Suriyan Ponnusamy

Tissue Selective Androgen Receptor Modulators (SARMs) Increase Pelvic Floor Muscle Mass in Ovariectomized Mice

Stress urinary incontinence (SUI), a prevalent condition, is represented by an involuntary leakage of urine that results, at least in part, from weakened or damaged pelvic floor muscles and is triggered by physical stress. Current treatment options are limited with no oral therapies available. The pelvic floor is rich in androgen receptor and molecules with anabolic activity including selective androgen receptor modulators (SARMs) may serve as therapeutic options for individuals with SUI. In this study, two SARMs (GTx‐024 and GTx‐027) were evaluated in a post‐menopausal animal model in order to determine their effect on pelvic floor muscles. Female C57BL/6 mice were ovariectomized and their pelvic muscles allowed to regress. The animals were then treated with vehicle or doses of GTx‐024 or GTx‐027. Animal total body weight, lean body mass, and pelvic floor muscle weights were measured along with the expression of genes associated with muscle catabolism. Treatment with the SARMs resulted in a restoration of the pelvic muscles to the sham‐operated weight. Coordinately, the induction of genes associated with muscle catabolism was inhibited. Although a trend was observed towards an increase in total lean body mass in the SARM‐treated groups, no significant differences were detected. Treatment of an ovariectomized mouse model with SARMs resulted in an increase in pelvic floor muscles, which may translate to an improvement of symptoms associated with SUI and serves as the basis for evaluating their clinical use. J. Cell. Biochem. 118: 640–646, 2017.

Androgen receptor agonists increase lean mass, improve cardiopulmonary functions and extend survival in preclinical models of Duchenne muscular dystrophy

Abstract Duchenne muscular dystrophy (DMD) is a neuromuscular disease that predominantly affects boys as a result of mutation(s) in the dystrophin gene. DMD is characterized by musculoskeletal and cardiopulmonary complications, resulting in shorter life‐span. Boys afflicted by DMD typically exhibit symptoms within 3‐5 years of age and declining physical functions before attaining puberty. We hypothesized that rapidly deteriorating health of pre‐pubertal boys with DMD could be due to diminished anabolic actions of androgens in muscle, and that intervention with an androgen receptor (AR) agonist will reverse musculoskeletal complications and extend survival. While castration of dystrophin and utrophin double mutant (mdx‐dm) mice to mimic pre‐pubertal nadir androgen condition resulted in premature death, maintenance of androgen levels extended the survival. Non‐steroidal selective‐AR modulator, GTx‐026, which selectively builds muscle and bone was tested in X‐linked muscular dystrophy mice (mdx). GTx‐026 significantly increased body weight, lean mass and grip strength by 60‐80% over vehicle‐treated mdx mice. While vehicle‐treated castrated mdx mice exhibited cardiopulmonary impairment and fibrosis of heart and lungs, GTx‐026 returned cardiopulmonary function and intensity of fibrosis to healthy control levels. GTx‐026 elicits its musculoskeletal effects through pathways that are distinct from dystrophin‐regulated pathways, making AR agonists ideal candidates for combination approaches. While castration of mdx‐dm mice resulted in weaker muscle and shorter survival, GTx‐026 treatment increased the muscle mass, function and survival, indicating that androgens are important for extended survival. These preclinical results support the importance of androgens and the need for intervention with AR agonists to treat DMD‐affected boys.

Novel, isoform-selective, cholecystokinin A receptor antagonist inhibits colon and pancreatic cancers in preclinical models through novel mechanism of action

Colon and pancreatic cancers contribute to 90,000 deaths each year in the USA. These cancers lack targeted therapeutics due to heterogeneity of the disease and multiple causative factors. One important factor that contributes to increased colon and pancreatic cancer risk is gastrin. Gastrin mediates its actions through two G-protein coupled receptors (GPCRs): cholecystokinin receptor A (CCK-A) and CCK-B/gastrin receptor. Previous studies have indicated that colon cancer predominantly expresses CCK-A and responds to CCK-A isoform antagonists. However, many CCK-A antagonists have failed in the clinic due to poor pharmacokinetic properties or lack of efficacy. In the present study, we synthesized a library of CCK-A isoform-selective antagonists and tested them in various colon and pancreatic cancer preclinical models. The lead CCK-A isoform, selective antagonist PNB-028, bound to CCK-A at 12 nM with a 60-fold selectivity towards CCK-A over CCK-B. Furthermore, it inhibited the proliferation of CCK-A-expressing colon and pancreatic cancer cells without affecting the proliferation of non-cancerous cells. PNB-028 was also extremely effective in inhibiting the growth of MAC-16 and LoVo colon cancer and MIA PaCa pancreatic cancer xenografts in immune-compromised mice. Genome‑wide microarray and kinase-array studies indicate that PNB-028 inhibited oncogenic kinases and angiogenic factors to inhibit the growth of colon cancer xenografts. Safety pharmacology and toxicology studies have indicated that PNB-028 is extremely safe and has a wide safety margin. These studies suggest that targeting CCK-A selectively renders promise to treat colon and pancreatic cancers and that PNB-028 could become the next-generation treatment option.

MP57-03 IDENTIFICATION AND CHARACTERIZATION OF SELECTIVE ANDROGEN RECEPTOR DEGRADERS (SARDS) FOR THE TREATMENT OF ENZALUTAMIDE UNRESPONSIVE AND/OR RESISTANT PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: Prostate cancer (PCa) is the second leading cause of cancer deaths in men. AR signaling is known to play a critical role in androgen responsive prostate cancer cells. Androgen deprivation therapy (ADT) is a standard of care for patients when prostate cancer has spread beyond the prostate. Almost all the prostate cancer (PCa) deaths result from castration resistant prostate cancer (CRPC). With the development of newer anti-androgen Enzalutamide (ENZ) there has been a marked improvement in CRPC. However, almost all patients develop resistance to ENZ in part due to expression of ARv7. Thus, to date, no acceptable treatment options are available for ENZ resistant CRPC. In the present study we evaluated the effects of TET, a derivative of bis-benzyly isoquinoline, Tetrandrine on two enzalutamide resistant prostate cancer cell lines on sensitizing these cells to Enzalutamide. We also evaluated the effects of TET on AR and ARv7 levels. METHODS: Enz resistant Prostate cancer cell lines (22rv1 and LNCaP-abl) were used in the present study. Cells were grown in supplemented media and maintained at 370C in a 5%CO2 incubator (as described elsewhere). Where indicated cells were treated with Enzalutamide or TET alone or in combination. Cell viability was measured by crystal violet and MTT assays. Protein levels were measured by Western Blot assays. mRNA expression measured in RTPCR assays. RESULTS: Treatment with Enz had only a marginal effect on growth and viability of 22rv1 cells. TET inhibited growth and proliferation of enzalutamide resistant prostate cancer cells in both dose and time dependent manner with an IC50 in the range of 5-10uM at 72 hr. However TET treatment did not result in death of RWPE cells, a line of normal prostate cells. Moreover, combination of TET and Enz was more effective than either treatment alone. Treatment with TET resulted in decreased levels of full length AR as well as ARv7 within 24-48h. We also observed that TET treatment was associated with decreased cyclin D1 and increased CDK inhibitors p21 and p27. Over all Tet alone and in combination with Enz promoted cell growth arrest and cell death in ENZ resistant CRPC cells and sensitized these cells to Enz. CONCLUSIONS: This study shows that TET sensitizes CRPC cells to Enz in part by decreasing protein levels of AR and ARv7.

An estrogen receptor β-selective agonist inhibits non-alcoholic steatohepatitis in preclinical models by regulating bile acid and xenobiotic receptors:

Non-alcoholic steatohepatitis (NASH) affects 8–10 million people in the US and up to 75% of obese individuals. Despite this, there are no approved oral therapeutics to treat NASH and therefore the need for novel approaches exists. The estrogen receptor β (ER-β)-selective agonist, β-LGND2, inhibits body weight and white adipose tissue, and increases metabolism, resulting in higher energy expenditure and thermogenesis. Due to favorable effects of β-LGND2 on obesity, we hypothesized that β-LGND2 will prevent NASH directly by reducing lipid accumulation in the liver or indirectly by favorably changing body composition. Male C57BL/6 mice fed with high fat diet (HFD) for 10 weeks or methionine choline-deficient diet for four weeks and treated with vehicle exhibited altered liver weights by twofold and increased serum transaminases by 2–6-folds. These changes were not observed in β-LGND2-treated animals. Infiltration of inflammatory cells and collagen deposits, an indication of fibrosis, were observed in the liver of mice fed with HFD for 10 weeks, which were effectively blocked by β-LGND2. Gene expression studies in the liver indicate that pregnane X receptor target genes were significantly increased by HFD, and the increase was inhibited by β-LGND2. On the other hand, metabolomics indicate that bile acid metabolites were significantly increased by β-LGND2. These studies demonstrate that an ER-β agonist might provide therapeutic benefits in NASH by directly modulating the function of xenobiotic and bile acid receptors in the liver, which have important functions in the liver, and indirectly, as demonstrated before, by inhibiting adiposity. Impact statement Over 75–90% of those classified as clinically obese suffer from co-morbidities, the most common of which is non-alcoholic steatohepatitis (NASH). While there are currently no effective treatment approaches for NASH, data presented here provide preliminary evidence that an estrogen receptor β-selective ligand could have the potential to reduce lipid accumulation and inflammation, and protect liver from NASH.

Novel Selective Agents for the Degradation of Androgen Receptor Variants to Treat Castration-Resistant Prostate Cancer

Androgen receptor (AR) mediates the growth of prostate cancer throughout its course of development, including in abnormal splice variants (AR-SV)-driven advanced stage castration-resistant disease. AR stabilization by androgens makes it distinct from other steroid receptors, which are typically ubiquitinated and degraded by proteasomes after ligand binding. Thus, targeting AR in advanced prostate cancer requires the development of agents that can sustainably degrade variant isoforms for effective therapy. Here we report the discovery and characterization of potent selective AR degraders (SARD) that markedly reduce the activity of wild-type and splice variant isoforms of AR at submicromolar doses. Three SARDs (UT-69, UT-155, and (R)-UT-155) bind the amino-terminal transcriptional activation domain AF-1, which has not been targeted for degradation previously, with two of these SARD (UT-69 and UT-155) also binding the carboxy-terminal ligand binding domain. Despite different mechanisms of action, all three SARDs degraded wild-type AR and inhibited AR function, exhibiting greater inhibitory potency than the approved AR antagonists. Collectively, our results introduce a new candidate class of next-generation therapeutics to manage advanced prostate cancer. Cancer Res; 77(22); 6282-98. ©2017 AACR.