Measurements of caffeine and plasma metabolite/caffeine ratios as a test for hepatic drug-oxidizing capacity in goats

1. The aim of this investigation was to determine the pharmacokinetics and demethylation of caffeine (CF) and the metabolite/CF ratios that correlated best with CF clearance, which were used to evaluate hepatic drug-oxidizing capacity of CF after a single intravenous dose (5?mg/kg) in hair goats (n?=?9).

2. Pharmacokinetic parameters of CF and its metabolites, theobromine (TB), paraxanthine (PX) and theophylline (TP), were calculated. The plasma metabolic ratios TB/CF, PX/CF, TP/CF and TB+PX+TP/CF were determined at 6, 8 and 10?h after CF administration to evaluate their hepatic drug-oxidizing capacity.

3. The plasma concentration–time data of CF were fit to a two-compartment model in all animals. The clearance of CF was 0.08?±?0.02?L/h/kg, and the volume of distribution was 0.91?±?0.16?L/kg. The demethylation fractions of CF to TB, PX and TP were 0.24, 0.37 and 0.39, respectively.

4. Correlations between the metabolic ratios and CF clearance were quite high, except for the PX/CF ratio, particularly at 6?h (r?=?0.650–0.750, P?<?0.01, 0.05) and 10?h (r?=?0.650–0.767, P?<?0.01, 0.05).

5. Plasma metabolite/CF ratios, except for the PX/CF ratio, may be useful as an alternative to measurements of CF clearance for the determination of the hepatic drug-oxidizing capacity in goats.

     

Effects of Phyllanthus amarus on the pharmacokinetics of midazolam and cytochrome P450 activities in rats

1. Phyllanthus amarus, a commonly used medicinal herb, was investigated for possible herb–drug interactions. The effect on CYP3A-mediated drug metabolism in rats after single dose administration of P. amarus extract was investigated using midazolam (MDZ) as a probe substrate. The effect of multiple dose administration of P. amarus extract on activity and expression of various CYP isoforms were studied.

2. Oral administration of P. amarus extract (800?mg/kg) 1?h before oral MDZ increased the C_max and AUC_0–-∞ of MDZ by 3.9- and 9.6-fold and decreased the clearance by 12%, but did not alter the pharmacokinetics of intravenous MDZ.

3. Daily administration of P. amarus extract (200 or 800?mg/kg/day) for 15 days in rats increased the activity and expression of CYP3A and CYP2B1/2. In contrast, the activities and expressions of CYP1A, CYP2C and CYP2E1 were not significantly changed.

4. The dual effects of P. amarus extract on CYP enzymes were demonstrated. Single dose administration of the extract increased oral bioavailability of MDZ through inhibition of intestinal CYP3A whereas repeated administration of the extract slightly induced hepatic CYP3A and CYP2B1/2 in rats, which suggested that herb–drug interactions by P. amarus may potentially occur via CYP3A and 2B.

     

In vitro metabolic interactions between black cohosh (Cimicifuga racemosa) and tamoxifen via inhibition of cytochromes P450 2D6 and 3A4

1. Women who experience hot flashes as a side effect of tamoxifen (TAM) therapy often try botanical remedies such as black cohosh to alleviate these symptoms. Since pharmacological activity of TAM is dependent on the metabolic conversion into active metabolites by the action of cytochromes P450 2D6 (CYP2D6) and 3A4, the objective of this study was to evaluate whether black cohosh extracts can inhibit formation of active TAM metabolites and possibly reduce its clinical efficacy.

2. At 50 μg/mL, a 75% ethanolic extract of black cohosh inhibited formation of 4-hydroxy- TAM by 66.3%, N-desmethyl TAM by 74.6% and α-hydroxy TAM by 80.3%. In addition, using midazolam and dextromethorphan as probe substrates, this extract inhibited CYP3A4 and CYP2D6 with IC₅₀ values of 16.5 and 50.1 μg/mL, respectively.

3. Eight triterpene glycosides were identified as competitive CYP3A4 inhibitors with IC₅₀ values ranging from 2.3–5.1 µM, while the alkaloids protopine and allocryptopine were identified as competitive CYP2D6 inhibitors with K_i values of 78 and 122?nM, respectively.

4. The results of this study suggests that co-administration of black cohosh with TAM might interfere with the clinical efficacy of this drug. However, additional clinical studies are needed to determine the clinical significance of these in vitro results.

     

Hepatic CYP2B6 is altered by genetic,physiologic, and environmental factors but plays little role in nicotine metabolism

1. Human cytochrome P4502B6 (CYP2B6) is predominantly expressed in the liver and it plays a major role in the metabolism of several therapeutically important drugs and environmental toxicants.

2. The objective was twofold: (1) to determine the role of genetic, physiological, and environmental factors in predicting hepatic CYP2B6 protein expression; and (2) to investigate the role of CYP2B6 in nicotine C-oxidation.

3. Human livers (n?=?40) were assessed for CYP2B6 protein and genotype.

4. Linear regression analyses indicated that CYP2B6 genotype (10%), gender (14%), and exposure to inducers (21%), but not age, were predictors of CYP2B6 protein amounts. Livers with at least one CYP2B6*5 or *6 allele were associated with lower CYP2B6. Female livers and livers exposed to inducers (phenobarbital and/or dexamethasone) were associated with higher CYP2B6.

5. A weak correlation between CYP2B6 and nicotine C-oxidation activity was observed, which was abrogated when controlling for CYP2A6 protein levels. CYP2B6*6 was not associated with different nicotine kinetics.

6. In summary, CYP2B6 protein expression was associated with genotype, gender, and exposure to inducers, but not with nicotine C-oxidation activity.

     

Baicalin,an emerging multi-therapeutic agent: pharmacodynamics,pharmacokinetics, and considerations from drug development perspectives

1. Baicalin was extensively researched for utility in a number of therapeutic areas owing to its anti-inflammatory, anti-oxidant, anti-bacterial, and anti-cancer properties.

2. A number of preclinical studies, in vitro work, and mechanistic studies were performed to understand the absorption, distribution, metabolism, and excretion profiles of baicalin.

3. The absorption of baicalin involved several complexities: the restriction to two distant sites; the conversion of baicalin to baicalein; the possible role of transporter(s); and enhanced absorption due to breakdown of conjugates by beta-glucuronidase.

4. Limited distribution data suggest that baicalin reached several sites such as the brain, eye lens, thymus, etc. Hepatobiliary recycling also served as a distribution phase for sustained delivery of baicalin.

5. Metabolism data suggest the rapid conversion of baicalin to baicalein, which was extensively subjected to Phase 2 metabolism, conjugates baicalein glucuronide/sulfate have been identified.

6. Limited excretion data suggest involvement of renal and faecal routes—glucuronide and sulfate conjugates were excreted in urine and faeces (via biliary excretion).

7. The published data on baicalin suggest imminent challenges for developing baicalin and/or during co-administration with other agents. These challenges are absorption related (transporter or changes in the microenvironment), metabolism related (CYP2B6 induction and/or CYP2E1 inhibition), and excretion/efflux related (competitive biliary pathway and/or OATP1B1 transport).

     

Inter-individual difference determinant of prednisolone pharmacokinetics for Japanese renal transplant recipients in the maintenance stage

1. The objective of this study was to elucidate the effects of CYP3A5, ABCB1, NR1I2 and NR3C1 BclI gene polymorphisms on prednisolone exposure for 65 Japanese renal transplant recipients in the maintenance stage one year after transplantation.

2. Prednisolone dosage ranged from 2.5 to 15.0?mg day^?1 based on individual immunosuppressive states.

3. The dose-adjusted area under the plasma concentration–time curve (AUC_0–24) and the maximal plasma concentration (C_max) of prednisolone in recipients with the BclI G allele were significantly higher than in those with the CC genotype (p?=?0.029 and 0.021, respectively), but there were no significant differences in the half-life and T_max of prednisolone between the two groups.

4. None of the CYP3A5, ABCB1 or NR1I2 allele variants had any significant influence on the dose-adjusted AUC_0–24 of prednisolone.

5. The NR3C1 BclI polymorphism was important in the inter-individual variability of prednisolone pharmacokinetics.

6. The transactivation of the CYP3A4 promoter by prednisolone via the glucocorticoid receptor might be especially responsive for intestinal CYP3A4.

     

CYP1A2 is the major isoform responsible for paeonol O-demethylation in human liver microsomes

1. Paeonol, the primary active component of a traditional Chinese medicine Moutan Cortex, has a wide range of pharmacological activities. In the present study, the metabolism of paeonol by cytochrome P450s (CYPs) was investigated in human liver microsomes.

2. One O-demethylated metabolite was detected in reaction catalysed by human liver microsomes, and was identified as resacetophenone by comparing the tandem mass spectra and the chromatographic retention time with that of the standard compound.

3. The study with a chemical selective inhibitor, cDNA-expressed human CYPs, a correlation assay, and a kinetics study demonstrated that CYP1A2 was the major isoform responsible for the paeonol O-demethylation in human liver microsomes.

     

Effects of Ginkgo biloba extracts on pharmacokinetics and efficacy of atorvastatin based on plasma indices

1. Ginkgo biloba extract (GBE) is one of the most widely used herbal medicines in the world. It is often administered in combination with statins to treat diseases, especially some nervous system disorders. We aimed to investigate the influences of GBE on pharmacokinetics and efficacy of atorvastatin, which are currently unclear.

2. Sixteen volunteers received a single oral dose of 40 mg atorvastatin, followed by a wash-out period of at least 5 days. Then the volunteers took 360 mg GBE daily for 14 days, followed by a single dose of 40 mg atorvastatin. Serial blood samples obtained over a period of 48 h after atorvastatin ingestion were subjected to determination of atorvastatin plasma concentrations and markers of cholesterol synthesis (lathosterol) and cholesterol absorption (sitosterol).

3. With GBE administration, AUC_0–48, AUC_0–∞ and C_max of atorvastatin were reduced by 14.27% (p = 0.005), 10.00% (p = 0.03) and 28.93% (p = 0.002), respectively; Vd/F and CL/F of atorvastatin were increased by 31.95% (p = 0.017) and 6.48% (p = 0.044). After 14 days of treatment, GBE has no significant effects on cholesterol-lowering efficacy of atorvastatin.

4. This study suggests that GBE slightly decreases the plasma atorvastatin concentrations, but has no meaningful effect on the cholesterol-lowering efficacy of atorvastatin.

     

Utility of multivariate analysis in support of in vitro metabolite identification studies: retrospective analysis using the antidepressant drug nefazodone

1. The utility of multivariate analysis in in vitro metabolite identification studies was examined with nefazodone, an antidepressant drug with a well-established metabolic profile.

2. The chromatographic conditions were purposefully chosen to reflect those utilized in high-throughput screening for microsomal stability of new chemical entities.

3. Molecular ion, retention time information on groups of human liver microsomal samples with/without nefazodone was evaluated by principal component analysis (PCA). Resultant scores and loadings plots from the PCA revealed the segregation and the ions of interest that designated the drug and its corresponding metabolites. Subsequent acquisition of tandem mass spectrometry (MS/MS) spectra for targeted ions permitted the interrogation and interpretation of spectra to identify nefazodone and its metabolites.

4. A comparison of nefazodone metabolites identified by PCA versus those found by traditional metabolite identification approaches resulted in very good correlation when utilizing similar analytical methods. Fifteen metabolites of nefazodone were identified in β-nicotinamide adenine dinucleotide phosphate (NADPH)-supplemented human liver microsomal incubations, representing nearly all primary metabolites previously reported.

5. Of the 15 metabolites, eight were derived from the N-dealkylation and N-dephenylation of the N-substituted 3-chlorophenylpiperazine motif in nefazodone, six were derived from mono- and bis-hydroxylation, and one was derived from the Baeyer Villiger oxidation of the ethyltriazolone moiety in nefazodone.

     

Intestinal absorption mechanisms of ginsenoside Rh2: stereoselectivity and involvement of ABC transporters

1. This study investigated the absorption mechanism of ginsenoside Rh2 to clarify the reasons for its poor absorption. Transepithelial transport across Caco-2 cell monolayers, cellular uptake, and in situ rat intestinal perfusion were examined.

2. Cellular uptake of Rh2 was linear from 1 to 50 μM at 4°C, whereas it was saturated when the concentration exceeded 10 μM at 37°C. At 37°C, the uptake at 10 μM was linear in 60?min. Intracellular exposure in 240?min was 2173.70 and 979.38 ng·min/μg for S and R isomers, respectively.

3. Transepithelial permeability of Rh2 was about 10^?8 to 10^?7 cm/s. Efflux ratios were above 1.5. Sodium dodecyl sulfate, sodium citrate, and sodium deoxycholate had no effect on Rh2 permeability.

4. After intestinal perfusion for 3?h, 9.1% of 20(R)-Rh2 and 15.7% of 20(S)-Rh2 were absorbed.

5. Cyclosporine, quercetin, and probenecid could improve the cellular uptake, absorptive permeability, and intestinal absorption. Carrier-mediated transport was the major absorption mechanism. Rh2 was a substrate of ABC transporters.

6. The ABC-transporter-mediated efflux and the poor permeability were the major reasons for Rh2 poor absorption.

7. The stereoselective absorption was significant. R isomer exhibited lower absorption profiles in all the experiments, possibly due to more potent efflux.

     

Prediction of clinical CYP3A4 induction using cryopreserved human hepatocytes

1. The purpose of this study was to construct a method to predict CYP3A4 induction in the clinical setting from in vitro data using cryopreserved human hepatocytes.

2. We recently developed an approach with in vitro assays of HepaRG cell lines for predicting CYP3A4 induction by using a novel value, termed the relative factor (RF), determined from the ratio of the concentration of an inducer to the reference standard. In this study, the applicability of the RF approach was expanded to cryopreserved human hepatocytes.

3. Induction assays were performed in vitro using hepatocytes from four individual donors and eight typical inducers. The obtained RF values were related to the free plasma concentration of each inducer (expressed as Css,u/RF).

4. A good relationship between the Css,u/RF values and the in vivo induction response was found for all donors. Inducers were classified by the Css,u/RF values into three categories for CYP3A4 induction risk (high, medium and low potency), and thereby the degree of CYP3A4 induction in vivo in humans could be predicted from the Css,u/RF values.

5. The RF approach is applicable to human cryopreserved hepatocytes. Thus, a method to predict the potency of CYP3A4 inducers was constructed using cryopreserved human hepatocytes.

     

Post-translational activation of human phenylalanine 4-monooxygenase from an endobiotic to a xenobiotic enzyme by reactive oxygen and reactive nitrogen species

1. An investigation into the post-translational activation of cDNA-expressed human phenylalanine 4-monooxygenase and human hepatic cytosolic fraction phenylalanine 4-monooxygenase activity with respect to both endobiotic metabolism and xenobiotic metabolism revealed that the reactive oxygen species (hydrogen peroxide and hydroxyl radical) and reactive nitrogen species (nitric oxide and peroxynitrite) could elicit the post-translational activation of the enzyme with respect to both of these biotransformation reactions.

2. In virtually all instances, the K_m values were decreased and the V_max values were increased; the only exceptions observed being with hydrogen peroxide and L-phenylalanine.

3. These effects were shown to occur at activator concentrations known to exist in physiological situations and, hence, suggest that reactive oxygen and reactive nitrogen species may cause, and may be involved with, the post-translational activation of phenylalanine 4-monooxygenase within the human body.

4. This mechanism, in response to free-radical bursts, may enable the enzyme to expand its substrate range and to process certain xenobiotics as and when required.

     

Metabolism and distribution of two highly potent and selective peptidomimetic inhibitors of matriptase

1. Matriptase is a serine protease expressed by several types of cancer cells and it participates in tumour growth and progression through the activation of hepatocyte growth factor (HGF) and urokinase-type plasminogen activator (uPA).

2. The metabolism of two potent and selective peptidomimetic inhibitors of matriptase (CJ-1737 and CJ-672) was examined in vitro with enzyme preparations (9000g supernatants, microsomes, and plasma) from dog, pig, rat, and human.

3. It was found that both compounds displayed interesting species-dependent differences. Though CJ-1737 was not metabolized by microsomes, by 9000g supernatants from all species, or by human or rat plasma, canine and porcine plasma enzymes rapidly hydrolysed this compound. In contrast, CJ-672 was metabolized exclusively by enzymes from human liver (microsomes and 9000g supernatants) via a two-step metabolic pathway.

4. Additionally, the distribution of both compounds was investigated in mice. The highest amounts were measured in the kidney and liver, followed by the spleen, lung, and heart. In contrast to CJ-1737, high concentrations of CJ-672 were detected in the colon, indicating an additional biliary excretion.

5. In summary, this work clarifies both the metabolism and distribution of two new matriptase inhibitors and demonstrates important metabolic differences between human enzymes and those from commonly used laboratory animals.

     

Trans-resveratrol-mediated inhibition of β-oestradiol conjugation in MCF-7 cells stably expressing human sulfotransferases SULT1A1 or SULT1E1, and human liver microsomes

1. High concentrations of endogenous oestradiol (E2) correlate with the proliferation of cancer cells. Resveratrol (a dietary chemopreventive agent) at high concentrations has an anti-oestrogenic effect. E2 and resveratrol are conjugated via common uridine diphosphoglucuronosyltransferase (UGT) and sulfotransferases (SULT) enzymes.

2. Experiments were conducted in MCF-7 mammalian cells stably expressing human SULT1A1 or SULT1E1 to observe the effect of resveratrol on E2-mediated cell proliferation. The combination of E2 and resveratrol did have a proliferative effect in cells expressing SULT1E1, but not in those expressing SULT1A1.

3. The effect of resveratrol (1–500 μM) on the glucuronidation of E2 (0.25–2.25 μM) was characterized in human liver microsomes. The highest resveratrol concentration significantly decreased the intrinsic clearance of E2 glucuronidation.

4. The results corroborate the reported significant inhibition of SULT1E1-mediated E2 sulfation in vitro by resveratrol. Thus, resveratrol may interact with E2 in vivo by inhibiting its conjugation.

     

Computational and in vitro studies on the inhibitory effects of herbal compounds on human cytochrome P450 1A2

1. Human CYP1A2 is an important enzyme for drug metabolism and procarcinogen activation. This study aimed to explore the binding mode of ligands with CYP1A2 and to screen potential inhibitors from a library of herbal compounds using computational and in vitro approaches.

2. The heme prosthetic group and six residues (Thr124, Phe125, Phe226, Phe260, Gly316, and Ala317) in the active site of CYP1A2 were identified as important residues for ligand binding using the LIGPLOT program. Ala317 in helix I immediately above heme was highly conserved in most human CYPs with known crystal structures.

3. In molecular docking, 19 of the 56 herbal compounds examined were identified as potential inhibitors of CYP1A2. Up to 21 of the 56 herbal compounds were hit by the pharmacophore model of CYP1A2 inhibitors developed and validated in this study.

4. In the in vitro inhibition study, 8 herbal compounds were identified as moderate to potent inhibitors of CYP1A2. Five of the 8 herbal compounds predicted to be potential inhibitors were confirmed as CYP1A2 inhibitors in the in vitro study.

5. A combination of computational and in vitro approaches, represent a useful tool to identify potential inhibitors for CYP1A2 from herbal compounds.

     

Microbial production of phase I and phase II metabolites of propranolol

1. The production in multimilligram amounts of 4- and 5-hydroxylated metabolites of (R)- or (S)-propranolol by biotransformation with two fungal strains, an Absidia sp. M50002 and a Cunninghamella sp. M50036, was carried out, starting from either the racemic drug or pure enantiomers.

2. While both enantiomers of propranolol were hydroxylated in the 5-position by incubation with strain M50002, the strain M50036 operated a chiral discrimination, resulting in the exclusive formation of the 4-hydroxy-(R)-enantiomer.

3. In addition, a Streptomyces sp. strain M52104, isolated from a soil sample, was selected for the high-yield regioselective β-glucuronidation of propranolol and its 4- and 5-hydroxylated derivatives.

4. NMR and mass spectroscopic data have been extensively used for the unambiguous characterization of 4- and 5-hydroxylated and glucuronidated derivatives, all of them corresponding to the major animal and human metabolites of propranolol, a typical substrate of CYP2D6.

     

Chan-Yu-Bao-Yuan-Tang induces apoptosis in NSCLC and SCLC cell lines via a mitochondria-mediated pathway

1. Previous clinical studies have shown efficacy and safety of the traditional Chinese medicinal herb extract Chan-Yu-Bao-Yuan-Tang (CYBYT) in lung cancer patients.

2. The effects of CYBYT on proliferation and apoptosis in the non-small cell lung cancer cell line NCI-H460 and the small cell lung cancer cell line NCI-H446 were investigated in vitro.

3. CYBYT significantly induced antiproliferative effects of NCI-H460 and NCI-H446 cells in a concentration- and time-dependent manner. The IC₅₀ values in NCI-H460 cells were 94.37 µg/mL (24?h) and 20.89 µg/mL (48?h), whereas in NCI-H446 cells IC₅₀ values were 214.72 µg/mL (24?h) and 114.58 µg/mL (48?h). Annexin V–FITC/PI staining showed CYBYT could significantly induce apoptosis in NCI-H460 and NCI-H446 cells, and the total apoptosis rates were positively correlated with the concentration and time of CYBYT treatment. Furthermore, treatment with the broad-spectrum caspase inhibitor (z-VAD-fmk) effectively protected NCI-H460 and NCI-H446 cells against CYBYT-triggered apoptosis. The apoptotic processes involved were a marked decrease in antiapoptotic protein Bcl-2 and an increase in proapoptotic protein Bax. The release of mitochondrial cytochrome c into the cytosol was also observed, which, in turn, resulted in the activation of caspase-9 and caspase-3.

4. CYBYT exerts antiproliferative and growth inhibition effects on NCI-H460 and NCI-H446 cells through the mitochondrial caspase-dependent cell death pathway.

     

The influence of probiotic treatment on sulfasalazine metabolism in rat

1. Probiotics are live microorganisms claimed to exert beneficial effects on the host. This study investigated their effect on the metabolism and pharmacokinetics of sulfasalazine (SSZ), a drug whose efficacy depends on metabolism by azoreductase (AR) in the gut microbiota to sulfapyridine (SP) and 5-acetylsalicylic acid (5-ASA).

2. The probiotic strains Lactobacillus acidophilus L10, Bifidobacterium lactis B94 and Streptococcus salivarius K12 possessed AR activity and a corresponding ability to metabolize SSZ.

3. Treatment of male Wistar rats (n = 5) with oral 2 g doses of a mixture of the three probiotics (total dose 1.8 × 10⁹ cfu) every 12 h for 3 days resulted in a significant increase (p < 0.05) in AR activity in ex vivo colon contents with a corresponding increase in SSZ metabolism.

4. Similar probiotic treatment of male Wistar rats (n = 8) followed by an oral 100 mg/kg dose of SSZ produced high plasma levels of SP, but pharmacokinetic parameters of SSZ and SP were not significantly different from control rats given SSZ.

5. These results indicate that probiotic strains possess AR activity and can metabolize SSZ. Treatment with probiotics increases AR activity in the gut microbiota but has no effect on plasma levels of SSZ and SP following a subsequent oral dose of SSZ.

     

Activation of CYP3A-mediated testosterone 6β-hydroxylation by tanshinone IIA and midazolam 1-hydroxylation by cryptotanshinone in human liver microsomes

1. This study evaluated the in vitro activation of CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation by tanshinone I, tanshinone IIA, and cryptotanshinone.

2. The abilities of tanshinones to activate CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation in human liver microsomes (HLMs) were tested. Substrate- and effector-dependent activation of CYP3A by tanshinones were both observed.

3. Cryptotanshinone was shown to activate CYP3A-mediated midazolam 1-hydroxylation in a concentration-dependent manner. In contrast, tanshinone IIA and tanshinone I did not activate this hydroxylation reaction. In addition, tanshinone IIA activated CYP3A-mediated testosterone 6β-hydroxylation, whereas cryptotanshinone and tanshinone I did not.

4. The results from our study enhance the understanding of CYP3A activation by tanshinone IIA and cryptotanshinone in HLMs. Additionally, these data allow for an accurate prediction of the magnitude and likelihood of Danshen-drug interactions.

     

Cocktail-substrate assay system for mechanism-based inhibition of CYP2C9, CYP2D6, and CYP3A using human liver microsomes at an early stage of drug development

1. We established a mechanism-based inhibition cocktail-substrate assay system using human liver microsomes and drug–probe substrates that enabled simultaneous estimation of the inactivation of main cytochrome P450 (CYP) enzymes, CYP2C9, CYP2D6, and CYP3A, in drug metabolism.

2. The inactivation kinetic parameters of typical mechanism-based inhibitors, tienilic acid, paroxetine, and erythromycin, for each enzyme in the cocktail-substrate assay were almost in agreement with the values obtained in the single-substrate assay.

3. Using this system, we confirmed that multiple CYP inactivation caused by mechanism-based inhibitors such as isoniazid and amiodarone could be detected simultaneously.

4. Mechanism-based inhibition potency can be estimated by the determination of the observed inactivation rate constants (k_obs) at a single concentration of test compounds because the k_obs of eleven CYP3A inactivators at 10?μM in the assay system nearly corresponded to k_inact/K_I values, an indicator of a compound’s propensity to alter the activity of a CYP in vivo (R²?=?0.97).

5. Therefore, this cocktail-substrate assay is considered to be a powerful tool for evaluating mechanism-based inhibition at an early stage of drug development.