Inhibition of cytochrome P450 and uridine 5′-diphospho-glucuronosyltransferases by MAM-2201 in human liver microsomes

MAM-2201, a synthetic cannabinoid, is a potent agonist of the cannabinoid receptors and is increasingly used as an illicit recreational drug. The inhibitory effects of MAM-2201 on major drug-metabolizing enzymes such as cytochrome P450s (CYPs) and uridine 5′-diphospho-glucuronosyltransferases (UGTs) have not yet been investigated although it is widely abused, sometimes in combination with other drugs. We evaluated the inhibitory effects of MAM-2201 on eight major human CYPs (CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and six UGTs (UGTs 1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) of pooled human liver microsomes; we thus explored potential MAM-2201-induced drug interactions. MAM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4′-hydroxylation, CYP3A4-catalyzed midazolam 1′-hydroxylation, and UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, with K _i values of 5.6, 5.4 and 5.0 µM, respectively. MAM-2201 exhibited mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-de-ethylation with K _i and k _inact values of 1.0 µM and 0.0738 min^?1, respectively. In human liver microsomes, MAM-2201 (50 µM) negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7. Based on these in vitro results, we conclude that MAM-2201 has the potential to trigger in vivo pharmacokinetic drug interactions when co-administered with substrates of CYP2C8, CYP2C9, CYP3A4, and UGT1A3.     

Comparison between recombinant P450s and human liver microsomes in the determination of cytochrome P450 Michaelis–Menten constants

1. Non-linear dose–exposure (supra-proportionality) occurs when plasma drug concentrations increase in a non-linear fashion with increasing dose. To predict the likelihood of this, an understanding is required of the K_M, which reflects a drug ability to saturate a specific enzyme involved in its metabolism.

2. This study assessed the accuracy of K_M and V_max determinations for compounds using a substrate-depletion approach with those determined using the product-formation approach, using both recombinant human cytochrome P450 (CYP) enzymes and human liver microsomes.

3. For the vast majority of the compounds studied, the K_M’s using recombinant CYPs and human liver microsomes in the two approaches predicted within two-fold. Further comparisons between the K_M and V_max-values were made between those measured using the product-formation approach and those estimated following simultaneous fitting of the Michaelis–Menten equation to all substrate depletion plots. In each case values were comparable.

4. In conclusion, the current study showed the substrate-depletion approach can be used to estimate K_M and V_max using both human liver microsomes and recombinant P450s. Estimation of these parameters during early discovery will aid in the understanding of dosages at which non-linearity may occur, but potentially aid predictions of likely clinical drug–drug interactions.

     

Drug–drug interactions in the metabolism of imidafenacin: Role of the human cytochrome P450 enzymes and UDP-glucuronic acid transferases,and potential of imidafenacin to inhibit human cytochrome P450 enzymes

Imidafenacin (IM), 4-(2-methyl-1H-imidazol-1-yl)-2,2-diphenylbutanamide, is a newly synthesized antimuscarinic drug developed for the treatment of overactive bladder. To predict clinically relevant drug interactions in the metabolism of IM, the paper investigated: (1) the major enzymes responsible for the metabolism of IM, (2) the effects of concomitant drugs on the inhibition of metabolism of IM, and (3) the effects of IM and its metabolites on the inhibition of human cytochrome P450 (CYP). The elimination of IM and production of oxidative metabolites were mainly catalysed by recombinant CYP3A4, and the elimination of IM by human liver microsomes (HLM) was markedly inhibited by co-incubation with ketoconazole. The production of the N-glucuronide metabolite was only catalysed by recombinant UGT1A4. Clinically established CYP3A4 inhibitors including itraconazole, ketoconazole, erythromycin and clarithromycin inhibited the elimination of IM in HLM. IM and its major metabolites did not affect the activities of CYP enzymes in vitro. The results suggest that the major enzymes responsible for the metabolism of IM are CYP3A4 and UGT1A4, and oxidative metabolism of IM is reduced by concomitant administration of CYP3A4 inhibitors. In contrast, IM and its metabolites have no inhibitory effect on the CYP-mediated metabolism of concomitant drugs.     

Determination of cytochrome P450 enzymes involved in the metabolism of (−)-terpinen-4-ol by human liver microsomes

1. The in vitro metabolism of (?)-terpinen-4-ol was examined in human liver microsomes and recombinant enzymes.

2. The biotransformation of (?)-terpinen-4-ol was investigated by gas chromatography–mass spectrometry. (?)-Terpinen-4-ol was found to be oxidized to (?)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol, major metabolic product by human liver microsomal P450 enzymes. The formation of metabolites of (?)-terpinen-4-ol was determined by relative abundance of mass fragments and retention times on GC.

3. CYP2A6 in human liver microsomes was a major enzyme involved in the oxidation of (?)-terpinen-4-ol by human liver microsomes, based on the following lines of evidence. First, of 11 recombinant human P450 enzymes tested, CYP2A6 had the highest activity for oxidation of (?)-terpinen-4-ol. Second, oxidation of (?)-terpinen-4-ol was inhibited by (+)-menthofuran. Finally, there was a good correlation between CYP2A6 maker activity and (?)-terpinen-4-ol oxidation activities in liver microsomes of 10 human samples.

4. Kinetic analysis showed that the V_max/K_m values for (?)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol catalysed by liver microsomes of human sample HH-18 was 2.49 μL/min/nmol.

5. Human recombinant CYP2A6 catalysed (?)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol with V_max values of 13.9 nmol/min/nmol P450 and apparent K_m values of 91 μM.

     

Enzyme kinetics of imperatorin and its cytochrome P450 phenotyping in liver microsomes北大核心CSCD

OBJECTIVE: To investigate enzyme kinetic characteristics of imperatorin in rat liver microsomes (RLM) or human liver microsomes (HLM), and to identify the reaction phenotyping of human recombinant cytochrome P450 enzyme (CYP) mediated phase I metabolism. METHODS: Imperatorin was incubated at 37°C with HLM or RLM in the presence or absence of nicotinamide adenine dinucleotide phosphate (NADPH) or uridine 5'-diphosphoglucuronic acid (UDGPA). The concentrations of imperatorin in the incubation systems were determined with LC-MS/MS to evaluate its metabolic stability and enzymatic kinetics. The CYP phenotyping of imperatorin was identified using a panel of human recombinant CYP isoforms (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) and also using a group of specific inhibitors in HLM. RESULTS: Imperatorin was metabolically eliminated in the presence of NADPH in HLM or RLM. The elimination rates for HLM and RLM in 30 min were 69.7% and 94.5%, respectively, and elimination half-life (t1/2) values were 18.9±0.6 and (2.8±0.4)min, respectively. The extrapolated hepatic clearance parameters (Clh) were 16.9±0.1 and (51.9±0.4)mL·min-1-kg-1. The Michaelism-Menten parameters (Km) were 13.60±0.16 and (14.00± 0.24)umol·L-1 and maximum velocity (Vmax) were (2928±96) and (8434±27)nmol·min-1·g-1, respectively. The metabolic elimination of imperatorin in RLM was quicker than in HLM. The results of CYP phenotyping indicated that CYP1A2, CYP2B6, CYP2C19 and CYP3A4 were the major CYP isoforms involved in the imperatorin metabolism. Their individual contributions assessed using the method of total normalized rate were 20.4%, 7.3%,10.5% and 61.8%, respectively. CONCLUSION: Imperatorin is mainly eliminated by CYP mediated metabolism in HLM and RLM. CYP1A2 and CYP3A4 are the major responsible enzymes with a contribution rate above 20%.     

Identification of cytochrome P450 enzymes involved in the metabolism of 3′,4′-methylenedioxy-α-pyrrolidinopropiophenone (MDPPP), a designer drug,in human liver microsomes

The metabolism of 3′,4′-methylenedioxy-α-pyrrolidinopropiophenone (MDPPP), a novel designer drug, to its demethylenated major metabolite 3′,4′-dihydroxy-pyrrolidinopropiophenone (di-HO-PPP) was studied in pooled human liver microsomes (HLM) and in cDNA-expressed human hepatic cytochrome P450 (CYP) enzymes. CYP2C19 catalysed the demethylenation with apparent K_m and V_max values of 120.0?±?13.4?µM and 3.2?±?0.1?pmol/min/pmol?CYP, respectively (mean?±?standard deviation). CYP2D6 catalysed the demethylenation with apparent K_m and V_max values of 13.5?±?1.5?µM and 1.3?±?0.1 pmol/min/pmol?CYP, respectively. HLM exhibited a clear biphasic profile with an apparent K_m,1 value of 7.6?±?9.0 and a V_max,1 value of 11.1?±?3.6?pmol/min/mg?protein, respectively. Percentages of intrinsic clearances of MDPPP by specific CYPs were calculated using the relative activity factor (RAF) approach with (S)-mephenytoin-4′-hydroxylation or bufuralol-1′-hydroxylation as index reactions for CYP2C19 or CYP2D6, respectively. MDPPP, di-HO-PPP and the standard 4′-methyl-pyrrolidinohexanophenone (MPHP) were separated and analysed by liquid chromatography-mass spectrometry in the selected-ion monitoring (SIM) mode. The CYP2D6-specific chemical inhibitor quinidine (3?µM) significantly (p?相似文献   

Mechanism-based inactivation of human cytochrome P450 enzymes: strategies for diagnosis and drug–drug interaction risk assessment

Among drugs that cause pharmacokinetic drug–drug interactions, mechanism-based inactivators of cytochrome P450 represent several of those agents that cause interactions of the greatest magnitude. In vitro inactivation kinetic data can be used to predict the potential for new drugs to cause drug interactions in the clinic. However, several factors exist, each with its own uncertainty, that must be taken into account in order to predict the magnitude of interactions reliably. These include aspects of in vitro experimental design, an understanding of relevant in vivo concentrations of the inactivator, and the extent to which the inactivated enzyme is involved in the clearance of the affected drug. Additionally, the rate of enzyme degradation in vivo is also an important factor that needs to be considered in the prediction of the drug interaction magnitudes. To address mechanism-based inactivation for new drugs, various in vitro experimental approaches have been employed. The selection of approaches for in vitro kinetic characterization of inactivation as well as in vitro–in vivo extrapolation should be guided by the purpose of the exercise and the stage of drug discovery and development, with an increase in the level of sophistication throughout the research and development process.     

Inhibition of human cytochrome P450 enzymes by 1,4-dihydropyridine calcium antagonists: prediction of in vivo drug–drug interactions

OBJECTIVE: 1,4-Dihydropyridine calcium antagonists such as nifedipine are potent vasodilators. It is now commonly agreed that the oxidation of 1,4-dihydropyridine into pyridine, which is one of the main metabolic pathways, is catalysed by the cytochrome P450 (CYP) 3A4 isoform. In the present study, the inhibitory effects of 13 kinds of 1,4-dihydropyridine calcium antagonists clinically used in Japan on human CYP-isoform-dependent reactions were investigated to predict the drug interactions using microsomes from human B-lymphoblast cells expressing CYP. RESULTS: The specific activities for human CYP isoforms included 7-ethoxyresorfin O-deethylation (CYP1A1), phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), 7-benzyloxyresorufin O-dealkylation (CYP2B6), S-warfarin 7-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylaion (CYP2C19), bufuralol 1'-hydroxylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), and testosterone 6beta-hydroxylation (CYP3A4). Benidipine and amlodipine competitively inhibited the CYP1A1 activity. Nifedipine, nisoldipine and aranidipine competitively inhibited the CYP1A2 activity. No 1,4-dihydropyridie calcium antagonists used in this study inhibited the CYP2A6 activity. Barnidipine and amlodipine inhibited the CYP2B6 activity. Nicardipine, benidipine, manidipine and barnidipine competitively inhibited the CYP2C9 and CYP2D6 activities. Inhibition extent of the CYP2E1 activity by nifedipine and aranidipine were weak. Nicardipine, benidipine and barnidipine inhibited the CYP2C19 and CYP3A4 activities. Among the human CYP isoforms investigated, the inhibitory effects of 1,4-dihydropyridine calcium antagonists were potent on human CYP1A2, CYP2B6, CYP2C9, CYP2C19 and CYP2D6 as well as CYP3A4. Furthermore, the isoform selectivity of inhibition by 1,4-dihydropyridine calcium antagonists was clarified. CONCLUSIONS: In consideration of the Ki values obtained in the in vitro inhibition study and the concentration of 1,4-dihydropyridine calcium antagonists in human liver, the possibility of in vivo drug interactions of nicardipine and other drugs which are mainly metabolised by CYP2C9 and/or CYP3A4 was suggested. The inhibition of human CYP isoforms by 1,4-dihydropyridine calcium antagonists except nicardipine might be clinically insignificant.     

Identification of cytochrome P450 enzymes involved in the metabolism of 4′-methoxy-α-pyrrolidinopropiophenone (MOPPP), a designer drug,in human liver microsomes

1. The metabolism of 4′-methoxy-α-pyrrolidinopropiophenone (MOPPP), a novel designer drug, to its demethylated major metabolite 4′-hydroxy-pyrrolidinopropio-phenone (HO-PPP) was studied in pooled human liver microsomes (HLM) and in cDNA-expressed human hepatic cytochrome P450 (CYP) enzymes.

2. CYP2C19 catalysed the demethylation with apparent K_m and V_max values of 373.4 ± 45.1?μM and 6.0 ± 0.3?pmol?min^?1?pmol^?1 CYP, respectively (mean ± SD). Both CYP2D6 and HLM exhibited clear biphasic profiles with apparent K_m,1 values of 1.3 ± 0.4 and 22.0 ± 6.5?μM, respectively, and V_max,1 values of 1.1 ± 0.1 pmol?min^?1?pmol^?1 CYP and 169.1 ± 20.5?pmol?min^?1?mg^?1 protein, respectively.

3. Percentages of intrinsic clearances of MOPPP by particular CYPs were calculated using the relative activity factor (RAF) approach with (S)-mephenytoin-4′-hydroxylation or bufuralol-1′-hydroxylation as index reactions for CYP2C19 or CYP2D6, respectively.

4. MOPPP, HO-PPP and the standard 3′,4′-methylenedioxy-pyrrolidinopropio-phenone (MDPPP) were separated and analysed by liquid chromatography–mass spectrometry in the selected-ion monitoring (SIM) mode.

5. The CYP2D6 specific chemical inhibitor quinidine (3?μM) significantly (?p<0.0001) inhibited HO-PPP formation by 91.8 ± 0.5% (mean ± SEM) in incubation mixtures with HLM and 2?μM MOPPP.

6. It can be concluded from the data obtained from kinetic and inhibition studies that polymorphically expressed CYP2D6 is the enzyme mainly responsible for MOPPP demethylation.     

In vitro drug–drug interactions of budesonide: inhibition and induction of transporters and cytochrome P450 enzymes

1.?Budesonide is a glucocorticoid used in the treatment of several respiratory and gastrointestinal inflammatory diseases. Glucocorticoids have been demonstrated to induce cytochrome P450 (CYP) 3A and the efflux transporter P-glycoprotein (P-gp). This study aimed to evaluate the potential of budesonide to act as a perpetrator or a victim of transporter- or CYP-mediated drug–drug interactions (DDIs).

2.?In vitro studies were conducted for P-gp, breast cancer resistance protein and organic anion and cation transporters (OATP1B1, OATP1B3, OAT1, OAT3, OCT2) in transporter-transfected cells. Changes in mRNA expression in human hepatocytes and enzyme activity in human liver microsomes by budesonide were determined for CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A.

3.?The data indicated that budesonide is a substrate of P-gp but is not a substrate or an inhibitor of the other transporters investigated. Budesonide is neither an inducer nor an inhibitor of major CYP enzymes. The effect of P-gp on budesonide disposition is anticipated to be low owing to CYP3A-mediated clearance.

4.?Collectively, our data indicate there is a low risk of budesonide perpetrating clinical DDIs mediated by the transporters or CYPs studied.     

Metabolic activation of 1 α-hydroxyvitamin D 3 in human liver microsomes

1. 1α -Hydroxyvitamin D 3 (1 α-OH-D 3) is a synthetic prodrug of the active form of vitamin D 3, and requires the hydroxylation at the C-25 position before eliciting its biological activity. 2. 25-Hydroxylation activities for 1 α-OH-D 3 were present in both microsomal and mitochondrial fractions of human liver. 3. To determine the P450 enzyme(s) involved in microsomal 25-hydroxylation, 14 P450s (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9-Arg, 2C9-Cys, 2C19, 2D6-Val, 2D6- Met, 2E1, 3A4, 4A11) were tested for their 25-hydroxylation activity of 1 α-OH-D 3. None catalysed the 25-hydroxylation reaction. 4. 1 α-OH-D 3 in a high concentration (2.5 ng ml -1) showed small but significant inhibition of the catalytic activities of CYP2C8, 2C9-Cys, 2C19, 2D6-Val and 2E1 for their typical substrates. However, 1 α-OH-D 3 in a clinically used low concentration will not significantly affect drug metabolism catalysed by the 14 P450s tested. 5. In summary, the 25-hydroxylation activity of 1 α-OH-D 3 that localizes in the microsomal fraction appears to be attributable to a cytochrome P450 other than the microsomal forms tested in this study.     

Roles of human cytochrome P450 3A4/5 in dexamethasone 6β-hydroxylation mediated by liver microsomes and humanized liver in chimeric mice metabolically suppressed with azamulin

The urinary metabolic ratio of 6β-hydroxydexamethasone to dexamethasone reportedly acts as a noninvasive marker for human cytochrome P450 (P450) 3A4/5, which is induced by rifampicin in humanized-liver mice. In the current study, the pharmacokinetics of dexamethasone in humanized-liver mice after intravenous administration (10 mg/kg) were investigated using azamulin (a time-dependent P450 3A4/5 inhibitor). After intravenous dexamethasone administration, significant differences were observed in the time-dependent plasma and 24-h urinary concentrations of 6β-hydroxydexamethasone between untreated humanized-liver mice and humanized-liver mice treated with azamulin (daily oral doses of 15 mg/kg for 3 days). The mean ratios of 6β-hydroxydexamethasone to dexamethasone for the maximum concentrations, the areas under the plasma concentration-versus-time curves, and urinary concentrations were significantly lower in the azamulin-treated group (59%, 58%, and 41% of the untreated values, respectively). 6β-Hydroxydexamethasone formation was suppressed by 93% by replacing control human liver microsomes with P450 3A4/5-inactivated liver microsomes. These results suggest that the oxidation of dexamethasone in humans is mediated mainly by P450 3A4/5 (which is suppressed by azamulin), and that humanized-liver mice orally treated with azamulin may constitute an in vivo model for metabolically inactivated P450 3A4/5 in human hepatocytes transplanted into chimeric mice.     

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.     

The effect of interferon-α on the expression of cytochrome P450 3A4 in human hepatoma cells

Esculetin (6,7-dihydroxy coumarin), is a potent antioxidant that is present in several plant species. The aim of this study was to investigate the mechanism of protection of esculetin in human hepatoma HepG2 cells against reactive oxygen species (ROS) induced by hydrogen peroxide. Cell viability, cell integrity, intracellular glutathione levels, generation of reactive oxygen species and expression of antioxidant enzymes were used as markers to measure cellular oxidative stress and response to ROS. The protective effect of esculetin was compared to a well-characterized chemoprotective compound quercetin. Pre-treatment of HepG2 cells with sub-lethal (10-25 μM) esculetin for 8 h prevented cell death and maintained cell integrity following exposure to 0.9 mM hydrogen peroxide. An increase in the generation of ROS following hydrogen peroxide treatment was significantly attenuated by 8 h pre-treatment with esculetin. In addition, esculetin ameliorated the decrease in intracellular glutathione caused by hydrogen peroxide exposure. Moreover, treatment with 25 μM esculetin for 8 h increased the expression of NAD(P)H: quinone oxidoreductase (NQO1) at both protein and mRNA levels significantly, by 12-fold and 15-fold, respectively. Esculetin treatment also increased nuclear accumulation of Nrf2 by 8-fold indicating that increased NQO1 expression is Nrf2-mediated. These results indicate that esculetin protects human hepatoma HepG2 cells from hydrogen peroxide induced oxidative injury and that this protection is provided through the induction of protective enzymes as part of an adaptive response mediated by Nrf2 nuclear accumulation.     

Determination of propofol UDP-glucuronosyltransferase (UGT) activities in hepatic microsomes from different species by UFLC–ESI-MS

Propofol O-glucuronidation has been used as probe reaction to phenotype UGT1A9 activity in human liver, thus a sensitive and specific method for determination of propofol O-glucuronide (PG) is urgently desirable. In the current study, a new LC–ESI-MS method for determination of PG in hepatic microsomes from human (HLM), monkey (CyLM), dog (DLM), minipig (PLM), rat (RLM) and mouse (MLM) was developed and validated using 4-methylumbelliferyl-β-d-glucuronide as an internal standard (IS). PG and IS was separated by a Shim-pack XR-ODS column (100 mm × 2.0 mm, 2.2 μm, Shimadzu) under gradient conditions with the mobile phase of acetonitrile and water containing 0.2% acetic acid (v/v). The mass spectrometric detection was performed under selected ion monitoring (SIM) for PG at m/z 353 and IS at m/z 351. The assay exhibited linearity over the range 0.05–30 μM for PG with the correlation coefficient of 0.9995. The intra- and inter-day precision was less than 7.2%, with accuracy in the range 93.8–107.5%. The developed method was successfully used for characterizing interspecies and human individual differences in the O-glucuronidation activity towards propofol, as well as investigating inhibitory effects of androsterone and phenylbutazone on propofol O-glucuronidation in HLM.     

Effects of Ganoderma sterols （GS） on hepatic cytochrome P450 in BCG-induced immunological hepatic injury in BALB／c mice

AIM: To investigate effects of Ganoderma sterols (GS) isolated from Ganoderma lucidum (Leyss ex fr) Karst on hepatic cytochrome P450 in BCG-induced immunological hepatic injury in BALB/c mice and its possible mechanism. METHODS: Immunological liver injury was induced by one intravenous injection of BCG (125 mg/kg) in BALB/c mice. One week later, successiveintragastric administration of GS (20, 40, 80 mg/kg, per day) and     

Role of human cytochrome P450 3A4 in the metabolism of DA–8159, a new erectogenic

1.?The purpose of this paper was to characterize cytochrome P450 (CYP) enzymes involved in N-dealkylation of a new oral erectogenic, DA-8159 to DA-8164, a major circulating active metabolite, in human liver microsomes and to investigate the inhibitory potential of DA-8159 on CYP enzymes.

2.?CYP3A4 was identified as the major enzyme responsible for DA-8159 N-dealkylation to DA-8164 based on correlation analysis and specific CYP inhibitor and antibody-mediated inhibition study in human liver microsomes, and DA-8159 metabolism in cDNA expressed CYP enzymes. There is the possibility of drug-drug interactions when prescribing DA-8159 concomitantly with known inhibitors or inducers of CYP3A4.

3.?DA-8159 was found to be only a very weak inhibitor of eight major CYPs (1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4), the largest inhibition occurring against CYP2D6 (IC₅₀ 67.7?μM) in human liver microsomes. Drug–drug interactions would not be predicted on the basis of DA-8159 inhibiting the metabolism of coadministered drugs.     

Assessment of the impact of CYP3A polymorphisms on the formation of α-hydroxytamoxifen and N-desmethyltamoxifen in human liver microsomes

Tamoxifen, an antiestrogen used in the prevention and treatment of breast cancer, is extensively metabolized by cytochrome P450 enzymes. Its biotransformation to α-hydroxytamoxifen (α-OHT), which may be genotoxic, and to N-desmethyltamoxifen (N-DMT), which is partially hydroxylated to 4-hydroxy-N-DMT (endoxifen), a potent antiestrogen, is mediated by CYP3A enzymes. However, the potential contribution of CYP3A5 and the impact of its low-expression variants on the formation of these metabolites are not clear. Therefore, we assessed the contributions of CYP3A4 and CYP3A5 and examined the impact of CYP3A5 genotypes on the formation of α-OHT and N-DMT, by using recombinant CYP3A4 and CYP3A5 and human liver microsomes (HLM) genotyped for CYP3A5 variants. We observed that the catalytic efficiency [intrinsic clearance (CL(int))] for α-OHT formation with recombinant CYP3A4 was 5-fold higher than that with recombinant CYP3A5 (0.81 versus 0.16 nl · min?1 · pmol cytochrome P450?1). There was no significant difference in CL(int) values between the three CYP3A5-genotyped HLM (*1/*1, *1/*3, and *3/*3). For N-DMT formation, the CL(int) with recombinant CYP3A4 was only 1.7-fold higher, relative to that with recombinant CYP3A5. In addition, the CL(int) for N-DMT formation by HLM with CYP3A5*3/*3 alleles was approximately 3-fold lower than that for HLM expressing CYP3A5*1/*1. Regression analyses of tamoxifen metabolism with respect to testosterone 6β-hydroxylation facilitated assessment of CYP3A5 contributions to the formation of the two metabolites. The CYP3A5 contributions to α-OHT formation were negligible, whereas the contributions to N-DMT formation ranged from 51 to 61%. Our findings suggest that polymorphic CYP3A5 expression may affect the formation of N-DMT but not that of α-OHT.     

Identification of human cytochrome P450 isoforms and esterases involved in the metabolism of mirabegron, a potent and selective β(3)-adrenoceptor agonist

Human cytochrome P450 (CYP) enzymes and esterases involved in the metabolism of mirabegron, a potent and selective human β(3)-adrenoceptor agonist intended for the treatment of overactive bladder, were identified in in vitro studies. Incubations of mirabegron with recombinant human CYP enzymes showed significant metabolism of mirabegron by CYP2D6 and CYP3A4 only. Correlation analyses showed a significant correlation between mirabegron metabolism and testosterone 6β-hydroxylation (CYP3A4/5 marker activity). In inhibition studies using antiserum against CYP3A4, a strong inhibition (at maximum 80% inhibition) of the metabolism of mirabegron was observed, whereas the inhibitory effects of monoclonal antibodies against CYP2D6 were small (at maximum 10% inhibition). These findings suggest that CYP3A4 is the primary CYP enzyme responsible for in vitro oxidative metabolism of mirabegron, with a minor role of CYP2D6. Mirabegron hydrolysis was catalyzed in human blood, plasma and butyrylcholinesterase (BChE) solution, but not in human liver microsomes, intestinal microsomes, liver S9, intestinal S9 and recombinant acetylcholinesterase solution. K(m) values of mirabegron hydrolysis in human blood, plasma and BChE solution were all similar (13.4-15.2 μM). The inhibition profiles in human blood and plasma were also similar to those in BChE solution, suggesting that mirabegron hydrolysis is catalyzed by BChE.     

Cytochrome P450 3A-mediated microsomal biotransformation of 1α,25-dihydroxyvitamin D3 in mouse and human liver: drug-related induction and inhibition of catabolism

The biological activities of vitamin D(3) are exerted through the dihydroxy metabolite of vitamin D(3) [1α,25(OH)(2)D(3)]. Hepatic biotransformation of 1α,25(OH)(2)D(3) by cytochrome P450 (P450) enzymes could be an important determinant of bioavailability in serum and tissues. In the present study, we investigated the comparative biotransformation of 1α,25(OH)(2)D(3) in mouse and human liver microsomes and determined the effects of commonly used drugs on the catabolism of 1α,25(OH)(2)D(3). Severe symptoms of vitamin D deficiency have historically been observed in patients who received dexamethasone. To compare the effects of clinically important glucocorticoids with hepatic biotransformation of 1α,25(OH)(2)D(3), adult male CD-1 mice were given intraperitoneal injections of either vehicle (50% ethanol), dexamethasone (80 mg/kg per day), or prednisone (80 mg/kg per day) for three consecutive days. Hydroxy metabolite formation pattern and the extent of substrate depletion were similar in mouse liver microsomes (MLM) from vehicle- or prednisone-treated mice, whereas treatment with dexamethasone led to the emergence of additional metabolites and increased substrate depletion, as determined by liquid chromatography/mass spectrometry. The metabolite formation profile in vehicle-treated mice was different from that of human liver microsomes (HLM). Selective P450 chemical inhibitors have demonstrated that CYP3A isoforms are responsible for the microsomal biotransformation of 1α,25(OH)(2)D(3) in MLM. Coincubation of 1α,25(OH)(2)D(3) with commonly used drugs led to approximately 60 to 100% inhibition of CYP3A4-mediated catabolism of 1α,25-(OH)(2)D(3) in HLM. A species-based difference was identified between CYP3A-mediated hepatic microsomal metabolism of 1α,25(OH)(2)D(3) in humans and mice. We have shown that the clinical importance of glucocorticoids differentially modulates catabolism of active vitamin D(3) and that commonly used drugs could affect vitamin D homeostasis.