Advertisement

Concentration-dependent metabolism of diazepam in mouse liver

  • Marie V. St-Pierre
  • K. Sandy Pang
Article

Abstract

Previous mouse liver studies with diazepam (DZ),N-desmethyldiazepam (NZ), and temazepam (TZ) confirmed that under first-order conditions, DZ formed NZ and TZ in parallel. Oxazepam (OZ) was generatedvia NZ and not TZ despite that preformed NZ and TZ were both capable of forming OZ. In the present studies, the concentration-dependent sequential metabolism of DZ was studied in perfused mouse livers and microsomes, with the aim of distinguishing the relative importance of NZ and TZ as precusors of OZ. In microsomal studies, theKms andVmaxs, corrected for binding to microsomal proteins, were 34 μM and 3.6 nmole/min per mg and 239 μM and 18 nmole/min per mg, respectively, forN-demthylation andC3-hydroxylation of DZ. TheKms andVmaxs forN-demethylation andC3-hydroxylation of TZ and NZ, respectively, to form OZ, were 58 μM and 2.5 nmole/min per mg and 311 μM and 2 nmole/min per mg, respectively. The constants suggest that at low DZ concentrations, NZ formation predominates and is a major source of OZ, whereas at higher DZ concentrations, TZ is the important source of OZ. In livers perfused with DZ at input concentrations of 13 to 35 μM, the extraction ratio of DZ (E{DZ}) decreased from 0.83 to 0.60. NZ was the major metabolite formed although its appearance was less than proportionate with increasing DZ input concentration. By contrast, the formation of TZ increased disporportionately with increasing DZ concentration, whereas that for OZ decreased and paralleled the behavior of NZ. Computer simulations based on a tubular flow model and thein vitro enzymatic parameters provided a poorin vitro-organ correlation. TheE{DZ}, appearance rates of the metabolites, and the extraction ratio of formed NZ (E{NZ, DZ}) were poorly predicted; TZ was incorrectly identified as the major precursor of OZ. Simulations with optimized parameters imporved the correlations and identified NZ as the major contributor of OZ. Saturation of DZN-demethylation at higher DZ concentrations increased the role of TZ in the formation of OZ. The poor aqueous solubility (limiting the concentration range of substrates usedin vitro), avid tissue binding and the coupling of enzymatic reactions in liver, favoring sequential metabolism, are possible explanations for the poorin vitro-organ correlation. This work emphasizes the complexity of the hepatic intracellular milieu for drug metabolism and the need for additional modeling efforts to adequately describe metabolite kinetics.

Key Words

benzodiazpines diazepam C3-hydroxylation andN-demethylation N-desmethyldiazepam nordiazepam temazepam oxazepam tubular flow-model Km andVmax metabolite kinetics perfused mouse liver mouse liver microsomes in vitro-organ correlation 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    M. A. Schwartz, B. A. Koechlin, E. Postma, S. Palmer, and G. Kroll. Metabolism of diazepam in rat, dog and man.J. Pharmacol. Exp. Ther. 149:423–435 (1965).PubMedGoogle Scholar
  2. 2.
    I. A. Zingales. Diazepam metabolism during chronic medication. Unbound fraction in plasma, erythrocytes and urine.J. Chromatogr. 75:55–78 (1973).PubMedCrossRefGoogle Scholar
  3. 3.
    F. Marcucci, R. Fanelli, E. Mussini, and S. Garattini. Further studies on species difference in diazepam metabolism.Eur. J. Pharmacol. 9:253–256 (1970).PubMedCrossRefGoogle Scholar
  4. 4.
    F. Marcucci, A. Guaitani, R. Fanelli, E. Mussini, and S. Garattini. Metabolism and anticonvulsant activity of diazepam in guinea pigs.Biochem. Pharmacol. 20:1711–1713 (1971).PubMedCrossRefGoogle Scholar
  5. 5.
    L. M. Fuccella, G. Tosolini, E. Moro, and V. Tamassia. Study of Physiological availability of temazepam in man.Int. J. Clin. Pharmacol. 6:303–309 (1972).PubMedGoogle Scholar
  6. 6.
    F. Marcucci, R. Fanelli, E. Mussini, and S. Garattini. Effect of phenobarbital on the in vitro metabolism of diazepam in several animal species.Biochem. Pharmacol. 19:1771–1776 (1970).PubMedCrossRefGoogle Scholar
  7. 7.
    T. Inaba, A. Tait, M. Nakano, M. A. Mahon, and W. Kalow. Metabolism of diazepam in vitro by human liver. Independent variability of N-demethylation and C3-hydroxylation.Drug Metab. Dispos. 16:605–608 (1988).PubMedGoogle Scholar
  8. 8.
    P. E. B. Reilly, D. A. Thompson, S. R. Mason, and W. D. Hooper. Cytochrome P450IIIA enzymes in rat liver microsomes: involvement in C3-hydroxylation of diazepam and nordiazepam but not N-dealkylation of diazepam and temazepam.Mol. Pharmacol. 37:767–774 (1990).PubMedGoogle Scholar
  9. 9.
    T. V. Beischlag, W. Kalow, W. A. Mahon, and T. Inaba. Diazepam metabolism by rat and human liverin vitro: inhibition by mephenytoin.Xenobiotica 22:559–567 (1992).PubMedCrossRefGoogle Scholar
  10. 10.
    M. A. Schwartz, P. Bommer, and F. M. Vane. Diazepam metabolites in the rat: characterization of high-resolution mass spectrometry and nuclear magnetic resonance.Arch. Biochem. Biosphys. 121:508–516 (1967).CrossRefGoogle Scholar
  11. 11.
    S. F. Sisenwine and C. O. Tio: The metabolic disposition of oxazepam in rats.Drug Metab. Dispos. 14:41–45 (1986).PubMedGoogle Scholar
  12. 12.
    S. F. Sisenwine, C. O. Tio, A. L. Liu, and J. F. Politowski. The metabolic fate of oxazepam in mice.Drug Metab. Dispos. 15:579–580 (1987).PubMedGoogle Scholar
  13. 13.
    M. V. St-Pierre, D. van den Berg, and K. S. Pang: Physiological modeling of drug and metabolite: disposition of oxazepam and oxazepam glucuronides in the recirculating perfused mouse liver preparation.J. Pharmacokin. Biopharm. 18:423–448 (1990).CrossRefGoogle Scholar
  14. 14.
    S. F. Sisenwine, C. O. Tio, S. R. Shrader, and H. W. Ruelius. The biotransformation of oxazepam (7-chloro-1,3-dihydro-3-hydroxy-5-phenyl-2H-1,4-benzodiazepin-2-one) in man, miniature swine and rat.Arzneim. Forsch. 22:682–687 (1972).Google Scholar
  15. 15.
    M. V. St-Pierre and K. S. Pang: Kinetics of sequential metabolism. I. Formation and metabiolism of oxazepam from nordiazepam and temazepam in the perfused murine liver.J. Pharmacol. Exp. Ther. 265:1429–1436 (1993).PubMedGoogle Scholar
  16. 16.
    M. V. St-Pierre and K. S. Pang. Kinetics of sequential metabolism. II. Formation and metabolism of nordiazepam and oxazepam from diazepam in the perfused murine liver.J. Pharmacol. Exp. Ther. 265:1437–1445 (1993).PubMedGoogle Scholar
  17. 17.
    K. S. Pang. Kinetics of sequential metabolism. Contribution of parallel, primary metabolic pathways to the formation of a common, secondary metabolite.Drug Metab. Dispos. 23:166–177 (1995).PubMedGoogle Scholar
  18. 18.
    T. Omura and R. Sato. The carbon monoxide-binding pigment of liver microsomes.J. Biol. Chem. 239:2370–2378 (1964).PubMedGoogle Scholar
  19. 19.
    B. Schoene, R. A. Fleischmann, H. Remmer, and H. F. Oldershausen. Determination of drug metabolizing enzymes in needle biopsies of human liver.Eur. J. Clin. Pharmacol. 4:65–73 (1972).PubMedCrossRefGoogle Scholar
  20. 20.
    D. J. Greenblatt, R. M. Arendt, D. R. Abernathy, H. G. Giles, E. M. Sellers, and R. I. Shrader.In vitro quantitation of benzodiazepine lipophilicity: relation toin vivo distribution.Br. J. Anaesth. 55:985–988 (1983).PubMedCrossRefGoogle Scholar
  21. 21.
    W. D. Hooper, J. A. Watt, G. E. McKinnon, and P. E. Reilly. Metabolism of diazepam and related benzodiazepines by human liver microsomes.Eur. J. Drug Metab. Pharmacokin. 17:51–59 (1992).CrossRefGoogle Scholar
  22. 22.
    Y. Igari, Y. Sugiyama, Y. Sawada, T. Iga, and M. Hanano. Tissue distribution of the14C-diazepam and its metabolites in rats.Drug Metab. Dispos. 10:676–679 (1982).PubMedGoogle Scholar
  23. 23.
    M. A. Bogeyevitch, E. M. Gillam, P. E. Reilly, and D. J. Winzor. Physical partitioning and the major source of metoprolol uptake by hepatic microsomes.Biochem. Pharmacol. 36:4167–4168 (1987).PubMedCrossRefGoogle Scholar
  24. 24.
    J. H. Lin, M. Hayashi, S. Awazu, and M. Hanano. Correlation between in vitro and in vivo drug metabolism rate: oxidation of ethoxybenzamide in rat.J. Pharmacokin. Biopharm. 6:327–337 (1978).CrossRefGoogle Scholar
  25. 25.
    M. V. St-Pierre and K. S. Pang. Determination of diazepam and its metabolites by HPLC and TLC.J. Chromatogr. 421:291–307 (1987).PubMedCrossRefGoogle Scholar
  26. 26.
    U. Klotz and I. Reimann. Clearance of diazepam can be impaired by its major metabolite desmethyldiazepam.Eur. J. Clin. Pharmacol. 21:161–163 (1981).PubMedCrossRefGoogle Scholar
  27. 27.
    E. M. Savenije-Chapel, A. Bast, and J. Noordhoek. Inhibition of diazepam metabolism in microsomal and perfused liver preparations of the rat by desmethyldiazepam, N-methyloxazepam and oxazepam:Eur. J. Drug Metab. Pharmacokin. 10:15–20 (1985).CrossRefGoogle Scholar
  28. 28.
    K. J. Laidler and P. S. Bunting.The Chemical Kinetics of Enzyme Action, Clarendon Press, Oxford, 1973, pp. 89–96.Google Scholar
  29. 29.
    J. T. McClave and F. H. Dietrich.Statistics, Dellen, San Francisco, 1979.Google Scholar
  30. 30.
    M. V. St-Pierre, P. I. Lee, and K. S. Pang. A comparative investigation of hepatic clearance models: predictions of metabolite formation and elimination.J. Pharmacokin. Biopharm. 20:105–145 (1992).CrossRefGoogle Scholar
  31. 31.
    K. S. Pang and M. Rowland. Hepatic clearance of drugs. I. Theoretical consideration of a “well-stirred” model and a “parallel-tube” model. Influence of hepatic blood flow, plasma and blood cell binding, and the hepatocellular activity on hepatic drug clearance.J. Pharmacokin. Biopharm. 5:625–653 (1977).CrossRefGoogle Scholar
  32. 32.
    R. S. Chhabra, R. J. Pohl, and J. R. Fouts. A comparative study of xenobioticmetabolizing enzymes in liver and intestine of various animal species.Drug Metab. Dispos. 2:443–447 (1974).PubMedGoogle Scholar
  33. 33.
    H. Souhaili-El Amri, A. M. Batt, and G. Siest. Comparison of cytochrome P-450 content and activities in liver microsomes of seven animal species, including man.Xenobiotica 16:351–358 (1986).PubMedCrossRefGoogle Scholar
  34. 34.
    R. Reiter and A. Wendel. Selenium and drug metabolism. Multiple modulations of mouse liver enzymes.Biochem. Pharmacol. 32:3063–3067 (1983).PubMedCrossRefGoogle Scholar
  35. 35.
    R. W. Estabrook: Cytochrome P-450 and oxygenation reactions: a status report. In J. A. Mitchell and M. G. Horning (eds.),Drug Metabolism and Drug Toxicity, Raven, New York, 1984, pp. 3–5.Google Scholar
  36. 36.
    E. M. Savenije-Chapel, A. Bast, and J. Noordhoek. Interaction of uridine 5′-diphosphoglucuronic acid (UDPGA) with cytochrome P-450.J. Pharm. Pharmacol. 35:522–523 (1983).PubMedCrossRefGoogle Scholar
  37. 37.
    F. Marcucci, R. Fanelli, E. Mussini, and S. Garattini. The metabolism of diazepam by liver microsomal enzymes of rats and mice.Eur. J. Pharmacol. 7:307–313 (1969).PubMedCrossRefGoogle Scholar
  38. 38.
    M. E. Morris and K. S. Pang. Competition between two enzymes for substrate removal in liver: modulating effects due to substrate recruitment of hepatocyte activity.J. Pharmacokin. Biopharm. 15:473–495 (1987).CrossRefGoogle Scholar
  39. 39.
    E. Ackermann and K. Richter: Diazepam metabolism in human foetal and adult liver.Eur. J. Clin. Pharmacol. 11:43–49 (1977).PubMedCrossRefGoogle Scholar
  40. 40.
    X. Xu, B. K. Tang, and K. S. Pang. Sequential metabolism of salicylamide exclusively to gentisamide-5-glucuronide and not gentisamide sulfate conjugates in the single passin situ perfused rat liver.J. Pharmacol. Exp. Ther. 253:965–973 (1990).PubMedGoogle Scholar
  41. 41.
    D. M. Himmelblau and B. B. Bischoff.Process Analysis and Simulation. Deterministic Systems, Wiley, New York, 1968, pp. 91–112.Google Scholar
  42. 42.
    K. K. Chan, M. B. Bolger, and K. S. Pang. Statistical moment theory in chemical kinetics. Anal. Chem.57:2145–2151 (1985).PubMedCrossRefGoogle Scholar
  43. 43.
    K. S. Pang and M. Chiba. Metabolism: Scaling-up fromin vitro to organ and whole body. In P. G. Welling and L. P. Balant (eds.),Handbook of Experimental Pharmacology, Springer-Verlag, Stuttgart, 1994, pp. 101–187.Google Scholar
  44. 44.
    Y. Igari, Y. Sugiyama, Y. Sawada, T. Iga, and M. Hanano.In vitro andin vivo assessment of hepatic and extrahepatic metabolism of diazepam in rats.J. Pharm. Sci. 73:826–282 (1984).PubMedCrossRefGoogle Scholar
  45. 45.
    M. Rowland, D. Leitch, G. Fleming, and B. Smith. Protein binding and hepatic clearance: discrimination between models of hepatic clearance with diazepam, a drug of high intrinsic clearance, in the isolated perfused rat liver preparation.J. Pharmacokin. Biopharm. 12:129–147 (1984).CrossRefGoogle Scholar
  46. 46.
    R. Tirona and K. S. Pang. Kinetics of primary and secondary metabolite formation in the perfused rat liver. Abstract, Exp. Biol. Atlanta, GA, 1995.Google Scholar

Copyright information

© Plenum Publishing Corporation 1995

Authors and Affiliations

  • Marie V. St-Pierre
    • 1
  • K. Sandy Pang
    • 1
    • 2
  1. 1.Faculty of PharmacyUniversity of TorontoTorontoCanada
  2. 2.Department of Pharmacology, Faculty of MedicineUniversity of TorontoTorontoCanada

Personalised recommendations