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

Abstract

1. Aggregates of the protein α-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 (C₈-6-I) or nicotine (C₈-6-N) bind α-synuclein and protect yeast cells from α-synuclein mediated toxicity.

2. A critical step in development of positron emission tomography (PET) probes for neurodegenerative diseases is evaluation of their metabolic stability. We determined that C₈-6-I, and C₈-6-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 ¹⁹F-C₈-6-I and ¹⁹F-C₈-6-N is stable to P450 enzymes.

3. 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 CL_int,u in mouse liver microsomes. We found that C₈-6-I and ¹⁹F-C₈-6-I have a two- to fourfold lower CL_int,u than C₈-6-N, and ¹⁹F-C₈-6-N. Our approach shows a simple, specific, and effective system to design and develop compounds as PET probes.

Keywords:

• Microsomal metabolism
• method validation
• positron emission tomography
• imaging probe design
• α-synuclein
• Parkinson’s disease
• enzyme kinetics

Disclosure statement

The authors report no declarations of interest.

Additional information

Funding

OPA is a recipient of a Department of Chemistry Scholarship, CKN is a recipient of Pharmacy and Nutrition Graduate Student Scholarship, MNO is a recipient of an NSERC-USRA scholarship, KJHA was funded by Sylvia Fedoruk Canadian Centre for Nuclear Innovation (CCNI) grant under grant #J2013-0124. This work was supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant (NSERC-DG) under grant #165915.