Ethylmorphine N-demethylase activity as a marker for cytochrome P450 CYP3A activity in rat hepatic microsomes

The purpose of this study was to evaluate the selectivity and sensitivity of ethylmorphine N-demethylase (EMD) as an indicator of chemically-induced cytochrome P450 CYP3A activity in liver microsomes of rats following treatment with selective enzyme inducers. Male and female Sprague–Dawley (CD®) rats were dosed with either pregnenolone-16α-carbonitrile (PCN; 50 mg/kg per day for 5 days), phenobarbital (PB; 100 mg/kg per day for 4 days), beta-naphthoflavone (βNF; 100 mg/kg per day for 3 days), clofibrate (CF; 300 mg/kg per day for 14 days), isoniazid (ISO; 100 mg/kg per day for 3 days), or dexamethasone (DEX; 50 mg/kg per day for 4 days). Microsomes were isolated, frozen and subsequently assayed for protein, cytochrome P450 content and EMD activity. In males, significant elevations (P<0.01) in EMD activity were observed in microsomes from PB-, DEX- and PCN-dosed animals compared with untreated controls. Microsomes from ISO- and βNF-dosed males showed a reduction (P<0.05) in EMD activity when compared with control microsomes, and CF was without effect. In females, EMD activities were significantly increased in microsomes from PCN, DEX and PB-dosed but not βNF, ISO, or CF-dosed animals. As expected on the basis of sex-related differences in gene expression, EMD activities in untreated animals were considerably higher in males than females, attributable to constitutive CYP3A and CYP2C11 activities. The selectivity of EMD for induced CYP3A was confirmed on the basis of inhibition studies with selected steroid substrates of CYP3A, polyclonal anti-CYP3A1 antibodies and triacetyloleandomycin (TAO), a selective inhibitor of CYP3A. In conclusion, for both sexes, the greatest elevations (≈3–13-fold) in EMD activity were observed in microsomes from rats dosed with DEX, a potent archetypal inducer with lesser but significant increases noted for PCN and PB, indicating that EMD is a reliable indicator of induced rat hepatic cytochrome P450 CYP3A activity.     

Monospecific antipeptide antibodies against human hepatic UDP-glucuronosyltransferase 1A subfamily (UGT1A) isoforms

Expression of UDP-glucuronosyltransferases (UGT) in mammals is thought to be regulated in both a tissue- and developmental-specific manner. Furthermore, induction of genes encoding UGT occurs after exposure to xenobiotics including drugs, environmental pollutants and dietary compounds. In human, isoforms of UGT 1A subfamily catalyze the glucuronidation of a greater proportion of drugs, suggesting that the expression of UGT1A isoforms is responsible for the clearance of a diverse range of drugs. To analyze the expression of human UGT1A isoforms, we have developed polyclonal antibodies against specific peptide regions within the isoforms (UGT1A1, 1A3, 1A4, 1A6 and 1A9). The prepared antipeptide antibodies were found to be highly monospecific for each UGT1A isoform and no cross-reactivity with UGT2B isoforms was detected. Analysis of UGT1A protein levels in hepatic microsomes using these antibodies demonstrated interindividual differential expression of each isoform. These highly specific antipeptide antibodies provide an important tool to analyze tissue distribution and interindividual expression levels of human UGT1As.     

Substrate-dependent modulation of UDP-glucuronosyltransferase 1A1 (UGT1A1) by propofol in recombinant human UGT1A1 and human liver microsomes

Our previous study has shown that propofol, a probe substrate for human UDP-glucuronosyltransferase (UGT) 1A9, activated the glucuronidation of 4-methylumbelliferone (4-MU) by recombinant UGT1A1 in a concentration-dependent manner. In the present study, we investigated the mechanism of activation, and whether the stimulatory effect occurs when another substrate is used with human liver microsomes. The glucuronidation of 4-MU followed Michaelis-Menten kinetics with a K(m) value of 101 microM in the absence of propofol. In the presence of 200 microM propofol, a concentration that causes heterotopic activation of 4-MU glucuronidation (4-MUG), the V(max) value increased to 1.5-fold, while the K(m) value decreased to 0.53-fold. In order to assess whether propofol activates UGT1A1 activity for a substrate other than 4-MU, the effect of propofol on oestradiol 3beta-glucuronidation by recombinant UGT1A1 and in human liver microsomes was evaluated. In contrast to 4-MUG activity, propofol inhibited UGT1A1-catalysed oestradiol 3beta-glucuronidation in recombinant UGT1A1 as well as in human liver microsomes with IC(50) values of 59 and 228 microM, respectively. In addition, a known UGT1A1 modulator, 17alpha-ethynyloestradiol, stimulated oestradiol 3beta-glucuronidation slightly at a concentration of 5 microM, while it inhibited 4-MUG in recombinant UGT1A1 at all concentrations tested (5-100 microM). These findings indicate that the modulation of UGT1A1 by propofol is substrate-dependent, and thus care should be taken when extrapolating the stimulatory effects of drugs for one glucuronidation substrate.     

Protein-protein interactions between rat hepatic cytochromes P450 (P450s) and UDP-glucuronosyltransferases (UGTs): evidence for the functionally active UGT in P450-UGT complex

The interaction between cytochrome P450s (CYP, P450) and UDP-glucuronosyltransferases (UGTs) was studied by co-immunoprecipitation. P450 isoform-selective antibody was used as a probe to co-precipitate UGTs with the P450s from solubilized rat liver microsomes. Antibodies toward CYP3A2, CYP2B2, CYP2C11/13 and CYP1A2 co-precipitated UGTs with corresponding P450s. However, calnexin, a type-I membrane protein, in the endoplasmic reticulum was not co-precipitated by anti-P450 antibodies. UGT activity toward 4-methylumbelliferone was detected in all co-precipitates, suggesting that UGT in the complex with P450s is functionally active. Repeated washing of co-immunoprecipitates revealed differences among P450 isoforms with regard to the affinity for UGT. Larger amounts of UGT1A1 and UGT1A6, compared with UGT2B1, were washed out from UGTs-CYP2C11/13 co-precipitates, whereas UGT-CYP3A2 and UGT-CYP2Bs complexes were resistant to thorough washing. Thus, CYP2C11/13 could associate with UGTs, but the affinity is assumed to be weaker than that of CYP2B/3As. These results suggest that there is isoform specificity in the interaction between P450s and UGTs.     

Food-drug interactions via human cytochrome P450 3A (CYP3A)

Food-drug interactions have been reported to occur in various systems in the body. The causes of these interactions are mainly divided into pharmacodynamic and pharmacokinetic processes. Among these processes, drug metabolism plays a crucial role in drug interactions. Metabolic food-drug interactions occur when a certain food alters the activity of a drug-metabolizing enzyme, leading to a modulation of the pharmacokinetics of drugs metabolized by the enzyme. A variety of interactions have been documented so far. Foods consisting of complex chemical mixtures, such as fruits, alcoholic beverages, teas, and herbs, possess the ability to inhibit or induce the activity of drug-metabolizing enzymes. According to results obtained thus far, cytochrome P450 3A4 (CYP3A4) appears to be a key enzyme in food-drug interactions. For example, interactions of grapefruit juice with felodipine and cyclosporine, red wine with cyclosporine, and St John's wort with various medicines including cyclosporine, have been demonstrated. The results indicate the requirement of dosage adjustment to maintain drug concentrations within their therapeutic windows. The CYP3A4-related interaction by food components may be related to the high level of expression of CYP3A4 in the small intestine, as well as its broad substrate specificity, as CYP3A4 is responsible for the metabolism of more than 50% of clinical pharmaceuticals. This review article summarizes the findings obtained to date concerning food-drug interactions and their clinical implications. It seems likely that more information regarding such interactions will accumulate in the future, and awareness is necessary for achieving optimal drug therapy.     

In vitro inhibition of human liver cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes by rose bengal: system-dependent effects on inhibitory potential

Abstract

1. Rose bengal (4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein) is being developed for the treatment of cutaneous melanoma and hepatocellular carcinoma. Interestingly, rose bengal can generate singlet oxygen species upon exposure to light.

2. We evaluated rose bengal as an in vitro inhibitor of cytochrome P450 (CYP) or UDP-glucuronosyltransferase (UGT) enzymes in both human liver microsomes (HLM) and cryopreserved human hepatocytes (CHHs) under both yellow light and dark conditions.

3. Rose bengal directly inhibited CYP3A4/5 and UGT1A6 in HLM under yellow light with inhibitor concentration that causes 50% inhibition (IC₅₀) values of 0.072 and 0.035?μM, respectively; whereas much less inhibition was observed in the dark with the IC₅₀ values increasing 43- and 120-fold, respectively. To determine if a more physiologically-relevant test system could be protected from such an effect, rose bengal was evaluated as an inhibitor of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4/5 and UGT enzymes in CHH. All IC₅₀ values were similar (64?±?8?μM) and little to no effect of light on inhibitory potential was observed.

4. Given the IC₅₀ values in CHH increased an order of magnitude compared to HLM and the atypical pharmacokinetics of the drug, the risk of rose bengal to cause clinically relevant drug–drug interactions is likely low, particularly when administered to cancer patients on an intermittent schedule.     

Cytochrome P450IIIA enzymes in rat liver microsomes: involvement in C3-hydroxylation of diazepam and nordazepam but not N-dealkylation of diazepam and temazepam

Microsomes prepared from livers of male and female rats of nine inbred and outbred strains and of male Sprague-Dawley rats pretreated with monooxygenase-inducing agents were used to study N-dealkylation of diazepam and temazepam and C3-hydroxylation of diazepam and nordazepam. Both C3-hydroxylation reactions were more rapid in male than in female liver preparations, but this gender-dependent pattern was not seen with the N-dealkylation reactions. These results indicate the lack of identity of the monooxygenases responsible for the two kinds of reaction and suggest that male-specific enzyme(s) are responsible for the C3-hydroxylations. Induction studies were undertaken to further define these enzymes. To do this, liver microsomes prepared from male Sprague-Dawley rats pretreated with a variety of agents known to have different monooxygenase induction effects were used. With triacetyloleandomycin, dexamethasone, and phenobarbital pretreatment, the specific activities of the C3-hydroxylation reactions were selectively elevated over corresponding control values. These particular xenobiotics are known to enhance the abundance of cytochrome P450IIIA family enzymes, and our results strongly suggest the involvement of these enzymes in the benzodiazepine B ring monooxygenations. Formation of temazepam was also shown to be inhibited by triacetyloleandomycin. This effect was demonstrated to be equal in both saline-treated and dexamethasone-treated male Sprague-Dawley rat liver microsomes, with the antibiotic present either with diazepam throughout the entire incubation period or initially with NADPH in a preincubation mix for 15 min, following which C3-hydroxylation was initiated by the addition of diazepam. These results confirm the uniformity of the involvement of cytochrome P450IIIA family enzymes in diazepam C3-hydroxylation in untreated and inducer-treated rat liver microsomes. Recent studies with human and rabbit liver microsomal preparations have shown that orthologues of these enzymes also catalyze an equivalent hydroxylation in the B ring of midazolam. These findings, considered with the present results showing that the adjacent methyl N-substituent (absent in nordazepam but present in diazepam) did not affect the selectivity of these enzymes for the C3-hydroxylation reaction, suggest that neither steric nor electronic factors markedly influence catalysis of this monooxygenation by these enzymes.     

Effects of cytochrome P450 (CYP) inducers and inhibitors on ondansetron pharmacokinetics in rats: involvement of hepatic CYP2D subfamily and 3A1/2 in ondansetron metabolism

The types of hepatic microsomal cytochrome P450 (CYP) isozymes responsible for the in-vivo metabolism of ondansetron in rats have not been reported. In this study, ondansetron at a dose of 8 mg kg(-1) was administered intravenously to rats pretreated with various inducers of CYP isozymes, such as 3-methylcholanthrene, orphenadrine citrate, isoniazid and dexamethasone phosphate (the main inducers of CYP1A1/2, 2B1/2, 2E1 and 3A1/2 in rats, respectively), and inhibitors, such as SKF-525A (a non-specific inhibitor of CYP isozymes), sulfaphenazole, quinine hydrochloride and troleandomycin (the main inhibitors of CYP2C6, 2D subfamily and 3A1/2 in rats, respectively). In rats pretreated with quinine hydrochloride and troleandomycin, the time-averaged non-renal clearance of ondansetron was significantly slower (48.9 and 13.2% decrease, respectively) than that in control rats. In rats pretreated with dexamethasone phosphate, the time-averaged non-renal clearance was significantly faster (18.2% increase) than that in control rats. The results suggest that ondansetron is primarily metabolized via the CYP2D subfamily and 3A1/2 in rats.     

Differential modulation of UDP-glucuronosyltransferase 1A1 (UGT1A1)-catalyzed estradiol-3-glucuronidation by the addition of UGT1A1 substrates and other compounds to human liver microsomes.

Previous results demonstrating homotropic activation of human UDP-glucuronosyltransferase (UGT) 1A1-catalyzed estradiol-3-glucuronidation led us to investigate the effects of 16 compounds on estradiol glucuronidation by human liver microsomes (HLM). In confirmation of previous work using alamethicin-treated HLM pooled from four livers, UGT1A1-catalyzed estradiol-3-glucuronidation demonstrated homotropic activation kinetics (S(50) = 22 microM, Hill coefficient, n = 1.9) whereas estradiol-17-glucuronidation (catalyzed by other UGT enzymes) followed Michaelis-Menten kinetics (K(m) = 7 microM). Modulatory effects of the following compounds were investigated: bilirubin, eight flavonoids, 17alpha-ethynylestradiol (17alpha-EE), estriol, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), anthraflavic acid, retinoic acid, morphine, and ibuprofen. Although the classic UGT1A1 substrate bilirubin was a weak competitive inhibitor of estradiol-3-glucuronidation, the estrogens and anthraflavic acid activated or inhibited estradiol-3-glucuronidation dependent on substrate and effector concentrations. For example, at substrate concentrations of 5 and 10 microM, estradiol-3-glucuronidation activity was stimulated by as much as 80% by low concentrations of 17alpha-EE but was unaltered by flavanone. However, at higher substrate concentrations (25-100 microM) estradiol-3-glucuronidation was inhibited by about 55% by both compounds. Anthraflavic acid and PhIP were also stimulators of estradiol 3-glucuronidation at low substrate concentrations. The most potent inhibitor of estradiol 3-glucuronidation was the flavonoid tangeretin. The UGT2B7 substrates morphine and ibuprofen had no effect on estradiol 3-glucuronidation, whereas retinoic acid was slightly inhibitory. Estradiol-17-glucuronidation was inhibited by 17alpha-EE, estriol, and naringenin but was not activated by any compound. This study demonstrates that the interactions of substrates and inhibitors at the active site of UGT1A1 are complex, yielding both activation and competitive inhibition kinetics.     

Effect of antifungal drugs on cytochrome P450 (CYP) 1A2, CYP2D6, and CYP2E1 activities in human liver microsomes

The effects of five antifungal drugs, fluconazole, itraconazole, micafungin, miconazole, and voriconazole, on cytochrome P450 (CYP) 1A2-mediated 7-ethoxyresorufin O-deethylation, CYP2D6-mediated debrisoquine 4-hydroxylation, and CYP2E1-mediated chlorzoxazone 6-hydroxylation activities in human liver microsomes were compared. In addition, the effect of preincubation was estimated in order to investigate the mechanism-based inhibition. IC50 values of miconazole against CYP1A2 and CYP2D6 activities were 2.90 and 6.46 microM, respectively, and miconazole at 10 microM concentration slightly inhibited CYP2E1 activity. On the other hand, other antifungal drugs neither inhibited nor stimulated all of the metabolic activities. The stimulation of the inhibition of the metabolic activities mediated by CYP1A2, CYP2D6, or CYP2E1 by 15-min preincubation was not observed for any of the antifungal drugs, suggesting that these antifungal drugs are not mechanism-based inhibitors. These results suggest that miconazole is the strongest inhibitor against CYP1A2, CYP2D6, and CYP2E1 among the antifungal drugs investigated.     

Stereoselective glucuronidation of 5-(4'-hydroxyphenyl)-5-phenylhydantoin by human UDP-glucuronosyltransferase (UGT) 1A1, UGT1A9, and UGT2B15: effects of UGT-UGT interactions.

5-(4'-Hydroxyphenyl)-5-phenylhydantoin (4'-HPPH), a major metabolite of phenytoin in human, is exclusively metabolized to a glucuronide. 4'-HPPH has a chiral center. (S)-4'-HPPH is a predominant form produced from phenytoin in humans, and (R)-4'-HPPH is an extremely toxic form with respect to gingival hyperplasia. In the present study, we investigated stereoselective 4'-HPPH O-glucuronide formation in human liver microsomes. Human liver microsomes predominantly formed (S)-4'-HPPH O-glucuronide rather than (R)-4'-HPPH O-glucuronide from racemic 4'-HPPH. Among human UDP-glucuronosyltransferase (UGT) enzymes, UGT1A1, UGT1A9, and UGT2B15 showed 4'-HPPH O-glucuronide formation. Interestingly, UGT1A1 stereoselectively formed (R)-4'-HPPH O-glucuronide, whereas UGT1A9 and UGT2B15 stereoselectively formed (S)-4'-HPPH O-glucuronide from racemic 4'-HPPH. By using UGT1A double-expression systems in HEK293 cells that we previously established, the effects of UGT-UGT interactions on 4'-HPPH O-glucuronide formation were investigated. It was demonstrated that coexpression of UGT1A4 increased the V(max) values of (S)- and (R)-4'-HPPH O-glucuronide formation catalyzed by UGT1A1 but decreased the V(max) values of (S)- and (R)-4'-HPPH O-glucuronide formation catalyzed by UGT1A9. Coexpression of UGT1A6 increased the S(50) values and decreased the V(max) values of (S)- and (R)-4'-HPPH glucuronide formation catalyzed by UGT1A1 and UGT1A9. However, the interaction did not alter the stereoselectivity. In conclusion, we found that 4'-HPPH O-glucuronide formation in human liver microsomes is catalyzed by UGT1A1, UGT1A9, and UGT2B15 in a stereoselective manner, being modulated by interaction with other UGT1A isoforms.     

Cytochrome P4503A (CYP3A) metabolism: Prediction ofIn Vivo activity in humans

CYP3A is one of the most important cytochrome P450 isoforms responsible for drug metabolism by humans because it is the major such enzyme in critical tissues such as the gastrointestinal tract and liver, and it is involved in the oxidative biotransformation of numerous clinically useful therapeutic agents. Many factors regulate CYP3A expression but these are being increasingly defined so that the disposition characteristics of a drug whose metabolism is importantly mediated by this isoform can be reasonably well predicteda priori. For example, metabolic clearance is distributed within a population in a unimodal fashion but marked (5- to 20-fold) interindividual variability is present as a consequence of both genetic and nongenetic factors. In addition, firstpass metabolism occurs following oral drug administration and this may be extensive so that bioavailability is low. CYP3A activity can also be readily modulated by inducers like rifampicin and several anticonvulsant agents, and many potent inhibitors exist such as azole antifungal agents and macrolide antibiotics. Accordingly, the potential for drug interactions with these drugs as well as other CYP3A substrates, when given concomitantly, is high. Metabolism involving CYP3A is also likely to be affected by liver disease as well as aging, and modest differences may be present between men and women but these are often clinically unimportant. Because of such predictability, knowledge of the role and importance of CYP3A in the metabolism of a putative drug candidate is becoming increasingly desirable at an early stage in the development process.In vitro studies using human liver preparations, including microsomes, cultured hepatocytes and heterologous expressed enzymes, can provide important insights in this regard. This is particularly the case for identifying potential drug interactions whose clinical significance can be subsequently assessed. Data with respect to terfenadine and cyclosporine obtained several years after their approval and marketing, indicate that, if available and applied during their development, the paradigm of usingin vitro studies to rationally direct and prioritize clinical studies would have prospectively prevented the serious adverse effects and inefficacy that were only recognized during their empiric clinical use. Such examples, along with those associated with the genetic polymorphism of CYP2D6, provide strong justification for establishing the role and importance of individual CYP isoforms in a candidate drug's metabolism at an early stage. Supported in part by grant GM31304 from the U.S. Public Health Service.     

Alteration in the Expression of Cytochrome P450s (CYP1A1, CYP2E1, and CYP3A11) in the Liver of Mouse Induced by Microcystin-LR

Microcystins (MCs) are cyclic heptapeptide toxins and can accumulate in the liver. Cytochrome P450s (CYPs) play an important role in the biotransformation of endogenous substances and xenobiotics in animals. It is unclear if the CYPs are affected by MCs exposure. The objective of this study was to evaluate the effects of microcystin-LR (MCLR) on cytochrome P450 isozymes (CYP1A1, CYP2E1, and CYP3A11) at mRNA level, protein content, and enzyme activity in the liver of mice the received daily, intraperitoneally, 2, 4, and 8 µg/kg body weight of MCLR for seven days. The result showed that MCLR significantly decreased ethoxyresorufin-O-deethylase (EROD) (CYP1A1) and erythromycin N-demthylase (ERND) (CYP3A11) activities and increased aniline hydroxylase (ANH) activity (CYP2E1) in the liver of mice during the period of exposure. Our findings suggest that MCLR exposure may disrupt the function of CYPs in liver, which may be partly attributed to the toxicity of MCLR in mice.     

Genetic variation of Cytochrome P450 1B1 (CYP1B1) and risk of breast cancer among Polish women

Four single nucleotide polymorphisms (SNPs) in CYP1B1 (Ex2 + 143 C > G, Ex2 + 356 G > T, Ex3 + 251 G > C, Ex3 + 315 A > G) cause amino acid changes (R48G, A119S, L432V and N453S, respectively) and are associated with increased formation of catechol estrogens; however, epidemiologic evidence only weakly supports an association between these variants and breast cancer risk. Because genetic variability conferring increased susceptibility could exist beyond these putative functional variants, we comprehensively examined the common genetic variability within CYP1B1. A total of eight haplotype-tagging (ht)SNPs (including Ex3 + 315 A > G), in addition to two putatively functional SNPs (Ex2 + 143 C > G and Ex3 + 251 G > C), were selected and genotyped in a large case-control study of Polish women (1995 cases and 2296 controls). Haplotypes were estimated using the expectation-maximization algorithm, and overall differences in the haplotype distribution between cases and controls were assessed using a global score test. We also evaluated levels of tumor CYP1B1 protein expression in a subset of 841 cases by immunohistochemistry, and their association with genetic variants. In the Polish population, we observed two linkage disequilibrium (LD)-defined blocks. Neither haplotypes (global P-value of 0.99 and 0.67 for each block of LD, respectively), nor individual SNPs (including three putatively functional SNPs) were associated with breast cancer risk. CYP1B1 was expressed in most tumor tissues (98%), and the level of expression was not related to the studied genetic variants. We found little evidence for modification of the estimated effect of haplotypes or individual SNPs by age, family history of breast cancer, or tumor hormone receptor status. The present study provides strong evidence against the existence of a substantial overall association between common genetic variation in CYP1B1 and breast cancer risk.     

A study for regulation of ethanol-inducible P450(CYP2E1) on CCl4-induced hepatic damage

Archives of Pharmacal Research - Previous study showed that CCl4 administration evoked a rapid decrease in cytochrome P450 2E1 protein soon after the exposure due to posttranslational...     

Cytochrome P450 2C8 (CYP2C8)-mediated hydroxylation of an endothelin ETA receptor antagonist in human liver microsomes.

In vitro studies were performed to identify the human cytochrome P450 enzyme(s) involved in the hydroxylation (isopropyl moiety) of a previously reported endothelin ET(A) receptor antagonist, compound A [(+)-(5S,6R,7R)-2-isopropylamino-7-(4-methoxy-2-[(2R)-3-methoxy-2-methylpropyl])-5-(3,4-methylenedioxyphenyl)cyclopenteno(1,2-b) pyridine 6-carboxylic acid]. Several lines of evidence indicated that the reaction was mainly catalyzed by CYP2C8. Of the 10 recombinant cytochrome P450 isoforms tested, only CYP2C8 exhibited hydroxylase activity. In agreement, inhibitory antibodies selective for CYP2C8 attenuated (>95%) the hydroxylase activity in human liver microsomes, whereas antibodies and chemical inhibitors selective for other cytochrome P450 isoforms had a minor or no effect on the reaction. In addition, the formation of the hydroxy metabolite correlated well with CYP2C8-selective paclitaxel 6alpha-hydroxylation (r(2) approximately 0.92; p < 0.0001) and amodiaquine N-de-ethylation (r(2) approximately 0.91; p < 0.0001) in a bank of human liver microsomes (n = 15 organ donors). Finally, compound A hydroxylase activity conformed to Michaelis-Menten kinetics, and the K(m) (Michaelis constant) in human liver microsomes was similar to that of CYP2C8 ( approximately 10 microM). It is concluded that the hydroxylation of compound A is mainly catalyzed by CYP2C8, and thus the reaction can possibly serve as an alternative marker assay for CYP2C8 in human liver microsomes.     

Artemisinin and thiabendazole are potent inhibitors of cytochrome P450 1A2 (CYP1A2) activity in humans

Objective To investigate the likelihood of artemisinin and thiabendazole causing pharmacokinetic interactions involving cytochrome P450 (CYP1A2) in humans given their potent inhibitory effects on the isoform in vitro. Methods Ten healthy volunteers received caffeine (136.5 mg), and after a washout period of 48 h, the volunteers were given a caffeine tablet (136.5 mg) together with thiabendazole (500 mg). After an additional 14 days, the volunteers received caffeine together with artemisinin (500 mg). After each treatment, plasma was obtained up to 24 h post-dose. The plasma concentrations of the drugs were measured by HPLC with UV and MS detection. Results Using the ratio of paraxanthine to caffeine after 4 h as an indicator of CYP1A2 activity, thiabendazole and artemisinin inhibited 92 and 66%, respectively, of the enzyme activity in vivo. In addition, the pharmacokinetics of caffeine were altered in the presence of the drugs; increases in AUC_0–24 of 1.6-fold (P<0.01) and 1.3-fold of caffeine in the presence of thiabendazole and artemisinin respectively were measured. The use of in vitro data to predict the effects of thiabendazole on the formation of paraxanthine yielded good results and underestimated the effects of artemisinin when total plasma concentrations were used. Corrections for protein binding resulted in underestimation of inhibitory effects on CYP1A2. Conclusions Co-administration of thiabendazole or artemisinin with CYP1A2 substrates could result in clinically significant effects. Our results highlight the validity of in vitro data in predicting in vivo CYP inhibition. The formation of paraxanthine seems to be a better indicator of in vivo CYP1A2 activity than caffeine levels.     

Effects of cytochrome b(5) on drug oxidation activities of human cytochrome P450 (CYP) 3As: similarity of CYP3A5 with CYP3A4 but not CYP3A7

Effects of cytochrome b(5) (b(5)) on catalytic activities of human cytochrome P450 (CYP) 3A5, CYP3A4, and CYP3A7 coexpressed with human NADPH-cytochrome P450 reductase in Escherichia coli membranes were investigated using 14 substrates. The activities of CYP3A5 were enhanced by addition of b(5) in approximately one third of the substrates employed in this study. Such enhancement by b(5) was roughly similar to that of CYP3A4, while the activities of CYP3A7 were not enhanced by b(5) with any substrates employed. V(max) values for midazolam 1'-hydroxylation and amitriptyline N-demethylation by CYP3A5 were increased about twice by addition of b(5), which was also seen with CYP3A4, although the extent of the effects of b(5) on S(50) (K(m)) and Hill coefficient differed dependent on substrates used. In contrast, b(5) did not alter any of these kinetic parameters of CYP3A7. The effects of b(5) on kinetic parameters of CYP3A5 were similar to those of CYP3A4 but not CYP3A7. These results suggest that roles of b(5) in drug oxidation activities of CYP3A5 and CYP3A4 are different from those of CYP3A7.     

Ephedra water decoction and cough tablets containing ephedra and liquorice induce CYP1A2 but not CYP2E1 hepatic enzymes in rats

1. Ephedra water decoction (EWD) and cough tablets containing ephedra and liquorice (maxing cough tablets, MXCT) have been widely used in the treatment of asthma. In the clinic, EWD and MXCT may be prescribed with theophylline, one of the most popular antiasthmatic drugs. CYP1A2 and CYP2E1 are mainly involved in the oxidative metabolism of theophylline in human liver. Drug interactions involving the cytochrome P450 (CYP) isoforms generally are of two types: enzyme induction or enzyme inhibition. Enzyme inhibition reduces metabolism, whereas induction can increase it.

2. To evaluate the pretreatment effect of EWD and MXCT on CYP1A2 and CYP2E1, CYP1A2 and CYP2E1 activity, the protein expression and mRNA expression levels were determined. After pretreatment with EWD or MXCT, the enzyme activity, mRNA expression and protein expression of CYP1A2 were increased significantly (p?<?0.05), but enzyme activity of CYP2E1 did not change compared with the control.

3. It was demonstrated that EWD or MXCT pretreatment obviously induced CYP1A2, therefore, in patients taking EWD or MXCT, possible CYP-induced drug interaction should be noted to decrease the risk of therapeutic failure or adverse effects resulting from the use of additional therapeutic agents.     

Progesterone metabolism in recombinant yeast simultaneously expressing bovine cytochromes P450c17 (CYP17A1) and P450c21 (CYP21B1) and yeast NADPH-P450 oxidoreductase.

Simultaneous expression plasmids were constructed for bovine adrenal cytochromes P450c17 and P450c21 (pA gamma alpha) and for both P450s together with NADPH-cytochrome P450 reductase (pAR gamma alpha). On introduction of each of the plasmids into Saccharomyces cerevisiae AH22 cells, the transformed yeast strains AH22/pA gamma alpha and AH22/pAR gamma alpha produced about 10(5) molecules per cell of P450c17 and 2 x 10(3) molecules per cell of P450c21. The expression levels of NADPH-cytochrome P450 reductase was about 3 x 10(4) and 6 x 10(5) molecules per cell in the strains AH22/pA gamma alpha and AH22/pAR gamma alpha, respectively. When progesterone was added to growing cell cultures of the transformed yeast strains, the substrate was metabolized more rapidly in the AH22/pAR gamma alpha cells than AH22/pA gamma alpha cells, probably due to overproduction of the reductase. In the AH22/pAR gamma alpha cells, progesterone was first converted into 17 alpha-hydroxyprogesterone to the extent of 82% by the catalysis of P450c17. 17 alpha-hydroxyprogesterone was further converted into 11-deoxycortisol by P450c21 to the extent of 60% of the added substrate. The conversion of progesterone into androstenedione through 17 alpha-hydroxyprogesterone was estimated to be less than 3%, suggesting very low C17,20-lyase activity of P450c17, although other hydroxylation products were detected. Androstenedione was further converted into testosterone by an unknown pathway present in S. cerevisiae cells.