Predicting drug pharmacokinetics in humans from in vitro metabolism studies

Biochem Soc Trans. 2001 May;29(Pt 2):135-9. doi: 10.1042/0300-5127:0290135.


The pharmaceutical industry is committed to market safer drugs with fewer side effects, predictable pharmacokinetic properties and quantifiable drug-drug interactions. There is an increasing need to develop robust, enhanced-throughput in vitro assays, which accurately extrapolate to humans. The major drug metabolizing human hepatic cytochrome P450s (CYPs; CYP1A2, 2C9, 2C19, 2D6 and 3A4) have been co-expressed functionally in Escherichia coli with human NADPH-cytochrome P450 reductase and validated as surrogates to their counterparts in human liver microsomes (HLM) with respect to their kinetic and inhibition properties. Using these recombinant enzymes, fully automated in vitro assays to assess CYP inhibition and determine the enzymology of drug oxidation have been developed and validated. IC(50) values determined for a series of test compounds in HLM and recombinant CYPs were similar (r(2)=0.9, P<0.001). There was a good correlation between the sum of individual CYP intrinsic clearance (Cl(int)) and HLM Cl(int) (r(2)=0.8, P<0.001) for ten prototypic substrates for which clearance was CYP-dependent. Several in vitro incubation milieu (e.g. CYPs, HLM, human hepatocytes) are routinely used and the level of non-specific binding was investigated with respect to effects on K(m) and K(i) determinations. There were clear correlations between binding and lipophilicity (logD(7.4)) for a selection of bases (r(2)=0.98, P<0.001) and acids (r(2)=0.79, P<0.001) that may allow prediction of this property. Our laboratory has shown that recombinant enzymes are suitable for "frontline" predictive human metabolism studies in early drug discovery.

MeSH terms

  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism*
  • Humans
  • Inhibitory Concentration 50
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Kinetics
  • Liver / cytology
  • Liver / enzymology
  • Liver / metabolism
  • Microsomes, Liver / enzymology
  • Microsomes, Liver / metabolism
  • Oxidation-Reduction
  • Pharmaceutical Preparations / metabolism*
  • Pharmacokinetics*
  • Predictive Value of Tests
  • Protein Binding
  • Recombinant Proteins / metabolism


  • Isoenzymes
  • Pharmaceutical Preparations
  • Recombinant Proteins
  • Cytochrome P-450 Enzyme System