Formulation and characterization of CMPI nanoparticles for enhanced targeting of brain nicotinic receptors by positive allosteric modulator

3-(2-Chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1 H-pyrazol-4-yl)isoxazole (CMPI) is a nicotinic acetylcholine receptor (nAChRs), one of the most subtype-selective a positive allosteric modulator (PAM) of nicotinic acetylcholine receptors (nAChRs). CMPI that preferentially potentiates the (α4)3(β2)2 nAChR, the major nAChR subtype in the cortex and as such carries potential experimental and therapeutic applications. Maximizing delivery of CMPI would enhance its interaction with brain nAChRs that are associated with the desired therapeutic effects while avoiding interactions with peripheral nAChRs that are associated with undesired side effects is critical to the development of nAChR PAM-based therapeutics. Towards this endeavor, this study aims to explore nanoformulation strategies to maximize delivery of CMPI. A biodegradable and biocompatible, the US-FDA-approved, poly(l-lactic-co-glycolic) acid (PLGA) was used to engineer nanoparticles (NPs) to solubilize CMPI in its hydrophobic core in an aqueous environment using the nanoprecipitation with the drug loading content of 10 ± 1.2% by weight of NPs. Thus, synthesized polymeric NPs were characterized for their colloidal properties and biological activities. The hydrodynamic size of these NPs was found to range from 60 to 150 nm and are stable for a prolonged period in biological media. An in-vitro drug release study was conducted to envision a sustained release of CMPI under physiological conditions, which shows distinct kinetics of CMPI under experimental conditions in which released drugs from NPs were collected using dialysis techniques. These NPs were found to be highly biocompatible when challenged against the human embryonic kidney-293 (HEK-293) cell line that stably expressed α4β2 (HEK-α4β2) nAChRs in a wide range of concentrations. In this pilot study, NPs were further labeled with Alexa fluorophore to track and study cellular uptake using fluorescence microscopy, which showed efficient uptake by HEK-α4β2 cells. Given the superiority of the nanoparticulate system in drug delivery and the unique role of CMPI, we hope this study will help in the development of nAChR PAM formulations that have superior pharmacokinetic profiles, especially their brain bioavailability.