Midazolam α‐Hydroxylation by Human Liver Microsomes <i>in vitro</i>: Inhibition by Calcium Channel Blockers, Itraconazole and Ketoconazole

469

380

ABSTRACT:The measurement of the effect of new chemical entities on human cytochrome P450 marker activities using in vitro experimentation represents an important experimental approach in drug development. In vitro drug interaction data can be used in guiding the design of clinical drug interaction studies, or, when no effect is observed in vitro, the data can be used in place of an in vivo study to claim that no interaction will occur in vivo. To make such a claim, it must be assured that the in vitro experiments are performed with absolute confidence in the methods used and data obtained. To meet this need, 12 semiautomated assays for human P450 marker substrate activities have been developed and validated using approaches described in the GLP (good laboratory practices) as per the code of U.S. Federal Regulations. The assays that were validated are: phenacetin O-deethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), bupropion hydroxylase (CYP2B6), amodiaquine N-deethylase (CYP2C8), diclofenac 4-hydroxylase and tolbutamide methylhydroxylase (CYP2C9), (S)-mephenytoin 4-hydroxylase (CYP2C19), dextromethorphan O-demethylase (CYP2D6), chlorzoxazone 6-hydroxylase (CYP2E1), felodipine dehydrogenase, testosterone 6␤-hydroxylase, and midazolam 1-hydroxylase (CYP3A4 and CYP3A5). High-pressure liquid chromatography-tandem mass spectrometry, using stable isotope-labeled internal standards, has been applied as the analytical method. This analytical approach, through its high sensitivity and selectivity, has permitted the use of very low incubation concentrations of microsomal protein (0.01-0.2 mg/ml). Analytical assay accuracy and precision values were excellent. Enzyme kinetic and inhibition parameters obtained using these methods demonstrated high precision and were within the range of values previously reported in the scientific literature. These methods should prove useful in the routine assessments of the potential for new drug candidates to elicit pharmacokinetic drug interactions via inhibition of cytochrome P450 activities.Drug-drug interactions are of great interest to scientists involved in drug research, regulatory authorities who are responsible for public safety, physicians, and their patients. Since "polypharmacy," or the practice of simultaneous prescription of more than one drug to treat one or more conditions in a single patient, has become a more common practice, drug interactions have been cited as one of the major reasons for hospitalization and even death (Lazarou et al., 1998). Thus, a great deal of effort is expended by researchers engaged in new drug research in avoiding the development of compounds that will cause drug-drug interactions.The most common mechanism underlying drug-drug interactions is the inhibition of cytochrome P450 activities. Several drugs in common use cause large increases in exposure to other drugs. Examples include ketoconazole, itraconazole, erythromycin, clarithromycin, diltiazem, and nefazodone (CYP3A); quinidine, paroxetine, and terbinafine (CYP2D6); ticlopidine (CYP2C19); ...

Section: Validated Assays For Human Cytochrome P450 Enzymessupporting

confidence: 81%

Validated Assays for Human Cytochrome P450 Activities

Walsky

Obach²

2004

469

380

“…For example, ketoconazole is more extensively used than itraconazole as a CYP3A index inhibitor, even though both ketoconazole and itraconazole have similar inhibitory potentials against CYP3A-mediated midazolam 19-hydroxylation (IC 50 @ 0.01 mM) (Racha et al, 2003). The reason for this preference is that studies have shown that ketoconazole has a strong inhibitory effect on various CYP3A substrates including midazolam, nifedipine, testosterone, and triazolam (Wang et al, 1999;Perloff et al, 2000;Racha et al, 2003;Greenblatt et al, 2011), while itraconazole has a weak inhibitory effect on CYP3A-mediated terfenadine metabolism (IC 50 . 50 mM) (Racha et al, 2003).…”

Section: Resultsmentioning

confidence: 99%

“…In general, P450 index inhibitors used in reaction phenotyping studies should inhibit each P450 isoform-specific activity in a substrate-independent manner. One example is ketoconazole, a representative CYP3A inhibitor that inhibits the CYP3A-mediated metabolism of several substrates including nifedipine, midazolam, terfenadine, testosterone, and triazolam (Wang et al, 1999;Perloff et al, 2000;Racha et al, 2003;Greenblatt et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Danazol Inhibits Cytochrome P450 2J2 Activity in a Substrate-independent Manner

Lee

Shon

et al. 2015

Cytochrome P450 2J2 (CYP2J2) is an enzyme responsible for the metabolism of endogenous substrates including arachidonic acid, as well as therapeutic drugs such as albendazole, astemizole, ebastine, and terfenadine. Selective inhibitors of CYP2J2 are essential for P450 reaction phenotyping studies. To find representative CYP2J2 index inhibitors, we evaluated the inhibitory potential of danazol, hydroxyebastine, telmisartan, and terfenadone against CYP2J2 activity for four representative CYP2J2 substrates (albendazole, astemizole, ebastine, and terfenadine) using recombinant CYP2J2. Of these four CYP2J2 inhibitors, danazol strongly inhibited CYP2J2-mediated albendazole, astemizole, ebastine, and terfenadine metabolism in a substrate-independent manner, with IC 50 values of 0.05, 0.07, 0.18, and 0.34 mM, respectively. Danazol noncompetitively inhibited CYP2J2-mediated astemizole O-demethylation activities with a K i value of 0.06 mM. Terfenadone strongly inhibited CYP2J2-mediated albendazole, astemizole, and terfenadine metabolism (IC 50 < 0.21 mM), whereas it showed weak inhibition against CYP2J2-catalyzed ebastine hydroxylase activity (IC 50 = 6.04 mM). Telmisartan had no inhibitory effect on CYP2J2-mediated ebastine and terfenadine hydroxylation (IC 50 > 20 mM). Taken together, these data suggest that danazol may be used as a CYP2J2 index inhibitor in reaction phenotyping studies.

“…It is likely that the decrease in inhibitory potency observed for relatively weaker inhibitors at high microsomal concentrations is attributable mainly to nonspecific binding. The findings suggest that large variability among laboratories in estimated inhibitory potency of a single inhibitor versus a single substrate (such as ketoconazole versus midazolam) could in part be explained by variations in protein and/or enzyme concentration (Gascon and Dayer, 1991;Hargreaves et al, 1994;Wrighton and Ring, 1994;Ghosal et al, 1996;von Moltke et al, 1996b;Gibbs et al, 1999a,b;Wang et al, 1999;Perloff et al, 2000;Venkatakrishnan et al, 2000b). In general, the impact of microsomal binding, both specific and nonspecific, may be minimized by use of the lowest possible microsomal concentration.…”

Section: Fig 3 Mean (ϯ Se) Fractional Decrement In Reaction Velocmentioning

confidence: 99%

Microsomal Protein Concentration Modifies the Apparent Inhibitory Potency of CYP3A Inhibitors

Tran¹,

Moltke²,

Venkatakrishnan³

et al. 2002

ABSTRACT:The effect of microsomal protein concentration on the inhibitory potency of a series of CYP3A inhibitors was assessed in vitro using Microsomal binding of drug substrates and/or metabolic inhibitors is increasingly recognized as a potential source of artifact arising in the course of in vitro studies of drug metabolism. Nonspecific binding of substrate to microsomal protein can influence availability of the substrate to the metabolizing enzyme or enzymes in vitro, and thereby yield biased estimates of enzyme kinetic parameters. These, in turn, may produce inaccurate predictions when in vitro data are used to estimate in vivo pharmacokinetics (Obach, 1996(Obach, , 1997Obach et al., 1997;McLure et al., 2000;Venkatakrishnan et al., 2000c Venkatakrishnan et al., , 2001Kalvass et al., 2001). Inhibitor binding to microsomal systems may likewise influence estimation of potency of metabolic inhibitors (Gibbs et al., 1999a). The influence of binding has been termed "inhibitor depletion" when the inhibitor interacts with the active site (Gibbs et al., 1999a). However, inhibitor depletion could also refer to nonspecific inhibitor binding to microsomal preparations, as well as to actual microsomal consumption of the inhibitor itself through biotransformation.The present study evaluated the influence of microsomal protein concentration on the inhibitory capacity of known CYP3A inhibitors. In vitro 3-hydroxylation of diazepam to temazepam was used as an index reaction to profile CYP3A activity. Two selective serotonin reuptake inhibitors (fluvoxamine and norfluoxetine), three antifungal azole agents (itraconazole, ketoconazole, and fluconazole), a metabolite of itraconazole (OH-itraconazole), and the human immunodeficiency virus protease inhibitor ritonavir were used as representative CYP3A inhibitors. These agents have different intrinsic inhibitory capacities and different binding affinities. Materials and MethodsIncubation Procedures and Index Reaction Characteristics. Liver samples from individual human donors with no known liver disease were provided by the International Institute for the Advancement of Medicine (Exton, PA), the Liver Tissue Procurement and Distribution System (University of Minnesota, Minneapolis, MN), or the National Disease Research Interchange (Philadelphia, PA). All samples were of the CYP2D6 and CYP2C19 normal metabolizer phenotype based on prior in vitro phenotyping studies. Microsomes were prepared by ultracentrifugation; microsomal pellets were suspended in 0.1 M potassium phosphate buffer containing 20% glycerol and stored at Ϫ80°C until use. Chemical reagents and drug entities were purchased from commercial sources or kindly provided by their pharmaceutical manufacturers (von Moltke et al., 1993(von Moltke et al., , 1994a(von Moltke et al., ,b, 1996aVenkatakrishnan et al., 2000a Venkatakrishnan et al., ,b,c, 2001a.Diazepam 3-hydroxylation to form temazepam was used as the index reaction for CYP3A activity (Andersson et al., 1994;Ono et al., 1996;Jung et al., 1997; Venkatakrishnan ...