Cytochrome P450 Protein Contents Research Articles

Roles of maize cytochrome P450 (CYP) enzymes in stereo-selective metabolism of hexabromocyclododecanes (HBCDs) as evidenced by in vitro degradation, biological response and in silico studies

In vitro biotransformation of HBCDs by maize cytochrome P450 (CYP) enzymes, responses of CYPs to HBCDs at protein and transcription levels, and in silico simulation of interactions between CYPs and HBCDs were investigated in order to elucidate the roles of CYPs in the metabolism of HBCDs in maize. The results showed that degradation reactions of HBCDs by maize microsomal CYPs followed the first-order kinetics and were stereo-selective, with the metabolic rates following the order (−)γ- > (+)γ- > (+)α- > (−)α-HBCD. The hydroxylated metabolites OH-HBCDs, OH-PBCDs and OH-TBCDs were detected. (+)/(−)-α-HBCDs significantly decreased maize CYP protein content and inhibited CYP enzyme activity, whereas (+)/(−)-γ-HBCDs had obvious effects on the induction of CYPs. HBCDs selectively mediated the gene expression of maize CYPs, including the isoforms of CYP71C3v2, CYP71C1, CYP81A1, CYP92A1 and CYP97A16. Molecular docking results suggested that HBCDs could bind with these five CYPs, with binding affinity following the order CYP71C3v2 < CYP81A1 < CYP97A16 < CYP92A1 < CYP71C1. The shortest distances between the Br-unsubstituted C atom of HBCD isomers and the iron atom of heme in CYPs were 4.18–11.7 Å, with only the distances for CYP71C3v2 being shorter than 6 Å (4.61–5.38 Å). These results suggested that CYP71C3v2 was an efficient catalyst for degradation of HBCDs. For (+)α- and (−)γ-HBCDs, their binding affinities to CYPs were lower and the distances to the iron atom of heme in CYPs were shorter than their corresponding antipodes, consistent with the in vitro experimental results showing that they had shorter half-lives and were more easily hydroxylated. This study provides solid evidence for the roles of maize CYPs in the metabolism of HBCDs, and gives insight into the molecular mechanisms of the enantio-selective metabolism of HBCDs by plant CYPs.

Liver lobe and strain differences in the activity of murine cytochrome P450 enzymes

The cytochrome P450 (CYP) enzyme superfamily is the most important enzyme system for phase I biotransformation. For toxico- and pharmacokinetic studies, use of liver-based microsomes, including those of mice, is state-of-the-art to study CYP-dependent metabolism. However, reproducibility and interpretation of these data is still very variable, partly because current testing guidelines do not cover details on organ sampling and potential liver lobe differences. Hence, we analyzed CYP activity, CYP protein content, mRNA expression of CYP1A, CYP2C, CYP2D and CYP3A isozymes, and cytochrome P450 reductase (CPR) activity of the four different liver lobes and processus papillaris of male C57BL/6J mice in comparison to whole liver. Additionally, we used whole liver of Balb/cJ and 129S1/SvImJ for strain comparison.Our data show significant differences in CYP activity, being most prominent in lobus sinister lateralis and lobus medialis, and lowest in processus papillaris. These differences were not caused by varying Cyp gene expression or CYP protein level, but partly correspond with lobe specific CPR activities. We also observed significant strain differences in CYP mRNA expression and activities with overall high activities in 129S1/SvImJ mice and low activities in Balb/cJ mice compared to C57BL/6J mice. In addition, strain specific differences in CYP2C and CYP2D activity seem to be reflected in strain dependent differences in CPR activity.In summary, our results indicate that in mice CYP activity and gene expression are strain dependent and may vary highly between liver lobes. To ensure reproducibility and comparability of different probes and studies, this should be taken into account when liver samples are collected for the analysis of CYP-dependent metabolism.

Evaluation of cytochrome P450 2C9 activity in normal, healthy, adult Western Indian population by both phenotyping and genotyping.

Objectives:Cytochrome P450 2C9 (CYP2C9) is a member of cytochrome P450 (CYP) family that accounts for nearly 18% of the total CYP protein content in the human liver microsomes and catalyzes almost 15–20% of the drugs. Considering the paucity of data on the polymorphisms of CYP2C9 in Western Indian population, the present study was conducted to evaluate the prevalence of CYP2C9 polymorphisms (*1, *2 and *3) and correlate it with the activity using flurbiprofen (FLB) as a probe drug.Materials and Methods:A 100 mg FLB capsule was administered to 298 healthy adult participants. Venous blood samples were analyzed at 2 h postdose for the estimation of FLB and 4-hydroxy FLB. Metabolic ratio (MR) was calculated to determine the extent of poor metabolizer (PM) and rapid metabolizer status using probit plot. Genotyping of CYP2C9 polymorphism was performed using polymerase chain reaction-restriction fragment length polymorphism technique.Results:Of the total 298 participants, phenotype was assessable in 288 and genotype was performed in 289 participants. The median (range) MR of the study population was 6.6 (1.65–66.05). Five participants were found to be PMs by phenotype. Of the total 289 participants, 209 (72.3%) (66.7, 77.2) had CYP2C9*1/*1, 25 (8.7%) (5.8, 12.7) with CYP2C9*1/*2, 55 (19%) (14.8, 24.1) had CYP2C9*1/*3, 3 (1%) (0.3, 3.3) had CYP2C9*2/*3 genotype. A significant association between phenotype and genotype was observed.Conclusion:To conclude, the present study found significant association of CYP2C9 activity by both phenotype and genotype and these findings have to be corroborated in different kinds of patients.

Quantitative Prediction of the Impact of Drug Interactions and Genetic Polymorphisms on Cytochrome P450 2C9 Substrate Exposure

Cytochrome P450 (CYP) 2C9 is the most common CYP2C enzyme and makes up approximately onethird of total CYP protein content in the liver. It metabolises more than 100 drugs. The exposure of drugs mainly eliminated by CYP2C9 may be dramatically modified by drug-drug interactions (DDIs) and genetic variations. The objective of this study was to develop a modelling approach to predict the impact of genetic polymorphisms and DDIs on drug exposure in drugs metabolised by CYP2C9. We then developed dosing recommendations based on genotypes and compared them to current Epar/Vidal dosing guidelines. We created two models. The genetic model was designed to predict the impact of CYP2C9 polymorphisms on drug exposure. It links the area under the concentration-time curve (AUC) ratio (mutant to wild-type patients) to two parameters: the fractional contribution of CYP2C9 to oral clearance in vivo (i.e. CR or contribution ratio), and the fractional activity of the allele combination with respect to the homozygous wild type (i.e. FA or fraction of activity). Data were available for 77 couples (substrate, genotype). We used a three-step approach: (1) initial estimates of CRs and FAs were calculated using a first bibliographic dataset; (2) external validation of these estimates was then performed through the comparison between the AUC ratios predicted by the model and the observed values, using a second published dataset; and (3) refined estimates of CRs and FAs were obtained using Bayesian orthogonal regression involving the whole dataset and initial estimates of CRs and FAs. Posterior distributions of AUC ratios, CRs and FAs were estimated using Monte-Carlo Markov chain simulation. The drug interaction model was designed to predict the impact of DDIs on drug exposure. It links the AUC ratio (ratio of drug given in combination to drug given alone) to several parameters: the CR, the inhibition ratio (IR) of an inhibitor, and the increase in clearance (IC) due to an inducer. Data were available for 80 DDIs. IRs and ICs were calculated using the interaction model and an external validation was performed. Doses adjustments were calculated in order to obtain equal values for drug exposure in extensive and poor metabolisers and then compared to Epar/Vidal dosing guidelines. CRs were assessed for 26 substrates, FAs for five genotype classes including CYP2C9*2 and *3 allelic variants, IRs for 27 inhibitors and ICs for two inducers. For the genetic model, the mean prediction error of AUC ratios was -0.01, while the mean prediction absolute error was 0.36. For the drug interaction model, the mean prediction error of AUC ratios was 0.01, while the mean prediction absolute error was 0.22. Of the 26 substrates and CYP2C9*2 and *3 variants investigated, 30 couples (substrate, genotype) lead to a dose adjustment, as opposed to only ten couples identified in the Epar/Vidal recommendations. These models were already used for CYP2D6. They are accurate at predicting the impact of drug interactions and genetic polymorphisms on CYP2C9 substrate exposure. This approach will contribute to the development of personalized medicine, i.e. individualized drug therapy with specific dosing recommendations based on CYP genotype or drug associations.

T1014 Bariatric Surgery-Induced Weight Loss Is Associated with Changes in Hepatic Cytochrome P-450 Protein Content

T1014 Bariatric Surgery-Induced Weight Loss Is Associated with Changes in Hepatic Cytochrome P-450 Protein Content

Noscapine may increase the effect of warfarin

The Drug Information Centre at the Karolinska University Hospital was contacted in February 2006 regarding a suspected case of adverse interaction between noscapine (antitussive drug) and warfarin (anticoagulant). This case was later reported to the Swedish adverse drug interactions register (SWEDIS). An 82-year-old male on stable maintenance warfarin was prescribed noscapine due to a difficult cough. International Normalized Ratio (INR) was regularly monitored every sixth week. The last INR (3 weeks before noscapine) was 3.0. After 6 days of noscapine (50 mg tid) INR was 7.2. Noscapine was stopped, phytonadione (vitamin K) was given, and warfarin withheld for 1 day. INR was 2.3 the day after and remained stable on a marginally reduced warfarin dose. A number of drugs may interact with warfarin, mostly due to its effect on the metabolism. S-warfarin is mainly metabolized by the cytochrome P450 (CYP) 2C9 enzyme. The less active R-warfarin is mainly metabolized by CYP3A4 and 1A2 [1]. So far it is unknown if noscapine may interact with warfarin. In order to investigate the possible interaction between noscapine and warfarin, literature search and an in vitro experiment were performed. In addition to the SWEDIS, PubMed, Embase, the Swedish–Finnish drug interaction database (SFINX), Stockley Online and other pharmacological literature were searched for interactions between warfarin and noscapine. Disproportionality analyses were conducted to determine whether the reports in SWEDIS indicated an adverse drug reaction (ADR) signal different from background reporting. Two methods were used: Bayesian confidence propagation neural network (BCPNN) and proportional reporting ratio (PRR) [2]. In vitro testing with recombinant human enzyme was conducted to examine whether noscapine inhibits CYP2C9 and/or CYP3A4 (Vivid® CYP2C9 and CYP3A4 Green Screening Kit; Invitrogen Corp., Carlsbad, CA, USA). Up to December 2006 a total of eight cases of suspected interaction between noscapine and warfarin had been reported to SWEDIS. The reported ADR was bleeding in one case and increased INR in the remaining cases. The ADR occurred after 3 days to 3 weeks of concomitant noscapine medication. INR returned to previous levels in the four cases where follow-up was reported (Table 1). Other drugs were present in most cases, but only noscapine was changed unless otherwise specified in Table 1. The signal showed to be significant using both BCPNN and PRR methods. The Bayesian method gave an information component (IC) value of 2.15, and IC − 2SD = 0.704 (IC > 0 indicates association, IC − 2SD > 0 indicates a statistically significant association), whereas the PRR was 11.9 (95% confidence interval 4.1, 34.4) (PRR > 1 indicates association). Results from in vitro testing indicate that noscapine may inhibit CYP2C9 and CYP3A4 with an IC50 of around 1 μm (data not shown). The maximum concentration after a single 50-mg dose of noscapine approximates to 0.15 μm[3]. Pharmacokinetic studies using repeated dosing of noscapine have not been found in the literature. The clinical relevance of these in vitro results remains to be explored. High-dose noscapine (200 mg kg−1 bodyweight) has previously been shown to reduce the CYP protein content in the liver of mice [4]. The relevance to man is unclear, as are the mechanisms of noscapine metabolism [3]. Noscapine is used against coughing, often related to inflammation. Inflammatory conditions have been associated with downregulation of certain CYP enzymes [5]. However, no relevant information has been found in the literature on the potential interactions between warfarin and noscapine or other antitussives. In summary, we have identified eight cases of a suspected interaction between warfarin and noscapine in the Swedish ADR database. All cases indicate that noscapine may increase the anticoagulative effects of warfarin. In vitro testing suggests that noscapine inhibits CYP2C9 and CYP3A4. As noscapine is a widely used and readily available over-the-counter antitussive, this potential interaction deserves attention. However, further studies are needed to confirm this and to elucidate further the possible mechanisms. This work was in part supported by grants from the Swedish Research Council (Medicine 04496/Rane).

Caffeine metabolism by human hepatic cytochromes p450: Contributions of 1A2, 2E1 and 3A isoforms

Caffeine (CA) N1-, N3- and N7-demethylase, CA 8-hydroxylase and phenacetin O-deethylase activities were measured in microsomes from 18 separate human livers which had been characterized previously for a range of cytochrome P450 (CYP) isoform-specific activities and immunoreactive CYP protein contents. Correlations between the high affinity components of the three separate CA N-demethylations were highly significant ( r = 0.77–0.91, P < 0.001) and each of the three high affinity CA N-demethylations correlated significantly ( r = 0.64–0.93, P < 0.05-0.001) with the high affinity phenacetin O-deethylase, 2-acetylaminofluorene N-hydroxylation and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5- f]quinoline (IQ) mutagenicity (all predominantly CYP1A2-mediated reactions). Consistent with these observations, cDNA-expressed human CYP1A2 catalyzed the N1-, N3- and N7-demethylation of CA and apparent K m values were similar (0.24–0.28 mM) for all three reactions and comparable to those observed previously with human liver microsomes. The low affinity components of CA N1- and N7-demethylation correlated significantly ( r = 0.55–0.85, P < 0.05-0.001) with immunoreactive CYP2E1 content and the CYP2E1-specific activities 4-nitrophenol and chlorzoxazone hydroxylation. Diethyldithiocarbamate, a selective inhibitor of CYP2E1, inhibited the low affinity CA N1- and N7-demethylation, with IC 50 values of 23 μM and 11 μM, respectively. The apparent K m values for CA N1- and N7-demethylation by cDNA-expressed CYP2E1 (namely 28 and 43 mM, respectively) were of a similar order to those calculated for the low affinity microsomal activities. Significant correlations ( r = 0.87–0.97, P < 0.001) were observed between CA 8-hydroxylation and immunoreactive CYP3A content and the CYP3A-mediated reactions benzo( a)pyrene hydroxylation, omeprazole sulfoxidation and aflatoxin B1 mutagenesis. Effects of α-naphthoflavone, erythromycin, troleandomycin and nifedipine on microsomal CA 8-hydroxylation were generally consistent with CYP3A involvement. Taken together with previous data, the results indicate a major involvement of CYP1A2 in the high affinity component of all three human hepatic CA N-demethylations. In contrast, CYP2E1 appears to be the main enzyme involved in the low affinity components of CA N1- and N7-demethylation while CA 8-hydroxylation is catalysed predominantly by a CYP3A isoform(s).