Paul A. Foster

Development of hormone-dependent prostate cancer models for the evaluation of inhibitors of 17β-hydroxysteroid dehydrogenase Type 3

17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) are responsible for the pre-receptor reduction/oxidation of steroids at the 17-position into active/inactive hormones, and the 15 known enzymes vary in their substrate specificity, localisation, and directional activity. 17beta-HSD Type 3 (17beta-HSD3) has been seen to be over-expressed in prostate cancer, and catalyses the reduction of androstenedione (Adione) to testosterone (T), which stimulates prostate tumour growth. Specific inhibitors of 17beta-HSD3 may have a role in the treatment of hormone-dependent prostate cancer and benign prostate hyperplasia, and also have potential as male anti-fertility agents. A 293-EBNA-based cell line with stable expression of transfected human 17beta-HSD3 was created and used to develop a whole cell radiometric TLC-based assay to assess the 17beta-HSD3 inhibitory potency of a series of compounds. STX2171 and STX2624 (IC(50) values in the 200-450nM range) were two of several active inhibitors identified. In similar TLC-based assays these compounds were found to be inactive against 17beta-HSD1 and 17beta-HSD2, indicating selectivity. A novel proof of concept model was developed to study the efficacy of the compounds in vitro using the androgen receptor positive hormone-dependent prostate cancer cell line, LNCaPwt, and its derivative, LNCaP[17beta-HSD3], transfected and selected for stable expression of 17beta-HSD3. The proliferation of the parental cell line was most efficiently stimulated by 5alpha-dihydrotestosterone (DHT), but the LNCaP[17beta-HSD3] cells were equally stimulated by Adione, indicating that 17beta-HSD3 efficiently converts Adione to T in this model. Adione-stimulated proliferation of LNCaP[17beta-HSD3] cells was inhibited in the presence of either STX2171 or STX2624. The compounds alone neither stimulated proliferation of the cells nor caused significant cell death, indicating that they are non-androgenic with low cytotoxicity. STX2171 inhibited Adione-stimulated growth of xenografts established from LNCaPwt cells in castrated mice in vivo. In conclusion, a primary screening assay and proof of concept model have been developed to study the efficacy of 17beta-HSD3 inhibitory compounds, which may have a role in the treatment of hormone-dependent cancer. Active compounds are selective for 17beta-HSD3 over 17beta-HSD1 and 17beta-HSD2, non-androgenic with low toxicity, and efficacious in both an in vitro proof of concept model and in an in vivo tumour model.

In vivo and in vitro properties of STX2484: a novel non-steroidal anti-cancer compound active in taxane-resistant breast cancer.

Background:STX2484 is a novel non-steroidal compound with potent anti-proliferative activity. These studies aimed to identify STX2484’s mechanism of action, in vivo efficacy and activity in taxane-resistant breast cancer models.Methods:Effects of STX2484 and paclitaxel on proliferation, cell cycle and apoptosis were assessed in vitro in drug-resistant (MCF-7DOX) and non-resistant cells (MCF-7WT). STX2484 efficacy in βIII tubulin overexpression in MCF-7 cells was also determined. Anti-angiogenic activity was quantified in vitro by a co-culture model and in vivo using a Matrigel plug assay. An MDA-MB-231 xenograft model was used to determine STX2484 efficacy in vivo.Results:STX2484 is a tubulin disruptor, which induces p53 expression, Bcl2 phosphorylation, caspase-3 cleavage, cell cycle arrest and apoptosis. In addition, STX2484 is a potent anti-angiogenic agent in vitro and in vivo. In breast cancer xenografts, STX2484 (20u2009mgu2009kg−1 p.o.) suppressed tumour growth by 84% after 35 days of daily dosing, with limited toxicity. In contrast to paclitaxel, STX2484 efficacy was unchanged in two clinically relevant drug-resistant models.Conclusions:STX2484 is an orally bioavailable microtubule-disrupting agent with in vivo anti-angiogenic activity and excellent in vivo efficacy with no apparent toxicity. Crucially, STX2484 has superior efficacy to paclitaxel in models of clinical drug resistance.

Abstract 2795: Safety profile and therapeutic potential in breast cancer of STX140, an original tubulin binding agent

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnSTX-140 is an anti-tubulin binding agent which has demonstrated a good anti-tumor efficacy profile. Although the binding site on tubulin differs from other classical tubulin binders, one could anticipate the same type of safety issues with such an agent, namely neurotoxicity. In order to differentiate this compound, we evaluated its efficacy in a transgenic model of mammary cancer, its neurotoxicity potential, and its therapeutic index in a breast cancer model. STX140 efficacy was evaluated in C3(1) Tag transgenic mice which develop mammary carcinomas. In those transgenic animals, the disease progresses in a similar fashion to human breast cancer, with the development of tumors which show the same histological characteristics as the human pathology. In these mice, STX140 was able to significantly increase the survival of the animals and to suppress the emergence of metastases, whereas paclitaxel was completely inactive on both parameters. Neurotoxicity potential was assessed in a thermal hyperalgesia model in comparison with paclitaxel. Continuous oral treatment of mice with STX-140 did not induce an increase sensibility to heat as measured by the latency of paw withdrawal after a heat stimulus. However, paclitaxel increased this heat sensitivity starting after the second i.v. administration, underlying the neurotoxicity of this compound, which is observed in the clinic. Finally, in MDA-MB-231 breast cancer xenografts, two different schedules of oral administration were evaluated in order to determine the therapeutic index and PK parameters were measured in parallel to the anti-tumor efficacy. Continuous (QD) and intermittent (Monday, Wednesday, Friday; MWF) treatments were evaluated and a large panel of doses were tested. The therapeutic index was determined as the ratio of the exposure observed between the first toxic dose and first active dose. The TI for the QD schedule was 2.1 and was 1.7 for the MWF schedule, showing the potential good safety index of this compound. Taking into account the previous data reported elsewhere and new data presented here, which underline the great efficacy and safety profile of STX140, this compound is considered for further preclinical and clinical development.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2795. doi:1538-7445.AM2012-2795

Abstract 4413: STX140 is an orally bioavailable microtubule targeting agent with no associated peripheral neuropathy and potent anti-metastasis efficacy in vivo

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnThere is a continued need for orally bioavailable anti-cancer compounds that exhibit good efficacy and with a more favourable toxicity profile than current ‘traditional’ cytotoxics. The microtubule disruptor, STX140 has previously been shown to have excellent oral bioavailability and significantly reduce tumor growth in vivo. These data presented here show STX140 to: lack significant neuropathy in a validated in vivo behavioural model and to significantly reduce metastasis in a highly aggressive orthotopic model of breast cancer.nnTo assess the potential for neurotoxicity animals were dosed with, vehicle (0.5% methlycellulose), STX140 (20 mg/kg p.o. 5/7 × 4) or Taxol (15 mg/kg i.v weekly x 4). Thermal hyperalgesia, as assessed by paw withdrawal latency, was measured every 3-4 days using Hargreaves Plantar equipment. The 4T1 murine tumor cell line was used to establish orthotopic tumors in the mammary fad pads of nude mice. After 28 days dosing, as above, tumor size, number of lung and liver metastasises were recorded. Additionally relative levels of circulating tumor cells (CTCs) were assessed by quantifying the number of viable tumor cell colonies grown from 30 µL of blood taken by cardiac puncture at day 28.nnIn the thermal hyperalgesia model of neuropathy Taxol caused a significant decrease in paw withdrawal latency from day 14 till the end of study, in contrast there were no significant changes in withdrawal times in either the vehicle or STX140 dosed groups. In the 4T1 xenograft study both Taxol and STX140 reduced, tumor volume, extent of lung metastasis and number of viable CTCs in the blood relative to vehicle. In a separate study STX140 but not Taxol caused a significant improvement in survival.nnThese studies highlight the excellent efficacy of the orally bioavailable anti-cancer agent STX140 in a highly aggressive model of breast cancer. STX140 may offer a major additional advantage over existing drugs as the most common dose limiting toxicity of neuropathy, is negated / minimised.nnCitation 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 4413.

Abstract 1558: STX140 but not paclitaxel inhibits mammary tumor initiation, progression and metastasis in C3(1)/SV40 T/t-antigen transgenic mice

New chemotherapeutic options for late-stage metastatic breast cancer are urgently required. Currently, the taxanes are considered one of the most effective treatments for late-stage cancer treatment. Paclitaxel and the novel cytotoxic agent STX140, compounds that have shown significant activity against numerous xenograft mouse models of cancers, are here investigated in the C3(1)/SV40 T/t-antigen mouse model of spontaneous breast cancer. This mouse model recapitulates important histopathological and molecular alterations in mammary cancer development over a highly predictable time-course. At 8 weeks of age, the female animals develop low-grade mammary intra-epithelial neoplasia (MIN) lesions. By 12 to 14 weeks of age this progresses to high-grade MIN, which is similar to human ductal carcinoma in situ. From 15 weeks of age onwards invasive, metastatic carcinomas are observed. STX140 and paclitaxel were administered to these mice at 12 weeks of age (early intervention) and when tumors had reached 0.5 cm (late intervention). Results demonstrated that all mice in the control and paclitaxel treated groups at early intervention developed palpable mammary cancer which grew to 3112 mm3 by 25 weeks of age and led to the termination of these animals at week at this time point. However, STX140, when administered at week 12 onwards, blocked disease progression and no palpable tumor develop in 47% of tumors and significantly inhibited the growth of tumors that did develop. This resulted in a significant (P To conclude, STX140 has a greater anti-cancer efficacy and conferred an improved survival outcome compared to paclitaxel in a clinically relevant transgenic mouse model of metastatic breast cancer. 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 1558.