Karen A. Veverka

Estrogen Receptor-β-selective Ligands Alleviate High-fat Diet- and Ovariectomy-induced Obesity in Mice

Obesity is an epidemic problem affecting millions of people in the Western hemisphere and costs the United States economy more than

Toremifene – a promising therapy for the prevention of prostate cancer and complications of androgen deprivation therapy

200 billion annually. Currently, there are no effective treatments to combat obesity. Recent studies have implicated the constitutive activity of estrogen receptor (ER) β as an important regulator of metabolic diseases. However, the potential of ER-β-selective ligands to offset obesity is not clear. We evaluated the pharmacological effect of ER-β-selective ligands (β-LGNDs) in animal models of high-fat diet- and ovariectomy-induced obesity. Ligand binding, transactivation, and uterotrophic studies with β-LGNDs demonstrated selectivity for ER-β over ER-α. Animals fed a high-fat diet showed a significant increase in body weight, and this weight gain was attenuated by β-LGNDs. High-fat diet-mediated increases in serum cholesterol, leptin, glucose, and fat accumulation in organs were also reduced by β-LGNDs. In addition, MRI scanning indicated that β-LGNDs altered body composition by reducing fat mass and increasing lean body mass. Organ weights and gene expression analyses demonstrated that adipose tissue is the center of action for β-LGNDs, and the reduction in body weight is likely due to increased energy expenditure. In vitro and in vivo mechanistic studies indicated that the anti-obesity effects of β-LGNDs were due to indirect peroxisome proliferator-activated receptor γ antagonistic actions requiring the ligand binding domain of ER-β and through abrogation of the ability of PGC-1 to coactivate peroxisome proliferator-activated receptor γ. In conclusion, these studies indicate that ligand-activated ER-β is a potential therapeutic target to combat obesity and obesity-related metabolic diseases.

Role and pharmacologic significance of cytochrome P-450 2D6 in oxidative metabolism of toremifene and tamoxifen†‡

Deregulation of the estrogen axis in humans prompts a series of tissue-specific events. In the breast and prostate, alterations in estrogen signalling lead to genotypic and phenotypic molecular alterations that result in dysplastic cellular appearance, deregulated cell growth and carcinoma. In bone, decreased estrogen leads to increased osteoclastogenesis and bone resorption, decreased bone mineral density and a significant fracture risk. Toremifene is a selective estrogen receptor modulator that exerts pharmacological activity in the breast, bone and prostate. An intense interest in developing this agent for prostate cancer chemoprevention is based on the reduction of premalignant and malignant prostate lesions in a transgenic model of prostate cancer. Biological and clinical activity was demonstrated in Phase II trials by the prevention of progression to prostate cancer in men with high-grade prostate intraepithelial neoplasia and through suppression of bone turnover biomarkers and increased bone mineral density in men on androgen deprivation therapy for prostate cancer.

Absorption, distribution, metabolism and excretion of the novel SARM GTx-024 [(S)-N-(4-cyano-3-(trifluoromethyl)phenyl)-3-(4-cyanophenoxy)-2-hydroxy-2-methylpropanamide] in rats.

We investigated the in vitro metabolism and estrogenic and antiestrogenic activity of toremifene (TOR), tamoxifen (TAM) and their metabolites to better understand the potential effects of cytochrome P‐450 2D6 (CYP2D6) status on the activity of these drugs in women with breast cancer. The plasma concentrations of TOR and its N‐desmethyl (NDM) and 4‐hydroxy (4‐OH) metabolites during steady‐state dosing with TOR were also determined. Unlike TOR, TAM and its NDM metabolite were extensively oxidized to 4‐OH TAM and 4‐OH‐NDM TAM by CYP2D6, and the rate of metabolism was affected by CYP2D6 status. 4‐OH‐NDM TOR concentrations were not measurable at steady state in plasma of subjects taking 80 mg of TOR. Molecular modeling provided insight into the lack of 4‐hydroxylation of TOR by CYP2D6. The 4‐OH and 4‐OH‐NDM metabolites of TOR and TAM bound to estrogen receptor (ER) subtypes with fourfold to 30‐fold greater affinity were 35‐ to 187‐fold more efficient at antagonizing ER transactivation and had antiestrogenic potency that was up to 360‐fold greater than their parent drugs. Our findings suggest that variations in CYP2D6 metabolic capacity may cause significant differences in plasma concentrations of active TAM metabolites (i.e., 4‐OH TAM and 4‐OH‐NDM TAM) and contribute to variable pharmacologic activity. Unlike TAM, the clinical benefits in subjects taking TOR to treat metastatic breast cancer would not likely be subject to allelic variation in CYP2D6 status or affected by coadministration of CYP2D6‐inhibiting medications.

Preclinical characterization of a novel diphenyl benzamide selective ERα agonist for hormone therapy in prostate cancer

Abstract 1. GTx-024, a novel selective androgen receptor modulator, is currently being investigated as an oral treatment for muscle wasting disorders associated with cancer and other chronic conditions. 2. Absorption of GTx-024 was rapid and complete, with high oral bioavailability. A wide tissue distribution of [14C]GTx-024 derived radioactivity was observed. [14C]GTx-024-derived radioactivity had a moderate plasma clearance (117.7 and 74.5 mL/h/kg) and mean elimination half-life of 0.6 h and 16.4 h in male and female rats, respectively. 3. Fecal excretion was the predominant route of elimination, with ∼70% of total radioactivity recovered in feces and 21–25% in urine within 48 h. Feces of intact rats contained primarily unchanged [14C]GTx-024 (49.3–64.6%). Metabolites were identified in urine and feces resulting from oxidation of the cyanophenol ring (M8, 17.6%), hydrolysis and/or further conjugation of the amide moiety (M3, 8–12%) and the cyanophenol ring (M4, 1.3–1.5%), and glucuronidation of [14C]GTx-024 at the tertiary alcohol (M6, 3.5–3.7%). There was no quantifiable metabolite in plasma. 4. In summary, in the rat GTx-024 is completely absorbed, widely distributed, biotransformed through several metabolic pathways, and eliminated in feces primarily as an unchanged drug.

Drug interaction potential of toremifene and N-desmethyltoremifene with multiple cytochrome P450 isoforms.

Androgen deprivation therapy (ADT) is the mainstay of treatment for advanced prostate cancer. ADT improves overall and disease-free survival rates, but long-term therapy is associated with severe side effects of androgen and estrogen depletion including hot flashes, weight gain, depression, and osteoporosis. Effective hormone reduction can be achieved without estrogen deficiency-related side effects by using therapy with estrogenic compounds. However, cardiovascular complications induced by estrogens coupled with the availability of LHRH agonists led to discontinuation of estrogen use for primary androgen deprivation therapy in the 1980s. New treatments for prostate cancer that improve patient outcomes without the serious estrogen deficiency-related toxicities associated with ADT using LHRH analogs are needed. Herein we describe a novel nonsteroidal selective estrogen receptor-α agonist designed for first-line therapy of advanced prostate cancer that in animal models induces medical castration and minimizes many of the estrogen deficiency-related side effects of ADT. The present studies show that orally administered GTx-758 reversibly suppressed testosterone to castrate levels and subsequently reduced prostate volume and circulating prostate-specific antigen in relevant preclinical models without inducing hot flashes, bone loss, thrombophilia, hypercoagulation, or increasing fat mass.

1448 ORAL ADMINISTRATION OF GTX-758, A SELECTIVE ERα AGONIST, INDUCES CHEMICAL CASTRATION BUT NOT GYNECOMASTIA IN MALE MONKEYS

Toremifene is an effective agent for the treatment of breast cancer in postmenopausal women and is being evaluated for its ability to prevent bone fractures in men with prostate cancer taking androgen deprivation therapy. Due to the potential for drug–drug interactions, the ability of toremifene and its primary circulating metabolite N-desmethyltoremifene (NDMT) to inhibit nine human cytochrome P450 (CYP) enzymes was determined using human liver microsomes. Induction of CYP1A2 and 3A4 by toremifene was also investigated in human hepatocytes. Toremifene did not significantly inhibit CYP1A2 or 2D6. However, toremifene is a competitive inhibitor of CYP3A4, non-competitive inhibitor of CYP2A6, 2C8, 2C9, 2C19 and 2E1 and mixed-type inhibitor of CYP2B6. CYP inhibition by NDMT was similar in magnitude to toremifene. Toremifene did not induce CYP1A2 but increased CYP3A4 monooxygenase activity and gene expression in drug-exposed human primary hepatocytes. Although clinical doses of toremifene produce steady state exposures to toremifene and NDMT that may be sufficient to cause pharmacokinetic drug–drug interactions with other drugs metabolised by CYP2B6, CYP2C8, CYP3A4, CYP2C9 and CYP2C19, these data indicate that toremifene is unlikely to play a role in clinical drug–drug interactions with substrate drugs of CYP1A2 and CYP2D6.

Pharmacodynamics of Selective Androgen Receptor Modulators

INTRODUCTION AND OBJECTIVES: Gynecomastia is a psychologically distressing and sometimes painful estrogen-related side effect experienced by men on androgen deprivation therapy (ADT) for prostate cancer (PCa). Gynecomastia affects 40–80% of men on estrogen therapy (e.g. diethylstilbesterol; DES) and is of high incidence in men on antiandrogen monotherapy and in combination with a luteinizing hormone releasing hormone (LHRH) agonist. GTx-758 is a novel oral selective estrogen receptor alpha (ER ) agonist designed as first or second line therapy for advanced PCa. We evaluated and compared the effects of GTx-758 and DES on serum hormones, prostate specific antigen (PSA) and male breast development (gynecomastia) in male nonhuman primates. METHODS: Male monkeys (n 4–7 /group, 4 years of age), received daily gavage with vehicle, DES (0.05 or 2 mg/kg) or GTx-758 (1, 10, or 100 mg/kg) for a period of at least 56 days. Nipple diameter and prepuce were examined at necropsy. Mammary gland thickness was measured as the greatest thickness of glandular elements perpendicular to skin from histologic sections. Serum testosterone and PSA concentrations were determined by enzyme immunoassay. RESULTS: GTx-758 and DES reduced serum testosterone to castrate levels (i.e., 50 ng/dL) and GTx-758 markedly suppressed PSA. Treatment with DES produced signs of hyperestrogenicity, including a highly significant bilateral increase in nipple diameter as well as swollen prepuce in 4/4 (100%) monkeys within 22 days of treatment initiation with 2 mg/kg DES. Nipple diameter was 1.6 and 2 fold (P 0.01) higher for 0.05 and 2 mg/kg DES, respectively, compared to vehicle control. Mammary gland hypertrophy was also evident by a 5.3 to 5.8 fold increase in thickness for DES-treated monkeys compared to vehicle control. Monkeys treated with GTx-758 did not exhibit similar magnitudes of change in mammary or scrotal tissue. CONCLUSIONS: Oral administration of GTx-758 and DES significantly reduced serum testosterone to castrate levels and GTx758 lowered serum PSA. However, unlike DES, monkeys treated with GTx-758 did not exhibit significant signs of hyperestrogenicity including gynecomastia. Future phase II clinical trials are designed to demonstrate the efficacy and safety of GTx-758 and provide evidence that GTx-758 is an attractive option for first-line hormone therapy in advanced prostate cancer.