies of these compounds suggested that a cyano group at the

para position of the A-ring is more stable than a nitro group. Therefore, we synthesized a

series of four compounds incorporating various halogens at the para -position of the B-

ring and cyano group in the A-ring, namely S-32, S-33, S-34, and S-35 (Table 2.1). The

Ki values for the 4-F, Cl, Br, and I substituted SARMs with a 4-cyano substituents in the

A-ring were 3.3 ± 0.1, 3.4 ± 0.1, 14 ± 10, and 20 ± 0.2, respectively. As electronegativity increased or/and the size of the atom decreased, AR binding affinities increased.

Inclusion of the cyano group on the A-ring maintained AR binding affinity of the SARMs to a similar extent to analogous SARMs substituted with a nitro group on the A-ring

[109].

Our previous study showed that incorporation of cyano group at the para -position of B-ring resulted in high binding affinity [111]. The binding affinities of S-20 (-NO 2 on

A-ring, -CN on B-ring) and S-21 (-CN on A-ring, -CN on B-ring) were 2.0 ± 0.2 and 3.8

± 0.5 nM, respectively. We modified these compounds by adding a chlorine atom to the meta -position of the B-ring. These compounds, S-34 and S-35, maintained or improved binding affinity and exerted K i values of 1.9 ± 0.5 and 2.1 ± 0.2, respectively.

Our previous study showed that the lowest DHT concentration to produce the maximal transcriptional activation of the AR was 1 nM [40]. Therefore, the abilities of the investigational compounds (100 nM) to induce AR-mediated transcriptional activation were quantified by comparing to that observed with 1 nM DHT. The antagonistic activities of compounds were determined by their ability to inhibit DHT

(1nM)-induced transcriptional activation at a concentration of 100 nM. The AR agonist and antagonist activities of S-25, S-27, S-34 and S-35 are shown in Figure 2.2.

Interestingly, the para -trifluoromethyl derivative (S-25) and meta -trifluoromethyl derivative (S-27) exerted similar binding affinity but showed different functional activity.

S-25 and S-27 alone stimulated transcription to 99% and 51%, respectively. With cotreatment of DHT, S-25 did not exert antagonistic activity but S-27 suppressed transcriptional activation to 78%. Although the changes were not dramatic, it suggests that the position of B-ring substitution could affect functional status. S-34 and S-35 stimulated transcription to 99%, 91%, and 96% at a 100 nM concentration and did not suppress DHT-induced transcription during cotreatment. This result suggests that multi- substitution on the B-ring might increase drug efficacy.

2.3.2. Linkage modification

Our previous SAR studies demonstrated that the linker (X) of bicalutamide pharmacophore plays a critical role in functional status (agonist and antagonist activity) of SARMs (Table 2.2). It is shown that AR antagonist (i.e., bicalutamide) was switched to AR agonist by changing the linkage group from sulfone to sulfide [55]. Moreover, previous study showed that a compound with oxygen linkage (X=O) and secondary amine linkage (X=NH) displayed agonist and antagonist activity, respectively (Table 2.2).

From these data, we hypothesized that only compounds having a single-atom, hydrogen- bond-acceptor linker act as agonists. To test this initial hypothesis, we synthesized compounds (R-7 (X=CH 2) and R-8 (X=null)) having a non-hydrogen bonding linker, and

determined their binding affinities and functional activities in this study. Results for R-2

(X=S), R-3 (X=SO), and R-4 (X=SO 2) are also reported herein. Table 2.2 shows binding

affinities and maximal transcriptional activation of these compounds. The modification of

the linkage group generally maintained AR binding affinities. R-8 and R-9 displayed low

in vitro transcriptional activation abilities of 28.9 % and 15.9%, respectively. R-8 and R-

9 suppressed the DHT-induced transcription activation by 33% and 18%, respectively

(Figure 2.3). These data suggests that R-8 and R-9 are weak partial agonists or

antagonists. Shortening the linker by one carbon seemed to aggravate both binding

affinity and transcriptional activation. Binding affinities and transcriptional activation of

R-2, R-3, and R-5 are also reported in Table 2.2. The Ki values (nM) of R-2, R-3, and R-

5 were 11 ± 2, 71 ± 10, and 28 ± 10, respectively. R-2, R-3, and R-5 induced AR- mediated transcriptional activation by 84%, 17%, and 17%, respectively. By addition of an oxygen atom(s) to the sulfide linker, the binding affinities and the abilities to induce

transcriptional activation of the compounds deteriorated. The x-ray crystallography of the

AR LBD bound to nonsteroidal AR ligand that was recently solved in our lab [54] helped to understand this effect of X-linkage on functional activities. The x-ray crystallography suggests that Met-895 is considerably displaced by bulk from the sulfonyl linkage group of R-bicalutamide as compared with the AR LBD-DHT structure and this displacement might result in improper folding of AR for interaction with coactivators. Single hydrogen bond acceptor atoms (e.g., S, O) form an intramolecular hydrogen bond with the amide nitrogen, resulting in a more bent ligand conformation and decreased bulk. In addition, the single atom linker decreases the bulk in that region and may allow accommodation of the Met-895 side chain in a similar region as with the AR LBD-DHT complex.

2.3. CONCLUSIONS

In this study, we modified the chemical structure of our lead compounds discovered in previous studies and tested AR binding affinities and transcriptional activation. We learned that minimal structural changes to the B-ring could make a significant difference in binding affinities and transcriptional activation. By B-ring modification, we identified S-34 and S-35 as efficacious agonists with high binding affinities. Moreover, we studied the SAR for linkage modification. This study demonstrated that compounds having a single atom and a hydrogen-bond accepting linker exert agonist activity.

O2N O

F3C N H

Flutamide

NC F O

* F3C N S H O2 H3C OH

Bicalutamide

O2N R O A B * F3C N O H H3COH

S-1 (R=F) S-4 (R=NHCOCH3)

Figure 2.1 Chemical structures of nonsteroidal AR antagonists (flutamide and bicalutamide) and nonsteroidal AR agonists (S-1 and S-4). Aromatic A and B are as shown.

140

120

100

(% of 1(%ofnM DHT) 40

Luciferase/Beta-galactosidase 20

0 Control DHT S-25 S-27 S-36 S-37 S-25 S-27 S-36 S-37

+ 1 nM DHT Drug Treatment

Figure 2.2 AR-mediated transcriptional activation of S-25, S-27, S-36, and S-37.

It is expressed as the ratio of luciferase and ß-galactosidase activities in the cotransfection assay and quantified by comparing to that observed with 1 nM DHT.

120

100

40 (%of 1 nM DHT)

20 Luciferase/Beta-galactosidase

0 Control DHT 1 10 100 500 1 10 100 500

L-10 + 1 nM DHT L-11 + 1 nM DHT

Drug Treatment

Figure 2.3 Antagonist activities of R-8 and R-9 on DHT-induced transcription.

The inhibitory activities of R-8 or R-9 on DHT-induced transcriptional activation were determined. It is expressed as the ratio of luciferase and ß-galactosidase activities in the co-transfection assay and quantified by comparing to those of 1 nM DHT.

A B X1 N O X2 H

H3C OH

F3C X3

Compounds X1 X2 X3 Ki (nM) a S-1 NO 2 F H 6.1 ± 0.2 a S-4 NO 2 NHC(O)CH 3 H 4.0 ± 0.7

S-23 CN C(O)OCH 3 H 25 ± 3 S-24 CN C(O)OH H > 770

S-25 NO 2 CF 3 H 40 ± 7

S-26 NO 2 OCF 3 H 32 ± 6

S-27 NO 2 H CF 3 49 ± 5

S-28 NO 2 NHC(S)NH 2 H 254 ± 12

S-29 NO 2 NHC(O)NH 2 H 345 ± 199

S-30 NO 2 NHC(S)NHCH 3 H 271 ± 37

S-31 NO 2 NHC(O)CH 2CH 3 H 14 ± 1 S-32 CN F H 3.3 ± 0.1 b S-33 CN Cl H 3.4 ± 0.1 b S-34 CN Br H 14 ± 10 S-35 CN I H 20 ± 0.2

S-36 NO 2 CN Cl 1.9 ± 0.5 S-37 CN CN Cl 2.1 ± 0.2

Table 2.1 In vitro AR binding affinities of B-ring modified SARMs. a previously reported in Yin et al.[45]. b previously reported in thesis of Scott Fisher at OSU, 2004

A B O2N N X Y H

H3C OH

F3C

Max Efficacy Compounds X Y K (nM) i (% of DHT ) a a S-4 O NHC(O)CH 3 4.0 ± 0.7 93 ± 7 S-1 O F 6.1 ± 0.2 a 43 ± 3 a b b R-1 S NHC(O)CH 3 4.9 ± 0.2 50 R-2 S F 11 ± 2 84 ± 4 R-3 SO F 71±10 17.3 ± 5.7 c c R-4 SO 2 NHC(O)CH 3 9.3 ± 0.8 45 ± 5

R-5 SO 2 F 28 ± 10 17.3 ± 0.7 a a R-6 NH NHC(O)CH 3 128 ± 6.0 5.2 ± 1.4 R-7 NH F 8.0 ± 0.4 a 21.3 ± 2.2 a

R-8 CH 2 F 10.5±0.3 28.9 ± 2.4 R-9 - F 47.9±2.9 15.9 ± 1.2

Table 2.2 AR binding affinities and transcriptional activation of linkage group modified compounds. a Previously reported Marhefka et al., 2004 [109] b Previously reported Yin et al., 2004 [46] c Previously reported He et al., 2004 [107]

CHAPTER 3

THE 4-PARA SUBSTITUENT OF S-3-(PHENOXY)-2-HYDROXY-2-METHYL-N-

(4-NITRO-3-TRIFLUOROMETHYL-PHENYL)-PROPIONAMIDES IS A MAJOR

STRUCTURAL DETERMINANT OF IN VIVO DISPOSITION AND ACTIVITY

OF SELECTIVE ANDROGEN RECEPTOR MODULATORS

3.1. INTRODUCTION

Androgens promote growth, differentiation, and function of male reproductive organs. In addition, androgens play an important role in the development and maintenance of extragenital aspects of the male phenotype including actions on hair growth, skeletal and muscle growth, fat distribution, and deepening of the voice [1, 56].

Physiological roles for androgens in women have been more difficult to define, but studies suggest that androgens have effects on libido, bone mineral density, and lean body mass in women [56, 57]. The broad spectrum of physiological actions of androgens suggests that a wide variety of therapeutic applications are possible. However, androgen therapy has been clinically used to a lesser degree than estrogen therapies.

More widely accepted use of androgen replacement therapy has been hampered by the lack of satisfactory androgen formulations. Current steroidal androgen formulations have serious limitations [102, 112]. Oral administration of unmodified testosterone results in low bioavailability due to hepatic inactivation and high variability in testosterone absorption, making acceptable plasma concentrations difficult to achieve.

17-alkylated testosterones, such as methyltestosterone and fluoxymesterone, are more resistant to hepatic inactivation and provide improved oral bioavailability. However, they are not commonly used due to their hepatotoxicity and low efficacy [102]. Testosterone esters are frequently used in intramuscular formulations. Esterification at the 17-hydroxy position of testosterone makes it hydrophobic, and results in a prodrug that is released gradually from oily drug containing vehicles at the site of injection. However, such formulations produce fluctuating plasma levels of testosterone and unpleasant side effects

[34, 113]. Skin patches and implants provide better plasma concentration profiles of testosterone. However, skin irritation at the site of patch application and the need for skill for administration and the risk of extrusion in implant delivery limit the usefulness of these formulations. Another common concern regarding steroidal androgen therapy is cross-reactivity of steroidal androgens and their in vivo metabolites with the other steroid hormone receptors, resulting in unfavorable side effects [102, 114].

The lack of satisfactory steroidal androgen receptor (AR) agonists has piqued interest in development of nonsteroidal AR agonists. Several laboratories, including ours, demonstrated that nonsteroidal AR agonists can be obtained by structural modification of existing nonsteroidal AR antagonists [40-43, 55, 106-108]. We were the first to report that structural modification of nonsteroidal AR ligands can produce selective androgen

receptor modulators (SARMs) with in vivo anabolic and androgenic activities [45]. In our early study, we discovered a group of nonsteroidal AR agonists that possess the ability to bind to AR and to induce AR-mediated gene transcription [107]. Acetothiolutamide

(Table 3.1) was chosen as a lead compound due to its in vitro AR agonist activity and

lack of interaction with other steroid hormone receptors. However, acetothiolutamide

showed little in vivo pharmacologic activity, resulting from poor pharmacokinetic

properties. In vivo metabolism studies showed that the thioether-linkage and B-aromatic

ring of acetothiolutamide are susceptible to oxidation, limiting its in vivo efficacy and

exposure [46]. We designed a series of ether-linked compounds bearing a variety of B-

ring substituents in an attempt to avoid metabolic inactivation [109]. Four halogen-

substituted analogs that bind the AR with high affinity and stimulate AR-mediated

transcription were identified (Table 3.1). Interestingly, in vivo pharmacologic activity

was not correlated with in vitro AR binding affinity. We examined the in vivo dose-

response relationship and in vivo pharmacokinetics of these 4-halogen substituted

SARMs in an attempt to delineate whether the observed discrepancy between in vitro and

in vivo pharmacologic activity was due to differences in intrinsic pharmacologic activity

or systemic exposure. We then used this data with previous structure-activity relationship

studies, molecular modeling studies, and in vivo metabolism studies to identify the most

potent, tissue-selective, and orally bioavailable SARM that we have observed to date.

The results of these studies are reported herein.

3.2. MATERIALS AND METHODS

3.2.1. Chemicals

The S-3-(4-Halophenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethylphenyl)

propionamides, denoted S-1 (4-fluoro), S-9 (4-chloro), S-10 (4-bromo), and S-11 (4-iodo)

hereafter, were prepared in our laboratories as previously described [109]. The internal

standard for HPLC analysis, a 2, 4-difluoro propionamide, and four (4-nitro/cyano

phenoxy) -2-hydroxy-2-methyl-N- (4-nitro/cyano-3-trifluromethylphenyl) derivatives, S-

19 through S-22, were synthesized using similar procedures. Reagent grade polyethylene

glycol 300 (PEG 300) and dimethyl sulfoxide (DMSO) were purchased from Sigma-

Aldrich (St. Louis, MO). Alzet osmotic pumps (model 2002) were purchased from

Durect Corporation (Cupertino, CA). Ethyl alcohol USP was purchased from Pharmco

Products, Inc., (Brookfield, CT). HPLC grade acetonitrile and water were purchased from

Fisher Scientific Co. (Fair Lawn, NJ).

3.2.2. In vitro pharmacologic activity

The AR binding affinity of these compounds was determined using an in vitro

3 competitive radioligand-binding assay with H-mibolerone (MIB) as described previously

[110]. Briefly, increasing concentrations (10 -2 nM to 5,000 nM) of each ligand were

3 incubated with rat cytosol, a saturating concentration of H-MIB (1 nM), and 1,000 nM triamcinolone acetonide to prevent interaction of MIB with progesterone receptors at 4 °C

3 for 18 h. At the end of incubation, free and bound H-MIB were separated using the

hydroxyapatite method. IC 50 values were determined by computer-fitting the data for 47

each ligand by nonlinear regression analysis (Pharsight Corporation, Mountain View,

CA). The apparent equilibrium dissociation constant of the inhibitor (i.e., K i) for each

compound was calculated as K i = K d × IC 50 / (K d + L), where K d was the dissociation

constant of 3H-MIB (0.19 ± 0.01 nM, previously determined by Mukherjee et al., 1999), and L was the concentration of 3H-MIB used in the experiment (1nM). Binding affinities of the ligands were then compared using the calculated K i values .

The abilities of the compounds to modulate the transcriptional activity of AR in a cellular context were measured using a cotransfection assay in CV-1 cells, as described previously [55]. CV-1 cells (American Type Culture Collection, Manassas, VA) cotransfected with a human AR expression vector, a luciferase reporter vector, and a control β-galactosidase vector were employed to evaluate the in vitro functional activity of these compounds. Transcriptional activation was assayed using a single concentration

(100 nM) of each ligand and expressed as a percentage of that induced by 1 nM DHT.

Transcriptional activation studies were supported by an NIH grant (1 RO1 DK59800).

3.2.3. Assay for In Vivo Pharmacological Activity in Rats

All animal protocols were reviewed and approved by the Institutional Laboratory

Animal Care and Use Committee of The Ohio State University. The in vivo pharmacological activities of these compounds were determined as the increase in weight of target tissues of castrated rats that received the drug of interest for 14 days, as described previously [45]. Briefly, immature male Sprague-Dawley rats (Harlan,

Indianapolis, IN) weighing 180g to 220g were randomly distributed into 23 groups of five animals. The castrated male rats in each group received increasing doses (0.1, 0.3,

0.5, 0.75 or 1 mg/day) of the designated compound via implantation of subdermal osmotic pumps (for S-9, S-10, and S-11) or daily subcutaneous injection (for S-22) for the dose-response analysis. For the in vivo pharmacologic activity assay of S-19, S-20, and S-21, the castrated rats received the designated compound at the dose rate of 1 mg/day via daily subcutaneous injections. After 14 days of drug treatment, the ventral prostates, seminal vesicles, and levator ani muscle were removed and weighed. Rats were weighed, anesthetized, and sacrificed. Plasma samples were collected and stored at –20

°C. The weights of all organs were normalized to body weight and compared with the historical data of intact control and castrated control animals. The weights of prostate and seminal vesicles were used as markers of androgenic effects and that of levator ani muscle was used as a marker of anabolic effects. The maximal pharmacologic effect

(E max ) and the dose required to elicit 50% of this effect (ED 50 ) were obtained by nonlinear regression analysis using the simple or sigmoid E max model in WinNonlin (Pharsight

Corporation, Mountain View, CA). The relative efficacy of each compound to

testosterone propionate (TP) was defined as the ratio of (E max of the compound) to (E max

of TP). The relative potency was defined as the ratio of (ED 50 of TP) to (ED 50 of the compound). Differences between groups were determined using ANOVA. P-values less than 0.05 were considered as statistically significant. The area under the curve (AUC) of compounds in the rats for pharmacodynamic studies was calculated using:

AUC = Dose rate / Clearance

The drug clearance after a single intravenous dose (10 mg/kg) was used for this calculation, assuming dose-independent clearance in the range of 0.4 mg/kg to 10 mg/kg dosing rate for these compounds [47].

3.2.4. Pharmacokinetic studies

The pharmacokinetics of 4-halogen substituted SARMs and cyano/nitro substituted SARMs were determined after an intravenous (i.v.) bolus or oral dose at 10 mg/kg body weight. Immature, male Sprague-Dawley rats weighing approximately 250g were randomly distributed into eight groups (i.v. and oral dose of S-1, S-9, S-10, and S-

11) of five animals and four groups (i.v. dose of S-19, S-20, S-21, and S-22) of four animals. A catheter was implanted in the right jugular vein of each animal one day prior to dosing. The compounds were dissolved in a solution consisting of 5% DMSO and 95%

PEG 300 and injected as a bolus dose via the jugular vein or administered orally by gavage. The jugular vein catheter was rinsed with saline (three times the volume of the dosing solution) after administration of each intravenous dose. Blood samples (250 µl)

were collected via the jugular vein at different predetermined time intervals after the

dose. Blood samples were centrifuged at 2,000 g, 4°C for 10 min and plasma fractions

were stored at -20°C until analysis.

3.2.5. Bioanalytical Methods

An aliquot (100 µl) of plasma from the pharmacokinetic study was placed in a

polypropylene tube with 100 µl of an internal standard solution (10 µg/ml solution of the

2, 4- difluoropropionamide derivative in acetonitrile for S-1, S-9, S-10, S-11, and S-19; 50

10 µg/ml solution of S-1 in acetonitrile for S-20, S-21, and S-22) and 800 µl of

acetonitrile. The mixture was briefly vortexed, and centrifuged at 10,000g, 4 °C for 2 min.

The supernatant was transferred to another tube and evaporated under nitrogen. The

residue was reconstituted with 150 µl of mobile phase and an aliquot (100 µl) was

injected on to a C 8 column (Symmetry, 3.9 ×150 mm; Waters Corporation, Milford, MA).

The mobile phase consisted of acetonitrile/H 2O [40:60 (v/v)] for S-1, acetonitrile/H 2O

[60:40 (v/v)] for S-9 and S-10, acetonitrile/H 2O [63:37 (v/v)] for S-11, or acetonitrile/H 2O [57:43 (v/v)] for S-19, S-20, S-21, and S-22 at a flow rate of 1 ml/min.

Analytes were detected by their UV absorbance at 297 nm (S-1), 282 nm (S-9 and S-10),

233 nm (S-11), 307 nm (S-19), and 272 nm (S-20, S-21, and S-22). The HPLC system consisted of a solvent pump, a degasser, an autosampler, and a UV detector (model 1100;

Agilent Technologies, Palo Alto, CA). The accuracy, precision, and recovery of each bioanalytical method were established according to FDA guidelines. The limits of detection were 0.5 mg/l (S-1, S-20 and S-21), 0.3 mg/l (S-9, S-11, S-19, and S-22) or 0.6 mg/l (S-10). The pharmacokinetic parameters were obtained by non-compartmental methods using WinNonlin. The area under the plasma concentration-time curve from time zero to infinity (AUC ∞) was calculated using the trapezoidal method with

extrapolation to time infinity. The plasma clearance (CL) was calculated as CL = Dose i.v.

/ AUC ∞ i.v. , where the Dose i.v. and AUC ∞ i.v. were the i.v. dose and corresponding AUC ∞

after i.v. administration, respectively. The terminal half-life (T 1/2 ) was calculated as T 1/2 =

0.693/ λ, where λ was the rate constant characterizing the terminal disposition phase. The apparent volume of distribution at equilibrium (V ss ) was calculated as V ss = CL•MRT,

where the MRT was the mean residence time following the i.v. dose. Oral bioavailability 51

(F p.o. ) was defined as F p.o. = (AUC ∞, p.o. • Dose i.v. ) / (AUC ∞, i.v. • Dose p.o.. ), where the Dose p.o. and AUC ∞ p.o. were the oral dose and corresponding AUC ∞ after oral administration,

respectively.

3.3. RESULTS

3.3.1. Halogen Substituted SARMs

3.3.1.1. In vivo Pharmacological Activity of 4-Halogen Substituted SARMs

The ability of the 4-halogen substituted SARMs to stimulate weight gain of

prostate, seminal vesicles, and levator ani muscle in castrated male rats was studied in

order to evaluate their anabolic and androgenic activity. The weights of prostate and

seminal vesicles were used as markers of androgenic effects and that of levator ani

muscle were used as a marker of anabolic effects. The pharmacological activity of S-1 in

these tissues was previously reported [45]. Table 3.1A shows that S-9 stimulated the

growth of prostate, seminal vesicles, and levator ani muscle in castrated rats in a dose-

dependent manner. S-9 maximally restored the weights of prostate, seminal vesicles, and

levator ani muscle to 33.8 ± 4.0, 28.5 ± 3.9, and 137 ± 9.4% (Mean ± S.D.), respectively,

of the intact control, as compared to 14.5, 12.7, and 74.9%, respectively, as previously

reported for S-1. S-9 fully maintained the levator ani muscle weight in castrated animals

at the same level as intact control, at a dose rate as low as 0.5 mg/day. At higher dose

rates, S-9 promoted growth of the levator ani muscle to a size significantly greater than

that observed in the intact control (Table 3.1A). Nonlinear regression analysis of the

dose-response relationships for S-9 showed that the ED50 values were 0.25 ± 0.06, 0.48

± 0.09, and 0.29 ± 0.09 in prostate, seminal vesicles, and levator ani muscle, respectively

(Table 3.1A, Table 3.2). The relative efficacy and potency of S-9 in androgenic and anabolic tissues were compared to those observed in the testosterone propionate-treated group (Table 3.2). The relative efficacy of S-9 in levator ani muscle was 1.31, which is much higher than its relative efficacies in prostate and seminal vesicles (0.28 and 0.41, respectively), clearly revealing the efficacious and selective activity of S-9 in anabolic tissues of the castrated male rat.

Table 3.1B shows the androgenic and anabolic activity of S-10 in castrated rats.

S-10 demonstrated the weakest pharmacologic activity within this series of compounds, with Emax values of 18.8 ± 3.0, 11.5 ± 0.6, and 64.4 ± 3.7 in the prostate, seminal vesicles, and levator ani muscle, respectively (Table 3.2). However, S-10 displayed lower

ED50 values (0.37 ± 0.25, 0.10 ± 0.04, and 0.11 ± 0.09 in prostate, seminal vesicles, and levator ani muscle, respectively) than the other 4-halogen substituted SARMs, suggesting that higher concentrations of S-10 might have been achieved in the tissues. However, it is important to note that these estimates were also generally more variable and may be low due to the limited response observed with this compound.

Despite its low AR binding affinity and limited in vitro activity (Table 3.1), S-11 demonstrated promising in vivo pharmacologic activity, maintaining the prostate, seminal vesicles, and levator ani muscle in castrated rats at 25.3 ± 2.4, 19.4 ± 2.0, and 95.3 ± 7.2, respectively, of the size of these organs observed in intact controls (Table 3.1C). This result suggested that slower drug metabolism and/or more favorable pharmacokinetic characteristics of S-11 might compensate for its poor AR binding affinity. The relative efficacy of S-11 in androgenic tissues was less than 0.3 as compared to TP, whereas its

relative efficacy in the levator ani muscle was 0.91, indicating a greater degree of activity in anabolic tissues (Table 3.2).

3.3.1.2. Pharmacokinetics of 4-Halogen Substituted SARMs

Mean plasma concentrations of S-1, S-9, S-10, and S-11 obtained after a single

intravenous dose (10 mg/kg) of each SARM are presented in Table 3.2A. Plasma

concentrations of these compounds declined rapidly after intravenous administration,

plateaued in some cases, and then declined in a monoexponential manner with terminal

half-lives ranging from 4 to 14.7 hours (Table 3.3). As the size of halogen atom on the B-

ring increased, volumes of distribution and clearances decreased. However, clearance

values decreased to a greater extent, resulting in prolonged terminal half-lives (i.e., S-11

> S-10 > S-9 > S-1). AUC ∞ was significantly larger for S-10 and S-11, indicating greater systemic drug exposure for these compounds. The AUC ∞ values of S-1, S-9, S-

10, and S-11 were 43 ± 5, 160 ± 33, 401 ± 21, and 1227 ± 206 µg•hr/ml, respectively.

The pharmacokinetics of these compounds after a single oral dose were also examined

(Table 3.2B). After oral dosing at 10 mg/kg, the plasma concentrations of S-1, S-9, S-10,

and S-11 increased gradually with peak concentrations (2.1 ± 0.2, 6.8 ± 0.8, 11.7 ± 2.9,

and 22 ± 4.2 mg/l, respectively) achieved at 4.8 ± 2.2, 9.2 ± 3.3, 9.7 ± 2.4, and 17.4 ± 6.4

h, respectively, after oral administration. Plasma concentrations then declined with

similar terminal half-lives as those observed after the intravenous dose. Oral

bioavailabilities of S-1, S-9, S-10, and S-11 at the 10 mg/kg dose were 55, 85, 69, and

84%, respectively.

3.3.1.3. Exposure-Response Relationship for SARMs

In vivo pharmacokinetic data suggested that the observed disparity between in vitro (i.e., AR binding affinity and ability to stimulate AR-mediated transcriptional activation) and in vivo pharmacologic activity might be due to differences in systemic exposure. We constructed AUC-response relationships in order to compare the in vivo activities of the halogen substituted SARMs at the same level of drug exposure. S-4, which showed the most efficacious in vivo activity in our historical data, also was compared. Table 3.3 shows the AUC-response relationship in levator ani muscle. Similar trends in the AUC-response relationship were observed in prostate and seminal vesicle

(data not shown). Interestingly, S-4, S-1, and S-9 exerted nearly identical in vivo pharmacologic activity when normalized to the same level of drug exposure. S-11, which was more efficacious than S-1 at comparable doses, was significantly less efficacious when compared at the same level of drug exposure. These results suggested that the in vivo pharmacologic activity of SARMS with high AR binding affinity (i.e., Ki < 10 nM) was largely governed by in vivo drug exposure, while sufficient in vivo exposure to elicit full in vivo agonist activity might be difficult to achieve for SARMs with lesser (i.e., Ki >

10 nM) AR binding affinity.

3.3.2. Cyano/Nitro group substituted SARMs

3.3.2.1. Binding Affinity and In Vitro Functional Activity of Cyano/Nitro Group

Substituted SARMs

The in vitro and in vivo pharmacologic activity and pharmacokinetic studies of halogen-substituted SARMs described above suggested that two simple criteria (i.e., AR binding affinity and in vivo clearance) could be used to select SARMs with promising in vivo pharmacologic activity. We tested this hypothesis using a series of four compounds incorporating either a nitro or cyano substituent at the para-position of the A- and B- aromatic rings (Table 3.4). Previous structure-activity relationship studies, molecular modeling studies, and in vivo metabolism studies suggested that cyano substituents at these positions might be structurally and metabolically favorable [55, 107, 110, 115]. All of these compounds (S-19, S-20, S-21, and S-22) bound to AR with high affinity and demonstrated agonist activity during in vitro cotransfection studies (Table 3.4), with no apparent differences to distinguish one compound from another.

3.3.2.2. Pharmacokinetics of Cyano/Nitro Group Substituted SARMs

We then determined the pharmacokinetics of S-19, S-20, S-21, and S-22 after administration of a single intravenous dose at 10 mg/kg (Table 3.4). Plasma concentrations of S-19, S-20, S-21, and S-22 declined in a biexponential manner after intravenous administration with terminal half-lives of 4.0, 3.7, 2.6, and 6.0 hr, respectively (Table 3.4 and Table 3.4). The Vss values of S-19, S-20, S-21, and S-22 were 1295 ± 171, 686 ± 42, 834 ± 88, and 635 ± 84 ml/kg, respectively. S-19 and S-21

(CL S-19 = 4.1 ± 0.1, CL S-20 = 4.0 ± 0.3 ml/min/kg), compounds bearing a nitro group in

the para-position of the B-ring, had higher CL values than S-20 and S-22 (CL S-20 = 2.4 ±

0.6, CL S-22 = 1.4 ± 0.3 ml/min/kg), compounds bearing a cyano group in the para-position of the B-ring. Corresponding to its lower CL value, S-22 showed the highest AUC ∞,

followed sequentially by S-20, S-21, and S-19. Based on the similar AR binding affinity but lower CL of S-22 as compared to S-19, S-20, and S-21, we predicted that S-22 would demonstrate the most promising in vivo activity.

3.3.2.3. In Vivo Pharmacologic Activity of Cyano/Nitro Group Substituted SARMs

We compared the in vivo androgenic and anabolic activities of this series of

cyano/nitro substituted SARMs at a 1 mg/day dose rate. Table 3.5 shows that these

compounds significantly stimulated the growth of prostate, seminal vesicles, and levator

ani muscle in castrated rats. S-19, S-20, S-21, and S-22 restored the weights of the

prostate to 25.2 ± 3.7, 34.2 ± 7.8, 31.2 ± 5.8 and 39.2 ± 8.7%, respectively, of that

observed in the intact control. The weight of seminal vesicle was also restored by S-19,

S-20, S-21, and S-22 to 19.0 ± 4.8, 47.4 ± 6.8, 21.5 ± 3.1 and 78.8 ± 9.1%, respectively,

of that observed in the intact control. S-19, S-20, S-21, and S-22 stimulated the growth of

levator ani muscle to a greater extent than androgenic organs, to 105.0 ± 13.4, 130.4 ±

4.9, 118.5 ± 15.8, and 141.9 ± 16.5, respectively. In vivo pharmacologic activity

correlated well with the AUC ∞ observed in pharmacokinetic studies within this series of

2 cyano/nitro substituted SARMs (r = 0.828), confirming that AUC ∞ is useful to predict in vivo activity for SARMs with high (i.e., Ki < 10 nM) AR binding affinity.

Expanded studies of the dose-response relationship for S-22 showed the highest in vivo anabolic activity of any compound that we, or anyone else, have examined.

Nonlinear regression analysis of dose-response relationships for S-22 showed that the

ED 50 values were 0.12 ± 0.05, 0.39 ± 0.15, and 0.03 ± 0.01 in prostate, seminal vesicles,

and levator ani muscle, respectively (Table 3.6, Table 3.2). S-22 maximally restored the 57

weights of prostate, seminal vesicles, and levator ani muscle to 51.1 ± 4.2, 98.0 ± 13.2,

and 136.3 ± 3.5%, respectively, of the intact control. S-22 exerted efficacious and

selective activity in anabolic tissues at dose rates as low as 0.03 mg/day, indicative of the

high potency of this compound in anabolic tissue (Relative potency = 4.41) and its

potential for clinical use.

3.4. DISCUSSION

In our early studies, we discovered a group of nonsteroidal AR ligands that

possess in vitro AR agonist activity and selected acetothiolutamide as a lead compound.

However, acetothiolutamide failed to show in vivo pharmacologic activity due to its poor

pharmacokinetic properties. Based on in vivo metabolism studies and structure-activity

relationships for AR binding, we found that structural modification to an ether linkage

instead of a thioether linkage reduced metabolic inactivation. S-4 and S-1 were chosen as

second-generation lead compounds. In subsequent studies, we have focused on discovery

of SARMs having efficacious and potent in vivo pharmacologic activity and favorable

pharmacokinetic properties by structural modification of the SARM pharmacophore,

focusing mainly on the B-ring since it is amenable to structural modification and critical

for agonist activity. 4-Halogen substituted SARMs that displayed potent in vitro and in

vivo activity were identified in our previous study [109]. In vitro AR binding affinity of

halogen-substituted SARMs increased as the size of the halogen atom in the B-ring

decreased and/or electronegativity increased; S-1 > S-9 > S-10 > S-11. Interestingly, in

vivo pharmacologic activity was not correlated with in vitro AR binding affinity; S-9 >

S-11 > S-1 > S-10. Pharmacokinetic studies of the halogen substituted SARMs in the 58

present study provided a valid explanation for this observed disparity, showing that the greater in vivo exposure of drug to target tissue results in greater in vivo pharmacologic effect than one would expect based on its AR binding affinity. Moreover, the AUC- response relationships suggest that the observed discrepancy between in vitro and in vivo pharmacologic activity of halogen substituted SARMs was due to differences in systemic exposure rather than intrinsic pharmacologic activity. Interestingly, the in vivo activity of compounds having binding affinity higher than an empirically determined cut-off (i.e., Ki

< 10 nM) was mainly governed by drug exposure (i.e., there were no apparent differences in intrinsic in vivo activity when normalized for exposure). Based on this finding, we hypothesized that two simple factors could be used to identify efficacious and potent

SARMs in this chemical class. Firstly, we selected compounds that possess Ki values lower than 10 nM. We then measured the in vivo CL of selected compounds as an additional selection criterion prior to dose-response analysis. A series of compounds with cyano or nitro substituents at the para positions of the A- and B-rings were selected for further study and demonstrated high AR binding affinity, suggesting that their in vivo activity would be closely related to their in vivo disposition. Pharmacokinetic studies of the selected compounds showed that the AUC ∞ of S-22 was the largest followed by S-20,

S-21, and S-19. In vivo pharmacologic studies of these compounds at the dose rate of 1

mg/day displayed the same rank order, as predicted based on the pharmacokinetic study.

The dose-response relationship of S-22, displaying the lowest CL among the series of

compounds, was examined. In fact, S-22 demonstrated the greatest in vivo androgenic

and anabolic activity of any AR nonsteroidal agonist examined to date and exceeded the

anabolic activity of TP. Moreover, S-22 was four times as potent as TP in levator ani muscle.

The present study demonstrates the importance of in vivo pharmacokinetics and

metabolism to predict and improve the in vivo response of SARMs. Structural

modification of our SARM pharmacophore resulted in various pharmacokinetic profiles.

Despite structural similarities, bicalutamide and S-4 exhibit vastly different

pharmacokinetic profiles. Bicalutamide, a clinically used antiandrogen, was eliminated

with a half-life of 18-21 hr in rats [104]. S-4 was eliminated with a much shorter half-life

of 3.7 hr in rats [47]. The clearance of bicalutamide (CL Bicalutamide = 0.8 ml/min/kg) was

slower than that of S-4 (CL S-4 = 1.5 ml/min/kg). Bicalutamide (Vss Bicalutamide = 1.2 - 1.3 l/kg) also has a larger volume of distribution than S-4 (Vss S-4 = 0.4 l/kg). S-1 is a structural intermediate with close similarity to both S-4 and bicalutamide. Fluoro- substitution of the B-ring, as compared to the acetamido group of S-4, produced a larger volume distribution (Vss S-1 = 1.5 l/kg) but faster clearance (CL S-1 = 4.0 ml/min/kg), resulting in a similar half-life (T 1/2 S-1 = 4 h) to S-4 (T 1/2 S-4 = 3.7 h). It is interesting to note that S-1 was cleared faster than S-4 despite the expectation that the fluoro substituent in the B-ring would be metabolically more stable than the acetamido group, suggesting that the presence of fluoro-substitution on the B-ring might increase the rate of hydroxylation or amide hydrolysis [46, 116] and/or increase the overall apparent CL due to an increase in the fraction unbound in plasma. When comparing the pharmacokinetics of S-1 to bicalutamide, which differ in the para-substituent of the A- ring and linkage (i.e., ether versus sulfonyl), S-1 demonstrated a similar volume of distribution but faster clearance (5-fold), resulting in a shorter half-life (5-fold).

Subsequent metabolism studies with S-1 (data not shown) suggested that the higher CL of S-1 was due to reduction of the nitro group in the A-ring, suggesting that replacement of the nitro group with a cyano-substituent on the A-ring would reduce clearance and enhance pharmacologic activity in vivo . These results demonstrate the significance of

A/B-ring substitution on the pharmacokinetic characteristics of SARMs. B-ring

substitution at the para position with halogens significantly affected the volume of

distribution and clearance. Compounds incorporating larger halogen substituents (e.g.,

bromine or iodine) produced slower clearance, resulting in prolonged terminal half-lives

in spite of a reduction in the volume of distribution, suggesting that larger halogens

and/or less electronegative substituents at this position might decrease the rate of B-ring

hydroxylation and/or affect the volume of distribution and CL by altering plasma protein

binding. In the series of compounds with cyano or nitro-substituents at the para-position

of the A- or B-ring, the compounds bearing a nitro group in the B-ring (CL S-19 = 4.1, CL S-

21 = 4.0) were cleared faster than their cyano-substituted counterpart (CL S-20 = 2.4, CL S-22

= 1.4). This result suggests that the presence of a nitro group on the B-ring increases drug metabolism via reduction of the nitro group to an amine [117]. Comparing the two compounds having a cyano group in the B-ring, S-22 (CL S-22 = 1.4), substituted with a cyano group at the para -position of the A-ring, was more metabolically stable than S-20

(CL S-20 = 2.4), its counterpart containing a nitro group in A-ring, indicating that the nitro group in A-ring was also more susceptible to in vivo metabolism than the cyano group. In the series of compounds having nitro group in B-ring (S-19 and S-21), incorporation of a cyano group in the A-ring was not helpful to reduce the clearance as S-19 and S-21 showed faster CL than the other compounds, suggesting that the CL induced by nitro-

substitution on B-ring was the major factor controlling the rate of metabolism in this class of compounds.

In summary, we examined the in vivo pharmacologic activity and pharmacokinetics of halogen-substituted SARMs in the present study. The results demonstrate the importance of in vivo pharmacokinetics and metabolism to SARM activity and the inability of in vitro models to predict in vivo response. With this understanding, we screened a series of AR nonsteroidal ligands. We identified S-22 as a compound with the most potent and tissue-selective in vivo activity that we have observed to date, along with favorable pharmacokinetic properties. Halogen-substituted

SARMs are promising for clinical use on androgen-deficiency related disorders. Our continuing studies will evaluate the pre-clinical and clinical value of identified SARMs and optimize the chemical structures based upon integrated structural, pharmacologic, and pharmacokinetic data.

Relative Compounds X X X K (nM) 1 2 3 i Activity (%)

DHT - - - 0.27 a 100 b c R-Bicalutamide CN SO 2 F 11.0 ± 1.5 8.3 ± 2.7 d d Acetothiolutamide CN S NHCOCH 3 4.9 ± 0.2 50 e e S-4 NO 2 O NHCOCH 3 4.0 ± 0.7 93 ± 7 a a S-1 NO 2 O F 6.1 ± 0.2 43.4 ± 2.6 a a S-9 NO 2 O Cl 9.6 ± 0.7 64.0 ± 11.2 a a S-10 NO 2 O Br 11.6 ± 0.4 17.0 ± 2.9 a a S-11 NO 2 O I 30.0 ± 2.7 16.0 ± 2.9

Table 3.1 Chemical structures for nonsteroidal AR ligands.

Previously reported in a Marhefka et al., 2004; b Mukherjee et al., 1996; c Dalton et al.,

1998; d Yin et al., 2003b; e Yin et al., 2003c. Relative activity was reported as a percentage of the transcriptional activation observed for 1 nM DHT. Transcriptional activation was measured using a single concentration of SARMs (10 nM) and R- bicalutamide (1,000 nM).

Emax (% of Relative ED Relative Organs Treatment 50 Intact Control) Efficacy (mg/day) Potency Androgenic Activity TP a 120.6 ± 13.4 1.00 0.13 ± 0.03 1.00 S-1 a 14.5 ± 0.7 0.12 0.42 ± 0.04 0.31 S-9 33.8 ± 4.0 0.28 0.25 ± 0.06 0.52 Prostate S-10 18.8 ± 3.0 0.16 0.37 ± 0.25 0.35 S-11 25.3 ± 2.4 0.21 0.28 ± 0.11 0.46 S-22 51.1 ± 4.2 0.42 0.12 ± 0.05 1.08 TP a 70.0 ± 18.8 1.00 0.12 ± 0.02 1.00 S-1 a 12.7 ± 3.1 0.18 0.38 ± 0.26 0.32 Seminal S-9 28.5 ± 3.9 0.41 0.48 ± 0.09 0.25 Vesicles S-10 11.5 ± 0.6 0.17 0.10 ± 0.04 1.20 S-11 19.4 ± 2.0 0.28 0.20 ± 0.13 0.60 S-22 98.0 ± 13.2 1.40 0.39 ± 0.15 0.31 Anabolic Activity TP a 104.2 ± 10.1 1.00 0.15 ± 0.03 1.00 S-1 a 74.9 ± 0.4 0.72 0.44 ± 0.01 0.34 Levator ani S-9 137 ± 9.4 1.31 0.29 ± 0.09 0.52 muscle S-10 64.4 ± 3.7 0.62 0.11 ± 0.09 1.36 S-11 95.3 ± 7.2 0.91 0.23 ± 0.11 0.65 S-22 136.3 ± 3.5 1.31 0.03 ± 0.01 4.41

Table 3.2 Mean (± S.D.) androgenic and anabolic activities of 4-halogen substituted

SARMs to TP in castrated male rats a In vivo androgenic anabolic activities of TP and S-1 were previously reported in Yin et al., 2003C.

Parameter S-1 S-9 S-10 S-11 Intravenous Administration

T1/2 (hr) 4.1 7.4 11.4 14.7 CL (ml/min/kg) 4.0 ± 0.5 1.1 ± 0.2 0.4 ± 0.02 0.2 ± 0.03

Vss (ml/kg) 1458 ± 352 818 ± 77 461 ± 101 240 ± 33

AUC ∞ ( µg·hr/ml) 43 ± 5 160 ± 33 401 ± 21 1027 ± 206 MRT (hr) 6.1 ± 0.2 13.2 ± 3.0 18.5 ± 4.5 24.5 ± 5.2 Oral Administration

T1/2 (hr) 3.7 8.2 11.1 17.1

AUC ∞ ( µg·hr/ml) 24 ± 4 136 ± 11 276 ± 74 860 ± 218

Fp.o. (%) 55% 85% 69% 84%

Table 3.3 Pharmacokinetic parameters of 4-halogen substituted SARMs in male rats after a single intravenous or oral administration at 10 mg/kg.

S-19 S-20 S-21 S-22 Chemical Structure

X1 NO 2 NO 2 CN CN

X2 NO 2 CN NO 2 CN In Vitro Pharmacologic Activity

Ki 2.2 ± 0.4 2.0 ± 0.2 2.5 ± 0.2 3.8 ± 0.5 Relative Activity (%) 97 ± 1 102 ± 20 106 ± 15 94 ± 27 Pharmacokinetics

T1/2 (hr) 4.0 3.7 2.6 6.0 CL (ml/min/kg) 4.1 ± 0.1 2.4 ± 0.6 4.0 ± 0.3 1.4 ± 0.3

Vss (ml/kg) 1295 ± 171 686 ± 42 834 ± 88 635 ± 84

AUC ∞ ( µg·hr/ml) 41 ± 1 73 ± 14 42 ± 3 127 ± 27 MRT (min) 317 ± 47 288 ± 58 210 ± 37 475 ± 46

Table 3.4 Chemical structures, AR relative binding affinity (Ki), in vitro transcriptional activities, and pharmacokinetic parameter of cyano/nitro group substituted SARMs.

Aromatic rings are designated as A and B as shown.

Figure 3.1 Dose-response relationships of S-9 (A), S-10 (B), and S-11 (C) in androgenic and anabolic organs of castrated male rats.

Continued

Figure 3.1 continued

Figure 3.2.1 Plasma concentrations of 4-halogen substituted SARMs after a single intravenous dose of 10 mg/kg to male rats.

Figure 3.2.2 Plasma concentrations of 4-halogen substituted SARMs after a single oral dose of 10 mg/kg to male rats.

Figure 3.3 AUC-response relationship of S-4 and halogen substituted SARMs in the levator ani muscle.

100

S-19 S-20 S-21 S-22 10

1 Plasma Concentration(mg/L)

0.1 0 500 1000 1500 2000 2500

Time (min)

Figure 3.4 Plasma concentrations of S-19, S-20, S-21, and S-22 after a single intravenous dose of 10 mg/kg to male rats.

Figure 3.5 In vivo pharmacologic activity of S-19, S-20, S-21, and S-22 in androgenic and anabolic organs of castrated male rats.

Figure 3.6 Dose-response relationships of S-22 in androgenic and anabolic organs of castrated male rats. The weights of prostate (closed circles) and seminal vesicles (open circles) were used as markers of androgenic effects and that of levator ani muscle

(triangles) was used as a marker of anabolic effects.

CHAPTER 4

EFFECT OF PARA HALOGEN MODIFICATION OF S-3-(PHENOXY)-2-

HYDROXY-2-METHYL-N-(4-NITRO-3-TRIFLUOROMETHYL-PHENYL)-

PROPIONAMIDES ON METABOLISM; FRACTION UNBOUND VS INTRINSIC

CLEARANCE

4.1. INTRODUCTION

Synthesized steroidal androgens have been used as valuable therapeutic agents for androgen deficiency. However it is unrealistic to use unmodified testosterone for clinical uses due to its rapid hepatic elimination. In order to improve pharmacokinetic properties, various formulations of androgens and their prodrugs were attempted. Unfortunately, virtually none of these attempts were successful. Introduction of an alkyl group or esterification on position 17 of testosterone prolong its half-life but other side effects (e.g., hepatotoxicity and variability of serum testosterone level) become problematic [30].

Androgen formulations administered via various routes (e.g., intramuscular injection, buccal administration, subcutaneous injection of microcapsules, transdermal delivery,

pellet implantation) have been approved or are being developed for clinical uses [30, 35-

38]. However unfavorable properties of these formulations such as patient compliance, early burst release, skin irritation and pellet extrusion are a concern. Moreover, the cross- reactivity of steroidal androgens and their metabolites with other steroid hormone receptors and the potential risks of androgens therapy in androgenic organ (i.e., prostate) hamper more widely accepted use of androgen therapy from more a widely accepted use.

Nonsteroidal selective androgen receptor modulators (SARMs) are theoretically advantageous over conventional steroidal therapies. Nonsteroidal ligands have greater flexibility in structural modification for optimal pharmacologic and pharmacokinetic properties. It is also expected that nonsteroidal ligands could possibly avoid cross- reactivity with steroid hormone receptors other than AR [118]. More importantly, the tissue selectivity of SARMs would offer great opportunities to treat androgen-deficiency related disorder with minimized side effects in androgenic organs. Our laboratory was the first to report SARMs with in vivo pharmacologic activity [45]. In our early study with affinity ligands, we identified R-1 (Table 4.1) as an efficacious AR agonist among a series of electrophilic nonsteroidal AR ligands. However, due to concern about the electrophilic property that can potentially alkylate many nucleophilic sites in a cell, we designed and synthesized acetothiolutamide (Table 4.1) [46]. In spite of the high AR binding affinity and in vitro AR agonist activity of the compound, acetothiolutamide displayed weak in vivo activity and subsequent pharmacokinetic and metabolism study demonstrated that this discrepancy was due to extensive hepatic metabolism of acetothiolutamide, mainly oxidation at the sulfur linkage. Therefore, we changed the sulfur linkage of acetothiolutamide to an ether linkage to improve metabolic stability [45].

Among these compounds, S-1 and S-4 (Table 4.1) were identified as SARMs and these first generation SARMs showed full agonistic activity in the anabolic organ (i.e., levator ani muscle) but partial agonistic activity in the androgenic organ (i.e., prostate and seminal vesicles). These SARMs displayed better pharmacokinetic properties than that of their steroidal counterpart, including oral bioavailability, long half-life, and linear kinetics [47, 48]. Our structure-activity relationship studies and in vivo metabolism studies demonstrated that the chemical modification of B-ring (Table 4.1) significantly changed in vitro binding affinities and transcriptional activation and that the B-ring is one of the susceptible sites for drug metabolism [46, 107]. Therefore, we further studied the in vitro and in vivo pharmacologic activities of various B-ring substituents bearing ether- linkage. Among a series of B-ring substituents, four halogen-substituted analogs exerted high AR binding affinities and ability to stimulate AR-mediated transcription [109].

Interestingly, in vivo pharmacologic activities did not correlate with in vitro pharmacologic activities. Further pharmacokinetic study on these compounds suggested that the discrepancy between in vitro and in vivo pharmacologic activities was due to differences in systemic exposure and demonstrated the importance of in vivo pharmacokinetics and metabolism to predict and improve the in vivo activities of SARMs

[111]. In the present study, we studied the effect of B-ring halogen substitution of

SARMs on the in vivo metabolism pathway. Moreover, we speculated which pharmacokinetic parameters play an important role in determining the clearance of halogen substituted SARMs and examined plasma protein binding of the compounds, which is one of determinants. The results of these are reported herein.

4.2. MATERIALS AND METHODS

4.2.1. Chemicals

The S-3-(4-halophenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethylphenyl)

propionamides (i.e., S-1, S-9, S-10, and S-11), the internal standard (i.e., a 2,4-

difluoropropionamide derivatives), and four (4-nitro/cyano phenoxy)-2-hydroxy-2-

methyl-N-(4-nitro/cyano-3-trifluromethylphenyl) derivatives (i.e., S-19, S-20, S-21, and

S-22) were prepared in our laboratories as previously described [Table 3.1, [109]. Ethyl

alcohol USP was purchased from Pharmco Products, Inc., (Brookfield, CT). HPLC grade

acetonitrile and water were purchased from Fisher Scientific Co. (Fair Lawn, NJ). All

other chemicals and reagents were purchased form Sigma-Aldrich (St. Louis, MO).

4.2.2. Animals

Sprague-Dawley rats were purchased from Harlan (Indianapolis, IN). The animals

were maintained on a 12-h light/dark cycle with food and water available ad libitum. All

animal protocols were reviewed and approved by the Institutional Laboratory Animal

Care and Use Committee of The Ohio State University.

4.2.3. Metabolism Studies

The in vivo metabolism of S-9, S-10, and S-11 was determined in rats using the

method described previously with modification [46]. Briefly, male Sprague-Dawley rats

(200 to 250 g) were catheterized in the jugular vein and housed in clean metabolism

cages one day prior to dosing. Urine and feces were collected for 24 hr before dosing.

The compounds were administered via jugular vein at a dose of 30 mg/kg. Urine and fecal samples were collected at 24 hr or 48 hr intervals for about five times of half-life of each compound after the dose. The rats were transferred to a clean cage at each collection interval. All samples were stored at -80 °C until analysis.

Urine samples including blank urine (urine sample before dosing) were thawed and centrifuged. An aliquot (5 ml) of urine sample was mixed with the same volume of ethyl acetate and vigorously shaken for 30 min. This extraction procedure was repeated three times and the organic phase was pooled after each extraction. The combined ethyl acetate extract and the aqueous phase of urine samples were evaporated separately under nitrogen gas purging. The residues of the ethyl acetate extract and aqueous phase were reconstituted in acetonitrile/water [50:50 (v/v)] and acetonitrile/water [10:90 (v/v)], respectively. The reconstituted solutions were then filtered using membrane filter (0.45

µm; Millipore Corporation), followed by LC-MS n analysis.

Fecal samples (about 9g) including blank fecal samples were thawed and homogenized using a mechanical homogenizer after adding 15 ml of distilled water. The fecal homogenates were extracted three times with a mixture of methanol /ethyl acetate

[2:1 (v/v)]. After centrifugation, the liquid phase after each extraction was pooled and evaporated under nitrogen gas. The residues were reconstituted in methanol and filtered using membrane filter, followed by LC-MS n analysis.

4.2.4. LC-MS n Analysis

The metabolites of S-9, S-10, and S-11 were qualitatively identified using LC-MS analysis. The LC/MS analyses were performed using a Thermo Finnigan LCQ Deca

quadrupole ion-trap mass spectrometer (San Jose, CA, USA) coupled with a surveyor

HPLC system and an electrospray ionization (ESI) source. An aliquot (10 µL) of each

sample was injected into a Waters XTerra C18 column (150 × 2.1 mm, 3.5 µm) at a flow rate of 0.2 mL/min using a gradient mobile phase comprised of acetonitrile and water.

The gradient was initiated with 0% of acetonitrile for the first 4 min and the flow was diverted to waste. The percentage of acetonitrile was increased to 65% (for S-9 and S-10) or 75% (for S-11) in a linear gradient from 4 to 40 min, then rapidly changed to 90 % from 40 to 41 min, and was maintained at this composition for 9 min. Finally, the composition of mobile phase returned to 0 % acetonitrile at 51 min and stayed at the initial composition for 9 min.

The general system parameters were tuned with the parent compounds (i.e., S-9,

S-10, and S-11) and operated in negative ion mode. The temperature of the heated capillary was set at 200 °C and spray voltage was 3.6 kV. The sheath and auxiliary gas flow rates were 60 and 20 mL/min, respectively. The scan range for full MS scans was

150-2000 m/z. For MS 2 and MS 3 analysis, and precursor ions were isolated with a width of 5 m/z. Data acquisition was controlled by Xcaliber software and mass spectrum data was analyzed using Metabolite ID software.

4.2.5 Plasma Protein Binding Study

Fresh blood was collected from male rats into vacutainers containing sodium heparin and centrifuged to obtain plasma and was stored at -20 °C until use. The binding of compounds to rat plasma proteins was measured by an equilibrium dialysis method using a 96-well dialysis block constructed of Teflon with a dialysis membrane (MWCO

12-14K , HTDialysis, Gales Ferry, CT) [119]. In order to hydrate dialysis membranes, the

membranes were soaked in phosphate buffer solution (PBS) for 1 hr and then in PBS

with 20% ethanol for 20 min. The membranes were rinsed twice with PBS and soaked in

PBS until time of use. The dialysis block was assembled according to manufacturer’s

instruction. 150 µl of PBS was pipetted into the dialysate side of dialysis block. And 150

µl of fresh plasma (99.5%) spiked with test compound solution in DMSO (0.5%) was

loaded into the sample side of dialysis block. The top surface of the dialysis block was

covered with an adhesive sealing film to prevent evaporation and pH change during the

incubation. The dialysis block was incubated at 250 rpm, 37 °C for 6 hr using an orbital

shaker. At the end of incubation, plasma and buffer volumes were recorded for all

samples and the drug concentrations of plasma and buffer were determined using the LC-

MS method described below. The fraction of drug bound was calculated using the

following equation [120].

Fb (fraction of drug bound) = [(D t-Df) * V pe /V pi ] / {[(D t-Df) * V pe /V pi ] + D f}* 100%

Where D t and D f represent the drug concentration in plasma side (total drug concentration at equilibrium) and buffer side (unbound drug concentration), respectively.

Vpi and V pe represent the initial and equilibrium plasma volume, respectively. As a

reference drug, bicalutamide was used. Whenever the plasma protein binding affinities of

SARMs were measured, bicalutamide was included and the measured fraction value of

unbound bicalutamide was compared to the published unbound fraction value (4% ~ 6%)

in order to validate our experimental method [104].

4.2.6 LC-MS Analysis

An aliquot (50 µl) of plasma or buffer from the plasma protein binding study was spiked with 50 µl of an internal standard (2, 4- difluoropropionamide derivative, Table

4.1) solution in acetonitrile at the designated concentration on Table 4.2. For the samples in plasma side, additional 1 ml of acetonitrile was added to precipitate plasma protein.

The samples from plasma side and buffer side were vortexed and centrifuged at 16,000g,

4°C for 5 min. The supernatant was transferred to another tube and the volume of each

® compound listed in Table 4.2 was injected to a C 8 column (XTerra ; 21 mm i.d. × 50 mm length, 3.5 µm particle size; Waters Corporation, Milford, MA) that connected to LC/MS system (Agilent 1100 series, Palo Alto, CA). Mobile phase was comprised of acetonitrile

(50%) and 0.1% acetic acid water (50%) at a flow rate of 0.2 ml/min. The column effluent was introduced into the electrospray interface, which was set at the following conditions: dry gas flow 8.0 l/min; nebulizer pressure 25 psi; drying gas temperature

350 °C; capillary voltage 3000 v; and fragmentor voltage 180 V. All other LC-MS parameters were set at default. All samples were analyzed using single ion monitoring

(SIM) in the negative ion mode. Analytical data were acquired by ChemStation software.

Calibration standards were measured at the concentration range as listed in Table 4.2.

The accuracy, precision, and recovery of the bioanalytical method for each compound were validated according to FDA guidelines.

4.3. RESULTS

The metabolite of S-9, S-10, and S-11 in the urine and feces after i.v. administration in rats were determined. In order to identify drug-related components, the 82

mass spectra of post-dose samples were compared to blank urine samples obtained prior to dosing. Molecular ions of interest were characterized using LC/MS n analysis.

Identification of the metabolites of halogen-substituted SARMs was based on the

understanding of the fragmentation pathways of the parent compounds. No metabolite

and parent compound from the feces samples was found. Therefore, urinary metabolites

of S-9, S-10, and S-11 are reported herein.

4.3.1. Identification of Metabolites of S-9, S-10, and S-11 in Rats

4.3.1.1. Metabolites of S-9 in rats

Urine samples were collected at 24, 48, and 96 hr after the dose. The urinary

samples collected at 24 and 48 hr showed similar metabolic profiles as listed at Table 4.3.

And the major drug-related molecular ions in these samples were found at m/z 229, 231,

301, 483, and 485. The drug-related molecular ions in the urine sample collected at 96 hr

were found only at m/z 229, 231, 301, 483, and 485.

Since a molecule containing a chlorine atom displays a unique mass spectrum

pattern in that two peaks are separated by 2 m/z unit and with a ratio of 3:1 in the peak

heights (Figure 4.1), this property aided in recognition and characterization of the

molecular anion and metabolite ions. All molecular ions and fragment ions of S-9 and its

metabolite having a 2 Da difference that are listed and discussed below showed this

unique pattern (i.e., 3:1 ratio in peak height) and this fact will not be mentioned from now

on. Proposed metabolite structure and fragmentation patterns of S-9 in rats are

summarized in Table 4.3 and discussed below.

Molecular ion at m/z 417 and 419 ([M-H] - of the S-9 parent compound). The theoretical mass of the S-9 is 418 and 420. The molecular ion at m/z 417 and 419 showed identical fragmentation patterns (m/z 261 and 289) and retention time (46.8 min) with those of the synthetic standard, confirming that the ions at m/z 417 and 419 is the molecular anion of the parent compound, S-9. The fragment ion at m/z 261 was the base peak in the spectrum and corresponds to a cleavage of the bond between the chiral carbon and methylene carbon, with a loss of the methyl group. The fragment ion at m/z 289 corresponds to a cleavage of the bond between the oxygen linkage and methylene carbon, with the loss of the hydrogen atom in the hydroxyl group attached to the chiral carbon.

Molecular ion at m/z 483 and 485. The [M-H] - at m/z 483 and 485 were eluted in two peaks at 24.8 (M9-1) and 23.0 min (M9-2). The fragmentation pattern for both peaks produced daughter ions at m/z 403 and 405. The loss of 80 Da from the molecular ions at m/z 483 and 485 to base fragment ions at m/z 403 and 405 correspond to an SO 3 moiety and suggested that these metabolites were a sulfate conjugate. Further fragmentation of the daughter ions (m/z 403 and 405) at each peak yielded the following fragmentation patterns: The [M-H] - at 23.0 min yielded the fragment ion at 275. The [M-H] - at 24.8 min yielded the fragment ions at 143 and 145.

The fragment ion at m/z 275 corresponds to reduction of nitro group of A-ring to hydroxyl amine group and cleavage of the bond between the oxygen linkage and methylene carbon with the loss of the hydrogen atom in the hydroxyl group attached to the chiral carbon. The fact that this proposed fragmentation pattern is identical to those of parent compound (see above) suggests that this fragmentation pathway is highly

favorable. Thus, M9-1 was most likely produced by the reduced metabolite in A-ring to hydroxyl amine and subsequent sulfate conjugation on this reduced moiety.

The fragment ions at m/z 143 and 145 correspond to mono-oxidized B-ring moiety from a cleavage of the bond between methylene carbon and oxygen linker, indicating that base fragment ion (m/z 403 and 405) at 23.0 min correspond to the reduction of the nitro group in A-ring to an amine group and oxidation in B-ring. The sulfate conjugation at the hydroxyl group in the B-ring of this proposed molecule seems to produce the M9-2.

Molecular ion at m/z 229 and 231. The retention time of this molecular ion was

17.4 min. This metabolite is thought to correspond to the hydrolysis product of S-9 at the amide bond. This [M-H] - ion produce fragment ions at m/z 127 and 129 (Table 4-3), which corresponds to the cleavage of the bond between methylene carbon and oxygen linker.

Molecular ion at m/z 301. The retention time of this molecular ion was 19.4 min.

This metabolite was proposed to be the sulfated product of 4-nitro-3- trifluoromethylphenylamine, which came from the hydrolysis metabolite other than M9-3.

Fragmentation of this metabolite produced base fragment ion at m/z 221 by the loss of 80

Da from the molecular ions (M9-4), indicating that M9-4 was a sulfate conjugate. Further fragmentation of ion at m/z 221 produced ions at 171 and 191. Our previous metabolisms study on S-1 (Table 4.1) showed same fragmentation pattern on this metabolite [121], supporting the proposed metabolite structure.

Molecular ion at m/z 403 and 405. The [M-H] - at m/z 403 and 405 were eluted in

two peaks at 38.8 min (M9-5) and 35.8 (M9-6). These molecular ions of M9-5 and M9-6

displayed very similar fragmentation patterns to those of base fragment ions of M9-1 and

M9-2, respectively (Table 4.3). These fragmentation patterns indicate that M9-5 was produced by the reduction of nitro group in A-ring to hydroxyl amine group. Moreover,

M9-6 was produced by the reduction of nitro group in A-ring to amine group and oxidation in B-ring. M9-5 and M9-6 had delayed retention times from those of M9-1 and

M9-2, confirming that M9-1 and M9-2 contain sulfate conjugate (i.e., hydrophilic moiety).

Molecular ion at m/z 387 and 389. The retention time of this molecular ion was

40.7 min and corresponds to reduction of nitro group to amine group in A-ring. The MS 2 spectrum of this molecular ion displayed fragment ions at m/z 127, 129, and 259, which corresponds to the cleavage of the bond between the oxygen linkage and methylene carbon in M9-7 (Table 4.3). Further fragmentation of base fragment ion at m/z 259 produced fragment ions at m/z 175, 189, 201, 209, and 229.

Molecular ion at m/z 499 and 501. The retention time of this molecular ion was

19.8 min. This molecular ion yielded product ions at m/z 159, 161, 277, 419, and 421.

The loss of 80 Da from the molecular ion to base fragment ion at m/z 419 and 421 suggested that this metabolite was a sulfate conjugate. The MS 3 spectrum of base

fragment ion (m/z 419 and 421) yielded fragment ions at m/z 159, 161, and 277. The

fragment ions at m/z 159 and 161 are assigned as double oxidized para-chlorophenol. The

proposed metabolism pathway of this metabolite is reduction at nitro group on A-ring to

amine group, double-oxidation on B-ring, and sulfate conjugation probably on a hydroxyl

group on B-ring.

Molecular ion at m/z 579 and 581. The [M-H] - at m/z 579 and 581 were eluted in

two separated peaks at 19.7 min (M9-9) and 23.3 min (M9-10). Both molecular ions

produced base fragment ions at m/z 403 and 405 (176 Da loss), indicating that these

metabolites were a glucuronide conjugate. Further fragmentations of the base ions at 19.7

min yield a very similar fragmentation pattern to M9-3 (ions at m/z 259, 143, and 145),

suggesting that M9-9 was produced by reduction of nitro group to amine group on A-ring,

oxidation on B-ring, and glucuronide conjugation. The base ions at 23.3 produced

fragment ions at m/z 275, which are same as those of M9-4. It suggests that M9-10 was

glucuronide conjugate of reduced metabolite in A-ring to hydroxyl amine.

4.3.1.2. Metabolites of S-10 in rats

Urine samples were collected at 48 and 96 hr after the dose. Drug-related molecular ions in urine sample collected at 48 hr were detected at m/z 527, 529, 273, 275,

301, 447 and 449 and molecular ion of parent compound was not found. Urine sample collected at 96 hr contained molecular ion at m/z 527 and 529. Similar to compound S-9, compound S-10 containing a bromine atom displays unique mass spectrum pattern with two peaks separated by 2 m/z unit but with ratio of 1:1 in the peak heights (Figure 4.1).

This unique characteristic played a critical role for structural identification of metabolites from S-10. All molecular ions and fragment ions of S-10 and its metabolite having a 2 Da difference that are listed in table 4-4 showed this unique pattern (i.e., 1:1 ratio in peak height). By characterization of these molecular ions with LC/MS n, the structures of six metabolites were identified (Table 4-4). As shown in table 4-4, the fragmentation patterns of M10-1 to M10-6 were almost identical to M9-1 to M9-6, respectively. Detailed

description about fragmentation would be redundant and is omitted. S-10 displayed a similar metabolism pathway to S-9, but with less metabolite species and no parent compound was found. The fragmentation data suggested that S-10 underwent hydrolysis at the amide bond (M10-3 and M10-4) and reduction of nitro group to hydroxyl amine

(M10-5) and single oxidation on B-ring (M10-6) followed by sulfate conjugation (M10-1 and M10-2). Metabolites in urine samples were eluted at 25.5 (M10-1), 23.7 (M10-2),

21.7 (M10-3), 19.6 (M10-4), 39.8 (M10-5), and 38.8 min (M10-6).

4.3.1.3. Metabolites of S-11 in rats

Urine samples were collected at 48 and 96 hr after the dose. Drug-related molecular ions in urine sample collected at 48 hr were detected at m/z 575, 321, and 301 and molecular ion of parent compound was not found in urine sample. Urine sample collected at 96 hr contained molecular ion at m/z 575. By characterization of these molecular ions with LC/MS n, the structures of four metabolites were identified (Table 4-

5). S-11 displayed similar metabolism pathway to S-9 and S-10, but less metabolite species were found. As shown in table 4-5, the fragmentation patterns of M11-1 to M11-4 were very similar to M9-1 to M9-4 (or M10-1 to M10-4), respectively. Detailed description about fragmentation would be redundant and is omitted. The fragmentation data suggested that S-11 underwent hydrolysis at the amide bond (M11-3 and M11-4) and reduction of nitro group to hydroxyl amine and single oxidation on B-ring followed by sulfate conjugation (M11-1 and M11-2). Metabolites in urine samples were eluted at

24.8 (M11-1), 22.9 (M11-2), 21.5 (M11-3), and 17.5 min (M11-4).

4.3.2. Plasma Protein Binding Study of Various SARMs

We chose the testing concentration range of this plasma protein binding study as

50 µM to 5 µM, based on plasma concentration range in our previous pharmacokinetic

study [111] and the detection limits of compounds interested in LC/MS assay. The

unbound fractions of various SARMs along with halogen-substituted SARMs are

reported in Table 4-1. The unbound S-11 at the concentration of 5 µM could not be measured, due to the drug concentration of unbound fraction being lower than the detection limit of the assay. The unbound fractions of 50 µM tested SARMs were similar

or slightly higher than those of the 5 µM SARMs. With exception of S-4, eight B-ring modified SARMs displayed unbound fractions less than 1%. It is shown that 6.27 % and

4.70 % of S-4 existed as unbound to plasma protein at 50 and 5 µM, respectively. Among halogen-substituted SARMs, S-1 showed the greatest unbound fraction (0.78% ± 0.17%, mean ± stdev), followed by S-9 (0.10% ± 0.04%), S-10 (0.03% ± 0.01%), and S-11 (0.01

± 0.00) at the concentration of 50 µM. In the cyano/nitro substituted SARMs, 50 µM of

S-19, S-20, S-21, and S-22 displayed the unbound fraction of 0.42 ± 0.14, 0.70 ± 0.01,

0.51 ± 0.02, and 0.96 ± 0.14, respectively. In the concentration of 5 µM, the unbound fraction of S-19, S-20, S-21, and S-22 were 0.35 ± 0.06, 0.34 ± 0.08, 0.27 ± 0.06, and

0.85 ± 0.00, respectively. These results seemed to suggest that the lipophilicity of substituted moiety in SARM correlate with plasma protein binding affinity. To see a more clear relationship, we used retention time in a C 8 column (described in 4.2.5. LC-

MS Analysis) as an indicator of lipophilicity and obtained the correlation coefficient

between unbound fraction vs retention time in a linear regression relationship. Figure 4.3

shows the relationship between retention time and unbound fraction of nine SARMs and bicalutamide at the concentration of 50 µM. The retention time and logarithm of unbound

fraction were highly correlated with R 2 = 0.954. Moreover, the plasma protein binding

affinities of S-22 at the large concentration range from 0.5 to 200 µM were measured. .

The fractions of unbound S-22 were ranging from 0.8% to 1.4% (Figure 4.4). The

fraction of unbound S-22 seemed to slightly increase as drug concentration increased, but

with vague trend.

4.4. DISCUSSION

Our previous study showed that in vitro AR binding affinity of halogen-

substituted SARMs increased as electronegativity increased and/or the size of the halogen

atom in the B-ring decreased; S-1 > S-9 > S-10 > S-11. However, in vivo pharmacologic activity was not correlated with in vitro binding affinity; S-9 > S-11 > S-1 >S-10 [109].

The subsequent pharmacokinetic studies of the halogen-substituted SARMs suggests that the greater drug exposure of the compound compensated the weak binding affinity and resulted in the discrepancy between in vitro binding affinity and in vivo pharmacologic activity [111]. These results demonstrated the importance of in vivo pharmacokinetics and metabolism to predict and improve the in vivo pharmacologic activities of SARMs.

In this study, we examined in vivo metabolic pathways of three halogen- substituted SARMs (i.e., S-9, S-10, S-11) and the metabolic pathways are outlined in

Figure 4.1. The in vivo metabolism of S-1 was reported in previous study [121]. Although the metabolite species of SARMs had differences, major metabolism pathways of halogen-substituted SARMs were the same. All four halogen-substituted SARMs

underwent three major phase I metabolism pathways; 1) hydrolysis of the amide bond, 2)

B-ring hydroxylation, 3) A-ring nitro reduction to an aromatic amine. On the other hand, bicalutamide (Table 4.1) exhibited two major metabolic pathways: 1) hydrolysis of amide bond and 2) B-ring hydroxylation without A-ring nitro reduction [122]. The comparison of metabolism pathways between halogen-substituted SARMs and bicalutamide suggests that substitution of nitro-group to cyano-group in the A-ring of SARMs could protect drugs from A-ring reduction, resulting in less clearance.

Furthermore, phase I metabolites of halogen-substituted SARMs underwent phase

II metabolisms including sulfation and/or glucuronidation. All four halogen-substituted

SARMs displayed sulfation metabolites but only S-1 and S-9 displayed glucuronidation metabolites. This phase II metabolism pattern might be due to the high affinity of sulfate- conjugation and the large capacity of glucuronide-conjugation. The phase I and II metabolism patterns suggest that the B-ring modification with halogen atom changes the rate of each metabolism pathway without the change of metabolism pathway. The question which pharmacokinetic factors affecting the rate of metabolism are changed by

B-ring substitution still remains. To answer this question, we returned to the basic equations explaining pharmacokinetic phenomenon. If we assumed that halogen- substituted SARMs are eliminated entirely via the liver [123], the hepatic clearance is equal to the systemic or total body clearance (Equation 4.1). With halogen-substituted

SARMs having very low clearance (hepatic extraction ratio; 0.07 ~ 0.004) [124], hepatic clearance becomes independent of hepatic blood flow and venous equilibration model equation (equation 4.1) can be simplified to the equation 4.2.

CL total ≈ (Q H × f u × CL int ) / (Q H + f u × CL int ) Equation 4.1

CL totoal ≈ f u × CL int Equation 4.2

Where CL total was total clearance, Q H was total hepatic blood flow, f u was fraction unbound, and CL int was free intrinsic clearance. This equation suggests that the CL total of

halogen substituted SARMs are mainly governed by f u and CL int . First, we examined

plasma protein bindings of halogen-substituted SARMs in this study. The result shows

that the f u significantly decreased, as the size of atom on B-ring increased. Based on the

equation 4.2, CL int of halogen-substituted SARMs were calculated using measured f u

(Table 4.1) and CL total (CL S-1 = 4.0 ± 0.5, CL S-9 = 1.1 ± 0.2, CL S-10 = 0.4 ± 0.02, CL S-11 =

0.2 ± 0.03 mL/min/kg) [111]. The calculated CL int values of S-1, S-9, S-10, and S-11

were 0.51, 1.13, 1.16, and 2.37 L/min/kg, respectively. Although the calculation of CL int using measured CL total and f u caused large standard deviations of CL int , it is obvious that

the CL total of halogen-substituted SARMs was significantly affected by the f u of drug. In

summary, as the size of halogen atom increase, the CL int increased or was similar without

the change of metabolism pathway but CL total decreased due to the protection by binding

of drug to plasma protein.

We also measured the plasma protein binding of SARMs other than halogen-

substituted SARMs. A series of SARMs displayed high plasma protein binding affinity.

For drugs having high plasma protein binding affinities, a small change of f u (e.g., a 0.5%

change from 0.5% to 1%) could cause a significant difference in the amount of unbound

drug (e.g., 2 fold increase). Therefore, it suggests that the small change of f u of SARMs would significantly affect the CL total . Additionally, this study demonstrated that the

logarithm of f u of B-ring substituted SARMs correlated with the retention time in a C 8 column (i.e., lipophilicity), suggesting that the fu of SARMs could be predicted by the

retention time and possibly other methods measuring lipophilicity.

This study demonstrates that the structural modification of SARMs significantly

affect pharmacokinetic behavior of the drugs. Further studies in our laboratories will

continue to explore the structure-pharmacokinetic relationship in laboratory animals and

human.

A B X1 N X2 X3 H

H3C OH

F3C X4

Unbound Fraction (%) Chemical structure Compounds (mean ± stdev) X1 X2 X3 X4 50 µM 5 µM

R-1 CN S NHC(O)CH 2Cl H - -

Acetothiolutamide CN S NHC(O)CH 3 H - -

S-1 NO 2 O F H 0.78 ± 0.17 0.69 ± 0.12

S-4 NO 2 O NHCOCH 3 H 6.27 ± 1.31 4.70 ± 0.14

S-9 NO 2 O Cl H 0.10 ± 0.04 0.11 ± 0.08

S-10 NO 2 O Br H 0.03 ± 0.01 0.03 ± 0.01

S-11 NO 2 O I H 0.01 ± 0.00 N/D

S-19 NO 2 O NO 2 H 0.42 ± 0.14 0.35 ± 0.06

S-20 NO 2 O CN H 0.70 ± 0.01 0.34 ± 0.08

S-21 CN O NO 2 H 0.51 ± 0.02 0.27 ± 0.06 S-22 CN O CN H 0.96 ± 0.14 0.85 ± 0.00

R-Bicalutamide CN SO 2 F H 6.22 ± 0.16 -

Internal standard NO 2 O F F - -

Table 4.1 Chemical structures and plasma protein binding of nonsteroidal AR ligands.

Aromatic rings are designated as A and B as shown.

N/D: Not determined due to low concentration of free drug.

Internal standard Standard curve Injection Compound concentration range volume Plasma side S-1, S-4, S-9, S-10, S-11, S-19, 10 µM 50 µM – 2 µM 1 µl S-20, S-21, S-22, Bicalutamide Buffer side S-1, S-20, S-21 100 nM 1,000 ~ 10 nM 3 µl S-4 1,000 nM 2,000 ~ 50 nM 1.5 µl S-9, S-10, S-11 50 nM 100 ~ 1 nM 15 µl S-19 50 nM 300 ~ 3 nM 5 µl S-22 50 nM 250 ~ 2.5 nM 5 µl

Table 4.2 LC-MS analysis condition to measure drug concentration in plasma side and buffer side.

Product ions Molecular ions Assigned structure and proposed observed ([M-H] -) fragmentation pattern (m/z values)

O 261 289

417, 419 * 289, 261 O2N N O Cl H (S-9) OH H3C

F3C O 275

* * OH 483, 485 405 , 403 , 275, HO3S N N O Cl H OH (M9-1) 255 H3C

403, 405 F3C

403, 405 O * * O SO H 483, 485 405 , 403 , 145, 3 H2N N O Cl (M9-2) 143 H OH H3C 143, 145 F3C O 229, 231 129, 127 HO O Cl (M9-3) H3C OH 127, 129

221

O SO3H 301 221 *, 191, 171 (M9-4) O2N NH2

F3C 275 O 403, 405 275, 255 HOHN N O Cl (M9-5) H OH H3C

F3C

Continued

Table 4.3 Proposed structure of S-9 and its urinary metabolites in rats.

* Base peak in the product ion mass spectrum. 96

Table 4.3 continued

259 O OH

403, 405 * * H2N N O Cl 259, 143 , 145 H (M9-6) OH H3C

F3C 143, 145

259 O 387, 389 259*, 189, 175, H2N N O Cl (M9-7) 129, 127 H H3C OH

F C 127, 129 3 419, 421 O 277 O SO3H 499, 501 421*, 419*, 277, NH2 NH O Cl

(M9-8) 161, 159 CH3 OH 159, 161 OH F3C 403, 405 259 O 579, 581 405*, 403*, 259, O C6O6H9 (M9-9) 145, 143 NH2 NH O Cl CH3 OH 143, 145 F3C O 275

579, 581 H9C6O6 OHN N O Cl H 405*, 403*, 275 OH (M9-10) H3C

403, 405 F3C

Product ions Molecular ions Assigned structure and proposed observed ([M-H] -) fragmentation pattern (m/z values)

O 261 289 461, 463 189, 261*, 173, O2N N O Br (S-10) 171 H H3C OH 171, 173 F3C O 275

527, 529 OH HO3S N N O Br 275, 447*, 449* H (M10-1) H C OH 447, 449 3 F3C 447, 449 O O SO H 527, 529 187, 189, 447*, 3 NH NH O Br (M10-2) 449* 2 CH3 OH 187, 189 F3C O 273, 275 113, 171*, 173*, HO O Br (M10-3) 217 H3C OH 171, 173

221

O SO3H 301 221 *, 191, 171 (M10-4) O2N NH2

F3C O 275 447, 449 HOHN N O Br 275 H (M10-6) OH H3C

F3C O OH 447, 449 187, 189 NH2 NH O Br (M10-5) CH3 OH 187, 189 F3C

Table 4.4 Proposed structure of S-10 and its urinary metabolites in rats 98

Product ions Molecular ions Assigned structure and proposed observed ([M-H] -) fragmentation pattern (m/z values) O 261 289

509 * O2N N O I 189, 261 , 219 H (S-11) H3C OH 219 F C 3 O 275

575 OH HO3S N N O I 275, 495* H OH (M11-1) H3C 495 F3C 495 O

O SO3H 575 235, 495* NH2 NH O I (M11-2) CH3 OH 235 F3C O 321 160, 219* HO O I (M11-3) H3C OH 219 221

O SO3H 301 221 *, 191, 171 (M11-4) O2N NH2

F3C

Table 4.5 Proposed structure of S-11 and its urinary metabolites in rats

EtOH-1st-C l-514 484 # 1 0 5 0 R T : 2 4 .8 7 A V : 1 N L : 9 .2 1 E 4 F : - c ESI Full m s2 484.00@ 40.00 [ 130.00-550.00] 4 0 3 .1 1 0 0

9 5

9 0 A

8 5

8 0

7 5

7 0

6 5

6 0

5 5

5 0

4 5 4 0 5 .0

RelativeAbundance 4 0

3 5

3 0

2 5 2 7 5 .1 2 0

1 5

1 0 3 8 3 .2 5 4 4 0 .1 4 5 1 .2 4 8 2 .8 1 7 0 .9 207.02 3 4 .9 272.1 2 9 8 .8 3 2 8 .2 3 6 6 .1 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 5 5 0 m /z

EtOH-1st-Br-528 608 # 1 1 1 3 R T: 2 5 .8 7 AV: 1 N L : 1 .6 2 E 5 F : - c ESI Full m s2 [email protected] [ 145.00-600.00] 4 4 7 .0 1 0 0

9 5

9 0 B 4 4 9 .0 8 5

8 0

7 5

7 0

6 5

6 0

5 5

5 0

4 5

RelativeAbundance 4 0

3 5 2 7 5 .1

3 0

2 5

2 0

1 5 4 4 9 .9

1 0

5 2 5 5 .2 2 7 5 .9 4 2 7 .0 1 9 1 .0 2 2 7 .0 3 0 3 .7 3 6 0 .0 4 0 6 .9 483.05 0 8 .4 527.4 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 5 5 0 6 0 0 m /z

Figure 4.1 Mass spectra of M9-1 (A) and M10-1 (B) isolated from rat urine.

100

Figure 4.2 Proposed major metabolism pathways of S-9 (X=Cl), S-10 (X=Br), and S-11

(X=I). Structural information was determined by LC/MS 2 and LC/MS 3 fragmentation.

101

100

0.1 UnboundFraction (%)

0.01

0.001 1 2 3 4 5 6

Lipophilicity (Retention Time)

Figure 4.3 Relationship between lipophilicity and plasma protein binding (50 µM).

102

1.6

1.4

1.2

1.0

0.8

0.6

Unbound(%) Fraction 0.4

0.2

0.0 0.5 2 5 50 100 200

S-22 Concentration (uM)

Figure 4.4 Unbound fraction of S-22 at various concentrations.

103

CHAPTER 5

ANABOLIC EFFECTS OF SELECTIVE ANDROGEN RECEPTOR

MODULATOR ALONE AND IN COMBINATION WITH SELECTIVE

ESTROGEN RECEPTOR MODULATOR ON BONE AND MUSCLE

5.1. INTRODUCTION

Osteoporosis and osteopenia are conditions that are characterized by low bone mass and structural deterioration of the bone, leading to bone fragility and an increased risk for fractures to the hip, spine, and wrist. Based on a report by the National Institutes of Health in 2000, 10 million individuals already have osteoporosis, and 18 million more have low bone mass, placing them at increased risk for this disorder [125]. Risk factors associated with low bone density include female gender, increased age, estrogen deficiency, low weight and body mass index, white race, family history of osteoporosis, smoking and history of prior fracture [125]. Especially, hormones play an important role in regulating bone mass. Estrogen deficiency in post-menopausal women and androgen deficiency in older men are known to associate with low bone mineral density (BMD)

104

and a higher incidence of osteoporosis-related fractures [126]. Although there is no cure for osteoporosis, the following medications are approved by the FDA for postmenopausal women to prevent and/or treat osteoporosis: selective estrogen receptor modulators

(SERMs), estrogens/progestins, calcitonin, bisphosphonates, and parathyroid hormone

(pTH). All the medications listed except PTH are categorized as antiresorptive drugs, which increase bone mass by reducing the rate of bone resorption. Unfortunately, antiresorptive agents are not sufficient to restore bone mass for patients who have already lost a significant amount of bone, since they only inhibit bone resorption and do not promote bone formation. Intermittent PTH treatment is the only clinically available option to promote bone formation. However, PTH treatment for osteoporosis is limited due to concerns related to side effects and osteosarcoma [81].

There is extensive data suggesting that androgens play an important role in skeletal development and have direct actions mediated through the androgen receptor

(AR), not via aromatization of testosterone to estrogen. In humans, androgen insensitive males that have AR mutations are associated with osteopenia [74]. Endogenous androgens increase BMD in both adolescent and adult premenopausal women. Women with excess endogenous androgen (i. e., those with hirsutism and polycystic ovary syndrome) have increased BMD compared with normal young women. In experimental animals, the non-aromatizable androgens significantly prevent osteopenia that results from orchidectomy [75]. DHT also prevents bone loss after ovariectomy in female rats

[76, 127]. Antiandrogens cause bone loss even in estrogen-replete female rats [77].

Further, estrogen alone is not enough for hormone replacement therapy in women. Oral estrogen increases sex hormone binding globulin (SHBG), which dramatically reduces

105

the endogenous and bioavailable androgen [128, 129]. Moreover, a number of clinical trials have shown that combined treatment with androgen and estrogen has an additive effect on BMD compared with estrogen-alone treatment [130].

Age-related muscle loss is thought to significantly contribute to the syndrome of

frailty and increase fracture risk [131, 132], suggesting that increase in muscle mass and

strength would be beneficial for treatment of osteoporosis. Administration of androgen

significantly increases muscle mass and strength in young hypogonadal men (physiologic

replacement dose) and eugonadal men (supraphysiologic dose) [86]. In elderly men, the

androgen effect on muscle mass and strength have not been consistent or impressive [28,

92, 94]. These disappointing results in elderly men are thought to be low doses of

androgen treatment, due to the concerns about side effects at higher doses, especially

accelerating the risk of prostate cancer. However, a dose-dependent relationship of

androgen treatment with muscle mass and strength [96] suggests that androgen

administration of higher doses in elderly men may significantly increase muscle mass and

strength. In women, although the information about the effects of androgen

administration is far more limited, there is agreement that androgen level in

postmenopausal women are associated with total lean mass and muscle strength [57, 133,

134].

In spite of the usefulness of androgen therapy in men and women, inconvenience

of existing androgen formulation and undesirable side effects, resulting from stimulation

of androgenic organs and cross-reactivity of androgens and their metabolites hamper

clinical uses of androgens for osteoporosis. In our early study, we identified S-1 and S-4

as nonsteroidal selective androgen receptor modulators (SARMs), which exert selectively

106

strong anabolic activity in vivo with partial agonistic activity in the androgenic organ [45,

47, 48]. These nonsteroidal SARMs displayed favorable pharmacokinetic properties, including oral bioavailability, long half-life, and linear kinetics [47, 48]. Moreover, these are more advantageous over steroidal counterpart, in terms of flexibility on structural modification and specificity to AR. Due to their favorable characteristics, SARMs are attractive as a new treatment of osteoporosis and we attempted to evaluate the clinical use of SARMs for osteoporosis. Our previous study demonstrated the beneficial effects of S-

4 (i.e., a novel SARM discovered in our laboratory) immediate or delayed treatment on skeleton of sexual hormone-depleted rats [135, 136]. Moreover, it is shown that S-4 is able to restore soleus muscle strength and levator ani muscle mass of orchidectomized rats to that seen in intact male rats [135]. Lately, we identified S-22 as a compound with the most potent and tissue-selective in vivo activity that we have observed to date and favorable pharmacokinetic properties [111]. In this study, we hypothesized that combined treatment of SARM and SERM will have additive positive effects on bone and muscle with minimized unwanted effects on reproductive tissues. To test this hypothesis, we examined (1) the effects of various treatments on BMD and trabecular bone microarchitecture, (2) the effects of SARM on muscle mass and strength, and (3) the effects of various treatments on uterus weight. Moreover, we conducted integrated studies of muscle mass, muscle strength, and bone mineral density in hindlimb suspended rat model to dissect the contribution, if any, of increase in muscle mass and strength to

SARM action on bone. The results of these are reported herein.

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5.2. MATERIALS AND METHODS

5.2.1. Materials

Compound S-22 was synthesized by Dr. Duane Miller’s research group at the

University of Tennessee. The purities of these compounds were greater than 99%, as determined by HPLC. Polyethylene glycol 300 (PEG 300, reagent grade), dimethylsulfoxide (DMSO, reagent grade), dihydrotestosterone (DHT) and were purchased from Sigma Chemical Company (St. Louis, MO).

5.2.2. Animals and Experimental Design

The animal protocol was reviewed and approved by the Institutional Laboratory

Animal Care and Use Committee of The Ohio State University. Intact, virgin, 21 week old female Sprague Dawley rats were obtained from Harlan (Indianapolis, IN). The animals were maintained on a 12-h light/dark cycle. Standard rat chow (22/5 rodent diet -

8640, Harlan Teklad, Madison, WI) and water were provided ad libitum throughout the

experiment.

Two weeks after arrival (i.e., Day 0) rats were randomly assigned to either

hindlimb unloading or weight-bearing group. Hindlimb unloading animals were further

divided into four groups (Table 1): (1) sham + vehicle, (2) ovariectomized (OVX) +

vehicle, (3) OVX + S-22 (3 mg/day), (4) OVX + DHT (3 mg/day). Weight-bearing

animals were also divided into eight groups as follows (Table 1): (5) Sham + vehicle, (6)

OVX + vehicle, (7) OVX + S-22 (0.5 mg/day), (8) OVX + S-22 (3 mg/day), (9) OVX +

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S-22 (0.5 mg/day) + Raloxifene (0.3 mg/day), (10) OVX + S-22 (3 mg/day) + Raloxifene

(0.3 mg/day), (11) OVX + Raloxifene (0.3 mg/day), (12) OVX + DHT (3 mg/day) + estradiol (0.03 mg/day). The rats were either OVX (group 2 to 4 and 6 to 12) or sham- operated (group 1 and 5) under anesthesia. On day 1, the weight-bearing groups (i.e., group 5 to 12) started to receive a 6 week-drug treatment. The compounds were dissolved in a mixture of dimethylsulfoxide and polyethylene glycol (20:80, v/v) and administered via daily subcutaneous injections in a volume of 0.2 ml. On day 15, unloading of hindlimb in group 1 to 4 was achieved by tail suspension and a 4 week-drug administration began.

Hindlimb unloading was achieved by tail suspension as previously described with slight modifications [137, 138]. Briefly, the tail was cleaned with 70% ethanol, removing all the dead or dirty skin. When the tail was thoroughly dried, the tail was sprayed with the tincture of benzoin and dried. A strip of traction tape (Skin-trac, Zimmer Inc.,

Warsaw, IN) was attached to approximately half of the tail along both sides of the tail.

Traction tape on the tail was secured by applying filament tape around tail over traction tape. Tape harness was attached to fishline swivel on the top of the cage. The height of suspended hindlimb was adjusted to prevent any contact with the bottom of the cage.

Animals were fully accessible to water and chow using their forelimbs.

On day 42, animals were anesthetized, weighed, and sacrificed. The soleus muscle from the left hindlimb was isolated for muscle strength measurement (Refer to

6.2.4.). The soleus muscle from right hindlimb was weighed. Uterus was dissected free of fat, and wet weight were measured. Bilateral tibiae and femurs were removed, cleaned of soft tissue, wrapped in gauze soaked in saline, and stored at – 20 ºC.

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5.2.3. DEXA Analysis

Total body BMD, bone mineral content (BMC), bone mineral area (BMA) and body composition (i.e., lean mass and fat mass) were determined by dual energy x-ray absorptiometry (DEXA) (GE, Lunar Prodigy™). To avoid potential errors associated with interday variability of instrument performance, all the animals in group 1 to 4 or group 5 to 12 were DEXA scanned at the same day. Animals were anesthetized with mixture of ketamine and xylazine (87:13 mg/kg) for scanning. Weight bearing groups were DEXA scanned the day before OVX and sacrifice and hindlimb unloading groups were DEXA scanned the day before OVX, tail suspension, and sacrifice. DEXA parameters were measured using the small animal software (Lunar enCORE, version

6.60.041) and the whole animal body area was selected as the region of interest.

5.2.4. Muscle Strength Measurement

Soleus muscle strength was measured using the methods that were described previously [135], with modifications. The muscle was carefully isolated and mounted vertically in a glass chamber which was filled with oxygenated (95% O 2 + 5% CO 2)

Krebs-Ringer solution (NaCl 137 mM, KCl 5 mM, CaCl 2 2 mM, MgSO 4 1 mM, NaHCO 3

13 mM, KH 2PO 4 1.8 mM, glucose 11 mM, and pH 7.4) at room temperature. The

chamber was held in an in-vitro muscle test apparatus (model 800A, Aurora Scientific).

One end of the muscle, with a short segment of tendon remaining, was attached to the

plastic rim of a syringe needle, using quick-setting glue. The needle was clamped to the

electrode assembly in the chamber. The other end of the muscle was glued to a fine wire,

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the other end of which was attached to the isometric force transducer (Kulite BG1000 transducer, Kulite Semiconductor products, Inc.). The muscle was stimulated using two parallel platinum plate electrodes connected to B-K Precision regulated DC power supply

(model 1601), using the output from a Grass S48 stimulator through a custom transistor switch for timing. The transducer output was digitized using a DaqBoard/2000 and

Daqview software (IOtech, Inc.). The force records were recorded and analyzed using

DASYLab 32 software (V. 5.5).

Measurement of twitch kinetics and amplitude were followed by the tetanus amplitude measurement. The muscle was stimulated using supramaximal voltage

(typically 15 volts) at 0.1 Hz frequency (pulse duration 2 ms) and the muscle was stretched, between contractions, to find the length (L o) at which optimal twitch force was

generated. Sixteen continuous twitches were then recorded at the same frequency and the

following parameters were measured in one out of every three twitches: maximal twitch

tension (P t), time to peak twitch tension (T Pt ), and time to one-half relaxation (T 1/2R ). The

average of five measurements for each parameter is reported for each muscle sample.

Tetanus amplitude (P o) was evoked with 7.0-s trains of stimuli (pulse duration 2 ms) at 40

Hz. Three tetanus measurements were obtained for each muscle and the average of the

three measurements of P o is reported. Pt and P o were normalized to the cross sectional

area (CSA) of the muscle. The CSA of the muscle was estimated using following

equation:

2 3 CSA (mm ) = muscle mass (mg) / {L o (mm) × muscle density (mg/mm )}

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Muscle density was assumed to be 1 mg/mm 3, as previously determined in rat

skeletal muscle [139].

5.2.5. MicroCT Measurement

µCT analysis was done on the left distal femur using the Skyscan 1072 scanner

(Skyscan N.V., Aartselaar, Belgium). Images were acquired with an x-ray tube voltage of

62 kV, a current of 150 mA, and a 4.5 s exposure with a 1 mm aluminum filter. The

angular scanning rotation was 180° with an angular increment of 0.68°. The

magnification was ×25 and the pixel size was 10.94 mm. Data sets were reconstructed

using a modified Feldkamp algorithm and segmented into binary images using global

thresholding. The region of analysis was selected as the trabecular bone within a volume

of interest approximately 1.5 mm proximal to the growth plate and extending

approximately 2.5 mm in the proximal direction. A total of 230 slices were analyzed

from each femur.

Statistical analysis was performed by single factor analysis of variance

(ANOVA). P-values of less than 0.05 were considered as statistically significant

differences.

5.3. RESULTS

5.3.1. Body Weight and Tissue Weights

Twenty three week old female rats were either OVX or sham-operated. In weight- bearing groups (Group 5 to 12), ovariectomized rat were immediately treated with

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assigned compounds or vehicle for 6 weeks. The body weights in the course of experiment and soleus muscle weights are reported in Table 5.2. There was no significant difference between the body weights of group 1 to 12 at week 0. Estrogen-depletion in

OVX rats for 6 weeks resulted in significant gain of body weight (+ 20%) compared with the sham-operated group (Group 5 and 6) and this gain of body weight was reversed by estrogen (in combination with DHT) or raloxifene treatment. Unexpectedly, the body weights of OVX animals treated with S-22 at 0.5 mg/day (360 ± 25 g) were significantly higher than those of OVX control (330 ± 13 g), but those of 3 mg/day treated groups

(331± 17 g) were not. The body weights of OVX animals treated with S-22 in

combination with raloxifene (302 ± 6 and 294 ± 14 g) were significantly reduced

compared with those treated with either S-22 alone (360 ± 25, 331 ± 17 g) or vehicle (330

± 13 g). In hindlimb unloaded groups (Group 1 to 4), 23 week old female rats were either

OVX or sham-operated at week 0 and were allowed to recover without any drug

treatment for 2 weeks.

At week 2, hindlimb unloading was achieved in the animals and the unloaded rats were immediately treated with assigned compounds or vehicle for 4 weeks. Most animals in hindlimb unloaded group gain body weight during the 6 week experimental period.

However, the hindlimb unloaded animals generally displayed lower body weight compared with weight bearing group and the OVX animals treated with either vehicle or

S-22 significantly lost weights by the unloading. Even in hindlimb unloading condition,

S-22 (297 ± 16 mg) or DHT (300 ± 20 mg) treatment to OVX animals significantly increased the body weight compared to vehicle control (277 ± 25 mg).

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The soleus muscle weights and soleus muscle weight to body weight ratios in treatment groups are presented in Table 5.2. In weight bearing groups, although the soleus muscle weights in OVX animals (123 ± 8 mg) were higher compared with intact control group (115 ± 7 mg), the difference was not significant. However, the soleus weight to body weight ratios in OVX group (0.37 ± 0.02 mg/g) were significantly lower compared with intact control group (0.42 ± 0.03 mg/g), due to the fact that body weight gain exceeded the muscle weight gain. The drug treatment in OVX did not significantly affect the soleus muscle weight to body weight ratios. Hindlimb unloading group dramatically decreased soleus muscle weight and soleus weight to body weight ratio compared with weight bearing groups. OVX and drug treatment with S-22 and DHT did not cause any significant changes in soleus muscle weight and soleus weight to body weight ratio.

The uterine weights were measured at the end of study and are presented in Figure

5.1. As expected, the uterine weight was clearly reduced by OVX both in weight bearing group and unloaded group. S-22 and raloxifene treatment in OVX rats partially reversed the OVX-induced weight loss of uterus both in unloaded group (Group 3) and weight bearing group (Group 7, 8, and 11). Moreover, the stimulating effects of S-22 and raloxifene in uterus were additive (Group 9 and 10). However, combined treatment of raloxifene and S-22 (274 ± 36 mg/100g) resulted in significantly lower uterine weight compared with the combined treatment of estrogen and DHT in OVX rats (344 ± 28 mg/100g) under the condition of weight bearing. In hindlimb unloading group, uterine weight treated with DHT (286 ± 68 mg/100g) was significantly higher compared with S-

22 treatment group (215 ± 49 mg/100g).

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5.3.2. BMD and Body Composition

The effects of drug treatment on the skeleton under the condition of weight bearing or hindlimb unloading were assessed by DEXA scans. The total body BMD is presented in table 5.3. All the animals in group 1 to 4 or group 5 to 12 were DEXA scanned at the same day in order to avoid potential errors associated with interday variability of instrument performance. At day 0, no significant difference in total body

BMD and the percentage of fat was observed between groups that were scanned at the same day. In weight bearing groups at week 6, total body BMD in OVX animals (0.158 g/cm 2) was significantly lower than that observed in intact animals (0.166 ± 0.005 g/cm 2).

S-22 treated group (group 7 and 8), sham operated animals gained 2.71 ± 5.82 mg/cm 2 of

total body BMD, whereas OVX animals lost 2.63 ± 2.26 mg/cm 2 of total body BMD and

the difference of the BMD changes was significant. Although S-22 treatment tended to

reduce the OVX-induced BMD loss, total body BMD of the animals treated with S-22

declined after the 6 week treatment and it resulted in 0.160 ± 0.005 (0.5 mg/day), 0.161 ±

0.005 g/cm 2 (3 mg/day), which were significantly lower than those observed in intact control. Raloxifene treatment in OVX animal increased total body BMD by 1.22 ± 6.70 mg/cm 2, but the increase was not significant. On the other hand, combined treatment of

S-22 and raloxifene or DHT and estradiol significantly increased total body BMD by 2.00

± 5.39 (group 9) and 2.11 ± 4.25 mg/cm 2 (group 12), reaching 0.166 ± 0.006 and 0.165 ±

0.005 g/cm 2, respectively.

In hindlimb unloading group, the 2 week treatment of OVX (group 2-4) did not significantly change total body BMD compared with intact control group (group 1). At

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the end of the study, total body BMD of OVX animals treated with vehicle (group 2,

0.156 ± 0.004 g/cm 2), S-22 (group 3, 0.154 ± 0.004 g/cm 2), or DHT (group 4, 0.156 ±

0.005 g/cm 2) was lower than that observed in intact control (group 1, 0.162 ± 0.007 g/cm 2), and total BMD of group 2 and 3 was significantly lower than that observed in

group 1. However, the changes in BMD from week 2 to week 6 were not significantly

different (Table 5.3).

The changes in body composition of animals are reported in figure 5.2. In weight bearing groups, intact animals gained both fat (2.4 ± 14.6 g) and lean (2.2 ± 7.8 g) mass for 6 weeks (Figure 5.2, B). The gains in fat mass (25.2 ± 10.0 g) and lean mass (35.4 ±

10.4 g) of OVX animals were significantly higher than those observed in intact control, respectively. The gain rates in both fat and lean mass by S-22 treatment were even higher than those observed in OVX control. On the other hand, raloxifene treatment to OVX animals significantly declined the OVX induced gains in fat and lean mass with a larger extent in fat mass loss than the lean mass loss. Interestingly, the combined treatment of S-

22 and raloxifene to OVX animals suppress fat mass increase (group 9, 2.5 ± 4.3; group

10, -2.1 ± 6.7 g) as observed in raloxifene treatment group (-2.4 ± 10.5 g) and maintained lean mass increase (group 9, 31.3 ± 5.9; group 10, 30.2 ± 9.0 g) as observed in OVX control (35.4 ± 10.4g) and S-22 treatment group (group 10, 32.3 ± 12.5 g), which is significantly higher than that observed in raloxifene treatment group (15.4 ± 10.7 g).

These effects of combined treatment of S-22 and raloxifene on fat and lean mass were similar to those observed in combined treatment of DHT and estradiol. The body composition of hindlimb unloaded groups is reported in figure 5.2. 2 week-estrogen depletion induced by OVX (group 2-4) increased both fat and lean mass (Figure 5.2, A)

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but to a smaller extent than observed in 6 week estrogen depletion (group 6). During 4 weeks of hindlimb unloading and drug treatment, the body composition was mainly governed by the condition of hindlimb suspension rather than OVX or drug treatment.

The hindlimb unloading allowed to gain lean mass but decreased fat mass.

5.3.3. Soleus Muscle Strength

The effects of S-22 and DHT on muscle strength under the condition of weight bearing or hindlimb unloading were determined and the results are presented in table 5.4.

Hindlimb unloading drastically decreased P t and P o of OVX animal treated with either

vehicle or S-22 and intact control (Table 5.4). Since hindlimb unloading significantly

decreased soleus muscle mass, the drastic drop of Pt and P o by hindlimb unloading

seemed to be due to decrease of muscle mass. However, the possibility that other changes

in soleus muscle by hindlimb unloading resulted in the decrease of P t and P o still remains.

Therefore, P t and P o are normalized by CSA and presented in Figure 5.2. P t/CSA and

Po/CSA significantly decreased by hindlimb unloading, suggesting that hindlimb unloading for 4 weeks might result in a decrease in myofilament density or changes in

MHC isoforms. However, OVX did not cause significant changes to Po in either weight

bearing or hindlimb unloading group. In OVX and hindlimb unloaded group, neither S-22

nor DHT treatment caused significant changes in the soleus muscle strength. In contrast,

S-22 (1.75 ± 0.19 N) significantly increased P o, which is commonly used for measuring

the contractile force of the soleus muscle in OVX animals under the condition of weight

bearing, compared with OVX control (1.55 ± 0.19 N) and intact control (1.52 ± 0.13 N)

[140]. When P o was normalized by the CSA, it showed a similar trend (Figure 5.2). 117

2 Po/CSA of OVX and weight bearing animal treated with S-22 (380 ± 29 kN/m ) was

significantly higher than that of OVX control (315 ± 45 kN/m 2) and that of intact control

2 2 (331 ± 31 kN/m ). Moreover P t/CSA of S-22 treated OVX group (58 ± 6 kN/m ) was

significantly higher than that of OVX control (49 ± 5 kN/m 2) and that of intact control

2 (49 ± 6 kN/m ). For TP t and T 1/2R after twitch and tetanus stimulus, hindlimb unloading

did not significantly affect T 1/2R after twitch stimulus, but significantly decreased TP t and

T1/2R after tetanus stimulus, consistent with a normal physiological response to weight unloading. On the other hand, OVX and drug treatment with S-22 or DHT did not result in any significant change of TP t and T 1/2R after twitch and tetanus stimulus in either

weight bearing or hindlimb unloaded group.

5.3.4. Structural Parameters of Trabecular Bone

The effects of drug treatment and weight unloading on trabecular bone microarchitecture were determined by 3D µCT analyses on the metaphyseal region of the

distal femur. The following parameters of 3D µCT analyses were reported in figure 5.3

and table 5.5; BMD, percent trabecular bone volume (BV/TV), bone surface density

(BS/TV), intersection surface (i.S), structure model index (SMI), trabecular pattern factor

(Tb.Pf), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation

(Tb.Sp), and degree of anisotropy (DA). In weight bearing groups, OVX resulted in

significant reduction in BV/TV, by reduced Tb.N and increased Tb.Sp without significant

reduction in Tb.Th. These changes are consistent with those observed in OVX group and

osteoporotic group in previous studies [141, 142]. Moreover, OVX significantly

decreased BMD, BS/TV and i.S and significantly increased SMI, Tb.Pf, and DA. The 118

treatment with S-22 and raloxifene alone partially prevented the OVX-induced negative changes in trabecular bone. S-22 treatment significantly reversed the OVX-induced changes in BV/TV, BS/TV, Tb.N, and Tb.Sp. Raloxifene treatment significantly reversed the OVX-induced changes in BMD, BV/TV, BS/TV, i.S, SMI, Tb.Pf, Tb.N, Tb.SP and

DA. The combined treatment of S-22 and raloxifene additively improve trabecular bone and fully recovered the OVX-induced changes in trabecular bones. The effects of S-22 in combination with raloxifene on BMD, BV/TV, BS/TV, i.S, SMI, Tb.Pf, Tb.Th, Tb.N, and Tb.SP were significantly higher than those observed in S-22 and raloxifene alone treatment group. The additive effect of S-22 and raloxifene was similar or higher compared with the additive effect of DHT and estradiol in this study. In hindlimb unloading groups, the parameters of trabecular bone were not significantly different from those observed in weight bearing groups except DA of OVX groups (Group 2 vs 6).

Effects of OVX and S-22 treatment on structural parameters of trabecular bone in hindlimb unloading groups were similar to those of weight bearing group.

5.4. DISCUSSION

It is reported that the AR and ER α activation pathways that exert in vivo bone-

sparing effects are clearly distinct from each other, supporting the rationale of combined

treatment of AR agonist and ER agonist as a treatment for osteoporosis [143]. Moreover,

in vivo additive effects of combined androgen and estrogen therapy on bone have been

reported previously [142, 144, 145]. Although the SERM (i.e., raloxifene) is already

clinically used for osteoporosis and the protective effect of SARM on bone was recently

reported [52, 136], the effects of combined treatment of SARM and SERM on bone are

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unknown. The results of BMD in distal femur demonstrated that raloxifene, but not S-22, significantly increased the BMD and the addition of S-22 (3 mg/day) to raloxifene increased the BMD even more than those observed in raloxifene alone treatment group.

In previous studies, it has been shown that BMD alone can be a useful predictor for average mechanical strength of trabecular bone over a large range of bone density [146,

147]. However, clinical study demonstrated that, within the critical BMD range where fracture often occurs, BMD alone is insufficient to accurately predict the mechanical strength of bone [148]. BV/TV and trabecular microarchitecture parameters (i.e., DA,

Tb.Pf, and SMI) are thought to be important factors in bone strength [32, 149-153] . In terms of BV/TV, OVX caused significant reduction of BV/TV by reduced Tb.N and increased Tb.Sp without significant reduction in Tb.Th [141, 142]. Both S-22 and raloxifene significantly reversed OVX induced changes in Tb.N and Tb.Sp, resulting in prevention of OVX-induced loss in trabecular BV/TV. The protective effects of S-22 and raloxifene for the BV/TV were additive, resulting in significant increase of BV/TV by the combinational treatment compared with single treatment. Tb.N, Tb.Th, Tb.Sp and bone volume in the combined treatment group of S-22 and raloxifene were not statistically different from those observed in intact group, while Tb.N and BV/TV in raloxifene treatment group and Tb.N, Tb.Sp, and BV/TV in S-22 group were significantly different from those of intact group. It suggests that treatments with S-22 or raloxifene alone are not sufficient to protect from the OVX-induced reduction of BV/TV caused by changes in Tb.N and Tb.Sp but combined treatment of S-22 and raloxifene is fully protective.

SMI is a morphometric parameter describing three-dimensional structure in terms of the amount of plates and rod structural component. The SMI value lies between 0 and

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3, which means ideal plate and rod structure, respectively. It was shown that the deterioration of trabecular bone structure by aging [154] and postmenopause [155] resulted in a conversion from ‘plate-like’ structure (stronger) to ‘rod-like’ structure

(weaker) [149]. In this study, OVX animal resulted in more rod-like trabecular structure and raloxifene, but not S-22, significantly but partially prevent this OVX-induced structural change. Interestingly, the combined treatment of S-22 and raloxifene to OVX animals additively protect from the structural change to rod-like structure and reserved

SMI value as observed in intact animals.

The direction of the trabecular (DA) and the connectivity of the trabecular structure (Tb.Pf) [152] were significantly affected by drug treatment with SARM and

SERM alone and in combination. S-22 and raloxifene alone or in combination significantly but partially prevent OVX-induced structural change without any significant difference between treatment groups. In terms of Tb.Pf, combined treatment of raloxifene and S-22 fully preserved connectivity of trabecular structure at the intact control level, while raloxifene alone partially prevent OVX-induced loss of connectivity to a lower extent than observed in intact control. As an overall summary for bone study, S-22 and raloxifene additively prevent OVX-induced trabecular bone loss and preserve the trabecular microarchitecture at the intact control level, while S-22 and raloxifene alone partially prevented the bone loss and preserved the microarchitecture. It suggests that the combination treatment of SARM and SERM for osteoporosis could be beneficial to preserve and, possibly, improve the mineral density and mechanical strength of bones in postmenopausal women. Although it was not determined in this study, SARM might have other beneficial property(s) in bone, such as promoting periosteal bone formation in

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cortical bone, while estrogens decrease it [84]. The cortical bone thickness is one of important factors associated with bone mechanical strength. In our future study, we would further study the effect of SARM alone and in combination with SERM on bone formation in cortical bone.

In body composition, treatment with raloxifene alone to OVX animals significantly suppressed the OVX-induced increases of total body fat mass and lean mass, while treatment with S-22 increased total body fat mass and lean mass, but only lean mass increase by treatment of S-22 0.5 mg/day was significant. The combined treatment of S-22 and raloxifene reduced OVX-induced total fat mass increase to the similar extent by raloxifene alone treatment and maintained total lean mass increase as observed in

OVX control and S-22 treatment group (3mg/day), which is significantly higher than that observed in raloxifene treatment group. As a result of body composition changes, S-22 and raloxifene treatment resulted in body weight changes. The body weights of OVX animals treated with combination of S-22 and raloxifene were significantly lower than those observed in OVX control (due to fat mass loss) but also significantly higher than those observed in raloxifene alone treatment group (due to lean mass gain). The changes in body composition reported in this study are consistent with the results of previous studies on the effects of combined treatment of testosterone and estradiol on body composition in postmenopausal women [145, 156-158]. The addition of SARM to raloxifene as a treatment agent for osteoporosis could be clinically beneficial for the prevention of muscle loss without counteracting the positive effect of raloxifene on total body fat. Along with total lean mass increase, S-22 alone or in combination with raloxifene significantly increases soleus muscle strength in OVX rats. Previous clinical

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study demonstrated that free testosterone level significantly correlated with total lean mass and lean leg mass in postmenopausal women treated with oral estrogen [133].

Moreover, it is shown that testosterone level is associated with muscle strength in postmenopausal women [134]. It suggests that SARM could increase total lean mass and muscle strength in postmenopausal women and would be beneficial for treatment of osteoporosis by (1) indirect increase in BMD due to increasing skeletal stress (lean mass increase) to stimulate bone formation [159] and/or (2) reduced the risk of falling and fracture [131, 132, 160].

Although the tissue selectivity of SARMs in male animal was already demonstrated [45, 52, 111, 135], the effect of SARM on reproductive tissues in female remains unclear. In this study, the stimulating effect of S-22 on uterus was significantly weaker than that observed in DHT treatment group. Although this result was obtained in hindlimb unloading group, the fact that there was no significant change in uterine weight caused by hindlimb unloading supports that a SARM is tissue selective in female [161,

162]. In weight bearing group, S-22 and raloxifene additively increased uterine weight but resulted in significantly lower uterine weights than those observed in combination treatment group with estradiol and DHT. It suggests that treatment with SARM alone or in combination with SERM can possibly minimize unwanted effects on reproductive tissues.

In summary, the combined S-22 and raloxifene treatment additively exerted protective effect on trabecular bone in OVX animals. S-22 alone or in combination with raloxifene promoted total lean mass and muscle strength with less stimulating effect on reproductive tissue than that of the steroidal counterpart. These beneficial properties of

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combined SARM and SERM treatment in bone, muscle, and uterus, suggest clinical promise of this treatment for osteoporosis.

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Group Dose Animal Status Treatment ID (mg/day) number 1 Unloaded + Sham Vehicle -- 6 2 Unloaded + OVX Vehicle -- 8 3 Unloaded + OVX S-22 3 8 4 Unloaded + OVX DHT 3 8 5 Wt bearing + Sham Vehicle -- 7 6 Wt bearing + OVX Vehicle -- 8 7 Wt bearing + OVX S-22 0.5 9 8 Wt bearing + OVX S-22 3 9 9 Wt bearing + OVX S-22 + Raloxifene 0.5 + 0.3 9 10 Wt bearing + OVX S-22 + Raloxifene 3 + 0.3 9 11 Wt bearing + OVX Raloxifene 0.3 9 12 Wt bearing + OVX DHT + Estradiol 3 + 0.03 9

Table 5.1 Animal status, treatment regimen, and animal number of treatment groups.

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126

127

128

129

500 Unloaded Wt bearing 400

300

200

100 UterineWeightWet (mg/100g BW) 0 - - 3 - - - 0.5 3 0.5 3 - - S-22 (mg/day) ------0.3 0.3 0.3 - Ral. (mg/day) - - - 3 ------3 DHT (mg/day) ------0.03 E2 (mg/day) OVX OVX

Figure 5.1 Effect of S-22, raloxifene (Ral), 5 α-dihydrotestosterone (DHT) and 17 β- estradiol (E2) in alone and/or combination on uterus in ovariectomized adult rats. Uterine wet weight (mg) was normalized by 100g of body weight. Data are presented as mean ±

SD. * p< 0.05; compared to the sham control group.

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A 40 Unloading, Fat Mass a Lean Mass 30 Drug treatment (4 weeks)

20 a a a 10

-10 OVX

-20 b b Changes in Body Composition (g) CompositioninBody Changes -30 (2 weeks) -40 1 2 3 4 1 2 3 4 Group

B 80 Fat Mass a,b Lean Mass 60

a a a a a 40 a a a a,b

20 b b b

b b -20 Changes in Body Composition (g) Composition inBody Changes a,b

-40 4 5 6 7 8 9 10 11 12 13 Group Figure 5.2 Body composition change in (A) hindlimb unloading groups (B) weight

bearing groups. . a P < 0.05, compare to sham operated group (group 1 or 5); b P < 0.05,

compare to OVX group (group 2 or 6). 131

A 70 Unloaded Wt bearing a, b 60

a, b

) 50 a, b 2 a, b a, b 40

30 / CSA (kN/m CSA / t

P 20

B 500 Unloaded Wt bearing a, b 400 ) 2

300 a, b a, b a, b a, b

200 /(kN/m CSA o P

100

0 - - 3 - - - 3 S-22 (mg/day) - - - 3 - - - DHT (mg/day)

OVX OVX

Figure 5.3 Pt/CSA (A) and P o/CSA (B) in different treatment groups. Data are presented as mean ± SD. a P < 0.05, compare to group 5; b P < 0.05, compare to group 6.

132

0.6 Unloaded Wt bearing b,c,d b b,c,d b,c 0.5 b,c a,b,c ) 3 0.4 a,b a,d a,b a a,d a,d 0.3 BMD (g/cm 0.2

0.1

0.0 B 60 Unloaded Wt bearing

50 b,c,d b b,c,d b,c 40 b,c a,b,c

30 a,b a,d a,b,d a a a,c,d BV/TV (%) BV/TV 20

0 - - 3 - - - 0.5 3 0.5 3 - - S-22 (mg/day) ------0.3 0.3 0.3 - Ral. (mg/day) - - - 3 ------3 DHT (mg/day) ------0.03 E2 (mg/day)

OVX OVX

Figure 5.4 BMD (A) and percent bone volume (B) measured by µCT.

133

CHAPTER 6

EFFECTS OF SELECTIVE ANDROGEN RECEPTOR MODULATOR ALONE

AND IN COMBINATION WITH SELECTIVE ESTROGEN RECEPTOR

MODULATOR ON OSTEOBLAST AND OSTEOCLAST

6.1. INTRODUCTION

Bone is a living tissue, which is continuously being broken down (i.e., bone resorption) and regenerated (i.e., bone formation) to maintain the mechanical integrity of the adult skeleton and to serve as a reservoir for calcium and phosphate ions balance in blood [163]. This process, called remodeling, occurs at a temporary anatomic structure, so-called basic multicellular units (BMUs) [58]. The bone remodeling consists of the removal of old bone and followed by the formation within the cavity, which is subsequently mineralized [163]. In normal adult skeleton, the resorption and formation step are tightly coupled in terms of time and space. Bone resorption always comes first and then bone formation follows it and the amount of bone resorbed and formed are similar and net change of bone is close to zero [163]. However, in the skeleton of

134

estrogen deficient women, whether from ovariectomy or menopause, the rate of bone loss is more rapid than that observed in estrogen sufficient women for following reason [164,

165] ; (1) the remodeling rate is higher, resulting in loss of connectivity and big drop in

BMD since bone resorption always comes first (2) more younger bone (less densely mineralized bone) is replaced by older bone (more densely mineralized bone), resulting in lower tissue mineral content, and (3) the lifespan of osteoclast increased, resulting in negative BMU balance [165].

It is generally agreed that estrogen administration reduces the rate of bone remodeling in postmenopausal women, resulting in increased BMD [166]. Although the mechanism of action of estrogen on bone is not fully understood, the consensus is that the protective effect of estrogen against bone loss in postmenopausal women is mainly due to via reduction of osteoclast number and activity [163, 166]. Estrogen suppresses the development and differentiation of osteoclast progenitors and induce apoptosis of osteoclast, resulting from direct or indirect action (via cytokine, growth factor or other hormone) [58, 164, 166]. On the other hand, the effects of estrogen on osteoblast are confusing and inconsistent [163]. It was reported in some studies that estrogen acted as a mitogen in osteoblasts and stimulated expression of alkaline phosphatase and type 1 collagen, which are related to osteoblastic differentiation [163, 167] while in other studies, estrogen had no proliferative effect and stimulatory effect on type 1 collagen and

Osteocalcin [163]. In others, estrogen suppressed cell proliferation and stimulated alkaline phosphatase expression [168].

Androgen displays a significant effect on the physiology of bone in men and women, by direct androgenic activity and converting to estrogens (i.e., aromatization

135

from testosterone to estradiol) [166]. As a direct androgenic effect on bone, nonaromatizable androgen (i.e., DHT) can prevent bone loss that follows orchidectomy or ovariectomy in rats [169]. Moreover, animal and human studies suggest that androgen has suppressive effect on bone remodeling in androgen deficient status, although androgen is less effective than estrogen in terms of suppression of bone remodeling [164,

169]. In addition, androgen increases periosteal bone formation, while estrogen depresses it [169]. In in vitro studies, the effects on osteoblast differentiation are rather

controversial, but most in vitro studies suggest that androgens have a stimulatory effect

on alkaline phosphatase, type I collagen, osteocalcin, and mineralization of extracellular

bone matrix [60]. Moreover, DHT has also been shown to suppress osteoclast

differentiation [62, 82].

SERMs are recently discovered and have been largely used for the treatment of osteoporosis. Those compounds exert the beneficial effect of estrogen in the bone without its unwanted stimulation in the breast and endometrium [163]. The major effect of raloxifene on bone seems to be prevention rather than restoration of bone loss [163], but the effects of this compounds on bone cells are still poorly understood. It was reported that raloxifene significantly reduced the osteoclast formation from bone marrow cells and increased the proliferation of osteoblast, which could be abolished by the estrogen antagonist [170]. SARMs have great therapeutic potential, including for osteoporosis, but are still in developing stage. Several studies previously showed that SARMs have beneficial effects on skeleton of animals (Kearbey, [52, 135]) and, in chapter 5, we reported that combined SARM and SERM additively restored bone loss induced by ovariectomy. However, none of study about in vitro activity of SARM alone or in

136

combination with SERM was reported yet. In this chapter, we studied the effects of S-22 and raloxifene alone or in combination on osteoblast and osteoclast and the results are reported herein.

6.2. MATERIALS AND METHODS

6.2.1. Experimental Animals and Materials

ICR mice were obtained from Harlan (Indianapolis, IN). The animals were

maintained on a 12-h light/dark cycle. Standard diet and water were provided ad libitum

throughout the experiment.

Charcoal-stripped fetal bovine serum (csFBS) was purchased from Hyclone

(Logan, Utah). Phenol-free Dulbecco’s Modified Eagle Medium/Ham’s F12 medium

(DMEM/HF12), alizarin red-S, geneticin, hygromycin B, Bovine serum albumin (BSA),

parathyroid hormone (1-34) (pTH), and other chemical reagents were obtained from

Sigma Chemical Company (St. Louis, MO). Protease inhibitor cocktail was purchased

from Roche applied science (Indianapolis, IN). EcoLite (+) scintillation cocktail was

purchased from ICN Pharmaceuticals (Costa Mesa, CA). Thymidine was purchased from

Amersham Biosciences (Buckinghamshire, U.K.). Alpha-modified essential medium was

purchased from ( α-MEM) GIBCO-BRL (Grand Island, NY, U.S.A.). Bicinchoninic acid

(BCA) protein assay kit was obtained from Pierce biotechnology, INC (Rockford, IL).

137

6.2.2. Cell Cultures

MC3T3-E1 and hFOB/AR6 cells were purchased from ATCC. The hFOB/AR6

cells were maintained in DMEM/HF12 containing 10% csFBS alternately supplemented

with either geneticin (300 µg/ml) or hygromycin B (100 µg/ml). Both hFOB/AR6 and

hFOB cells were cultured either at 33.5 °C to be maintained or at 39.5 °C to be

differentiated. The hFOB and hFOB/AR6 cells under passages 12 were used for

experiments [171]. MC3T3-E1 cells were maintained at 37°C under a humidified 5%

CO 2 atmosphere in α-MEM supplemented with 10% csFBS (v/v), penicillin (100 U/ml),

and streptomycin (100 µg/ml). Saos-2 cells were maintained in McCoy’s 5A media with

1.5 mM L-glutamine containing 10% cs FBS, penicillin (100 U/ml), and streptomycin

(100 µg/ml). For differentiation and mineralization study, MC3T3-E1 cells were seeded at a density of 1x10 4 cells per well of 24-well plates of differentiation study, or at a density of 4x10 4 cells per well of 6-well plates for mineralization study. After reaching confluence, cells were treated with assigned compound(s) in α-MEM supplemented with

10% csFBS, penicillin, streptomycin, β-glycerophosphate (10 nM) and ascorbic acid (50

µg/ml) to stimulate the differentiation of the osteoblast. Drug containing media was

refreshed every 4-5 days.

Mouse bone marrow cells for osteoclast formation study were isolated by the

method previously described with modifications [172-174]. Briefly, nine-wk-old ICR

mice were sacrificed by cervical dislocation and the femur were removed aseptically.

Bone marrow cells were eluted by flushing with α-MEM using 27-gauge needle. The

bone marrow cells were washed with α-MEM once and one million cells per well of a 24 well plate in MEM-alpha supplemented with 10% cs FBS, penicillin (100 U/ml),

138

streptomycin (100 µg/ml), and fungizone (300 ng/ml). On the next day, cells were treated

with assigned compounds in 10% cs FBS, penicillin, streptomycin, fungizone (300

mg/ml), and pTH (100 ng/ml) to induce osteoclastogenesis. Drug treatment maintained

for 10 days and drug containing media was refreshed every 3 days.

6.2.3. Proliferation Studies

Cell proliferation in hFOB/AR6 was assessed by [ 3H] thymidine incorporation

assay that previously reported with modification [175, 176]. Briefly, cells were plated

into 24-well plates at 40,000 cells/well in DMEM/HF12 media containing 10% csFBS

and geneticin and incubated at 33.5 °C. After attachment, cells were rinsed with serum-

free media twice and cultured in 0.1% (w/v) BSA supplemented DMEM/HF12 media

containing hygromycin for 24 hours at 33.5 °C. After one day, the cells were treated with

the media containing 0.1% BSA, hygromycin, and assigned compound and cultured at

39.5 °C for 2 days. To determine the [3H] thymidine incorporation for DNA synthesis,

0.5 µCi [ 3H] thymidine was added to each well for the last 48 hours of incubation. At the

end of incubation, the media was removed and cell layer was rinsed twice with PBS to

remove unincorporated [3H] thymidine. DNA was then precipitated with 1 ml of 5%

(w/v) trichloroacetic acid (TCA) at 4 ºC for 15 min and precipitates were washed twice

with 95% ethanol. In order to dissolve [ 3H] thymidine, 0.5 ml of 0.25 M NaOH was

added to each well and incubated at room temperature for 30 min. The supernatant was

transferred to 5 ml of scintillation cocktail and the radioactivity was counted in a

Beckman LS6500 liquid scintillation counter (Beckman Instruments Inc., Irvine, CA).

139

6.2.4. Alkaline Phosphatase Activity

Compound-induced differentiation in MC3T3-E1 cells was assessed by measuring

alkaline phosphatase (ALP) activity [177, 178]. Culture media was removed at the end of

drug treatment and cell layer was rinsed with ice-cold PBS. Ice-cold 200 µl lysis buffer

(20 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100 and protease inhibitor cocktail at pH

7.6) was added to each cell layer and cell layer detached by scraping on ice. The cell

lysate in each well was transferred to a eppendorf tube and one cycle of freezing (-80 ºC

for 30 min) and thawing (37 ºC) and sonification (15 sec.) was performed [177]. The cell

lysate was centrifuged at 16,000g for 10 min at 4 ºC and the supernatant was transferred

into new tube to measure ALP activity and total protein content. The ALP activity in the

lysate was assessed by spectrophotometrically measuring the release of p-nitrophenol (9

mM) from p-nitrophenylphosphate at 37 ºC, pH 10.2 and normalized with the protein

content in the lysate. The protein content was determined using the BCA protein assay as

suggested by the manufacturer.

6.2.5. Alizarin Red-S Assay

To determine the degree of mineralization, the calcium depositions of cell cultures were stained with alizarin red-S as described previously [179]. Briefly, culture media was removed at the end of drug treatment and cell layer was rinsed with PBS followed by fixation (70% ethanol at -20 ˚C for 1 hr). The cultures were then rinsed five times with distillated water and stained for 10 min with Alizarin red-S solution (40 mM at pH 4.2) at room temperature with shaking. The culture was rinsed five times with water followed by shaking with PBS for 15 min to reduce nonspecific stain. After the culture was dried, ti

140

was destained using cetylpyridinium chloride solution (100 mM cetylpyridinium chloride in 10 mM sodium phosphate solution at pH 7.0) for 15 min at room temperature.

Concentration of alizarin red-S in extract was determined by measuring the absorbance at

540 nm was normalized by protein content in cell lysate, which was obtained from cell culture grown separately but same condition with alizarin red-S measurement group. The protein content was measured by using BCA protein assay. The mineralization was expressed as units of alizarin red-S released per mg of protein in lysate.

6.2.6. TRAP assay

For the determination of osteoclastogenesis, cells were stained for tartrate-

resistant acid phosphatase (TRAP), a marker enzyme of osteoclasts [174]. Briefly, as a

cell fixation step, cells were treated with 3.7% (v/v) formaldehyde for 10 min, followed

by treatment with mixture of ethanol and acetone (1:1, v/v) for 1 min. The fixed cell layer

was then incubated with TRAP staining solution for 10 min at room temperature. After

staining, cells were rinsed with distillated water and TRAP positive cells were quantified

by analyzed the stained area of scanned cell layer using ImageJ. The minimum and

maximum threshold value to analyze the area was 3 and 70, respectively.

6.2.7. Statistical analysis

All experimental data were obtained by replication three to five times and

expressed as a mean ± stdev. Statistical analysis was performed using single factor

analysis of variance (ANOVA). P-values of less than 0.05 were considered as statistically

significant differences.

141

6.3. RESULTS

6.3.1. Effects on proliferation, differentiation, and mineralization of osteoblast

Previously, the effects of androgen and estrogen on proliferation of osteoblast were studied and these studies suggest that the effects of the hormones could be various due to difference in species, the origin and culture technique, and stage of differentiation of osteoblastic system [100]. In this study, we assessed the proliferation of osteoblastic cells in Saos-2 (human osteosarcoma cell line), MC3T3-E1 (murine clonal calvaria preosteoblast cell line), and FOB-AR cell line (subclone of the hFOB cells, which is conditionally immortalized human fetal osteoblastic cells, transfected with the human AR gene) by measuring [3H] thymidine incorporation into the cells [100]. Androgen, S-22, and estradiol at the range of 1 nM to 1 µM did not show any significant effect on

MC3T3-E1 cell (Data were not shown). In the later study using Saos-2 and FOB-AR

cells, we increased testing drug concentration up to 10 µM. DHT and S-22 at the

concentration of 10 µM significantly suppressed the proliferation of Saos-2 cells to the

extent of 78% ± 9% and 5% ± 9% of vehicle control, respectively, while these

compounds at lower concentration range (1 nM to 1 µM) did not exert any significant

effect on it. Figure 6.2 shows that proliferation of FOB-AR cell significantly decreased

by treatment with S-22 (75% ± 3%), raloxifene (3% ± 1%), DHT (55% ± 8%), and

estradiol (59% ± 1%) at the concentration of 10 µM, but was not affected by treatment

with S-22 and raloxifene at lower concentration (1 nM to 1 µM).

142

The effects of AR and ER ligands on differentiation and mineralization of osteoblast were studied using MC3T3-E1 and Saos-2 cell line. First, it was determined the effect of the treatment of DHT at the concentration range of 1 nM to 10 µM for 7, 14,

and 21 days on ALP activities of MC3T3-E1 and Saos-2 cells. In Saos-2 cells, ALP

activities of the cells did not increased by DHT treatment but their activities were

significantly higher than those observed in MC3T3-E1 cells treated with DHT,

suggesting that majority of osteoblast in Saos-2 were already mature and differentiated

before drug treatment and Saos-2 cell line is not suitable for differentiation and

mineralization study. In MC3T3-E1 cell line, DHT increased the ALP activity up to 21

days in cultures in a dose-dependent manner (Figure 6.3). The DHT treatment for 14 or

21 days significantly increased ALP activity at a concentration range of 10 nM to 10 µM,

while only 10 µM of DHT significantly increased it on day 7. Therefore, we selected 14

days as a drug treatment period to determine the effects of various compounds on ALP

activity in MC3T3-E1 cells. The effects of S-22 (10nM to 10 µM) and raloxifene (10nM

to 10 µM) alone or in combination on ALP activity in MC3T3-E1 were determined. The effects of DHT (10 µM) and estradiol (10 µM) on ALP activity also measured as a

positive control. The results are shown in Figure 6.4.A and B. S-22 increased ALP

activities in MC3T3-E1 cells in a dose-dependent manner. The increases in ALP activity

stimulated by 1 and 10 µM of S-22 were significant and 10 µM of S-22 (173% ± 10%)

increased ALP activity to the similar extent of that observed in DHT (10 µM) treatment

group (181% ± 13%). On the other hand, raloxifene did not exert any significant effect on

the ALP activity, while estradiol (10 µM) significantly stimulated the enzyme activity to

199% ± 23 % of vehicle control. When MC3T3-E1 cells were treated with S-22 and

143

raloxifene in combination, raloxifene displayed antagonistic effect against stimulatory effect exerted by S-22 on ALP activity in MC3T3-E1 (Figure 6.4. B). Although S-22 (10 nM to 10 µM) alone significantly enhance ALP activity in a dose-dependent manner and

10 µM of S-22 stimulated the enzyme activity up to 173% of vehicle control, combined

S-22 (10 nM to 10 µM) and raloxifene (10 µM) treatment could not stimulate ALP activity. The antagonistic activity of raloxifene seems to increase as the concentration of raloxifene increased. In the concentration range of 10 nM to 1 µM of raloxifen, the

stimulatory effect of S-22 (10 µM) on ALP activity remained significant.

During the 21 day incubation period, MC3T3-E1 cells underwent a significant change in cellular morphology, including calcium deposition on surface of cell layer that was confirmed by alizarin red-S assay. Figure 6.5 shows the effects of various treatments on calcium deposition in MC3T3-E1 cells, an indicator of mineralization. S-22 increased osteoblast mineralization in a dose-dependent manner and the increase of mineralization by 100 nM to 10 µM of S-22 was significant. At the same concentration (10 µM),

mineralization effect in S-22 treatment group (236% ± 4%) was significantly higher than

those observed in DHT (133% ± 13%) and estradiol group (135% ± 10%). On contrast,

raloxifene significantly suppressed osteoblast mineralization in a dose-dependent manner.

The mineralization of MC3T3-E1 cell treated with 1 µM and 10 µM was 56% ± 5% and

39% ± 2% of vehicle control, respectively.

6.3.2. Effects on osteoclastogenesis

The effects of S-22, raloxifene, DHT, and estradiol alone and in combination on

the differentiation of bone marrow cell toward osteoclast were determined and the result

144

is presented in Figure 6.6. Raloxifene suppressed pTH induced TRAP activities at dose- dependent manner and decrease at the concentration of 10 µM was significant, while S-

22 did not exert any significant effect on it. DHT and estradiol at the concentration of 10

µM significantly suppressed pTH induced TRAP activities to the extent of 76% ± 15% and 37% ± 15%, respectively. In the combination treatment, the suppression of pTH induced TRAP activity by 10 µM of raloxifene was maintained under the cotreatment

with S-22 at the concentration range of 10 nM to 10 µM.

6.4. DISCUSSION

In chapter 5, we found that S-22 and raloxifene have preventive effects on bone

loss induced by estrogen depletion and their beneficial effects are additive. In this study,

we determined the effects of S-22, raloxifene, DHT, and estradiol on the proliferation,

differentiation, and mineralization of osteoblast and osteoclastogenesis. S-22 and DHT

showed similar effects on proliferation and differentiation of osteoblast. S-22 was more

effective to induce the mineralization of MC3T3-E1 cells than observed in DHT

treatment group, while DHT was more effective to suppress the pTH induced

differentiation of bone marrow cells toward osteoclast than that observed in S-22 group.

Previous studies (chapter 5 and [136]) about in vivo activities of S-22 and androgen in

bone demonstrated that SARM (i.e, S-4 and S-22) have similar or slightly stronger effect

on skeleton than observed with DHT, suggesting that it might be due to pharmacokinetic

difference (resulting in higher drug exposure in S-22 treatment group) and difference in

intrinsic activities. Overally, DHT and SARM exerted similar effect on bone cells in this

study and the protective effects of S-22 on bone loss might be resulted from its simulative

145

effect on osteoblastic cells, especially, via increase in their differentiation and mineralization not increase in the number of osteoblast.

Effects of raloxifene and estradiol on the osteoblast and osteoclast were also studied. Interestingly, the effect of raloxifene and estradiol on the differentiation and mineralization of MC3T3-E1 were quite different. Raloxifene exerted no effect or suppressed the differentiation and mineralization of osteoblast while estradiol significantly increased those activities. On the other hand, raloxifene was more effective to suppress the pTH induced differentiation of bone marrow cells toward osteoclast than that observed in estradiol treatment group. These results suggest that protective effect of raloxifene on bone loss induced by estrogen deficiency is mainly via suppression of osteoclast activity. Moreover, the beneficial effects of combined treatment with SARM and SERM on bone are possibly due to different major mechanism of action, which is stimulation of osteoblastic activity (SARM) or suppression of osteoclastic number and/or activity (SERM). Further studies about the effects of SARM and SERM on the osteoblastic and osteoclastic apoptosis and cell signaling in non-genomic pathway would be helpful for better understanding of their mechanism of action.

146

140

120

100 * 80

20 *

* H]-Thymidine (% Incoporation of Control)

3 0 [ 0 0.001 0.01 0.1 1 10 Drug Treatment ( µµµM)

DHT DHT: 100 S-22

Figure 6.1 Effect of DHT and S-22 on the proliferation of Saos-2 cells.

147

140

120

100

80 *

* 60 *

* H]-Thymidine H]-Thymidine Incoporation of(% Control)

3 0 [ 0 0.01 0.1 1 10 0.01 0.1 1 10 10 10 S-22 Raloxifene DHT E2

Drug Treatment ( µµµM)

Figure 6.2 Effect of DHT and S-22 on the proliferation of hFOB-AR6 cells.

148

180

* 160 *

140 * * * * * 120 * *

ALP (%) Activity 100

80 20 0 0 0.001 0.01 0.1 1 10 Drug Treatment ( µµµM)

7 days 14 days 21 days

Figure 6.3 Time course study of DHT (1 nM to 10 µM) in MC3T3-E1 cells.

149

A 250 * 200 * *

150 *

100 ALP Activity ALP Activity (%)

0 0 0.01 0.1 1 10 0.01 0.1 1 10 10 10

S-22 Raloxifene DHT E2 Drug Treatment ( µµµM)

150

B 300

* 250 *

200 * * 150

100 ALP ALP Activity (%)

0 Raloxifene 0 0 0.01 0.1 1 10 0 10 10 10 10 10 S-22 0 10 10 10 10 10 0 0 0.01 0.1 1 10

Drug Treatment ( µµµM)

Figure 6.4 ALP activities of MC3T3-E1 cells treated with S-22, raloxifene, estradiol, and

DHT alone (A), and S-22 and raloxifene in combination (B).

151

300

250 *

200 * 150 * **

100 Mineralization (%) Mineralization * * * 50 *

0 0 0.01 0.1 1 10 0.01 0.1 1 10 10 10

S-22 Raloxifene DHT E2 Drug Treatment ( µµµM)

Figure 6.5 Mineralization of MC3T3-E1 cells treated with S-22, raloxifene, estradiol, and DHT.

152

180

160

140

120

100 * 80

60 * TRAP Activity Activity TRAP (%) 40 * 20 * * * * 0 0 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 10 10

S-22 RaloxifeneS-22 DHT E2 + Raloxifene (10 µM) Drug Treatment (uM)

Figure 6.6 Effect of S-22 and raloxifene alone or in combination on differentiation of

bone morrow cell toward osteoclast.

153

CHAPTER 7

SUMMARY

Although abundant data suggest that androgens are useful for the treatment for osteoporosis, undesirable side effects and pharmacokinetic properties limit their clinical uses. SARMs display higher anabolic activity than androgenic activity in vivo without androgen-related side effects. These nonsteroidal SARMs are more amenable to structural modification and demonstrate high oral bioavailability with long half-life. Due to their favorable characteristics, SARMs are promising as a new treatment of osteoporosis. The specific aims of this project were to: analyze in vitro structure-activity relationship by determining in vitro AR binding affinity and AR-mediated transcriptional activation of novel SARMs modified in the aromatic B-ring (Chapter 2); determine the in vivo pharmacologic activity and pharmacokinetics of B-ring substituted SARMs with halogen atom, discover the major factor(s) determining in vivo pharmacologic activities of SARMs, and based on this information, select a potent SARM (S-22) for osteoporosis studies (Chapter 3); examine the in vivo metabolism and plasma protein binding of a series of SARM in male rats (Chapter 4); determine effects of a SARM (S-22) alone or in

154

combination with SERM (i.e., raloxifene) in bone, muscle, uterus, and body composition in order to evaluate a SARM as a treatment of osteoporosis (Chapter 5); determine in vitro activities of SARM (S-22) alone or in combination with SERM (raloxifene) in bone cells (Chapter 6).

As a result, we observed that B-ring modification of SARM plays a significant

role in binding affinities and transcriptional activation and learned the favorable structure

for AR binding (Chapter 2). In chapter 3, in vivo pharmacologic activity and

pharmacokinetics of 4-halogen substituted SARMs were determined and we found that

the greater in vivo exposure of those compounds to target tissue results in greater in vivo pharmacologic effect than one would expect based on its AR binding affinity. Moreover, the AUC-response relationships suggest that the observed discrepancy between in vitro and in vivo pharmacologic activity of halogen substituted SARMs was due to differences in systemic exposure rather than intrinsic pharmacologic activity. Based on this finding, we hypothesized that two simple criteria (i.e., Ki < 10 nM and lower in vivo clearance) could be used to identify efficacious and potent SARMs in this chemical class. Using these criteria, S-22 was identified as a compound with the most potent and tissue- selective in vivo activity that we have observed to date and favorable pharmacokinetic properties (Chapter 3). In vivo metabolism and plasma protein binding of a series of

SARM in male rats were determined. Four halogen-substituted SARMs exhibited similar

metabolite pattern and their major phase I metabolism pathways including 1) hydrolysis

of the amide bond, 2) B-ring hydroxylation, and 3) A-ring nitro reduction to an aromatic

amine. Some of the phase I metabolites underwent phase II metabolisms including

sulfation and/or glucuronidation. Plasma protein binding study showed that a series of

155

SARMs displayed high plasma protein binding affinity (f u < 1%) and drug unbound

decreased as the size of halogen atom increased (f u, I < fu, Br < fu, Cl < fu,F ). These results

suggest that the total clearance of halogen-substituted SARMs was significantly affected

by the fraction of drug unbound rather than intrinsic clearance (Chapter 4). The effects of

a SARM (S-22) alone or in combination with SERM (i.e., raloxifene) in bone, muscle,

uterus, and body composition were determined. S-22 and raloxifene additively increase

bone density and improve microarchitecture of bone with less stimulatory effect on uterus

than steroidal counterpart. Moreover, S-22 increased total lean mass and muscle strength

in OVX rats, suggesting that SARMs would be beneficial for treatment of osteoporosis

by (1) indirect increase in BMD due to increasing skeletal stress (lean mass increase) to

stimulate bone formation and/or (2) reduced the risk of falling and fracture (Chapter 5).

In Chapter 6, in vitro bone cell study suggests that the beneficial effect of SARM on bone

is mainly due to stimulation of osteoblastic activity SARM while suppression of

osteoclastic number and/or activity is major mechanism of action for SERM.

156

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171

APPENDIX A

DATA RELATED TO CHAPTER 2

172

Competitive binding affinity of S-23 (K i of DHT = 0.47 ± 0.05) Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 59.8 76.2 68.4 1.867 3.210 2.571 1 220.6 235.0 173.6 15.033 16.212 11.185 0.5 356.4 350.0 267.6 26.152 25.628 18.882 0.1 996.4 1079.0 787.4 78.556 85.319 61.443 0.05 1348.2 1243.0 984.4 107.361 98.747 77.573 0.01 1538.8 1465.2 1070.2 122.967 116.941 84.598 0.005 1645.4 1506.6 1207.8 131.696 120.331 95.865 0.001 1586.0 1529.4 1217.8 126.832 122.198 96.684 0.0001 1649.8 1524.6 1251.6 132.056 121.805 99.451 0.00001 1652.4 1546.0 1247.2 132.269 123.557 99.091

IC 50 0.15108 0.17870 0.15230 Ki 23.262 27.515 23.450

Competitive binding affinity of S-25 (K i of DHT = 0.69 ± 0.21) Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 44.8 45.0 40.0 2.152 2.213 0.676 1 99.6 86.8 89.2 18.998 15.063 15.801 0.5 161.2 149.6 141.8 37.934 34.368 31.970 0.1 274.4 259.0 312.8 72.733 67.999 84.537 0.05 312.4 311.8 338.2 84.414 84.230 92.346 0.01 319.4 310.8 387.4 86.566 83.923 107.470 0.005 418.8 299.8 391.2 117.123 80.541 108.638 0.001 336.0 357.6 399.6 91.669 98.309 111.220 0.0001 347.6 324.2 427.8 95.235 88.042 119.889 0.00001 360.6 322.8 414.6 99.231 87.611 115.832

IC 50 0.27822 0.29604 0.21070

Ki 42.839 45.583 32.442

Table A.1. Competitive binding affinity of S-23 and S-25 to the AR

173

Competitive binding affinity of S-26 (K i of DHT = 0.69 ± 0.21) Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 45.0 36.4 31.0 2.213 -0.430 -2.090 1 68.8 74.4 59.6 9.530 11.251 6.702 0.5 123.6 126.6 147.8 26.376 27.298 33.815 0.1 250.4 236.6 214.4 65.355 61.113 54.288 0.05 382.4 260.4 274.4 105.933 68.429 72.733 0.01 333.2 319.2 469.2 90.808 86.505 132.616 0.005 345.6 345.8 351.6 94.620 94.682 96.465 0.001 375.0 347.4 350.8 103.658 95.174 96.219 0.0001 305.0 308.2 295.0 82.140 83.123 79.065 0.00001 320.4 385.8 336.2 86.874 106.978 91.731

IC 50 0.25136 0.17363 0.18832 Ki 38.704 26.735 28.997

Competitive binding affinity of S-27 (K i of DHT = 0.69 ± 0.21) Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 43.8 47.0 50.8 1.844 2.828 3.996 1 108.8 93.4 81.4 21.826 17.092 13.403 0.5 145.4 176.8 142.6 33.077 42.730 32.216 0.1 273.8 319.4 278.4 72.548 86.566 73.962 0.05 297.8 283.6 391.0 79.926 75.561 108.577 0.01 363.4 350.4 297.4 100.092 96.096 79.803 0.005 327.8 370.0 393.0 89.148 102.121 109.192 0.001 303.4 307.4 319.6 81.648 82.877 86.628 0.0001 333.8 401.8 285.4 90.993 111.897 76.114 0.00001 394.0 369.4 313.8 109.499 101.937 84.845

IC 50 0.28405 0.32292 0.34651

Ki 43.736 49.722 53.354

Table A.2. Competitive binding affinity of S-26 and S-27 to the AR

174

Competitive binding affinity of S-28 (K i of DHT = 0.47 ± 0.05) Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 440.6 414.0 438.6 33.047 30.869 32.883 1 1019.8 990.0 1061.4 80.472 78.032 83.878 0.5 1272.6 1237.0 1333.0 101.171 98.256 106.116 0.1 1617.2 1513.0 1634.2 129.387 120.855 130.779 0.05 1856.0 1497.0 1620.4 148.940 119.545 129.649 0.01 1441.0 1628.0 1624.4 114.959 130.271 129.976 0.005 1760.0 1506.6 1694.6 141.079 120.331 135.724 0.001 1620.0 1591.0 1584.0 129.616 127.241 126.668 0.0001 1670.0 1607.0 1674.6 133.710 128.552 134.087 0.00001 1566.0 1685.0 1717.2 125.194 134.938 137.575

IC 50 1.63897 1.57662 1.73637 Ki 252.362 242.763 267.360

Competitive binding affinity of S-29 (DHT K i = 0.62 ± 0.24) Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 251.4 233.8 221.0 42.500 39.041 36.524 1 473.2 430.2 430.4 86.102 77.649 77.688 0.5 512.8 495.8 528.4 93.886 90.545 96.953 0.1 531.6 574.6 571.0 97.582 106.035 105.327 0.05 546.2 539.6 575.4 100.452 99.155 106.192 0.01 560.2 565.6 535.2 103.204 104.266 98.290 0.005 584.0 578.0 591.6 107.883 106.703 109.377 0.001 571.4 585.6 555.2 105.406 108.197 102.221 0.0001 568.0 563.2 587.6 104.738 103.794 108.591 0.00001 559.6 566.8 561.8 103.086 104.502 103.519

IC 50 3.69670 1.79280 1.22840

Ki 569.204 276.049 189.145

Table A.3. Competitive binding affinity of S-28 and S-29 to the AR

175

Competitive binding affinity of S-30 (K i of DHT = 0.21 ± 0.03) Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 219.6 221.0 198.4 26.548 26.763 23.283 1 538.4 507.8 495.6 75.639 70.927 69.048 0.5 501.6 613.0 565.8 69.972 87.127 79.858 0.1 659.6 707.8 733.6 94.302 101.725 105.698 0.05 711.4 695.4 676.2 102.279 99.815 96.859 0.01 740.0 719.6 730.6 106.683 103.542 105.236 0.005 726.8 741.0 743.0 104.650 106.837 107.145 0.001 774.2 787.4 735.6 111.949 113.982 106.006 0.0001 744.4 680.2 681.2 107.361 97.475 97.629 0.00001 790.2 690.0 772.4 114.413 98.984 111.672

IC 50 1.58117 2.03569 1.66615 Ki 243.462 313.449 256.548

Competitive binding affinity of S-31 (K i of DHT = 0.18 ± 0.02) Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 66.2 46.4 59.8 3.521 0.427 2.521 1 120.0 96.6 100.2 11.928 8.272 8.834 0.5 141.6 125.8 121.2 15.304 12.835 12.116 0.1 368.8 348.6 340.2 50.807 47.651 46.338 0.05 398.0 392.2 392.4 55.370 54.464 54.495 0.01 657.4 577.4 570.8 95.906 83.405 82.373 0.005 581.6 582.0 601.4 84.061 84.123 87.155 0.001 639.6 629.4 639.6 93.124 91.530 93.124 0.0001 617.2 629.4 627.2 89.624 91.530 91.187 0.00001 631.2 617.8 644.6 91.812 89.718 93.906

IC 50 0.09771 0.09168 0.08131

Ki 15.045 14.117 12.519

Table A.4. Competitive binding affinity of S-30 and S-31 to the AR

176

Competitive binding affinity of S-34 (DHT K i = 0.62 ± 0.24) Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 40.4 39.0 46.7 1.022 0.747 2.265 1 70.6 78.4 72.8 6.959 8.492 7.391 0.5 100.6 141.2 140.8 12.856 20.837 20.759 0.1 243.8 374.4 375.4 41.006 66.680 66.876 0.05 334.6 489.6 496.8 58.856 89.326 90.741 0.01 337.6 595.2 605.8 59.446 110.085 112.168 0.005 383.8 622.0 620.8 68.528 115.353 115.117 0.001 593.6 609.2 610.0 109.770 112.837 112.994 0.0001 657.8 644.0 604.2 122.390 119.678 111.854 0.00001 631.4 629.2 607.4 117.201 116.768 112.483

IC 50 0.01495 0.13068 0.13420

Ki 2.302 20.121 20.664

Competitive binding affinity of S-35 (DHT K i = 0.62 ± 0.04) Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 84.4 80.2 82.6 1.550 1.145 1.376 1 179.4 208.2 159.4 10.716 13.495 8.786 0.5 261.8 307.8 251.2 18.666 23.104 17.643 0.1 737.4 713.6 685.8 64.553 62.256 59.574 0.05 903.4 890.2 925.0 80.569 79.295 82.653 0.01 1097.0 1161.8 1128.8 99.247 105.499 102.316 0.005 1191.6 1202.4 1079.8 108.375 109.417 97.588 0.001 1200.4 1174.8 1141.8 109.224 106.754 103.570 0.0001 1209.4 1176.4 1158.8 110.092 106.908 105.210 0.00001 1201.2 1214.4 1168.4 109.301 110.574 106.136 IC50 0.13044 0.13279 0.13106 Ki 20.085 20.447 20.180

Table A.5. Competitive binding affinity of S-32 and S-33 to the AR

177

Competitive binding affinity of S-36 (K i of DHT = 1.77 ± 0.16) Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 75.2 54.6 52.0 4.786 1.646 1.250 1 135.0 61.4 60.6 13.900 2.683 2.561 0.5 98.2 79.4 70.8 8.291 5.426 4.115 0.1 146.6 128.6 124.0 15.668 12.925 12.224 0.05 208.4 179.6 209.2 25.088 20.698 25.210 0.01 385.4 427.4 477.0 52.065 58.467 66.027 0.005 562.0 591.0 563.0 78.982 83.402 79.134 0.001 712.2 719.0 727.0 101.875 102.911 104.130 0.0001 797.4 811.4 690.6 114.861 116.994 98.583 0.00001 741.0 720.0 770.4 106.264 103.064 110.745

IC 50 0.00897 0.01202 0.01529 Ki 1.381 1.850 2.355

Competitive binding affinity of S-37 (K i of DHT = 1.77 ± 0.16) Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 55.8 58.8 63.4 1.829 2.286 2.987 1 64.4 50.8 59.4 3.140 1.067 2.378 0.5 62.2 71.0 75.4 2.804 4.146 4.816 0.1 128.8 131.2 126.2 12.955 13.321 12.559 0.05 213.8 219.8 214.8 25.911 26.825 26.063 0.01 501.8 444.6 481.6 69.806 61.088 66.728 0.005 627.8 599.3 562.2 89.011 84.667 79.012 0.001 767.4 754.0 724.0 110.288 108.246 103.673 0.0001 775.8 763.4 776.8 111.568 109.678 111.721 0.00001 809.4 783.4 788.0 116.690 112.727 113.428

IC 50 0.01515 0.01323 0.01307

Ki 2.332 2.036 2.012

Table A.6. Competitive binding affinity of S-34 and S-35 to the AR

178

Competitive binding affinity of R-2 (K i of DHT = 1.48 ± 0.11) Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 130.0 139.8 155.4 11.833 13.105 15.129 1 301.8 286.0 304.0 34.125 32.075 34.410 0.5 331.6 379.1 426.6 37.991 44.155 50.318 0.1 700.6 708.8 681.0 85.870 86.934 83.327 0.05 636.6 707.2 690.8 77.566 86.726 84.598 0.01 742.6 749.6 746.1 91.320 92.228 91.774 0.005 773.2 744.4 760.0 95.290 91.553 93.571 0.001 736.8 784.8 773.8 90.567 96.795 95.368 0.0001 801.8 765.2 801.0 99.001 94.252 98.897 0.00001 722.8 771.4 680.4 88.750 95.056 83.249

IC 50 0.40263 0.46734 0.52836 Ki 61.995 71.960 81.355

Competitive binding affinity of R-3 (K i of DHT = 0.87 ± 0.28) Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 89.0 103.6 103.6 3.939 6.610 6.610 1 206.2 196.4 192.6 25.380 23.587 22.892 0.5 255.4 316.8 322.2 34.380 45.613 46.600 0.1 436.0 408.0 529.2 67.419 62.296 84.469 0.05 400.2 539.8 466.6 60.870 86.408 73.017 0.01 463.8 493.6 535.4 72.504 77.956 85.603 0.005 451.4 510.8 506.8 70.236 81.103 80.371 0.001 461.8 559.0 540.6 72.139 89.920 86.554 0.0001 465.6 571.6 495.4 72.834 92.225 78.285 0.00001 471.8 605.2 638.6 73.968 98.372 104.482

IC 50 0.50164 0.38972 0.49941

Ki 77.241 60.008 76.898

Table A.7. Competitive binding affinity of R-2 and R-3 to the AR

179

Competitive binding affinity of R-5 (K i of DHT = 0.87 ± 0.28) Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 98.6 95.0 41.2 5.695 5.037 -4.805 1 121.2 108.2 123.6 9.830 7.452 10.269 0.5 254.0 167.0 157.4 34.124 18.208 16.452 0.1 364.0 317.2 343.8 54.247 45.686 50.552 0.05 455.2 361.0 458.4 70.931 53.698 71.517 0.01 465.2 429.6 506.8 72.761 66.248 80.371 0.005 624.4 615.0 548.4 101.884 100.165 87.981 0.001 523.2 574.2 502.4 83.371 92.701 79.566 0.0001 516.2 456.0 444.2 82.090 71.078 68.919 0.00001 529.6 448.4 486.8 84.542 69.687 76.712

IC 50 0.21922 0.11063 0.21927 Ki 33.754 17.034 33.763

Competitive binding affinity of R-8 (K i of DHT = 1.37 ± 0.08) Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 39.6 38.0 39.0 1.006 0.731 0.903 1 75.6 77.4 78.6 7.177 7.485 7.691 0.5 115.0 106.6 112.2 13.931 12.491 13.451 0.1 274.2 262.2 255.0 41.221 39.163 37.929 0.05 349.6 341.0 361.8 54.145 52.671 56.237 0.01 475.0 505.4 540.2 75.641 80.853 86.818 0.005 508.6 510.8 549.4 81.401 81.778 88.395 0.001 541.6 581.8 574.4 87.058 93.949 92.680 0.0001 587.4 548.8 620.8 94.909 88.292 100.634 0.00001 578.2 596.6 602.6 93.332 96.486 97.514

IC 50 0.07030 0.06800 0.06658

Ki 10.824 10.471 10.252

Table A.8. Competitive binding affinity of R-5 and R-8 to the AR

180

Competitive binding affinity of R-9 (K i of DHT = 1.37 ± 0.08) Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 68.8 70.2 63.8 6.011 6.251 5.154 1 167.6 170.8 164.8 22.947 23.496 22.467 0.5 248.8 237.6 244.2 36.866 34.947 36.078 0.1 452.6 439.0 463.2 71.802 69.470 73.619 0.05 512.8 530.8 549.0 82.121 85.207 88.326 0.01 572.0 566.2 568.8 92.269 91.275 91.720 0.005 578.0 570.0 597.4 93.298 91.926 96.623 0.001 588.4 586.2 446.8 95.080 94.703 70.807 0.0001 561.2 572.0 613.4 90.418 92.269 99.366 0.00001 578.0 592.2 644.2 93.298 95.732 104.645

IC 50 0.31694 0.29035 0.32580 Ki 48.802 44.707 50.165

Table A.9. Competitive binding affinity of R-9 to the AR

181

APPENDIX B

DATA RELATED TO CHAPTER 3

182

Competitive binding affinity of S-9 Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 5 41.6 35.6 3.222 1.786 1.308 1 1 55.6 45.0 6.571 4.035 4.945 0.5 0.5 64.6 77.2 8.725 11.739 10.304 0.1 0.1 181.4 177.8 36.670 35.808 38.201 0.05 0.05 259.0 246.4 55.236 52.221 61.552 0.01 0.01 487.4 413.8 109.881 92.272 84.472 0.005 0.005 523.8 492.8 118.590 111.173 97.631 0.001 0.001 357.6 272.8 78.826 58.537 110.264 0.0001 0.0001 587.4 545.4 133.807 123.758 120.169 0.00001 0.00001 419.6 504.8 93.660 114.044 77.965

IC 50 0.04925 0.05416 0.06487

Ki 7.583 8.339 9.988

Competitive binding affinity of S-10 Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 38.8 31.2 43.6 2.552 0.734 3.700 1 49.2 52.6 56.0 5.040 5.854 6.667 0.5 70.4 77.4 81.2 10.112 11.787 12.696 0.1 234.6 236.2 228.2 49.398 49.781 47.867 0.05 333.8 333.2 294.2 73.132 72.988 63.657 0.01 465.2 472.4 491.6 104.570 106.292 110.886 0.005 481.2 486.5 491.8 108.398 109.666 110.934 0.001 498.2 481.2 461.2 112.465 108.398 103.613 0.0001 546.2 450.8 262.0 123.949 101.124 55.953 0.00001 560.6 472.4 376.4 127.395 106.292 83.324

IC 50 0.06802 0.08660 0.09018

Ki 10.473 13.335 13.886

Table B.1. Competitive binding affinity of S-9 and S-10 to the AR

183

Competitive binding affinity of S-11 Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 44.4 46.4 41.0 2.025 2.393 1.399 1 78.2 71.2 74.8 8.247 6.959 7.622 0.5 138.6 122.8 126.2 19.367 16.458 17.084 0.1 414.8 411.4 391.2 70.214 69.588 65.869 0.05 516.2 489.2 497.8 88.881 83.910 85.493 0.01 599.0 622.2 613.0 104.124 108.395 106.701 0.005 612.2 590.8 592.8 106.554 102.614 102.982 0.001 611.0 693.6 594.4 106.333 121.539 103.277 0.0001 609.8 578.0 607.4 106.112 100.258 105.670 0.00001 625.4 640.6 622.0 108.984 111.782 108.358

IC 50 0.15860 0.13917 0.14160

Ki 24.421 21.428 21.803

Table B.2. Competitive binding affinity of S-11 to the AR.

184

PK parameters of S-9 in rats after i.v. administration Parameters Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Dosing_time 0 0 0 0 0 Rsq 1.0 1.0 0.9 1.0 1.0 Rsq(adjusted) 1.0 1.0 0.9 1.0 1.0 Corr(x:y) -1.0 -1.0 -1.0 -1.0 -1.0 Tmax 0.0 0.0 0.0 0.0 0.0 Cmax 31.4 25.5 27.3 49.1 41.3 No.points_Lambda_z 4.0 3.0 3.0 3.0 3.0 Tlast 1920.0 1920.0 1920.0 1920.0 2895.0 Clast 1.4 0.5 1.3 0.4 0.6 AUClast 9738.9 7447.4 8865.5 7360.7 11592.9 Lambda_z 0.0 0.0 0.0 0.0 0.0 Lambda_z_lower 480.0 720.0 720.0 720.0 1440.0 Lambda_z_upper 1920.0 1920.0 1920.0 1920.0 2895.0 t1/2_Lambda_z 538.6 350.9 567.5 330.8 560.9 AUCall 9738.9 7447.4 8865.5 7360.7 11592.9 AUCINF(observed) 10826.8 7715.7 9929.9 7551.6 12094.6 AUCINF(observed)/D 1082.7 771.6 993.0 755.2 1209.5 AUC_%Extrap(obs.) 10.0 3.5 10.7 2.5 4.1 Vz(observed) 717.7 656.2 824.5 632.1 669.1 Cl(observed) 0.9 1.3 1.0 1.3 0.8 AUCINF(predicted) 10728.8 7689.5 10090.6 7558.4 12084.8 AUCINF(predicted)/D 1072.9 769.0 1009.1 755.8 1208.5 AUC_%Back_Ext obs 1.2 1.4 1.2 2.6 1.5 AUC%Back_Ext pred 1.3 1.5 1.2 2.6 1.5 AUC_%Extrap(pred) 9.2 3.1 12.1 2.6 4.1 Vz(predicted) 724.3 658.4 811.4 631.5 669.7 Cl(predicted) 0.9 1.3 1.0 1.3 0.8 AUMClast 5983295 4072160 6478275 3973090 9960647 1181926 AUMCINF(observed) 8917495 4723199 9393208 4430790 1 AUMC_%Extrap(obs) 32.9 13.8 31.0 10.3 15.7 1178290 AUMCINF(predicted) 8653344 4659628 9833510 4447028 0 AUMC_%Extrap pred 30.9 12.6 34.1 10.7 15.5 MRTlast 614.4 546.8 730.7 539.8 859.2 MRTINF(observed) 823.7 612.2 946.0 586.7 977.2 Vss(observed) 760.8 793.4 952.6 777.0 808.0 MRTINF(predicted) 806.6 606.0 974.5 588.4 975.0 Vss (predicted) 751.8 788.1 965.8 778.4 806.8

Table B.3. Pharmacokinetic parameters of S-9 in rats after i.v. administration.

185

PK parameters of S-9 in rats after p.o. administration Parameters Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Rsq 0.9987 0.9899 0.9997 0.9978 0.9999 Rsq_adjusted 0.9983 0.9849 0.9994 0.9956 0.9998 Corr_XY -0.9994 -0.995 -0.9999 -0.9989 -1 No_points_lambda_z 5 4 3 3 3 Lambda_z 0.0017 0.0015 0.0009 0.0016 0.0013 Lambda_z_lower 540 720 1440 1440 1440 Lambda_z_upper 2160 2160 3100 2160 2160 HL_Lambda_z 419 451 746 436 522 Tlag 0 0 0 0 0 Tmax 540 720 720 240 540 Cmax 7.6 7.7 5.7 6.5 6.6 Tlast 2160 2160 3100 2160 2160 Clast 0.5 0.9 0.7 0.7 1 AUClast 7673.5 8098 8057.0 6723.0 7379.8 AUCall 7673.5 8098 8057.0 6723.0 7379.8 AUCINF_obs 7975.9 8684.0 8810.5 7163.1 8133.3 AUCINF_D_obs 797.5934 868.4015 881.0 716.3 813.3 AUC_%Extrap_obs 3.7918 6.7482 8.6 6.1 9.3 Vz_F_obs 758.366 749.800 1221.739 877.757 926.469 Cl_F_obs 1.2538 1.1515 1.135 1.396 1.2295 AUCINF_pred 7992.51 8652.53 8808.06 7169.99 8131.33 AUCINF_D_pred 799.251 865.252 880.805 716.999 813.132 AUC_%Extrap_pred 3.9914 6.4088 8.5269 6.2342 9.243 Vz_F_pred 756.793 752.529 1222.076 876.916 926.691 Cl_F_pred 1.2512 1.1557 1.1353 1.3947 1.2298 AUMClast 5374522 6741003 9814540 5105092 6360701 AUMCINF_obs 6210712 8388367 12961419 6332487 8556108 AUMC_%Extrap_obs 13.4637 19.6387 24.2788 19.3825 25.6589 AUMCINF_pred 6256547 8299850 1295126 6351648 8550442 AUMC_%Extrap_pred 14.0976 18.7816 24.2195 19.6257 25.6097 MRTlast 700.40 832.428 1218.13 759.34 861.91 MRTINF_obs 778.68 965.95 1471.13 884.03 1051.98 MRTINF_pred 782.80 959.24 1470.38 885.86 1051.54

Table B.4. Pharmacokinetic parameters of S-9 in rats after p.o. administration.

186

PK parameters of S-10 in rats after i.v. administration Parameters Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Rsq 0.9833 0.9855 0.9907 0.9937 0.9975 Rsq_adjusted 0.9667 0.971 0.9861 0.9874 0.9963 Corr_XY -0.9916 -0.9927 -0.9953 -0.9969 -0.9988 No_points_lambda_z 3 3 4 3 4 Lambda_z 0.0011 0.0007 0.0007 0.0014 0.0012 Lambda_z_lower 1480 1480 540 720 720 Lambda_z_upper 2880 2880 2880 2040 2940 HL_Lambda_z 614 1005 1062 490 579 Tmax 5 9 14 5 5 Cmax 42.4 40.5 65.6 62 29 C0 49.4752 52.8129 208.9358 72.8407 31.728 Tlast 2880 2880 2880 2040 2940 Clast 1.4 2.4 2.1 2.2 1.1 AUClast 22130.7 21054.1 21282.5 20671.9 24598.6 AUCall 22130.7 21054.1 21282.5 20671.9 24598.6 AUCINF_obs 23371.6 24534.1 24498.8 22228.5 25517.0 AUCINF_D_obs 2337.2 2453.4 2449.9 2222.9 2551.7 AUC_%Extrap_obs 5.3097 14.1847 13.1 7.0 3.6 AUC%Back_Ext_ob 0.9828 1.7115 7.8443 1.5165 0.595 Vz_obs 379.3 591.0 625.2 318.3 327.199 Cl_obs 0.43 0.41 0.41 0.45 0.39 AUCINF_pred 23428.7 24624.3 24390.8 22157.5 25518.42 AUCINF_D_pred 2342.9 2462.4 2439.1 2215.8 2551.843 AUC_%Extrap_pred 5.5402 14.499 12.7438 6.705 3.6047 AUC%Back_Extpred 0.9804 1.7053 7.879 1.5214 0.5949 Vz_pred 378.3 588.9 627.9 319.3 327.2 Cl_pred 0.4268 0.4061 0.4 0.5 0.4 AUMClast 20502.2 21040.3 16850.2 13067.4 21176.5 AUMCINF_obs 25176.1 36109.3 31039.0 17344.5 24643.4 AUMC%Extrap_obs 18.565 41.7314 45.7128 24.6598 14.0682 AUMCINF_pred 25390.9 36499.7 30562.7 17149.3 24648.8 AUMC%Extrap_pre 19.2539 42.3548 44.8668 23.8023 14.0873 MRTlast 926.4154 999.3508 791.7408 632.136 860.8845 MRTINF_obs 1077.209 1471.798 1266.963 780.283 965.7659 MRTINF_pred 1083.756 1482.266 1253.044 773.976 965.9256 Vss_obs 460.9043 599.8977 517.1534 351.0276 378.4798 Vss_pred 462.5766 601.9516 513.7359 349.3065 378.5208

Table B.5. Pharmacokinetic parameters of S-10 in rats after i.v. administration. 187

PK parameters of S-10 in rats after p.o. administration Parameters Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Rsq 0.9955 0.9991 0.9996 0.991 0.9974 Rsq_adjusted 0.9941 0.9983 0.9995 0.9819 0.9961 Corr_XY -0.9978 -0.9996 -0.9998 -0.9955 -0.9987 No_points_lambda_z 5 3 4 3 4 Lambda_z 0.0009 0.0007 0.0008 0.0012 0.0015 Lambda_z_lower 720 720 720 1440 720 Lambda_z_upper 3150 2880 2880 2910 2910 HL_Lambda_z 770 942 816 591 453 Tlag 20 0 0 0 0 Tmax 720 540 380 720 540 Cmax 8.1 9.3 14.3 14.3 12.3 Tlast 3150 2880 2880 2910 2910 Clast 0.9 1.8 2.2 0.7 0.4 AUClast 12430 15237 21254.0 13843.5 13128.0 AUCall 12430 15237 21254.0 13843.5 13128.0 AUCINF_obs 13429.3 17682.2 23843.8 14439.8 13389.3 AUCINF_D_obs 1342.9 1768.2 2384.4 1444.0 1338.9 AUC_%Extrap_obs 7.4414 13.829 10.9 4.1 2.0 Vz_F_obs 826.82 768.28 493.69 589.98 487.88 Cl_F_obs 0.7446 0.5655 0.4194 0.6925 0.7469 AUCINF_pred 13476.2 17659.4 23816.9 14417.1 13377.1 AUCINF_D_pred 1347.61 1765.94 2381.69 1441.71 1337.71 AUC_%Extrap_pred 7.7633 13.7174 10.7611 3.9789 1.8624 Vz_F_pred 823.9463 769.2753 494.2541 590.9116 488.326 Cl_F_pred 0.742 0.5663 0.4199 0.6936 0.7475 AUMClast 14436445 16052180 22864730 13256505 11511720 AUMCINF_obs 18693955 26416537 33371806 15499922 12442781 AUMC_%Extrap_obs 22.7748 39.2344 31.4849 14.4737 7.4827 AUMCINF_pred 18893631 26319589 33263107 15414511 12399473 AUMC_%Extrap_pred 23.5909 39.0105 31.261 13.9998 7.1596 MRTlast 1161.41 1053.5 1075.78 957.59 876.88 MRTINF_obs 1392.02 1493.95 1399.60 1073.41 929.30 MRTINF_pred 1402.00 1490.39 1396.61 1069.17 926.91

Table B.6. Pharmacokinetic parameters of S-10 in rats after p.o. administration.

188

PK parameters of S-11 in rats after i.v. administration Parameters Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Rsq 0.9921 0.9959 0.9722 0.9905 1 Rsq_adjusted 0.9841 0.9938 0.9682 0.9873 0.9999 Corr_XY -0.996 -0.9979 -0.986 -0.9952 -1 No_points_lambda_z 3 4 9 5 3 Lambda_z 0.0007 0.0009 0.0005 0.0008 0.001 Lambda_z_lower 1030 720 60 540 3120 Lambda_z_upper 1990 1990 2880 4560 4560 HL_Lambda_z 955 753 1329 877 721 Tmax 5 10 8 10 10 Cmax 86.7 77.1 70.6 63.4 60.9 C0 107.2154 111.8931 81.3272 79.9395 84.0582 Tlast 1990 1990 2880 4560 4560 Clast 16.7 7.7 8.4 1.2 1.2 AUClast 55895.3 42318.4 54121.4 54626.5 50816.6 AUCall 55895.3 42318.4 54121.4 54626.5 50816.6 AUCINF_obs 78900.4 50685.0 70231.3 56144.8 52064.6 AUCINF_D_obs 7890.04 5068.50 7023.1 5614.5 5206.5 AUC_%Extrap_obs 29.1571 16.507 22.9 2.7 2.4 AUC_%Back_Ext_obs 0.6144 1.8644 0.8653 1.2765 1.3921 Vz_obs 174.6 214.4 273.1 225.3 199.8 Cl_obs 0.13 0.20 0.14 0.18 0.19 AUCINF_pred 78380.3 50410.5 69423.3 56191.0 52062.5 AUCINF_D_pred 7838.0 5041.1 6942.3 5619.1 5206.2 AUC%Extrap_pred 28.687 16.0523 22.0415 2.7841 2.3931 AUC%Back_Ext_pred 0.6185 1.8745 0.8754 1.2755 1.3922 Vz_pred 175.75 215.54 276.25 225.16 199.76 Cl_pred 0.1276 0.1984 0.1 0.2 0.2 AUMClast 48539.8 29255.0 57031.3 65768.4 66966.3 AUMCINF_obs 126010.6 54995.4 134324.3 74612.5 73955.2 AUMC_%Extrap_obs 60.9 46.0 56.3 12.2 9.4 AUMCINF_pred 868.4 691.3 1053.8 1204.0 1317.8 AUMC%Extrap_pred 1597.1 1085.0 1912.6 1328.9 1420.5 MRTlast 1585.3 1074.2 1879.0 1332.6 1420.3 MRTINF_obs 202.4 214.1 272.3 236.7 272.8 MRTINF_pred 202.3 213.1 270.7 237.2 272.8 Vss_obs 60.9 46.0 56.3 12.2 9.4 Vss_pred 868.4 691.3 1053.8 1204.0 1317.8

Table B.7. Pharmacokinetic parameters of S-11 in rats after i.v. administration.

189

PK parameters of S-11 in rats after p.o. administration Parameters Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Rsq 0.9959 0.9984 0.9502 0.9941 0.9137 Rsq_adjusted 0.9946 0.9969 0.9378 0.993 0.8705 Corr_XY -0.998 -0.9992 -0.9748 -0.9971 -0.9559 No_points_lambda_z 5 3 6 7 4 Lambda_z 0.0007 0.0007 0.0006 0.0007 0.0007 Lambda_z_lower 1890 2960 760 760 1440 Lambda_z_upper 4890 4890 4890 4890 2900 HL_Lambda_z 960 1027 1160 1060 955 Tlag 0 0 0 0 0 Tmax 760 1490 760 760 1440 Cmax 25.9 21.8 20.1 26.2 16.2 Tlast 4890 4890 4890 4890 2900 Clast 2.3 3.2 1.9 1.9 5.2 AUClast 62874 58952 38995 47146 28939 AUCall 62874 58952 38995 47146 28939 AUCINF_obs 66058 63692 42175 50051 36102 AUCINF_D_obs 6606 6369 4217 5005 3610 AUC_%Extrap_obs 5 7 8 6 20 Vz_F_obs 210 233 397 306 382 Cl_F_obs 0.15 0.16 0.24 0.20 0.28 AUCINF_pred 66062 63646 41611 49797 36277 AUCINF_D_pred 6606 6365 4161 4980 3628 AUC_%Extrap_pred 5 7 6 5 20 Vz_F_pred 210 233 402 307 380 Cl_F_pred 0.15 0.16 0.24 0.20 0.28 AUMClast 111536.0 119345.2 59656.0 77312.9 42666.3 AUMCINF_obs 131514.2 149541.4 80528.2 95967.6 73306.4 AUMC%Extrap_obs 15 20 26 19 42 AUMCINF_pred 131543.0 149250.1 76828.3 94334.9 74055.8 AUMC_%Extrap_pr 15 20 22 18 42 MRTlast 1774 2024 1530 1640 1474 MRTINF_obs 1991 2348 1909 1917 2031 MRTINF_pred 1991 2345 1846 1894 2041

Table B.8. Pharmacokinetic parameters of S-11 in rats after p.o. administration.

190

Competitive binding affinity of S-19 Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 53.4 48.6 47.8 0.817 0.043 -0.086 1 85.8 55.4 70.0 6.041 1.139 3.494 0.5 101.6 62.4 88.2 8.589 2.268 6.428 0.1 242.8 253.6 230.2 31.357 33.099 29.325 0.05 373.0 350.4 360.0 52.352 48.707 50.255 0.01 707.0 691.8 662.8 106.208 103.757 99.081 0.005 753.8 759.2 716.4 113.754 114.625 107.724 0.001 881.6 826.4 811.8 134.362 125.461 123.107 0.0001 907.4 852.2 726.2 138.522 129.621 109.304 0.00001 929.0 891.4 782.4 142.005 135.942 118.366

IC 50 0.02862 0.03212 0.03961

Ki 4.406 4.945 6.100

Corrected K i 1.88 2.11 2.61

Ki (mean ± stdev) 2.20 ± 0.37

Competitive binding affinity of S-20 Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 111.0 48.4 39.4 5.643 0.668 0.000 1 63.6 71.4 56.2 1.876 2.496 1.288 0.5 98.6 75.0 70.8 4.658 2.782 2.448 0.1 252.6 207.6 220.0 16.898 13.322 14.307 0.05 302.4 328.0 315.0 20.857 22.892 21.858 0.01 772.8 789.0 773.0 58.247 59.534 58.262 0.005 922.2 999.2 957.8 70.122 76.242 72.951 0.001 1363.2 1387.2 1368.4 105.174 107.082 105.588 0.0001 1414.4 1447.6 1539.8 109.244 111.883 119.212 0.00001 1438.6 1445.8 1555.8 111.168 111.740 120.483

IC 50 0.00953 0.01106 0.00882

Ki 1.468 1.703 1.358

Corrected K i 2.01 2.33 1.86

Ki (mean ± stdev) 2.05 ± 0.24

Table B.9. Competitive binding affinity S-19 and S-20 to the AR.

191

Competitive binding affinity of S-21 Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 47.8 60.2 57.4 -0.086 1.913 1.462 1 67.6 77.8 67.2 3.107 4.751 3.042 0.5 83.0 101.4 97.4 5.590 8.557 7.912 0.1 229.8 233.2 243.6 29.261 29.809 31.486 0.05 366.0 349.6 335.8 51.223 48.578 46.353 0.01 690.8 681.4 502.0 103.596 102.080 73.152 0.005 780.8 692.0 623.2 118.108 103.789 92.696 0.001 843.0 773.6 688.4 128.138 116.947 103.209 0.0001 847.6 693.6 611.6 128.879 104.047 90.825 0.00001 824.2 778.4 685.8 125.106 117.721 102.790

IC 50 0.03539 0.04092 0.04144

Ki 5.449 6.300 6.381

Corrected K i 2.29 2.65 2.68

Ki (mean ± stdev) 2.54 ± 0.22

Competitive binding affinity of S-22 Concentration Scintillation Counting (DPM) % Specific Binding (µM) 1 2 3 1 2 3 5 57.8 54.0 49.8 1.415 1.113 0.779 1 70.2 74.0 65.0 2.400 2.702 1.987 0.5 94.2 98.6 85.8 4.308 4.658 3.640 0.1 132.8 270.2 291.2 7.376 18.297 19.967 0.05 403.8 419.8 390.0 28.917 30.188 27.820 0.01 940.2 936.0 911.4 71.552 71.219 69.263 0.005 1044.0 1007.8 1074.8 79.803 76.926 82.251 0.001 1312.8 1375.6 1327.0 101.168 106.160 102.297 0.0001 1454.8 1277.0 1372.0 112.455 98.323 105.874 0.00001 1408.0 1315.8 1334.8 108.735 101.407 102.917

IC 50 0.01575 0.02023 0.01896

Ki 2.425 3.114 2.920

Corrected K i 3.30 4.23 3.97

Ki (mean ± stdev) 2.54 ± 0.22

Table B.10. Competitive binding affinity S-21 and S-22 to the AR.

192

Control DHT S-19 S-20 S-21 S-22 1 62805 1904202 1311596 1466986 1656859 1573595 Reading for 2 38983 1401127 1125392 1710392 1380586 1313281 luciferase activity 3 45525 966882 1386789 1808636 1164743 1324267 4 46399 1393961 1445092 1551808 1730535 1207831 1 0.928 0.883 0.877 0.85 0.783 0.709 Reading for 2 1.146 0.689 0.584 0.852 0.661 0.566 β-gal activity 3 0.983 0.589 0.737 0.752 0.817 0.774 4 0.942 0.682 0.755 0.694 0.731 1.057 1 67678 2156514 1495548 1725866 2116040 2219457 Luciferase 2 34017 2033566 1927041 2007502 2088632 2320284 activity / β- gal activity 3 46312 1641565 1881668 2405101 1425634 1710939 4 49256 2043931 1914029 2236035 2367353 1142697 % of DHT 3 100 97 102 106 94 CV (%) 1 11 1 20 15 27

Table B.11. Transcriptional activational activities of various SARMs

193

PK parameters of S-19 Parameters Unit Rat 1 Rat 2 Rat 3 Rat 4 Rsq 0 0.9992 0.9997 0.9993 0.9962 Rsq_adjusted 0 0.9987 0.9993 0.999 0.9952 Corr_XY 0 -0.9996 -0.9998 -0.9996 -0.9981 No_points_lambda_z 0 4 3 5 6 Lambda_z 1/min 0.0024 0.0035 0.0026 0.003 Lambda_z_lower min 240 360 120 60 Lambda_z_upper min 720 720 720 720 HL_Lambda_z min 287.386 199.186 263.879 229.562 Tmax min 5 5 5 5 Cmax ug/mL 18.6 23.6 17.4 15.3 C0 ug/mL 28.5401 39.5575 26.328 21.4284 Tlast min 720 720 720 720 Clast ug/mL 0.9 0.6 0.9 0.8 AUClast min*ug/mL 2087.1 2224.64 2186.07 2110.07 AUCall min*ug/mL 2087.1 2224.64 2186.07 2110.07 AUCINF_obs min*ug/mL 2460.25 2397.06 2528.7 2375.02 AUCINF_D_obs min*kg*ug/mL/mg 246.025 239.706 252.87 237.502 AUC_%Extrap_obs % 15.1671 7.1929 13.5495 11.1557 AUC_%Back_Ext_obs % 4.7902 6.587 4.3232 3.8661 Vz_obs mL/kg 1685.24 1198.82 1505.51 1394.46 Cl_obs mL/min/kg 4.0646 4.1718 3.9546 4.2105 AUCINF_pred min*ug/mL 2460.16 2395.9 2535.04 2361.11 AUCINF_D_pred min*kg*ug/mL/mg 246.016 239.59 253.504 236.111 AUC_%Extrap_pred % 15.1641 7.148 13.766 10.6321 AUC_%Back_Ext_pred % 4.7903 6.5902 4.3123 3.8889 Vz_pred mL/kg 1685.3 1199.4 1501.74 1402.68 Cl_pred mL/min/kg 4.0648 4.1738 3.9447 4.2353 AUMClast min*min*ug/mL 457691 433440 495158 456671 AUMCINF_obs min*min*ug/mL 881070 607129 872286 735184 AUMC_%Extrap_obs % 48.0528 28.6082 43.2345 37.8834 AUMCINF_pred min*min*ug/mL 880971 605961 879272 720557 AUMC_%Extrap_pred % 48.0469 28.4706 43.6855 36.6224 MRTlast min 219.295 194.836 226.506 216.425 MRTINF_obs min 358.122 253.28 344.955 309.548 MRTINF_pred min 358.095 252.915 346.847 305.178 Vss_obs mL/kg 1455.63 1056.63 1364.16 1303.35 Vss_pred mL/kg 1455.57 1055.62 1368.21 1292.52

Table B.12. Pharmacokinetic parameters of S-19 after i.v. administration.

194

PK parameters of S-20 Parameters Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Rsq 0.9968 0.991 0.9901 0.9989 0.9996 Rsq_adjusted 0.9961 0.9895 0.9881 0.9977 0.9993 Corr_XY -0.9984 -0.9955 -0.995 -0.9994 -0.9998 No_points_lambda_z 7 8 7 3 3 Lambda_z 0.003 0.003 0.003 0.004 0.003 Lambda_z_lower 60 30 32 360 360 Lambda_z_upper 720 720 720 600 720 HL_Lambda_z 231 203 258 177 257 Tmax 5 5 8 5 5 Cmax 35.2 26.7 17.8 20.3 33.1 C0 54 35 22 30 70 Tlast 720 720 720 600 720 Clast 1.4 1.2 1.8 0.9 1.7 AUClast 4111 4017 4399 2708 4198 AUCall 4111 4017 4399 2708 4198 AUCINF_obs 4578 4369 5069 2938 4829 AUCINF_D_obs 458 437 507 294 483 AUC_%Extrap_obs 10 8 13 8 13 AUC_%Back_Ext_obs 5 4 3 4 5 Vz_obs 728 672 735 871 769 Cl_obs 2 2 2 3 2 AUCINF_pred 4602 4380 5118 2936 4826 AUCINF_D_pred 460 438 512 294 483 AUC_%Extrap_pred 11 8 14 8 13 AUC_%Back_Extpred 5 4 3 4 5 Vz_pred 725 670 728 871 769 Cl_pred 2 2 2 3 2 AUMClast 899640 847061 1074482 442405 927180 AUMCINF_obs 1391551 1204098 1806970 639417 1615678 AUMC_%Extrap_obs 35 30 41 31 43 AUMCINF_pred 1416494 1215540 1860105 637632 1613256 AUMC_%Extrap_pred 36 30 42 31 43 MRTlast 219 211 244 163 221 MRTINF_obs 304 276 356 218 335 MRTINF_pred 308 277 363 217 334 Vss_obs 664 631 703 741 693 Vss_pred 669 633 710 740 693

Table B.13. Pharmacokinetic parameters of S-20 after i.v. administration.

195

PK parameters of S-21 Parameters Unit Rat 1 Rat 2 Rat 3 Rat 4 Rsq 0 0.9969 0.9996 0.9966 0.9982 Rsq_adjusted 0 0.9963 0.9995 0.9959 0.9976 Corr_XY 0 -0.9985 -0.9998 -0.9983 -0.9991 No_points_lambda_z 0 7 6 7 5 Lambda_z 1/min 0.005 0.004 0.005 0.004 Lambda_z_lower min 15 60 15 60 Lambda_z_upper min 540 720 540 540 HL_Lambda_z min 128 193 149 161 Tmax min 5 5 5 5 Cmax ug/mL 24.7 20.5 18 18.6 C0 ug/mL 37 29 24 23 Tlast min 540 720 540 540 Clast ug/mL 0.6 0.7 0.9 1 AUClast min*ug/mL 2233 2571 2223 2256 AUCall min*ug/mL 2233 2571 2223 2256 AUCINF_obs min*ug/mL 2344 2766 2417 2488 AUCINF_D_obs min*kg*ug/mL/mg 234 277 242 249 AUC_%Extrap_obs % 5 7 8 9 AUC_%Back_Ext_obs % 7 4 4 4 Vz_obs mL/kg 786 1007 890 935 Cl_obs mL/min/kg 4 4 4 4 AUCINF_pred min*ug/mL 2349 2764 2412 2480 AUCINF_D_pred min*kg*ug/mL/mg 235 276 241 248 AUC_%Extrap_pred % 5 7 8 9 AUC_%Back_Ext_pred % 7 4 4 4 Vz_pred mL/kg 785 1008 892 938 Cl_pred mL/min/kg 4 4 4 4 AUMClast min*min*ug/mL 312315 514298 344351 349084 AUMCINF_obs min*min*ug/mL 392422 709060 490617 528863 AUMC_%Extrap_obs % 20 27 30 34 AUMCINF_pred min*min*ug/mL 395744 707170 486855 522330 AUMC_%Extrap_pred % 21 27 29 33 MRTlast min 140 200 155 155 MRTINF_obs min 167 256 203 213 MRTINF_pred min 169 256 202 211 Vss_obs mL/kg 714 927 840 854 Vss_pred mL/kg 717 926 837 849

Table B.14. Pharmacokinetic parameters of S-21 after i.v. administration. 196

PK parameters of S-22 Parameters Unit Rat 1 Rat 2 Rat 3 Rat 4 Rsq 0 0.9981 0.9964 0.9988 0.9933 Rsq_adjusted 0 0.9978 0.9955 0.9985 0.9921 Corr_XY 0 -0.9991 -0.9982 -0.9994 -0.9966 No_points_lambda_z 0 9 6 7 8 Lambda_z 1/min 0.0018 0.0017 0.0022 0.002 Lambda_z_lower min 30 180 30 60 Lambda_z_upper min 2040 2040 1440 2040 HL_Lambda_z min 392.238 401.991 311.924 349.149 Tmax min 5 5 5 5 Cmax ug/mL 26.4 58.9 15.8 40.2 C0 ug/mL 30.9567 90.0651 17.2862 61.4575 Tlast min 2040 2040 1440 2040 Clast ug/mL 0.4 0.3 0.5 0.2 AUClast min*ug/mL 9312.14 7337.21 5429.72 7693.14 AUCall min*ug/mL 9312.14 7337.21 5429.72 7693.14 AUCINF_obs min*ug/mL 9538.49 7511.2 5654.72 7793.89 AUCINF_D_obs min*kg*ug/mL/mg 953.849 751.12 565.472 779.389 AUC_%Extrap_obs % 2.373 2.3163 3.9791 1.2926 AUC_%Back_Ext_obs % 1.5033 4.9581 1.4628 3.2608 Vz_obs mL/kg 593.259 772.114 795.815 646.295 Cl_obs mL/min/kg 1.0484 1.3313 1.7684 1.2831 AUCINF_pred min*ug/mL 9561.98 7522.45 5650.22 7818.13 AUCINF_D_pred min*kg*ug/mL/mg 956.198 752.245 565.022 781.813 AUC_%Extrap_pred % 2.6128 2.4625 3.9026 1.5987 AUC_%Back_Ext_pred % 1.4996 4.9507 1.4639 3.2507 Vz_pred mL/kg 591.802 770.959 796.449 644.291 Cl_pred mL/min/kg 1.0458 1.3294 1.7698 1.2791 AUMClast min*min*ug/mL 4528688 3067764 1990493 3368303 AUMCINF_obs min*min*ug/mL 5118533 3523596 2415756 3624564 AUMC_%Extrap_obs % 11.5237 12.9366 17.6037 7.0701 AUMCINF_pred min*min*ug/mL 5179727 3553082 2407254 3686236 AUMC_%Extrap_pred % 12.569 13.6591 17.3127 8.6249 MRTlast min 486.321 418.11 366.592 437.832 MRTINF_obs min 536.619 469.112 427.21 465.052 MRTINF_pred min 541.701 472.33 426.046 471.498 Vss_obs mL/kg 562.582 624.551 755.493 596.688 Vss_pred mL/kg 566.515 627.894 754.034 603.083

Table B.15. Pharmacokinetic parameters of S-22 after i.v. administration.

197

Normalized organ weight in S-9 treatment group 0.1 mg/day 0.3 mg/day 0.5 mg/day 0.75 mg/day 1 mg/day Rat 1 10.6 18.6 27.5 31.1 22.2 Rat 2 6.5 23.2 37.7 32.3 29.7 Prostate Rat 3 9.5 19.4 23.8 24.6 28.8 Rat 4 13.7 19.6 39.4 35.2 23.1 Rat 5 18.3 20.7 29.3 44.0 30.4 Rat 1 11.7 10.9 18.8 29.0 21.2 Rat 2 5.9 15.1 20.7 24.6 17.8 Seminal Rat 3 7.8 10.5 14.0 19.1 29.8 vesicle Rat 4 7.5 8.1 19.4 24.4 30.5 Rat 5 9.0 13.2 22.7 23.1 38.1 Rat 1 64.2 79.9 102.8 114.7 104.3 Rat 2 63.9 84.6 120.1 107.9 119.6

Levator ani Rat 3 63.8 88.4 93.6 101.1 115.2 muscle Rat 4 73.9 75.6 99.8 113.5 117.9 Rat 5 78.5 115.4 103.2 115.2 123.7

Table B.16. Normalized tissue weights of androgenic and anabolic organs in S-9 administered male rats

198

Normalized organ weight in S-10 treatment group 0.1 mg/day 0.3 mg/day 0.5 mg/day 0.75 mg/day 1 mg/day Rat 1 12.6 9.9 13.0 12.7 16.2 Rat 2 8.4 9.9 9.6 12.5 16.8 Prostate Rat 3 6.8 11.9 13.1 13.4 11.7 Rat 4 8.9 11.5 15.3 16.6 15.4 Rat 5 7.2 17.5 16.6 15.0 18.9 Rat 1 9.9 8.8 9.9 10.9 11.0 Rat 2 10.8 11.6 10.8 12.4 10.7 Seminal Rat 3 9.4 10.5 12.5 10.2 9.1 vesicle Rat 4 8.0 11.4 11.9 13.9 10.4 Rat 5 10.8 13.1 16.4 15.0 8.4 Rat 1 45.6 49.4 56.7 66.5 73.1 Rat 2 57.4 48.8 52.8 60.1 66.7

Levator Rat 3 51.2 55.5 59.8 59.2 58.5 ani muscle Rat 4 61.3 57.4 58.1 61.3 59.2 Rat 5 50.0 72.7 75.6 57.4 57.4

Table B.17. Normalized tissue weights of androgenic and anabolic organs in S-10 administered male rats

199

Normalized organ weight in S-11 treatment group 0.1 mg/day 0.3 mg/day 0.5 mg/day 0.75 mg/day 1 mg/day Rat 1 12.6 14.5 19.9 22.6 20.9 Rat 2 13.2 14.9 20.3 24.5 22.1 Prostate Rat 3 8.1 14.1 17.1 16.0 21.2 Rat 4 10.3 19.1 16.8 21.7 16.6 Rat 5 10.8 16.1 20.3 21.5 - Rat 1 13.5 11.5 14.1 22.4 16.6 Rat 2 11.3 12.7 17.3 18.8 18.2 Seminal Rat 3 12.1 22.7 12.0 21.8 19.1 vesicle Rat 4 12.3 16.4 14.1 14.0 16.3 Rat 5 11.5 13.7 18.4 16.3 - Rat 1 56.4 78.8 79.3 - 78.6 Rat 2 63.3 72.2 71.3 73.7 83.2

Levator ani Rat 3 64.9 68.2 72.8 91.7 98.0 muscle Rat 4 70.3 68.4 61.8 97.9 90.4 Rat 5 63.2 66.1 78.1 84.4 88.9

Table B.18. Normalized tissue weights of androgenic and anabolic organs in S-11 administered male rats

200

Time post Concentration of S-9 after i.v. administration ( µg/ml) dose (min) Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Average stdev 5 23 19 21 28 29 24 4.4 10 16 14 16 16 21 17 2.2 20 15 12 11 12 20 14 3.4 30 13 11 10 11 15 12 1.9 60 10 9 8 10 14 10 2.5 95 9 8 7 7 9 8 1.0 120 9 7 6 6 9 7 1.4 240 7 6 5 6 6 6 0.5 480 8 6 5 6 6 6 1.1 720 7 5 6 5 6 6 0.6 1440 2 1 3 1 4 2 1.3 1920 1 1 1 0 2 1 0.6

Time post Concentration of S-9 after p.o. administration ( µg/ml) dose (min) Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Average stdev 15 0 1 1 0 1 1 0.1 35 2 2 1 1 2 1 0.6 60 2 3 1 1 2 2 0.8 120 6 4 1 2 5 4 2.2 240 7 4 1 3 6 4 2.3 360 6 4 2 5 6 5 1.5 540 8 5 4 7 - 6 1.7 720 6 8 6 6 4 6 1.4 1440 2 3 3 3 2 3 0.6 1800 1 1 2 2 1 2 0.5 2160 1 1 - 1 1 1 0.2

Table B.19. Plasma concentration of S-9 after i.v. or p.o. administration.

201

Time post Concentration of S-10 after i.v. administration ( µg/ml) dose (min) Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Average stdev 5 48 40 66 62 29 49 15.2 30 22 22 26 28 18 23 3.8 60 17 16 18 19 13 16 2.4 120 17 11 18 15 11 14 3.2 240 14 13 15 15 18 15 1.8 360 15 10 12 15 15 13 2.5 540 12 - 9 - 17 13 3.9 720 13 9 9 14 15 12 2.9 1440 8 6 5 5 7 6 1.4 2010 5 5 - 2 3 4 1.5 2880 2 2 2 - 1 2 0.6

Time post Concentration of S-10 after p.o. administration ( µg/ml) dose (min) Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Average stdev 30 0 3 2 0 0 1 1.2 60 1 4 4 1 - 2 1.9 120 2 6 6 1 3 4 2.3 240 5 6 7 3 6 5 1.4 360 6 9 14 6 9 9 3.4 540 8 9 13 10 12 11 2.2 720 8 9 14 14 11 11 2.8 1440 4 5 7 4 4 5 1.4 2000 4 2 1 - - 3 1.7 2880 2 2 0.65 0.43 - 1 0.9

Table B.20. Plasma concentration of S-10 after i.v. or p.o. administration.

202

Concentration of S-11 after i.v. administration ( µg/ml) Time Time Time Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 (min) (min) (min) 10 77 64 71 10 63 10 61 30 54 48 44 33 37 34 28 60 46 35 37 60 29 60 25 180 30 22 18 190 22 190 18 360 35 31 33 360 20 360 18 540 27 28 20 540 28 540 20 720 24 30 23 720 28 720 27 1450 17 12 15 1440 12 1440 15 1990 8 16.66 11 2160 10 2160 10 4560 1 1 - 4560 1 3120 5 3600 3 4560 1

Time post Concentration of S-11 after p.o. administration ( µg/ml) dose (min) Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Average stdev 30 2 0 1 1 0 1 0.6 60 2 1 2 1 1 2 0.5 120 3 3 7 3 4 4 1.5 300 12 7 12 8 7 9 2.2 480 21 11 20 17 9 16 5.2 760 26 19 20 26 - 23 3.8 1490 23 22 13 16 16 18 4.1 1890 20 18 10 13 9 14 4.9 2160 17 17 7 10 10 12 4.7 2960 8 12 3 6 5 7 3.3 3590 6 7 1.9 4 - 5 2.5 4890 2 3 - 2 - 2 0.7

Table B.21. Plasma concentration of S-11 after i.v. or p.o. administration.

203

Time post Concentration of S-19 ( µg/ml) dose (min) Rat 1 Rat 2 Rat 3 Rat 4 Average stdev 5 18.6 23.6 17.4 15.3 18.7 3.5 15 7.9 8.4 7.6 7.8 7.9 0.3 40 6.8 6.3 6.3 6.5 6.5 0.3 60 5.5 6.1 5.6 5.7 5.7 0.2 120 4.1 4.9 4.4 4.6 4.5 0.3 240 2.9 3.4 3.2 3.2 3.2 0.2 360 2.1 2.1 2.4 2.3 2.2 0.1 540 1.4 1.1 1.5 1.2 1.3 0.2 720 0.9 0.6 0.9 0.8 0.8 0.2

Time post Concentration of S-20 ( µg/ml) dose (min) Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Average stdev 5 35.2 26.7 17.8 20.3 33.1 26.6 7.7 15 14.8 16.1 14.5 13.9 15.7 15.0 0.9 30 12.6 14.1 12.6 12.2 13.2 12.9 0.7 60 10.8 11.8 11.2 9.5 11.5 11.0 0.9 120 8.5 9.7 9.3 7.2 8.6 8.7 1.0 240 6.1 5.8 6.7 4.0 6.0 5.7 1.0 360 4.6 3.7 5.1 2.3 4.5 4.0 1.1 480 3.0 2.9 4.2 1.4 3.2 2.9 1.0 720 1.4 1.2 1.8 - 1.7 1.5 0.3

Table B.22. Plasma concentration of S-19 and S-20 after i.v. administration.

204

Time post Concentration of S-21 ( µg/ml) dose (min) Rat 1 Rat 2 Rat 3 Rat 4 Average stdev 5 24.7 20.5 18.0 18.6 20.4 3.0 15 11.1 10.5 10.5 12.1 11.0 0.7 30 10.5 8.8 9.7 10.1 9.8 0.7 60 7.8 7.4 8.2 7.8 7.8 0.3 120 5.8 5.9 5.9 5.9 5.9 0.0 240 3.2 4.0 3.2 3.4 3.5 0.4 360 1.8 2.5 2.1 2.0 2.1 0.3 540 0.6 1.3 0.9 1.0 1.0 0.3 720 0.2 0.7 0.4 0.4 0.5 0.2

Time post Concentration of S-22 ( µg/ml) dose (min) Rat 1 Rat 2 Rat 3 Rat 4 Average stdev 5 26.4 58.9 15.8 40.2 35.3 18.6 15 19.2 13.9 13.2 17.2 15.9 2.8 30 16.1 12.6 11.4 12.0 13.1 2.1 60 14.5 11.6 10.7 9.9 11.7 2.0 180 11.5 8.4 7.7 7.0 8.6 2.0 360 8.5 5.8 5.7 5.0 6.2 1.6 540 6.1 4.2 3.6 3.2 4.3 1.3 720 4.4 2.8 2.3 1.0 2.6 1.4 1440 1.4 1.3 0.5 0.5 0.9 0.5 1740 0.8 0.6 - 0.2 0.5 0.3 2040 0.4 0.3 - - 0.3 0.1

Table B.23. Plasma concentration of S-21 and S-22 after i.v. administration.

205

Normalized organ weight in rats treated with cyano/nitro group substituted SARM Intact Castrated S-19 S-20 S-21 S-22 Control Control Rat 1 101 3 24 35 27 28 Rat 2 91 7 23 34 34 42 Prostate Rat 3 89 8 27 38 32 32 Rat 4 124 4 31 42 39 45 Rat 5 96 8 21 22 24 49 Rat 1 68 11 15 36 19 75 Rat 2 96 7 27 51 20 74 Seminal Rat 3 92 8 19 49 25 78 vesicle Rat 4 141 7 19 47 25 73 Rat 5 104 7 15 54 19 95 Rat 1 101 45 102 124 94 126 Rat 2 92 36 124 130 135 150 Levator Rat 3 97 54 87 128 127 145 ani muscle Rat 4 113 41 110 134 113 164 Rat 5 97 28 102 136 123 125

Table B.24. Normalized tissue weights of anabolic and adrogenic organs in male rats administered with cyano/nitro substituted SARMs.

206

Normalized organ weight in S-22 treatment group Dose rate (mg/day) Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Prostate 0.005 4.9 4.9 4.1 3.6 3.9 0.01 4.6 3.5 5.5 6.2 8.4 0.05 14.4 23.2 16.5 19.2 14.7 0.1 15.7 31.1 25.2 22.2 21.2 0.3 37.9 47.1 29.0 36.8 44.5 0.5 43.1 54.1 39.0 42.6 40.0 0.75 43.0 53.4 45.2 59.7 41.3 1 28.5 41.7 32.1 44.9 49.1 Seminal vesicle 0.005 8.7 7.5 8.2 7.5 7.0 0.01 5.3 8.7 6.7 6.4 7.0 0.05 14.1 21.2 12.7 14.0 16.2 0.1 26.3 51.0 54.9 52.4 26.3 0.3 27.3 67.5 69.3 61.1 27.3 0.5 31.4 54.8 44.7 71.2 31.4 0.75 26.6 48.3 51.0 65.5 26.6 1 74.7 73.8 77.5 73.3 94.8 Levator ani muscle 0.005 51.0 49.7 54.3 52.4 45.8 0.01 62.9 66.0 50.1 61.3 74.2 0.05 90.9 112.6 94.6 104.1 98.5 0.1 91.2 121.5 118.8 114.6 114.6 0.3 122.1 131.1 116.4 116.5 125.7 0.5 134.2 121.9 111.4 118.4 124.7 0.75 124.2 126.1 139.5 126.6 142.4 1 126.1 150.4 144.8 163.5 124.7

Table B.25. Normalized tissue weights of anabolic and adrogenic organs in S-22 administered male rats.

207

APPENDIX C

DATA RELATED TO CHAPTER 4

208

Plasma protein binding study

Compounds CB ( µM) CU ( µM) FB (%) CB ( µM) CU ( µM) FB (%) 50.1 0.36 99.33 4.21 0.037 99.17 S-1 49.6 0.37 99.30 4.13 0.027 99.39 49.5 0.52 99.02 4.18 0.029 99.36 43.9 2.62 94.39 4.28 N/D - S-4 46.6 2.68 94.58 4.49 0.22 95.40 45.3 3.74 92.22 4.62 0.24 95.20 46.0 0.071 99.85 4.55 0.003 99.93 S-9 45.6 0.037 99.92 4.49 0.010 99.80 44.0 0.033 99.93 4.73 0.003 99.94 44.6 0.018 99.96 4.02 0.0011 99.97 S-10 44.3 0.011 99.98 4.00 0.0013 99.97 43.9 0.020 99.96 3.95 0.0018 99.96 48.7 - - 4.33 N/D - S-11 48.1 0.0041 99.992 4.38 N/D - 47.5 0.0045 99.991 4.35 N/D - 46.4 0.19 99.61 4.19 0.014 99.69 S-19 46.7 0.29 99.42 4.13 0.018 99.59 45.9 0.15 99.70 4.17 0.015 99.67 66.0 0.48 99.31 3.77 0.011 99.73 S-20 67.0 0.50 99.30 3.77 0.013 99.67 66.6 0.50 99.30 3.78 0.017 99.57 47.3 0.26 99.48 4.90 0.017 99.67 S-21 48.6 0.27 99.47 4.91 0.013 99.76 48.4 0.25 99.51 4.89 0.012 99.77 46.2 0.41 99.16 4.43 0.040 99.15 S-22 46.0 0.54 98.89 4.42 0.040 99.14 45.0 0.45 99.06 4.32 0.039 99.15 44.2 2.89 93.83 - - - Bicalutamide 43.5 2.82 93.91 - - - 44.7 3.04 93.60 - - -

Table C.1. Concentration of drug bound (C B) and unbound (C U) in plasma protein binding study. F B, Fraction bound; N/D, Not determined.

209

Plasma protein binding study Drug / Concentration Drug Bound ( µM) Drug Unbound ( µM) Fraction Bound (%) 0.45 N/D - S-22 / 0.5 µM 0.45 0.0050 98.97 0.45 0.0046 99.02 1.73 0.016 99.15 S-22 / 2 µM 1.73 0.025 98.63 1.71 0.015 99.15 95.6 1.06 98.96 S-22 / 100 µM 95.8 1.04 98.98 95.7 1.17 98.85 190.5 2.46 98.79 S-22 / 200 µM 189.8 2.64 98.70 191.0 2.51 98.77

Table C.2. Concentration of drug bound (C B) and unbound (C U) of S-22 in plasma protein binding study.

210

APPENDIX D

DATA RELATED TO CHAPTER 5

211

Body, soleus, and uterus weight Group Animal Soleus weight Uterus weight Body weight (g) number number (mg) (mg) 1 238 52.1 883 2 282 69.8 643.2 3 258 55.3 515.5 1 4 270 66.7 376 5 259 - - 6 270 51.9 595.5 1 244 55 110.5 2 276 59 172.3 3 293 54.2 178.2 4 321 69.4 332.2 2 5 264 57 98.6 6 256 63.4 133.8 7 295 67.7 119.5 8 268 70.3 141.6 1 283 74.3 778 2 312 48.7 695.5 3 288 69.2 664.2 4 297 62.7 561.7 3 5 284 59.8 545.1 6 295 74.7 636 7 291 77 785 8 329 64.1 398.9 1 278 1010 61.4 2 308 770.2 53.5 3 300 670.5 65.9 4 315 882.1 49.9 4 5 325 680 70.3 6 264 680 7 304 906 53 8 302 1223.6 59

Continued

Table D.1. Body, soleus, and uterus weight of OVX rats administered with various compounds.

212

Table D.1. continued

Group Animal Soleus weight Uterus weight Body weight (g) number number (mg) (mg) 1 256 111.8 718.5 2 276 119.3 580.7 3 281 107.6 427 5 4 263 119.5 1222 5 277 105 760 6 305 125 426 7 275 117.3 1305 1 323 121.2 152.6 2 312 119 108.3 3 337 139 122.8 4 336 118.1 124.4 6 5 320 118.3 128 6 323 113 124 7 337 130.6 104.1 8 354 127.2 150 1 370 125.9 644.1 2 413 168.1 759.4 3 328 123.1 524 4 359 136.7 599 7 5 358 145.3 564.8 6 342 124 549 7 377 131.8 624.1 8 338 142.3 647.6 9 355 - 615.1 1 340 139 667.6 2 350 117 560.3 3 329 124.6 612.5 4 309 116.5 667.6 8 5 306 99.7 528.3 6 345 142.4 645.7 7 330 139.5 553.2 8 352 120.2 651.5 9 344 122.8 538

Continued

213

Table D.1. continued

Group Animal Soleus weight Uterus weight Body weight (g) number number (mg) (mg) 1 300 107 970 2 297 120 850 3 302 125 550 4 295 117 750 9 5 299 131 560 6 304 105 720 7 312 112 750 8 299 114 660 9 311 109 1230 1 316 130 790 2 297 112 880 3 279 103 770 4 282 103 910 10 5 283 98 740 6 287 114 690 7 292 101 660 8 291 114 960 9 318 117 840 1 272 112 310 2 298 119 270 3 278 96 230 4 269 98 270 11 5 295 127 270 6 262 99 260 7 305 97 210 8 273 116 260 9 304 125 440 1 311 140 1160 2 297 135 930 3 286 104 1070 4 260 107 930 12 5 249 101 850 6 288 105 960 7 269 98 800 8 290 108 8750 9 302 110 1090

214

DEXA parameters Group Animal Tissue mass BMD (g/cm 2) BMC (g) Fat (%) (Week) number (g) 1 0.170 8.59 23.6 244 2 0.165 9.03 25.9 266 Group 1 3 0.163 8.20 29.4 256 (Week 0) 4 0.158 8.43 32.0 262 5 0.160 8.60 38.0 255 6 0.165 8.73 30.9 260 1 0.165 8.86 28.9 251 3 0.166 9.02 26.2 253 Group 1 4 0.164 8.60 27.4 258 (Week 2) 5 0.153 8.23 28.5 272 6 0.158 8.90 27.2 246 1 0.168 8.97 26.2 232 2 0.162 9.37 21.4 271 Group 1 3 0.159 8.31 25.1 246 (Week 6) 4 0.169 9.19 23.6 263 5 0.149 8.29 22.7 251 6 0.162 8.82 21.9 260 1 0.156 8.01 33.4 247 2 0.169 8.54 29.5 245 3 0.164 8.81 30.8 261 Group 2 4 0.168 8.71 31.8 251 (Week 0) 5 0.161 8.63 31.6 257 6 0.169 8.77 29.9 258 7 0.161 8.26 27.7 257 8 0.168 8.66 29.1 243 1 0.152 8.42 36.4 274 2 0.154 8.51 29.9 267 3 0.153 8.73 30.0 294 Group 2 4 0.161 8.89 30.3 283 (Week 2) 5 0.157 8.68 33.0 272 6 0.160 8.82 28.5 284 7 0.158 8.68 29.7 283 8 0.158 9.12 27.8 273

Continued

Table D.2. DEXA parameters of OVX rats administered with various compounds.

215

Table D.2. continued

Group Animal Tissue mass BMD (g/cm 2) BMC (g) Fat (%) (Week) number (g) 1 0.150 7.89 30.3 234 2 0.152 8.30 23.8 256 3 0.156 9.25 22.8 280 Group 2 4 0.163 9.45 29.2 312 (Week 6) 5 0.158 8.84 23.8 257 6 0.156 8.58 23.5 245 7 0.152 8.51 25.6 289 8 0.159 9.06 25.4 257 1 0.168 8.96 27.3 253 2 0.158 8.58 34.4 258 3 0.168 8.81 28.4 268 Group 3 4 0.173 9.11 28.9 268 (Week 0) 5 0.165 8.47 34.4 242 6 0.166 8.48 27.1 247 7 0.166 8.91 31.5 269 8 0.167 8.98 25.4 260 1 0.159 8.63 26.9 275 2 0.154 8.43 27.8 284 3 0.161 8.66 26.7 285 Group 3 4 0.165 8.98 31.2 286 (Week 2) 5 0.159 8.38 32.1 269 6 0.152 8.47 28.7 282 7 0.156 8.89 31.1 297 8 0.163 8.72 26.0 278 1 0.155 8.93 24.5 274 2 0.152 8.78 25.2 297 3 0.160 8.88 23.7 276 Group 3 4 0.158 8.94 28.0 285 (Week 6) 5 0.152 8.56 28.9 273 6 0.150 8.34 27.3 287 7 0.151 8.69 22.1 282 8 0.158 9.59 27.0 319

Continued

216

Table D.2. continued

Group Animal Tissue mass BMD (g/cm 2) BMC (g) Fat (%) (Week) number (g) 1 0.152 8.33 32.7 235 2 0.168 9.05 28.2 257 3 0.174 9.08 30.4 263 Group 4 4 0.172 9.42 35.5 277 (Week 0) 5 0.162 8.26 28.5 251 6 0.166 8.73 29.4 257 7 0.168 8.38 24.4 255 8 0.171 8.68 25.0 261 1 0.150 8.15 28.6 260 2 0.159 9.20 27.0 284 3 0.163 9.15 27.4 302 Group 4 4 0.164 9.39 31.0 297 (Week 2) 5 0.150 8.40 28.4 283 6 0.157 8.91 29.7 295 7 0.157 8.54 26.5 285 8 0.164 8.79 23.9 288 1 0.154 8.55 30.7 262 2 0.162 9.56 24.0 296 3 0.159 9.37 25.6 289 Group 4 4 0.156 9.30 27.8 313 (Week 6) 5 0.146 8.34 24.0 310 6 0.158 8.50 22.4 254 7 0.157 8.63 20.2 294 8 0.159 9.02 22.3 291 1 0.157 8.83 33.6 250 2 0.162 8.73 32.1 237 3 0.159 8.99 29.7 261 Group 5 4 0.167 9.26 32.1 261 (Week 0) 5 0.169 8.79 30.1 264 6 0.166 8.68 24.4 261 7 0.164 8.58 28.8 259

Continued

217

Table D.2 continued

Group Animal Tissue mass BMD (g/cm 2) BMC (g) Fat (%) (Week) number (g) 1 0.162 8.25 29.9 242 2 0.172 9.89 34 262 3 0.163 8.76 29 264 Group 5 4 0.166 8.92 28.9 244 (Week 6) 5 0.165 9.20 32.3 256 6 0.174 9.57 31.1 297 7 0.160 8.90 27.7 260 1 0.166 9.01 31.4 244 2 0.163 8.26 32.7 249 3 0.153 8.05 30.1 264 Group 6 4 0.166 8.77 30.7 255 (Week 0) 5 0.160 8.13 30.6 254 6 0.167 8.46 32.8 248 7 0.156 8.26 31.9 251 8 0.152 8.16 30.6 264 1 0.159 9.35 34.6 310 2 0.159 9.09 33.7 301 3 0.153 9.23 35.2 320 Group 6 4 0.162 9.60 32.2 312 (Week 6) 5 0.159 9.45 32.5 299 6 0.165 9.63 28.4 313 7 0.157 9.36 34.2 317 8 0.149 9.04 35.4 342 1 0.167 9.32 32.0 259 2 0.155 8.82 31.9 296 3 0.159 7.92 27.6 237 4 0.156 8.68 29.5 272 Group 7 5 0.163 8.35 26.3 250 (Week 0) 6 0.166 8.76 32.0 250 7 0.158 8.53 28.7 266 8 0.165 8.72 31.0 252 9 0.151 7.72 33.4 243

Continued

218

Table D.2 continued

Group Animal Tissue mass BMD (g/cm 2) BMC (g) Fat (%) (Week) number (g) 1 0.165 10.56 31.3 352 2 0.161 10.85 30.3 399 3 0.154 9.24 31.5 320 4 0.161 9.58 33.8 337 Group 7 5 0.157 9.41 31.4 337 (Week 6) 6 0.163 9.60 36.0 331 7 0.160 10.43 33.6 357 8 0.165 9.77 34.5 322 9 0.150 9.28 32.2 333 1 0.165 9.22 30.1 269 2 0.162 8.86 32.6 256 3 0.156 8.13 30.8 249 4 0.158 8.40 31.6 253 Group 8 5 0.166 8.05 28.5 240 (Week 0) 6 0.160 8.45 27.6 251 7 0.163 8.83 25.5 273 8 0.165 8.75 29.0 253 9 0.164 9.02 32.5 275 1 0.170 10.19 33.5 327 2 0.158 9.67 35.7 340 3 0.156 9.58 38.1 318 4 0.157 9.04 33.4 302 Group 8 5 0.162 9.18 31.9 292 (Week 6) 6 0.162 9.82 30.5 334 7 0.164 10.25 32.8 320 8 0.158 10.12 33.3 337 9 0.160 9.38 34.5 326

Continued

219

Table D.2 continued

Group Animal Tissue mass BMD (g/cm 2) BMC (g) Fat (%) (Week) number (g) 1 0.164 8.60 31.0 244 2 0.163 8.90 32.8 262 3 0.164 8.81 31.8 263 4 0.180 9.42 28.1 251 Group 9 5 0.158 8.58 32.5 259 (Week 0) 6 0.163 8.82 30.1 254 7 0.163 9.08 30.8 264 8 0.162 8.46 30.5 254 9 0.158 8.36 31.5 253 1 0.166 8.96 27.4 289 2 0.166 9.04 29.5 288 3 0.161 8.51 30.8 287 4 0.173 9.51 26.1 286 Group 9 5 0.165 8.86 28.4 286 (Week 6) 6 0.164 9.11 28.3 287 7 0.174 9.80 26.3 293 8 0.167 9.50 28.2 288 9 0.156 9.17 30.2 295 1 0.157 8.90 30.3 262 2 0.161 8.66 32.7 259 3 0.162 8.10 29.8 253 4 0.160 8.11 31.3 246 Group 10 5 0.157 8.37 34.0 253 (Week 0) 6 0.164 8.74 35.7 246 7 0.172 8.66 32.5 266 8 0.158 8.19 26.9 253 9 0.161 8.43 32.7 261

Continued

220

Table D.2 continued

Group Animal Tissue mass BMD (g/cm 2) BMC (g) Fat (%) (Week) number (g) 1 0.162 9.15 28.6 299 2 0.165 9.16 25.4 288 3 0.166 8.67 27.2 267 4 0.160 8.44 26.1 266 Group 10 5 0.164 8.66 27.8 283 (Week 6) 6 0.157 9.19 33.9 275 7 0.168 9.09 29.0 285 8 0.165 9.12 26.1 288 9 0.159 9.10 26.9 301 1 0.159 8.54 28.7 253 2 0.157 8.39 26.0 263 3 0.172 9.09 28.9 259 4 0.161 8.45 32.9 246 Group 11 5 0.168 8.33 28.4 249 (Week 0) 6 0.160 8.27 31.2 259 7 0.159 8.16 31.8 254 8 0.165 8.48 35.7 248 9 0.169 9.39 33.2 278 1 0.155 8.43 28.2 267 2 0.158 8.80 28.3 287 3 0.166 9.22 29.2 268 4 0.156 8.36 30.6 260 Group 11 5 0.163 9.23 26.2 280 (Week 6) 6 0.172 9.06 27.7 244 7 0.163 8.68 31.1 274 8 0.167 8.89 27.9 266 9 0.177 9.82 26.3 280

Continued

221

Table D.2 continued

Group Animal Tissue mass BMD (g/cm 2) BMC (g) Fat (%) (Week) number (g) 1 0.167 9.39 29.4 253 2 0.162 8.45 32.2 252 3 0.161 8.49 31.6 274 4 0.164 8.63 31.3 246 Group 12 5 0.158 7.99 33.2 242 (Week 0) 6 0.163 8.21 30.6 251 7 0.164 8.26 29.4 260 8 0.157 8.16 28.9 260 9 0.174 8.97 31.5 271 1 0.167 9.75 29.8 295 2 0.169 8.93 25.2 289 3 0.163 8.81 25.7 280 4 0.161 8.35 23.5 250 Group 12 5 0.168 8.68 24.4 253 (Week 6) 6 0.162 8.95 27.5 274 7 0.163 8.53 25.1 275 8 0.159 8.60 27.3 282 9 0.177 9.50 24.7 286

222

Muscle strength measurement Animal CSA Twitch Tetanus Group 2 number (mm ) Pt (N) TPt (ms) T1/2R (ms) Po (N) T1/2R (ms) 1 2.17 0.09 0.18 0.65 0.42 0.27 2 3.03 0.17 0.17 0.32 0.48 0.25 Group 3 2.51 0.25 0.15 0.28 0.70 0.31 1 4 3.03 0.09 0.15 0.23 0.66 0.23 5 2.36 0.10 0.11 0.22 0.72 0.25 1 2.04 0.09 0.19 0.57 0.49 0.24 2 2.46 0.11 0.09 0.20 0.56 0.21 3 3.15 0.11 0.11 0.23 0.60 0.16 Group 4 2.59 0.08 0.10 0.13 0.26 0.30 2 5 2.76 0.08 0.17 0.35 0.68 0.23 6 3.19 0.13 0.15 0.35 0.66 0.22 7 2.67 0.15 0.25 0.57 0.61 0.36 1 2.21 0.09 0.19 0.70 0.42 0.28 2 2.85 0.08 0.17 0.60 0.46 0.22 Group 3 2.72 0.12 0.15 0.46 0.63 0.25 3 4 3.40 0.11 0.14 0.23 0.71 0.25 5 3.50 0.07 0.12 0.28 0.61 0.16 6 2.91 0.08 0.09 0.30 0.69 0.18 1 - - 0.17 0.57 - - 2 2.43 0.07 0.16 0.45 0.61 0.30 3 3.00 0.11 0.11 0.47 0.07 - Group 4 2.27 0.10 0.10 0.21 0.55 0.19 4 5 - - 0.22 0.65 - - 6 2.41 0.07 0.07 0.24 0.44 0.15 7 2.68 0.08 0.09 0.22 0.52 0.17

Continued

Table D.3. Parameters for muscle strength measurement

223

Table D.3. continued

Animal CSA Twitch Tetanus Group 2 number (mm ) Pt (N) TPt (ms) T1/2R (ms) Po (N) T1/2R (ms) 1 4.66 0.20 0.44 0.89 1.48 0.57 2 4.48 0.25 0.20 0.47 1.71 0.42 3 4.98 0.27 0.15 0.30 1.53 0.29 Group 5 4 3.89 0.19 - - 1.38 - 5 5.21 0.22 0.28 0.50 1.62 0.43 6 4.51 0.24 0.18 0.35 1.40 0.37 1 4.66 0.18 0.35 0.49 1.31 0.43 2 5.06 0.28 0.22 0.46 1.57 0.43 3 5.79 0.28 0.18 0.34 1.94 0.38 4 4.72 0.23 0.16 0.35 1.53 0.34 Group 6 5 4.55 0.23 0.19 0.45 1.41 0.40 6 4.19 0.22 0.22 0.44 1.63 0.34 7 4.84 0.27 0.16 0.31 1.64 0.31 8 6.06 0.27 0.39 0.76 1.42 0.64 1 5.72 0.33 0.30 0.54 1.82 0.57 2 7.00 0.30 0.26 0.28 1.82 0.37 3 5.86 0.27 0.25 0.32 1.44 0.39 4 5.06 0.23 0.27 0.33 1.74 0.48 Group 7 5 5.81 0.19 0.25 0.60 1.81 0.46 6 5.17 0.29 0.18 0.27 1.57 0.33 7 5.07 0.23 0.22 0.34 1.77 0.28 8 5.47 0.27 0.22 0.42 1.79 0.45 1 4.96 0.28 0.20 0.42 1.93 0.33 2 4.33 0.28 0.18 0.39 1.79 0.39 3 4.45 0.23 0.22 0.33 1.55 0.35 4 4.48 0.24 0.18 0.40 1.65 0.45 Group 8 5 3.56 0.24 0.22 0.43 1.49 0.41 6 5.48 0.33 0.13 0.24 1.88 0.42 7 4.98 0.25 0.18 0.30 2.01 0.31 8 4.72 0.28 0.17 0.32 1.70 0.39

224

Micro CT parameters Parameter Unit Group 1 Group 1 BMD g/cm 3 - 0.503 TV Tissue volume mm 3 24.44 27.21 BV Bone volume mm 3 9.812 11.81 BV/TV Percent bone volume 40.16 43.41 TS Tissue surface mm 2 60.67 64.86 BS Bone surface mm 2 421.4 502 BS/BV Bone surface/volume ratio mm -1 42.95 42.5 T.Ar Mean total crossectional tissue area mm 2 9.67 10.77 T.Pm Mean total crossectional tissue perimeter mm 12.67 13.58 B.Ar Mean total crossectional bone area mm 2 3.883 4.674 B.Pm Mean total crossectional bone perimeter mm 111.5 133.1 Obj.N Mean number of objects per slice 144.4 176.5 Av.Obj.Ar Average object area per slice mm 2 0.027 0.026 2D Av.Obj.Le Average object size per slice mm 0.185 0.184 MMI(polar) Mean polar moment of inertia mm 2 7.651 9.581 Ecc Mean eccentricity 0.712 0.736 Cs.Th Crossectional thickness mm 0.07 0.07 Tb.Th(pl) Trabecular thickness (plate model) mm 0.047 0.047 Tb.Sp(pl) Trabecular separation (plate model) mm 0.069 0.061 Tb.N(pl) Trabecular number (plate model) mm -1 8.623 9.225 Tb.Dm(rd) Trabecular diameter (rod model) mm 0.093 0.094 Tb.Sp(rd) Trabecular separation (rod model) mm 0.037 0.032 Tb.N(rd) Trabecular number (rod model) mm -1 7.677 7.9 Tb.Pf Mean trabecular pattern factor mm -1 -7.89 -9.58 Po Percent porosity 16.15 20.44 TV Tissue volume mm 3 24.42 27.19 BV Bone volume mm 3 9.65 11.62 BV/TV Percent bone volume % 39.51 42.73 TS Tissue surface mm 2 54.98 59.02 BS Bone surface mm 2 350.9 418 i.S Intersection surface mm 2 23.74 25.18 BS/BV Bone surface/volume ratio mm -1 36.36 35.98 3D BS/TV Bone surface density mm -1 14.37 15.37 Tb.Pf Trabecular pattern factor mm -1 -7.2 -8.26 SMI 0.602 0.506 Tb.Th mm 0.101 0.102 Tb.N mm -1 3.929 4.195 Tb.Sp mm 0.178 0.151 DA 1.595 1.64

Table D.4. Micro CT parameters of group 1.

225

Micro CT parameters Parameter Unit Group 1 Group 1 Group 1 Group 2 Group 2 BMD g/cm 3 0.476 0.38 0.315 0.28 0.225 TV mm 3 22.16 23.44 19.81 26.1 25.29 BV mm 3 8.917 7.142 4.691 5.4 4.034 BV/TV 40.24 30.47 23.68 20.7 15.95 TS mm 2 58.4 58.52 57.17 65.3 63.28 BS mm 2 373.4 370.3 265 301 251.6 BS/BV mm -1 41.87 51.85 56.49 55.8 62.37 T.Ar mm 2 8.769 9.274 7.839 10.3 10.01 T.Pm mm 12.14 12.6 11.8 13.3 13.04 B.Ar mm 2 3.529 2.826 1.856 2.14 1.596 B.Pm mm 98.43 97.4 68.91 80.5 66.83 Obj.N 138.2 150.8 127.1 158 132.4 Av.Obj.Ar mm 2 0.026 0.019 0.015 0.01 0.012 2D Av.Obj.Le mm 0.18 0.154 0.136 0.13 0.124 MMI(polar) mm 2 6.335 5.263 3.256 4.58 3.476 Ecc 0.796 0.786 0.744 0.81 0.809 Cs.Th mm 0.072 0.058 0.054 0.05 0.048 Tb.Th(pl) mm 0.048 0.039 0.035 0.04 0.032 Tb.Sp(pl) mm 0.071 0.088 0.114 0.14 0.169 Tb.N(pl) mm -1 8.425 7.9 6.69 5.78 4.974 Tb.Dm(rd) mm 0.096 0.077 0.071 0.07 0.064 Tb.Sp(rd) mm 0.038 0.047 0.058 0.07 0.078 Tb.N(rd) mm -1 7.493 8.074 7.756 7.16 7.027 Tb.Pf mm -1 -6.18 -3.31 0.687 2.69 4.718 Po 14.63 9.99 3.024 4.49 0.651 TV mm 3 22.14 23.42 19.79 26 25.27 BV mm 3 8.77 6.991 4.578 5.27 3.922 BV/TV % 39.61 29.85 23.13 20.2 15.52 TS mm 2 52.83 53.09 51.09 58.4 57.25 BS mm 2 310.3 306.6 219.1 250 208.5 i.S mm 2 20.77 15.52 12.89 11.8 9.257 BS/BV mm -1 35.38 43.85 47.85 47.4 53.16 3D BS/TV mm -1 14.01 13.09 11.07 9.6 8.25 Tb.Pf mm -1 -4.82 -0.67 4.287 7.69 9.222 SMI 0.821 1.092 1.47 1.76 1.681 Tb.Th mm 0.106 0.085 0.082 0.09 0.075 Tb.N mm -1 3.743 3.492 2.826 2.36 2.071 Tb.Sp mm 0.168 0.19 0.227 0.24 0.282 DA 1.624 1.535 1.686 1.89 1.622

Table D.5. Micro CT parameters of group 1 and 2.

226

Micro CT parameters Parameter Unit Group 2 Group 2 Group 2 Group 2 Group 2 BMD g/cm 3 0.324 0.219 0.229 0.237 0.257 TV mm 3 25.84 26.92 23.82 26.7 23.03 BV mm 3 6.387 4.134 3.749 4.526 4.287 BV/TV 24.72 15.36 15.74 16.95 18.61 TS mm 2 62.9 65.32 61.19 66.49 61.23 BS mm 2 305.8 248 210.4 262.7 223.4 BS/BV mm -1 47.88 59.98 56.1 58.05 52.11 T.Ar mm 2 10.22 10.65 9.425 10.57 9.115 T.Pm mm 13.17 13.52 12.73 13.57 12.73 B.Ar mm 2 2.528 1.636 1.484 1.791 1.696 B.Pm mm 79.8 64.65 55.77 69.33 58.78 Obj.N 126.5 136.4 115.6 138.7 100.9 Av.Obj.Ar mm 2 0.02 0.012 0.013 0.013 0.017 2D Av.Obj.Le mm 0.16 0.124 0.128 0.128 0.146 MMI(polar) mm 2 5.273 3.549 2.938 3.627 3.33 Ecc 0.736 0.741 0.813 0.726 0.813 Cs.Th mm 0.063 0.051 0.053 0.052 0.058 Tb.Th(pl) mm 0.042 0.033 0.036 0.034 0.038 Tb.Sp(pl) mm 0.127 0.184 0.191 0.169 0.168 Tb.N(pl) mm -1 5.918 4.606 4.416 4.92 4.85 Tb.Dm(rd) mm 0.084 0.067 0.071 0.069 0.077 Tb.Sp(rd) mm 0.065 0.084 0.088 0.079 0.081 Tb.N(rd) mm -1 6.715 6.631 6.279 6.742 6.342 Tb.Pf mm -1 0.379 5.445 4.816 3.077 3.864 Po 5.535 1.154 0.719 3.644 2.507 TV mm 3 25.82 26.9 23.8 26.69 23.02 BV mm 3 6.261 4.024 3.655 4.41 4.192 BV/TV % 24.25 14.96 15.36 16.53 18.21 TS mm 2 57.37 58.95 55.27 59.26 56.31 BS mm 2 252.7 204.6 174.3 217.5 185.1 i.S mm 2 14.94 8.397 8.25 8.873 10.14 BS/BV mm -1 40.36 50.85 47.68 49.31 44.15 3D BS/TV mm -1 9.788 7.606 7.323 8.149 8.04 Tb.Pf mm -1 4.301 10.02 10.17 7.857 7.959 SMI 1.539 1.853 1.942 1.733 1.73 Tb.Th mm 0.096 0.081 0.085 0.082 0.089 Tb.N mm -1 2.519 1.856 1.806 2.009 2.05 Tb.Sp mm 0.245 0.294 0.298 0.288 0.298 DA 1.556 1.544 1.738 1.627 1.594

Table D.6. Micro CT parameters of group 2.

227

Micro CT parameters Parameter Unit Group 2 Group 2 Group 3 Group 3 Group 3 BMD g/cm 3 0.185 0.303 0.30 0.22 0.35 TV mm 3 23.36 25.87 31.2 27.1 27.0 BV mm 3 2.985 5.899 6.93 4.28 7.60 BV/TV 12.78 22.8 22.2 15.8 28.1 TS mm 2 61.73 66.99 75.0 66.8 66.7 BS mm 2 172.9 294.1 367 253 369 BS/BV mm -1 57.91 49.86 53 59 49 T.Ar mm 2 9.245 10.24 12.3 10.7 10.7 T.Pm mm 12.84 13.53 14.8 13.3 13.8 B.Ar mm 2 1.181 2.335 2.7 1.7 3.0 B.Pm mm 46.73 77.82 97 67 98 Obj.N 98.16 136.6 166 146 155 Av.Obj.Ar mm 2 0.012 0.017 0.017 0.012 0.019 2D Av.Obj.Le mm 0.124 0.148 0.145 0.121 0.157 MMI(polar) mm 2 2.431 4.962 6.69 3.73 6.79 Ecc 0.84 0.838 0.81 0.79 0.82 Cs.Th mm 0.051 0.06 0.056 0.051 0.061 Tb.Th(pl) mm 0.035 0.04 0.038 0.034 0.041 Tb.Sp(pl) mm 0.236 0.136 0.13 0.18 0.11 Tb.N(pl) mm -1 3.7 5.685 5.88 4.67 6.83 Tb.Dm(rd) mm 0.069 0.08 0.076 0.068 0.082 Tb.Sp(rd) mm 0.102 0.069 0.066 0.083 0.055 Tb.N(rd) mm -1 5.839 6.717 7.04 6.63 7.26 Tb.Pf mm -1 6.359 0.998 -0.02 5.08 -1.33 Po 0.439 5.444 6.15 1.63 7.50 TV mm 3 23.35 25.86 31.2 27.1 27.0 BV mm 3 2.906 5.772 6.8 4.2 7.4 BV/TV % 12.45 22.33 21.7 15.4 27.6 TS mm 2 55.35 60.03 67.6 60.5 60.4 BS mm 2 143.9 243.5 305 210 307 i.S mm 2 6.785 11.48 13.7 9.3 15.7 BS/BV mm -1 49.52 42.18 45 50 41 3D BS/TV mm -1 6.164 9.418 9.8 7.7 11.3 Tb.Pf mm -1 11 5.261 3.8 10.4 2.0 SMI 1.946 1.653 1.48 1.91 1.37 Tb.Th mm 0.081 0.095 0.087 0.082 0.094 Tb.N mm -1 1.535 2.344 2.50 1.87 2.92 Tb.Sp mm 0.382 0.251 0.270 0.295 0.217 DA 1.963 1.679 1.60 1.60 1.75

Table D.7. Micro CT parameters of group 2 and 3.

228

Micro CT parameters Parameter Unit Group 3 Group 3 Group 3 Group 3 Group 3 BMD g/cm 3 0.41 0.31 0.29 0.36 0.30 TV mm 3 24.5 24.6 22.8 24.6 24.4 BV mm 3 8.14 5.77 5.06 7.00 5.41 BV/TV 33.2 23.5 22.2 28.4 22.2 TS mm 2 62.7 62.1 60.7 62.3 63.4 BS mm 2 355 283 296 332 284 BS/BV mm -1 44 49 58 47 52 T.Ar mm 2 9.7 9.7 9.0 9.8 9.6 T.Pm mm 13.0 13.4 12.6 13.1 13.4 B.Ar mm 2 3.2 2.3 2.0 2.8 2.1 B.Pm mm 94 75 77 88 76 Obj.N 143 117 155 156 148 Av.Obj.Ar mm 2 0.023 0.019 0.013 0.018 0.014 2D Av.Obj.Le mm 0.169 0.158 0.128 0.150 0.136 MMI(polar) mm 2 6.16 4.73 3.70 6.11 4.70 Ecc 0.80 0.85 0.78 0.82 0.86 Cs.Th mm 0.069 0.061 0.052 0.063 0.057 Tb.Th(pl) mm 0.046 0.041 0.034 0.042 0.038 Tb.Sp(pl) mm 0.09 0.13 0.12 0.11 0.13 Tb.N(pl) mm -1 7.24 5.75 6.49 6.74 5.82 Tb.Dm(rd) mm 0.092 0.082 0.068 0.084 0.076 Tb.Sp(rd) mm 0.049 0.068 0.060 0.056 0.067 Tb.N(rd) mm -1 7.09 6.70 7.77 7.14 6.97 Tb.Pf mm -1 -3.11 1.09 0.64 -1.69 3.07 Po 7.90 4.50 2.56 7.72 2.26 TV mm 3 24.5 24.6 22.7 24.6 24.3 BV mm 3 8.0 5.7 4.9 6.9 5.3 BV/TV % 32.6 23.0 21.7 27.9 21.7 TS mm 2 56.4 56.3 54.7 56.4 57.4 BS mm 2 294 234 244 275 235 i.S mm 2 17.6 11.8 11.5 15.8 12.0 BS/BV mm -1 37 41 50 40 45 3D BS/TV mm -1 12.0 9.5 10.8 11.2 9.7 Tb.Pf mm -1 -0.3 4.9 4.7 0.8 7.4 SMI 1.25 1.54 1.54 1.42 1.81 Tb.Th mm 0.104 0.094 0.081 0.099 0.091 Tb.N mm -1 3.14 2.45 2.68 2.80 2.39 Tb.Sp mm 0.203 0.249 0.230 0.232 0.236 DA 1.64 1.73 1.61 1.57 1.83

Table D.8. Micro CT parameters of group 3.

229

Micro CT parameters Parameter Unit Group 4 Group 4 Group 4 Group 4 Group 4 BMD g/cm 3 0.30 0.32 0.35 0.29 0.27 TV mm 3 30.0 30.4 25.3 27.4 25.7 BV mm 3 6.83 7.48 7.08 6.06 5.03 BV/TV 22.8 24.6 27.9 22.1 19.5 TS mm 2 71.8 72.9 63.4 67.3 64.7 BS mm 2 378 391 358 343 285 BS/BV mm -1 55 52 51 57 57 T.Ar mm 2 11.9 12.0 10.0 10.8 10.2 T.Pm mm 14.4 14.5 13.2 13.6 13.5 B.Ar mm 2 2.7 3.0 2.8 2.4 2.0 B.Pm mm 100 103 95 91 75 Obj.N 178 167 149 170 148 Av.Obj.Ar mm 2 0.015 0.018 0.019 0.014 0.013 2D Av.Obj.Le mm 0.139 0.150 0.155 0.134 0.131 MMI(polar) mm 2 6.46 7.49 5.81 5.02 4.17 Ecc 0.80 0.75 0.77 0.76 0.78 Cs.Th mm 0.054 0.057 0.059 0.053 0.053 Tb.Th(pl) mm 0.036 0.038 0.040 0.035 0.035 Tb.Sp(pl) mm 0.12 0.12 0.10 0.12 0.15 Tb.N(pl) mm -1 6.31 6.43 7.06 6.27 5.53 Tb.Dm(rd) mm 0.072 0.077 0.079 0.071 0.071 Tb.Sp(rd) mm 0.062 0.060 0.054 0.062 0.071 Tb.N(rd) mm -1 7.46 7.31 7.54 7.52 7.05 Tb.Pf mm -1 0.85 -0.83 -1.21 0.72 3.18 Po 4.52 6.19 6.00 4.09 1.88 TV mm 3 30.0 30.4 25.3 27.3 25.7 BV mm 3 6.7 7.3 6.9 5.9 4.9 BV/TV % 22.2 24.1 27.3 21.6 19.1 TS mm 2 64.9 65.6 57.0 60.4 58.2 BS mm 2 314 324 297 284 235 i.S mm 2 12.7 15.4 16.1 12.5 10.7 BS/BV mm -1 47 44 43 48 48 3D BS/TV mm -1 10.5 10.7 11.7 10.4 9.1 Tb.Pf mm -1 4.8 2.6 2.2 5.3 7.4 SMI 1.52 1.37 1.33 1.56 1.70 Tb.Th mm 0.084 0.088 0.091 0.083 0.084 Tb.N mm -1 2.65 2.75 3.02 2.61 2.28 Tb.Sp mm 0.232 0.238 0.213 0.230 0.256 DA 1.70 1.68 1.77 1.66 1.71

Table D.9. Micro CT parameters of group 4.

230

Micro CT parameters Parameter Unit Group 4 Group 4 Group 4 Group 5 Group 5 BMD g/cm 3 0.29 0.31 0.32 0.452 0.489 TV mm 3 23.4 23.9 25.3 19.6 21.9 BV mm 3 5.06 5.74 5.99 7.5 9.1 BV/TV 21.6 24.0 23.7 38 41 TS mm 2 62.7 59.6 63.3 56 57 BS mm 2 287 336 306 339 381 BS/BV mm -1 57 59 51 45.0 41.9 T.Ar mm 2 9.3 9.5 10.0 7.76 8.68 T.Pm mm 12.8 12.7 13.5 11.5 12.0 B.Ar mm 2 2.0 2.3 2.4 2.98 3.60 B.Pm mm 75 89 81 90 101 Obj.N 151 162 136 115 154 Av.Obj.Ar mm 2 0.013 0.014 0.017 0.026 0.023 2D Av.Obj.Le mm 0.130 0.134 0.149 0.181 0.173 MMI(polar) mm 2 3.94 4.52 4.98 4.75 6.40 Ecc 0.80 0.80 0.85 0.804 0.809 Cs.Th mm 0.053 0.051 0.058 0.066 0.072 Tb.Th(pl) mm 0.035 0.034 0.039 0.044 0.048 Tb.Sp(pl) mm 0.13 0.11 0.13 0.071 0.067 Tb.N(pl) mm -1 6.12 7.02 6.05 8.64 8.68 Tb.Dm(rd) mm 0.071 0.068 0.078 0.089 0.096 Tb.Sp(rd) mm 0.064 0.055 0.064 0.038 0.036 Tb.N(rd) mm -1 7.43 8.09 7.01 7.87 7.61 Tb.Pf mm -1 2.34 0.10 1.21 -6.60 -6.89 Po 2.56 4.60 5.05 18.0 16.1 TV mm 3 23.4 23.9 25.3 20 22 BV mm 3 4.9 5.6 5.9 7.4 8.9 BV/TV % 21.1 23.4 23.2 38 41 TS mm 2 56.4 54.3 57.4 49 51 BS mm 2 237 279 253 282 316 i.S mm 2 11.7 12.1 11.4 18 20 BS/BV mm -1 48 50 43 38 35 3D BS/TV mm -1 10.1 11.7 10.0 14 14 Tb.Pf mm -1 7.0 4.0 5.4 -5.6 -5.4 SMI 1.71 1.42 1.60 0.65 0.85 Tb.Th mm 0.084 0.079 0.091 0.094 0.106 Tb.N mm -1 2.51 2.97 2.55 4.00 3.87 Tb.Sp mm 0.231 0.208 0.229 0.17 0.16 DA 1.68 1.64 1.70 1.73 1.73

Table D.10. Micro CT parameters of group 4 and 5.

231

Micro CT parameters Parameter Unit Group 5 Group 5 Group 5 Group 5 Group 5 BMD g/cm 3 0.397 0.399 0.446 0.482 0.498 TV mm 3 23.8 18.7 23.0 22.5 22.4 BV mm 3 8.0 6.1 8.6 9.5 9.5 BV/TV 34 33 37 42 42 TS mm 2 62 54 59 61 60 BS mm 2 376 309 383 381 384 BS/BV mm -1 47.2 50.2 44.6 40.2 40.6 T.Ar mm 2 9.40 7.41 9.09 8.92 8.85 T.Pm mm 13.0 11.6 12.3 12.7 12.4 B.Ar mm 2 3.15 2.43 3.40 3.75 3.74 B.Pm mm 100 82 101 100 101 Obj.N 141 139 140 122 144 Av.Obj.Ar mm 2 0.022 0.018 0.024 0.031 0.026 2D Av.Obj.Le mm 0.169 0.149 0.175 0.198 0.182 MMI(polar) mm 2 6.27 4.00 6.17 6.97 6.76 Ecc 0.813 0.814 0.762 0.812 0.796 Cs.Th mm 0.063 0.059 0.067 0.075 0.074 Tb.Th(pl) mm 0.042 0.040 0.045 0.050 0.049 Tb.Sp(pl) mm 0.084 0.082 0.075 0.069 0.067 Tb.N(pl) mm -1 7.91 8.24 8.33 8.45 8.59 Tb.Dm(rd) mm 0.085 0.080 0.090 0.100 0.099 Tb.Sp(rd) mm 0.045 0.044 0.040 0.036 0.036 Tb.N(rd) mm -1 7.71 8.12 7.69 7.35 7.45 Tb.Pf mm -1 -4.93 -3.99 -5.65 -9.07 -7.92 Po 16.8 7.9 13.4 22.4 19.4 TV mm 3 24 19 23 23 22 BV mm 3 7.8 6.0 8.4 9.3 9.3 BV/TV % 33 32 37 41 42 TS mm 2 56 48 53 54 54 BS mm 2 312 257 318 316 319 i.S mm 2 19 17 18 22 22 BS/BV mm -1 40 43 38 34 34 3D BS/TV mm -1 13 14 14 14 14 Tb.Pf mm -1 -2.8 -2.2 -3.9 -8.3 -6.1 SMI 0.94 1.15 0.85 0.34 0.76 Tb.Th mm 0.093 0.092 0.098 0.105 0.106 Tb.N mm -1 3.53 3.51 3.76 3.96 3.92 Tb.Sp mm 0.19 0.19 0.17 0.17 0.16 DA 1.84 1.68 1.68 1.75 1.52

Table D.11. Micro CT parameters of group 5.

232

Micro CT parameters Parameter Unit Group 6 Group 6 Group 6 Group 6 Group 6 BMD g/cm 3 0.209 0.272 0.256 0.234 0.267 TV mm 3 24.7 22.0 24.1 24.6 20.3 BV mm 3 3.7 4.6 4.7 4.5 4.1 BV/TV 15 21 19 18 20 TS mm 2 64 57 63 63 57 BS mm 2 188 231 251 239 203 BS/BV mm -1 50.3 50.6 53.7 52.6 49.3 T.Ar mm 2 9.76 8.70 9.52 9.74 8.03 T.Pm mm 13.5 12.1 12.9 13.2 12.2 B.Ar mm 2 1.48 1.80 1.85 1.80 1.62 B.Pm mm 51 62 66 64 53 Obj.N 93 100 153 110 101 Av.Obj.Ar mm 2 0.016 0.018 0.012 0.016 0.016 2D Av.Obj.Le mm 0.142 0.152 0.124 0.144 0.143 MMI(polar) mm 2 3.53 3.56 3.64 3.70 3.34 Ecc 0.866 0.832 0.829 0.841 0.863 Cs.Th mm 0.058 0.058 0.056 0.056 0.061 Tb.Th(pl) mm 0.040 0.039 0.037 0.038 0.041 Tb.Sp(pl) mm 0.223 0.151 0.155 0.168 0.160 Tb.N(pl) mm -1 3.80 5.25 5.21 4.85 4.99 Tb.Dm(rd) mm 0.079 0.079 0.074 0.076 0.081 Tb.Sp(rd) mm 0.102 0.075 0.075 0.081 0.079 Tb.N(rd) mm -1 5.52 6.50 6.67 6.37 6.26 Tb.Pf mm -1 4.78 1.18 0.37 2.47 3.07 Po 1.4 5.2 3.7 3.9 2.3 TV mm 3 25 22 24 25 20 BV mm 3 3.6 4.5 4.6 4.4 4.0 BV/TV % 15 20 19 18 20 TS mm 2 58 51 56 57 51 BS mm 2 156 192 208 198 168 i.S mm 2 8 11 11 10 11 BS/BV mm -1 43 43 46 45 42 3D BS/TV mm -1 6 9 9 8 8 Tb.Pf mm -1 8.8 4.6 5.0 6.4 6.4 SMI 1.91 1.54 1.84 1.65 1.82 Tb.Th mm 0.094 0.090 0.091 0.087 0.096 Tb.N mm -1 1.57 2.25 2.07 2.08 2.06 Tb.Sp mm 0.39 0.30 0.30 0.30 0.32 DA 1.99 1.91 1.73 1.87 1.90

Table D.12. Micro CT parameters of group 6.

233

Micro CT parameters Parameter Unit Group 6 Group 6 Group 7 Group 7 Group 7 BMD g/cm 3 0.333 0.202 0.327 0.272 0.258 TV mm 3 20.6 23.6 24.7 27.6 22.4 BV mm 3 5.3 3.3 6.3 5.7 4.3 BV/TV 26 14 26 21 19 TS mm 2 57 61 64 69 61 BS mm 2 254 176 306 291 227 BS/BV mm -1 47.9 53.6 48.5 50.8 52.7 T.Ar mm 2 8.15 9.32 9.79 10.94 8.85 T.Pm mm 12.1 13.1 13.1 14.2 13.0 B.Ar mm 2 2.10 1.30 2.50 2.27 1.71 B.Pm mm 67 47 82 80 59 Obj.N 105 87 116 153 111 Av.Obj.Ar mm2 0.020 0.015 0.021 0.015 0.015 2D Av.Obj.Le mm 0.159 0.137 0.165 0.137 0.140 MMI(polar) mm 2 4.02 2.76 5.48 6.01 3.66 Ecc 0.834 0.835 0.854 0.888 0.856 Cs.Th mm 0.062 0.056 0.061 0.057 0.057 Tb.Th(pl) mm 0.042 0.037 0.041 0.039 0.038 Tb.Sp(pl) mm 0.120 0.231 0.120 0.150 0.159 Tb.N(pl) mm -1 6.18 3.73 6.19 5.27 5.08 Tb.Dm(rd) mm 0.083 0.075 0.082 0.079 0.076 Tb.Sp(rd) mm 0.062 0.103 0.062 0.074 0.077 Tb.N(rd) mm -1 6.86 5.64 6.91 6.53 6.53 Tb.Pf mm -1 0.26 5.24 -1.22 1.19 2.95 Po 6.4 1.4 9.2 3.6 2.5 TV mm 3 21 24 25 28 22 BV mm 3 5.2 3.2 6.2 5.6 4.2 BV/TV % 25 14 25 20 19 TS mm 2 51 55 58 62 54 BS mm 2 211 146 254 244 187 i.S mm 2 13 8 14 13 11 BS/BV mm -1 41 46 41 44 44 3D BS/TV mm -1 10 6 10 9 8 Tb.Pf mm -1 3.9 9.1 2.2 4.5 7.6 SMI 1.51 1.85 1.32 1.69 1.80 Tb.Th mm 0.095 0.087 0.091 0.092 0.090 Tb.N mm -1 2.66 1.56 2.74 2.21 2.10 Tb.Sp mm 0.24 0.43 0.24 0.35 0.28 DA 1.89 1.96 1.92 2.16 1.80

Table D.13. Micro CT parameters of group 6 and 7.

234

Micro CT parameters Parameter Unit Group 7 Group 7 Group 7 Group 7 Group 7 BMD g/cm 3 0.280 0.286 0.344 0.389 0.364 TV mm 3 24.2 27.7 24.3 22.9 22.9 BV mm 3 5.2 6.4 6.9 7.2 6.6 BV/TV 21 23 28 32 29 TS mm 2 63 70 62 60 60 BS mm 2 274 351 343 320 321 BS/BV mm -1 53.1 54.8 49.5 44.4 48.8 T.Ar mm 2 9.58 10.98 9.63 9.04 9.05 T.Pm mm 13.0 14.3 13.0 13.0 12.7 B.Ar mm 2 2.05 2.54 2.74 2.85 2.60 B.Pm mm 74 93 91 85 85 Obj.N 122 183 140 122 140 Av.Obj.Ar mm 2 0.017 0.014 0.020 0.023 0.019 2D Av.Obj.Le mm 0.146 0.133 0.158 0.172 0.154 MMI(polar) mm 2 4.29 6.20 5.97 5.71 5.38 Ecc 0.837 0.845 0.840 0.838 0.868 Cs.Th mm 0.056 0.054 0.061 0.067 0.061 Tb.Th(pl) mm 0.038 0.036 0.040 0.045 0.041 Tb.Sp(pl) mm 0.139 0.121 0.101 0.098 0.102 Tb.N(pl) mm -1 5.67 6.33 7.05 6.99 7.01 Tb.Dm(rd) mm 0.075 0.073 0.081 0.090 0.082 Tb.Sp(rd) mm 0.069 0.062 0.053 0.052 0.054 Tb.N(rd) mm -1 6.92 7.43 7.46 7.03 7.38 Tb.Pf mm -1 1.39 -0.19 -2.05 -3.61 -2.13 Po 4.3 4.1 6.6 11.2 10.1 TV mm 3 24 28 24 23 23 BV mm 3 5.1 6.3 6.8 7.1 6.4 BV/TV % 21 23 28 31 28 TS mm 2 56 63 55 54 53 BS mm 2 228 292 284 266 266 i.S mm 2 12 14 15 16 15 BS/BV mm -1 45 47 42 38 41 3D BS/TV mm -1 9 11 12 12 12 Tb.Pf mm -1 5.2 4.3 0.9 -0.8 1.5 SMI 1.54 1.60 1.26 1.16 1.37 Tb.Th mm 0.086 0.086 0.092 0.101 0.093 Tb.N mm -1 2.44 2.62 3.04 3.07 3.01 Tb.Sp mm 0.26 0.24 0.21 0.22 0.21 DA 1.87 1.78 1.73 1.91 2.02

Table D.14. Micro CT parameters of group 7.

235

Micro CT parameters Parameter Unit Group 7 Group 8 Group 8 Group 8 Group 8 BMD g/cm 3 0.250 0.358 0.290 0.287 0.256 TV mm 3 24.5 23.7 23.6 18.7 26.7 BV mm 3 4.6 7.0 5.3 6.1 5.0 BV/TV 19 30 22 33 19 TS mm 2 63 62 61 54 67 BS mm 2 272 326 290 309 283 BS/BV mm -1 59.6 46.3 54.6 50.2 57.0 T.Ar mm 2 9.71 9.40 9.34 7.41 10.58 T.Pm mm 13.1 13.0 12.9 11.6 13.6 B.Ar mm 2 1.81 2.79 2.10 2.43 1.96 B.Pm mm 72 87 77 82 74 Obj.N 144 131 129 139 147 Av.Obj.Ar mm 2 0.013 0.021 0.016 0.018 0.013 2D Av.Obj.Le mm 0.127 0.165 0.144 0.149 0.130 MMI(polar) mm 2 4.02 5.92 4.27 4.00 4.32 Ecc 0.823 0.844 0.843 0.814 0.797 Cs.Th mm 0.050 0.064 0.054 0.059 0.053 Tb.Th(pl) mm 0.034 0.043 0.037 0.040 0.035 Tb.Sp(pl) mm 0.147 0.102 0.126 0.082 0.154 Tb.N(pl) mm -1 5.55 6.87 6.14 8.24 5.28 Tb.Dm(rd) mm 0.067 0.086 0.073 0.080 0.070 Tb.Sp(rd) mm 0.071 0.054 0.064 0.044 0.074 Tb.N(rd) mm -1 7.26 7.12 7.31 8.12 6.92 Tb.Pf mm -1 3.18 -2.47 0.44 -3.99 2.75 Po 2.4 9.8 4.9 7.9 1.8 TV mm 3 25 24 24 19 27 BV mm 3 4.4 6.9 5.2 6.0 4.8 BV/TV % 18 29 22 32 18 TS mm 2 56 56 55 48 60 BS mm 2 226 271 241 257 233 i.S mm 2 11 15 12 17 10 BS/BV mm -1 51 39 46 43 48 3D BS/TV mm -1 9 11 10 14 9 Tb.Pf mm -1 7.5 0.1 4.5 -2.2 7.7 SMI 1.68 1.23 1.46 1.15 1.74 Tb.Th mm 0.079 0.097 0.083 0.092 0.083 Tb.N mm -1 2.30 3.00 2.63 3.51 2.18 Tb.Sp mm 0.26 0.22 0.24 0.19 0.27 DA 1.76 1.87 1.88 1.68 1.66

Table D.15. Micro CT parameters of group 7 and 8.

236

Micro CT parameters Parameter Unit Group 8 Group 8 Group 8 Group 8 Group 8 BMD g/cm 3 0.351 0.315 0.032 0.332 0.324 TV mm 3 21.8 20.7 26.1 25.4 25.7 BV mm 3 5.9 5.1 6.6 6.7 6.4 BV/TV 27 25 25 26 25 TS mm 2 57 58 66 63 63 BS mm 2 272 243 328 325 312 BS/BV mm -1 45.7 47.8 49.9 48.5 48.9 T.Ar mm 2 8.64 8.18 10.34 10.07 10.19 T.Pm mm 12.4 12.1 13.7 13.3 13.3 B.Ar mm 2 2.35 2.01 2.60 2.65 2.52 B.Pm mm 72 62 86 87 83 Obj.N 111 102 182 154 131 Av.Obj.Ar mm 2 0.021 0.020 0.014 0.017 0.019 2D Av.Obj.Le mm 0.164 0.158 0.135 0.148 0.157 MMI(polar) mm 2 4.39 3.91 6.00 5.64 5.48 Ecc 0.848 0.820 0.871 0.833 0.800 Cs.Th mm 0.065 0.064 0.060 0.061 0.061 Tb.Th(pl) mm 0.044 0.042 0.040 0.041 0.041 Tb.Sp(pl) mm 0.117 0.128 0.119 0.115 0.124 Tb.N(pl) mm -1 6.22 5.87 6.27 6.39 6.05 Tb.Dm(rd) mm 0.088 0.084 0.080 0.082 0.082 Tb.Sp(rd) mm 0.061 0.066 0.062 0.060 0.064 Tb.N(rd) mm -1 6.73 6.68 7.06 7.03 6.87 Tb.Pf mm -1 -1.08 0.18 -0.72 -0.41 -0.06 Po 7.8 4.8 4.7 7.1 7.3 TV mm 3 22 21 26 25 26 BV mm 3 5.8 5.0 6.4 6.6 6.2 BV/TV % 27 24 25 26 24 TS mm 2 51 52 59 57 57 BS mm 2 225 200 272 270 259 i.S mm 2 14 14 14 14 15 BS/BV mm -1 39 40 42 41 41 3D BS/TV mm -1 10 10 10 11 10 Tb.Pf mm -1 2.7 3.2 4.2 3.7 3.8 SMI 1.47 1.50 1.80 1.60 1.50 Tb.Th mm 0.100 0.095 0.097 0.097 0.092 Tb.N mm -1 2.68 2.54 2.53 2.66 2.63 Tb.Sp mm 0.24 0.27 0.23 0.23 0.25 DA 1.90 1.66 1.80 1.78 1.72

Table D.16. Micro CT parameters of group 8.

237

Micro CT parameters Parameter Unit Group 9 Group 9 Group 9 Group 9 Group 9 BMD g/cm 3 0.496 0.416 0.398 0.457 0.356 TV mm 3 22.1 18.9 18.7 18.7 20.2 BV mm 3 9.4 6.3 6.1 6.1 5.9 BV/TV 43 34 33 33 29 TS mm 2 60 54 54 54 56 BS mm 2 399 292 309 309 274 BS/BV mm -1 42.5 46.1 50.2 50.2 46.3 T.Ar mm 2 8.73 7.47 7.41 7.41 8.01 T.Pm mm 12.5 11.5 11.6 11.6 12.0 B.Ar mm 2 3.72 2.51 2.43 2.43 2.34 B.Pm mm 105 77 82 82 73 Obj.N 157 121 139 139 100 Av.Obj.Ar mm 2 0.024 0.021 0.018 0.018 0.023 2D Av.Obj.Le mm 0.174 0.162 0.149 0.149 0.172 MMI(polar) mm 2 6.98 4.02 4.00 4.00 4.21 Ecc 0.827 0.836 0.814 0.814 0.862 Cs.Th mm 0.071 0.065 0.059 0.059 0.064 Tb.Th(pl) mm 0.047 0.043 0.040 0.040 0.043 Tb.Sp(pl) mm 0.063 0.086 0.082 0.082 0.105 Tb.N(pl) mm -1 9.05 7.73 8.24 8.24 6.76 Tb.Dm(rd) mm 0.094 0.087 0.080 0.080 0.086 Tb.Sp(rd) mm 0.034 0.046 0.044 0.044 0.055 Tb.N(rd) mm -1 7.82 7.53 8.12 8.12 7.06 Tb.Pf mm -1 -8.50 -3.14 -3.99 -3.99 -1.90 Po 19.1 9.4 7.9 7.9 10.5 TV mm 3 22 19 19 19 20 BV mm 3 9.2 6.2 6.0 6.0 5.8 BV/TV % 42 33 32 32 29 TS mm 2 54 48 48 48 50 BS mm 2 332 242 257 257 227 i.S mm 2 22 15 17 17 15 BS/BV mm -1 36 39 43 43 39 3D BS/TV mm -1 15 13 14 14 11 Tb.Pf mm -1 -7.0 -0.1 -2.2 -2.2 0.9 SMI 0.69 1.24 1.15 1.15 1.20 Tb.Th mm 0.102 0.098 0.092 0.092 0.095 Tb.N mm -1 4.10 3.37 3.51 3.51 3.02 Tb.Sp mm 0.15 0.19 0.19 0.19 0.23 DA 1.78 1.81 1.68 1.68 1.84

Table D.17. Micro CT parameters of group 9.

238

Micro CT parameters Parameter Unit Group 9 Group 9 Group 9 Group 9 Group 10 BMD g/cm 3 0.470 0.333 0.402 0.468 0.373 TV mm 3 23.9 23.2 20.1 20.7 23.6 BV mm 3 9.6 6.1 6.7 8.2 7.3 BV/TV 40 26 33 40 31 TS mm 2 62 60 56 56 63 BS mm 2 388 341 315 338 363 BS/BV mm -1 40.5 55.9 47.3 41.0 49.7 T.Ar mm 2 9.47 9.18 7.94 8.18 9.35 T.Pm mm 12.9 12.8 12.0 11.9 13.0 B.Ar mm 2 3.79 2.42 2.64 3.26 2.89 B.Pm mm 103 91 83 90 96 Obj.N 144 179 149 113 141 Av.Obj.Ar mm 2 0.026 0.013 0.018 0.029 0.020 2D Av.Obj.Le mm 0.183 0.131 0.150 0.192 0.161 MMI(polar) mm 2 7.47 5.14 4.68 5.40 5.96 Ecc 0.804 0.857 0.851 0.792 0.834 Cs.Th mm 0.074 0.053 0.064 0.072 0.060 Tb.Th(pl) mm 0.049 0.036 0.042 0.049 0.040 Tb.Sp(pl) mm 0.074 0.100 0.085 0.074 0.090 Tb.N(pl) mm -1 8.10 7.36 7.86 8.16 7.69 Tb.Dm(rd) mm 0.099 0.072 0.084 0.098 0.080 Tb.Sp(rd) mm 0.040 0.052 0.045 0.039 0.048 Tb.N(rd) mm -1 7.22 8.09 7.70 7.30 7.80 Tb.Pf mm -1 -6.32 -1.01 -4.58 -5.90 -3.75 Po 15.8 4.8 11.6 16.5 10.3 TV mm 3 24 23 20 21 24 BV mm 3 9.4 6.0 6.5 8.1 7.2 BV/TV % 39 26 33 39 30 TS mm 2 56 54 50 51 56 BS mm 2 322 284 262 281 302 i.S mm 2 22 13 16 20 17 BS/BV mm -1 34 48 40 35 42 3D BS/TV mm -1 13 12 13 14 13 Tb.Pf mm -1 -4.5 2.8 -1.3 -4.2 -1.3 SMI 0.87 1.48 1.28 0.78 1.05 Tb.Th mm 0.107 0.085 0.096 0.105 0.089 Tb.N mm -1 3.68 3.03 3.40 3.73 3.40 Tb.Sp mm 0.18 0.20 0.19 0.17 0.20 DA 1.62 1.79 1.76 1.86 1.95

Table D.18. Micro CT parameters of group 9 and 10.

239

Micro CT parameters Parameter Unit Group 10 Group 10 Group 10 Group 10 Group 10 BMD g/cm 3 0.602 0.428 0.416 0.411 0.524 TV mm 3 22.8 18.7 18.7 23.1 23.2 BV mm 3 12.5 6.1 6.1 8.1 10.5 BV/TV 55 33 33 35 45 TS mm 2 59 54 54 60 60 BS mm 2 454 309 309 370 409 BS/BV mm -1 36.3 50.2 50.2 45.8 39.1 T.Ar mm 2 9.02 7.41 7.41 9.13 9.18 T.Pm mm 12.6 11.6 11.6 12.7 13.0 B.Ar mm 2 4.95 2.43 2.43 3.19 4.15 B.Pm mm 119 82 82 98 109 Obj.N 152 139 139 136 151 Av.Obj.Ar mm 2 0.033 0.018 0.018 0.023 0.027 2D Av.Obj.Le mm 0.203 0.149 0.149 0.173 0.187 MMI(polar) mm 2 8.31 4.00 4.00 6.03 8.02 Ecc 0.765 0.814 0.814 0.824 0.843 Cs.Th mm 0.083 0.059 0.059 0.065 0.076 Tb.Th(pl) mm 0.055 0.040 0.040 0.044 0.051 Tb.Sp(pl) mm 0.045 0.082 0.082 0.081 0.062 Tb.N(pl) mm -1 9.97 8.24 8.24 8.01 8.82 Tb.Dm(rd) mm 0.110 0.080 0.080 0.087 0.102 Tb.Sp(rd) mm 0.022 0.044 0.044 0.044 0.033 Tb.N(rd) mm -1 7.59 8.12 8.12 7.65 7.40 Tb.Pf mm -1 -15.57 -3.99 -3.99 -5.90 -9.19 Po 23.9 7.9 7.9 16.7 16.9 TV mm 3 23 19 19 23 23 BV mm 3 12.3 6.0 6.0 7.9 10.3 BV/TV % 54 32 32 34 45 TS mm 2 54 48 48 54 54 BS mm 2 378 257 257 307 341 i.S mm 2 25 17 17 18 23 BS/BV mm -1 31 43 43 39 33 3D BS/TV mm -1 17 14 14 13 15 Tb.Pf mm -1 -16.0 -2.2 -2.2 -4.2 -7.4 SMI -0.32 1.15 1.15 0.81 0.65 Tb.Th mm 0.114 0.092 0.092 0.095 0.113 Tb.N mm -1 4.75 3.51 3.51 3.61 3.94 Tb.Sp mm 0.12 0.19 0.19 0.20 0.15 DA 1.67 1.68 1.68 1.72 1.76

Table D.19. Micro CT parameters of group 10.

240

Micro CT parameters Parameter Unit Group 10 Group 10 Group 10 Group 11 Group 11 BMD g/cm 3 0.399 0.518 0.447 0.244 0.330 TV mm 3 21.1 22.2 23.9 17.4 18.5 BV mm 3 7.0 10.2 8.9 3.1 4.8 BV/TV 33 46 37 18 26 TS mm 2 56 61 61 50 53 BS mm 2 324 401 384 180 249 BS/BV mm -1 46.6 39.2 43.1 58.7 52.0 T.Ar mm 2 8.33 8.80 9.48 6.90 7.31 T.Pm mm 12.2 12.9 12.9 10.9 11.4 B.Ar mm 2 2.75 4.05 3.52 1.22 1.90 B.Pm mm 86 105 102 48 66 Obj.N 130 147 158 108 102 Av.Obj.Ar mm 2 0.021 0.027 0.022 0.011 0.019 2D Av.Obj.Le mm 0.164 0.187 0.168 0.120 0.153 MMI(polar) mm 2 4.95 7.51 7.08 1.91 3.07 Ecc 0.835 0.843 0.836 0.813 0.853 Cs.Th mm 0.064 0.077 0.069 0.051 0.058 Tb.Th(pl) mm 0.043 0.051 0.046 0.034 0.038 Tb.Sp(pl) mm 0.087 0.060 0.078 0.159 0.110 Tb.N(pl) mm -1 7.69 9.01 8.01 5.17 6.75 Tb.Dm(rd) mm 0.086 0.102 0.093 0.068 0.077 Tb.Sp(rd) mm 0.047 0.031 0.042 0.076 0.057 Tb.N(rd) mm -1 7.55 7.50 7.41 6.95 7.48 Tb.Pf mm -1 -3.68 -10.94 -5.55 3.94 -1.03 Po 12.2 19.7 12.0 1.2 8.8 TV mm 3 21 22 24 17 18 BV mm 3 6.8 10.1 8.8 3.0 4.7 BV/TV % 32 45 37 17 25 TS mm 2 50 55 55 44 47 BS mm 2 269 333 319 150 207 i.S mm 2 17 23 20 8 12 BS/BV mm -1 39 33 36 50 44 3D BS/TV mm -1 13 15 13 9 11 Tb.Pf mm -1 -1.5 -10.1 -3.3 7.4 1.3 SMI 1.08 0.37 1.06 1.80 1.20 Tb.Th mm 0.095 0.111 0.107 0.082 0.086 Tb.N mm -1 3.39 4.10 3.41 2.08 2.97 Tb.Sp mm 0.20 0.16 0.18 0.30 0.23 DA 1.80 1.65 1.91 1.76 1.82

Table D.20. Micro CT parameters of group 10 and 11.

241

Micro CT parameters Parameter Unit Group 11 Group 11 Group 11 Group 11 Group 11 BMD g/cm 3 0.362 0.354 0.433 0.447 0.324 TV mm 3 18.7 18.7 22.9 18.6 19.0 BV mm 3 6.1 6.1 8.3 7.0 4.9 BV/TV 33 33 36 37 26 TS mm 2 54 54 61 53 54 BS mm 2 309 309 367 288 253 BS/BV mm -1 50.2 50.2 44.2 41.3 51.8 T.Ar mm 2 7.41 7.41 9.07 7.37 7.53 T.Pm mm 11.6 11.6 12.7 11.4 11.5 B.Ar mm 2 2.43 2.43 3.29 2.76 1.93 B.Pm mm 82 82 96 75 67 Obj.N 139 139 145 114 123 Av.Obj.Ar mm 2 0.018 0.018 0.023 0.024 0.016 2D Av.Obj.Le mm 0.149 0.149 0.170 0.175 0.141 MMI(polar) mm 2 4.00 4.00 5.98 4.02 3.06 Ecc 0.814 0.814 0.792 0.731 0.793 Cs.Th mm 0.059 0.059 0.069 0.074 0.058 Tb.Th(pl) mm 0.040 0.040 0.045 0.048 0.039 Tb.Sp(pl) mm 0.082 0.082 0.080 0.081 0.112 Tb.N(pl) mm -1 8.24 8.24 8.01 7.73 6.64 Tb.Dm(rd) mm 0.080 0.080 0.091 0.097 0.077 Tb.Sp(rd) mm 0.044 0.044 0.043 0.043 0.058 Tb.N(rd) mm -1 8.12 8.12 7.50 7.13 7.40 Tb.Pf mm -1 -3.99 -3.99 -5.62 -4.28 -0.79 Po 7.9 7.9 13.9 10.9 5.7 TV mm 3 19 19 23 19 19 BV mm 3 6.0 6.0 8.2 6.9 4.8 BV/TV % 32 32 36 37 25 TS mm 2 48 48 55 47 48 BS mm 2 257 257 304 238 210 i.S mm 2 17 17 18 17 12 BS/BV mm -1 43 43 37 35 44 3D BS/TV mm -1 14 14 13 13 11 Tb.Pf mm -1 -2.2 -2.2 -3.2 -2.0 2.9 SMI 1.15 1.15 1.01 1.09 1.50 Tb.Th mm 0.092 0.092 0.102 0.106 0.089 Tb.N mm -1 3.51 3.51 3.50 3.47 2.82 Tb.Sp mm 0.19 0.19 0.19 0.19 0.23 DA 1.68 1.68 1.60 1.58 1.88

Table D.21. Micro CT parameters of group 11.

242

Micro CT parameters Parameter Unit Group 11 Group 12 Group 12 Group 12 Group 12 BMD g/cm 3 0.411 0.471 0.467 0.322 0.392 TV mm 3 20.5 22.2 20.8 18.7 18.7 BV mm 3 6.9 9.1 7.9 6.1 6.1 BV/TV 34 41 38 33 33 TS mm 2 57 58 56 54 54 BS mm 2 313 377 351 309 309 BS/BV mm -1 45.5 41.6 44.3 50.2 50.2 T.Ar mm 2 8.11 8.77 8.24 7.41 7.41 T.Pm mm 12.1 12.3 11.8 11.6 11.6 B.Ar mm 2 2.72 3.59 3.14 2.43 2.43 B.Pm mm 82 100 92 82 82 Obj.N 125 141 181 139 139 Av.Obj.Ar mm 2 0.022 0.025 0.017 0.018 0.018 2D Av.Obj.Le mm 0.167 0.180 0.149 0.149 0.149 MMI(polar) mm 2 4.99 6.74 5.07 4.00 4.00 Ecc 0.855 0.827 0.764 0.814 0.814 Cs.Th mm 0.066 0.072 0.068 0.059 0.059 Tb.Th(pl) mm 0.044 0.048 0.045 0.040 0.040 Tb.Sp(pl) mm 0.087 0.069 0.074 0.082 0.082 Tb.N(pl) mm -1 7.63 8.51 8.43 8.24 8.24 Tb.Dm(rd) mm 0.088 0.096 0.090 0.080 0.080 Tb.Sp(rd) mm 0.047 0.037 0.039 0.044 0.044 Tb.N(rd) mm -1 7.43 7.51 7.71 8.12 8.12 Tb.Pf mm -1 -3.66 -7.72 -5.78 -3.99 -3.99 Po 10.9 16.7 16.2 7.9 7.9 TV mm 3 20 22 21 19 19 BV mm 3 6.7 8.9 7.8 6.0 6.0 BV/TV % 33 40 37 32 32 TS mm 2 51 52 50 48 48 BS mm 2 259 314 292 257 257 i.S mm 2 17 21 18 17 17 BS/BV mm -1 38 35 37 43 43 3D BS/TV mm -1 13 14 14 14 14 Tb.Pf mm -1 -0.9 -6.4 -2.5 -2.2 -2.2 SMI 1.16 0.72 1.31 1.15 1.15 Tb.Th mm 0.098 0.105 0.102 0.092 0.092 Tb.N mm -1 3.36 3.82 3.67 3.51 3.51 Tb.Sp mm 0.19 0.17 0.17 0.19 0.19 DA 1.68 1.86 1.61 1.68 1.68

Table D.22. Micro CT parameters of group 11 and 12.

243

Micro CT parameters Parameter Unit Group 12 Group 12 Group 12 Group 12 Group 12 BMD g/cm 3 0.360 0.478 0.395 0.374 0.550 TV mm 3 16.7 21.2 17.7 16.2 21.2 BV mm 3 4.7 8.8 5.9 4.8 10.0 BV/TV 28 41 33 30 47 TS mm 2 48 56 50 48 58 BS mm 2 232 386 276 229 380 BS/BV mm -1 49.4 44.1 47.0 47.6 38.1 T.Ar mm 2 6.61 8.41 7.01 6.42 8.40 T.Pm mm 10.6 12.1 10.9 10.7 12.1 B.Ar mm 2 1.86 3.47 2.33 1.90 3.94 B.Pm mm 60 103 74 60 100 Obj.N 111 148 110 96 156 Av.Obj.Ar mm 2 0.017 0.023 0.021 0.020 0.025 2D Av.Obj.Le mm 0.146 0.173 0.164 0.158 0.179 MMI(polar) mm 2 2.48 5.96 3.36 2.40 6.77 Ecc 0.767 0.800 0.798 0.777 0.791 Cs.Th mm 0.061 0.067 0.063 0.063 0.079 Tb.Th(pl) mm 0.040 0.045 0.043 0.042 0.052 Tb.Sp(pl) mm 0.104 0.065 0.086 0.100 0.059 Tb.N(pl) mm -1 6.93 9.09 7.80 7.06 8.94 Tb.Dm(rd) mm 0.081 0.091 0.085 0.084 0.105 Tb.Sp(rd) mm 0.054 0.034 0.046 0.053 0.031 Tb.N(rd) mm -1 7.38 7.99 7.64 7.31 7.37 Tb.Pf mm -1 -0.72 -8.08 -3.51 -1.50 -9.15 Po 5.2 20.0 11.5 9.2 16.8 TV mm 3 17 21 18 16 21 BV mm 3 4.6 8.6 5.8 4.7 9.8 BV/TV % 27 41 33 29 46 TS mm 2 44 51 45 43 52 BS mm 2 192 322 230 190 315 i.S mm 2 12 20 14 12 24 BS/BV mm -1 42 37 40 40 32 3D BS/TV mm -1 11 15 13 12 15 Tb.Pf mm -1 2.2 -6.8 -1.2 1.0 -7.8 SMI 1.48 0.69 1.11 1.28 0.68 Tb.Th mm 0.094 0.098 0.095 0.094 0.114 Tb.N mm -1 2.92 4.12 3.42 3.08 4.05 Tb.Sp mm 0.22 0.16 0.19 0.21 0.15 DA 1.62 1.91 1.75 1.84 1.63

Table D.23. Micro CT parameters of group 12.

244

APPENDIX E

DATA RELATED TO CHAPTER 6

245

Concentration Drug Well 1 Well 2 Well 3 Well 4 (nM) [3H]-thymidine incorporation (CPM) in Saos-2 cell - 0 5175 5077 6064 5178 1 4464 5197 6365 5522 10 4989 5272 6517 4983 DHT 100 4469 5663 5868 5747 1,000 4517 4761 6232 5701 10,000 3696 4330 4612 4199 - 0 6063 6501 6592 5239 1 4361 5858 6164 6281 10 7019 5816 5714 6253 S-22 100 4576 4126 7429 5755 1,000 5069 5331 6057 5667 10,000 306 311 370 331 [3H]-thymidine incorporation (CPM) in FOB-AR6 cell - 0 10904 10185 9887 9400 10 10186 9706 5891 9885 100 8953 10122 12210 9349 S-22 1,000 9953 9402 9541 9446 10,000 7411 7411 7827 7727 DHT 10,000 6005 5805 5449 4989 0 10851 10204 9626 10245 10 11396 10330 10452 11359 Raloxifene 100 11142 9952 11556 11637 1,000 10589 10740 10331 11617 10,000 451 177 435 288 Estradiol 10,000 6221 6119 5912 5941

Table E.1. Proliferation of osteoblast.

246

Concentration Drug Well 1 Well 2 Well 3 Well 4 (nM) - 0 40.1 51.4 59.9 47 10 52.1 63.9 48.2 54 100 58.5 59.3 52.7 65 S-22 1,000 66.71 64.95 64.19 71 10,000 85.75 97.11 75.85 85 DHT 10,000 78.88 106.8 90.33 84 0 25.98 39.69 55.8 40.19 10 28.2 38.44 49.94 38.15 Raloxifene 100 28.32 38.19 39.33 38.83 1,000 33.16 43.68 45.68 33.69 10,000 24.15 27.8 32.75 27.92 Estradiol 10,000 53.91 83.61 96.56 88.21

Table E.2. ALP activities in MC3T3-E1 cells.

247

Concentration Drug Well 1 Well 2 Well 3 (nM) - 0 28.4 24.6 29.6 10 35.4 20.5 22.8 100 38.0 35.4 35.5 S-22 1,000 43.1 36.4 39.0 10,000 57.0 54.1 58.4 DHT 10,000 28.6 36.3 35.4 0 28.2 30.5 34.0 10 20.7 19.2 21.0 Raloxifene 100 20.3 18.6 20.7 1,000 16.9 17.1 18.4 10,000 9.8 9.5 9.7 Estradiol 10,000 31.0 37.1 32.0

Table E.3. Alizarin Red-S activities in MC3T3-E1 cells.

248

Concentration Drug Well 1 Well 2 Well 3 (nM) - 0 6.4 5.4 7.3 6.3 10 4.1 8.5 7.4 6.5 100 7.5 6.9 - 7.1 S-22 1,000 9.9 7.5 - 5.8 10,000 6.9 8.1 7.8 8.5 DHT 10,000 6.2 3.9 4.6 4.7 0 4.4 5.3 7 3.9 10 4.5 5.4 8 7.3 Raloxifene 100 5.8 4.9 7.9 6.9 1,000 10.3 7.1 5.4 2.4 10,000 1.4 0.3 0.1 1 Estradiol 10,000 2.8 1.5 2.3 1.1

Table E.4. TRAP activities in MC3T3-E1 cells.

249