Metabolism of a liver-selective nitric oxide-releasing agent, V-PYRRO/NO, by human microsomal cytochromes P450.
Endogenously generated nitric oxide (NO) mediates a host of important physiological functions, playing roles in the vascular, immunological, and neurological systems. As a result, exogenous agents that release NO have become important therapeutic interventions and research tools. O(2)-Vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) is a prodrug designed with the hypothesis that it might release nitric oxide via epoxidation of the vinyl group by cytochrome P450, followed by enzymatic and/or spontaneous epoxide hydration to release the ultimate NO-donating moiety, 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (PYRRO/NO) ion. In this study, we investigated this hypothetical activation mechanism quantitatively for V-PYRRO/NO using cDNA-expressed human cytochrome P450 (CYP)2E1. Incubation with CYP2E1 and an NADPH-regenerating system resulted in a time-dependent decomposition of V-PYRRO/NO, with a turnover rate of 2.0 nmol/min/pmol CYP2E1. Nitrate and nitrite were detected in high yield as metabolites of NO. The predicted organic metabolites pyrrolidine and glycolaldehyde were also detected in near-quantitative yields. The enzymatic decomposition of V-PYRRO/NO was also catalyzed, albeit at lower rates, by CYP2A6 and CYP2B6. We conclude that the initial step in the metabolism of V-PYRRO/NO to NO in the liver is catalyzed efficiently but not exclusively by the alcohol-inducible form of cytochrome P450 (CYP2E1). The results confirm the proposed activation mechanism involving enzymatic oxidation of the vinyl group in V-PYRRO/NO followed by epoxide hydration and hydrolytic decomposition of the resulting PYRRO/NO ion to generate nitric oxide.
Chemistry Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, MD 21702, USA.
Keiko
K
Inami
Raymond W
RW
Nims
Aloka
A
Srinivasan
Michael L
ML
Citro
Joseph E
JE
Saavedra
Arthur I
AI
Cederbaum
Larry K
LK
Keefer
D000079:Acetaldehyde / Q000031:analogs & derivatives / Q000378:metabolism; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D002384:Catalysis; D065637:Cytochrome P-450 CYP2A6; D065702:Cytochrome P-450 CYP2B6; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D011759:Pyrrolidines / Q000378:metabolism* / Q000494:pharmacology
D018076:DNA, Complementary; D009566:Nitrates; D009573:Nitrites; C107010:O(2)-vinyl-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate; D011759:Pyrrolidines; D009569:Nitric Oxide; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019392:Cytochrome P-450 CYP2E1; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; C585599:CYP2B6 protein, human; D065637:Cytochrome P-450 CYP2A6; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating; D000079:Acetaldehyde; C010972:glycolaldehyde