Abstract
(+)- And (−)-amphetamine and methamphetamine wereN-oxygenated by the cDNA expressed adult human flavin-containing monooxygenase form 3 (FMO3), their corresponding hydroxylamines. Two major polymorphic forms of human FMO3 were studied, and the results suggested preferentialN-oxygenation by only one of the two enzymes. Chemically synthesized (±)-amphetamine hydroxylamine was also a substrate for the human FMO3 and it was converted to phenylpropanone oxime with a stereoselectivity ratio of trans/cis of 5:1. Human FMO3 also N-oxygenated methamphetamine to produce methamphetamine hydroxylamine. Methamphetamine hydroxylamine was alsoN-oxygenated by human FMO3, and the ultimate product observed was phenylpropanone. For amphetamine hydroxylamine, studies of the biochemical mechanism of product formation were consistent with the production of an N,N-dioxygenated intermediate that lead to phenylpropanone oxime. This was supported by the observation that α-deutero (±)-amphetamine hydroxylamine gave an inverse kinetic isotope effect on product formation in the presence of human FMO3. For methamphetamine, the data were consistent with a mechanism of human FMO3-mediated N,N-dioxygenation but the immediate product, a nitrone, rapidly hydrolyzed to phenylpropanone. The pharmacological activity of amphetamine hydroxylamine, phenylpropanone oxime, and methamphetamine hydroxylamine were examined for effects at the human dopamine, serotonin, and norepinephrine transporters. Amphetamine hydroxylamine and methamphetamine hydroxylamine were apparent substrates for the human biogenic amine transporters but phenylpropanone oxime was not. Presumably, phenylpropanone oxime or nitrone formation from amphetamine and methamphetamine, respectively, represents a detoxication process. Because of the potential toxic nature of amphetamine hydroxylamine and methamphetamine hydroxylamine metabolites and the polymorphic nature of N-oxygenation, human FMO3-mediated metabolism of amphetamine or methamphetamine may have clinical consequences.
Footnotes
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Send reprint requests to: John R. Cashman, Human BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, CA. E-mail: ledcash{at}aol.com
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↵1 This work was financially supported by National Institutes of Health Grants GM 36426, DA 11547 and DA 00269 (to J.R.C.) and Veterans Administration Merit Review and Career Scientist Program and NIH/NIDA Contract Number N01DA7-8071 (to A.J.).
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↵2 Present address: Portland Veterans Administration Medical Center and Department of Psychiatry and Physiology, Pharmacology and Behavioral Neuroscience, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Rd. Portland, OR 97201.
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↵3 Throughout the text, the synthetic AMPH hydroxylamine and METH hydroxylamine described is racemic.
- Abbreviations:
- AMPH
- amphetamine
- METH
- methamphetamine
- FMO3
- flavin-containing monooxygenase form 3
- NE
- norepinephrine
- DA
- dopamine
- MBP
- maltose binding protein
- NaCNBH3
- sodium cyanoborohydride
- FAB
- fast atom bombardment
- THF
- tetrahydrofuran
- TLC
- thin-layer chromatography
- 5-DPT
- 10-[(N,N-dimethylamino)pentyl]-2-(trifluoromethyl)phenothiazine
- 5-DPT N-oxide
- 10-[(N,N-dimethylamino)pentyl]-2-(trifluoromethyl)phenothiazineN-oxide
- hDAT
- human DA transporter
- hSERT
- human serotonin transporter
- hNET
- human NE transporter
- C6-hDAT cells
- C6 glioma cells transfected with hDAT
- DAT
- DA transporter
- 5-HT
- 5-hydroxytryptamine or serotonin
- MPP+
- 1-methyl-4-phenylpyridinium
- Received June 29, 1998.
- Accepted September 29, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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