Automated Assessment of Time-Dependent Inhibition of Human Cytochrome P450 Enzymes Using Liquid Chromatography-Tandem Mass Spectrometry Analysis

Abstract

ABSTRACT:Increasing reports of time-dependent inhibition of cytochrome P450 (P450) suggest further emphasis on interpreting the consequences, either from a pharmacokinetic or toxicological perspective. Two automated, time-dependent inhibition assays with a liquid chromatography-tandem mass spectrometric endpoint are presented. The initial assay utilizes human liver microsomes, a single concentration of inhibitor, and a single preincubation time of 30 min. Phenacetin, diclofenac, S-mephenytoin, bufuralol, and midazolam are used as substrates for CYP1A2, 2C9, 2C19, 2D6, and 3A4, and the assay differentiates between reversible and irreversible inhibition. The second assay uses individual recombinant human P450s, six inhibitor concentrations, and three time points to accurately define k inact and K I . A good correlation is demonstrated between k inact /K I and partition ratio, indicating that both terms are related in describing the efficiency of enzyme inactivation. Despite the single preincubation time point of 30 min used in the initial assay, a good relationship has been found to exist between the unbound IC 50 estimated from this initial screen and the k inact /K I ratio derived from the more extensive subsequent single P450 assay. The higher throughput human liver microsomal assay can therefore generate IC 50 values that can be used to predict the pharmacokinetic impact on cotherapies from the estimated k inact /K I ratio, predicted human dose, and pharmacokinetics.Drug-drug interactions (DDIs) following drug therapy, which result in a high number of hospital admissions and even deaths (Lazarou et al., 1998;Kohler et al., 2000), are primarily caused by macromolecule binding of reactive species or drug cotherapy resulting in plasma concentrations of one of the coadministered drugs being elevated to toxic levels (Hollenberg, 2002). The mechanism is frequently competition at the active site of drug-metabolizing enzymes. Since multiple drug therapy is a very common practice, the possibility of DDI therefore exists in the majority of patients. This is a high profile issue for drug discovery and development programs, and there is even an on-line DDI database that shows the number of drugs currently implicated (Carlson et al., 2002). Great importance is now placed on in vitro studies as tools for predicting in vivo DDIs, particularly those resulting from cytochrome P450 (P450) inhibition (Lin and Lu, 1998), since the metabolic elimination of a large number of drugs is dependent on the P450 family of enzymes.To date, 57 human cytochrome P450 genes have been identified (http://drnelson.utmem.edu/CytochromeP450.html), but remarkably, only three human P450s (3A4, 2C9, and 2D6) perform the majority of biotransformations involving pharmaceuticals (Smith et al., 1998). CYP1A2, 2B6, 2C8, 2C19, and 2E1 are also involved, but to a much lesser extent. Inhibition of P450-dependent metabolism can generally be classified into three categories: reversible, quasi-irreversible (when compounds complex the heme prosthetic group and le...

Inhibitory Effects of Seven Components of Danshen Extract on Catalytic Activity of Cytochrome P450 Enzyme in Human Liver Microsomes

“…Although danshensu decreased the percentage of at ASPET Journals on May 9, 2018 dmd.aspetjournals.org remaining activity of CYP2C9 in HLMs preincubated for 10 min with NADPH approximately 17% compared with that without NADPH, further studies are required to confirm whether danshensu had timedependent inhibitory effect on CYP2C9 and estimate the parameters of time-dependent inhibition (K i , k incat , portioning ratio, etc.) (Jushchyshyn et al, 2003;Atkinson et al, 2005). The other five components of danshen extract were not found to inhibit CYP2C9 activity.…”

CYP3A Time-Dependent Inhibition Risk Assessment Validated with 400 Reference Drugs

“…The characteristic parameters for TDI, k inact , the maximal inactivation rate, and K I , the concentration at half k inact , and their ratio k inact /K I are typically used for risk evaluation. Results of attempts to use excerpts of these experiments to screen a large number of compounds have been published (Atkinson et al, 2005;Lim et al, 2005;Obach et al, 2007;Perloff et al, 2009). The IC 50 shift method, which is essentially an extension of the classic highthroughput testing for reversible inhibition, is the most popular because of its easy setup.…”

A Comprehensive in Vitro and in Silico Analysis of Antibiotics That Activate Pregnane X Receptor and Induce CYP3A4 in Liver and Intestine

“…Spheres represent hydrophobic features (cyan), hydrogen bond acceptor and vector (green), and hydrogen bond donor and vector (purple). Original (n ϭ 12) Pharmacophore (Ekins and Erickson, 2002;Bachmann et al, 2004) Diverse (n ϭ 31) Pharmacophore (Ekins et al, 2007) Steroidal (n ϭ 30) Pharmacophore (Ekins et al, 2007) Docking FlexX Score (Khandelwal et al, 2007) Although the macrolide antibiotics erythromycin and troleandomycin were efficacious PXR activators, having both increased mRNA expression of CYP3A4, they both failed to induce CYP3A4 activity, consistent with their ability to also act as CYP3A4 inhibitors (McConn et al, 2004;Atkinson et al, 2005). Interestingly, TAO had previously been shown to have an EC 50 value of 8.9 M, whereas erythromycin was inactive in a PXR transactivation assay (Sinz et al, 2006).…”