In vitro and in vivo studies on novel pcsk9 inhibitors as pharmacological approach for the treatment of alzheimer’s disease

Abstract

Aim: Impairment of cholesterol homeostasis is one of the multiple etiopathological mechanisms at the origin of both cardiovascular and neurodegenerative diseases. The PCSK9 protein, known for its role in the degradation of hepatic LDLR and plasma cholesterol regulation, is expressed also in the CNS, where it exacerbates -amyloid neurotoxicity and reduces neuronal cholesterol uptake, suggesting an involvement in AD. This study proposes an in vitro screening of molecules (MR) with inhibitory activity on PCSK9, selecting the best compounds to test their activity on cerebral cell models and their in vivo tolerability.
Methods: 30 newly synthesized compounds were tested at increasing concentrations on human hepatoma cells (HepG2) to evaluate their cytotoxicity and efficacy in inhibiting PCSK9. MR-3 was tested on human neuroblastoma cells (SH-SY5Y) overexpressing PCSK9 to assess neurotoxicity and cholesterol uptake. Cytotoxicity was determined through MTT assay; PCSK9 secretion was quantified with an ELISA kit; and radioisotopic techniques measured cholesterol uptake . Three compounds were selected to be tested in vivo on C57BL/6 mice at a dose of 40 mg/Kg for 7 days to evaluate: tolerability with SHIRPA test; plasma lipid profile by ELISA assay; biodistribution in plasma and brain through LC-MS/MS.
Results: Among the tested compounds, MR-3, MR-532, MR-533 demonstrated no sign of cytotoxicity and the greatest efficacy on HepG2 cells (IC50=1.7μM; 5.7μM; 6.1μM). Neuronal cholesterol uptake was restored after treatment with MR-3 at 10μM (p<0,05). MR-3, MR-532, and MR-533 exhibited good in vivo tolerability; MR-3 and MR-532 were detected in plasma and brain tissue.
Conclusions: Preliminary in vitro screening allowed the identification of MR-3, MR-532, MR-533 as promising PCSK9 inhibitors. The outcome of MR-3 on neuronal cholesterol uptake may suggest a neuroprotective effect to be further investigated. In vivo treatment with selected inhibitors shown absence of toxicity, however, it is necessary to bring proof of efficacy.