Purpose of this investigation is to develop a small molecule PCSK9i, and demonstrate its cholesterol lowering, anti-platelet and neuroprotective efficacy in in vitro intracerebral hemorrhage model.

Stroke is a medical emergency and currently the only available treatment is the intravenous injection of tissue-type plasminogen activator (tPA) or removal of blood clots. Hypertension, blood clotting and hypercholesterolemia are the main causative factors for cerebral thrombosis and ischemia. Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) plays a major role in LDL receptor (LDLR) regulation, atherosclerotic plaque formation and platelet activation. A meta-analysis study based on data from the clinical trial (IMPROVEIT), confirms that cholesterol-lowering interventions (statins and PCSK9 inhibitors) are expected to reduce stroke. As statins are reported to elevate serum PCSK9 levels, PCSK9i would be a better choice for the prevention and treatment of stroke. Currently there are no approved small molecule PCSK9i.

The crystal structure of PCSK9 enzyme was obtained from the Protein Data Bank. The FDA-approved Drug Library containing a collection of 2992 compounds were screened using molecular docking and the top five molecules with high affinity to PCSK9 were selected. The selected molecules were screened for PCSK9 inhibition assay using ELISA method (CircuLex). Effect of PCSK9i on fluorescently labeled LDL uptake was tested using caco-2 cells. Anti-platelet activity was tested by flow cytometry using mouse platelets, and neuroprotective activity was tested in cerebral vascular endothelial (CVE) cells and human astrocytes (HA) after challenging with hemin (75 μM) using MTT assay. All the experiments were repeated in triplicate (n=3. Statistical analysis of the data was done using ANOVA. p<0.05 was considered significant.

Among the inhibitors tested, proanthocyanidin (PAC) was determined to be the best PCSK9 inhibitor. PAC showed dose-dependent PCSK9 inhibition (sigmoid shape) in the concentration range tested (10 to 1000 μM), increased cellular uptake of LDL in Caco-2 cells. PAC showed the highest platelet inhibitory activity with IC50 of <3 μM. When CVE and HA were treated with PAC (100 μM), cell viability increased to 90% in 24 h as compared to hemin (30% cell viability) (p<0.05). As PAC is not water-soluble, a nanoemulsion was prepared with globule size <136 nm for improving absorption across the blood-brain-barrier (BBB).

PAC is the best PCSK9i among the various PCSK9 modulators identified by molecular docking. PAC has also shown increased LDL uptake, high antiplatelet activity and excellent neuroprotection. Collectively, the results indicate the great potential of PAC for prevention and treatment of stroke warranting in vivo testing in stroke models.