Comparative study of the metabolism of drug substrates by human cytochrome P450 3A4 expressed in bacterial, yeast and human lymphoblastoid cells

1. The aim was to compare the metabolic activity of human CYP3A4 expressed in bacteria (E. coli), yeast (S. cerevisiae) and human lymphoblastoid cells (hBl), with the native CYP3A4 activity observed in a panel of human livers. 2. Three CYP3A4 substrates were selected for study: dextromethorphan (DEM), midazolam (MDZ) and diazepam (DZ). The substrate metabolism in each of the four systems was characterized by deriving the kinetic parameters Km or S50, Vmax and intrinsic clearance (CLint) or maximum clearance (CLmax) from the kinetic profiles; the latter differing by 100-fold across the three substrates. 3. The Km or S50 for the formation of metabolites 3-methoxymorphinan (MEM), 1'-hydroxymidazolam (1'-OH MDZ) and 3-hydroxydiazepam (3HDZ) compared well in all systems. For CYP3A4-mediated metabolism of DEM, MDZ and DZ, the Vmax for hBl microsomes were generally 2-9-fold higher than the respective yeast and human liver microsomes and E. coli membrane preparations, resulting in greater CLint or CLmax. In the case of 3HDZ formation, non-linear kinetics were observed for E. coli, hBl microsomes and human liver microsomes, whereas the kinetics observed for S. cerevisiae were linear. 4. The use of native human liver microsomes for drug metabolic studies will always be preferable. However, owing to the limited availability of human tissues, we find it is reasonable to use any of the recombinant systems described herein, since all three recombinant systems gave good predictions of the native human liver enzyme activities.

[1]  J. Houston,et al.  Diazepam-omeprazole inhibition interaction: an in vitro investigation using human liver microsomes. , 1996, British journal of clinical pharmacology.

[2]  A. Somogyi,et al.  The role of CYP2D6 in primary and secondary oxidative metabolism of dextromethorphan: in vitro studies using human liver microsomes. , 1994, British journal of clinical pharmacology.

[3]  P. Thomas,et al.  Inhibition and kinetics of cytochrome P4503A activity in microsomes from rat, human, and cdna-expressed human cytochrome P450. , 1996, Drug metabolism and disposition: the biological fate of chemicals.

[4]  B. Ring,et al.  Human cytochrome P450 3A (CYP3A) mediated midazolam metabolism: the effect of assay conditions and regioselective stimulation by alpha-naphthoflavone, terfenadine and testosterone. , 1998, Pharmacogenetics.

[5]  H. Yamazaki,et al.  Enhancement of cytochrome P-450 3A4 catalytic activities by cytochrome b(5) in bacterial membranes. , 1999, Drug metabolism and disposition: the biological fate of chemicals.

[6]  A. Somogyi,et al.  Simultaneous Determination of Dextromethorphan and Three Metabolites in Plasma and Urine Using High‐Performance Liquid Chromatography with Application to Their Disposition in Man , 1990, Therapeutic drug monitoring.

[7]  K. Korzekwa,et al.  Role of cDNA-expressed human cytochromes P450 in the metabolism of diazepam. , 1998, Biochemical pharmacology.

[8]  T. Baillie,et al.  Sigmoidal kinetic model for two co-operative substrate-binding sites in a cytochrome P450 3A4 active site: an example of the metabolism of diazepam and its derivatives. , 1999, The Biochemical journal.

[9]  C. Wolf,et al.  Effects of human cytochrome b5 on CYP3A4 activity and stability in vivo. , 1999, Archives of biochemistry and biophysics.

[10]  Aiming Yu,et al.  Comparative contribution to dextromethorphan metabolism by cytochrome P450 isoforms in vitro: can dextromethorphan be used as a dual probe for both CTP2D6 and CYP3A activities? , 2001, Drug metabolism and disposition: the biological fate of chemicals.

[11]  J. Miners,et al.  Diazepam metabolism by human liver microsomes is mediated by both S-mephenytoin hydroxylase and CYP3A isoforms. , 1994, British journal of clinical pharmacology.

[12]  D. Mansuy,et al.  Optimization of yeast-expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH-cytochrome P450 reductase and cytochrome b5. , 1992, European journal of biochemistry.

[13]  K. Korzekwa,et al.  Cytochromes P450 Expression Systems , 1995 .

[14]  J. Houston,et al.  Sigmoidal kinetics of CYP3A substrates: an approach for scaling dextromethorphan metabolism in hepatic microsomes and isolated hepatocytes to predict in vivo clearance in rat. , 1999, The Journal of pharmacology and experimental therapeutics.

[15]  C. Wolf,et al.  Recombinant DNA technology as an investigative tool in drug metabolism research , 1996 .

[16]  T. Omura,et al.  THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. I. EVIDENCE FOR ITS HEMOPROTEIN NATURE. , 1964, The Journal of biological chemistry.

[17]  C. Funck-Brentano,et al.  CYP2D6- and CYP3A-dependent metabolism of dextromethorphan in humans. , 1993, Pharmacogenetics.

[18]  T. Kost,et al.  CYP3A4 expressed by insect cells infected with a recombinant baculovirus containing both CYP3A4 and human NADPH-cytochrome P450 reductase is catalytically similar to human liver microsomal CYP3A4. , 1995, Archives of biochemistry and biophysics.

[19]  R. Amrein,et al.  Pharmacology of Dormicum (midazolam) and Anexate (flumazenil) , 1990, Acta anaesthesiologica Scandinavica. Supplementum.

[20]  S D Hall,et al.  Regioselective biotransformation of midazolam by members of the human cytochrome P450 3A (CYP3A) subfamily. , 1994, Biochemical pharmacology.

[21]  H. Iwata,et al.  High catalytic activity of human cytochrome P450 co-expressed with human NADPH-cytochrome P450 reductase in Escherichia coli. , 1998, Biochemical pharmacology.

[22]  F. Guengerich,et al.  Expression of mammalian cytochrome P450 enzymes using yeast-based vectors. , 1991, Methods in enzymology.

[23]  M. J. Coon,et al.  Purified liver microsomal NADPH-cytochrome P-450 reductase. Spectral characterization of oxidation-reduction states. , 1978, The Journal of biological chemistry.

[24]  C. Wolf,et al.  Coexpression of a human P450 (CYP3A4) and P450 reductase generates a highly functional monooxygenase system in Escherichia coli , 1996, FEBS letters.

[25]  D. Thompson,et al.  Cytochrome P450IIIA enzymes in rat liver microsomes: involvement in C3-hydroxylation of diazepam and nordazepam but not N-dealkylation of diazepam and temazepam. , 1990, Molecular pharmacology.

[26]  J. Houston,et al.  Prediction of in vivo disposition from in vitro systems: clearance of phenytoin and tolbutamide using rat hepatic microsomal and hepatocyte data. , 1995, The Journal of pharmacology and experimental therapeutics.

[27]  R J Riley,et al.  Rapid characterization of the major drug-metabolizing human hepatic cytochrome P-450 enzymes expressed in Escherichia coli. , 1999, Drug metabolism and disposition: the biological fate of chemicals.

[28]  H. Yamazaki,et al.  Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. , 1994, The Journal of pharmacology and experimental therapeutics.

[29]  T. Kronbach,et al.  Oxidation of midazolam and triazolam by human liver cytochrome P450IIIA4. , 1989, Molecular pharmacology.

[30]  S. Wrighton,et al.  Characterization of dextromethorphan N-demethylation by human liver microsomes. Contribution of the cytochrome P450 3A (CYP3A) subfamily. , 1994, Biochemical pharmacology.

[31]  C. Crespi.  Xenobiotic-metabolizing human cells as tools for pharmacological and toxicological research , 1995 .

[32]  T. Richardson,et al.  Diazepam metabolism by cDNA-expressed human 2C P450s: identification of P4502C18 and P4502C19 as low K(M) diazepam N-demethylases. , 1997, Drug metabolism and disposition: the biological fate of chemicals.

[33]  Y Wang,et al.  Evaluation of the selectivity of In vitro probes and suitability of organic solvents for the measurement of human cytochrome P450 monooxygenase activities. , 1998, Drug metabolism and disposition: the biological fate of chemicals.

[34]  F. Guengerich,et al.  Expression of modified human cytochrome P450 3A4 in Escherichia coli and purification and reconstitution of the enzyme. , 1993, Archives of biochemistry and biophysics.

[35]  I. Wainer,et al.  Dextromethorphan as an in vivo probe for the simultaneous determination of CYP2D6 and CYP3A activity. , 1996, Journal of chromatography. B, Biomedical applications.

[36]  J B Houston,et al.  In vitro-in vivo scaling of CYP kinetic data not consistent with the classical Michaelis-Menten model. , 2000, Drug metabolism and disposition: the biological fate of chemicals.

[37]  B. W. Penman,et al.  Use of cDNA-expressed human cytochrome P450 enzymes to study potential drug-drug interactions. , 1997, Advances in pharmacology.

[38]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[39]  K. Korzekwa,et al.  Cytochromes P450 expression systems. , 1995, Annual review of pharmacology and toxicology.