Discovery of the First Potent and Selective Inhibitor of Centromere-Associated Protein E: GSK923295.

Inhibition of mitotic kinesins represents a novel approach for the discovery of a new generation of anti-mitotic cancer chemotherapeutics. We report here the discovery of the first potent and selective inhibitor of centromere-associated protein E (CENP-E) 3-chloro-N-{(1S)-2-[(N,N-dimethylglycyl)amino]-1-[(4-{8-[(1S)-1-hydroxyethyl]imidazo[1,2-a]pyridin-2-yl}phenyl)methyl]ethyl}-4-[(1-methylethyl)oxy]benzamide (GSK923295; 1), starting from a high-throughput screening hit, 3-chloro-4-isopropoxybenzoic acid 2. Compound 1 has demonstrated broad antitumor activity in vivo and is currently in human clinical trials.

Involvement of intestinal uptake transporters in the absorption of azithromycin and clarithromycin in the rat.

Macrolide antibiotics azithromycin (AZI) and clarithromycin (CLARI) are large molecular weight compounds and are substrates for apically polarized efflux transporters such as P-glycoprotein, which can potentially restrict intestinal absorption. However, despite these undesired physicochemical and biopharmaceutical properties, AZI and CLARI exhibit moderate to excellent p.o. bioavailability in preclinical species and humans. Intestinal uptake transporters, such as organic anion transporting polypeptides (OATPs), can facilitate the uptake of drugs that are substrates and hence increase p.o. absorption. The present study was designed to determine whether the intestinal Oatps are involved in absorption of these macrolides. AZI or CLARI was dosed p.o. to Sprague-Dawley rats after p.o. administration with vehicle or rifamycin SV (RIF), an OATP inhibitor. The p.o. exposures of AZI and CLARI were reduced 65 and 45%, respectively, when coadministered with an optimized RIF regimen. The p.o. RIF had no affect on the total blood clearance of these macrolides and most likely did not cause induction of metabolizing enzymes and/or transporters. Therefore, the results suggest that inhibition of an RIF-sensitive uptake transporter such as Oatp along the rat gastrointestinal tract was responsible for reduced p.o. exposure of AZI and CLARI. In addition, AZI and CLARI caused inhibition of taurocholate uptake in rat Oatp1a5-transfected Madin-Darby canine kidney cell monolayers. The in vitro and in vivo results suggest that the intestinal Oatps are involved in the p.o. absorption of AZI and CLARI in the rat.

Novel ATP-competitive kinesin spindle protein inhibitors.

Kinesin spindle protein (KSP), an ATPase responsible for spindle pole separation during mitosis that is present only in proliferating cells, has become a novel and attractive anticancer target with potential for reduced side effects compared to currently available therapies. We report herein the discovery of the first known ATP-competitive inhibitors of KSP, which display a unique activity profile as compared to the known loop 5 (L5) allosteric KSP inhibitors that are currently under clinical evaluation. Optimization of this series led to the identification of biphenyl sulfamide 20, a potent KSP inhibitor with in vitro antiproliferative activity against human cells with either wild-type KSP (HCT116) or mutant KSP (HCT116 D130V). In a murine xenograft model with HCT116 D130V tumors, 20 showed significant antitumor activity following intraperitoneal dosing, providing in vivo proof-of-principle of the efficacy of an ATP-competitive KSP inhibitor versus tumors that are resistant to the other known KSP inhibitors.

Automated analysis of polyethylene glycol-induced inhibition of P-glycoprotein activity in vitro.

Previous studies in our laboratories have shown that commonly used polyethoxylated pharmaceutical excipients inhibit P-glycoprotein activity in cell culture models of the intestinal mucosa. The results presented in this technical note show that the TECAN Genesis robotic workstation can be utilized to automate cellular transport studies for evaluating excipient effects on P-glycoprotein activity in vitro and for estimating the permeation of drug-like molecules across cell monolayers.

Effects of poly(ethylene glycol) on efflux transporter activity in Caco-2 cell monolayers.

Poly(ethylene glycol) (PEG) is an excipient commonly used in pharmaceutical formulations to increase the aqueous solubility of drugs intended for oral administration. High concentrations of PEG are often used to solubilize drug candidates for in vitro experiments in cell culture (e.g., Caco-2 cell permeability studies) and/or for in vivo pharmacokinetic and safety studies in animals. Although PEG is often deemed safe in these studies based on gross morphological studies, changes on a molecular level may be overlooked. The purpose of this study was to determine the possible effects of PEG on efflux transporter activity in Caco-2 cell monolayers, an in vitro model of the intestinal mucosa. In these studies, relatively high, yet clinically achievable, concentrations of PEG-300 did not significantly change the passive paracellular or transcellular permeation of model solutes across Caco-2 cell monolayers. More importantly, PEG-300 inhibited efflux transporter activity in Caco-2 cell monolayers, which is probably mediated by P-gp and/or MRP. Such PEG-induced inhibition of efflux transporter activity is most likely caused by changes in the microenvironment of the Caco-2 cell membranes, which perturbs the ability of these transporters to efflux substrates such as taxol and doxorubicin.

A comparison of commonly used polyethoxylated pharmaceutical excipients on their ability to inhibit P-glycoprotein activity in vitro.

P-glycoprotein (P-gp), a multidrug resistance (MDR) protein encoded by the MDR1 gene in humans, is responsible for the efflux of structurally diverse drugs. Previous studies in our laboratory have shown that excipients such as poly(ethylene)glycol (PEG)-300, Cremophor EL, and Tween 80 inhibit P-gp activity in Caco-2 cell monolayers. The objective of this study was to determine the effects of these excipients in an MDR1- transfected Madin Darby Canine Kidney (MDR1-MDCK) cell line and to compare the results with those obtained from Caco-2 cells. The results presented herein show that PEG-300 (20%, v/v) causes almost complete inhibition of P-gp activity in both Caco-2 and MDR1-MDCK cell monolayers, whereas Cremophor EL (0.1%, w/v) and Tween 80 (0.05%, w/v) only partially inhibit P-gp activity in Caco-2 cells. Cremophor EL (0.1%, w/v) and Tween 80 (0.05%, w/v) were inactive as P-gp inhibitors in MDR1-MDCK cell monolayers. This inability of Tween 80 and Cremphor EL to inhibit P-gp activity in MDR1-MDCK cells may be related to differences in the interactions of the surfactants with these different cell membranes. PEG-induced changes in P-gp activity are probably related to changes in the fluidity of the polar head group regions of cell membranes.