In vitro metabolism of quinidine: the (3S)-3-hydroxylation of quinidine is a specific marker reaction for cytochrome P-4503A4 activity in human liver microsomes

The aim of this study was to evaluate the (3S)-3-hydroxylation and the N-oxidation of quinidine as biomarkers for cytochrome P-450 (CYP)3A4 activity in human liver microsome preparations. An HPLC method was developed to assay the metabolites (3S)-3-hydroxyquinidine (3-OH-Q) and quinidine N-oxide (Q-N-OX) formed during incubation with microsomes from human liver and from Saccharomyces cerevisiae strains expressing 10 human CYPs. 3-OH-Q formation complied with Michaelis-Menten kinetics (mean values of Vmax and Km: 74.4 nmol/mg/h and 74.2 microM, respectively). Q-N-OX formation followed two-site kinetics with mean values of Vmax, Km and Vmax/Km for the low affinity isozyme of 15.9 nmol/mg/h, 76.1 microM and 0.03 ml/mg/h, respectively. 3-OH-Q and Q-N-OX formations were potently inhibited by ketoconazole, itraconazole, and triacetyloleandomycin. Isozyme specific inhibitors of CYP1A2, -2C9, -2C19, -2D6, and -2E1 did not inhibit 3-OH-Q or Q-N-OX formation, with Ki values comparable with previously reported values. Statistically significant correlations were observed between CYP3A4 content and formations of 3-OH-Q and Q-N-OX in 12 human liver microsome preparations. Studies with yeast-expressed isozymes revealed that only CYP3A4 actively catalyzed the (3S)-3-hydroxylation. CYP3A4 was the most active enzyme in Q-N-OX formation, but CYP2C9 and 2E1 also catalyzed minor proportions of the N-oxidation. In conclusion, our studies demonstrate that only CYP3A4 is actively involved in the formation of 3-OH-Q. Hence, the (3S)-3-hydroxylation of quinidine is a specific probe for CYP3A4 activity in human liver microsome preparations, whereas the N-oxidation of quinidine is a somewhat less specific marker reaction for CYP3A4 activity, because the presence of a low affinity enzyme is demonstrated by different approaches.     

Role of a potent inhibitory monoclonal antibody to cytochrome P-450 3A4 in assessment of human drug metabolism.

Cytochrome P-450 (CYP) 3A4 is an inordinately important CYP enzyme that catalyzes the metabolism of a vast array of clinically used drugs. Microsomal proteins of Spodoptera frugiperda (Sf21) insect cells infected with recombinant baculoviruses encoding CYP3A4 cDNA were used to immunize mice and to develop a monoclonal antibody (mAb(3A4a)) specific to CYP3A4 through the use of hybridoma technology. The mAb is both a potent inhibitor and a strong binder of CYP3A4. One and 5 microl (0.5 and 2.5 microM IgG(2a)) of the mAb mouse ascites in 1-ml incubation containing 20 pmol of CYP3A4 strongly inhibited the testosterone 6beta-hydroxylation by 95 and 99%, respectively, and, to a lesser extent, cross-inhibited CYP3A5 and CYP3A7 activity. mAb(3A4a) exhibited no cross-reactivity with any of the other recombinant human CYP isoforms (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1) in the course of CYP reaction phenotyping and Western immunoblot analyses. The potency of mAb-induced inhibition is insensitive to substrate concentration in human liver microsomes. Therefore, mAb(3A4a) was used to assess the quantitative role of CYP3A4/5 to the metabolism of testosterone and diazepam in five human liver microsomes. The results showed that CYP3A4 and CYP3A5 contribute >95% to both testosterone 6beta-hydroxylation and diazepam 3-hydroxylation and 52 to 73% to diazepam N-demethylation, respectively. In addition, mAb(3A4a) significantly inhibited testosterone 6beta-hydroxylase activity in rhesus monkey liver microsomes to a degree equal to that observed with CYP3A4 in human liver microsomes. By comparison, no inhibition of testosterone 6beta-hydroxylase activity was observed in the presence of dog, rat, and mouse liver microsomes. The selectivity of ketoconazole, a chemical inhibitor of CYP3A4, was probed with mAb(3A4a) and was shown to be highly concentration dependent in the diazepam N-demethylation by human liver microsomes. The results demonstrate that inhibitory and immunoblotting mAb(3A4a) can offer a precise and useful tool for quantitative identification of CYP3A4/5 in the metabolism of drugs in clinical use and drugs in development.     

Application of the relative activity factor approach in scaling from heterologously expressed cytochromes p450 to human liver microsomes: studies on amitriptyline as a model substrate

The relative activity factor (RAF) approach is being increasingly used in the quantitative phenotyping of multienzyme drug biotransformations. Using lymphoblast-expressed cytochromes P450 (CYPs) and the tricyclic antidepressant amitriptyline as a model substrate, we have tested the hypothesis that the human liver microsomal rates of a biotransformation mediated by multiple CYP isoforms can be mathematically reconstructed from the rates of the biotransformation catalyzed by individual recombinant CYPs using the RAF approach, and that the RAF approach can be used for the in vitro-in vivo scaling of pharmacokinetic clearance from in vitro intrinsic clearance measurements in heterologous expression systems. In addition, we have compared the results of two widely used methods of quantitative reaction phenotyping, namely, chemical inhibition studies and the prediction of relative contributions of individual CYP isoforms using the RAF approach. For the pathways of N-demethylation (mediated by CYPs 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and E-10 hydroxylation (mediated by CYPs 2B6, 2D6, and 3A4), the model-predicted biotransformation rates in microsomes from a panel of 12 human livers determined from enzyme kinetic parameters of the recombinant CYPs were similar to, and correlated with the observed rates. The model-predicted clearance via N-demethylation was 53% lower than the previously reported in vivo pharmacokinetic estimates. Model-predicted relative contributions of individual CYP isoforms to the net biotransformation rate were similar to, and correlated with the fractional decrement in human liver microsomal reaction rates by chemical inhibitors of the respective CYPs, provided the chemical inhibitors used were specific to their target CYP isoforms.     

Diltiazem inhibition of cytochrome P-450 3A activity is due to metabolite intermediate complex formation.

Diltiazem (DTZ) N-demethylation occurs by cytochrome P-450 (CYP) 3A based on the following observations: 1) a single enzyme Michaelis-Menten model of metabolite formation, 2) high correlations of DTZ N-demethylation activity to other CYP3A activities, 3) inhibition of DTZ N-demethylation activity by triacetyloleandomycin, and 4) DTZ N-demethylation activity by expressed CYP3A enzymes only. The mean K(m)s for DTZ N-demethylation in human liver microsomes and expressed CYP3A4(+b(5)) were 53 and 16 microM, respectively. A 30-min preincubation of DTZ in expressed CYPs inhibited CYP3A4(+b(5)) by 100%, of which 55% was due to formation of a metabolite intermediate complex (MIC), which is an inactive form of CYP. MIC was observed in human liver microsomes and cDNA-expressed CYP3A only. In experiments to assess simultaneous MIC formation and loss of CYP3A activity, DTZ caused greater than 80% inhibition of midazolam hydroxylation after a 60-min preincubation in human liver microsomes. The rate constants for MIC formation and loss of midazolam hydroxylation activity were equivalent for the line of best fit for both data sets, which illustrates that MIC formation causes the inhibition of CYP3A activity. The mechanistic inhibition was characterized in expressed CYP3A4(+b(5)), which exhibited a concentration-dependent formation of MIC by DTZ (1-100 microM) with an estimated k(inact) of 0.17 min(-1) and K(I) of 2.2 microM. The partition ratio for expressed CYP3A4(+b(5)) was substrate concentration dependent and varied from 13 to 86. This study showed that DTZ inhibition of CYP3A substrate metabolism occurs primarily by MIC formation.     

Activation of human cytochrome P-450 3A4-catalyzed meloxicam 5'-methylhydroxylation by quinidine and hydroquinidine in vitro.

In humans, meloxicam is metabolized mainly by cytochrome P-450 (CYP)-dependent hydroxylation of the 5'-methyl group. The predominant P-450 enzyme involved in meloxicam metabolism is CYP 2C9, with a minor contribution of CYP 3A4. Quinidine, a CYP 3A4 substrate commonly used as a selective in vitro inhibitor of CYP 2D6, was found to markedly increase the rate of meloxicam hydroxylation during in vitro experiments with human liver microsomes. A similar activation was observed with other compounds that are structurally related to quinidine. Besides quinidine, quinine and hydroquinidine were the most potent activators of meloxicam hydroxylation. Using expressed cytochrome P-450 enzymes and selective chemical inhibitors of CYP 2C9 and CYP 3A4, it was found that quinidine markedly increased the rate of CYP 3A4-mediated meloxicam hydroxylation but was virtually without effect on CYP 2C9. Kinetic analysis was performed to obtain insight into the possible mechanism of activation of CYP 3A4 and into the mutual interaction of quinidine/hydroquinidine and meloxicam. Quinidine and hydroquinidine decreased Km and increased Vmax of meloxicam hydroxylation, which was consistent with a mixed-type nonessential activation. Meloxicam, in turn, decreased both Km and Vmax of quinidine metabolism by CYP 3A4, indicating an uncompetitive inhibition mechanism. These results support the assumption that CYP 3A4 possesses at least two different substrate-binding sites. A clinically relevant effect on meloxicam drug therapy is not expected, because the most likely outcome in practice is moderately decreased meloxicam plasma concentrations.     

Metabolic activation of pradefovir by CYP3A4 and its potential as an inhibitor or inducer

Metabolic activation of pradefovir to 9-(2-phosphonylmethoxyethyl)adenine (PMEA) was evaluated by using cDNA-expressed CYP isozymes in portal vein-cannulated rats following oral administration and in human liver microsomes. The enzyme induction potential of pradefovir was evaluated in rats following multiple oral dosing and in primary cultures of human hepatocytes. The results indicated that CYP3A4 is the only cDNA-expressed CYP isozyme catalyzing the conversion of pradefovir to PMEA. Pradefovir was converted to PMEA in human liver microsomes with a K(m) of 60 microM, a maximum rate of metabolism of 228 pmol/min/mg protein, and an intrinsic clearance of about 359 ml/min. Addition of ketoconazole and monoclonal antibody 3A4 significantly inhibits the conversion of pradefovir to PMEA in human liver microsomes, suggesting the predominant role of CYP3A4 in the metabolic activation of pradefovir. Pradefovir at 0.2, 2, and 20 microM was neither a direct inhibitor nor a mechanism-based inhibitor of CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP2E1, and CYP1A2 in human liver microsomes. In rats, the liver was the site of metabolic activation of pradefovir, whereas the small intestine did not play a significant role in the metabolic conversion of pradefovir to PMEA. Daily oral dosing (300 mg/kg of body weight) to rats for 8 days showed that pradefovir was not an inducer of P450 enzymes in rats. Furthermore, pradefovir at 10 microg/ml was not an inducer of either CYP1A2 or CYP3A4/5 in primary cultures of human hepatocytes.     

Chloramphenicol is a potent inhibitor of cytochrome P450 isoforms CYP2C19 and CYP3A4 in human liver microsomes

The inhibitory effect of chloramphenicol on human cytochrome P450 (CYP) isoforms was evaluated with human liver microsomes and cDNA-expressed CYPs. Chloramphenicol had a potent inhibitory effect on CYP2C19-catalyzed S-mephytoin 4′-hydroxylation and CYP3A4-catalyzed midazolam 1-hydroxylation, with apparent 50% inhibitory concentrations (inhibitory constant [K_i] values are shown in parentheses) of 32.0 (7.7) and 48.1 (10.6) μM, respectively. Chloramphenicol also weakly inhibited CYP2D6, with an apparent 50% inhibitory concentration (K_i) of 375.9 (75.8) μM. The mechanism of the drug interaction reported between chloramphenicol and phenytoin, which results in the elevation of plasma phenytoin concentrations, is clinically assumed to result from the inhibition of CYP2C9 by chloramphenicol. However, using human liver microsomes and cDNA-expressed CYPs, we showed this interaction arises from the inhibition of CYP2C19- not CYP2C9-catalyzed phenytoin metabolism. In conclusion, inhibition of CYP2C19 and CYP3A4 is the probable mechanism by which chloramphenicol decreases the clearance of coadministered drugs, which manifests as a drug interaction with chloramphenicol.     

The cytochrome P450 2B6 (CYP2B6) is the main catalyst of efavirenz primary and secondary metabolism: implication for HIV/AIDS therapy and utility of efavirenz as a substrate marker of CYP2B6 catalytic activity

We used human liver microsomes (HLMs) and recombinant cytochromes P450 (P450s) to identify the routes of efavirenz metabolism and the P450s involved. In HLMs, efavirenz undergoes primary oxidative hydroxylation to 8-hydroxyefavirenz (major) and 7-hydroxyefavirenz (minor) and secondary metabolism to 8,14-dihydroxyefavirenz. The formation of 8-hydroxyefavirenz in two HLMs showed sigmoidal kinetics (average apparent Km, 20.2 micro M; Vmax, 140 pmol/min/mg protein; and Hill coefficient, 1.5), whereas that of 7-hydroxyefavirenz formation was characterized by hyperbolic kinetics (Km, 40.1 micro M and Vmax, 20.5 pmol/min/mg protein). In a panel of 10 P450s, CYP2B6 formed 8-hydroxyefavirenz and 8,14-dihydroxyefavirenz from efavirenz (10 micro M) at the highest rate. The Km value for the formation of 8-hydroxyefavirenz in CYP2B6 derived from hyperbolic Eq. 12.4 micro M) was close to that obtained in HLMs (Km, 20.2 micro M). None of the P450s tested showed activity toward 7-hydroxylation of efavirenz. When 8-hydroxyefavirenz (2.5 micro M) was used as a substrate, 8,14-dihydroxyefavirenz was formed by CYP2B6 at the highest rate, and its kinetics showed substrate inhibition (Ksi, approximately 94 micro M in HLMs and approximately 234 micro M in CYP2B6). In a panel of 11 HLMs, 8-hydroxyefavirenz and 8,14-dihydroxyefavirenz formation rates from efavirenz (10 micro M) correlated significantly with the activity of CYP2B6 and CYP3A. N,N',N"-Triethylenethiophosphoramide (thioTEPA; 50 micro M) inhibited the formation rates of 8-hydroxyefavirenz and 8,14-dihydroxyefavirenz from efavirenz (10 micro M) by > or = 60% in HLMs) and CYP2B6, with Ki values < 4 micro M. In conclusion, CYP2B6 is the principal catalyst of efavirenz sequential hydroxylation. Efavirenz systemic exposure is likely to be subject to interindividual variability in CYP2B6 activity and to drug interactions involving this isoform. Efavirenz may be a valuable phenotyping tool to study the role of CYP2B6 in human drug metabolism.     

Microsomal binding of amitriptyline: effect on estimation of enzyme kinetic parameters in vitro

The effect of binding of amitriptyline to human liver microsomes and to microsomes from human B-lymphoblastoid cells on the estimation of enzyme kinetic parameters describing N-demethylation to nortriptyline was investigated using a combination of microsomal binding and in vitro enzyme kinetic studies. Quantitative binding in both matrices increased with higher microsomal protein concentrations (free fractions 0.88-0.32 at 100-500 microg protein/ml in human liver microsomes and 0.82-0.26 at 250-1000 microg protein/ml in microsomes from B-lymphoblastoid cells) and was independent of amitriptyline concentration over a concentration range of 0.2 to 200 microM. Addition of heat-inactivated microsomal protein (50-450 microg/ml) to native human liver microsomes (50 microg/ml) reduced the amitriptyline N-demethylation rate in a protein concentration dependent manner. This effect was greater at lower substrate concentrations and was overcome by saturating concentrations of substrate, thereby decreasing the apparent affinities of the high- and low-affinity components of the N-demethylation process, with minimal effect on the net V(max). Addition of metabolically inactive microsomes from untransfected human lymphoblastoid cells (750 microg/ml) to CYP2C19 (250 microg/ml protein) increased the apparent K(m) value for amitriptyline N-demethylation by 3.5-fold and increased the uncompetitive substrate inhibition constant (K(s)) by 2.2-fold, making substrate inhibition essentially undetectable. A similar effect was seen with CYP3A4, with a 1.8-fold increase in the S(50) (substrate concentration at which half-maximal velocity of a Hill enzyme is achieved). Microsomal binding did not alter the V(max) of either CYP isoform to any appreciable extent. These findings emphasize the importance of incorporating microsomal binding in the estimation of enzyme kinetic parameters in vitro and making appropriate corrections for unbound drug concentrations.     

Midazolam and triazolam biotransformation in mouse and human liver microsomes: relative contribution of CYP3A and CYP2C isoforms

Midazolam (MDZ) and triazolam (TRZ) hydroxylation, reactions considered to be cytochrome P-4503A (CYP3A)-mediated in humans, were examined in mouse and human liver microsomes. In both species, alpha- and 4-hydroxy metabolites were the principal products. Western blotting with anti-CYP3A1 antibody detected a single band of immunoreactive protein in both human and mouse samples: 0.45 +/- 0. 12 and 2.02 +/- 0.24 pmol/mg protein (mean +/- S.E., n = 3), respectively. Ketoconazole potently inhibited MDZ and TRZ metabolite formation in human liver microsomes (IC(50) range, 0.038-0.049 microM). Ketoconazole also inhibited the formation of both TRZ metabolites and of 4-OH-MDZ formation in mouse liver microsomes (IC(50) range, 0.0076-0.025 microM). However, ketoconazole (10 microM) did not produce 50% inhibition of alpha-OH-MDZ formation in mouse liver microsomes. Anti-CYP3A1 antibodies produced concentration-dependent inhibition of MDZ and TRZ metabolite formation in human liver microsomes and of TRZ metabolite and 4-OH-MDZ formation in mouse liver microsomes to less than 20% of control values but reduced alpha-OH-MDZ formation to only 66% of control values in mouse liver microsomes. Anti-CYP2C11 antibodies inhibited alpha-OH-MDZ metabolite formation in a concentration-dependent manner to 58% of control values in mouse liver microsomes but did not inhibit 4-OH-MDZ formation. Thus, TRZ hydroxylation appears to be CYP3A specific in mice and humans. alpha-Hydroxylation of MDZ has a major CYP2C component in addition to CYP3A in mice, demonstrating that metabolic profiles of drugs in animals cannot be assumed to reflect human metabolic patterns, even with closely related substrates.     

Major role of human liver microsomal cytochrome P450 2C9 (CYP2C9) in the oxidative metabolism of celecoxib, a novel cyclooxygenase-II inhibitor

In vitro studies were conducted to identify the cytochromes P450 (CYP) involved in the oxidative metabolism of celecoxib. The hydroxylation of celecoxib conformed to monophasic Michaelis-Menten kinetics (mean +/- S.D., n = 4 livers, K(m) = 3.8 +/- 0.95 microM, V(max) = 0.70 +/- 0.45 nmol/min/mg protein) in the presence of human liver microsomes, although substrate inhibition was significant at higher celecoxib concentrations. The treatment of a panel of human liver microsomal samples (n = 16 subjects) with antibodies against CYP2C9 and CYP3A4 inhibited the formation of hydroxy celecoxib by 72 to 92% and 0 to 27%, respectively. The presence of both antibodies in the incubation suppressed the activity by 90 to 94%. In addition, the formation of hydroxy celecoxib significantly correlated with CYP2C9-selective tolbutamide methyl hydroxylation (r = 0.92, P <. 001) and CYP3A-selective testosterone 6beta-hydroxylation (r = 0.55, P <.02). In contrast, correlation with activities selective for other forms of CYP was weak (r 相似文献   

CYP2J2 and CYP2C19 Are the Major Enzymes Responsible for Metabolism of Albendazole and Fenbendazole in Human Liver Microsomes and Recombinant P450 Assay Systems

Albendazole and fenbendazole are broad-spectrum anthelmintics that undergo extensive metabolism to form hydroxyl and sulfoxide metabolites. Although CYP3A and flavin-containing monooxygenase have been implicated in sulfoxide metabolite formation, the enzymes responsible for hydroxyl metabolite formation have not been identified. In this study, we used human liver microsomes and recombinant cytochrome P450s (P450s) to characterize the enzymes involved in the formation of hydroxyalbendazole and hydroxyfenbendazole from albendazole and fenbendazole, respectively. Of the 10 recombinant P450s, CYP2J2 and/or CYP2C19 was the predominant enzyme catalyzing the hydroxylation of albendazole and fenbendazole. Albendazole hydroxylation to hydroxyalbendazole is primarily mediated by CYP2J2 (0.34 μl/min/pmol P450, which is a rate 3.9- and 8.1-fold higher than the rates for CYP2C19 and CYP2E1, respectively), whereas CYP2C19 and CYP2J2 contributed to the formation of hydroxyfenbendazole from fenbendazole (2.68 and 1.94 μl/min/pmol P450 for CYP2C19 and CYP2J2, respectively, which are rates 11.7- and 8.4-fold higher than the rate for CYP2D6). Correlation analysis between the known P450 enzyme activities and the rate of hydroxyalbendazole and hydroxyfenbendazole formation in samples from 14 human liver microsomes showed that albendazole hydroxylation correlates with CYP2J2 activity and fenbendazole hydroxylation correlates with CYP2C19 and CYP2J2 activities. These findings were supported by a P450 isoform-selective inhibition study in human liver microsomes. In conclusion, our data for the first time suggest that albendazole hydroxylation is primarily catalyzed by CYP2J2, whereas fenbendazole hydroxylation is preferentially catalyzed by CYP2C19 and CYP2J2. The present data will be useful in understanding the pharmacokinetics and drug interactions of albendazole and fenbendazole in vivo.     

In vivo age-related changes in hepatic drug-oxidizing capacity in humans

Very few studies have been carried out looking at how the effects of drugs and their toxicity in humans change during their lifespan (developing and ageing). The purpose of this study is to review the literature on the changes in probe-drug metabolism, classified by cytochrome P450 (P450 or CYP) at five stages in life: neonates < 4 weeks, infants < 12 months, children < 19 years, young/mature adults 20–64 years, and elderly adults >65 years. The main probe drugs include caffeine and theophylline, whose metabolism is catalysed by CYP1A2, tolbutamide, phenytoin and ibuprofen, catalysed by CYP2C9, amitriptyline and nortriptyline, catalysed by CYP2C19, acetaminophen, catalysed by CYP2E1 and lidocaine, midazolam and terfenadine, catalysed by 3A3/4. From the published in vivo studies two different patterns of drug metabolism can be identified: (i) activity is low immediately after birth, increases, then peaks at the young/mature adult level and, finally, decreases in old age (drugs catalysed by CYPlA2, CYP2C9, CYP2C19, CYP2D6 and CYP3A3/4) and (ii) activity increases rapidly after birth to reach a level equivalent to that in the young/mature adult, then gradually decreases and finally decreasing faster in old age (drugs catalysed by CYP2E1). Further study of the changes in P450 with age is warranted to help prevent adverse reactions and to guide us in tailoring therapy better for the individual patient.     

Clinical importance of non-genetic and genetic cytochrome P450 function tests in liver disease

Liver disease is associated with reduced metabolic capacity for drugs that are metabolized by oxidative biotransformation. Three cytochrome P450 (P450 or CYP) gene families in liver microsomes (CYP 1, CYP2 and CYP3) appear to be responsible for much of the drug metabolism that takes place. The genetic polymorphism of the CYPs responsible for debrisoquine/sparteine (CYP2D6) metabolism and S-mephenytoin (CYP2C19) metabolism has been well documented, but information on the impairment of each isoform in liver disease is still limited. There are two types of hepatic P450 function tests. One type consists of non-genetic P450 function tests (CYP1A2, 2A6, 2C9/10, 2E1 and 3A3/4), and probe drugs include caffeine, catalysed by CYP1A2, coumarin by CYP2A6, phenytoin by CYP2C6, chlorzoxazone by CYP2E1, and nifedipine, erythromycin and lidocaine by CYP3A3/4. The second type of genetic P450 function tests (CYP2C19 and CYP2D6) involves probe drugs such as S-mephenytoin, catalysed by CYP2C19, and debrisoquine and sparteine, catalysed by CYP2D6. The metabolism of the probe drugs used in non-genetic P450 function tests in patients with liver disease falls into two categories: reduced (CYP1A2, CYP2C, 2E1 and 3A) and unchanged (CYP2C). In genetic P450 function tests there seems to be a lesser degree of inhibition in poor metabolizers (PMs) than extensive metabolizers (EMs) among patients with liver disease. There have been very few reports on changes in metabolism of the probe drugs used in genetic P450 function tests in liver disease. In this paper the subject is reviewed     

Heterogeneity of CYP3A isoforms metabolizing erythromycin and cortisol.

The N-demethylation of erythromycin and 6 beta-hydroxylation of cortisol are both functions of the glucocorticoid-inducible CYP3A in human liver microsomes. To determine whether 6 beta-hydroxylation and erythromycin N-demethylation are catalyzed by similar or distinct CYP3A isoforms, erythromycin N-demethylase activity, as reflected by the recently described 14[C]-erythromycin breath test, was compared with urinary 6 beta-hydroxycortisol/cortisol ratios, a measure of cortisol 6 beta-hydroxylase activity, in nine patients. Erythromycin N-demethylation varied fourfold and 6 beta-hydroxycortisol/cortisol ratios varied sevenfold among the subjects; no correlation was found between these activities (r2 = 0.065). New noninvasive tests of CYP3A strongly suggest cortisol 6 beta-hydroxylation and erythromycin N-demethylation are performed by distinct CYP3A isoforms.     

Prediction of the likelihood of drug interactions with kinase inhibitors based on in vitro and computational studies

Kinase inhibitors (KIs) represent an important group of anticancer drugs, and many of them are substrates and inhibitors of human cytochrome P450s (CYPs), raising the potential of harmful drug interactions. This study investigated the effect of a library of KIs (n = 91) including 11 FDA‐approved KIs on human CYP1A2, 2D6, 2C9, and 3A4 using high‐throughput screening kits and the binding modes with CYPs using the Discovery Studio program 3.1. The KIs exhibited differential inhibitory effect on CYP1A2, 2D6, 2C9, and 3A4, while some of them showed activating effect on CYP2C9 and 3A4. For example, SP 600125 was a potent inhibitor for CYP1A2, but enhanced the activity of CYP2C9 fourfolds. Among the 80 KIs that are not used clinically, about 13% showed significant inhibition to CYPs. Nilotinib, sunitinib, and imatinib were found to be potent CYP1A2 inhibitor. Our docking studies have demonstrated the importance of multiple amino acid residues in the active sites of CYP1A2, 2C9, 2D6, and 3A4 in binding with various KIs. Finally, the in vitro data were used to predict potential KI–drug interactions. These findings indicate that many KIs can serve as CYP inhibitors, and further studies are needed to examine the clinical impact.     

Effect of sinomenine on human cytochrome P450 activity

BACKGROUND: Cytochrome P450s superfamily expressed widely in organisms are known to play an important role in the biotransformation of many endogenous and exogenous substances. Inhibition or induction of cytochrome P450 isozymes is one of the major causes for clinical drug-drug interactions. Sinomenine can be metabolized to at least 2 metabolites in human, rat in vivo and in human liver microsomes. The major metabolite was identified to be N-demethylsinomenine. However, which CYP450 isozymes mediated by sinomenine in vivo and in vitro is not known. METHOD: In vitro study, 6 probe drugs were incubated with or without sinomenine respectively to study the effect of sinomenine on different cytochrome P450s activities in human microsomes. In vivo study, a 5-drug cocktail approach was used to study the inhibitive and inducing effect of sinomenine at normal clinical dose on cytochrome P450s activities. RESULTS: Sinomenine (50 micromol/l) had no significant effects on the activities of CYP1A2, CYP3A4, CYP2C9, CYP2E1, and CYP2D6, but it decreased the activity of CYP2C19 by 69% (p=0.012) in human microsomes. In vivo, sinomenine showed almost no significant effects on the activities of CYP1A2, CYP3A4, CYP2E1, and CYP2D6, but enhanced the elimination of mephenytoin by 73% (p=0.032). CONCLUSION: Sinomenine (50 micromol/l) inhibited the activity of CYP2C19 in human microsomes, but in vivo sinomenine at normal clinical dose enhanced the elimination of mephenytoin.     

Stereoselective metabolism of bufuralol racemate and enantiomers in human liver microsomes

A new HPLC method was developed using a chiral column to efficiently separate four 1"-hydroxybufuralol (1"-OH-BF) diastereomers that are major metabolites of bufuralol (BF). Employing this method, we examined diastereomer selectivity in the formation of 1"-OH-BF from BF racemate or enantiomers in four individual samples of human liver microsomes. Three different human liver microsomes showed a selectivity of 1"R-OH < 1"S-OH for BF enantiomers, which was similar to that of recombinant CYP2D6 expressed in insect cell microsomes, whereas one human liver microsomal fraction yielded a selectivity of 1"R-OH > 1"S-OH for BF enantiomers, which was similar to those of recombinant CYP2C19 expressed in insect cell microsomes. Recombinant CYP1A2 and CYP3A4 showed a selectivity similar to that of CYP2D6, but their BF 1"-hydroxylase activities were much lower than those of CYP2D6. In inhibition studies, quinidine, a known CYP2D6 inhibitor, markedly inhibited BF 1"-hydroxylation in the fractions of human liver microsomes that showed the CYP2D6-type selectivity. Furthermore, omeprazole, a known CYP2C19 inhibitor, efficiently suppressed the formation of 1"-OH-BF diastereomers from BF in the microsomal fraction that showed the CYP2C19-type selectivity. From these results, we concluded that the diastereomer selectivity in the formation of 1"-OH-BF from BF differs between CYP2D6 and CYP2C19, both of which can be determinant enzymes in the diastereoselective 1"-hydroxylation of BF in human liver microsomes.     

Evidence for the involvement of human liver microsomes CYP1A2 in the mono-hydroxylation of daidzein

BACKGROUND: In vitro studies with rats and human liver microsomes (HLM) demonstrated that daidzein is readily metabolized to mono-hydroxylated compounds. In this study, daidzein mono-hydroxylated metabolites was investigated using human liver microsomes to identify the cytochrome P450 (CYP) isoform(s) involved in this metabolic pathway. METHODS: Kinetic analysis for the formation rates of mono-hydroxylated metabolites of daidzein, including 7,8,4'-trihydroxyisoflavone (7,8,4'-THI), 7,3,4'-trihydroxyisoflavone (7,3,4'-THI) and 6,7,4'-trihydroxyisoflavone (6,7,4'-THI), were performed using human liver microsomes (HLM) and recombinant enzymes at substrate concentrations ranging from 0.5 to 400 micromol/l. Nine selective inhibitors or substrate probes specific for different CYP isoforms were applied for screening the isoform(s) responsible for mono-hydroxylated metabolism of daidzein. RESULTS: Michaelis-Menten kinetic parameters were best fitted to a one-component enzyme kinetic model. The mean K(m) (micromol/l) and V(max) (micromol/g min) values (+/-S.D.) were 26.86 (10.45) and 4.76 (2.07), 53.83 (22.25) and 2.29 (1.04), 51.48 (29.32) and 2.21(0.82), for the formation rates of 7,8,4'-THI, 7,3',4'-THI and 6,7,4'-THI, respectively. Furafylline, the CYP1A2-specific inhibitor, estrogen and monoclonal antibody raised against human CYP1A2 (MAB-1A2) substantially inhibited the formation rates of mono-hydroxylated metabolites. The IC(50) of Fur for the formation of 7,3',4'-THI, 6,7,4'-THI and 7,8,4'-THI was 1.0, 0.9 and 0.8 micromol/l, respectively. The IC(50) of estrogen for the formation of 7,3',4'-THI, 6,7,4'-THI and 7,8,4'-THI was 51, 60 and 64 micromol/l, respectively. The IC(50) of MAB-1A2 for the formation of the mono-hydroxylated products was 1 micromol/l, but neither other selective inhibitor nor substrate probes, including coumarin (CYP2D6), sulphaphenzole (CYP2C9/10), omeprazole (CYP2C19), quinidine (CYP2D6), diethyldithiocarbamate (CYP2E1), troleandomycin (CYP3A4) and keteconazole (CYP3A4), did so with human liver microsomes. CONCLUSION: Daidzein mono-hydroxylated products are principally metabolized by CYP1A2 in human.