Xiaohong Cai

Molecular and cellular pharmacology of the hypoxia-activated prodrug TH-302

TH-302 is a 2-nitroimidazole triggered hypoxia-activated prodrug (HAP) of bromo-isophosphoramide mustard currently undergoing clinical evaluation. Here, we describe broad-spectrum activity, hypoxia-selective activation, and mechanism of action of TH-302. The concentration and time dependence of TH-302 activation was examined as a function of oxygen concentration, with reference to the prototypic HAP tirapazamine, and showed superior oxygen inhibition of cytotoxicity and much improved dose potency relative to tirapazamine. Enhanced TH-302 cytotoxicity under hypoxia was observed across 32 human cancer cell lines. One-electron reductive enzyme dependence was confirmed using cells overexpressing human NADPH:cytochrome P450 oxidoreductase and radiolytic reduction established the single-electron stoichiometry of TH-302 fragmentation (activation). Examining downstream effects of TH-302 activity, we observed hypoxia-dependent induction of γH2AX phosphorylation, DNA cross-linking, and cell-cycle arrest. We used Chinese hamster ovary cell–based DNA repair mutant cell lines and established that lines deficient in homology-dependent repair, but not lines deficient in base excision, nucleotide excision, or nonhomologous end-joining repair, exhibited marked sensitivity to TH-302 under hypoxia. Consistent with this finding, enhanced sensitivity to TH-302 was also observed in lines deficient in BRCA1, BRCA2, and FANCA. Finally, we characterized TH-302 activity in the three-dimensional tumor spheroid and multicellular layer models. TH-302 showed much enhanced potency in H460 spheroids compared with H460 monolayer cells under normoxia. Multicellular layers composed of mixtures of parental HCT116 cells and HCT116 cells engineered to express an oxygen-insensitive bacterial nitroreductase showed that TH-302 exhibits a significant bystander effect. Mol Cancer Ther; 11(3); 740–51. ©2011 AACR.

A novel class of tubulin inhibitors that exhibit potent antiproliferation and in vitro vessel-disrupting activity

PurposeSince anticancer agents that interfere with microtubule function are in widespread use and have a broad spectrum of activity against both hematological malignancies and solid tumors, there is an urgent need to develop novel tubulin inhibitors with broader activities and avoiding drug resistance.Methods and resultsIn this study, we describe the characterization of select lead compounds from a novel class of indazole-based tubulin inhibitors. Three lead compounds, TH-337, TH-482 and TH-494, exhibit potent antiproliferative activity against cell lines derived from human pancreatic carcinoma, human breast adenocarcinoma and human colorectal adenocarcinoma cells. The three compounds were also tested for cytotoxicity against a panel of clinically relevant drug resistant cancer cell lines that either overexpress the drug resistance pumps MDR-1, MRP-1 and BCRP-1 or have altered Topoisomerase II activity. TH-482 and -494 retained cytotoxic activities against all of the resistant cell lines tested; however, TH-337 exhibited decreased cytotoxicity in the cell line overexpressing BCRP-1, indicating that TH-337 is a substrate of that pump. We show that TH-482’s antiproliferative activity is due to cell cycle arrest at the G2/M phase. We demonstrate that TH-482 binds specifically to the colchicine site of tubulin and that it inhibits tubulin polymerization in vitro in a concentration-dependent manner. The in vitro anti-vascular activities of TH-482 were assessed using the HUVEC-C cell line. TH-482 inhibits in vitro neovessel formation and disrupts pre-established vessels using HUVEC-C cells. TH-482 also increases permeability of vascular endothelial cells in a concentration- and time-dependent manner.ConclusionsTH-482 demonstrates potent in vitro efficacy as a novel tubulin-targeted anti-proliferative and anti-vascular agent and notably is more potent in antiproliferative assays than the benchmark compound combretastatin A-4. These results identify TH-482 as a potent tubulin inhibitor, and support the investigation of its in vivo efficacy and pharmacokinetic properties as the prototype of a new class of anti-tubulin agents.

14-Aminocamptothecins: their synthesis, preclinical activity, and potential use for cancer treatment.

14-Aminocamptothecins were synthesized in good yields by treating camptothecin (1a) and 7-ethylcamptothecin (1b) with 90% fuming nitric acid either neat or in acetic anhydride and then followed by reduction of the resulting 14-nitrocamptothecins (2). 14-Aminocamptothecin (3a) and 7-ethyl-14-aminocamptothecin (3b) demonstrated excellent cytotoxic potency against human tumor cell lines in vitro, and they are not substrates for any of the major clinically relevant efflux pumps (MDR1, MRP1, and BCRP). 3a and 3b showed similar cytotoxicity against human and mouse bone marrow progenitor cells. This is in contrast to many camptothecin analogues, which are substrates for efflux pumps and are dramatically more toxic to human marrow cells relative to murine. 3a and 3b demonstrated significant brain penetration when dosed orally in mice. 3b showed significantly better efficacy relative to topotecan when dosed orally in the three ectopic xenograft models, H460, HT29, and PC-3. On the basis of its favorable in vitro and in vivo profile, 3b warrants future development.