Drug-like property profiling of novel neuroprotective compounds to treat acute ischemic stroke: guidelines to develop pleiotropic molecules

Abstract

The development of novel neuroprotective compounds to treat acute ischemic stroke (AIS) has been problematic and quite complicated, since many candidates that have been tested clinically lacked significant pleiotropic activity, were unable to effectively cross the blood brain barrier (BBB), had poor bioavailability or were toxic. Moreover, the compounds did not confer significant neuroprotection or clinical efficacy measured using standard behavioral endpoints, when studied in clinical trials in a heterogeneous population of stroke patients. To circumvent some of the drug development problems describe above, we have used a rational funnel approach to identify and develop promising candidates. Using a step-wise approach, we have identified a series of compounds based upon two different neuroprotection assays. We have then taken the candidates and determined their "drug-like" properties. This guidelines article details in vitro screening assays used to show pleiotropic activity of a series of novel compounds; including enhanced neuroprotective activity compared to the parent compound fisetin. Moreover, for preliminary drug de-risking or risk reduction during development, we used compound assessment in the CeeTox assay, ADME toxicity using the AMES test for genotoxicity and interaction with Cytochrome P450 using CYP450 inhibition analysis against a spectrum of CYP450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) as a measure of drug interaction. Moreover, the compounds have been studied using a transfected Madin Darby canine kidney (MDCK) cell assay to assess blood brain barrier penetration (BBB). Using this series of assays, we have identified 4 novel molecules to be developed as an AIS treatment.

Keywords: ADME; Ames test; MDCK; chalcone; flavone; flavonoid; pleiotropic; screening; toxicity.

Figure 1. HT22 IAA Neuroprotection Profiles

Flavones promote cell survival following IAA challenge. Pharmacological effects of the 10 primary candidates on cultured HT22 mouse hippocampal cells treated with 20 μM iodoacetic acid (IAA) for 2 hr alone or in the presence of varying concentrations of drug. At 24 hours, cell survival was measured using a standard MTT assay (1, 2). In the absence of a neuroprotective, >95% of the cell population dies off within 24 hours (3, 4). The graph shows that the candidates are neuroprotective up to 1 μM, the maximum concentration used in the assay and cell survival was maximally increased to 75 – 85% of control.

Figure 2

Representative Ceetox analysis curves (Compound CSMC-6)