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Inhibition and kinetics of cytochrome P4503A activity in microsomes from rat, human, and cdna-expressed human cytochrome P450.
Drug Metab Dispos 1996; 24(9):940-7DM

Abstract

Midazolam (MDZ) is metabolized in human liver microsomes by the cytochrome P450 (CYP) 3A subfamily to 1'-hydroxy (1'-OH) and 4-hydroxy (4-OH) metabolites. MDZ is metabolized in the rat primarily to 4-OH MDZ, 1'-OH MDZ, and 1',4-dihydroxy (1',4-diOH) MDZ. The kinetics of 4-OH and 1'-OH metabolite formation were determined using hepatic microsomes from control, Ro 23-7637 and dexamethasone-treated male rats. KM values for the major metabolite, 4-OH MDZ, were 24.5, 43.1, and 32.8 microM, and the corresponding Vmax values were 5.9, 28.9, and 13 nmol/mg/min for the control, DEX, and Ro 23-7637-treated animals, respectively KM values for 1'-hydroxylation of MDZ (the major metabolite) after incubation with human liver microsomes from three individuals were 5.57, 2.50, and 3.56 microM, and the corresponding Vmax values were 4.38, 0.49, and 0.19 nmol/mg/min, respectively. In parallel studies using cDNA-expressed human CYP3A4 microsomes, the KM for 1'-OH formation was 1.56 microM, and the corresponding Vmax was 0.16 nmol/mg/min. MDZ was not metabolized by cDNA-expressed human CYP2D6, CYP2E1, or CYP1A2, thus confirming that these isoforms were not responsible for its biotransformation. The formation of 1',4-diOH metabolite in rat and 1'-OH formation in cDNA-expressed human CYP3A4 microsomes showed a decrease in velocity at high substrate concentrations. Inhibition studies showed that MDZ hydroxylation was strongly inhibited by ketoconazole and Ro 23-7637 in rat, human, and cDNA-expressed human CYP3A4 microsomes. alpha-Naphthoflavone stimulated 1'-OH metabolite formation in human and cDNA-expressed human CYP3A4 microsomes at low concentration (10 microM). Naringenin, a flavonoid present in grapefruit juice, also inhibited MDZ metabolism in human liver microsomes. Immunoinhibition studies revealed that polyclonal anti-rat CYP3A2 antibody inhibited MDZ metabolism 80-90% in rat, human, and cDNA-expressed human CYP3A4 microsomes, thus suggesting that members of the CYP3A4 subfamily were involved in the metabolism.

Authors+Show Affiliations

Hoffmann-La Roche, Inc., Nutley, NJ 07110-1199, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article

Language

eng

PubMed ID

8886602

Citation

Ghosal, A, et al. "Inhibition and Kinetics of Cytochrome P4503A Activity in Microsomes From Rat, Human, and Cdna-expressed Human Cytochrome P450." Drug Metabolism and Disposition: the Biological Fate of Chemicals, vol. 24, no. 9, 1996, pp. 940-7.
Ghosal A, Satoh H, Thomas PE, et al. Inhibition and kinetics of cytochrome P4503A activity in microsomes from rat, human, and cdna-expressed human cytochrome P450. Drug Metab Dispos. 1996;24(9):940-7.
Ghosal, A., Satoh, H., Thomas, P. E., Bush, E., & Moore, D. (1996). Inhibition and kinetics of cytochrome P4503A activity in microsomes from rat, human, and cdna-expressed human cytochrome P450. Drug Metabolism and Disposition: the Biological Fate of Chemicals, 24(9), pp. 940-7.
Ghosal A, et al. Inhibition and Kinetics of Cytochrome P4503A Activity in Microsomes From Rat, Human, and Cdna-expressed Human Cytochrome P450. Drug Metab Dispos. 1996;24(9):940-7. PubMed PMID: 8886602.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Inhibition and kinetics of cytochrome P4503A activity in microsomes from rat, human, and cdna-expressed human cytochrome P450. AU - Ghosal,A, AU - Satoh,H, AU - Thomas,P E, AU - Bush,E, AU - Moore,D, PY - 1996/9/1/pubmed PY - 1996/9/1/medline PY - 1996/9/1/entrez SP - 940 EP - 7 JF - Drug metabolism and disposition: the biological fate of chemicals JO - Drug Metab. Dispos. VL - 24 IS - 9 N2 - Midazolam (MDZ) is metabolized in human liver microsomes by the cytochrome P450 (CYP) 3A subfamily to 1'-hydroxy (1'-OH) and 4-hydroxy (4-OH) metabolites. MDZ is metabolized in the rat primarily to 4-OH MDZ, 1'-OH MDZ, and 1',4-dihydroxy (1',4-diOH) MDZ. The kinetics of 4-OH and 1'-OH metabolite formation were determined using hepatic microsomes from control, Ro 23-7637 and dexamethasone-treated male rats. KM values for the major metabolite, 4-OH MDZ, were 24.5, 43.1, and 32.8 microM, and the corresponding Vmax values were 5.9, 28.9, and 13 nmol/mg/min for the control, DEX, and Ro 23-7637-treated animals, respectively KM values for 1'-hydroxylation of MDZ (the major metabolite) after incubation with human liver microsomes from three individuals were 5.57, 2.50, and 3.56 microM, and the corresponding Vmax values were 4.38, 0.49, and 0.19 nmol/mg/min, respectively. In parallel studies using cDNA-expressed human CYP3A4 microsomes, the KM for 1'-OH formation was 1.56 microM, and the corresponding Vmax was 0.16 nmol/mg/min. MDZ was not metabolized by cDNA-expressed human CYP2D6, CYP2E1, or CYP1A2, thus confirming that these isoforms were not responsible for its biotransformation. The formation of 1',4-diOH metabolite in rat and 1'-OH formation in cDNA-expressed human CYP3A4 microsomes showed a decrease in velocity at high substrate concentrations. Inhibition studies showed that MDZ hydroxylation was strongly inhibited by ketoconazole and Ro 23-7637 in rat, human, and cDNA-expressed human CYP3A4 microsomes. alpha-Naphthoflavone stimulated 1'-OH metabolite formation in human and cDNA-expressed human CYP3A4 microsomes at low concentration (10 microM). Naringenin, a flavonoid present in grapefruit juice, also inhibited MDZ metabolism in human liver microsomes. Immunoinhibition studies revealed that polyclonal anti-rat CYP3A2 antibody inhibited MDZ metabolism 80-90% in rat, human, and cDNA-expressed human CYP3A4 microsomes, thus suggesting that members of the CYP3A4 subfamily were involved in the metabolism. SN - 0090-9556 UR - https://www.unboundmedicine.com/medline/citation/8886602/Inhibition_and_kinetics_of_cytochrome_P4503A_activity_in_microsomes_from_rat_human_and_cdna_expressed_human_cytochrome_P450_ L2 - http://dmd.aspetjournals.org/cgi/pmidlookup?view=long&pmid=8886602 DB - PRIME DP - Unbound Medicine ER -