Effect of Nonspecific Binding to Microsomes and Metabolic Elimination of Buprenorphine on the Inhibition of Cytochrome P4502D6

Abstract

The inhibition of drug-metabolizing enzymes, such as cytochrome P450 (CYP) often can lead to alterations in the extent of exposure to coadministered drug. Such drug interactions can limit the use of a drug because of adverse clinical effects, depending upon the potential for the toxicity of affected drug. Since CYP2D6 is one of the important human CYP isozymes and metabolizes more than 20% of commercially available drugs, 1) a profound understanding of the inhibitory potency against CYP2D6 of the drug candidates is important in the discovery and development of new drugs. The pharmaceutical industry routinely has assessed the risk of the drug interaction by determining the inhibitory effect of the candidates on the rate of a probe reaction that represents of a specific CYP enzyme activity in human liver microsomes (HLM).2,3) The in vitro findings obtained with one probe substrate are usually extrapolated to the potential of compound to affect all substrates of the same enzyme. Bufuralol (BF) 1Ј-hydroxylation and dextromethorphan (DM) O-demethylation have generally been used as probe reactions for the evaluation of CYP2D6 inhibition. Buprenorphine (BN) is a semi-synthetic, highly lipophilic opioid derivative of the morphine alkaloid tebaine and is often prescribed for the treatment of moderate to severe pain in cases of postoperative and terminal cancer. 7) BN is metabolized by N-dealkylation of its cyclopropyl methyl group to norbuprenorphine (NBN). 8) N-Dealkylation of BN in HLM is mainly catalyzed by CYP3A4. 9,10) Uehara et al. and Zhang et al. reported that BN competitively inhibits not only CYP3A4 but also CYP2D6. According to their reports, BN competitively inhibits BF 1Ј-hydroxylation (K i ϭ21 mM) and DM O-demethylation (K i ϭ10 mM) in HLM respectively (Table 1). 11,12)When we evaluated the inhibition by BN against CYP2D6 using the high throughput method in the present study, BN strongly inhibited it (IC 50 ϭ0.25 mM). Although the substrate concentration (1.5 mM) in this assay was near to the reported K m value (1.0 mM) for this reaction, 4) this IC 50 value showed a large discrepancy with the above reported K i values in HLM.Margolis et al. reported that the inhibitory potency of some highly lipophilic drugs against CYP2D6 decreased with an increase in microsomal concentration. This is caused by the decrease of free concentrations of inhibitors due to an increase in the nonspecific binding to microsomes.13) As BN shows high lipophilicity, it could have high binding affinity to microsomes. Thus, we considered nonspecific binding to be a probable cause of the discrepancy.On the other hand, BN is rapidly metabolized by CYP3A4, but not by CYP2D6 at low substrate concentrations. 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 pot...

Prediction of Cyp2c9-Mediated Drug-Drug Interactions: A Comparison Using Data From Recombinant Enzymes and Human Hepatocytes

“…Inhibitor K i or IC 50 has been well documented for the CYP3A4 inhibitor ketoconazole (Riley, 2001) and the CYP2D6 inhibitor buprenorphine (Umeda et al, 2005). To this end, in P450 inhibition screening assays, this laboratory routinely uses rP450s expressed in E. coli at the lowest acceptable protein concentration where, for compounds of moderate or less lipophilicity, fu inc approaches unity.…”

Modelling Species Selectivity in Rat and Human Cytochrome P450 2D Enzymes

“…However, we do not observe a close approach to the iron in our docking studies. This may suggest that as both quinine and quinidine are inhibitors (at least in vitro [26]), their roles are to occupy space in the binding site thereby denying other ligands access to the active site.…”

Validated method for rapid inhibition screening of six cytochrome P450 enzymes by liquid chromatography–tandem mass spectrometry

“…inhibition study using cDNA expressed CYPs might be suitable to minimize the effect of nonspecific binding, and therefore, a more precise estimation of the risk of adverse drug interaction can be achieved [25,26]. The assay of pooling sample after individual incubation instead of "cocktail" incubation could further eliminate mutual drug interactions among substrates.…”

“…Therefore, as a minimum requirement for in vitro inhibition studies, it was recommended that at least two CYP3A4 substrates should be used [22][23][24]6]. Additionally, human hepatic microsomes used in these "cocktail" experiments is the greater contributor to the effect of nonspecific binding compared with cDNA-expressed CYPs, which may result in false negative errors in prediction of the risk of drug interaction [25,26].…”