Abstract

The major mode of metabolism of nicotine is via the formation of cotinine by the enzyme cytochrome P450 (CYP) 2A6. Cotinine undergoes further CYP2A6-mediated metabolism by hydroxylation to 3-hydroxycotinine and norcotinine but can also form cotinine-N-glucuronide and cotinine-N-oxide (COX). The goal of the present study was to investigate the enzymes that catalyze COX formation and determine whether genetic variation in these enzymes may affect this pathway. Specific inhibitors of major hepatic cytochrome P450 (CYP) enzymes were used in cotinine-N-oxidation reactions using pooled human liver microsomes (HLM). COX formation was monitored by ultra-high pressure liquid chromatography-mass spectrometry and enzyme kinetic analysis was performed using microsomes from CYP-overexpressing HEK293 cell lines. Genotype-phenotype analysis was performed in a panel of 113 human liver specimens. Inhibition of COX formation was only observed in HLM when using inhibitors of CYPs 2A6, 2B6, 2C19, 2E1, and 3A4. Microsomes from cells overexpressing CYPs 2A6 or 2C19 exhibited similar N-oxidation activity against cotinine, with V_max/K_M values of 4.4 and 4.2 nL/min/mg, respectively. CYP2B6-, CYP2E1-, and CYP3A4-overexpressing microsomes were also active in COX formation. Significant associations (p<0.05) were observed between COX formation and genetic variants in CYPs 2C19 (*2 and *17 alleles) in HLM. These results demonstrate that genetic variants in CYP2C19 are associated with decreased COX formation, potentially affecting the relative levels of cotinine in the plasma or urine of smokers and ultimately affecting recommended smoking cessation therapies.

Significance Statement This study is the first to elucidate the enzymes responsible for cotinine-N-oxide formation and genetic variants that affect this biological pathway. Genetic variants in CYP2C19 have the potential to modify NMR in smokers and could affect pharmacotherapeutic decisions for smoking cessation treatments.