Benzydamine N-oxidation As an Index Reaction Reflecting FMO Activity in Human Liver Microsomes and Impact of FMO3 Polymorphisms on Enzyme Activity

Overview

Journal Br J Clin Pharmacol

Specialty Pharmacology

Date 2001 Jan 3

PMID 11136294

Citations 26

Authors

E Stormer

I Roots

J Brockmoller

Affiliations

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Abstract

Aims: The role of flavin containing monooxygenases (FMO) on the disposition of many drugs has been insufficiently explored. In vitro and in vivo tests are required to study FMO activity in humans. Benzydamine (BZD) N-oxidation was evaluated as an index reaction for FMO as was the impact of genetic polymorphisms of FMO3 on activity.

Methods: BZD was incubated with human liver microsomes (HLM) and recombinant enzymes. Human liver samples were genotyped using PCR-RFLP.

Results: BZD N-oxide formation rates in HLM followed Michaelis-Menten kinetics (mean Km = 64.0 microM, mean Vmax = 6.9 nmol mg-1 protein min-1; n = 35). N-benzylimidazole, a nonspecific CYP inhibitor, and various CYP isoform selective inhibitors did not affect BZD N-oxidation. In contrast, formation of BZD N-oxide was almost abolished by heat treatment of microsomes in the absence of NADPH and strongly inhibited by methimazole, a competitive FMO inhibitor. Recombinant FMO3 and FMO1 (which is not expressed in human liver), but not FMO5, showed BZD N-oxidase activity. Respective Km values for FMO3 and FMO1 were 40.4 microM and 23.6 microM, and respective Vmax values for FMO3 and FMO1 were 29.1 and 40.8 nmol mg-1 protein min-1. Human liver samples (n = 35) were analysed for six known FMO3 polymorphisms. The variants I66M, P135L and E305X were not detected. Samples homozygous for the K158 variant showed significantly reduced Vmax values (median 2.7 nmol mg-1 protein min-1) compared to the carriers of at least one wild type allele (median 6.2 nmol mg-1 protein min-1) (P < 0.05, Mann-Whitney-U-test). The V257M and E308G substitutions had no effect on enzyme activity.

Conclusions: BZD N-oxidation in human liver is mainly catalysed by FMO3 and enzyme activity is affected by FMO3 genotype. BZD may be used as a model substrate for human liver FMO3 activity in vitro and may be further developed as an in vivo probe reflecting FMO3 activity.

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References

Cashman J, Park S, Berkman C, Cashman L . Role of hepatic flavin-containing monooxygenase 3 in drug and chemical metabolism in adult humans. Chem Biol Interact. 1995; 96(1):33-46. DOI: 10.1016/0009-2797(94)03581-r. View

Baldock G, Brodie R, Chasseaud L, Taylor T . Determination of benzydamine and its N-oxide in biological fluids by high-performance liquid chromatography. J Chromatogr. 1990; 529(1):113-23. DOI: 10.1016/s0378-4347(00)83812-8. View

Dolphin C, Cullingford T, Shephard E, Smith R, Phillips I . Differential developmental and tissue-specific regulation of expression of the genes encoding three members of the flavin-containing monooxygenase family of man, FMO1, FMO3 and FM04. Eur J Biochem. 1996; 235(3):683-9. DOI: 10.1111/j.1432-1033.1996.00683.x. View

Rostami-Hodjegan A, Nurminen S, Jackson P, Tucker G . Caffeine urinary metabolite ratios as markers of enzyme activity: a theoretical assessment. Pharmacogenetics. 1996; 6(2):121-49. DOI: 10.1097/00008571-199604000-00001. View

Fuhr U, Rost K, Engelhardt R, Sachs M, Liermann D, Belloc C . Evaluation of caffeine as a test drug for CYP1A2, NAT2 and CYP2E1 phenotyping in man by in vivo versus in vitro correlations. Pharmacogenetics. 1996; 6(2):159-76. DOI: 10.1097/00008571-199604000-00003. View

Grothusen A, Hardt J, Brautigam L, Lang D, Bocker R . A convenient method to discriminate between cytochrome P450 enzymes and flavin-containing monooxygenases in human liver microsomes. Arch Toxicol. 1996; 71(1-2):64-71. DOI: 10.1007/s002040050359. View

Chung W, CHA Y . Oxidation of caffeine to theobromine and theophylline is catalyzed primarily by flavin-containing monooxygenase in liver microsomes. Biochem Biophys Res Commun. 1997; 235(3):685-8. DOI: 10.1006/bbrc.1997.6866. View

Kawaji A, Isobe M, TAKABATAKE E . Differences in enzymatic properties of flavin-containing monooxygenase in brain microsomes of rat, mouse, hamster, guinea pig and rabbit. Biol Pharm Bull. 1997; 20(8):917-9. DOI: 10.1248/bpb.20.917. View

Overby L, Carver G, Philpot R . Quantitation and kinetic properties of hepatic microsomal and recombinant flavin-containing monooxygenases 3 and 5 from humans. Chem Biol Interact. 1997; 106(1):29-45. DOI: 10.1016/s0009-2797(97)00055-0. View

10.

Dolphin C, Janmohamed A, Smith R, Shephard E, Phillips I . Missense mutation in flavin-containing mono-oxygenase 3 gene, FMO3, underlies fish-odour syndrome. Nat Genet. 1997; 17(4):491-4. DOI: 10.1038/ng1297-491. View

11.

Dolphin C, Riley J, Smith R, Shephard E, Phillips I . Structural organization of the human flavin-containing monooxygenase 3 gene (FMO3), the favored candidate for fish-odor syndrome, determined directly from genomic DNA. Genomics. 1998; 46(2):260-7. DOI: 10.1006/geno.1997.5031. View

12.

Treacy E, Akerman B, CHOW L, Youil R, Bibeau C, Lin J . Mutations of the flavin-containing monooxygenase gene (FMO3) cause trimethylaminuria, a defect in detoxication. Hum Mol Genet. 1998; 7(5):839-45. DOI: 10.1093/hmg/7.5.839. View

13.

Lang D, Yeung C, Peter R, Ibarra C, Gasser R, Itagaki K . Isoform specificity of trimethylamine N-oxygenation by human flavin-containing monooxygenase (FMO) and P450 enzymes: selective catalysis by FMO3. Biochem Pharmacol. 1998; 56(8):1005-12. DOI: 10.1016/s0006-2952(98)00218-4. View

14.

Akerman B, Forrest S, Chow L, Youil R, Knight M, Treacy E . Two novel mutations of the FMO3 gene in a proband with trimethylaminuria. Hum Mutat. 1999; 13(5):376-9. DOI: 10.1002/(SICI)1098-1004(1999)13:5<376::AID-HUMU5>3.0.CO;2-A. View

15.

Park C, Chung W, Kang J, Roh H, Lee K, CHA Y . Phenotyping of flavin-containing monooxygenase using caffeine metabolism and genotyping of FMO3 gene in a Korean population. Pharmacogenetics. 1999; 9(2):155-64. View

16.

Ubeaud G, Schiller C, Hurbin F, Jaeck D, Coassolo P . Estimation of flavin-containing monooxygenase activity in intact hepatocyte monolayers of rat, hamster, rabbit, dog and human by using N-oxidation of benzydamine. Eur J Pharm Sci. 1999; 8(4):255-60. DOI: 10.1016/s0928-0987(99)00016-0. View

17.

Akerman B, Lemass H, CHOW L, Lambert D, Greenberg C, Bibeau C . Trimethylaminuria is caused by mutations of the FMO3 gene in a North American cohort. Mol Genet Metab. 1999; 68(1):24-31. DOI: 10.1006/mgme.1999.2885. View

18.

Ziegler D . Recent studies on the structure and function of multisubstrate flavin-containing monooxygenases. Annu Rev Pharmacol Toxicol. 1993; 33:179-99. DOI: 10.1146/annurev.pa.33.040193.001143. View

19.

von Moltke L, Greenblatt D, Harmatz J, Shader R . Alprazolam metabolism in vitro: studies of human, monkey, mouse, and rat liver microsomes. Pharmacology. 1993; 47(4):268-76. DOI: 10.1159/000139107. View

20.

Lawton M, Cashman J, Cresteil T, Dolphin C, Elfarra A, Hines R . A nomenclature for the mammalian flavin-containing monooxygenase gene family based on amino acid sequence identities. Arch Biochem Biophys. 1994; 308(1):254-7. DOI: 10.1006/abbi.1994.1035. View