Application of substrate depletion assay for early prediction of nonlinear pharmacokinetics in drug discovery: assessment of nonlinearity of metoprolol, timolol, and propranolol

J Pharm Sci. 2005 Dec;94(12):2656-66. doi: 10.1002/jps.20490.


The purpose of this study was to investigate the advantages of the substrate depletion assay for evaluating linearity of pharmacokinetics compared with the metabolite formation assay. For propranolol, metoprolol, and nisoldipine with multiple and/or sequential metabolisms, the Michaelis constant (Km) and maximum metabolic intrinsic clearance obtained from the depletion assay using rat and human liver microsomes showed a good correlation with relevant parameters with the formation assay. In vitro kinetics and in vivo pharmacokinetic profiles after oral administration of timolol, metoprolol, and propranolol, were investigated in rats using the depletion assay. The same rank order was found between nonlinearities based on dose-normalized areas under the plasma concentration curve (AUC/Dose) and Km values. Using the kinetic parameters of these compounds, AUC was predicted based on a physiological based pharmacokinetic model incorporated saturable metabolism. The AUCs predicted for propranolol and metoprolol had a good relationship with those observed in the in vivo studies, implying that the depletion assay could be useful for assessing linearity of pharmacokinetics.

MeSH terms

  • Adrenergic beta-Antagonists / blood
  • Adrenergic beta-Antagonists / pharmacokinetics*
  • Animals
  • Area Under Curve
  • Blood Proteins / metabolism
  • Drug Design*
  • Female
  • Male
  • Metabolic Clearance Rate
  • Metoprolol / blood
  • Metoprolol / pharmacokinetics*
  • Microsomes, Liver / metabolism
  • Nonlinear Dynamics*
  • Predictive Value of Tests
  • Propranolol / blood
  • Propranolol / pharmacokinetics*
  • Rats
  • Rats, Wistar
  • Substrate Specificity
  • Timolol / blood
  • Timolol / pharmacokinetics*


  • Adrenergic beta-Antagonists
  • Blood Proteins
  • Timolol
  • Propranolol
  • Metoprolol