Purpose The anti-apoptotic function of the 70 kDa family of heat shock proteins and their role in cancer is well documented. Dual targeting of Hsc70 and Hsp70 with siRNA induces proteasome-dependent degradation of Hsp90 client proteins and extensive tumor specific apoptosis as well as the potentiation of tumor cell apoptosis following pharmacological Hsp90 inhibition. Methods We have previously described the discovery and synthesis of novel adenosine-derived inhibitors of the 70 kDa family of heat shock proteins; the first inhibitors described to target the ATPase binding domain. The in vitro activity of VER-155008 was evaluated in HCT116, HT29, BT474 and MDA-MB-468 carcinoma cell lines. Cell proliferation, cell apoptosis and caspase 3/7 activity was determined for VER-155008 in the absence or presence of small molecule Hsp90 inhibitors. Results VER-155008 inhibited the proliferation of human breast and colon cancer cell lines with GI 50 s in the range 5.3-14.4 lM, and induced Hsp90 client protein degradation in both HCT116 and BT474 cells. As a single agent, VER-155008 induced caspase-3/7 dependent apoptosis in BT474 cells and non-caspase dependent cell death in HCT116 cells. VER-155008 potentiated the apoptotic potential of a small molecule Hsp90 inhibitor in HCT116 but not HT29 or MDA-MB-468 cells. In vivo, VER-155008 demonstrated rapid metabolism and clearance, along with tumor levels below the predicted pharmacologically active level. Conclusion These data suggest that small molecule inhibitors of Hsc70/Hsp70 phenotypically mimic the cellular mode of action of a small molecule Hsp90 inhibitor and can potentiate the apoptotic potential of a small molecule Hsp90 inhibitor in certain cell lines. The factors determining whether or not cells apoptose in response to Hsp90 inhibition or the combination of Hsp90 plus Hsc70/ Hsp70 inhibition remain to be determined.

Synthetic routes to a series of mono- and difluorinated 2-(4-amino-3-substituted-phenyl)benzothiazoles have been devised. Whereas mixtures of regioisomeric 5- and 7-fluoro-benzothiazoles were formed from the established Jacobsen cyclization of precursor 3-fluoro-thiobenzanilides, two modifications to this general process have allowed the synthesis of pure samples of these target compounds. Fluorinated 2-(4-aminophenyl)benzothiazoles were potently cytotoxic (GI(50) < 1 nM) in vitro in sensitive human breast MCF-7 (ER+) and MDA 468 (ER-) cell lines but inactive (GI(50) > 10 microM) against PC 3 prostate, nonmalignant HBL 100 breast, and HCT 116 colon cells. The biphasic dose-response relationship characteristically shown by the benzothiazole series against sensitive cell lines was exhibited by the 4- and 6-fluoro-benzothiazoles (10b,d) but not by the 5- and 7-fluoro-benzothiazoles (10h,i). The most potent broad spectrum agent in the NCI cell panel was 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (10h) which, unlike the 6-fluoro isomer (10d), produces no exportable metabolites in the presence of sensitive MCF-7 cells. Induction of cytochrome P450 CYP1A1, a crucial event in determining the antitumor specificity of this series of benzothiazoles, was not compromised. 10h is currently the focus of pharmaceutical and preclinical development.

Novel 2-(4-aminophenyl)benzothiazoles possess highly selective, potent antitumour properties in vitro and in vivo. They induce and are biotransformed by cytochrome P450 (CYP) 1A1 to putative active as well as inactive metabolites. Metabolic inactivation of the molecule has been thwarted by isosteric replacement of hydrogen with fluorine atoms at positions around the benzothiazole nucleus. The lipophilicity of these compounds presents limitations for drug formulation and bioavailability. To overcome this problem, water soluble prodrugs have been synthesised by conjugation of alanyl-and lysyl-amide hydrochloride salts to the exocyclic primary amine function of 2-(4-aminophenyl)benzothiazoles. The prodrugs retain selectivity with significant in vitro growth inhibitory potency against the same sensitive cell lines as their parent amine, but are inactive against cell lines inherently resistant to 2-(4-aminophenyl)benzothiazoles. Alanyl and lysyl prodrugs rapidly and quantitatively revert to their parent amine in sensitive and insensitive cell lines in vitro. Liberated parent compounds are sequestered and metabolised by sensitive cells only; similarly, CYP1A1 activity and protein expression are selectively induced in sensitive carcinoma cells. Amino acid prodrugs meet the criteria of aqueous solubility, chemical stability and quantitative reversion to parent molecule, and thus are suitable for in vivo preclinical evaluation.

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Background and ObjectiveThe inhibition of fatty acid amide hydrolase 1 (FAAH) has been proposed as a novel mechanism for treating pain syndromes by increasing the levels of endogenous cannabinoids (ECs). This study describes the safety, tolerability, pharmacokinetics and pharmacodynamics of V158866, a reversible FAAH inhibitor, after first administration to man.Methods51 healthy male subjects were recruited into this double-blind, randomised, placebo-controlled, adaptive dose, phase I single (Part A) and repeated ascending dose (Part B) study. The primary outcome was the safety and tolerability of V158866. Secondary outcomes were (1) pharmacokinetics of V158866 and (2) pharmacodynamics of V158866, as assessed by changes in plasma EC concentrations.ResultsSingle oral doses of 5–300 mg and repeated oral doses of 50–500 mg were evaluated. V158866 was well tolerated, with no apparent treatment-related effects on laboratory variables. V158866 was rapidly absorbed with a mean terminal elimination half-life of 9.6–18.3 h (Day 7; Part B). V158866 reached steady state within 2–3 days of administration, with an accumulation ratio, based on AUC0–24h, of approximately 2 on Day 7. V158866 showed a linear relationship between dose and AUC across the entire dose range. V158866 caused reversible, dose-related increases in plasma ECs. At hemi-equilibrium, there was a sigmoidal maximum effect relationship between plasma V158866 concentrations and changes in plasma ECs.ConclusionsV158866 is well tolerated, with linear pharmacokinetics suitable for once-daily administration, and reversible effects on plasma ECs. Maximum increases in plasma ECs occur with V158866 doses of 300–500 mg/day.Electronic supplementary materialThe online version of this article (doi:10.1007/s40268-016-0127-y) contains supplementary material, which is available to authorized users.