Effect of nonspecific binding to microsomes and metabolic elimination of buprenorphine on the inhibition of cytochrome P4502D6

Biol Pharm Bull. 2005 Feb;28(2):212-6. doi: 10.1248/bpb.28.212.


Recently, the pharmaceutical industry has employed the high-throughput method for the evaluation of cytochrome P450 (CYP) inhibition, using a combination of the heterologously expressed enzyme and a fluoregenic substrate. When buprenorphine (BN), a potent mixed agonist-antagonist analgesic, was evaluated by this method, it exhibited potent inhibition of CYP2D6 with an IC50 value of 0.25 microM in recombinant CYP2D6-expressing insect cell microsomes (rCYP2D6 microsomes). In contrast, the IC50 value was 22.7 microM in human liver microsomes (HLM) using a classical method. Although the substrate concentrations in each study were set to near the Km values, there was a large discrepancy in IC50 values. When we investigated the effect of nonspecific binding to microsomes on the inhibitory potency, with a view to clarifying this discrepancy, the unbound fraction in microsomes (fu,mic) was 0.06-0.21 and 0.99 in HLM and rCYP2D6 microsomes, respectively. The corrected IC50 value (1.74 microM) using free BN concentrations was much smaller than the uncorrected value. On the other hand, it was observed that the concentration of BN in HLM decreased rapidly due to metabolism by CYP3A4 while that in rCYP2D6 microsomes decreased only slightly. We then investigated the effect of incubation time on the inhibitory potency, since the rapid elimination of BN in HLM could have been a cause of the discrepancy. The IC50 value for BN was observed to decrease slightly from 22.7 to 17.1 microM, following the shortening of the incubation time from 10 to 2 min in HLM. We conclude that nonspecific binding to microsomes of the inhibitor could affect the inhibitory potency against CYP2D6. If this factor is considered, a more precise estimate of the risk of adverse drug interaction could be achieved.

MeSH terms

  • Animals
  • Buprenorphine / metabolism*
  • Buprenorphine / pharmacology
  • Cytochrome P-450 CYP2D6 / metabolism*
  • Cytochrome P-450 CYP2D6 Inhibitors
  • Dose-Response Relationship, Drug
  • Enzyme Inhibitors / metabolism
  • Enzyme Inhibitors / pharmacology
  • Female
  • Humans
  • Insecta
  • Male
  • Microsomes, Liver / drug effects
  • Microsomes, Liver / metabolism*
  • Protein Binding / drug effects
  • Protein Binding / physiology


  • Cytochrome P-450 CYP2D6 Inhibitors
  • Enzyme Inhibitors
  • Buprenorphine
  • Cytochrome P-450 CYP2D6