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.

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 3ß-(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 3ß-(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 3ß-(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.

Murine toxicology and pharmacokinetics evaluation of retinoic acid metabolism blocking agent (RAMBA), VN/12-1.

PURPOSE: Novel retinoic acid metabolism blocking agent (RAMBA), VN/12-1, is a highly potent anti-cancer agent that induces autophagy. Its combination with autophagy inhibitor chloroquine (CHL) has been shown to synergistically enhance apoptosis in breast cancer cells. The purpose of this study was to determine the toxicity and pharmacokinetic profile of VN/12-1 and its combination with CHL. METHODS: Preliminary toxicology of VN/12-1 was determined using female SCID mice (n = 4 for each group). ATRA was used for comparison. We selected four different doses of VN/12-1 and ATRA. Two of the doses were low and less frequent (2.5 and 5 mg/kg twice a week), and the remaining doses were high and more frequent (10 and 20 mg/kg every day). The dose of CHL was 50 mg/kg twice a week. For pharmacokinetic (PK) study, 20 mg/kg of VN/12-1 was injected subcutaneously (s.c.) into the mice, and their plasma was collected at various intervals (n = 2) and analyzed by HPLC. RESULTS: The lower and less frequent doses of VN/12-1 and ATRA were found to be least toxic. However, high and more frequent doses of these compounds were toxic to the mice. PK results showed that VN/12-1 has a half-life of 6 h. The area under the curve (AUC) for VN/12-1 was 83.78 h µg/ml. CONCLUSIONS: VN/12-1 and ATRA are non-toxic when used as 5 mg/kg twice a week as single agents or in combination with CHL. The favorable PK properties of VN/12-1 can potentially be used for its further advanced pre-clinical and clinical development.

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 G(1)-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.

Anti-tumor effects of a novel retinoic acid metabolism blocking agent VN/14-1 in the N-methyl-N-nitrosourea-induced rat mammary carcinoma model and its effects on the uterus.

VN/14-1 [4-(±)-(1H-Imidazol-1-yl)-(E)-retinoic acid], a novel retinoic acid metabolism blocking agent (RAMBA), works by inhibiting the breakdown of all-trans-retinoic acid. The purpose of this study was to evaluate the anti-tumor effects of VN/14-1 on the N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinoma model, and peripheral organ effects on the uteri of immature ovariectomized (OVX) rats. In tumor burden experiments, after 56 days of administration of VN/14-1 5, 10, and 20 mg/kg/day, significant tumor reductions in mean tumor weight of 19.1, 34.4, and 44.3%, compared to tumors in control animals occurred. Cumulative tumor growth was also significantly slower in a dose-dependent manner in groups receiving 5, 10, and 20 mg/kg/day of VN/14-1 compared to growth rates in the control group. Tumor apoptosis was significant increases in animals treated with 5, 10, and 20 mg/kg/day of VN/14-1. In uterotrophic experiments, immature OVX rats given VN/14-1 significantly reduced uterine weight and blocked endometrial stimulation induced by unopposed ß-estradiol (E2). In both rat models, adverse toxicities included weakness, anorexia, and reduction in body weight in the groups given the highest dose of 20 mg/kg/day. In summary, VN/14-1 inhibited tumor growth in the MNU-induced estrogen receptor (ER)-positive rat mammary tumor model, and antagonized the stimulatory effect of estrogens on the uterus. The studies suggest that VN/14-1 may be a useful novel therapy for ER-positive breast cancer.

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.

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 (3ß-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.