Screening of drug metabolizing enzymes for fusidic acid and its interactions with isoform-selective substrates in vitro

1.?Fusidic acid (FA) is widely used for the treatment of infections of sensitive osteomyelitis or skin and soft tissue caused by bacteria. However, the role of cytochrome P450s (CYPs) in the metabolism of FA is unclear. In the present study, we screened the main CYPs for the metabolism of FA and studied its interactions with isoform-selective substrates in vitro.

2.?The main CYP450s were screened according to the inhibitory effect of specific inhibitors on the metabolism of FA in human liver microsomes (HLMs) or recombinant CYP isoforms. Enzyme kinetic parameters including K_i, K_i′, V_max, and IC₅₀ were calculated to determine the potential of FA to affect CYP-mediated metabolism of isoform-selective substrates.

3.?FA metabolism rate was inhibited by 49.8% and 83.1% under CYP2D6, CYP3A4 selective inhibitors in HLMs. In recombinant experiment, the inhibitory effects on FA metabolism were 83.3% for CYP2D6 and 58.9% for CYP3A4, respectively. FA showed inhibition on CYP2D6 and CYP3A4 with K_is of 13.9 and 38.6?μM, respectively. Other CYP isoforms including CYP1A2, CYP2A6, CYP2C9, CYP2E1, and CYP2C19 showed minimal or no effect on the metabolism of FA.

4.?FA was primarily metabolized by CYP2D6 and CYP3A4 and showed a noncompetitive inhibition on CYP2D6 and a mixed competitive inhibition on CYP3A4. Drug–drug interactions between FA and other chemicals, especially with substrates of CYP2D6 and CYP3A4, are phenomena that clinicians need to be aware of and cautious about.     

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.

     

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

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

The paraoxonase-1 pathway is not a major bioactivation pathway of clopidogrel in vitro

BACKGROUND AND PURPOSE

Clopidogrel is a prodrug bioactivated by cytochrome P450s (CYPs). More recently, paraoxonase-1 (PON1) has been proposed as a major contributor to clopidogrel metabolism. The purpose of this study was to assess the relative contribution of CYPs and PON1 to clopidogrel metabolism in vitro.

EXPERIMENTAL APPROACH

Clopidogrel metabolism was studied in human serum, recombinant PON1 enzyme (rePON1), pooled human liver microsomes (HLMs), HLMs with the CYP2C19*1/*1 genotype and HLMs with the CYP2C19*2/*2 genotype. Inhibition studies were also performed using specific CYP inhibitors and antibodies. Clopidogrel and its metabolites were measured using LC/MS/MS method.

KEY RESULTS

PON1 activity was highest in the human serum and there was no difference in PON1 activity between any of the HLM groups. The production of clopidogrel''s active metabolite (clopidogrel-AM) from 2-oxo-clopidogrel in pooled HLMs was approximately 500 times that in serum. When 2-oxo-clopidogrel was incubated with rePON1, clopidogrel-AM was not detected. Clopidogrel-AM production from 2-oxo-clopidogrel was lower in CYP2C19*2/*2 HLMs compared with CYP2C19*1/*1 HLMs, while PON1 activity in HLMs with both genotypes was similar. Moreover, incubation with inhibitors of CYP3A, CYP2B6 and CYP2C19 significantly reduced clopidogrel bioactivation while a PON1 inhibitor, EDTA, had only a weak inhibitory effect.

CONCLUSION AND IMPLICATIONS

This in vitro study shows that the contribution of PON1 to clopidogrel metabolism is limited at clinically relevant concentrations. Moreover, CYP2C19, CYP2B6 and CYP3A play important roles in the bioactivation of clopidogrel.     

Metabolism of novel anti-HIV agent 3-cyanomethyl-4-methyl-DCK by human liver microsomes and recombinant CYP enzymes

Aim:

To investigate the metabolism of 3-cyanomethyl-4-methyl-DCK (CMDCK), a novel anti-HIV agent, by human liver microsomes (HLMs) and recombinant cytochrome P450 enzymes (CYPs).

Methods:

CMDCK was incubated with HLMs or a panel of recombinant cytochrome P450 enzymes including CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, and 3A5. LC-ion trap mass spectrometry was used to separate and identify CMDCK metabolites. In the experiments with recombinant cytochrome P450 enzymes, specific chemical inhibitors combined with CYP antibodies were used to identify the CYP isoforms involved in CMDCK metabolism.

Results:

CMDCK was rapidly and extensively metabolized by HLMs. Its intrinsic hepatic clearance estimated from the in vitro data was 19.4 mL·min⁻¹·kg⁻¹, which was comparable to the mean human hepatic blood flow rate (20.7 mL·min⁻¹·kg⁻¹). The major metabolic pathway of CMDCK was oxidation, and a total of 14 metabolites were detected. CYP3A4 and 3A5 were found to be the principal CYP enzymes responsible for CMDCK metabolism.

Conclusion:

CMDCK was metabolized rapidly and extensively in human hepatic microsomes to form a number of oxidative metabolites. CYP3A4 and 3A5 were the predominant enzymes responsible for the oxidation of CMDCK.     

Impact of CYP2C8*3 polymorphism on in vitro metabolism of imatinib to N-desmethyl imatinib

Abstract

1.?Imatinib is metabolized to N-desmethyl imatinib by CYPs 3A4 and 2C8. The effect of CYP2C8*3 genotype on N-desmethyl imatinib formation was unknown.

2.?We examined imatinib N-demethylation in human liver microsomes (HLMs) genotyped for CYP2C8*3, in CYP2C8*3/*3 pooled HLMs and in recombinant CYP2C8 and CYP3A4 enzymes. Effects of CYP-selective inhibitors on N-demethylation were also determined.

3.?A single-enzyme Michaelis–Menten model with autoinhibition best fitted CYP2C8*1/*1 HLM (n?=?5) and recombinant CYP2C8 kinetic data (median?±?SD K_i?=?139?±?61?µM and 149?µM, respectively). Recombinant CYP3A4 showed two-site enzyme kinetics with no autoinhibition. Three of four CYP2C8*1/*3 HLMs showed single-enzyme kinetics with no autoinhibition. Binding affinity was higher in CYP2C8*1/*3 than CYP2C8*1/*1 HLM (median?±?SD K_m?=?6?±?2 versus 11?±?2?µM, P=0.04). CYP2C8*3/*3 (pooled HLM) also showed high binding affinity (K_m?=?4?µM) and single-enzyme weak autoinhibition (K_i?=?449?µM) kinetics. CYP2C8 inhibitors reduced HLM N-demethylation by 47–75%, compared to 0–30% for CYP3A4 inhibitors.

4.?In conclusion, CYP2C8*3 is a gain-of-function polymorphism for imatinib N-demethylation, which appears to be mainly mediated by CYP2C8 and not CYP3A4 in vitro in HLM.     

Characterization of human cytochrome P450 enzymes catalyzing domperidone N-dealkylation and hydroxylation in vitro

AIMS: To confirm the identity of the major metabolites of domperidone and to characterize the cytochrome P450s (CYPs) involved in their formation. METHODS: Human liver microsomes (HLMs) were used to characterize the kinetics of domperidone metabolism and liquid chromatography-mass spectrometry to identify the products. Isoform-specific chemical inhibitors, correlation analysis and expressed human CYP genes were used to identify the CYPs involved in domperidone oxidation. RESULTS: In HLMs, domperidone underwent hydroxylation to form 5-hydroxydomperidone (MIII) and N-dealkylation to form 2,3-dihydro-2-oxo-1H-benzimidazole-1-propionic acid (MI) and 5-chloro-4-piperidinyl-1,3-dihydro-benzimidazol-2-one (MII). The formation of all three metabolites (n = 4 HLMs) followed apparent Michaelis-Menten kinetics. The mean Km values for MI, MII and MIII formation were 12.4, 11.9, and 12.6 micro m, respectively. In a panel of HLMs (n = 10), the rate of domperidone (5 microm and 50 microm) metabolism correlated with the activity of CYP3A (r > 0.94; P < 0.0001). Only ketoconazole (1 microm) (by 87%) and troleandomycin (50 microm) (by 64%) inhibited domperidone (5 microm) metabolism in HLMs. Domperidone (5 and 50 microm) hydroxylation and N-dealkylation was catalyzed by expressed CYP3A4 at a higher rate than the other CYPs. CYP1A2, 2B6, 2C8 and 2D6 also hydroxylated domperidone CONCLUSIONS: CYP3A-catalyzed N-dealkylation and aromatic hydroxylation are the major routes for domperidone metabolism. The drug would be expected to demonstrate highly variable bioavailability due to hepatic, and possibly intestinal first-pass metabolism after oral administration. Increased risk of adverse effects might be anticipated during concomitant administration with CYP3A inhibitors, as well as decreased efficacy with inducers of this enzyme.     

Identification of the human liver cytochrome P450 isoenzymes responsible for the 5-methylhydroxylation of the novel anti-fibrotic drug AKF-PD

1. Identification of cytochrome P450 isoforms (CYPs) involved in flourofenidone (5-methyl-1-(3-fluorophenyl)-2-[1H]-pyridone, AKF-PD) 5-methylhydroxylation was carried out using human liver microsomes and cDNA-expressed human CYPs (supersomes). The experiments were performed in the following in vitro models: (A) a study of AKF-PD metabolism in liver microsomes: (a) correlations study between the rate of AKF-PD 5-methylhydroxylation and activity of CYPs; (b) the effect of specific CYPs inhibitors on the rate of AKF-PD 5-methylhydroxylation; (B) AKF-PD biotransformation by cDNA-expressed human CYPs (1A2, 2D6, 2C9, 2C19, 2E1, 3A4).

2. In human liver microsomes, the formation of AKF-PD 5-methylhydroxylation metabolite significantly correlated with the caffeine N3-demethylase (CYP1A2), chlorzoxazone 6-hydroxylase (CYP2E1), midazolam 1’- hydroxylase (CYP3A4), tolbutamide 4-hydroxylase (CYP2C9), and debrisoquin 4-hydroxylase (CYP2D6) activities. The production of AKF-PD 5-methylhydroxylation metabolite was completely inhibited by a-naphthoflavone (a CYP1A2 inhibitor) with the IC50 value of 0.12 μM in human liver microsomes. The cDNA-expressed human CYPs generated different amounts of AKF-PD 5-methylhydroxylation metabolites, but the preference of CYP isoforms to catalyze AKF-PD metabolism was as follows: 2D6?>?2C19?>?1A2?>?2E1?>?2C9?>?3A4.

3. The results demonstrated that CYP1A2 is the main isoform catalyzing AKF-PD 5-methylhydroxylation while CYP3A4, CYP2C9, CYP2E1, CYP2C19, and CYP2D6 are engaged to a lesser degree. Potential drug–drug interactions involving CYP1A2 may be noticed when AKF-PD is used combined with CYP1A2 inducers or inhibitors.

     

Stereo‐Selective Metabolism of Methadone by Human Liver Microsomes and cDNA‐Expressed Cytochrome P450s: A Reconciliation

Abstract: In vitro metabolism of methadone was investigated in cytochrome P450 (CYP) supersomes and phenotyped human liver microsomes (HLMs) to reconcile past findings on CYP involvement in stereo‐selective metabolism of methadone. Racaemic methadone was used for incubations; (R)‐ and (S)‐methadone turnover and (R)‐ and (S)‐EDDP formation were determined using chiral liquid chromatography–tandem mass spectrometry. CYP supersome activity for methadone use and EDDP formation ranked CYP2B6 > 3A4 > 2C19 > 2D6 > 2C18, 3A7 > 2C8, 2C9, 3A5. After abundance scaling, CYP3A4, 2B6 and 2C19 accounted for 63–74, 12–32 and 1. 4–14% of respective activity. CYP2B6, 2D6 and 2C18 demonstrated a preference for (S)‐EDDP formation; CYP2C19, 3A7 and 2C8 for (R)‐EDDP; 3A4 none. Correlation analysis with 15 HLMs supported the involvement of CYP2B6 and 3A. The significant correlation of S/R ratio with CYP2B6 activity confirmed its stereo‐selectivity. CYP2C19 and 2D6 inhibitors and monoclonal antibody (mAb) did not inhibit EDDP formation in HLM. Chemical and mAb inhibition of CYP3A in high 3A activity HLM reduced EDDP formation by 60–85%; inhibition of CYP2B6 in 2B6 high‐activity HLM reduced (S)‐EDDP formation by 80% and (R)‐EDDP formation by 55%. Inhibition changed methadone metabolism in a stereo‐selective manner. When CYP3A was inhibited, 2B6 mediated (S)‐EDDP formation predominated; S/R stereo‐selectivity increased. When 2B6 was inhibited (S)‐EDDP formation fell and stereo‐selectivity decreased. The results confirmed the primary roles of CYPs 3A4 and 2B6 in methadone metabolism; CYP2C8 and 2C9 did not appear involved; 2C19 and 2D6 have minimal roles. CYP2B6 is the primary determinant of stereo‐selective metabolism; stereo‐selective inhibition might play a role in varied plasma concentrations of the two enantiomers.     

Effect of sodium tanshinone II A sulfonate on the activity of CYP1A2 in healthy volunteers

1. Sodium tanshinone IIA sulfonate (STS) is a water-soluble derivative of tanshinone IIA, a famous Chinese medicine which has been used in the treatment of cardiovascular disorders for many years. Using caffeine as a probe drug, this project was designed to investigate the effect of STS on the activity of CYP1A2 in humans.

2. Sixteen unrelated healthy volunteers were recruited for this two-phase, randomized and crossover study. The volunteers received either placebo or 60?mg day^?1 of STS injections through vein for 13 days. Pharmacokinetics of caffeine and the metabolite paraxanthine was determined by high-performance liquid chromatography. CYP1A2 activity was monitored by the ratio of paraxanthine to caffeine at 6?h in plasma.

3. Enzyme activity analysis showed that STS significantly increased the activity of CYP1A2 by 41.1% [90% confidence interval (CI), 17.4–64.8%] (p = 0.036). The area under the curve [AUC_(0–24h)] of caffeine significantly decreased by 13.3% [90% CI = 7.0–19.6%] (p = 0.005) with 13 days of treatment of STS. AUC_(0–24h) of paraxanthine significantly increased by 17.4% [90% CI = 4.3–30.5%] (p = 0.035). No significant difference was found for other parameters of caffeine and paraxanthine between two phases.

4. STS has significantly induced the activity of CYP1A2 in vivo. Simultaneously, AUC_(0–24h) of caffeine and paraxanthine were significantly affected by STS. The findings have provided some useful information for safe and effective usage of STS in clinic.

     

In vitro characterization of 4′-(p-toluenesulfonylamide)-4-hydroxychalcone using human liver microsomes and recombinant cytochrome P450s

1.?4′-(p-Toluenesulfonylamide)-4-hydroxychalcone (TSAHC) is a synthetic sulfonylamino chalcone compound possessing anti-cancer properties. The aim of this study was to elucidate the metabolism of TSAHC in human liver microsomes (HLMs) and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of TSAHC.

2.?TSAHC was incubated with HLMs or recombinant P450 isoforms (rP450) in the presence of an nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-regenerating system. The metabolites were identified and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). P450 isoforms, responsible for TSAHC metabolite formation, were characterized by chemical inhibition and correlation studies in HLMs and enzyme kinetic studies with a panel of rP450 isoforms.

3.?Two hydroxyl metabolites, that is M1 and M2, were produced from the human liver microsomal incubations (K_m and V_max values were 2.46?µM and 85.1?pmol/min/mg protein for M1 and 9.98?µM and 32.1?pmol/min/mg protein for M2, respectively). The specific P450 isoforms responsible for two hydroxy-TSAHC formations were identified using a combination of chemical inhibition, correlation analysis and metabolism by expressed recombinant P450 isoforms. The known P450 enzyme activities and the rate of TSAHC metabolite formation in the 15 HLMs showed that TSAHC metabolism is correlated with CYP2C and CYP3A activity. The P450 isoform-selective inhibition study in HLMs and the incubation study of cDNA-expressed enzymes also showed that two hydroxyl metabolites M1 and M2 biotransformed from TSAHC are mainly mediated by CYP2C and CYP3A, respectively. These findings suggest that CYP2C8, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 isoforms are major enzymes contributing to TSAHC metabolism.     

Identification of human cytochrome P450 enzymes involved in the metabolism of IN-1130, a novel activin receptor-like kinase-5 (ALK5) inhibitor

1.?The in vitro metabolism of 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide (IN-1130), a selective activin receptor-like kinase-5 (ALK5) inhibitor and a candidate drug for fibrotic disease, was studied.

2.?The cytochrome P450s (CYPs) responsible for metabolism of IN-1130 in liver microsomes of rat, mouse, dog, monkey and human, and in human CYP supersomes?, were identified using specific CYP inhibitors. The order of disappearance of IN-1130 in various liver microsomal systems studied was as follows: monkey, mouse, rat, human, and dog.

3.?Five distinct metabolites (M1–M5) were identified in all the above microsomes and their production was substantially inhibited by CYP inhibitors such as SKF-525A and ketoconazole. Among nine human CYP supersomes? examined, CYP3A4, CYP2C8, CYP2D6*1, and CYP2C19 were involved in the metabolism of IN-1130, and the production of metabolites were significantly inhibited by specific CYP inhibitors. IN-1130 disappeared fastest in CYP2C8 supersomes. CYP3A4 produced four metabolites of IN-1130 (M1–M4), whereas supersomes expressing human FMO cDNAs, such as FMO1, FMO3, and FMO5, produced no metabolites.

4.?Hence, it is concluded that metabolism of IN-1130 is mediated by CYP3A4, CYP2C8, CYP2D6*1, and CYP2C19.     

Cynomolgus monkey CYPs: a comparison with human CYPs

1. Characteristics of twelve cytochromes P450 (CYPs) from cynomolgus monkeys were compared with those of human CYPs that play an important role in drug metabolism.

2. Eleven members of CYP1A, CYP2A, CYP2C, CYP2D, CYP2E, and CYP3A subfamilies from cynomolgus monkeys exhibited a high degree of homologies (more than 90%) in cDNA and amino acid sequences with corresponding human CYPs, and catalysed typical reactions of corresponding human CYPs.

3. One member of the cynomolgus monkey CYP2C subfamily, CYP2C76, exhibited a lower homology (around 70%) in amino acid sequences with other cynomolgus monkey and human CYP2C subfamilies. CYP2C76 catalysed typical CYP2C substrates with low activities, and has not been found in humans.

4. CYPs identified in cynomolgus monkeys were similar to CYP1A1, CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 in humans.

5. These results indicate that cynomolgus monkeys express CYPs similar to human CYPs that are important in drug metabolism.

     

Direct and metabolism‐dependent cytochrome P450 inhibition assays for evaluating drug–drug interactions

We developed methods for evaluating the ntial inhibition of human cytochrome P450 (CYP) enzymes, including CYP1A2, CYP2A6, CYP2B6, CYP2 C9, CYP2 C19, CYP2D6, CYP2E1 and CYP3A4, using pooled human liver microsomes (HLMs). The CYP inhibition assay used substrate cocktail sets [set A: phenacetin for CYP1A2, coumarin for CYP2A6, (S)‐(+)‐mephenytoin for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4; set B: bupropion for CYP2B6, tolbutamide for CYP2C9, chlorzoxazone for CYP2E1, and testosterone for CYP3A4] with quantitation by liquid chromatography–tandem mass spectrometry. A direct inhibition assay was performed with the substrate cocktails without β‐nicotinamide adenine dinucleotide phosphate (NADPH) pre‐incubation, and a metabolism‐dependent inhibition (MDI) assay was performed after 30 min of pre‐incubation with NADPH in HLMs. MDI was identified based on the half‐maximal inhibitory concentration (IC₅₀) shifts. The IC₅₀ values of the direct inhibitors determined using the probe substrate cocktails were in good agreement with previously reported values. Eight metabolism‐dependent inhibitors including furafylline, 8‐methoxypsoralen, tienilic acid, ticlopidine, fluoxetine, paroxetine, disulfiram and verapamil against CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, respectively, resulted in significant IC₅₀ shifts (≥2.5‐fold) after pre‐incubation. Thus, these CYP inhibition assays are considered to be useful tools for evaluating both direct inhibition and MDI at an early stage of the drug discovery and development process. Copyright © 2011 John Wiley & Sons, Ltd.     

Characterization of in vitro metabolites of cudratricusxanthone A in human liver microsomes

Cudratricusxanthone A (CTXA), isolated from the roots of Cudrania tricuspidata, exhibits several biological activities; however, metabolic biotransformation was not investigated. Therefore, metabolites of CTXA were investigated and the major metabolic enzymes engaged in human liver microsomes (HLMs) were characterized using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). CTXA was incubated with HLMs or human recombinant CYPs and UGTs, and analysed by an LC‐MS/MS equipped electrospray ionization (ESI) to qualify and quantify its metabolites. In total, eight metabolites were identified: M1–M4 were identified as mono‐hydroxylated metabolites during Phase I, and M5–M8 were identified as O‐glucuronidated metabolites during Phase II in HLMs. Moreover, these metabolite structures and a metabolic pathway were identified by elucidation of MSⁿ fragments and formation by human recombinant enzymes. M1 was formed by CYP2D6, and M2–M4 were generated by CYP1A2 and CYP3A4. M5–M8 were mainly formed by UGT1A1, respectively. While investigating the biotransformation of CTXA, eight metabolites of CTXA were identified by CYPs and UGTs; these data will be valuable for understanding the in vivo metabolism of CTXA. Copyright © 2015 John Wiley & Sons, Ltd.     

Investigation of selective inhibitory effects of glycyrol on human CYP 1A1 and 2C9

1.?Glycyrol is a coumarin derivative isolated from the roots of Glycyrrhiza uralensis called Gamcho in Korea and commonly used as a sweetener in oriental medicine. Glycyrol shows several biological activities, including anti-oxidative, anti-inflammatory, antibacterial, anti-angiogenic, and anti-allergenic properties. Although there have been studies on the biological effects of glycyrol, the inhibitory effects of glycyrol on cytochrome P450 (CYP) activities have not been investigated.

2.?We investigated the inhibitory effects of glycyrol on the activities of CYP isoforms using a cocktail of probe substrates in pooled human liver microsome (HLM) and human recombinant cDNA-expressed CYPs. Glycyrol strongly inhibited CYP1A-mediated phenacetin O-deethylation and CYP2C9-mediated diclofenac 4′-hydroxylation in HLMs, which were the result of competitive inhibition as revealed by a Dixon plot. In addition, glycyrol showed selective inhibition of CYP1A1- and CYP1A2-catalyzed phenacetin O-deethylase activity with a half-maximal inhibitory concentration of (IC₅₀) 1.3 and 16.1?μM in human recombinant cDNA-expressed CYP1A1 and CYP1A2, respectively.

3.?Glycyrol decreased CYP2C9-catalyzed diclofenac 4′-hydroxylation activity with IC₅₀ values of 0.67?μM in human recombinant cDNA-expressed CYP2C9. This is the first investigation of competitive inhibitory effects on CYP1A1 and CYP2C9 in HLMs.     

Metoclopramide is metabolized by CYP2D6 and is a reversible inhibitor,but not inactivator,of CYP2D6

Abstract

1. Metoclopramide is a widely used clinical drug in a variety of medical settings with rare acute dystonic events reported. The aim of this study was to assess a previous report of inactivation of CYP2D6 by metoclopramide, to determine the contribution of various CYPs to metoclopramide metabolism, and to identify the mono-oxygenated products of metoclopramide metabolism.

2. Metoclopramide interacted with CYP2D6 with Type I binding and a K_s value of 9.56?±?1.09?µM. CYP2D6 was the major metabolizer of metoclopramide and the two major products were N-deethylation of the diethyl amine and N-hydroxylation on the phenyl ring amine. CYPs 1A2, 2C9, 2C19, and 3A4 also metabolized metoclopramide.

3. While reversible inhibition of CYP2D6 was noted, CYP2D6 inactivation by metoclopramide was not observed under conditions of varying concentration or varying time using Supersomes^TM or pooled human liver microsomes.

4. The major metabolites of metoclopramide were N-hydroxylation and N-deethylation formed most efficiently by CYP2D6 but also formed by all CYPs examined. Also, while metoclopramide is metabolized primarily by CYP2D6, it is not a mechanism-based inactivator of CYP2D6 in vitro.     

CYP2D plays a major role in berberine metabolism in liver of mice and humans

1. Berberine is a widely used plant extract for gastrointestinal infections, and is reported to have potential benefits in treatment for diabetes and hypercholesterolemia. It has been suggested that interactions between berberine-containing products and cytochromes P450 (CYPs) exist, but little is known about which CYPs mediate the metabolism of berberine in vivo.

2. In this study, berberine metabolites in urine and feces of mice were analyzed, and the role that CYPs play in producing these metabolites were characterized in liver microsomes from mice (MLM) and humans (HLM), as well as recombinant human CYPs. Eleven berberine metabolites were identified in mice, including 5 unconjugated metabolites, mainly in feces, and 6 glucuronide and sulfate conjugates, predominantly in urine. Three novel berberine metabolites were observed. Three unconjugated metabolites of berberine were produced by MLM, HLM, and recombinant human CYPs. CYP2D6 was the primary recombinant human CYP producing these metabolites, followed by CYP1A2, 3A4, 2E1 and CYP2C19. The metabolism of berberine in MLM and HLM was decreased the most by a CYP2D inhibitor, and moderately by inhibitors of CYP1A and 3A.

3. CYP2D plays a major role in berberine biotransformation, therefore, CYP2D6 pharmacogenetics and potential drug-drug interactions should be considered when berberine is used.

     

In vitro metabolism of nobiletin,a polymethoxy-flavonoid,by human liver microsomes and cytochrome P450

1. Cytochrome P450 enzymes (CYPs) in the liver metabolize drugs prior to excretion, with different enzymes acting at different molecular motifs. At present, the human CYPs responsible for the metabolism of the flavonoid, nobiletin (NBL), are unidentified. We investigated which enzymes were involved using human liver microsomes and 12 cDNA-expressed human CYPs.

2. Human liver microsomes metabolized NBL to three mono-demethylated metabolites (4′-OH-, 7-OH- and 6-OH-NBL) with a relative ratio of 1:4.1:0.5, respectively, by aerobic incubation with nicotinamide adenine dinucleotide phosphate (NADPH). Of 12 human CYPs, CYP1A1, CYP1A2 and CYP1B1 showed high activity for the formation of 4′-OH-NBL. CYP3A4 catalyzed the formation of 7-OH-NBL with the highest activity and of 6-OH-NBL with lower activity. CYP3A5 also catalyzed the formation of both metabolites but considerably more slowly than CYP3A4. In contrast, seven CYPs (CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1) were inactive for NBL.

3. Both ketoconazole and troleandomycin (CYP3A inhibitors) almost completely inhibited the formation of 7-OH- and 6-OH-NBL. Similarly, α-naphthoflavone (CYP1A1 inhibitor) and furafylline (CYP1A2 inhibitor) significantly decreased the formation of 4′-OH-NBL.

4. These results suggest that CYP1A2 and CYP3A4 are the key enzymes in human liver mediating the oxidative demethylation of NBL in the B-ring and A-ring, respectively.

     

Regulation of human pregnane X receptor and its target gene cytochrome P450 3A4 by Chinese herbal compounds and a molecular docking study

1. The pregnane X receptor (PXR) plays a critical role in the regulation of human cytochrome P450 3A4 (CYP3A4) gene. In this study, we investigated the effect of an array of compounds isolated from Chinese herbal medicines on the activity of PXR using a luciferase reporter gene assay in transiently transfected HepG2 and Huh7 cells and on the expression of PXR and CYP3A4 in LS174T cells. Furthermore, molecular docking was performed to investigate the binding modes of herbal compounds with PXR.

2. Praeruptorin A and C, salvianolic acid B, sodium danshensu, protocatechuic aldehyde, cryptotanshinone, emodin, morin, and tanshinone IIA significantly transactivated the CYP3A4 reporter gene construct in either HepG2 or Huh7 cells. The PXR mRNA expression in LS174T cells was significantly induced by physcion, protocatechuic aldehyde, salvianolic acid B, and sodium danshensu. However, epifriedelanol, morin, praeruptorin D, mulberroside A, tanshinone I, and tanshinone IIA significantly down-regulated the expression of PXR mRNA in LS174T cells.

3. All the herbal compounds tested can be readily docked into the ligand-binding cavity of PXR mainly through hydrogen bond and aromatic interactions with Ser247, Gln285, His407, and Arg401.

4. These findings suggest that herbal medicines can significantly regulate PXR and CYP3A4 and this has important implication in herb–drug interactions.