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Xenobiotica
the fate of foreign compounds in biological systems
Volume 52, 2022 - Issue 8: 50th year of the DMDG
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Research Articles

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

, , , , , & show all
Pages 916-927 | Received 29 Jul 2022, Accepted 22 Oct 2022, Published online: 20 Dec 2022
 

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 Vmax, KM and CLint,u in mouse liver microsomes. We found that C8-6-I and 19F-C8-6-I have a two- to fourfold lower CLint,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.

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.

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