Michael A. Gentile

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.

The effects of the aminobisphosphonate alendronate on thyroid hormone-induced osteopenia in rats

SummaryHyperthyroidism, either endogenous or iatrogenic, leads to increased bone turnover and osteopenia. This study was conducted to examine (1) whether thyroid hormone excess in rats causes bone changes similar to those seen in patients with hyperthyroidism, and (2) the effects of the aminobisphosphonate alendronate on the thyroid hormone-induced bone changes. Sprague-Dawley male rats, divided into four groups, received L-thyroxine (T4) 250 μg/kg/day (+T4) or vehicle (-T4) subcutaneously six times per week and alendronate 1.75 mg/kg (+ALN) or vehicle (-ALN) orally twice a week. Rats were sacrificed after 3 weeks of treatment, blood samples were analyzed for serum T4, triiodo-L-thyronine (T3), and osteocalcin, and the proximal tibiae were processed for histomorphometric analysis. Serum T4 and T3 levels measured 20–24 hours after the last injection were 2 to 2.5-fold higher in +T4 groups than in-T4 groups. Serum osteocalcin was significantly (P < 0.05) higher in +T4/-ALN group than in the other groups, which were not statistically different from each other. T4 treatment (+T4/-ALN) significantly decreased the amount of cancellous bone volume (-45%) and increased osteoid surface (+254%), osteoblast surface (+111%), and osteoclast surface (+176%) relative to control values. Alendronate increased the bone volume above control values in both T4-treated (+ T4/ +ALN) and untreated (-T4/ +ALN) rats, and prevented the T4-induced increase in bone turnover in +T4/+ALN rats. It is concluded that (1) excess thyroid hormone induces cancellous bone loss associated with high bone turnover in the rat, and (2) this bone loss can be prevented by alendronate through the inhibition of osteoclastic activity.

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.

Identification of Anabolic Selective Androgen Receptor Modulators with Reduced Activities in Reproductive Tissues and Sebaceous Glands

Androgen replacement therapy is a promising strategy for the treatment of frailty; however, androgens pose risks for unwanted effects including virilization and hypertrophy of reproductive organs. Selective Androgen Receptor Modulators (SARMs) retain the anabolic properties of androgens in bone and muscle while having reduced effects in other tissues. We describe two structurally similar 4-aza-steroidal androgen receptor (AR) ligands, Cl-4AS-1, a full agonist, and TFM-4AS-1, which is a SARM. TFM-4AS-1 is a potent AR ligand (IC50, 38 nm) that partially activates an AR-dependent MMTV promoter (55% of maximal response) while antagonizing the N-terminal/C-terminal interaction within AR that is required for full receptor activation. Microarray analyses of MDA-MB-453 cells show that whereas Cl-4AS-1 behaves like 5α-dihydrotestosterone (DHT), TFM-4AS-1 acts as a gene-selective agonist, inducing some genes as effectively as DHT and others to a lesser extent or not at all. This gene-selective agonism manifests as tissue-selectivity: in ovariectomized rats, Cl-4AS-1 mimics DHT while TFM-4AS-1 promotes the accrual of bone and muscle mass while having reduced effects on reproductive organs and sebaceous glands. Moreover, TFM-4AS-1 does not promote prostate growth and antagonizes DHT in seminal vesicles. To confirm that the biochemical properties of TFM-4AS-1 confer tissue selectivity, we identified a structurally unrelated compound, FTBU-1, with partial agonist activity coupled with antagonism of the N-terminal/C-terminal interaction and found that it also behaves as a SARM. TFM-4AS-1 and FTBU-1 represent two new classes of SARMs and will allow for comparative studies aimed at understanding the biophysical and physiological basis of tissue-selective effects of nuclear receptor ligands.

Effects of pharmacological inhibition of cathepsin K on fracture repair in mice.

Cathepsin K inhibitors (CatK-I) have been developed and established to restore bone mass in both animal models of bone loss and postmenopausal osteoporotic patients. We investigated the effects of a CatK-I L-006235 on bone repair and compared to alendronate (ALN) for its known effects on fracture healing in preclinical models. Femoral fractures were performed on wild type mice that were given vehicle (CON), CatK-I or ALN from day 0 post-fracture until euthanasia. Radiologic and micro-CT analyses demonstrated that CatK-I enhanced mineralization within the calluses at day 21 post-fracture, but to a lesser degree than ALN. Histological analyses showed residual unmineralized and mineralized cartilage in the calluses of CatK-I and ALN treated groups at day 21 post-fracture compared to that in CON. CatK-I enhanced the number of tartrate-resistant acid phosphatase positive (TRAP+) osteoclasts in the fracture calluses compared to ALN and CON treated groups. However, relative levels of serum C-terminal telopeptides of type I collagen (CTX) normalized to the number of TRAP+ osteoclasts within the calluses were significantly decreased in both CatK-I and ALN groups compared to CON. Additionally, the percentages of osteoblast surface over mineralized calluses and levels of the bone formation marker serum N-terminal propeptide of type I procollagen (P1NP) were comparable between CatK-I versus CON groups, while these bone formation parameters were decreased by ALN. Taken together, these results indicate that unlike ALN, CatK-I inhibits osteoclastic activity without changing bone formation, and the inhibition of CatK delayed but did not abrogate callus remodeling during bone repair.

Parathyroid hormone and prostaglandin E2 preferentially increase luciferase levels in bone of mice harboring a luciferase transgene controlled by elements of the pro-α1(I) collagen promoter

Abstract Type 1 collagen is the major extracellular protein in bone, tendons, ligaments, and skin. DNA elements of the mouse pro-α1 (I) collagen promoter were shown to drive the bone-selective expression of a luciferase transgene. We examined whether this expression can be used to evaluate the effect of anabolic agents on bone formation in vivo. Treatment of either intact males, intact females, or ovariectomized (ovx) mice with 80 μg/kg/day of human parathyroid hormone (hPTH), for 5 to 11 days increased luciferase levels in tibiae by two- to threefold compared with vehicle-treated mice. The increases were tissue specific, as no changes in skin luciferase expression were observed. Treatment with prostaglandin E 2 , a potent bone anabolic agent, for 11 days also increased expression of the transgene in bone, but not in skin. Treatment with dihydrotestosterone (DHT) for 11 days increased luciferase activity in skin, but not in bone. Histomorphometric analysis revealed that 28-day treatment with PTH increased bone formation; 60-day treatment of OVX mice with DHT did not. These findings show a correlation between bone formation and the expression of a transgene driven by DNA elements of the mouse pro-α1 (I) collagen promoter, suggesting that this expression can be used as an indicator and provide a faster readout for the ability of agents to stimulate bone formation in this mouse strain.

The aminobisphosphonate alendronate inhibits bone loss induced by thyroid hormone in the rat. Comparison between effects on tibiae and vertebrae.

The aims of this study were to develop a rat model of hyperthyroidism and to determine the efficacy of alendronate in the prevention of thyroid hormone-induced bone loss. Ten week-old Sprague-Dawley rats injected with thyroxine 250 micrograms/kg/day (+T4) or vehicle (-T4) were treated with alendronate (+ALN) or vehicle (-ALN) orally 0.5 mg/kg/day. After 3 weeks of treatment histomorphometric parameters of cancellous bone remodeling were assessed in the proximal tibia and in the first lumbar vertebra. In the secondary spongiosa of the tibia T4 treatment caused significant bone loss, associated with increased bone turnover; trabecular bone volume, trabecular thickness and trabecular number were significantly decreased. Osteoid and osteoclast surfaces increased in +T4/-ALN as compared to control. Alendronate prevented the increase in bone turnover and increased bone volume above control values without interfering with the recruitment of osteoclasts. These changes were not apparent in the vertebra. It is concluded that excess thyroid hormone in the rat induces high turnover bone loss in the tibia which can be prevented by alendronate through an inhibition of osteoclastic activity. The lack of effects of thyroid hormone on the vertebra may be ascribed to a lower rate of basal bone turnover at that site.

Increased fracture callus mineralization and strength in cathepsin K knockout mice.

Cathepsin K (CatK) is a cysteine protease, expressed predominantly in osteoclasts (OC) which degrades demineralized bone matrix. Novel selective inhibitors of CatK are currently being developed for the treatment of postmenopausal osteoporosis. Pharmacological inhibition of CatK reduces OC resorption activity while preserving bone formation in preclinical models. Disruption of the CatK gene in mice also results in high bone mass due to impaired bone resorption and elevated formation. Here, we assessed mid-shaft femoral fracture healing in 8-10week old CatK knock-out (KO) versus wild type (WT) mice. Fracture healing and callus formation were determined in vivo weekly via X-ray, and ex vivo at days 14, 18, 28 and 42 post-fracture by radiographic scoring, micro-computed tomography (μCT), histomorphometry and terminal mechanical four point bend strength testing. Radiological evaluation indicated accelerated bone healing and remodeling for CatK KO animals based on increased total radiographic scores that included callus opacity and bridging at days 28 and 42 post-fracture. Micro-CT based total callus volume was similar in CatK KO and WT mice at day 14. Callus size in CatK KO mice was 25% smaller than that in WT mice at day 18, statistically significant by day 28 and exhibited significantly higher mineralized tissue volume and volumetric BMD as compared to WT by day 18 onward. Osteoclast surface and osteoid surface trended higher in CatK KO calluses at all time-points and osteoblast number was also significantly increased at day 28. Increased CatK KO callus mineral density was reflected in significant increases in peak load and stiffness over WT at day 42 post-fracture. Regression analysis indicated a positive correlation (r=0.8671; p<0.001) between callus BMC and peak load indicating normal mineral properties in CatK KO calluses. Taken together, gene deletion of cathepsin K in mice accelerated callus size resolution, significantly increased callus mineralized mass, and improved mechanical strength as compared to wild type mice.

Design, synthesis, and biological evaluation of 16-substituted 4-azasteroids as tissue-selective androgen receptor modulators (SARMs).

A novel series of 16-substituted-4-azasteroids has been identified as potential tissue-selective androgen receptor modulators. These ligands display potent hAR binding and agonist activity, low virilizing potential, and good pharmacokinetic profiles in dogs. On the basis of its in vitro profile, 21 was evaluated in the OVX and ORX rat models and exhibited an osteoanabolic, tissue-selective profile.

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.