Cell-specific activation of the human skeletal alpha-actin by androgens.

Although it is evident that androgens increase muscle mass and strength, little is known about the critical molecular targets of androgens in skeletal muscle. In rodents, the skeletal alpha-actin gene is a tissue-specific gene expressed only in the levator ani and other skeletal muscles but not in the prostate or preputial gland, the well-known androgen target tissue. We identified tissue-specific androgen-regulated genes in the skeletal muscle in rats after oral administration of androgens and focused on androgen-dependent up-regulation of the skeletal alpha-actin gene. To investigate the mechanism of action, an in vitro system with various cell lines and a series of deletion mutants of the alpha-actin promoter were used. The human skeletal alpha-actin promoter was activated by androgens in the muscle cell line C2C12 but not in the liver, prostate, or breast cancer cell lines in which exogenous human androgen receptor is expressed. The sequence of the promoter is sufficient for cell-specific androgen response, providing a model for the tissue specificity demonstrated in vivo. Using a series of deletion mutants, the androgen response can be maintained using just the proximal promoter region. The importance of androgen regulation of this small portion of the human skeletal alpha-actin promoter was demonstrated by the correlation between muscle and the alpha-actin promoter activity for an array of selective androgen receptor modulators (SARMs), including an orally active SARM LGD2226. Taken together, the results suggest that the regulation of skeletal alpha-actin by androgens/SARMs may represent an important model system for understanding androgen anabolic action in the muscle.

Anticancer activity of the intraperitoneal-delivered DFP-10825, the cationic liposome-conjugated RNAi molecule targeting thymidylate synthase, on peritoneal disseminated ovarian cancer xenograft model.

INTRODUCTION: Peritoneal disseminated ovarian cancer is one of the most difficult cancers to treat with conventional anti-cancer drugs and the treatment options are very limited, although an intraperitoneal (ip) paclitaxel has shown some clinical benefit. Therefore, treatment of peritoneal disseminated ovarian cancer is a highly unmet medical need and it is urgent to develop a new ip delivered drug regulating the fast DNA synthesis. METHODS: We developed a unique RNAi molecule consisting of shRNA against the thymidylate synthase (TS) and a cationic liposome (DFP-10825) and tested its antitumor activity and PK profile in peritoneally disseminated human ovarian cancer ascites models by the luciferase gene-transfected SCID mice. DFP-10825 alone, paclitaxel alone or combination with DFP-10825 and paclitaxel were administered in an ip route to the tumor-bearing mice. The TS expression level was measured by conventional RT-PCR. The anti-tumor activity and host survival benefit by DFP-10825 treatment on tumor-bearing mice were observed as resulting from the specific TS mRNA knock-down in tumors. RESULTS: DFP-10825 alone significantly suppressed the growth of SKOV3-luc tumore ascites cells and further extended the survival time of these tumor-bearing mice. Combination with the ip paclitaxel augmented the antitumor efficacy of DFP-10825 and significantly prolonged the survival time in the tumor-bearing mice. Short-hairpin RNA for TS (TS shRNA) levels derived from DFP-10825 in the ascetic fluid were maintained at a nM range across 24 hours but not detected in the plasma, suggesting that TS shRNA is relatively stable in the peritoneal cavity, to be able to exert its anti-tumor activity, but not in blood stream, indicating little or no systemic effect. CONCLUSION: Collectively, the ip delivery of DFP-10825, TS shRNA conjugated with cationic liposome, shows a favorable antitumor activity without systemic adverse events via the stable localization of TS shRNA for a sufficient time and concentration in the peritoneal cavity of the peritoneally disseminated human ovarian cancer-bearing mice.

An orally active selective androgen receptor modulator is efficacious on bone, muscle, and sex function with reduced impact on prostate.

A number of conditions, including osteoporosis, frailty, and sexual dysfunction in both men and women have been improved using androgens. However, androgens are not widely used for these indications because of the side effects associated with these drugs. We describe an androgen receptor (AR) ligand that maintains expected anabolic activities with substantially diminished activity in the prostate. LGD2226 is a nonsteroidal, nonaromatizable, highly selective ligand for the AR, exhibiting virtually no affinity for the other intracellular receptors. We determined that AR bound to LGD2226 exhibits a unique pattern of protein-protein interactions compared with testosterone, fluoxymesterone (an orally available steroidal androgen), and other steroids, suggesting that LGD2226 alters the conformation of the ligand-binding domain. We demonstrated that LGD2226 is fully active in cell-based models of bone and muscle. LGD2226 exhibited anabolic activity on muscle and bone with reduced impact on prostate growth in rodent models. Biomechanical testing of bones from animals treated with LGD2226 showed strong enhancement of bone strength above sham levels. LGD2226 was also efficacious in a sex-behavior model in male rats measuring mounts, intromissions, ejaculations, and copulation efficiency. These results with an orally available, nonaromatizable androgen demonstrate the important role of the AR and androgens in mediating a number of beneficial effects in bone, muscle, and sexual function independent from the conversion of androgens into estrogenic ligands. Taken together, these results suggest that orally active, nonsteroidal selective androgen receptor modulators may be useful therapeutics for enhancing muscle, bone, and sexual function.

Sexual differentiation of the spinal nucleus of the bulbocavernosus is not mediated solely by androgen receptors in muscle fibers.

The spinal nucleus of the bulbocavernosus (SNB) neuromuscular system is a highly conserved and well-studied model of sexual differentiation of the vertebrate nervous system. Sexual differentiation of the SNB is currently thought to be mediated by the direct action of perinatal testosterone on androgen receptors (ARs) in the bulbocavernosus/levator ani muscles, with concomitant motoneuron rescue. This model has been proposed based on surgical and pharmacological manipulations of developing rats as well as from evidence that male rats with the testicular feminization mutation (Tfm), which is a loss of function AR mutation, have a feminine SNB phenotype. We examined whether genetically replacing AR in muscle fibers is sufficient to rescue the SNB phenotype of Tfm rats. Transgenic rats in which wild-type (WT) human AR is driven by a human skeletal actin promoter (HSA-AR) were crossed with Tfm rats. Resulting male HSA-AR/Tfm rats express WT AR exclusively in muscle and nonfunctional Tfm AR in other tissues. We then examined motoneuron and muscle morphology of the SNB neuromuscular system of WT and Tfm rats with and without the HSA-AR transgene. We observed feminine levator ani muscle size and SNB motoneuron number and size in Tfm males with or without the HSA-AR transgene. These results indicate that AR expression in skeletal muscle fibers is not sufficient to rescue the male phenotype of the SNB neuromuscular system and further suggest that AR in other cell types plays a critical role in sexual differentiation of this system.

New and improved glucocorticoid receptor ligands.

The therapeutic and prophylactic use of glucocorticoids is widespread due to their powerful anti-inflammatory, antiproliferative and immunomodulatory activity. However, long-term use of these drugs can result in severe dose-limiting side effects. One of the most critical and debilitating side effects is osteoporosis, which leads to increased risk of fractures. Glucocorticoids damage bone through several different mechanisms. The search for novel glucocorticoids that have reduced side effects in bone and other tissues is being driven by the identification of new mechanisms of action of the glucocorticoid receptor. This may facilitate the detection of new, safer therapies with efficacies equivalent to currently prescribed steroids.