In vivo MTD study of new potential inhibitors of Proprotein Convertase Subtilisin/Kexin 9 (PCSK9)

Abstract

Aim: This study aims to determine the in vivo safety of newly synthesized PCSK9 inhibitors (MR-compounds). PCSK9 controls peripheral and central cholesterol levels and it plays a crucial role in hypercholesterolemia, being a well-established pharmacological target to treat this pathology, while a possible involvement in the aetiopathogenesis of AD has been postulated. Currently, the available PCSK9i to counteract hypercholesterolemia are very expensive biotechnological drugs and only subcutaneously administrated. Based on these premises, orally bioavailable small-molecules may be a valuable addition to existing treatments.
Methods: After preliminary in vitro screening in human hepatocyte and neuroblastoma cells of 30 compounds, 4 compounds were selected to test tolerability and bioavailability in vivo in wild-type mice (C57BL/6J) at 12.5mg/kg, 25mg/kg, 50mg/kg, and 100mg/kg for 5 days. MR-532 and MR-533 were administered subcutaneously, while MR-3 and MR-644 both subcutaneously and orally. Body weight and phenotype analysis were assessed daily to evaluate tolerability and macroscopic toxicity. After the sacrifice, hepatic toxicity (histological analysis and ALT activity) and biodistribution (LC-MS/MS) were evaluated.
Results: All doses of compounds were well tolerated (no changes in body weight, food intake, coat; no lethargy was observed). The MR-532 and MR-533 at 100mg/kg did not show elevated levels of ALT activity compared to vehicle (66mU±55, 76mU±127, and 130mU±203, respectively) or inflammatory cell infiltration or necrosis in liver sections (histological analysis). Interestingly, MR-532 and MR-533 were detected at all doses in plasma (261-318nM; 159-192nM), liver (522-1063pmol/g; 2824-3135mol/g) and brain (513-779pmol/g; 457-380mol/g), respectively, without a dose-dependent trend. MR-3 and MR-644 analyses are in progress.
Conclusion: All tested compounds proved to be safe. MR-532 and MR-533 showed plasma and hepatic bioavailability. They can reach the CNS, although at low concentrations. Further investigations are needed to understand how the route of administration affects biodistribution and to evaluate the efficacy of these compounds in cardiovascular and neurodegenerative diseases.