17α-Alkynyl 3α, 17β-androstanediol non-clinical and clinical pharmacology, pharmacokinetics and metabolism

PRECLINICAL STUDIES

17α-Alkynyl 3α, 17β-androstanediol non-clinicaland clinical pharmacology, pharmacokineticsand metabolism

Clarence Ahlem & Michael Kennedy & Theodore Page & David Bell & Evelyn Delorme &

Sonia Villegas & Chris Reading & Steven White & Dwight Stickney & James Frincke

Received: 27 May 2010 /Accepted: 6 August 2010 /Published online: 3 September 2010# Springer Science+Business Media, LLC 2010

Summary 17α-ethynyl-5α-androstane-3α, 17β-diol (HE3235,Apoptone) is an orally bioavailable synthetic analogue of3β-androstanediol, that is active in rodent models ofprostate and breast cancer, and is in Phase IIa clinicaltrials for the treatment of early- and late-stage prostatecancer. In preparation for clinical studies, nuclearhormone receptor and P450 interactions for HE3235and major metabolites were characterized in vitro, andpharmacokinetics and metabolite profiles were studied inrodents, dogs, and monkeys. Four-week safety studiesconducted in rats and dogs indicated a substantial marginof safety for clinical use, and no evidence of electrocar-diographic or neurological effects, although anorexia,thrombocytopenia, and hypokalemia were identified aspotentially dose-limiting toxicities at superpharmacolog-ical exposures. Pharmacokinetics and drug metabolismhave been studied in prostate cancer patients.

Keywords Prostate cancer . Hormone . Toxicology .

Pharmacology . HE3235

Introduction

Hormone-responsive cancers (HRC) are the most commontypes of malignancy, representing 30% and 38% of all newcancer cases in men and women, respectively [1]. In theUnited States, prostate cancer is the second leading cause of

death in men from cancer, following lung cancer [1]. It isestimated that one in six men will develop prostate cancerand one in 30 will die from their disease [2]. HRC requirethe presence of a hormone receptor for growth, but growthdependence on the cognate hormone may vary with diseaseprogression, and the disease may evolve to use otherhormones and/or other hormone receptors, or no hormone atall, stimulating growth through ligand independent pathways.Blocking the production of androgen effectively treats earlystage disease, but patients ultimately relapse, their diseaseprogresses, and the only remaining option is chemotherapythat does not provide a durable benefit, and is associated withsignificant morbidity. A well-tolerated treatment that has anew and perhaps complementary mechanism of action toother medications could greatly enhance the treatment ofHRC and meet an unmet medical need.

17α-Ethynyl-5α-androstane-3α, 17β-diol (HE3235, Apop-tone) is a synthetic analogue of 3β-androstanediol, which is anaturally occurring metabolite of dihydrotestosterone that isformed in prostate tissue [3, 4]. In a castration-sensitiveprostate xenograft tumor model (LNCaP), HE3235 treatmentproduced a dose-dependent reduction in tumor incidence, andsignificantly suppressed the rate of established tumor growth.In castration-resistant mouse tumor models, HE3235 signifi-cantly suppressed the growth of LuCaP 35 V human prostatecancer mouse xenografts, and a C42B intra-tibial tumorxenograft with reduction in intra-tumoral concentrations oftestosterone and dihydrotestosterone [5]. In a carcinogen-induced rat breast cancer model, HE3235 ablated establishedtumors, suppressed tumor growth, and blocked developmentof new tumors (manuscript submitted for publication).HE3235 is presently in clinical trials for the treatment ofpatients with late-stage castration-resistant prostate cancer.Here we describe non-clinical and clinical studies of thepharmacology, pharmacokinetics and metabolism of HE3235.

C. Ahlem (*) :M. Kennedy : T. Page :D. Bell : E. Delorme :S. Villegas : C. Reading : S. White :D. Stickney : J. FrinckeHarbor BioSciences, Inc.,9171 Towne Centre Drive, Suite 181,San Diego, CA 92122, USAe-mail: [email protected]

Invest New Drugs (2012) 30:59–78DOI 10.1007/s10637-010-9517-0

Materials and methods

All experimental procedures in animals were conductedwith the approval of institutional animal care and usecommittees (IACUC), and in accordance with local, state,and federal animal welfare regulations. Clinical studieswere performed in accordance with the guidelines of theInternational Conference on Harmonization (ICH) and theDeclaration of Helsinki and approved by the associatedInstitutional Review Boards (IRB).

Reagents and chemicals

HE3235, 17α-ethynyl-5α-androstane-3α, 17β-diol(C21H3202, MW=316.48), and metabolites described in thispublication were synthesized by Harbor BioSciences, Inc.Except as noted, all reagents and chemicals were obtainedfrom Sigma-Aldrich (St. Louis, MO).

HE3235 was formulated for oral administration or intra-peritoneal injection in nonclinical studies as a 20mg/mL aqueoussolution in 40% 2-hydroxypropyl-β-cyclodextrin (CTD, Inc.,High Springs, FL), or an aqueousmicro-suspension in 0.1% (w:v)carboxymethylcellulose sodium (CMC), 2% (w:v) polysorbate-80, 0.9% (w:v) NaCl and 0.05% (w:v) phenol (CMC and PS-80were purchased from Spectrum, Gardena, CA).

HE3235 for monkey absolute bioavailability studies wasformulated as a 10 mg/mL solution in 30% aqueous β-cyclodextrin sulfobutyl ether (Cydex Pharmaceuticals,Lenexa, KS).

HE3235 was administered to humans as a solid oraldosage form.

Nuclear hormone receptor interaction profile

Assessment of binding and transactivation for various nuclearreceptors was performed as previously described [6].

13

149810

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CH3191

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HE3235

HE3759

HE3562

HE3539

HE3598 HE3601

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149810

171211

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756

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CH3191

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HE3758

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149810

171211

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CH3191

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HE3660

Fig. 1 HE3235 metabolites detected in vivo. HE3235 is metabolizedprimarily by oxidation (HE3562) and inversion (HE3539) of thehydroxyl at C-3, or hydroxylation at C-2 (HE3759, major; HE3758,

minor). Minor metabolites (HE3598, HE3660, and HE3601) areformed by hydroxylation at C-7 and C-16

60 Invest New Drugs (2012) 30:59–78

1A2 2C9 2C19 2D6 3A4a 3A4b

DirectInhibition VehicleControl 0 0 0 0 0 0

InhibitorControl 99 94 98 91 99 99

HE3235100 μM 41 84 75 91 99 95

HE323510 μM 45 35 50 8 90 77

HE32351 μM <1 <1 8 7 35 29

HE353960 μM <1 11 16 3 34 28

HE356220 μM <1 0 14 1 18 19

TimeDependentInhibition VehicleControl 0 0 0 0 0 0

InhibitorControl 96 99 98 93 99 98

3235100 μM 84 96 92 89 99 99

323510 μM 39 31 68 11 98 94

32351 μM 26 4 10 4 66 75

32350.1 μM NDc NDc NDc NDc 19 20

32350.01 μM NDc NDc NDc NDc 0 0

353960 μM 40 23 33 3 86 70

35396 μM 22 11 0 0 70 59

35390.6 μM 7 0 17 0 15 12

356220 μM 39 12 18 0 38 38

35622 μM 20 0 0 0 41 23

35620.2 μM 28 1 0 0 9 12

Table 1 Pooled human livermicrosomes were incubated withvarious concentrations of HE3235and major metabolites HE3539and HE3562 in the presence ofindicator substrates and controlinhibitors for specific P450isoenzymes to measure directinhibition and time dependentinhibition. Each isozyme wasassessed by pre-incubation of theHE3235 and metabolites withmicrosomes and a cofactorsolution for 30min prior to theaddition of the substrate to initiatethe reaction. Enzyme productswere quantified by LC-MS/MS.Values shown are percent P450inhibition relative to vehicle(0.4% acetonitrile)

a testosterone 6β-hydroxylaseactivityb midazolam 1-hydroxylaseactivityc Not Determined

Table 2 Mouse, rat, dog, monkey, and human liver microsomes were incubated at 37°C with HE3235 and an NADPH regenerating system for10, 30, and 90min. Samples were analyzed by GC-MS. Putative general structures were assigned from mass transitions assuming enzymaticoxidation and/or reduction of the parent. “X” indicates the molecular species was detected. Gas-chromatography retention times (GC RT) areshown for each molecular species detected. The results were not quantitative. F: free steroid; G: glucuronide conjugate; S: sulfate conjugate

Trimethylsilyl parent ion MW Metabolite GC RT Species

Human Monkey Dog Rat Mouse

456 17a-ethynyl-17b-hydroxy-androst-4-en-3-one 6.54 X X X X

458 17a-ethynyl-17b-hydroxy-5a-androstan-3-one 7.11 X X X X X

460 HE3235 (parent) 6.53 X X X X X

460 17α-ethynyl-5α-androstane-3β,17β-diol 7.05 X X X X X

546 17α-ethynyl-5α-androstane-diol-one 7.76 X X X X

546 17α-ethynyl-5α-androstane-diol-one 7.84 X X X X X

548 17α-ethynyl-5α-androstane-triol 6.86 X

548 17α-ethynyl-5α-androstane-triol 6.96 X X X


548 17α-ethynyl-5α-androstane-triol 7.14 X X X X X





548 17α-ethynyl-5α-androstane-triol 7.89 X

634 17α-ethynyl-5α-androstane-triol-one 8.31

636 17α-ethynyl-5α-androstane-tetrol 7.75 X X X

636 17α-ethynyl-5α-androstane-tetrol 7.83 X

636 17α-ethynyl-5α-androstane-tetrol 7.90 X X

636 17α-ethynyl-5α-androstane-tetrol 8.24 X X

636 17α-ethynyl-5α-androstane-tetrol 8.38 X

Table 2 Mouse, rat, dog, monkey, and human liver microsomes wereincubated at 37°C with HE3235 and an NADPH regenerating system for10, 30, and 90min. Samples were analyzed by GC-MS. Putative generalstructures were assigned from mass transitions assuming enzymatic

oxidation and/or reduction of the parent. “X” indicates the molecularspecies was detected. Gas-chromatography retention times (GC RT) areshown for each molecular species detected. The results were notquantitative. F: free steroid; G: glucuronide conjugate; S: sulfate conjugate

Invest New Drugs (2012) 30:59–78 61

HE3235 metabolizing enzymes

P450 inhibition studies

HE3235 reactions with microsomal enzymes CYP1A2,2C9, 2C19, 2D6, and 3A4 were studied with pooledmixed-sex human liver microsomes (In Vitro Technolo-gies, Baltimore, MD). Probe substrates (Phenacetin,Tolbutamide, S-Mephenytoin, Dextromethorphan, Testos-terone and Midazolam [both tested with 3A4]) andstandard inhibitors (Furafylline, Sulfaphenazole, Tranyl-cypromine, Quinidine, and Ketoconazole [for bothtestosterone and midazolam]) used in the assays werein accord with draft FDA guidance for drug interactionstudies [7]. HE3235 (1, 10, and 100 μM), HE3539 (0.6, 6,and 60 μM), and HE3562 (0.2, 2, and 20 μM) wereincubated at 37°C with microsomes for 10–30 min, asdetermined specifically for each enzyme to consume lessthan 10% of the substrate in the absence of inhibitors. The

time dependent inhibition for each isozyme was assessedby pre-incubation of the test compound with microsomesand cofactor solution for 30 min prior to the addition ofthe substrate to initiate the reaction. Reactions wereterminated with cold acetonitrile containing internalstandard (5α-pregnane-3α-11β-17α-20β-tetrol), clarifiedby centrifugation, and analyzed by LC/MS-MS, using aSynergi 4 μ POLAR-RP 80 column (150×2 mm, Pheno-monex, Torrance CA), and a Varian 1200 LC-MS/MSspectrometer (Varian, Palo Alto, CA).

Identification of CYP isozymes that metabolize HE3235

To identify which P450 enzymes are primarily responsiblefor HE3235 metabolism, HE3235 (1 μM) was incubatedwith pooled human liver microsomes (1 mg/mL) in thepresence or absence of CYP-specific inhibitors at 37°C for90 min, and analyzed by LC/MS-MS as described above.Metabolizing enzymes were identified by a decreased rate

Table 3 HE3235 metabolites were profiled by GC-MS in the plasma orserum from mice, rats, dogs, and monkeys dosed orally with HE3235.General structure assignments were made from mass transitions

assuming enzymatic oxidation and/or reduction of the parent. “X”indicates that the molecular species was detected. Gas-chromatographyretention times (GC RT) are shown for each molecular species

Metabolite GC RT Monkey Dog Rat Mouse

F G S F G S F G S F G S

HE3562 7.1 X X X X X

HE3235 6.51 X X X X X X X X X

HE3539 7.05 X X X X X X X

17α-ethynyl-5α-androstane-diol-one 7.72 X X X

17α-ethynyl-5α-androstane-diol-one 7.85 X X X X X X X X

17α-ethynyl-5α-androstane-triol 7.05 X X X

17α-ethynyl-5α-androstane-triol 7.12 X X X X X X X

17α-ethynyl-5α-androstane-triol 7.29 X X X X X X X X X

17α-ethynyl-5α-androstane-triol 7.37 X X X X X X X X X

17α-ethynyl-5α-androstane-triol 7.47 X

17α-ethynyl-5α-androstane-triol 7.54 X X

17α-ethynyl-5α-androstane-triol 7.69 X X X X





17α-ethynyl-5α-androstane-triol-one 8.15 X X



17α-ethynyl-5α-androstane-tetrol 7.59 X X

17α-ethynyl-5α-androstane-tetrol 7.67 X X X


17α-ethynyl-5α-androstane-tetrol 7.83 X X X X X X X

17α-ethynyl-5α-androstane-tetrol 7.9 X



of HE3235 metabolism in the presence of an inhibitor(compared to control reaction).

In vitro induction of CYP1A2 and CYP3A4 by HE3235and select metabolites

Human hepatocytes were cultured with HE3235 to measurethe potential for HE3235 to induce the two major inducibleP450 enzymes, CYP1A2 and CYP3A4. Pooled cryopre-served human hepatocytes and hepatocyte growth medium(In Vitro Technologies, Baltimore, MD) were prepared forP450 induction studies according to the manufacturer’sinstructions. Cells were plated and adhered in 24-wellplates (0.35×106 viable cells in 0.5 mL per well, 37°C, 5%

CO2, saturating humidity), and treated with vehicle (0.4%acetonitrile), control CYP inducer (50 μM omeprazole forCYP1A2; 25 μM rifampicin for CYP3A4) or test com-pounds (HE3235, HE3539 and HE3562 at 0.3 μM and10 μM). The media was removed after 48 h, and replacedwith media containing enzyme substrate (50 μM phenacetinfor CYP1A2; 25μM testosterone for CYP3A4), incubated for4 h, and assayed for enzyme products (acetaminophen forCYP1A2; 6β-hydroxytestosterone for CYP3A4) by LC/MS/MS as described above. Enzyme induction was quantified bycomparing the amounts of enzyme product in uninduced andinduced cultures.

Interspecies comparative liver microsomal metabolism(in vitro)

The metabolite profile of HE3235 was characterized invitro with pooled liver microsomes from humans (male andfemale combined), and male monkeys, dogs, rats, and mice.HE3235 (10 μM) and microsomes (0.5 mg/mL) wereincubated with an NADPH regeneration system (glucose-6-phosphate, glucose-6-phosphate dehydrogenase, andNADP) at 37°C. Samples were collected at 0, 10, 30, and90 min, clarified by centrifugation, extracted with methyl-t-butyl-ether (MTBE), and derivatized with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) for GC-MSanalysis, using a Varian CP-Sil 5 CB column in a VarianCP-3800 gas chromatograph with helium carrier gas, and aVarian 1200 L mass spectrometer. Because the exactstructures of most of the analytes were unknown, datawas collected without internal standards and standardcurves for quantification. The activity of the microsomalpreparation was confirmed using 5-androstenediol as acontrol substrate. Metabolites were identified by theirmolecular weight and retention time, and in comparison toHE3539 and HE3562 analytical standards.

Interspecies comparative metabolism (in vivo)

Plasma metabolites were characterized in human males,male and female cynomolgus monkeys (20 mg/kg,), beagledogs (200 mg/kg), and Sprague–Dawley rats (200 mg/kg,)and male CD-1 mice (40 mg/kg) following oral adminis-tration of HE3235. Plasma samples were collected near thetime of maximum HE3235 concentration (Tmax, 1-2 h formonkeys, dogs, and rats, and 0.25 h for mice), and storedfrozen until analyzed by GC-MS. Steroid conjugates werehydrolyzed by sequential treatment with glucuronidase,sulfatase, and sulfuric acid and analyzed by GC-MS.

Plasma samples were also analyzed by LC/MS-MS. Prior toanalysis, internal standard (17α-methyl-androst-5-ene-3β,17β-diol) was added, and the samples were extracted with MTBE.The organic phase was evaporated to dryness, and reconstituted

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3235

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Fig. 2 The pharmacokinetics of HE3235 and major metabolites weremeasured in two male and two female cynomolgus monkeysfollowing oral-gastric administration of 20mg/kg (1mL/kg) formulat-ed as an aqueous microsuspension of a stable crystalline form. Plasmawas collected from each animal at 0.25, 0.5, 1, 2, 3, 4, 8, 12 and 24h afteradministration. HE3235 and metabolites were quantified by LC/MS-MS


in LC/MS-MS mobile phase buffer. HE3235 and metaboliteswere quantified, using reversed-phase high-performance liquidchromatography (Agilent, Palo Alto, CA and Leap Technolo-gies, Carrboro, NC) with an Xbridge Phenyl column (Waters,Beverly, MA) coupled to a tandem mass spectrometer in ESI+mode (Waters, Beverly, MA).

Nonclinical pharmacokinetics

Single and multiple oral dose pharmacokinetics (PK)were measured in CD-1 mice, Sprague–Dawley rats,beagle dogs, cynomolgus monkeys, and humans. Drugconcentrations in serum or plasma were determined byLC/MS-MS, and PK parameters were calculated withWinNonlin 5.2 (Pharsight, St. Louis, Missouri) using themean values for each collection time.

Safety studies

HE3235 was evaluated for genotoxic effects in accordancewith FDA guidelines for testing new chemical entities insupport of clinical trials [8].

The genotoxic potential was assessed in vitro using both abacterial reverse mutation assay [9–12] and a cytogeneticchromosomal aberration assay that evaluates damage inmetaphase cells [13, 14]. An in vivo test for the occurrenceof chemically-induced micronucleated polychromatic eryth-rocytes in bone marrow cells was conducted in mice[15–17].

Systemic toxicity

The systemic toxicity of HE3235 was evaluated in GLP-compliant studies in Sprague–Dawley rats and Beagle dogs.

Rats

Male and female rats received 28-daily oral gavagesoluble HE3235 doses at 20, 100, and 400 mg/kg. The400 mg/kg dose was administered as 200 mg/kg BID for13 days, and then reduced to 200 mg/kg QD. On day 29,animals were sacrificed and necropsied. Blood and urinewere collected for clinical pathology, and tissues werecollected for histopathology. Ophthalmic examinationswere performed prior to treatment and terminal necropsy.A satellite group of animals were allowed to recover for28 days and then sacrificed and evaluated in the samemanner. A neurological assessment (functional observa-tion battery) was performed for piloerection, exophthal-mus, polyuria/diarrhea, pupillary function, convulsions,tremors, degree of palpebral closure, reactivity to generalstimuli, and alertness pre-study and on days 1 and 25,using methods described by in Moser [18, 19], Meyer[20], Edwards and Parker [21], and Ankier [22].

Dogs

Male and female dogs received 28-daily oral doses ofsoluble HE3235 at 20, 60, and 200 mg HE3235 per kg.

Day Sex Cmax (ng/mL) AUC(0–24) (ng/mL*h) Parent ratio

HE3235

1 M 938 598014 1432 10147

28 1365 10394

1 F 2978 1464014 2712 19117

28 2640 16176

HE3539

1 M 943 10957 1.8

14 2415 17715 1.7

28 2018 16101 1.5

1 F 788 8545 0.6

14 1801 13518 0.7

28 2180 16383 1.0

HE3562

1 M 3726 26003 4.3

14 8621 41632 4.1

28 5475 39324 3.8

1 F 2300 22583 1.5

14 4533 29680 1.6

28 5044 32786 2.0

Table 4 Pharmacokinetics weremeasured in groups of five maleand female beagle dogs followinga single or 28 daily HE3235 oralgavage doses of 60mg/kg bodyweight, formulated in cyclodextrin.Plasma (lithium heparin) was col-lected from a peripheral vein fromeach animal at each time point foreach dose level at 0, 0.25, 0.5, 1, 2,3, 4, 8, 12, and 24h after drugadministration. Samples werestored frozen prior to analysis.HE3235 was quantified by LC/MS-MS and pharmacokineticsparameters were calculated withWinNonlin. The “parent ratio”listed is the AUC(0–24) of themetabolite divided by HE3235AUC(0–24)


Main study and satellite recovery animals were evaluated fortoxicological endpoints as described for rats with the exclusionof a functional observation battery. Electrocardiographic exams(ten-lead measuring RR, PR, and QTc intervals, and QRSduration), were performed pre-study and on days 1 and 25.

Measurement of sex steroids and luteinizing hormonein HE3235-treated dogs

Plasma concentrations of testosterone, dehydroepiandros-terone (DHEA), androstenedione, and luteinizing hormone

Table 5 Pharmacokinetics were measured in male and femaleSprague–Dawley rats following a single or 28 daily HE3235 oralgavage doses of 20, 100, and 200 mg/kg body weight, formulated incyclodextrin. Plasma (lithium heparin) was collected from a peripheralvein from three animals per gender at each time point for each doselevel. Five cohorts were used for each gender with each cohort

sampled only twice in the 24-hour period (0, 0.25, 0.5, 1, 2, 3, 4, 8,12, and 24 h). Samples were stored frozen prior to analysis. HE3235,HE3539, and HE3562 were quantified by LC/MS-MS and pharma-cokinetics parameters were calculated with WinNonlin. The “parentratio” listed is the AUC(0–24) of the metabolite divided by HE3235AUC(0–24). BQL Below Quantitation Limit

Analyte Day Sex Dose mg/kg Cmax ng/mL Tmax hr AUC(0–24) ng*hr/mL Parent ratio

HE3235 1 M 25 71 1 131

28 M 25 121 1 336

1 F 25 1360 0.25 2390

28 F 25 291 3 949

1 M 100 1390 0.25 4080

28 M 100 1120 2 4670

1 F 100 2150 0.25 7450

28 F 100 999 2 5710

1 M 200 1210 2 4940

28 M 200 2320 2 10700

1 F 200 3680 0.5 11700

28 F 200 4070 0.5 14800

HE3539 1 M 25 76 0.50 112 0.85

28 M 25 12 0.50 50 0.15

1 F 25 22 0.25 35 0.01

28 F 25 BQL N/A N/A 0.01

1 M 100 2117 0.25 6572 1.61

28 M 100 224 0.50 943 0.20

1 F 100 48.0 12.0 621 0.08

28 F 100 28 2.00 80 0.01

1 M 200 2249 2.00 7839 1.59

28 M 200 1044 2.00 2398 0.22

1 F 200 44 0.50 285 0.02

28 F 200 143 0.50 454 0.03

HE3562 1 M 25 BQL N/A N/A 0.04

28 M 25 BQL N/A N/A 0.01

1 F 25 46 0.25 N/A 0.00

28 F 25 BQL N/A N/A 0.01

1 M 100 120 0.25 464 0.11

28 M 100 78 2.00 108 0.02

1 F 100 72 0.25 68 0.01

28 F 100 56 2.00 N/A 0.00

1 M 200 192 2.00 393 0.08

28 M 200 217 3.00 533 0.05

1 F 200 131 0.50 151 0.01

28 F 200 241 0.50 476 0.03


were measured in male beagle dogs from the systemictoxicity study using LC/MS-MS.

Luteinizing hormone was measured in duplicate wells withELISA kits according to the manufacturer’s instructions(USCN Life,Wuhan, China), using an 8-point standard curve.

Assay for HE3235 inhibition of steroidogenic enzymes

The potential for HE3235 to inhibit the formation oftestosterone was measured in vitro in LNCaP cells byfollowing the conversion of 3H-androst-5-ene-3β, 17β-diolto testosterone and other androgenic metabolites using radio-HPLC. Confluent LNCaP cells in T75 flasks (approximately1.7 million cells per flask) were incubated with 10 nMradiolabeled androstenediol (60 Ci/mmol, American Radio-labeled Chemicals, Inc., St. Louis, MO) and either 50 nMHE3235 or vehicle for 1, 6, or 24 h. Steroids in culturemedia were isolated separately from cells, and analyzed byradio-HPLC using a Supelco Discovery C18 column

(2×250 mm, Sigma Chemical Company, St. Louis, MO) usinga water:acetonitrile gradient. Steroid conjugates were hydro-lyzed with sulfatase/glucuronidase (Sigma Chemical Company,St. Louis, MO) for 3 h at 52°C and analyzed as above.

The potential for HE3235 to inhibit CYP17 was exploredin vitro by following the conversion of 3H-pregnenolone todehydroepiandrosterone (DHEA), testosterone, and other 19-carbon steroids in H295R adrenal tumor cells (AmericanType Culture Collection, Manassas, VA). Confluent H295Rcells in T25 flasks (approximately 1 million cells/flask) wereincubated (8 h) with 50 nM 3H-pregnenolone (20 Ci/mmol,American Radiolabeled Chemicals, Inc., St. Louis, MO) andeither 100 ng/mL HE3235 or vehicle. Cells and mediumwere prepared and analyzed for steroids as described above.

Clinical pharmacology

A multi-site Phase I/IIa clinical study was conducted incastrate-resistant prostate cancer patients that failed at

Metabolite Dose (mg) AUC(0–8) ng*hr/mL AUC(0–24) ng*hr/mL

Mean (SD) Parent ratio Mean (SD) Parent ratio

HE3539 5 ND – ND –

10 7 (4.5) 0.12 21 (12.3) 0.20

15 6 (4.7) 0.05 22 (12.4) 0.10

25 3 (3.7) 0.03 N/A 0.02

50 7 (3.6) 0.02 23 (19.3) 0.04

HE3562 5 10 (6.3) 0.37 15 (10.4) 0.32

10 15 (5.9) 0.26 34 (6.8) 0.32

15 23 (8.8) 0.19 51 (15.2) 0.22

25 20 (21.7) 0.17 64 (56.5) 0.23

50 32 (42.1) 0.11 98 (138.4) 0.16

HE3759 5 NA – NA –

10 12 (4.8) 0.20 21 (4.6) 0.20

15 27 (12.6) 0.23 61 (33.8) 0.26

25 46 (21.8) 0.40 68 (45.2) 0.24

50 43 (35.0) 0.14 115 (39.9) 0.19

Table 6 Patients with late-stageCRPC received oral doses ofHE3235 on a BID schedule. Onday 28 (presumed steady statebased on 14-hour T1/2) themorning dose was administeredand the evening dose in the BIDschedule was omitted to permitelimination phase sampling forthe morning dose. The dose indi-cated in the table is the morningdose on day 28. Plasma (lithiumheparin) was collected immedi-ately prior to the morning dosingand at 1, 1.5, 2, 3, 4, 8, and24h post dose. HE3235, HE3539,and HE3562 were quantified byLC/MS-MS and pharmacokineticsparameters were calculated withWinNonlin. The “parent ratio”listed is the AUC(0–8) of themetabolite divided by HE3235AUC(0–8). ND Not Detected,NA Not Assayed

Table 7 Patients with late-stage CRPC received oral doses of HE3235 on a BID schedule. On day 28 (presumed steady state based on 14-hourT1/2) the morning dose was given and the evening dose in the BID schedule was omitted to permit elimination phase PK sampling for the morningdose. The dose indicated in the table is the morning dose on day 28. Plasma (lithium heparin) was collected immediately prior to dosing and at 1,1.5, 2, 3, 4, 8, and 24 h post dose. HE3235 was quantified by LC/MS-MS. The AUC was calculated with WinNonlin 5.2

Dose 5mg, n=4 10mg, n=6 15mg, n=5 25mg, n=3 50mg, n=3

Mean (SD) Median Mean (SD) Median Mean (SD) Median Mean (SD) Median Mean (SD) Median

Cmax, ng/mL 10 (6.6) 9 19.5 (8.2) 18 34 (11.2) 34 31 (6.0) 28 110 (81.4) 81

Tmax, hr 0.9 (0.6) 1.0 1.3 (0.8) 1.5 1.2 (0.3) 1.0 1.8 (1.0) 1.5 1.3 (0.3) 1.5

AUC(0–8), ng*hr/mL 28 (19.6) 26 59.5 (27.2) 51 119 (27.3) 123 115 (55.1) 124 299 (88.8) 348

AUC(0–24), ng*hr/mL 46 (32.0) 44 106.0 (51.1) 88 230 (69.5) 246 282 (35.4) 282 596 (190.3) 579

Table 7 Patients with late-stage CRPC received oral doses of HE3235on a BID schedule. On day 28 (presumed steady state based on14-hour T1/2) the morning dose was given and the evening dose in theBID schedule was omitted to permit elimination phase PK samplingfor the morning dose. The dose indicated in the table is the morning

dose on day 28. Plasma (lithium heparin) was collected immediatelyprior to dosing and at 1, 1.5, 2, 3, 4, 8, and 24 h post dose. HE3235was quantified by LC/MS-MS. The AUC was calculated withWinNonlin 5.2


least one prior taxane-based chemotherapy regimen.Patients received a solid oral dosage form of 5, 10, 15,25, 50, or 100 mg twice daily (BID) in hard gelatincapsules. Blood was collected for pharmacokinetics,metabolite profiling, and endogenous hormone concen-trations on days 1 and 28.

Results

HE3235 metabolites

Our primary objectives for metabolite profiling were tocompare the humans to rodents for the purpose oftranslating results in rodent efficacy models, and tocompare humans to rodents and canines for the purposeof measuring exposure in safety studies. Two dioxygenatedmetabolites were identified, and GC-MS analyses ofmicrosomal incubates and plasma samples did not suggestthe existence of additional dioxygenated species. Inaddition to the parent drug, these dioxygenated metabolitesmay be important to the activity of HE3235 throughinteractions with the sex steroid signaling and metabolicpathways. Additional higher oxidized forms including triand tetra oxidized forms were also detected in variousanimal species, but no tetrols were detected in humanplasma. The metabolic tree for the identified HE3235metabolites is shown in Fig. 1.

Effects on metabolizing CYP enzymes

HE3235 inhibition of CYP enzymes

All major P450 enzymes were inhibited with 100 μMHE3235 and also 10 μM except for CYP2D6 (Table 1).Pharmacologically-significant time-dependent enzyme in-hibition was also observed at 10 and 100 μM HE3235, and

R2 = 0.9605

R2 = 0.9993

0

50

100

150

200

250

300

350

400

0 20 40 60 80 100 120mg/day

HE

3235

AU

C(0

-8),

ng*

hr/m

L

Day 1Day 28Linear (Day 28)Linear (Day 1)

Fig. 3 Patients with late-stage CRPC received oral doses of HE3235on a BID schedule. On day 28 (presumed steady state based on 14-hour T1/2) the morning dose was given and the evening dose in theBID schedule was omitted to permit elimination phase PK samplingfor the morning dose. The dose indicated in the table is the morningdose on day 28. Plasma (lithium heparin) was collected immediatelyprior to dosing and at 1, 1.5, 2, 3, 4, 8, and 24 h post dose. HE3539and HE3562 were quantified by LC/MS-MS. The AUC wascalculated with WinNonlin 5.2. The “parent ratio” listed is theAUC(0–24) of the metabolite divided by HE3235 AUC(0–24)

Table 8 Patients with late-stage CRPC received oral doses of HE3235 on a BID schedule. On day 28 (presumed steady state based on 14-hourT1/2) the morning dose was given and the evening dose in the BID schedule was omitted to permit elimination phase PK sampling for the morningdose. The dose indicated in the table is the morning dose on day 28. Plasma (lithium heparin) was collected immediately prior to dosing and at 1,1.5, 2, 3, 4, 8, and 24 h post dose. HE3539 and HE3562 were quantified by LC/MS-MS. The AUC was calculated with WinNonlin 5.2. The“parent ratio” listed is the AUC(0-t) of the metabolite divided by HE3235 AUC(0-t). ND Not Detected, NA Not Assayed

Metabolite Dose mg AUC(0–8)(SD) ng*hr/mL Parent ratio AUC(0–24) (SD) ng*hr/mL Parent ratio

HE3539 5 ND ND ND ND

10 7 (4.5) 0.12 21 (12.3) 0.20

15 6 (4.7) 0.05 22 (12.4) 0.10

25 3 (3.7) 0.03 NA 0.02

50 7 (3.6) 0.02 23 (19.3) 0.04

HE3562 5 10 (6.3) 0.37 15 (10.4) 0.32

10 15 (5.9) 0.26 34 (6.8) 0.32

15 23 (8.8) 0.19 51 (15.2) 0.22

25 20 (21.7) 0.17 64 (56.5) 0.23

50 32 (42.1) 0.11 98 (138.4) 0.16

HE3759 5 N/A – N/A –

10 12 (4.8) 0.20 21 (4.6) 0.20

15 27 (12.6) 0.23 61 (33.8) 0.26

25 46 (21.8) 0.40 68 (45.2) 0.24

50 43 (35.0) 0.14 115 (39.9) 0.19

Table 8 Patients with late-stage CRPC received oral doses of HE3235on a BID schedule. On day 28 (presumed steady state based on 14-hour T1/2) the morning dose was given and the evening dose in theBID schedule was omitted to permit elimination phase PK samplingfor the morning dose. The dose indicated in the table is the morningdose on day 28. Plasma (lithium heparin) was collected immediately

prior to dosing and at 1, 1.5, 2, 3, 4, 8, and 24 h post dose. HE3539and HE3562 were quantified by LC/MS-MS. The AUC was calculatedwith WinNonlin 5.2. The “parent ratio” listed is the AUC(0-t) of themetabolite divided by HE3235 AUC(0-t). ND Not Detected, NA NotAssayed


in contrast to the direct inhibition studies, time-dependentinhibition was also observed at 1 μM for CYP3A4.

The major P450 enzymes were not directly inhibited bymetabolites HE3539 and HE3562. Metabolites HE3539 andHE3562 are less potent inhibitors, and are present at lowerconcentrations than HE3235 in humans. HE3759 is abun-dantly produced by CYP3A4 and its presence in HE3235P450 assays indicates it is not likely a potent enzyme inhibitor.

Identification of HE3235 metabolizing enzymes and CYPenzyme induction

Inhibition of CYP 2C19 and CYP 3A4 decreased HE3235metabolism by 30 and 38%, respectively. Neither CYP1A2nor CYP 3A4 were induced by HE3235 or its metabolitesat drug concentrations up to 10 μM.

In vitro interspecies comparative metabolism

The HE3235 metabolites produced by liver microsomesderived from human, monkey, dog, rat and mouse wereprofiled by GC-MS. All species produced HE3539 andHE3562. All metabolites produced by human liver micro-somes were produced by microsomes from all other species,except for 17α-ethynyl-17β-hydroxy-androst-4-en-3-one andthe unknown metabolite RT7.76, which were not present inthe mouse (Table 2). All species also have the microsomalmetabolic capacity to form trioxy species although theidentity varies with primate forming only two species bothof which are formed in dog and rat but not mouse. Onlydogs and rodents appear to form tetrols with five tetrolmolecular species observed. No aromatic compounds orpentoxy metabolites were detected.

Table 9 The pharmacokinetics of HE3235 and major metaboliteswere measured in two male and two female cynomolgus monkeysfollowing oral-gastric administration of 20mg/kg (1mL/kg) formulat-ed as an aqueous microsuspension of a stable crystalline form. Plasma

was collected from each animal at 0.25, 0.5, 1, 2, 3, 4, 8, 12 and24 h after administration. HE3235 and metabolites were quantified byLC/MS-MS; pharmacokinetics parameters calculated with WinNonlin5.2. N/A Not Applicable

Analyte Cmax (ng/mL) Tmax (hr) AUC(0–24) (ng*hr/mL) AUC(0-inf) (ng*hr/mL) T1/2 (hr)

HE3235 1647 1.0 7890 8994 7.5

HE3539 63 7.3 776a N/A N/A

HE3562 85 1.0 520b N/A N/A

a 9.8% of parentb 6.6% of parent

Day Sex Dose, mg/kg Cmax (SD) ng/mL Tmax hr AUC(0–24) (SD) ng*hr/mL T1/2hr

1 M 20 692 (446) 0.8 3154 (1672) 7.5

14 M 20 775 (383) 0.9 4756 (2817) 6.3

28 M 20 1030 (350) 0.7 5188 (2071) 8.7

1 F 20 467 (133) 0.9 2082 (1070) 4.8

14 F 20 566 (103) 0.9 3173 (858) 6.3

28 F 20 702 (96) 0.6 3877 (530) 6.3

1 M 60 938 (777) 0.9 5980 (3826) 7.2

14 M 60 1432 (685) 1.2 10147 (3800) 7.2

28 M 60 1365 (529) 0.9 10394 (4361) 8.5

1 F 60 2978 (2420) 0.7 14640 (12674) 8.9

14 F 60 2712 (2067) 0.7 19117 (18665) 6.2

28 F 60 2640 (1646) 1.0 16176 (7893) 5.5

1 M 200 8152 (8064) 0.9 56540 (59646) 7.2

14 M 200 9756 (10662) 1.8 63445 (56797) 7.6

28 M 200 6029 (3111) 1.1 47440 (18410) 6.2

1 F 200 6209 (8178) 1.1 50955 (78965) 7.5

14 F 200 8018 (10943) 1.4 57017 (64418) 7.4

28 F 200 4372 (1719) 3.0 37466 (10560) 5.8

Table 10 Pharmacokineticswere measured in groups offive male and female beagledogs following a single or 28daily HE3235 oral gavage dosesof 20, 60, and 200mg/kg bodyweight, formulated in cyclodex-trin. Plasma (lithium heparin)was collected from a peripheralvein from each animal at eachtime point for each dose level at0, 0.25, 0.5, 1, 2, 3, 4, 8, 12, and24 h after drug administration.Samples were stored frozenprior to analysis. HE3235 wasquantified by LC/MS-MS andpharmacokinetics parameterswere calculated with WinNonlin


In vivo interspecies comparative metabolism

The species distribution from in vitro microsome studieswas reflected by the in vivo results (Table 3). Rodentsformed a plethora of more highly oxidized metabolitescompared to dogs and monkeys. A tetrol was detected inmonkey plasma with GC-MS, even though no tetrols werefound in monkey microsomal incubates. The majority offree steroid species were also detected as conjugates inmonkeys, dogs, and rats, with glucuronides predominatingover sulfates. No aromatic analogues were detected.

Quantitative analyses of the major known metaboliteswere performed by LC-MS/MS. No gender differenceswere apparent in monkeys (Fig. 2) and dogs (Table 4).Metabolite HE3539 was present in low abundance infemale rats relative to males (Table 5), perhaps reflectiveof the limited array of androgen-specific aldo-keto-reductases in rodents [23]. The dominant unconjugatedmolecular species was parent HE3235 in rats and monkeys.In dogs HE3235 was more extensively metabolized byapproximately one-half of the animals of both genders, andin this species HE3539 and HE3562 frequently dominatedthe molecular profile. The relative abundance of the majormetabolites in humans (Table 6) is similar to rodents, which

is potentially important to the translation of preclinical anti-tumor activity into humans.

Pharmacokinetics

HE3235 is orally bioavailable and rapidly absorbed whenadministered as a cyclodextrin solution, an aqueoussuspension or a solid oral dosage form. Drug exposureincreased with increasing dose, but was frequently not doseproportional. The terminal half-life increased phylogeneti-cally. The integrated drug plasma concentration of HE3235parent and the known dioxygenated metabolites wascalculated.

Clinical pharmacokinetics

HE3235 was orally bioavailable in humans when adminis-tered in 5–50 mg doses as a solid oral dosage form. Parentdrug was the dominant unconjugated molecular species anddrug exposure was dose proportional (Table 7, Fig. 3). Theterminal half-life was approximately 14 h, and modest doseaccumulation was observed on day 28. All unconjugatedmetabolites were present in low abundance compared toparent (Table 8).

Table 11 Pharmacokinetics were measured in groups of five male andfemale beagle dogs following a single or 28 daily HE3235 oral gavagedoses of 20, 60, and 200mg/kg body weight, formulated incyclodextrin. Plasma (lithium heparin) was collected from a peripheralvein from each animal at each time point for each dose level at 0, 0.25,

0.5, 1, 2, 3, 4, 8, 12, and 24 h after drug administration. Samples werestored frozen prior to analysis. HE3539 and HE3562 were quantifiedby LC/MS-MS and pharmacokinetics parameters were calculated withWinNonlin. The “parent ratio” listed is the AUC(0–24) of themetabolite divided by HE3235 AUC(0–24)

HE3539 HE3562

Day Sex Dose(mg/kg)

Cmax (SD),(ng/mL)

Tmax, hr AUC (0–24)(SD), ng/mL*h

Parentratio

Cmax (SD),(ng/mL)

Tmax, hr AUC (0–24)(SD), ng/mL*h

Parentratio

1 M 20 531 (101) 1.6 3,783 (1,167) 1.2 2,377 (356) 1.2 11,542 (3,140) 3.7

14 M 20 939 (365) 2 7,440 (3,909) 1.6 2,494 (383) 1 16,256 (3,908) 3.4

28 M 20 978 (349) 1.8 7,140 (2,758) 1.4 2,361 (173) 1.1 16,574 (2,526) 3.2

1 F 20 606 (94) 1.1 3,278 (1,247) 1.6 2,567 (283) 0.5 9,881 (4,233) 4.7

14 F 20 903 (204) 1.1 5,757 (2,248) 1.8 2,485 (587) 0.5 13,214 (2,915) 4.2

28 F 20 849 (107) 1.6 5,560 (1,712) 1.4 2,351 (350) 1.7 14,794 (2,924) 3.8

1 M 60 943 (122) 1.8 10,957 (4,347) 1.8 3,726 (1,803) 1.6 26,003 (15,470) 4.3

14 M 60 2,415 (1,100) 1.4 17,715 (5,136) 1.7 8,621 (5,939) 1.6 41,632 (17,227) 4.1

28 M 60 2,018 (818) 1.6 16,101 (5,999) 1.5 5,475 (4,111) 1.4 39,324 (16,788) 3.8

1 F 60 788 (462) 2.5 8,545 (5,171) 0.6 2,300 (1,397) 2.6 22,583 (10,828) 1.5

14 F 60 1,801 (1,093) 2.5 13,518 (7,483) 0.7 4,533 (3,120) 2.6 29,680 (11,326) 1.6

28 F 60 2,180 (1,663) 1.8 16,383 (9,886) 1.0 5,044 (3,718) 2.6 32,786 (21,182) 2.0

1 M 200 1,122 (827) 2.6 14,447 (1,1030) 0.3 2,184 (904) 2.1 24,935 (12,472) 0.4

14 M 200 1,561 (909) 2.6 21,474 (1,3824) 0.3 2,160 (410) 3.3 30,908 (15,451) 0.5

28 M 200 6,219 (5,581) 2.3 48,160 (3,3562) 1.0 9,556 (7,918) 2.3 60,942 (40,296) 1.3

1 F 200 1,107 (363) 3 15,117 (5,922) 0.3 3,661 (3,239) 4.8 36,331 (13,782) 0.7

14 F 200 2,158 (1,163) 4.6 25,016 (10,995) 0.4 4,987 (2,961) 1.9 43,511 (16,607) 0.8

28 F 200 5,223 (3,145) 3 38,686 (14,780) 1.0 10,684 (6,406) 2 61,774 (17,034) 1.6


Pharmacokinetics in monkeys

PK results from monkeys are shown in Table 9 and Fig. 2.The percent absolute oral bioavailability of HE3235 was54% in the fasted state and 37% in the fed state. Thenormalized AUC(0-24) was 519 ng*hr/mL/mg/kg and394 ng*hr/mL/mg/kg for the soluble and particulateformulations respectively. The terminal half-life was ap-proximately 8 h with no apparent gender differences. Therelative abundance of HE3235 and deoxygenated metabo-lites resembled rat more than dog and HE3539 was equallyabundant in both sexes.

Pharmacokinetics in dogs

PK results from dogs are shown in Table 10. There wasconsiderable variability in exposure between individuals inall dose groups, apparently resulting from differentialmetabolism within dogs, but without correlation to gender.Dioxygenated metabolites were present in significant,although variable, abundance in both males and females(Table 11).

Pharmacokinetics in rats

Pharmacokinetics results from rats (Table 5) show thatexposure was approximately dose proportional over the25–200 mg/kg experimental range in females, but onlybetween the 100 and 200 mg/kg dose levels in males. Themean AUC(0–24) normalized to dose for all groupscombined was 48±26 ng*hr/mL/mg/kg. However, thevalue in males for the 25 mg/kg dose was appreciablylower (9.3 ng*hr/mL/mg/kg) than females (66.8 ng*hr/mL/mg/kg). The pronounced gender difference in expo-sure (overall mean of 234 ng*hr/mL in males vs.1,670 ng*hr/mL in females) could not be attributed todifferential production of the major dioxygenated metab-olites, which was small in comparison to HE3235.HE3759 was not quantified in the analysis due tointerfering substances in rat plasma.

Pharmacokinetics in mice

In mice, HE3235 has approximately 15% oral bioavailabil-ity. The 40 mg/kg Cmax was 1,818±693 ng/mL at a Tmax of

Table 12 Groups of five male beagle dogs received HE3235 by oralgavage for 28 days. Blood was collected on day 29 for automatedevaluation of hematology (Seimen Advia 120) and coagulation

(Diagnostica Stago Compact) parameters. Hgb hemoglobin, HCThematocrit, APTT activated partial prothrombin time

Table 12 Groups of five male beagle dogs received HE3235 by oralgavage for 28 days. Blood was collected on day 29 for automatedevaluation of hematology (Seimen Advia 120) and coagulation



Dose(mg/kg)

AnimalID

Leukocytes(103/μL)

RBC(106/μL)

Hgb(%)

HCT (%) Platelets(103/μL)

Reticulocytes(103/μL)

Reticulocytes(%)

APTT(sec)

Prothrombin(sec)

0 106 8.5 7.07 16.8 48.8 295 72.4 1 11.6 6.7

107 7.4 6.57 15 44.4 380 35.2 0.5 11.7 7.4

108 8.9 7.23 15.1 44.5 238 65.7 0.9 10.6 7.1

mean 8.3 7.0 15.6 45.9 304 57.8 0.8 11.3 7.1

SD 0.78 0.34 1.01 2.51 72 19.83 0.27 0.61 0.35

20 111 15.4 6.27 14.3 42.4 521 56.6 0.9 16.6 7.1

112 12.4 6.94 15.1 45.1 401 65.4 0.9 11.7 7.3

113 15.4 6.59 15.2 45.2 382 56.1 0.9 10.8 7.1

mean 14.4 6.6 14.9 44.2 435 59.4 0.9 13.0 7.2

SD 1.73 0.34 0.49 1.59 75 5.23 0.00 3.12 0.12

60 121 19.6 6.48 14.8 43.5 374 68.5 1.1 13.1 7.4

122 20.3 6.55 15.7 46.5 251 34.2 0.5 15.6 7.3

123 30.9 7.88 17.8 53 360 95.4 1.2 13.2 7.3

mean 23.6 7.0 16.1 47.7 328 66.0 0.9 14.0 7.3

SD 6.33 0.79 1.54 4.86 67 30.67 0.38 1.42 0.06

200 131 36.3 6.45 14.2 41.7 77 11.8 0.2 13.4 7.7

132a 22.4 6.83 16 47 72 5.3 0.1 12.7 7.2

133 31.3 5.59 13 38.7 310 28.7 0.5 11.9 7

134 30.8 6.17 14.1 42.9 140 23.4 0.4 12.1 6.9

mean 30.2 6.3 14.3 42.6 150 17.3 0.3 12.5 7.2

SD 5.76 0.52 1.24 3.44 111 10.67 0.18 0.68 0.36

a samples collected day 15

0.5 h. The drug was cleared quickly from circulation with aterminal half-life of approximately 0.5 h and very little wasdetectable in serum after 8 h (10±11 ng/mL). Plasmaexposure was 47±7.1 ng*hr/mL/mg/kg and was doseproportional over the 40–160 mg/kg experimental range.HE3235 was the dominant unconjugated species in circu-lation, although significant amounts of HE3539 andHE3562 were formed.

Safety studies

Genotoxicity

HE3235 did not cause chromosome damage in the mouseerythrocyte micronucleus test, and was negative in thebacterial reverse mutation assay. The in vitro mammalianchromosome aberration assay gave evidence of clastoge-nicity in CHO cells when the compound was used withoutmetabolic activation at high concentrations (70 μg/mL orgreater).

Systemic toxicity studies in rats

Chromodacryorrhea was observed in most high doseanimals, and severe anorexia was dose limiting in approx-imately one-half of the animals in the 200 mg/kg BID highdose groupAfter 13 days, the high dose was reduced to200 mg/kg QD and the anorexia resolved. There was notreatment effect on ophthalmology or evidence of neuro-toxicity in the functional observation battery. The highestdose without severe toxicity was 200 mg/kg.

Systemic toxicity studies in dogs

Severe anorexia was also observed in a subset of dogs in thehigh dose group, and three animals were sacrificed in extremis,but no specific pathologies were identified in association withthe observed morbidities. Surviving high dose animals showedlittle signs of toxicity and only mild anorexia.

Electrocardiographic and ophthalmologic examinations werenormal. Hematology measurements revealed highly variableplatelet counts at the high dose in both sexes with grade 3 and 4

Table 13 Groups of five female beagle dogs received HE3235 by oral gavage for 28 days. Blood was collected on day 29 for automatedevaluation of hematology (Seimen Advia 120) and coagulation (Diagnostica Stago Compact) parameters. Hgb hemoglobin, HCT hematocrit,APTT activated partial prothrombin time

Dose(mg/kg)

AnimalID

Leukocytes(103/μL)

RBC(106/μL)

Hgb(%)

HCT(%)

Platelets(103/μL)

Reticulocytes(103/μL)

Reticulocytes(%)

APTT(sec)

Prothrombin(sec)

0 106 9.7 6.35 14.6 42.5 425 44.4 0.7 10.6 7.2

107 6.9 6.3 14.9 43.7 169 46.2 0.7 11.7 7.0

108 7.0 6.38 14.9 44 331 26.1 0.4 11.6 7.1

mean 7.9 6.3 14.8 43.4 309 38.9 0.6 11.3 7.1

SD 1.59 0.04 0.17 0.79 130 11.1 0.173 0.61 0.10

20 116 8.4 6.5 16.1 46.6 255 37.9 0.6 12.5 7.7

117 12.2 7.23 16.2 47.6 393 69.5 1.0 12.3 6.9

118 11.2 6.59 15.5 45.1 305 37.3 0.6 12.2 7.1

mean 10.6 6.8 15.9 46.4 318 48.2 0.7 12.3 7.2

SD 1.97 0.40 0.38 1.26 70 18.4 0.231 0.15 0.42

60 126 13.8 6.77 15.6 45.5 357 66.1 1.0 13.7 7.2

127 14.8 5.98 14.5 42.7 286 55.3 0.9 14.2 7.4

128 12.5 7.19 15.9 46.7 444 53.1 0.7 12.2 7.1

mean 13.7 6.6 15.3 45.0 362 58.2 0.9 13.4 7.2

SD 1.15 0.61 0.74 2.05 79 6.96 0.153 1.04 0.15

200 136a 28.6 6.1 13.5 41.4 195 22 0.4 12.8 9.5

137b 18.6 7.17 15.6 46.9 620 42.3 0.6 10.8 6.9

138 28.2 6.0 15 42.5 346 28.1 0.5 12.8 7.4

139 52.2 6.28 14.8 42.9 27 13.8 0.2 14.4 6.9

140 34.7 5.18 12 36.3 127 53.3 1.0 12.8 7.7

mean 32.5 6.2 14.2 42.0 263 31.9 0.5 12.7 7.7

SD 12.45 0.71 1.44 3.81 231 15.9 0.297 1.28 1.07

a sample collected Day 20b sample collected Day 15

Table 13 Groups of five female beagle dogs received HE3235 by oralgavage for 28 days. Blood was collected on day 29 for automatedevaluation of hematology (Seimen Advia 120) and coagulation



thrombocytopenia observed in some animals, but red blood cellparameters were not affected. Dose dependent neutrophilia wasobserved in both sexes (Tables 12 and 13), although this wasnot considered an adverse response to treatment. Clinicalchemistry measurements showed modest liver enzymeincreases (Table 14) and serum potassium decreases (Table 15)at the highest dose. The mean testes and ovary weights weredecreased with HE3235 treatment (Table 16).

Histopathology revealed hormone effects that includedmild prostate hyperplasia, moderate degeneration ofseminiferous tubules, mammary gland hyperplasia inboth males and females, and epithelial hyperplasia ofthe uterus and vagina consistent with an estrogeniceffect. The reproductive organs (ovaries, uterus, andvagina) were immature and not actively cycling in amajority of the treated female animals in contrast tomature and cycling ovaries in the female control group.Moderate cholestasis, possibly associated with 17-glucronide conjugates of HE3235 and metabolites [24],and moderate to severe depletion of the thymus, consistent

with an estrogenic effect [25], was found in severalanimals in the high dose group. Dose dependent granulo-cytic hyperplasia was observed in the rib and sternumbone marrow, and minimal to moderate extramedullaryhematopoiesis was found in the liver and spleen. Alleffects showed evidence of reversibility in the survivingmale recovery animal. No dose-limiting toxicity wasobserved at the 20 or 60 mg/kg/day doses.

Nuclear hormone receptor interaction profile

Experiments to measure the nuclear receptor binding profileof HE3235 were conducted using AR, ERα and ERβ, GRand PR. The IC50 value for competition with a fluorescentreference ligand indicates that wild type AR does not havea strong affinity for HE3235 when compared to DHT.Although ERα has a higher affinity for HE3235 than AR, itis relatively weak compared to estradiol (Table 17). TheIC50 values for ERβ, GR and PR were greater than10,000 nM. HE3235 transactivated AR, ERα, and ERβ

Table 14 Beagle dogs received HE3235 by oral gavage for 28 days. Blood was collected on day 29 for automated evaluation of clinicalchemistries (Olympus AU 2700)

Male Female

AnimalNo.

GGT(U/L)

AST(U/L)

ALT(U/L)

SDH(U/L)

Total protein(g/dL)

Animalno.

GGT(U/L)

AST(U/L)

ALT(U/L)

SDH(U/L)

Total protein(g/dL)

0 101 3 33 29 5.3 5.6 106 4 31 24 5.7 5.5

102 4 29 23 3.3 5.7 107 3 24 27 4 5.3

103 4 27 23 3.5 5.4 108 3 33 28 6.2 5.3

mean 3.7 29.7 25.0 4.0 5.6 mean 3.3 29.3 26.3 5.3 5.4

SD 0.6 3.1 3.5 1.1 0.2 SD 0.6 4.7 2.1 1.2 0.1

20 111 5 32 52 4.8 6 116 4 26 44 4 6.4

112 4 28 50 4.2 6 117 3 34 41 6.6 5.9

113 4 22 28 2.4 5.6 118 4 35 34 3.9 6.2

mean 4.3 27.3 43.3 3.8 5.9 mean 3.7 31.7 39.7 4.8 6.2

SD 0.6 5.0 13.3 1.3 0.2 SD 0.6 4.9 5.1 1.5 0.3

60 121 4 25 30 5.1 5.9 126 3 34 41 4 6.3

122 4 40 79 5 7 127 3 34 49 5.8 6.4

123 5 28 81 4.7 6.3 128 5 31 37 3.3 6.1

mean 4.3 31.0 63.3 4.9 6.4 mean 3.7 33.0 42.3 4.4 6.3

SD 0.6 7.9 28.9 0.2 0.6 SD 1.2 1.7 6.1 1.3 0.2

200 136b 9 44 111 4.4 4.1

131 6 26 52 5.8 5.2 137a 3 31 30 5.3 5.5

132a 3 27 21 4.7 4.6 138 4 30 57 6.8 5.7

133 4 26 34 3.9 5.7 139 5 121 110 5.2 5.1

134 4 27 69 6 5.6 140 4 30 66 3.6 5.6

mean 3.7 26.7 41.3 4.9 5.3 mean 5.0 51.2 74.8 5.1 5.2

SD 0.6 0.6 24.8 1.1 0.6 SD 2.4 39.5 35.2 1.2 0.7

a samples collected day 15b sample collected day 20


with a greater potency than expected from the ligandaffinity measurements indicating a possible alternativepathway to transcription control (Table 18).

Metabol i te HE3539, 17α -e thynyl 3β , 17β -androstanediol, bound AR (277 nM), ERα (16 nΜ) andERβ (126 nΜ) with affinities that are potentially relevant toconcentrations that are achieved in human plasma. HE3539transactivates ERα/ERβ (0.9 nM) with high potency butnot AR, suggesting it may be an antagonist. HE3562, 17α-ethynyl-17β-hydroxy androstan-3-one, bound AR (15 nM),ERα (229 nM), GR (7 nM) and PR (130 nM), but not ERβ.

HE3235 treatment effects on plasma sex steroidconcentrations

The endogenous plasma concentrations of testosterone andits metabolic precursors were substantially reduced inHE3235-treated male dogs in a dose- and time-dependentmanner (Fig. 4, Table 19).

HE3235 effects on the enzymatic activity of CYP17and enzymes that convert DHEA to androgens

Testosterone, dihydrotestosterone, and DHEA formedfrom 5-androstenendiol were independent of 100 ng/mL HE3235 in H295R cells (data not shown), indicat-ing that direct inhibition of the metabolic pathways thatconverts 19-carbon precursors to androgens is not causalfor decreased plasma concentrations. Similarly, DHEA,testosterone, dihydrotestosterone, 5-androstene-3β,7β-diol, 4-androstenedione, and progesterone formedfrom pregnenolone were not decreased by HE3235,indicating that direct inhibition of CYP17 was also notcausal for decreased plasma sex steroid concentrations.These studies measured only direct enzyme inhibition,but did not measure steroidogenic enzyme expression,with potential effects on expression remaining an openquestion regarding suppression of sex steroidconcentrations.

Table 15 Beagle dogs received HE3235 by oral gavage for 28 days. Blood was collected on day 29 for automated evaluation of clinicalchemistries (Olympus AU 2700)

Dose (mg/kg) Male Female

Animal no. Na+ K+ Cl− Ca+2 PO4−3 APc Animal no. Na+ K+ Cl− Ca+2 PO4

−3 APc

0 101 146 4.7 111 11.2 6.2 88 106 146 4.2 110 11.0 4.8 131

102 146 4.6 110 11.2 6.3 78 107 145 4.3 110 10.7 5.2 63

103 145 4.8 111 11.0 7.2 76 108 145 4.8 111 11.0 5.2 87

mean 145.7 4.7 110.7 11.1 6.6 81 mean 145.3 4.4 110.3 10.9 5.1 94

SD 0.58 0.10 0.58 0.12 0.55 6.4 SD 0.58 0.32 0.58 0.17 0.23 34.5

20 111 150 4.9 111 10.9 6.6 491 116 148 4.8 109 11.6 7.2 75

112 146 4.9 109 11.8 7.3 84 117 147 4.8 110 11.4 6.6 73

113 146 5.1 111 11.3 6.1 78 118 148 4.9 111 11.4 6.8 80

mean 147.3 5.0 110.3 11.3 6.7 218 mean 147.7 4.8 110.0 11.5 6.9 76

SD 2.31 0.12 1.15 0.45 0.60 236.7 SD 0.58 0.06 1.00 0.12 0.31 3.6

60 121 146 4.3 111 11.0 6.7 78 126 146 4.3 108 11.4 6.7 86

122 145 4.5 106 12.2 7.1 82 127 147 4.5 109 11.8 6.5 107

123 151 3.9 110 11.8 6.8 101 128 146 4.3 111 11.0 6.0 71

mean 147.3 4.2 109.0 11.7 6.9 87 mean 146.3 4.4 109.3 11.4 6.4 88

SD 3.21 0.31 2.65 0.61 0.21 12.3 SD 0.58 0.12 1.53 0.40 0.36 18.1

200 136b 152 2.4 112 9.0 5.4 173

131 150 2.6 108 10.3 5.6 136 137a 151 3.8 108 10.3 6.0 129

132a 151 3.2 111 9.7 4.5 205 138 149 3.6 111 11.2 6.1 86

133 150 3.6 111 10.5 5.4 63 139 146 1.6 95 9.4 4.1 177

134 153 3.5 114 11.1 5.6 109 140 149 2.9 107 10.7 7.6 113

mean 151.3 3.4 112.0 10.4 5.2 126 mean 149.4 2.9 106.6 10.1 5.8 136

SD 1.53 0.21 1.73 0.70 0.59 72.5 SD 2.30 0.90 6.80 0.91 1.27 39.1

a sample collected Day 15b sample collected Day 20c alkaline phosphatase


Table 16 Groups of five beagle dogs per gender received HE3235 byoral gavage for 28 days. Animals were sacrificed on day 29 withsodium pentobarbital solution, and subjected to a complete necropsy.Organs were blotted dry and weighed; paired organs were weighed

together. Body weight is expressed in kilograms; all other organs areexpressed in grams. Statistical significance was determined byanalysis of variance (ANOVA), *p<0.05 vs. vehicle. The ratio is thetreatment value divided by the vehicle control value

0mg/kg 20mg/kg 60mg/kg 200mg/kg

Mean SD Mean SD Ratio Mean SD Ratio Mean SD Ratio

Male Body weight 9.18 0.78 10.1 0.89 1.100 9.78 0.4 1.065 8.99 0.97 0.979

Adrenal 0.808 0.327 0.794 0.154 0.983 0.847 0.298 1.048 1.178 0.561 1.458

Brain 74.339 12.115 75.681 5.336 1.018 70.412 7.402 0.947 73.148 3.478 0.984

Epididymides 2.105 0.18 2.377 0.346 1.129 2.343 0.42 1.113 2.659 0.481 1.263

Heart 66.502 5.614 76.453 13.903 1.150 72.605 2.582 1.092 63.239 8.792 0.951

Kidneys 46.664 8.181 53.914 11.548 1.155 48.719 6.177 1.044 47.952 6.786 1.028

Liver 226.442 28.834 244.02 9.709 1.078 224.134 19.497 0.990 237.522 19.744 1.049

Lung 72.936 8.868 76.307 7.054 1.046 71.87 1.622 0.985 78.029 5.96 1.070

Pituitary 0.065 0.001 0.068 0.006 1.046 0.049* 0.005 0.754 0.062 0.007 0.954

Prostate 4.938 1.94 7.784 4.993 1.576 8.719 1.79 1.766 10.122 3.5 2.050

Testes 11.996 1.743 9.043 1.368 0.754 4.421* 1.219 0.369 7.045* 1.798 0.587

Thymus 5.55 2.637 5.494 2.782 0.990 4.578 1.261 0.825 4.54 2.154 0.818

Female Body weight 6.5 0.96 8 0.84 1.231 7.49 0.45 1.152 6.47 1.11 0.995

Brain 62.54 4.834 65.239 3.281 1.043 69.354 4.92 1.109 61.012 3.15 0.976

Adrenal 1.194 0.095 0.729* 0.065 0.611 0.754* 0.155 0.631 0.79* 0.131 0.662

Heart 54.661 7.382 59.035 8.501 1.080 61.339 9.67 1.122 51.647 3.484 0.945

Kidneys 28.54a 3.385 45.107* 3.526 1.580 42.207* 2.57 1.479 42.605* 5.362 1.493

Liver 179.03 26.319 205.371 13.96 1.147 205.738 43.499 1.149 185.688 22.985 1.037

Lung 58.629 0.673 64.786 4.218 1.105 62.733 11.53 1.070 59.961 5.459 1.023

Ovaries 0.834 0.383 0.497 0.156 0.596 0.485 0.041 0.582 0.359* 0.093 0.430

Pituitary 0.052 0.014 0.065 0.017 1.250 0.043 0.013 0.827 0.049 0.002 0.942

Thymus 5.92 2.437 4.961 2.214 0.838 3.583 1.703 0.605 2.765 2.424 0.467

Uterus w/cervix 11.724 7.243 7.281 2.7 0.621 7 1.564 0.597 6.182 3.464 0.527

a The mean female kidney weight was approximately 50% lower than historical controls; there was no apparent effect of treatment on kidney function, orevidence of histopathology in the kidneys of HE3235 treated animals

Table 17 Binding activity for nuclear receptors was measured in homogeneous competition assays using the PolarScreen™ fluorescencepolarization system (InVitrogen, Carlsbad, CA). Serial dilutions of test compounds or reference competitor were incubated for 2 h at roomtemperature with recombinant human nuclear receptors. Reference ligands used for each receptor are listed. Results are expressed as IC50 valuesin nM, representing the mean ± SEM. The numbers in parenthesis represent the number of independent experiments performed (n value). ARAndrogen receptor, ERα Estrogen receptor α, ERβ Estrogen receptor β, GR Glucocorticoid receptor, PR Progesterone receptor, DHTDihydrotestosterone, E2 17β-estradiol, ND Not determined

Ligand AR ERα ERβ GR PR

DHT 15±4.2 (4) ND ND ND ND

E2 ND 8±5.4 (3) 7±1.6 (3) ND ND

Dexamethasone ND ND ND 8.2±0.83 (4) ND

Progesterone ND ND ND ND 15.5±2.1 (4)

HE3235 1,500 (3) 200 (3) >10,000 (3) >10,000 (3) >10,000 (3)

HE3539 277 16 126 5,000 2,640

HE3562 15 229 >10,000 7 130

Table 17 Binding activity for nuclear receptors was measured inhomogeneous competition assays using the PolarScreen™ fluores-cence polarization system (InVitrogen, Carlsbad, CA). Serial dilutionsof test compounds or reference competitor were incubated for 2 h atroom temperature with recombinant human nuclear receptors. Refer-ence ligands used for each receptor are listed. Results are expressed as

IC50 values in nM, representing the mean ± SEM. The numbers inparenthesis represent the number of independent experiments per-formed (n value). AR Androgen receptor, ERα Estrogen receptor α,ERβ Estrogen receptor β, GR Glucocorticoid receptor, PR Progester-one receptor, DHT Dihydrotestosterone, E2 17β-estradiol, ND Notdetermined


Discussion

HE3235 is in clinical trials for the treatment of castration-resistant prostate cancer. The results of the pharmacologicalprofiling have complemented previous work regarding themechanisms of action of this novel drug [5, 26]. Preclinicalsafety studies with HE3235 indicate a low potential for adose-limiting toxicity at therapeutically relevant exposures(Table 20). Anorexia and hypokalemia, which are easilymonitored in humans, were observed only at 10-fold greater

exposure than found with a 100 mg dose in clinical studies.Thymic depletion in dogs may be attributed to the estrogenicityof metabolite HE3539, which was probably accentuated by thehigh-administered dose and the unusually high prevalence ofthis metabolite in canines [25].

Neither HE3235 nor its metabolites were potent inhib-itors or inducers of the major P450 enzymes. HE3235 doesnot inhibit the major P450 enzymes at clinically relevantconcentrations, which is a highly desirable characteristic fora drug that is likely to be used in combination with

Table 18 Transactivation of nuclear hormone receptors for AR, ERα,ERβ, and GR was measured in stably-transfected human cancer celllines expressing nuclear receptor-sensitive luciferase reporter genes.Luciferase assays were performed as previously described [16], with20,000 cells per well in 100μL of phenol red-free RPMI supple-mented with 4mM L-glutamine and 10% charcoal-stripped FBS in96-well clear bottom white microtiter plates. The cells were incubatedwith serial dilutions of test compounds overnight at 37°C prior to

measuring luciferase activity. Transactivation assays for ERβ and amutated form of AR (mt-AR) found in LNCaP cells were performedby transient transfection of HEK293 fibroblasts using expressionplasmids encoding full-length human GR or ERβ and appropriateluciferase reporter vectors. Results are expressed as EC50 values (nM),representing the mean ± SEM. The numbers in parenthesis representthe number of independent experiments performed (n value). Noparentheses indicate n=1

Ligand Mutated ARa AR/GRb ERα/ERβc ERβd

DHT 0.5 0.06±0.03 (9) 112 408±224 (6)

E2 1.1 987±293 (5) 0.002±0.002 (10) 0.04±0.04 (17)

AED 144±171 (5) 2969+790 (7) 2.5±0.76 1.7+0.26 (7)

HE3235 0.48+0.69 (4) 11.4±9.3 (3) 0.8 164 (3)

HE3539 ND >10,000 0.9 ND

HE3562 ND ND 11 ND

aMtAR-HEK293 cells are HEK293 fibroblasts transiently co-transfected with an ARE/luciferase promoter/reporter construct and a cDNA expression vectorencoding the full-length LNCaP AR, with virtually undetectable levels of endogenous sex steroid receptorsbMDA-kb2 cells are stably transfected with a promoter/reporter construct sensitive to sex steroid receptor stimulation (MMTV promoter) fused upstream ofa luciferase reporter gene, and endogenously express both AR and GRc T47D-kBluc cells are stably transfected with a synthetic promoter/reporter construct sensitive to estrogenic stimulation, consisting of 3 copies of theestrogen response element (ERE) fused upstream of a luciferase reporter gene, and endogenously express both ERα and ERβd ERβ-HEK293 cells are HEK293 fibroblasts transiently co-transfected with an ERE/luciferase promoter/reporter construct and a cDNA expression vectorencoding the full-length human ERβ, with virtually undetectable levels of endogenous sex steroid receptors

Vehicle

00.10.20.30.40.50.60.70.80.9

1

14 28Study Day

Rat

io t

o D

ay 1

TA4DHEA

HE3235, 20 mg/kg

00.10.20.30.40.50.60.70.80.9

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14 28Study Day

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io t

o D

ay 1

TA4DHEA

HE3235, 60 mg/kg

00.10.20.30.40.50.60.70.80.9

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14 28Study Day

Rat

io t

o D

ay 1

TA4DHEA

HE3235, 200 mg/kg

00.10.20.30.40.50.60.70.80.9

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14 28Study Day

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io t

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ay 1

TA4DHEA

Fig. 4 Groups of five male bea-gle dogs received 0 (vehicle), 20,60, and 200mg/kg HE3235 byoral gavage for 28days. Plasma(lithium heparin) was collectedprior to dosing on day 1, 14, and28. Samples were analyzed fortestosterone (T), androstenedione(A4), and DHEA by LC/MS-MS.The y-axis is of the mean con-centration on day 14 and 28divided by day 1


numerous co-medications, as is usually the case incancer therapy.

Like most steroids, HE3235 is subject to extensiveprimary and secondary metabolism. The 3α-hydroxylfunction, but not the 17b-hydroxyl, can be oxidized andinverted by aldo-keto-reductases that are present in sexhormone-responsive tissues and the liver, forming the majormetabolites, HE3562 (oxidation of HE3235) and HE3539(HE3235 inversion or HE3562 reduction), both of whichhave LNCaP anti-proliferative activity in vitro [23]. Thesedi-oxgenated metabolites likely have the most relevantpharmacology to sex hormone-responsive cancers, consideringtheir capacity to interact within structurally related steroidsignaling pathways. Such interactions are likely to be concen-tration dependent, and thereby responsive to dose escalation.Several metabolites are triols, and one such, HE3759 (2α-hydroxy derivative), is present in high abundance, but itspharmacology has not been explored. The other triol metabo-lites are likely formed by CYP7B, 11β-hydroxysteroid

dehydrogenase, and CYP3A, enzymes known to form 7-α, 7-β, and 16α-hydroxy steroid metabolites respectively [27, 28].

In vitro metabolism formed all primary metabolitesfound in vivo except one, a tetrol observed in monkeys.None of the metabolites detected in vivo were speciesspecific, although canine metabolism produced relativelylarge proportions of HE3539 and HE3562, which differedquantitatively from other species (Tables 6, 7, and 8). Therelative abundance of unconjugated metabolites in rodentswas similar to primates where the dominant molecularspecies was HE3235. This was an important factor whenevaluating effects in preclinical efficacy models, andprojecting the potential outcome of these compounds inthe treatment of human disease. Rodents produce more trioland tetrol metabolites from HE3235 than primates orcanines, which are a concern when projecting activity fromrodents into higher species. However, HE3235 was shownto have low nanomolar potency (EC50=6 nM) againstLNCaP cells in vitro, suggesting that highly oxidized

Table 19 Groups of five male beagle dogs received HE3235 by oral gavage for 28 days. Plasma (lithium heparin) was collected prior to dosingon day 1, 14, and 28. Samples were analyzed for testosterone (T), androstenedione (A4), and DHEA by LC/MS-MS

HE3235mg/kg Day T, pg/mL A4, pg/mL DHEA, pg/mL

Mean SD Day 1 ratio Mean SD Day 1 ratio Mean SD Day 1 ratio

0 1 2181 1407 1.000 642 394 1.000 1889 1957 1.000

14 1802 1328 0.826 504 321 0.785 1172 991 0.621

28 1846 1456 0.846 501 283 0.781 4540 6459 2.403

20 1 1463 744 1.000 464 200 1.000 2058 1744 1.000

14 130 122 0.089 100 90 0.216 189 182 0.092

28 80 75 0.055 71 71 0.154 449 603 0.218

60 1 903 631 1.000 383 182 1.000 975 924 1.000

14 161 111 0.178 132 92 0.346 193 127 0.198

28 8 6 0.009 13 6 0.033 62 4 0.064

200 1 1140 827 1.000 360 178 1.000 813 855 1.000

14 77 101 0.067 68 74 0.188 159 59 0.196

28 8 3 0.007 30 15 0.084 71 11 0.087

Table 20 Total daily HE3235 AUC from a BID dosing schedule at steady-state was estimated as the sum of the day 28 AUC(0–8) plus AUC(0–24).The AUC for the 200mg dose was estimated from the median exposure at 100mg assuming dose proportionality. The safety margin wascalculated for each dose level by dividing the mean daily AUC achieved in dogs at the highest dose that did not produce dose-limiting toxicity(8,830 ng*hr/mL at 60mg/kg) by the mean steady state daily HE3235 AUC in humans

Daily dose, mg Median SS daily HE3235 AUC (ng*hr/mL) Safety margin

10 70 126

20 139 63.5

30 368 24

50 406 22

100 927 9.5

200 2060a 4.3

a estimated

Table 20 Total daily HE3235 AUC from a BID dosing schedule atsteady-state was estimated as the sum of the day 28 AUC(0–8) plusAUC(0–24). The AUC for the 200mg dose was estimated from themedian exposure at 100mg assuming dose proportionality. The safety

margin was calculated for each dose level by dividing the mean dailyAUC achieved in dogs at the highest dose that did not produce dose-limiting toxicity (8,830 ng*hr/mL at 60mg/kg) by the mean steadystate daily HE3235 AUC in humans


metabolites were not responsible for the anti-cancer activityfound in rodent models [26].

Although the in vitro transactivation data may portendestrogenic potential, both from HE3235 and its majormetabolites, estrogenic effects in rodent and canine modelswere mild in light of the very high exposure levels. Therewere no adverse systemic toxicological effects at 60 mg/kg(HE3235 exposure of approximately 8,300 ng*hr/mL). Asthere were high concentrations of both HE3539 andHE3562, the sum of the mean daily exposure of unconju-gated ethynylated metabolites was elevated to greater than54,000 ng*hr/mL. The corresponding steady-state exposurefrom ethynylated metabolites in humans is only about1,200 ng*hr/mL in patients receiving 100 mg of HE3235daily, which at this dose establishes a margin of safety ofgreater than 40-fold for this exposure parameter.

The interactions of HE3235 and metabolites HE3539and HE3562 with nuclear hormone receptors in vitro appearto be complex. Collectively they exhibit the potential forboth agonist and antagonist activity for AR as well asagonist activity for ERα/ERβ. The potential for HE3235metabolites to transactivate ERβ is of significant impor-tance considering the well-established anti-proliferative/pro-apoptotic role of ERβ in prostate tissue [29]. The corestructure of metabolite HE3539 (17α-ethynyl-5α-andros-tane-3β,17β-diol) is androstanediol (3β5α-diol), an ERβselective ligand that is naturally formed in the prostate bythe action of 3β-hydroxysteroid dehydrogenase on dihy-drotestosterone [29]. In humans, the relative abundance offree HE3539 is very low, but significant amounts of theconjugate are formed, and the potential impact on ERβ isan important question considering that free metabolites inthe plasma may not reflect the intracellular milieu.

The ability to interfere with sex hormone steroidogenesisthrough a mechanism that is complementary to existingprostate cancer treatments would be a significant advance-ment. Koreckij, et al., reported that HE3235 decreasedintratumoral androgen concentrations [5]. Here we report asubstantial reduction in the endogenous plasma concen-trations of testosterone and androstenedione in HE3235-treated male dogs. HE3235 does not appear to interferewith the hypothalamus-pituitary-gonadal axis. Endogenouslevels of DHEAwere also diminished, but not as drastically(90% reduction), suggesting that the compound’s actionwas independent in gonadal and adrenal tissue, becausetestosterone production in the testes is not dependent onadrenal DHEA [30]. Investigation of the steroidogenicenzymes indicated there was no HE3235 mediated enzymeinhibition. We note that a preliminary experiment in femalerats indicated that HE3235 also dramatically reducedplasma concentrations of estradiol and estrone, without anapparent effect on plasma levels of LH, FSH, and ACTH(unpublished observations).

HE3235 pharmacokinetics in humans is amenable toeither a once or twice daily dosing schedule, although twicedaily dosing (approximately one half-life intervals) toreduce concentration fluctuations and maintain anti-tumor“pressure” is preferred. Dose accumulation of HE3235 upto approximately 2-fold was observed, which is consistentwith the half-life, from which it is expected that steady-statein humans is achieved in a few days.

HE3235 has an unusual combination of desirablepharmaceutical properties. Potent preclinical activity andthe similarity of metabolite profiles in efficacy models andhumans provide a basis for anticipating significant anti-neoplastic activity in clinical trials. Although the initialclinical focus was on late-stage patients, benefit may alsobe anticipated for earlier stage patients, considering thepotential for greater mechanistic contributions from inhibi-tion of sex steroid production and ERβ mediated anti-proliferative effects. Activity in prostate cancer wouldsuggest that clinical investigations in breast cancer andcancers of other hormone-responsive tissues might alsoelicit clinical benefit.

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Documents

17α-Alkynyl 3α, 17β-androstanediol non-clinical and clinical pharmacology, pharmacokinetics and metabolism