121 - Development of Protease Proprotein Convertase Subtilisin-Like Kexin Type 9 Antagonists

Abstract
Protease proprotein convertase subtilisin-like kexin type 9 (PCSK9) is an important target for the treatment of hypercholesterolemia. Several pharmaceutical companies have focused on the development of mAbs. Yet, our approach has focused on developing orally bioavailable small molecule antagonists. PCSK9 is a serine protease that is synthesized as a zymogen that undergoes autocatalytic processing and secretion. Secreted PCSK9 binds to the LDL-receptor (LDLR) and chaperones it to the degradation pathway. We have successfully identified small molecule antagonists against PCSK9 through a virtual screen coupled with in vitro and cell-based assays. Our lead compound, P-4, exhibited a concentration-dependent inhibition of the PCSK9/LDLR interaction with an IC50 in the nanomolar range, an increase in the level of LDLR in recombinant cell-based assays, and a significant increase in the fluorescently labeled DiI-LDL uptake in the nanomolar range. In addition, P-4 exhibited over 20% reduction in LDL-C when administered at 10 mg/kg orally to mice fed a high fat/high cholesterol diet. This study focused on the development of P-4 as a preclinical candidate for an IND filling. P-4 exhibited a good safety profile as demonstrated in vitro, in cell-based assays as well as in an animal model. In vitro analysis of P-4 revealed no inhibition of P-4 against 8 cytochrome P-450 forms up to >100 µM. P-4 showed good stability in both mouse and human liver microsomes, with in vitro half-life values ≥ 2 hours. P-4 exhibited higher turnover rate in monkey microsomes, with a half-life of 34 minutes. Formulation strategies in early-stage drug development are critical and we focused our strategy on applying the right formulation early in our drug development process to avoid costly late-stage failures. We tested the effect of many soluble and nano-formulations on P-4 pharmacokinetics and its oral bioavailability. Our data showed that formulations P-4/PEG 300 liquid formulation and P-4/PVP/Alginic acid Core Shell Chitosan Nanoparticles were among the best formulations tested. These formulations improved significantly the PK and oral bioavailability of P-4. To our knowledge, no small molecule orally bioavailable antagonist against PCSK9 has been reported, only Injectable mAbs.