Discovery of the selective androgen receptor modulator MK-0773 using a rational development strategy based on differential transcriptional requirements for androgenic anabolism versus reproductive physiology.

Selective androgen receptor modulators (SARMs) are androgen receptor (AR) ligands that induce anabolism while having reduced effects in reproductive tissues. In various experimental contexts SARMs fully activate, partially activate, or even antagonize the AR, but how these complex activities translate into tissue selectivity is not known. Here, we probed receptor function using >1000 synthetic AR ligands. These compounds produced a spectrum of activities in each assay ranging from 0 to 100% of maximal response. By testing different classes of compounds in ovariectomized rats, we established that ligands that transactivated a model promoter 40-80% of an agonist, recruited the coactivator GRIP-1 <15%, and stabilized the N-/C-terminal interdomain interaction <7% induced bone formation with reduced effects in the uterus and in sebaceous glands. Using these criteria, multiple SARMs were synthesized including MK-0773, a 4-aza-steroid that exhibited tissue selectivity in humans. Thus, AR activated to moderate levels due to reduced cofactor recruitment, and N-/C-terminal interactions produce a fully anabolic response, whereas more complete receptor activation is required for reproductive effects. This bimodal activation provides a molecular basis for the development of SARMs.

Applicability of in vitro-in vivo translation of cathepsin K inhibition from animal species to human with the use of free-drug hypothesis.

The correlation of in vitro inhibition of cathepsin K (CatK) activity and in vivo suppression of collagen I biomarkers was examined with three selective CatK inhibitors to explore the potential translatability from animal species to human. These inhibitors exhibited good in vitro potencies toward recombinant CatK enzymes across species, with IC50 values ranging from 0.20 to 6.1 nM. In vivo studies were conducted in animal species following multiple-day dosing of the CatK inhibitors to achieve steady-state plasma drug concentration-time profiles. Measurement of urinary bone resorption biomarkers (cross-linked N-terminal telopeptide and helical peptide of type I collagen) revealed drug concentration-dependent suppression of biomarkers, with EC50 values estimated to be 12 to 160 nM. Marked improvement in the correlation between in vitro and in vivo CatK activities was observed with the application of unbound (free) fraction in plasma, consistent with the conditions stipulated by the free-drug hypothesis. These results indicate that the in vitro-in vivo translation of CatK inhibition observed in animal species can translate to humans when the unbound fraction of the inhibitor is considered. Interestingly, residual levels of urinary bone resorption marker were detected as the suppression reached saturation (at an average of 82% inhibition), an apparent phenomenon observed regardless of the species, biomarker, or compound examined. Since cathepsin enzymes other than CatK were reported to catalyze cleavage of collagen I, it is hypothesized that CatK-mediated degradation of collagen I in bone represents ~82% of overall collagen I turnover in the body.

Identification of an anabolic selective androgen receptor modulator that actively induces death of androgen-independent prostate cancer cells.

Prostate cancer (PCa) initially responds to inhibition of androgen receptor (AR) signaling, but inevitably progresses to hormone ablation-resistant disease. Much effort is focused on optimizing this androgen deprivation strategy by improving hormone depletion and AR antagonism. However we found that bicalutamide, a clinically used antiandrogen, actually resembles a selective AR modulator (SARM), as it partially regulates 24% of endogenously 5α-dihydrotestosterone (DHT)-responsive genes in AR(+) MDA-MB-453 breast cancer cells. These data suggested that passive blocking of all AR functions is not required for PCa therapy. Hence, we adopted an active strategy that calls for the development of novel SARMs, which induce a unique gene expression profile that is intolerable to PCa cells. Therefore, we screened 3000 SARMs for the ability to arrest the androgen-independent growth of AR(+) 22Rv1 and LNCaP PCa cells but not AR(-) PC3 or DU145 cells. We identified only one such compound; the 4-aza-steroid, MK-4541, a potent and selective SARM. MK-4541 induces caspase-3 activity and cell death in both androgen-independent, AR(+) PCa cell lines but spares AR(-) cells or AR(+) non-PCa cells. This activity correlates with its promoter context- and cell-type dependent transcriptional effects. In rats, MK-4541 inhibits the trophic effects of DHT on the prostate, but not the levator ani muscle, and triggers an anabolic response in the periosteal compartment of bone. Therefore, MK-4541 has the potential to effectively manage prostatic hypertrophic diseases owing to its antitumor SARM-like mechanism, while simultaneously maintaining the anabolic benefits of natural androgens.

Cathepsin K inhibitors prevent bone loss in estrogen-deficient rabbits.

Two cathepsin K inhibitors (CatKIs) were compared with alendronate (ALN) for their effects on bone resorption and formation in ovariectomized (OVX) rabbits. The OVX model was validated by demonstrating significant loss (9.8% to 12.8%) in lumbar vertebral bone mineral density (LV BMD) in rabbits at 13-weeks after surgery, which was prevented by estrogen or ALN. A potent CatKI, L-006235 (L-235), dosed at 10 mg/kg per day for 27 weeks, significantly decreased LV BMD loss (p < .01) versus OVX-vehicle control. ALN reduced spine cancellous mineralizing surface by 70%, whereas L-235 had no effect. Similarly, endocortical bone-formation rate and the number of double-labeled Haversian canals in the femoral diaphysis were not affected by L-235. To confirm the sparing effects of CatKI on bone formation, odanacatib (ODN) was dosed in food to achieve steady-state exposures of 4 or 9 µM/day in OVX rabbits for 27 weeks. ODN at both doses prevented LV BMD loss (p < .05 and p < .001, respectively) versus OVX-vehicle control to levels comparable with sham or ALN. ODN also dose-dependently increased BMD at the proximal femur, femoral neck, and trochanter. Similar to L-235, ODN did not reduce bone formation at any bone sites studied. The positive and highly correlative relationship of peak load to bone mineral content in the central femur and spine suggested that ODN treatment preserved normal biomechanical properties of relevant skeletal sites. Although CatKIs had similar efficacy to ALN in preventing bone loss in adult OVX rabbits, this novel class of antiresorptives differs from ALN by sparing bone formation, potentially via uncoupling bone formation from resorption.