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[O-methyl 14C]naproxen O-demethylase activity in human liver microsomes: evidence for the involvement of cytochrome P4501A2 and P4502C9/10.

Abstract

Cytochrome P450 (CYP) activity in human liver microsomes was measured after the O-demethylation of [O-methyl 14C]naproxen (NAPase). The formation of [14C]formaldehyde in the presence of microsomes was described by an apparent KM(1) and Vmax(1) of 0.16 +/- 0.09 mM and 4.1 +/- 2.8 nmol HCHO/min/mg protein (mean +/- SD; N = 5 different livers), respectively, over a relatively wide naproxen concentration (5-1600 microM) range. With two sets of microsomes, a high KM NAPase component was also detected (mean KM2 = 2.7 mM; mean Vmax2 = 23 nmol HCHO/min/mg). As expected, the O-demethylation of naproxen (0.4 mM) was found to be highly correlated with tolbutamide hydroxylase (TOLase) activity in a panel of human liver microsomes (r = 0.82, p < 0.01, N = 10) and was inhibited (32-54%) by a number of purported CYP2C (CYP2C9/10) inhibitors/substrates (e.g. phenytoin, sulfaphenazole, tienilic acid, tolbutamide, and ibuprofen). Only marginal decreases in activity (< or = 14%) were observed with inhibitors of other CYP proteins. However, NAPase activity was also found to correlate significantly with CYP1A2 [ethoxyresorufin O-deethylase (ERODase)] activity (r = 0.68, p < 0.05, n = 11). In addition, the reaction was inhibited (36-75%, N = 11 different livers) by furafylline (FURA), a CYP1A2-selective mechanism-based inhibitor. The effect of FURA and tienilic acid was additive, leading to 90 +/- 4.2% inhibition of NAPase activity. FURA-inhibited activity also significantly correlated with ERODase activity (r = 0.78, p < 0.01, N = 11), whereas tienilic acid-inhibited activity correlated with TOLase activity (r = 0.63, p < 0.05, N = 10). In human B-lymphoblast microsomes, cDNA-expressed CYP1A2 exhibited relatively high activity (KM = 0.25 mM; Vmax = 24 nmol/min/nmol CYP), when compared with CYP2A6, CYP2D6, CYP2E1, CYP2B6, and CYP3A4. The kinetic parameters for reconstituted purified human liver microsomal CYP2C9 (KM = 0.43 mM; Vmax = 11 nmol/min/nmol CYP) were comparable with those of CYP1A2. It is concluded that the O-demethylation of naproxen (< or = 0.4 mM) is catalyzed by CYP2C subfamily members (CYP2C9/10) and CYP1A2 in human liver microsomes.

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  • Authors+Show Affiliations

    ,

    Drug Metabolism Department, Abbott Laboratories, Abbott Park, IL 60064, USA.

    , , ,

    Source

    MeSH

    B-Lymphocytes
    Carbon Radioisotopes
    Cell Line
    Cytochrome P-450 Enzyme System
    DNA, Complementary
    Humans
    Isoenzymes
    Kinetics
    Microsomes, Liver
    Naproxen
    Oxidoreductases, O-Demethylating
    Recombinant Proteins

    Pub Type(s)

    Journal Article

    Language

    eng

    PubMed ID

    8825200

    Citation

    Rodrigues, A D., et al. "[O-methyl 14C]naproxen O-demethylase Activity in Human Liver Microsomes: Evidence for the Involvement of Cytochrome P4501A2 and P4502C9/10." Drug Metabolism and Disposition: the Biological Fate of Chemicals, vol. 24, no. 1, 1996, pp. 126-36.
    Rodrigues AD, Kukulka MJ, Roberts EM, et al. [O-methyl 14C]naproxen O-demethylase activity in human liver microsomes: evidence for the involvement of cytochrome P4501A2 and P4502C9/10. Drug Metab Dispos. 1996;24(1):126-36.
    Rodrigues, A. D., Kukulka, M. J., Roberts, E. M., Ouellet, D., & Rodgers, T. R. (1996). [O-methyl 14C]naproxen O-demethylase activity in human liver microsomes: evidence for the involvement of cytochrome P4501A2 and P4502C9/10. Drug Metabolism and Disposition: the Biological Fate of Chemicals, 24(1), pp. 126-36.
    Rodrigues AD, et al. [O-methyl 14C]naproxen O-demethylase Activity in Human Liver Microsomes: Evidence for the Involvement of Cytochrome P4501A2 and P4502C9/10. Drug Metab Dispos. 1996;24(1):126-36. PubMed PMID: 8825200.
    * Article titles in AMA citation format should be in sentence-case
    TY - JOUR T1 - [O-methyl 14C]naproxen O-demethylase activity in human liver microsomes: evidence for the involvement of cytochrome P4501A2 and P4502C9/10. AU - Rodrigues,A D, AU - Kukulka,M J, AU - Roberts,E M, AU - Ouellet,D, AU - Rodgers,T R, PY - 1996/1/1/pubmed PY - 1996/1/1/medline PY - 1996/1/1/entrez SP - 126 EP - 36 JF - Drug metabolism and disposition: the biological fate of chemicals JO - Drug Metab. Dispos. VL - 24 IS - 1 N2 - Cytochrome P450 (CYP) activity in human liver microsomes was measured after the O-demethylation of [O-methyl 14C]naproxen (NAPase). The formation of [14C]formaldehyde in the presence of microsomes was described by an apparent KM(1) and Vmax(1) of 0.16 +/- 0.09 mM and 4.1 +/- 2.8 nmol HCHO/min/mg protein (mean +/- SD; N = 5 different livers), respectively, over a relatively wide naproxen concentration (5-1600 microM) range. With two sets of microsomes, a high KM NAPase component was also detected (mean KM2 = 2.7 mM; mean Vmax2 = 23 nmol HCHO/min/mg). As expected, the O-demethylation of naproxen (0.4 mM) was found to be highly correlated with tolbutamide hydroxylase (TOLase) activity in a panel of human liver microsomes (r = 0.82, p < 0.01, N = 10) and was inhibited (32-54%) by a number of purported CYP2C (CYP2C9/10) inhibitors/substrates (e.g. phenytoin, sulfaphenazole, tienilic acid, tolbutamide, and ibuprofen). Only marginal decreases in activity (< or = 14%) were observed with inhibitors of other CYP proteins. However, NAPase activity was also found to correlate significantly with CYP1A2 [ethoxyresorufin O-deethylase (ERODase)] activity (r = 0.68, p < 0.05, n = 11). In addition, the reaction was inhibited (36-75%, N = 11 different livers) by furafylline (FURA), a CYP1A2-selective mechanism-based inhibitor. The effect of FURA and tienilic acid was additive, leading to 90 +/- 4.2% inhibition of NAPase activity. FURA-inhibited activity also significantly correlated with ERODase activity (r = 0.78, p < 0.01, N = 11), whereas tienilic acid-inhibited activity correlated with TOLase activity (r = 0.63, p < 0.05, N = 10). In human B-lymphoblast microsomes, cDNA-expressed CYP1A2 exhibited relatively high activity (KM = 0.25 mM; Vmax = 24 nmol/min/nmol CYP), when compared with CYP2A6, CYP2D6, CYP2E1, CYP2B6, and CYP3A4. The kinetic parameters for reconstituted purified human liver microsomal CYP2C9 (KM = 0.43 mM; Vmax = 11 nmol/min/nmol CYP) were comparable with those of CYP1A2. It is concluded that the O-demethylation of naproxen (< or = 0.4 mM) is catalyzed by CYP2C subfamily members (CYP2C9/10) and CYP1A2 in human liver microsomes. SN - 0090-9556 UR - https://www.unboundmedicine.com/medline/citation/8825200/[O_methyl_14C]naproxen_O_demethylase_activity_in_human_liver_microsomes:_evidence_for_the_involvement_of_cytochrome_P4501A2_and_P4502C9/10_ L2 - http://dmd.aspetjournals.org/cgi/pmidlookup?view=long&amp;pmid=8825200 DB - PRIME DP - Unbound Medicine ER -