The preclinical pharmacology of novel inhibitors of perforin function

Abstract

A novel series of chemical inhibitors of perforin mediated cytolytic function is currently in preclinical development as specific inhibitors of the granule exocytosis pathway of the immune response. This thesis focussed on the aryl-substituted isobenzofuran-1(3H)-one series of inhibitors. There are no published reports regarding the preclinical pharmacology of this novel series. Therefore the aim of this thesis was to investigate the pharmacokinetics, metabolic stability and disposition in preclinical models (mouse in vitro and in vivo) as well as the mechanism of inhibition (in vitro biosensor analysis) of the aryl-substituted isobenzofuran-1(3H)-one inhibitors of perforin function. Following an intravenous dose (5 mg/kg) to mice, plasma concentrations were observed to be higher than the concentration required for inhibition of perforin function in vitro (IC50 values) for eleven of sixteen compounds. The compounds were found to be well tolerated by mice and have suitable pharmacokinetic characteristics for further development as potential drugs. The metabolic stability of the series of perforin inhibitor compounds was determined by incubation with mouse liver subcellular fractions, in the presence of various cofactors. The compounds were observed to be substrates for oxidative metabolism, with considerable metabolic loss observed in microsomal incubations for all compounds. Following multiple intraperitoneal dosing (60 mg/kg, b.i.d) to mice, a lead compound (SN 33614) was observed to be well tolerated. The drug did not accumulate in plasma or tissues following multiple doses and, importantly, the preclinical efficacy of SN 33614 was demonstrated to relate to the systemic exposure of the drug. The compounds were shown to reversibly and specifically bind to perforin through the use of surface plasmon resonance biosensor technology. A range of binding was observed and a significant rank order correlation between in vitro inhibitory activity and binding to perforin monomers was observed. The results in this thesis provide an understanding of the preclinical pharmacology of novel small chemical inhibitors of perforin function. This may provide insights which could result in clinical development of the first drug to inhibit perforin function for use as an immunosuppressive agent.