An HPLC-UV validated bioanalytical method for measurement of in vitro phase 1 kinetics of oc-synuclein binding bifunctional compounds

Aggregates of the protein alpha-synuclein are associated with pathophysiology of Parkinson's disease and are present in Lewy Bodies found in the brains of Parkinson's patients. We previously demonstrated that bifunctional compounds composed of caffeine linked via a six carbon chain to either 1-aminoindan (C-8-6-I) or nicotine (C-8-6-N) bind alpha-synuclein and protect yeast cells from alpha-synuclein mediated toxicity.A critical step in development of positron emission tomography (PET) probes for neurodegenerative diseases is evaluation of their metabolic stability. We determined that C-8-6-I, and C-8-6-N both undergo phase 1 P450 metabolism in mouse, rat, and human liver microsomes. We utilised this metabolic information to guide the design of fluorinated analogues for use as PET probes and determined that the fluorine in F-19-C-8-6-I and F-19-C-8-6-N is stable to P450 enzymes.We have developed and validated an analytical HPLC-UV method following FDA and EMA guidelines to measure in vitro phase 1 kinetics of these compounds and determine their V-max, K-M and CLint,u in mouse liver microsomes. We found that C-8-6-I and F-19-C-8-6-I have a two- to fourfold lower CLint,u than C-8-6-N, and F-19-C-8-6-N. Our approach shows a simple, specific, and effective system to design and develop compounds as PET probes.