Abhijit M. Godbole

Synthesis and biological evaluations of putative metabolically stable analogs of VN/124-1 (TOK-001): Head to head anti-tumor efficacy evaluation of VN/124-1 (TOK-001) and abiraterone in LAPC-4 human prostate cancer xenograft model

In a continuing study of our clinical candidate 5 VN/124-1 (TOK-001) and analogs as potential agents for prostate cancer therapy, putative metabolites (10, 15 and 18) of compound 5 were rationally designed and synthesized. However, none of these agents were as efficacious as 5 in several in vitro studies. Using western blot analysis, we have generated a preliminary structure-activity relationship (SAR) of 5 and related analogs as androgen receptor ablative agents (ARAAs). In vivo using the androgen-dependent LAPC-4 prostate cancer xenograft model, we demonstrated for the first time that 5 is more efficacious than the 17-lyase inhibitor 3 (abiraterone)/4 (abiraterone acetate) that is currently in phase III clinical trials. In our desire to optimize the potency of 5, compounds 6 (3ξ-fluoro-) and 9 (3β-sulfamate-) designed to increase the stability and oral bioavailability of 5, respectively were evaluated in vivo. We showed, that on equimolar basis, compound 6 was ∼2-fold more efficacious versus LAPC-4 xenografts than 5, but the toxicity observed with 6 is of concern. These studies further demonstrate the efficacy of 5 in a clinically relevant prostate cancer model and justify its current clinical development as a potential treatment of prostate cancer.

Systematic Structure Modifications of Multitarget Prostate Cancer Drug Candidate Galeterone To Produce Novel Androgen Receptor Down-Regulating Agents as an Approach to Treatment of Advanced Prostate Cancer

As part of our program to explore the influence of small structural modifications of our drug candidate 3β-(hydroxy)-17-(1H-benzimidazol-1-yl)androsta-5,16-diene (galeterone, 5) on the modulation of the androgen receptor (AR), we have prepared and evaluated a series of novel C-3, C-16, and C-17 analogues. Using structure activity analysis, we established that the benzimidazole moiety at C-17 is essential and optimal and also that hydrophilic and heteroaromatic groups at C-3 enhance both antiproliferative (AP) and AR degrading (ARD) activities. The most potent antiproliferative compounds were 3β-(1H-imidazole-1-carboxylate)-17-(1H-benzimidazol-1-yl)androsta-5,16-diene (47), 3-((EZ)-hydroximino)-17-(1H-benzimidazol-1-yl)androsta-4,16-diene (36), and 3β-(pyridine-4-carboxylate)-17-(1H-benzimidazol-1-yl)androsta-5,16-diene (43), with GI50 values of 0.87, 1.91, and 2.57 μM, respectively. Compared to 5, compound 47 was 4- and 8-fold more potent with respect to AP and ARD activities, respectively. Importantly, we also discovered that our compounds, including 5, 36, 43, and 47, could degrade both full-length and truncated ARs in CWR22rv1 human prostate cancer cells. With these activities, they have potential for development as new drugs for the treatment of all forms of prostate cancer.

Autophagy Inhibition Synergistically Enhances Anticancer Efficacy of RAMBA, VN/12-1 in SKBR-3 Cells, and Tumor Xenografts

VN/12-1 is a novel retinoic acid metabolism blocking agent discovered in our laboratory. The purpose of the study was to elucidate the molecular mechanism of anticancer activity of VN/12-1 in breast cancer cell lines and in tumor xenografts. We investigated the effects of VN/12-1 on induction of autophagy and apoptosis in SKBR-3 cells. Furthermore, we also examined the impact of pharmacologic and genomic inhibition of autophagy on anticancer activity of VN/12-1. Finally, the antitumor activity of VN/12-1 was evaluated as a single agent and in combination with autophagy inhibitor chloroquine in an SKBR-3 mouse xenograft model. Short exposure of low dose (<10 μmol/L) of VN/12-1 induced endoplasmic reticulum stress, autophagy, and inhibited G1–S phase transition and caused a protective response. However, a higher dose of VN/12-1 initiated apoptosis in vitro. Inhibition of autophagy using either pharmacologic inhibitors or RNA interference of Beclin-1 enhanced anticancer activity induced by VN/12-1 in SKBR-3 cells by triggering apoptosis. Importantly, VN/12-1 (5 mg/kg twice weekly) and the combination of VN/12-1 (5 mg/kg twice weekly) + chloroquine (50 mg/kg twice weekly) significantly suppressed established SKBR-3 tumor growth by 81.4% (P < 0.001 vs. control) and 96.2% (P < 0.001 vs. control), respectively. Our novel findings suggest that VN/12-1 may be useful as a single agent or in combination with autophagy inhibitors for treating human breast cancers. Our data provides a strong rationale for clinical evaluation of VN/12-1 as single agent or in combination with autophagy inhibitors. Mol Cancer Ther; 11(4); 898–908. ©2012 AACR.

New Insights into the Androgen-Targeted Therapies and Epigenetic Therapies in Prostate Cancer

Prostate cancer is the most common cancer in men in the United States, and it is the second leading cause of cancer-related death in American men. The androgen receptor (AR), a receptor of nuclear family and a transcription factor, is the most important target in this disease. While most efforts in the clinic are currently directed at lowering levels of androgens that activate AR, resistance to androgen deprivation eventually develops. Most prostate cancer deaths are attributable to this castration-resistant form of prostate cancer (CRPC). Recent work has shed light on the importance of epigenetic events including facilitation of AR signaling by histone-modifying enzymes, posttranslational modifications of AR such as sumoylation. Herein, we provide an overview of the structure of human AR and its key structural domains that can be used as targets to develop novel antiandrogens. We also summarize recent findings about the antiandrogens and the epigenetic factors that modulate the action of AR.

VN/14-1 induces ER stress and autophagy in HP-LTLC human breast cancer cells and has excellent oral pharmacokinetic profile in female Sprague Dawley rats

Resistance to aromatase inhibitors is a major concern in the treatment of breast cancer. Long-term letrozole cultured (LTLC) cells represent a model of resistance to aromatase inhibitors. The LTLC cells were earlier generated by culturing MCF-7Ca, the MCF-7 human breast cancer cell line stably transfected with human placental aromatase gene for a prolonged period in the presence of letrozole. In the present study the effect of RAMBA, VN/14-1 on the sensitivity of LTLC cells upon multiple passaging and the mechanisms of action of VN/14-1 in such high passage LTLC (HP-LTLC) cells was investigated. We report that multiple passaging of LTLC cells (HP-LTLC cell clones) led to profound decrease in their sensitivity to VN/14-1. Additionally, microarray studies and protein analysis revealed that VN/14-1 induced marked endoplasmic reticulum (ER) stress and autophagy in HP-LTLC cells. We further report that VN/14-1 in combination with thapsigargin exhibited synergistic anti-cancer effect in HP-LTLC cells. Preliminary pharmacokinetics in rats revealed that VN/14-1 reached a peak plasma concentration (Cmax) within 0.17h after oral dosing. Its absolute oral bioavailability was >100%. Overall these results indicate potential of VN/14-1 for further clinical development as a potential oral agent for the treatment of breast cancer.

Prostate Cancer: Current and Emerging Therapies

Prostate cancer (PC) is the second most prevalent cause of death in men in the USA and Europe. The dependence of PC on androgens has been recognized for more than 7 decades. Medical and surgical androgen deprivation therapy (ADT) has been a standard palliative therapy for metastatic PC. However, an estimated 217,730 new cases and 32,050 PC related deaths in the USA alone in 2010 despite ADT, make the need for finding new targets and novel therapies an absolute priority [1]. Despite medical treatment, the vast majority of patients with metastatic prostate cancer inevitably progress and die from their disease. While initially majority of metastatic prostate cancers rely on the availability of androgens for growth and survival, in their final stages of disease, these patients eventually progress clinically under androgen-deprived conditions. Under the selective pressure of drug treatment, prostate cancer cells are then able to acquire molecular changes that allow them to survive androgen-deprived conditions, gain a selective growth advantage, and finally, result in progression of disease. Our knowledge about this disease is increasing. However, the cellular and molecular events that are necessary to cause progression of prostate cancer from an androgen-dependent (AD) to an androgen-independent (AI) state of disease are not completely understood. With a 9% response rate, chemotherapy was once thought to play a clinically insignificant role in metastatic and castration resistant prostate cancer (CRPC) [2]. More recently, however, a role has emerged for systemic chemotherapy after the demonstration of a small but significant survival benefit for taxane-based chemotherapy in the two landmark studies, TAX-327 and SWOG-9916 [3, 4]. Since median survival for patients with metastatic CRPC is still only about 18 months, there is plenty room for further improvement. Moreover, there is a strong need for second and third-line regimen for patients progressing after docetaxel, and these patients should be enrolled into clinical trials.

Abstract 659: Inhibiting autophagy improves the efficacy of VN/12-1 over all-trans retinoic acid in a HER2 over-expressing SK-BR3 breast cancer xenograft model

Purpose: Autophagy is a critical physiological process of cell survival in the presence of stress and is an important cause of resistance to anti-cancer therapy. Drugs that inhibit autophagy (e.g. chloroquine) deprive the cells of this protective mechanism and therefore are promising agents in a combination therapy of breast cancer. Our group has designed and synthesized a novel retinoic acid derivative, VN/12-1. We reported previously that VN/12-1 is a potent inducer of autophagy in estrogen independent, HER2 over-expressing breast cancer cell line SK-BR3 in vitro. In the present study, we used chloroquine in combination with VN/12-1 in an SK-BR3 xenograft model to test the hypothesis that the combination of VN/12-1 and chloroquine would result in greater efficacy against SK-BR3 tumors compared to VN/12-1 alone or all-trans retinoic acid (ATRA) alone. Experimental Design: To test this hypothesis, 64 female SCID mice (8 mice per group) were inoculated with SK-BR3 cells. Once the tumors formed, the mice were treated with vehicle (control), VN/12-1 and ATRA alone or in combination with chloroquine for 30 days. The two dosages of VN/12-1 for the groups involving VN/12-1 were 2.5 mg/kg and 5 mg/kg. The doses of ATRA and chloroquine were 5 mg/kg and 50 mg/kg, respectively. All the agents were administered subcutaneously (s.c) twice a week. Results: Out of all the treatment groups, both the dosages of VN/12-1 (2.5 mg/kg and 5mg/kg) when used in combination with chloroquine were the most effective in inhibiting the growth of SK-BR3 tumors (70% and 80% growth inhibition respectively compared to vehicle control). The combination of ATRA with chloroquine did not show statistically significant inhibition of tumor growth. The effect of each treatment on the body weight and the weights of uteri was also determined. No toxicities were observed as noted by no significant changes in the weight of the mice. The data about the tumor samples’ analysis for the effect of each treatment on the expression of various tumor markers and autophagy markers and pharmacokinetic studies on VN/12-1 will be presented. Conclusion: Our studies have demonstrated for the first time that the combination of VN/12-1 and chloroquine can be used to treat estrogen independent breast cancer. It is hoped that this combination therapy would solve the problem of the development of acquired resistance due to autophagy that typically follows therapeutic treatment of breast cancer. We envision further advanced preclinical development of this combination therapy as potential therapy for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 659. doi:10.1158/1538-7445.AM2011-659

Abstract LB-340: Inhibition of autophagy enhances the anticancer activity of the retinoic acid derivative VN/12-1 in endocrine resistant human breast cancer cell lines

Breast cancer is the most common cancer and the second leading cause of cancer related deaths among women in the United States. Although endocrine therapies such as anti-estrogens and aromatase inhibitors have improved the overall survival, resistance to this therapy remains a major concern. Autophagy is a unique, regulated mechanism of cell survival by catabolism of proteins under nutrient deprivation or chemotherapeutic stress. It is an important cause of resistance to chemotherapeutic agents. VN/12-1, a novel 4-imidazolyl methyl ester of retinoic acid synthesized in our laboratory, showed excellent anti-proliferative activity (as shown by MTT assay) in endocrine resistant human breast cancer cell lines SK-BR-3 and MDA-MB-231. Its IC 50 in SK-BR-3 cells was 5.2 μ M compared to 15.2 μ M (all-trans retinoic acid-ATRA) and 45.6 μ M (letrozole). For MDA-MB-231 cells, IC 50 of VN/12-1 was 7.3 μ M compared to 53.7 μ M (ATRA) and 74.3 μ M (letrozole). At low micromolar concentrations of VN/12-1 treatment on these cells, western blot analysis and imaging studies confirmed the presence of autophagosomes. This was associated with up-regulation of proteins involved in endoplasmic reticulum (ER) stress, markers for DNA damage and downregulation of cyclin D1 and phosphorylated Akt. Co-treatment with chloroquine (an autophagy inhibitor) resulted in increased sensitivity of cells to VN/12-1 as indicated by decrease in cell viability from 71.22% for VN/12-1 (3 μM) alone, 90.18% chloroquine (3 μM) alone to 34.31% for the combination of VN/12-1 (3 μM) and chloroquine (3 μM) in SK-BR-3 cells. It also resulted in induction of apoptosis which was caspase mediated. In summary, ER stress and DNA damage induced by VN/12-1 resulted in cell cycle arrest and induction of autophagy. Inhibition of autophagy by chloroquine overwhelmed this cellular stress and resulted in apoptotic cell death in endocrine resistant breast cancer cell lines. Significance: VN/12-1 and its combination with autophagy inhibitor chloroquine is a novel therapeutic alternative to treat endocrine resistant breast cancer and hence warrants further pre-clinical and clinical development. This is the first in vitro study which showed an enhancement of anti-cancer activity of a retinoic acid derivative by an autophagy inhibitor in endocrine resistant human breast cancer cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-340.