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The Functional Role of CYP2B6 in Human Drug Metabolism: Substrates and Inhibitors In Vitro, In Vivo and In Silico
Abstract
CYP2B6 metabolizes a number of drug substrates, that are usually non-planar, neutral or weakly basic, fairly lipophilic with one or two hydrogen bond acceptors, on which it catalyses various oxidative reactions. For bupropion, cyclophosphamide, ifosfamide, pethidine, ketamine and propofol, these reactions represent major metabolic or activation pathways and for their kinetics CYP2B6 function is of considerable importance. For the rest of the substrates found, CYP2B6 contributes to overall metabolism or to a single pathway, but probably not to a materially significant extent. Among inhibitors, thiotepa, ticlopidine and clopidogrel have been characterised extensively in terms of selectivity and potency. Thiotepa is the most selective of the inhibitors, but is not useful as an in vivo inhibitor, whereas ticlopidine and clopidogrel can be used as CYP2B6-selective probes in human clinical studies. Bupropion hydroxylation is a selective, and consequently useful, in vivo probe for CYP2B6. Computational approaches are being developed to the extent that predictions on affinity of chemicals to CYP2B6 are becoming reliable enough as a first screen of new drug molecules and other chemicals. With validated in vitro and in vivo substrates (e.g. bupropion) and inhibitors (e.g. ticlopidine), it is expected that pharmacological (including pharmacogenetic) and clinical significance of CYP2B6 will be delineated more fully in the near future.
... The human cytochrome CYP2B6 is also a highly polymorphic enzyme, which plays a major role in the biotransformation of an increasing number of endogenous and exogenous substrates (Hodgson and Rose, 2007;Turpeinen et al., 2006;. CYP2B6 is the only functional isozyme of the 2B subfamily in humans and the gene is located, together with the expressed pseudogene CYP2B7, within a 350 kb CYP2ABFGST gene cluster on chromosome 19 that contains genes and pseudogenes of the CYP2A, 2B, 2F, 2G, 2S and 2T subfamilies (Hoffman et al., 2001). ...
... Clinically used drug substrates include cytostatics (cyclophosphamide (Chang et al., 1993;Roy et al., 1999)), HIV drugs (efavirenz (Ward et al., 2003), nevirapine (Erickson et al., 1999)), antidepressants (bupropion (Kirchheiner et al., 2003)), antimalarias (artemisinin (Svensson and Ashton, 1999)), anaesthetics (propofol (Court et al., 2001)), and synthetic opioids (methadone (Kharasch et al., 2004)) and several others (Turpeinen et al., 2006). Variability of CYP2B6 expression in liver is high and protein levels can be 100-fold different between individuals (Gervot et al., 1999). ...
... Like the rodent phenobarbital-inducible CYP2B genes, human CYP2B6 is strongly inducible by numerous drugs and chemicals including rifampicin, barbiturates, cyclophosphamide, artemisinin, carbamazepine, metamizole, and efavirenz (EFV) as well as nevirapine (Faucette et al., 2007;Saussele et al., 2007;Wang and Negishi, 2003;Zanger et al., 2007). Potent inhibitors of CYP2B6 are the platelet aggregation inhibitor clopidogrel and the cytostatic triethylenethiophosphoramide (thioTEPA) (Turpeinen et al., 2006;Rae et al., 2002). ...
This study was devoted to the detection of alternative splicing within the Cytochrome P450 enzymes 2D6 and 2B6, mapping of the most common splice variants and to draw connections to certain single nucleotide polymorphisms (SNPs) and alleles. For both enzymes a splicing sensitive microarray was developed. The microarray was produced and optimized in all steps including the oligonucleotide probe design, microarray processing and target preparation, optimization of hybridization conditions and the development of a new data quantification method for the used probe design. For the developed splicing platform a design was chosen based on 5 different probes.
Within the CYP2D6 gene it was known that the SNP 2988G>A (allele *41) in intron 6 shifts splicing towards a variant lacking exon 6, what explains the intermediate phenotype within allele *41. The splicing platform verified this splicing aberration in allele *41. Using the microarry specific splicing patterns were monitored in human liver tissue within the most common alleles of CYP2D6 *1, *2, *4 and *41. It could be observed that within mRNA from allele *41 carriers additionally to the known transcript variant, which is lacking exon 6, total or partial retention of intron 5 and 6 was enhanced. Transcript patterns of CYP2D6*1 and *2 were similar with 5 times higher amount of the full functional transcript (NP), including all nine exons, compared to allele *41. The splicing array showed to be a valuable tool not only for detection of splicing variants in human liver tissue but additionally for detection for allele specific splicing patterns.
The existence of highly homologous Cytochrome P450 pseudogenes, which in some cases, as in CYP2D7 also express alternative splicing variants, results in a major problem of interpreting the data from splicing arrays. The developed splicing platform is the first existing array with which gene and pseudogene specific transcript patterns can be monitored individually.
The microarray platform can be easily transferred to other genes as shown for the second gene CYP2B6. Alternative splicing in this gene was so far only reported descriptive. CYP2B6 is a polymorphic human drug metabolizing cytochrome P450 with clinical relevance for several drug substrates including cyclophosphamide, bupropion and efavirenz. The common allele CYP2B6*6 [c. 516G>T, Q172H and c.785A>G, K262R] has previously been associated with lower expression in human liver and with increased plasma levels of efavirenz in HIV patients, but the molecular mechanism has remained unclear. With the developed splicing array for CYP2B6 allele specific splicing patterns were observed comparing CYP2B6*6 and CYP2B6*1. This lead to the idea that alternative splicing might play an important role in allele *6. This was investigated in more detail using RNA originating from well-documented human liver tissue. Analysis of mRNA in this tissue demonstrated that additional unknown splicing variants exist (SV8, SV7, SV9).
Investigations in human liver tissue using RT-PCR and sequencing showed that the most common transcript in CYP2B6*6 was not the normal transcript (NP) but an alternative splicing transcript lacking exons 4 to 6 (SV1). SV1 was tightly associated with the allele*6 and apparently also with the rare variant c.777C>A (CYP2B6*3). The observations lead to the assumptions that alternative splicing might explain the decreased function observed in allele CYP2B6*6.
Further investigations in this direction were performed by cloning CYP2B6 minigene constructs including all nine exons and additional intronic regions. Minigenes carrying the single c.785A>G polymorphism or the rare c.777C>A variant resulted in normal and intermediate expression phenotypes, respectively. In conclusion, the mechanism of the common allele*6 involves predominantly a pretranslational mechanism resulting in decreased enzyme expression. Aberrant splicing is leading to reduce functional mRNA, protein and activity. These results establish the SNP c.516G>T, a nonsynonymous exonic mutation, as the causal sequence variation for severely decreased expression and function associated with CYP2B6*6.
This work emphasizes the role of SNPs in non-consensus splicing elements such as exonic and intronic splicing enhancers as well as the clinical relevance of alternative splicing in context of adverse drug reactions. In both investigated genes CYP2D6 as well as in CYP2B6 there exists a common allele (CYP2D6*41 and CYP2B6*6, respectively) in which aberrant splicing results in reduced amounts of functional transcript, reduced amount of protein and enzyme activity. The findings establishes the SNP c.516G>T as the causal sequence variation that can now be reliably used in pharmacogenetic studies in various clinical settings including prediction of drug plasma concentration, toxicity, drug effectiveness and dose adjustment.
... The in silico approach is used to facilitate the process of finding drug candidate compounds with low, fast, and affordable failure rates. Then the best compounds (drug candidates) obtained from the in silico approach are followed by testing in vitro, in vivo, and clinical tests to evaluate the activity and toxicity of the drug candidates [14]. ...
... Moreover, the in silico approach can be predicted by ADME/T (Absorption, Distribution, Metabolism, Excretion, and Toxicity) and interactions that will affect solubility, stability, bioavailability, and permeability of ligands to be candidates for drug [20,21]. Then the best drug candidates obtained by the in silico method can be continued with in vitro, in vivo, and clinical tests in the wet laboratory to evaluate the toxicity of the compounds used [14]. ...
Diabetes is one of the top causes of death in the world, with 425 million sufferers reported in 2017. About 90% of diabetics suffer from Type 2 Diabetes Mellitus (T2DM). Recent studies show that inhibiting the α-amylase enzyme can significantly decrease the postprandial blood glucose levels through blocking carbohydrate hydrolysis. Therefore, it can be a promising strategy for T2DM treatment. This research was aimed to find the new potential inhibitor for the α-amylase from lead-like compounds Molecular Operating Environment (MOE) database through fragment-based drug design, combining with structure-based pharmacophore design method to obtain new drug candidate for T2DM. There were 653,214 lead-like compounds which were obtained from MOE database and screened based on the Astex Rules of Three along with toxicity filter to gain lead-like fragments. The filtered fragments were docked into the binding site of the α-amylase utilizing MOE 2014.09 software. Potential lead-like fragments were grown to generate 25,600 new ligands by utilizing DataWarrior v5.0.0 software, based on the Lipinski’s Rule of Five and toxicity filter. Molecular docking simu-lation and pharmacological test was performed on the ligand libraries to acquire the best ligand, namely BGOJI which were chosen according to the lowest ΔG binding score, RMSD value < 2, good molecular interaction, ADME/T test result.
... Finally, the interest in CYP2B6 metabolism has increased recently as its role in the metabolism of substrates (e.g. bupropion, cyclophosphamide, ifosfamide, methadone) has been demonstrated [9,10]. ...
... Furthermore, our sample preparation uses a simple isolation procedure. Numerous CYP450 phenotyping methods employed extensive extraction procedures for sample preparation such as dual liquid extraction or solid phase extraction which are costly and time consuming [10,[17][18][19][20][23][24][25]30]. A new method was recently reported by Tanaka et al. [17] for probe drug extraction i.e. "the Ostro Pass-Through Sample preparation" which showed a significant improvement in bioanalysis compared to the use of protein precipitation solely. ...
Cocktails composed of several Cytochrome P450 (CYP450)-selective probe drugs have been shown of value to characterize in vivo drug-metabolism activities. Our objective was to develop and validate highly sensitive and selective LC-MS/MS assays allowing the determination of seven major human CYP450 isoenzyme activities following administration of low oral doses of a modified CYP450 probe-drug cocktail in patients. The seven-drug cocktail was composed of caffeine, bupropion, tolbutamide, omeprazole, dextromethorphan, midazolam (all administered concomitantly) and chlorzoxazone (administered separately) to phenotype for CYP1A2, 2B6, 2C9, 2C19, 2D6, 3A4/5 and 2E1, respectively. Serial plasma and urine samples were collected over an 8 hour period. The probe-drugs and their respective metabolites were measured in both human plasma and urine, except for omeprazole (plasma only) and chlorzoxazone (urine only). Samples were analyzed by high performance liquid chromatography with heated electrospray ionization tandem mass spectrometry (HPLC-HESI-MS/MS) using a Phenomenex Luna PFP (2) analytical column (3 μm PFP(2) 150 × 3 mm) for chromatographic separation. Optimal detection was achieved based on 3 different analytical methods; (1) isocratic elution with a mobile phase consisting of acetonitrile and water both fortified with 0.01% formic acid for the analysis of bupropion, tolbutamide, chlorzoxazone and their respective metabolites; (2) isocratic elution with a mobile phase composed of acetonitrile and ammonium formate (pH 3; 10 mM) for omeprazole, dextromethorphan, midazolam and their metabolites; (3) for caffeine and paraxanthine, gradient elution using acetonitrile and 0.01% formic acid in water was used. All calibration functions were linear for all probe drugs and metabolites in both matrices over wide analytical ranges. The main advantages of our methods are the use of specific probe drugs available in most countries, the administration of small doses of probe drugs, small volume of plasma required for the analyses and simple and rapid extraction procedures. The methods met all requirements of specificity, sensitivity, linearity, precision and accuracy and stability generally accepted in bioanalytical chemistry. Determination of CYP450 phenotype in patients will permit characterization of their capacities to metabolize drugs through CYP450 s under specific conditions at a definite time. This tool will be highly clinically relevant since wide intersubject variability observed in drug response is largely explained by variation in drug metabolism; it will be particularly useful in polymedicated patients with multiple comorbidities. So far, our CYP450 cocktail assays have been successfully applied to phenotype CYP450 activities in patients.
... Accurate assessment of hepatic CYP2B6 activity has been hampered by the lack of a selective and easy-to-use phenotyping probe. The hydroxylation of bupropion, predominantly catalyzed by CYP2B6 (18), was an early and frequently used in vitro and in vivo probe of CYP2B6 activity (19). However, its usefulness in vivo to quantitatively assess induction of drug interactions (20) and to phenotype genetic variants of CYP2B6 (21) has been limited due to the significant competition from non-CYP2B6 parallel elimination pathways (22,23) and the complex disposition of bupropion and 4-hydroxybupropion (24)(25)(26). ...
... It is well known that variants in the CYP2B6 gene are associated with EFV metabolism and effect (10,17,34,(52)(53)(54). Together, EFV is a reliable probe to assess the impact of CYP2B6 genetic polymorphisms and induction and inhibition drug interactions on CYP2B6 activity in vivo. Because the fraction of the EFV dose metabolized via the CYP2B6-mediated 8-hydroxylation is close to unity (37,49; present data), this probe is superior to other CYP2B6 phenotypic probe drugs used currently (19,27). The proposed single-point sampling strategy (ϳ3-hour concentration metabolic ratio), along with the validation of a low dose devoid of common CNS side effects observed at therapeutic doses (40), should facilitate the utility of EFV alone or included in a cocktail as an easy-to-use, safe, and cost-effective CYP2B6 phenotypic probe. ...
Cytochrome P450 (CYP) 2B6 metabolizes clinically important drugs and other compounds. Its expression and activity varies widely among individuals, but quantitative estimation is hampered by the lack of safe and selective in vivo probe of CYP2B6 activity. Efavirenz, a non-nucleoside HIV-1 reverse transcriptase inhibitor, is mainly cleared by CYP2B6, an enzyme strongly inhibited in vitro by voriconazole. To test efavirenz metabolism as an in vivo probe of CYP2B6 activity, we quantified the inhibition of CYP2B6 activity by voriconazole in sixty-one healthy volunteers administered a single 100 mg oral dose of efavirenz with and without voriconazole administration. The kinetics of efavirenz metabolites demonstrated formation rate-limited elimination. Compared to control, voriconazole prolonged the elimination half-life (t 1/2 ), increased both the C max and AUC 0-t of efavirenz (mean change by 51%, 36%, and 89%, respectively) ( P < 0.0001) with marked intersubject variability (e.g., the percent change in efavirenz AUC 0-t ranged from 0.4% to ∼224 %). Voriconazole decreased efavirenz 8-hydroxylation by greater than 60% ( P < 0.0001) whereas its effect on 7-hydroxylation was marginal. The plasma concentration ratio of efavirenz to 8-hydroxyefavirenz, determined 1 to 6 hour after dosing was significantly increased by voriconazole and correlated with the efavirenz AUC 0-t (Pearson r = >0.8; p<0.0001). This study demonstrates the mechanisms of voriconazole-efavirenz interaction, establishes the use of a low dose of efavirenz as a safe and selective in vivo probe for phenotyping CYP2B6 activity, and identified several easy-to-use indices that should enhance understanding of the mechanisms of CYP2B6 inter-individual variability.
... The cytochrome P450 (CYP) 2B6 represents on average~3e5% of the total hepatic P450 protein content and plays a more important role than previously estimated in the detoxification or activation of a growing list of clinically important drugs, endogenous compounds, and other compound of toxicological relevance, including procarcinogens and environmental toxicants (reviewed in and references therein: [1e6]). The protein expression and activity of CYP2B6 are highly variable among human livers in vitro, in part due to CYP2B6 genetic variation, with distinct ethnic and racial frequencies [7,8], and exposure to structurally diverse inducer [3] or inhibitor drugs [5,6,9]. This variability likely reflects large changes in activity in vivo. ...
... Progress towards quantitative determination and prediction of the in vivo consequences of the wealth of in vitro data has been greatly hampered by the lack of selective and easy to use clinical phenotyping probe. Bupropion 4-hydroxylation, a reaction exclusively catalyzed by CYP2B6 [10], has been frequently used to assess the impact of genetic and nongenetic factors on CYP2B6 activity [9]. However, the utility of bupropion in assessing in vivo induction drug interactions mediated by CYP2B6 [11] and functional consequences of CYP2B6 genetic variants [12] appear to be limited. ...
The effect of rifampin on the in vivo metabolism of the antiretroviral drug efavirenz was evaluated in healthy volunteers. In a cross-over placebo control trial, healthy subjects (n = 20) were administered a single 600 mg oral dose of efavirenz after pretreatment with placebo or rifampin (600 mg/day for 10 days). Plasma and urine concentrations of efavirenz, 8-hydroxyefavirenz and 8,14-dihydroxyefavirenz were measured by LC–MS/MS. Compared to placebo treatment, rifampin increased the oral clearance (by ∼2.5-fold) and decreased maximum plasma concentration (Cmax) and area under the plasma concentration–time curve (AUC0–∞) of efavirenz (by ∼1.6- and ∼2.5-fold respectively) (p < 0.001). Rifampin treatment substantially increased the Cmax and AUC0–12h of 8-hydroxyefavirenz and 8,14-dihydroxyefavirenz, metabolic ratio (AUC0–72h of metabolites to AUC0–72h efavirenz) and the amount of metabolites excreted in urine (Ae0–12hr) (all, p < 0.01). Female subjects had longer elimination half-life (1.6–2.2-fold) and larger weight-adjusted distribution volume (1.6–1.9-fold) of efavirenz than male subjects (p < 0.05) in placebo and rifampin treated groups respectively. In conclusion, rifampin enhances CYP2B6-mediated efavirenz 8-hydroxylation in vivo. The metabolism of a single oral dose of efavirenz may be a suitable in vivo marker of CYP2B6 activity to evaluate induction drug interactions involving this enzyme.
... Although historically regarded as a minor cytochrome P450 enzyme in the human liver, accumulating evidence has demonstrated that CYP2B6 plays an important role in human drug metabolism. Many drugs with inhibitory effects on CYP2B6 activity may affect the metabolism of CYP2B6 substrates [27,28]. Ji et al. [13] proposed that psoralen is a mechanism-based CYP2B6 inactivator that can moderately inhibit the activity of the recombinant human CYP2B6 enzyme. ...
Ciprofol is a novel short-acting intravenous anaesthetic developed in China that is mainly metabolized by cytochrome P450 2B6 (CYP2B6) and uridine diphosphate glucuronosyltransferase 1A9 (UGT1A9). Currently, insufficient evidence is available to support drug‒drug interactions between ciprofol and CYP2B6 inactivators. Here, we established a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method to assess the concentration of ciprofol and investigated the effects of psoralen and clopidogrel on the metabolism of ciprofol in liver microsomes and rats. In rat and human liver microsomes, the median inhibitory concentration (IC50) values of psoralen were 63.31 μmol·L⁻¹ and 34.05 μmol·L⁻¹, respectively, showing mild inhibitory effects on ciprofol metabolism, whereas the IC50 values of clopidogrel were 6.380 μmol·L⁻¹ and 2.565 μmol·L⁻¹, respectively, with moderate inhibitory effects. SD rats were randomly divided into three groups: psoralen (27 mg·kg⁻¹), clopidogrel (7.5 mg·kg⁻¹), and the same volume of 0.5% carboxy methyl cellulose. After 7 days, all rats were injected with 2.4 mg·kg⁻¹ ciprofol. Compared with the control group, the AUC and MRT values of ciprofol in the psoralen and clopidogrel groups were significantly greater, whereas the CL values were significantly lower. In addition, the durations of loss of righting reflex (LORR) in the psoralen and clopidogrel groups were 16.1% and 23.0% longer than that in the control group, respectively. In conclusion, psoralen and clopidogrel inhibit ciprofol metabolism to different degrees and prolong the duration of LORR in rats.
... The P450 heme active site was modeled using a methoxy radical, as described in literature (Turpeinen et al., 2006;Li et al., 2012;Delaine et al., 2014;Niklasson et al., 2014). Reactivity calculations were carried out at the B3LYP-D3/6-31+G** (Ditchfield et al., 1971;Slater and James, 1974;Becke, 1988;Stephens et al., 1994) level of theory in Jaguar (Schrodinger, 2009), part of the Schrödinger suite of programs. ...
Background
Cinnamic alcohol is a natural compound, widely used in fragrances, which can cause allergic contact dermatitis. Cinnamic alcohol lacks intrinsic reactivity and autoxidation or metabolic activation is necessary for it to act as a sensitizer.
Methods
Bioactivation of cinnamic alcohol was explored using human liver microsomes, human liver S9 and SkinEthic™ Reconstructed Human Epidermis. A targeted multiple reaction monitoring mass spectrometry method was employed to study and quantify cinnamic alcohol along with eight potential phase I or phase II metabolites. The reconstructed human epidermis model, treated with cinnamic alcohol, was also analyzed with a non-targeted high-resolution mass spectrometry method to identify metabolites not included in the targeted method.
Results
Two metabolites identified with the targeted method, namely, pOH-cinnamic alcohol and pOH-cinnamic aldehyde, have not previously been identified in a metabolic in vitro system. Their reactivity toward biologically relevant nucleophiles was investigated and compared to their sensitizing potency in vivo in the murine local lymph node assay (LLNA). According to the LLNA, the pOH-cinnamic alcohol is non-sensitizing and pOH-cinnamic aldehyde is a moderate sensitizer. This makes pOH-cinnamic aldehyde less sensitizing than cinnamic aldehyde, which has been found to be a strong sensitizer in the LLNA. This difference in sensitizing potency was supported by the reactivity experiments. Cinnamic sulfate, previously proposed as a potential reactive metabolite of cinnamic alcohol, was not detected in any of the incubations. In addition, experiments examining the reactivity of cinnamic sulfate toward a model peptide revealed no evidence of adduct formation. The only additional metabolite that could be identified with the non-targeted method was a dioxolan derivative. Whether or not this metabolite, or one of its precursors, could contribute to the sensitizing potency of cinnamic alcohol would need further investigation.
Discussion
Cinnamic alcohol is one of the most common fragrance allergens and as it is more effective to patch test with the actual sensitizer than with the prohapten itself, it is important to identify metabolites with sensitizing potency. Further, improved knowledge of metabolic transformations occurring in the skin can improve prediction models for safety assessment of skin products.
... CYP2B6 accounts for about 2-5% of the total CYP in liver, but it shows about 300 times of variability [16]. CYP2B6 516G > T variant changes the amino acid residue of glutamine to histidine, and this substitution may decrease protein expression levels of the CYP2B6 in human liver [17]. This in turn impedes hydroxylation of testosterone, which may affect prostate development and the progression of cancer. ...
Background: Recently, many clinical studies have demonstrated that Cytochrome P450 (CYP) is related to the occurrence and development of many cancers. The purpose of our study is to clarify the relevance of CYP2B6 single nucleotide polymorphisms (SNPs) to lung cancer susceptibility.
Methods: Five SNPs in CYP2B6 were genotyped in the Han population of China including 507 cases and 505 controls using Agena MassARRAY. Using odds ratio, 95% confidence interval, and χ2 test to calculate the relationship between the SNPs and LC susceptibility. The SNP-SNP interactions were analyzed by performing multifactor dimensionality reduction (MDR).
Results: We found that rs2099361 was associated with an increased susceptibility to LC in the codominant model (OR = 1.31, p = 0.045). After stratification analysis, the results found that CYP2B6 rs4803418 (BMI > 24 kg/m2), rs4803420 (BMI > 24 kg/m2, No-smoking), rs12979270 (BMI > 24 kg/m2), and rs1038376 (III-IV versus I-II) were significantly associated with decreased susceptibility of LC. Inversely, rs4803418 (BMI > 24 kg/m2), rs4803420 (III-IV versus I-II) and rs12979270 (No-smoking) showed increased susceptibility to LC. Furthermore, the result of MDR showed that a synergistic relationship might exist between rs12979270, rs4803420, rs2099361, rs1038376, and rs4803418 in CYP2B6 gene and the pathophysiology and genetics of LC.
Conclusions: The results suggest that SNPs of CYP2B6 have crucial roles in LC susceptibility, and its correlation is related to the patients’ BMI, smoking status, and clinical stages.
... The major biotransformation enzyme CYP2B6, member of the cytochrome P450 family of relevant pharmacogenes, is responsible for the metabolism of 4% of the top 200 prescription drugs (22), including anesthetics, anticancer drugs, antidepressants, antiretrovirals, and antismoking agents (23,24). CYP2B6 gene is one of the most polymorphic ADME genes in humans. ...
Aim:
To compare the Croatian and European population in terms of allele frequencies of clinically relevant polymorphisms in drug absorption, distribution, metabolism, and excretion (ADME) genes.
Methods:
In 429 Croatian participants, we genotyped 27 loci in 20 ADME genes. The obtained frequencies were merged with the published frequencies for the Croatian population by sample size weighting. The study sample obtained in this way was compared with the average data for the European population from the gnomAD database.
Results:
Variant allele frequencies in the Croatian population were higher in three and lower in two polymorphisms (Benjamini-Hochberg-corrected P values: 0.0027 for CYP2B6*4 rs2279343, CYP2C9*2 rs1799853, and VKORC1 rs9923231; 0.0297 for GSTP1 rs1695; 0.0455 for CYP2A6 rs1801272) compared with the European population. The most marked difference was observed for CYP2B6*4 (9.3% in Europe vs 24.3% in Croatia). The most clinically relevant findings were higher variant allele frequencies in two polymorphisms related to lower warfarin requirements: VKORC1*2 (34.9% in Europe vs 40.1% in Croatia) and CYP2C9*2 (12.3% in Europe vs 14.7% in Croatia). This indicates that three-quarters of Croatian people have at least one variant allele at these loci. Variants in genes GSTP1 and CYP2A6 were significantly less frequently observed in Croatia.
Conclusions:
Croatian population has a higher bleeding and over-anticoagulation risk, which is why we recommend the prescription of lower doses of anticoagulation drugs such as warfarin and acenocoumarol. Lower phenytoin, and higher bupropion and efavirenz doses are also recommended in the Croatian population.
... A good example includes bupropion, cyclophosphamide, ifosfamide, pethidine, ketamine and propofol [89]. This is because this enzyme metabolizes a number of drug substrates which are usually nonplanar, neutral or weakly basic, with one or two hydrogen-bonding acceptors [98]. Paroxetine also has the highest inhibitory constant of all antidepressants for CYP2B6 (K i = 1.03 µM). ...
In the 21st century and especially during a pandemic, the diagnosis and treatment of depression is an essential part of the daily practice of many family doctors. It mainly affects patients in the age category 15–44 years, regardless of gender. Anxiety disorders are often diagnosed in children and adolescents. Social phobias can account for up to 13% of these diagnoses. Social anxiety manifests itself in fear of negative social assessment and humiliation, which disrupts the quality of social functioning. Treatment of the above-mentioned disorders is based on psychotherapy and pharmacotherapy. Serious side effects or mortality from antidepressant drug overdose are currently rare. Recent studies indicate that paroxetine (ATC code: N06AB), belonging to the selective serotonin reuptake inhibitors, has promising therapeutic effects and is used off-label in children and adolescents. The purpose of this review is to describe the interaction of paroxetine with several molecular targets in various points of view including the basic chemical and pharmaceutical properties. The central point of the review is focused on the pharmacodynamic analysis based on the molecular mechanism of binding paroxetine to various therapeutic targets.
... 2). W wyniku opisanej interakcji farmakokinetycznych w surowicy utrzymuje się duże stężenie bupropionu przy niskim stężeniu hydroksybupropionu, co w konsekwencji zwiększa ryzyko drgawek [48]. ...
Objectives:
To evaluate harmful interactions between antidepressants and medications used in treatment of cardiovascular disorders.
Methods:
The analysis of 66 cases of adverse reactions with a clinical picture indicating, to a degree that is probable or certain, that they were the result of the combination of antidepressant with cardiovascular medication.
Results:
The most common side effect (n = 25, 37.9%) was bradycardia (and other side effects of beta blockers) as a consequence of addition of metoprolol or propranolol to SSRI or bupropion. In one case combination of fluoxetine with propranolol resulted in cardiac arrest. We observed 8 cases of intensified side effects of amlodipine (swelling of lower limbs, headaches) after its combination with: fluoxetine, sertraline and paroxetine, and occurrence of myalgia, elevated aminotransferase levels, polyuria and hypotension after combination of lercanidipine with some of the SSRIs. We also found i.a. worsening of propafenone tolerance in combination with venlafaxine or bupropion, 2 cases of granulopenia associated with duloxetine-propafenone combination, 2 cases of hemorrhagic complications associated with the combination of vortioxetine-warfarin, 1 case of hyponatremia associated with the combination of vortioxetine and hydrochlorothiazide, as well as antagonizing clonidine's hypotensive effect by mirtazapine, and peripheral thrombosis following the combination of warfarin with trazodone.
Conclusions:
Because of ahigh risk of interactions and related adverse effects, especially in older patients, each decision regarding combination of a particular antidepressant with a medication used in treatment of cardiovascular disorders should be preceded by a detailed analysis of safety and risk-benefit ratio, and also be associated with the search for the safest, alternative combinations of the above-mentioned medications.
... Furthermore, previous research has shown that CYP2B6 plays an important role in propofol hydroxylation [36,37,40,63,64]. Additionally, it has been shown before that CYP2B6 activity is low in HLMs, mainly caused by its very low abundance [33,40,65,66]. For these three reasons, AAFs were calculated for CYP2B6 and CYP2C9 (see supplementary information S-2). ...
The project SAFEPEDRUG aims to provide guidelines for drug research in children, based on bottom-up and top-down approaches. Propofol, one of the studied model compounds, was selected because it is extensively metabolized in liver and kidney, with an important role for the glucuronidation pathway. Besides, being a lipophilic molecule, it is distributed into fat tissues, from where it redistributes into the systemic circulation. In the past, both bottom-up (Physiologically based pharmacokinetic, PBPK) and top-down approaches (population pharmacokinetic, popPK) were applied to describe its pharmacokinetics (PK). In this work, a combination of the two was used to check their performance to describe PK in children and neonates (both term and preterm) using propofol as a case compound. First, in vitro data was generated in human liver microsomes and recombinant enzymes and used to develop an adult PBPK model in Simcyp®. Activity adjustment factors (AAFs) were calculated to account for differences between in vitro and in vivo enzyme activity. Clinical data were analyzed using a 3-compartment model in NONMEM. These data were used to construct a retrograde PBPK model and for qualification of the PBPK models. Once an accurate in vivo clearance was obtained accounting for the contribution of the different metabolic pathways, the resulting PBPK models were challenged with new data for qualification. After that, the constructed adult PPBK model for propofol was extrapolated to the pediatric population. Both the default built-in and in vivo derived ontogeny functions were used to do so. The models were qualified by comparing their predicted PK parameters to published values, and by comparison of predicted concentration–time profiles to available clinical data. Clearance values were predicted well, especially when compared with values obtained from trials where long-term sampling was applied, whereas volume of distribution was lower compared to the most common popPK model predictions. Concentration–time profiles were predicted well up until and including the preterm neonatal population. In this work, it was thus shown that PBPK can be used to predict the PK up to and including the preterm neonatal population without the use of pediatric in vivo data. This work adds weight to the need for further development of PBPK models, especially regarding distribution modeling and the use of in vivo derived ontogeny functions.
... The enzyme contributes to 19.4% of the transformation of Clopidogrel to the 2-oxo-Clopidogrel reaction, and 32.9% of the transformation of this intermediate metabolite to the pharmacologically active form, responsible for the inhibitory effect of platelet aggregation (Fig. 5). Polymorphisms in CYP2B6 gene have been reported as interesting predictors of pharmacokinetics and drugs heterogeneity of response (Zanger et al., 2007;Telenti and Zanger, 2008;Rakhmanina and van den Anker, 2010); they were shown to act on several levels of gene expression (mRNA transcription and expression, splice variants generation) and production of proteins; they have also been reported as responsible for complex substrate-dependent and substrate-independent effects (Zanger and Klein, 2013;Zanger et al., 2007;Ekins and Wrighton, 1999;Turpeinen et al., 2006;Hodgson and Rose, 2007;Wang and Tompkins, 2008;Mo et al., 2009;Turpeinen and Zanger, 2012). ...
... CYP2B6 is responsible for the metabolism of 4 % of the top 200 drugs and is known to be highly inducible by xenobiotics [14]. Substrates of CYP2B6 include artemisinin (an antimalarial drug), bupropion and methoxetamine (antidepressants), cyclophosphamide, ifosphamide, and tamoxifen (antitumor drugs), efavirenz and nevirapine (nonnucleoside reverse transcriptase inhibitors of HIV), ketamine and propofol (anesthesia agents), meperidine and methadone (analgesics), mephenytoin and valproic acid (anticonvulsants), prasugrel (antiplatelet agent), and selegiline (Parkinsonʼs disease drug) [15][16][17][18]. Considering recent trends in the interest of weight loss and ease of access to dietary supplements for that purpose, it is likely that some of the aforementioned drugs are taken concomitantly with G. cambogia extract. ...
This study assessed the inhibitory effects of Garcinia cambogia extract on the cytochrome P450 enzymes in vitro. G. cambogia extract was incubated with cytochrome P450 isozyme-specific substrates in human liver microsomes and recombinant CYP2B6 isozyme, and the formation of the marker metabolites was measured to investigate the inhibitory potential on cytochrome P450 enzyme activities. The results showed that G. cambogia extract has significant inhibitory effects on CYP2B6 activity in a concentration-dependent manner. Furthermore, the inhibition was potentiated following preincubation with NADPH, indicating that G. cambogia extract is a time-dependent inhibitor of CYP2B6. Meanwhile, hydroxycitric acid, the major bioactive ingredient of G. cambogia extract, did not exhibit significant inhibition effects on cytochrome P450 enzyme activities. G. cambogia extract could modulate the pharmacokinetics of CYP2B6 substrate drugs and lead to interactions with those drugs. Therefore, caution may be required with respect to concomitant intake of dietary supplements containing G. cambogia extract with CYP2B6 substrates.
... CYP2B6 is known to metabolize some xenobiotics, such as the anti-cancer drugs cyclophosphamide and ifosphamide. [24] Our results suggest that CYP2B6 has a clinically important effect on pioglitazone metabolism. By regression analysis, CYP2B6 gene has significant effect on T max and AUC inf . ...
Pioglitazone is known to have antidiabetic effects through decreasing peripheral, hepatic and vascular insulin resistance by the stimulation of PPAR gamma. To address the possible genetic factors affecting the pharmacokinetics (PK) of pioglitazone, 27 male Korean volunteers were enrolled from two separate bioequivalence studies. Each subject was administered 15 mg pioglitazone and reference drug PK parameters were used. We used Illumina Human610 Quad v1.0 DNA Analysis BeadChip for whole genome SNPs analysis and whole genome genotyping data was processed by linear regression analysis for PK parameters. We found 35 significant SNPs (P < 0.0001) in Cmax, 1,118 significant SNPs (P < 0.0001) in Tmax and 1,259 significant SNPs (P < 0.0001) in AUCinf from whole genome analysis. For clinical pharmacological purpose, we selected SNPs from several phase I and II drug metabolizing enzyme and analyzed PK parameters with genotypes. Four SNPs (rs7761731 and rs3799872 from CYP39A1; rs156697 from GSTO2; rs1558139 from CYP4F2) showed significant associations with pioglitazone Cmax. In the Tmax group, seven SNPs from 3 genes (rs3766198 from CYP4B1; rs2270422 from GSTZ1; rs2054675, rs10500282, rs3745274, rs8192719, and rs11673270 from CYP2B6) had significant associations. In the AUCinf group, seven SNPs from 4 genes (rs11572204 from CYP2J2; rs4148280 from UGT2A1, rs4646422 from CYP1A1; rs3745274, rs8192719, rs11673270, and rs707265 from CYP2B6) showed significant associations with pioglitazone absorption. These results showed that genetic makeup could affect the PK parameters and these informations could be provide information for personalized pioglitazone therapy.
... Approximately 3e8% of the marketed drugs are metabolized by CYP2B6 which include efavirenz, sibutramine and bupropion [36e38]. Moreover, induction or inhibition of CYP2B6 has resulted in serious DDIs in cases where the substrate drugs have narrow therapeutic index such as cyclophosphamide and (S)-methadone [39]. ...
... Polychlorinated dibenzo-p-dioxin metabolism by CYP2B6 has not been described, most likely because CYP2B6 is limited in metabolizing only non-planar molecules (Turpeinen et al., 2006). Nevertheless, some studies demonstrated these polyhalogenated compounds can be metabolized by cytochrome P450 monooxygenase systems (Hu and Bunce, 1999), especially by CYP1A1 and CYP1A2, as demonstrated by Inouye et al. (2002). ...
... Bupropion 4-hydroxylation is exclusively catalyzed by CYP2B6 [17,18] and this reaction has been widely used as a marker of CYP2B6 activity in vitro [30][31] and in vivo [13,15,29,32]. In addition, recent studies have focused on whether reduced plasma exposure to 4hydroxybupropion as a result of CYP2B6 genetic variations or non-genetic factors results in altered clinical outcomes following administration of bupropion [11]. ...
Bupropion metabolites formed via oxidation and reduction exhibit pharmacological activity, but little is known regarding their stereoselective disposition. A novel stereoselective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to separate and quantify enantiomers of bupropion, 4-hydroxybupropion, and erythro- and threo-dihydrobupropion. Liquid-liquid extraction was implemented to extract all analytes from 50μL human plasma. Acetaminophen (APAP) was used as an internal standard. The analytes were separated on a Lux 3μ Cellulose-3 250×4.6mm column by methanol: acetonitrile: ammonium bicarbonate: ammonium hydroxide gradient elution and monitored using an ABSciex 5500 QTRAP triple-quadrupole mass spectrometer equipped with electrospray ionization probe in positive mode. Extraction efficiency for all analytes was ≥70%. The stability at a single non-extracted concentration for over 48h at ambient temperature resulted in less than 9.8% variability for all analytes. The limit of quantification (LOQ) for enantiomers of bupropion and 4-hydroxybupropion was 0.3ng/mL, while the LOQ for enantiomers of erythro- and threo-hydrobupropion was 0.15ng/mL. The intra-day precision and accuracy estimates for enantiomers of bupropion and its metabolites ranged from 3.4% to 15.4% and from 80.6% to 97.8%, respectively, while the inter-day precision and accuracy ranged from 6.1% to 19.9% and from 88.5% to 99.9%, respectively. The current method was successfully implemented to determine the stereoselective pharmacokinetics of bupropion and its metabolites in 3 healthy volunteers administered a single 100mg oral dose of racemic bupropion. This novel, accurate, and precise HPLC-MS/MS method should enhance further research into bupropion stereoselective metabolism and drug interactions.
... Cytochrome P450 enzymes (CYP450) are responsible for the metabolism of nearly 75% of phase I-dependent metabolism of clinically used drugs [21]. Relative expression of CYP2B6 ranges from 2% to 10% of the total hepatic content [22][23][24] and contributes to a significant proportion of drug metabolism [25]. Genetic polymorphism in CYP2B6 have been implicated in variations in the activity of the enzyme in metabolizing xenobiotics [26]. ...
Highly active antiretroviral therapy (HAART) has greatly improved health parameters of HIV infected individuals. However, there are several challenges associated with the chronic nature of HAART administration. For populations in health transition, dual use of medicinal plant extracts and conventional medicine poses a significant challenge. There is need to evaluate interactions between commonly used medicinal plant extracts and antiretroviral drugs used against HIV/AIDS. Efavirenz (EFV) and nevirapine (NVP) are the major components of HAART both metabolized by CYP2B6, an enzyme that can potentially be inhibited or induced by compounds found in medicinal plant extracts. The purpose of this study was to evaluate the effects of extracts of selected commonly used medicinal plants on CYP2B6 enzyme activity. Recombinant human CYP2B6 was used to evaluate inhibition, allowing the assessment of herb-drug interactions (HDI) of medicinal plants Hyptis suaveolens, Myrothamnus flabellifolius, Launaea taraxacifolia, Boerhavia diffusa and Newbouldia laevis. The potential of these medicinal extracts to cause HDI was ranked accordingly for reversible inhibition and also classified as potential time-dependent inhibitor (TDI) candidates. The most potent inhibitor for CYP2B6 was Hyptis suaveolens extract (IC50 = 19.09 ± 1.16 µg/mL), followed by Myrothamnus flabellifolius extract (IC50 = 23.66 ± 4.86 µg/mL), Launaea taraxacifolia extract (IC50 = 33.87 ± 1.54 µg/mL), and Boerhavia diffusa extract (IC50 = 34.93 ± 1.06 µg/mL). Newbouldia laevis extract, however, exhibited weak inhibitory effects (IC50 = 100 ± 8.71 µg/mL) on CYP2B6. Launaea taraxacifolia exhibited a TDI (3.17) effect on CYP2B6 and showed a high concentration of known CYP450 inhibitory phenolic compounds, chlorogenic acid and caffeic acid. The implication for these observations is that drugs that are metabolized by CYP2B6 when co-administered with these herbal medicines and when adequate amounts of the extracts reach the liver, there is a high likelihood of standard doses affecting drug plasma concentrations which could lead to toxicity.
... NVP is administered orally as a 200 mg twice daily dose and mainly metabolized to 3-and 8-hydroxynevirapine by CYP2B6 and 2-and 12hydroxynevirapine by CYP3A4/5 before undergoing glucuronidation and excretion (>60%) through feces (Riska et al., 1999). CYP2B6 exhibits genetic polymorphisms that affect hepatic expression and the activity of the coded enzyme, which in turn leads to inter-individual variability in the metabolism of substrate drugs (Turpeinen et al., 2006). ...
The extremely high prevalence of HIV/AIDS in sub-Saharan Africa and limitations of current antiretroviral medicines demand new tools to optimize therapy such as pharmacogenomics for person-to-person variations. African populations exhibit greater genetic diversity than other world populations, thus making it difficult to extrapolate findings from one population to another. Nevirapine, an antiretroviral medicine, displays large plasma concentration variability which adversely impacts therapeutic virological response. This study, therefore, aimed to identify sources of variability in nevirapine pharmacokinetics and pharmacodynamics, focusing on genetic variation in CYP2B6 and CYP1A2. Using a cross-sectional study design, 118 HIV-infected adult Zimbabwean patients on nevirapine-containing highly active antiretroviral therapy (HAART) were characterized for three key functional single nucleotide polymorphisms (SNPs), CYP2B6 c.516G>T (rs3745274), CYP2B6 c.983T>C (rs28399499), and CYP1A2 g.-163C>A (rs762551). We investigated whether genotypes at these loci were associated with nevirapine plasma concentration, a therapeutic biomarker, and CD4 cell count, a biomarker of disease progression. CYP2B6 and CYP1A2 were chosen as the candidate genes based on reports in literature, as well as their prominence in the metabolism of efavirenz, a drug in the same class with nevirapine. Nevirapine plasma concentration was determined using LC-MS/MS. The mean nevirapine concentration for CYP2B6 c.516T/T genotype differed significantly from that of 516G/G (p < 0.001) and 516G/T (p < 0.01) genotypes, respectively. There were also significant differences in mean nevirapine concentration between CYP2B6 c.983T > C genotypes (p = 0.04). Importantly, the CYP1A2 g.-163C>A SNP was significantly associated with the pharmacodynamics endpoint, the CD4 cell count (p = 0.012). Variant allele frequencies for the three SNPs observed in this Zimbabwean group were similar to other African population groups but different to observations among Caucasian and Asian populations. We conclude that CYP2B6 c.516G>T and CYP2B6 c.983T>C could be important sources of nevirapine pharmacokinetic variability that could be considered for dosage optimization, while CYP1A2 g.-163C>A seems to be associated with HIV disease progression. These inter- and intra-population pharmacokinetic and pharmacodynamics differences suggest that a single prescribed dosage may not be appropriate for the treatment of disease. Further research into a personalized nevirapine regimen is required.
... Clinically used drug substrates include cytostatics (cyclophosphamide) 23 , HIV drugs (efavirenz, nevirapine), 24,25 antidepressants (bupropion), 26 antimalarials (artemisinin), 27 anaesthetics (propofol), 28 and synthetic opioids (methadone) 29 and several others. 30 Variability of CYP2B6 expression in liver is high and protein levels can be 100-fold different between individuals. 31,32 Like the rodent phenobarbital-inducible CYP2B genes, human CYP2B6 is strongly inducible by numerous drugs and chemicals including rifampicin, barbiturates, cyclophosphamide, artemisinin, carbamazepine, metamizole, efavirenz and nevirapine. ...
Recent work on functional polymorphisms in drug metabolizing cytochromes P450 CYP2B6 and CYP2D6 emphasizes the role of single nucleotide polymorphisms (SNPs) in non-consensus splicing elements such as exonic and intronic splicing enhancers. In the CYP2D6 gene the intron 6 SNP 2988G > A (allele *41) shifts the balance of spliced transcripts towards a variant that lacks exon 6. In the CYP2B6 gene, the 516G > T SNP, a marker of allele CYP2B6*6, encodes an amino acid change in exon 4 [Q172H] but also leads to increased amounts of a transcript lacking exons 4 to 6. In both cases aberrant splicing results in reduced amounts of functional transcript and reduced amounts of functional protein in the liver. Although expression of functional protein is only partially diminished, reduced activity phenotypes arise in homozygous genotypes or in compound heterozygotes carrying other severely affected alleles. We here describe the elucidation of these genetic polymorphisms and their mechanisms as well as their clinical relevance.
... In the present studies, there are many CYP gene polymorphisms which increase the risk of AL geneses [1,12,13] . CYP2B6 c.516G>T polymorphism change glutamine (Glu) amino acid to histidine (His) amino acid [14][15][16] , which can reduce the CYP2B6 enzyme activity and expression in the liver [11] , and can block the transformation of carcinogen substrates to harmless metabolites, and can be a risk factor for AL nascence because AL is the type of cancer that is most sensitive to exogenous chemical exposure [7] . ...
Objective: To investigate whether any association exists between genetic polymorphism in CYP2B6 c.516G>T and individual susceptibility to acute lymphoblastic leukemia (ALL). Methods: Our study group consisted of 96 ALL patients(T-ALL 17 cases, B-ALL 79 cases) and 348 unrelated healthy newborn volunteers as a control group. Genomic DNA was extracted from peripheral blood and cord blood leukocytes. We genotyped CYP2B6 c.516G>T polymorphism by use of PCR with sequence-specific primers (PCR-SSP). The data were analyzed statistically using chi-square and logistic regression analyses. Results: The frequencies of GG genotype were 74.14%, 57.29%, 29.41% and 63.29%, and GT genotype were 23.85%, 37.50%, 64.71% and 31.65%, and TT genotype were 2.01%, 5.21%, 5.88% and 5.06% in control group, ALL, T-ALL, and B-ALL cases, respectively. Chi-square test showed a significant correlation between the CYP2B6 c.516G>T polymorphism GT genotype and ALL patients (OR=2.035, 95%CI=1.249-3.313, P=0.004); and T-ALL patients (OR=6.839, 95%CI=2.309-20.252, P=0.000); whereas and B-ALL patients (OR=1.554, 95%CI=0.906-2.667, P=0.108). Conclusions: This study revealed the CYP2B6 c.516GT genotype may be a risk factor to the development of ALL, especially T-ALL.
... Considerable interindividual variability as well as ethnic differences in expression level have contributed to an increased attention to this enzyme [51,52]. An increasing number of clinically important drugs, including bupropion, ketamine, propofol, cyclophosphamide, nevirapine and efavirenz, have been recognized as CYP2B6 substrates [53]. ...
In recent years our understanding of molecular mechanisms of drug action and interindividual variability in drug response has grown enormously. Meanwhile, the practice of anesthesiology has expanded to the preoperative environment and numerous locations outside the OR. Anesthetic Pharmacology: Basic Principles and Clinical Practice, 2nd edition, is an outstanding therapeutic resource in anesthesia and critical care: Section 1 introduces the principles of drug action, Section 2 presents the molecular, cellular and integrated physiology of the target organ/functional system and Section 3 reviews the pharmacology and toxicology of anesthetic drugs. The new Section 4, Therapeutics of Clinical Practice, provides integrated and comparative pharmacology and the practical application of drugs in daily clinical practice. Edited by three highly acclaimed academic anesthetic pharmacologists, with contributions from an international team of experts, and illustrated in full colour, this is a sophisticated, user-friendly resource for all practitioners providing care in the perioperative period.
Human CYP2B6 enzyme although constitutes relatively low proportion (1–4%) of hepatic cytochrome P450 content, it is the major catalyst of metabolism of several clinically important drugs (efavirenz, cyclophosphamide, bupropion, methadone). High interindividual variability in CYP2B6 function, contributing to impaired drug-response and/or adverse reactions, is partly elucidated by genetic polymorphisms, whereas non-genetic factors can significantly modify the CYP2B6 phenotype. The influence of genetic and phenoconverting non-genetic factors on CYP2B6-selective activity and CYP2B6 expression was investigated in liver tissues from Caucasian subjects (N = 119). Strong association was observed between hepatic S-mephenytoin N-demethylase activity and CYP2B6 mRNA expression (P < 0.0001). In less than one third of the tissue donors, the CYP2B6 phenotype characterized by S-mephenytoin N-demethylase activity and/or CYP2B6 expression was concordant with CYP2B6 genotype, whereas in more than 35% of the subjects, an altered CYP2B6 phenotype was attributed to phenoconverting non-genetic factors (to CYP2B6-specific inhibitors and inducers, non-specific amoxicillin + clavulanic acid treatment and chronic alcohol consumption, but not to the gender). Furthermore, CYP2B6 genotype–phenotype mismatch still existed in one third of tissue donors. In conclusion, identifying potential sources of CYP2B6 variability and considering both genetic variations and non-genetic factors is a pressing requirement for appropriate elucidation of CYP2B6 genotype–phenotype mismatch.
Failure to evaluate actual toxicities of investigational molecules in drug discovery is majorly due to inadequate evaluation of their pharmacokinetics. Limitation of conventional drug metabolism profiling procedure demands advancement of existing approaches. Various techniques such as 3D cell culture system, bio microfluidic OoC model, sandwich culture model is in pipeline to be employed at their full potential in drug discovery phase. Although they outweigh the conventional techniques in various aspects, a more detailed exploration of applicability in terms of automation and high throughput analysis is required. This review extensively discusses various ongoing innovations in bioanalytical techniques. The review also proposed various scientific strategies to be adopted for prior assessment of interaction possibilities in translational drug discovery research.
The cytochrome P450 (CYP) enzyme family is the most important enzyme system catalyzing the phase 1 metabolism of pharmaceuticals and other xenobiotics such as herbal remedies and toxic compounds in the environment. The inhibition and induction of CYPs are major mechanisms causing pharmacokinetic drug–drug interactions. This review presents a comprehensive update on the inhibitors and inducers of the specific CYP enzymes in humans. The focus is on the more recent human in vitro and in vivo findings since the publication of our previous review on this topic in 2008. In addition to the general presentation of inhibitory drugs and inducers of human CYP enzymes by drugs, herbal remedies, and toxic compounds, an in-depth view on tyrosine-kinase inhibitors and antiretroviral HIV medications as victims and perpetrators of drug–drug interactions is provided as examples of the current trends in the field. Also, a concise overview of the mechanisms of CYP induction is presented to aid the understanding of the induction phenomena.
Background
Cancer-related fatigue is a significant problem and is associated with poor quality of life. Behavioral interventions include exercise and cognitive-behavioral therapy, which survivors may be unwilling or unable to adopt. Pharmacologic interventions (e.g., selective serotonin reuptake inhibitors) have been disappointing. One potential therapy is the antidepressant bupropion, a norepinephrine-dopamine reuptake inhibitor that targets both inflammation and the hypothalamic-pituitary-adrenal axis. The current study is intended to provide a rigorous test of the efficacy and tolerability of bupropion for cancer-related fatigue.
Methods
A randomized, double-blind, placebo-controlled trial will examine the effects of bupropion on cancer-related fatigue. The trial will be conducted nationwide through the University of Rochester Medical Center (URMC) National Cancer Institute Community Oncology Research Program (NCORP). Disease-free breast cancer survivors (n = 422) who completed chemotherapy and/or radiotherapy 12–60 months previously and report significant fatigue will be randomized 1:1 to receive bupropion (300 mg/day) or placebo. Outcomes will be assessed at baseline and the 12-week follow-up. The primary outcome, fatigue, will be measured with the Functional Assessment of Chronic Illness Therapy – Fatigue (FACIT-F). Secondary outcomes include quality of life, depression, and drug tolerability. Exploratory outcomes include cognition and symptomatology. Potential biological mechanisms and genetic moderators of cancer-related fatigue will also be explored.
Discussion
This study is the first placebo-controlled trial to our knowledge to evaluate bupropion for cancer-related fatigue. Positive results could revolutionize the treatment of cancer-related fatigue, as bupropion is safe, inexpensive, widely-available, and may be more tolerable and acceptable for many patients than current, limited treatment options.
Bupropion is an atypical antidepressant of the aminoketone group, structurally related to cathinone, associated with a wide interindividual variability. An extensive pharmacokinetics review of bupropion was performed, also focusing on chemical, pharmacological, toxicological, clinical and forensic aspects of this drug without a limiting period. Bupropion is a chiral, basic, highly lipophilic drug, clinically used as racemate that undergoes extensive stereoselective metabolism. Its major active metabolites, hydroxybupropion, threohydrobupropion and erythrohydrobupropion reach higher plasma concentrations than bupropion. Bupropion exerts its effects mainly by inhibiting dopamine and norepinephrine reuptake and inhibiting several nicotinic receptors. Recent reports highlight recreational use of bupropion via intranasal insufflation and intravenous use. Seizures, insomnia, agitation, headache, dry mouth, nausea are some of the reported adverse effects. Neurologic effects are major signs of intoxication that should be carefully managed. Finally, the characterization of the polymorphic enzymes involved in the metabolism of bupropion is essential to understand factors that may influence the interindividual and intraindividual variability in bupropion metabolite exposure, including the evaluation of potential drug-drug interactions and pharmacogenetic implications.
Biotransformation or metabolism is responsible for elimination of 70% of drugs available in the market today [1]. Drug-metabolizing enzymes (DMEs) are an assorted group of enzymes responsible for metabolizing xenobiotics such as drugs, carcinogens, pesticides, and food toxicants as well as endogenous compounds such as prostaglandins, steroids, and bile acids [2, 3]. R.T. Williams coined the concept of two-phase elimination of xenobiotics; reactions such as oxidation, reduction, and hydrolysis are categorized as phase I or activating reactions, while conjugation reactions constitutes phase II reactions and are generally detoxifying in nature [4]. Cytochrome P450 (CYP) families of enzymes are responsible for catalyzing majority of phase I reactions. Phase I reactions convert lipophilic molecules to their water-soluble counterparts [4]. Phase II reactions are catalyzed by enzymes such as uridine diphosphate glucuronosyltransferase (UGT), glutathione transferases (GSTs), N-acetyltransferase (NAT), and sulfotransferases (SULTs) [4]. Phase II enzymes catalyze conjugation of water-soluble molecules to intermediates of phase I reactions for the purpose of improving water solubility. In most cases, the net outcome of both phase I and phase II types of reactions is to impart hydrophilicity to xenobiotics and facilitate their elimination from the body. However, phase I and phase II reactions can also activate inert compounds to pharmacologically active entities [5], toxic end products, and procarcinogens into carcinogenic compounds [4, 6, 7]. CYPs and phase II metabolizing enzymes are known to exhibit polymorphism and have been associated with interindividual variability in drug response and toxicity.
The onset, intensity and duration of therapeutic response to a compound depend on the intrinsic pharmacological activity of the drug and pharmacokinetic factors related to its absorption, distribution, metabolism and elimination that are inherent to the biological system. The process of drug transfer from the site of administration to the systemic circulation and the interspecies factors that impact this process are the scope of this review. In general, the factors that influence oral drug bioavailability via absorption and metabolism can be divided into physicochemical/biopharmaceutical and physiological factors. Physicochemical and biopharmaceutical factors that influence permeability and solubility tend to be species independent. Although there are significant differences in the anatomy and physiology of the gastrointestinal tract, these are not associated with significant differences in the rate and extent of drug absorption between rats and humans. However, species differences in drug metabolism in rats and humans did result in significant species differences in bioavailability. Overall, this review provides a better understanding of the interplay between drug physicochemical/biopharmaceutical factors and species differences/similarities in the absorption and metabolism mechanisms that affect oral bioavailability in rats and humans. This will enable a more rational approach to perform projection of oral bioavailability in human using available rat in vivo data.
Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase solubility and facilitate excretion. On some occasions those structural modifications lead to the formation of new toxic products. In order to reduce animal testing, chemical risk can be assessed using metabolically competent cells. The expression of metabolic enzymes, however, is not stable over time in many in vitro primary culture systems and is often partial or absent in cell lines. Therefore, the study of medicines, additives, and environmental pollutants metabolism in vitro should ideally be conducted in cell systems where metabolic activity has been characterized. We explain here an approach to measure the activity of a class of metabolic enzymes (Human Phase I) in 2D cell lines and primary 3D cultures using chemical probes and their metabolic products quantifiable by UPLC mass spectrometry and luminometry. The method can be implemented to test the metabolic activity in cell lines and primary cells derived from a variety of tissues. © 2017 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.
CYP2B subfamily accounts for 2–10% of total hepatic CYP450 enzymes and participate in the metabolism of around 8% of clinical drugs. Borneol has been widely used in traditional Chinese medicine for thousands of years. There are many studies about borneol-induced promoting penetration role for a number of drugs through various physiologic barriers, whereas there is no report involved the effect of borneol on hepatic CYP2B. The present work studied the in vivo effect of borneol on the expression and activity of rat hepatic CYP2B. The results indicated that the oral administration of borneol (33, 100 and 300 mg/kg/d) to rats for consecutive 7 days increased the hepatic CYP2B1/2 activity by 1.4-, 1.7- and 2.8-fold, hepatic CYP2B1 mRNA expression by 6.3-, 8.7- and 18.1-fold, and hepatic CYP2B1/2 protein expression by 1.2-, 1.9- and 2.6-fold, respectively compared to the control. Additionally, in the borneol pre-dosing (300 mg/kg/d for consecutive 7 days) rats, the increased Clint and decreased AUC0–24 of bupropion were observed as compared to the control. Moreover, there were no obvious effects on CAR protein level in rat liver microsome and nucleus following the borneol treatment. Taken together, our observations indicate that borneol is an in vivo inducer of rat hepatic CYP2B with different regulatory mechanism from phenobarbital-like inducers which caused CYP2B induction with CAR activation.
The crystal structures of human P450 2B6 (CYP2B6) indicate that Phe206 and Val367 are in close proximity to the substrate binding site and suggest that both residues may play important roles in substrate metabolism and inhibitor binding. To test this hypothesis, we investigated the effects of mutating these residues to Ala on the regiospecificity of CYP2B6 for the metabolism of testosterone (Testo) and androstenedione (Andro). For Testo metabolism, 16β-OH-Testo formation by F206A was <5% of wild type (WT), whereas the V367A mutant exhibited a doubling of 16α-OH-Testo formation with a 50% decrease in 16β-OH-Testo formation compared to WT. Significant alterations in the regiospecificity for Andro metabolism were also observed. To investigate the roles of these two residues in the metabolic activation of mechanism-based inactivators, tert-butylphenylacetylene (BPA) and bergamottin (BG) were used to test susceptibility to inactivation. Although the rates of inactivation of both mutants by BG were not significantly decreased compared to WT, the efficiency of inactivation by BPA of both mutants was more than an order of magnitude lower. Our results demonstrate that Phe206 plays a crucial role in determining the specificity of 2B6 for the 16β-hydroxylation of Testo and Andro and that it also plays an important role in BG binding and mechanism-based inactivation by BPA. In addition, Val367 dramatically enhances the catalytic activity of CYP2B6 toward Andro and plays an important role in mechanism-based inactivation by BPA. The results presented here show the important roles of Phe206 and Val367 in interactions of CYP2B6 with substrates and inactivators/inhibitors and are consistent with the crystal structures.
A selective and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for quantitation of bupropion hydrochloride in rat plasma using triazolam as an internal standard. Chromatographic separation was achieved on a SB-C18 column at 30 degrees C, with 50:50 (v/v) acetonitrile-0.1 % formic acid in water as mobile phase. The flow rate was 0.3 mL/min. The determination of bupropion was performed in MRM mode, m/z 239.9 -> 183.7 for bupropion and m/z 343.0 -> 308.0 for triazolam (IS) and positive ion electrospray ionization interface. Calibration curve was linear over range of 1.2 to 480 ng/mL. The intra- and inter-run relative standard deviations of the assay were less than 10 %. The mean absolute recoveries determined at the concentrations of 2.4, 48 and 360 ng/mL were 91.00%, 92.06%, 91.71%, respectively. The validated method is successfully used to analyze the drug in samples of rat plasma for pharmacokinetic study.
This chapter discusses the genetics, metabolic actions, substrates, inducers, and inhibitors of cytochrome P450 2B6.
Polymorphisms in genes encoding cytochrome P450
s (CYPs) cause interindividual variability in the metabolism of a wide range of structurally diverse substrates including drugs, hormones, lipids, and environmental pollutants. Because P450s account for approximately 75 % of the total drug metabolisms, genetic polymorphisms in P450-encoding genes have garnered a good deal of attention in the context of pharmacogenomics research, resulting in the discovery of a number of clinically relevant P450 genetic polymorphisms. Advances in our understanding of P450 pharmacogenomics enhance the therapeutic efficacy of drugs and reduce their toxicities. In this chapter, we review the pharmacogenomics of major P450s from molecular discovery to clinical application, and discuss the clinical implications of genotype-guided personalized pharmacotherapy.
OBJECTIVE: To investigate the effects of myocardial ischemia/reperfusion injury (MIRI) on the metabolizing ability and related change in oxidative/antioxidative capacity in rats. METHODS: Male SD rats were divided into 5 groups, and received the operation to establish model of the myocardial ischemia except the sham operation control group. After myocardial ischemia for 40 min, rats were respectively sacrificed at 15, 60 and 180 min of reperfusion. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities as well as hepatic malondialdehyde(MDA) content and superoxide dismutase(SOD) activity were detected. The catalytic activities of hepatic cytochrome P450(CYP) 3A, CYP2B1 and CYP2E1 were measured by erythromycin N-demethylase, pentoxyresorufin O-deethylase and aniline hydroxylase, respectively. CYP3A1, CYP2B1/2, CYP2E1, phase II detoxicating enzyme NAD(P)H quinone oxidoreductase 1 (NQO1) and its upstream factor NF-E2-related factor (Nrf2) mRNA were determined by RT-PCR. RESULTS: At reperfusion for 60 min, hepatic MDA content began to elevate (P<0.05); meanwhile, SOD activity decreased (P<0.01). Plasma ALT and AST activities increased at 180 min of reperfusion (P<0.05). Nrf2 mRNA level was elevated at 60 min of reperfusion (P<0.05), and its downstream factors NQO1 gene expression also increased after 180 min of reperfusion (P<0.05). CYP3A activity levels and mRNA obviously decreased, respectively, at 60 min and 180 min of reperfusion (P<0.05). The levels of CYP2B mRNA and activity decreased, too, respectively, at 15 min and 180 min of reperfusion (P<0.05). No change in the catalytic activity of CYP2E1 was observed at any time points of reperfusion. CONCLUSION: MIRI results in the hepatic oxidative stress which is followed by the reduction of liver function. At early stage of reperfusion, NQO1 gene is significantly up-regulated at transcription level, which may be due to the activation of its upstream factor Nrf2. CYP3A and CYP2B catalytic activities are down-regulated at transcription and/or post-transcription level during the period of the reperfusion.
Licoricidin isolated from Glycyrrhiza uralensis is known to have anticancer, anti-nephritic, anti-Helicobacter pylori, and antibacterial effects. In this study, a cocktail probe assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to investigate the modulating effect of licoricidin on cytochrome P450 (CYP) enzymes in human liver microsomes. When licoricidin was incubated at 0-25?m with CYP probes for 60 min at 37^{\circ}C, it showed potent inhibitory effects on CYP2B6-catalyzed bupropion hydroxylation and CYP2C9-catalyzed diclofenac 4`-hydroxylation with half maximal inhibitory concentration (IC_{50}) values of 3.4 and 4.0?m, respectively. The inhibition mode of licoricidin was revealed as competitive, dose-dependent, and non-time-dependent, and following the pattern of Lineweaver-Burk plots. The inhibitory effect of licoricidin has been confirmed in human recombinant cDNA-expressed CYP2B6 and 2C9 with IC_{50} values of 4.5 and 0.73?m, respectively. In conclusion, this study has shown the potent inhibitory effect of licoricidin on CYP2B6 and CYP2C9 activity could be important for predicting potential herb-drug interactions with substrates that mainly undergo CYP2B- and CYP2C9-mediated metabolism.
The role of membrane transporters for the pharmacokinetic (PK) and pharmacodynamic (PD) properties of a drug is becoming evident in many aspects. Membrane transporters are major determinants for the absorption, distribution, and elimination of many drugs or at least of their more hydrophilic metabolites. Thereby, the overall effect of transporters is thoughtlessly underestimated, if only the systemic concentration of a drug is monitored clinically. The role of drug transporters on cellular and tissue distribution, especially with regard to the brain, the heart, the liver, the kidney, and other peripheral tissues seems to be of much higher importance. Unfortunately, the clinical investigation of tissue distribution phenomena requires the development of new techniques like PET (positron emission tomography), but already today, there is a clinical example of 11C-verapamil crossing the blood-brain barrier in the presence of a strong P-gp inhibitor (Eyal et al. 2010, pp. 579–585). Another impressive example is a significant and growing body of data showing a correlation between long-term response to imatinib chemotherapy in CML patients with functional OCT1 activity in the cancer cells (Engler et al. 2011, pp. 608–611) (imatinib is described as substrate of OCT1). More examples like this, illustrating the importance of transporters for cellular or tissue exposure, will be experienced in the near future with the development of PET-labeled transporter probe substrates and approaches of personalized medicine. The understanding of the role of pharmacogenetics in drug metabolism expanded greatly in the 1990s. This is mainly due to technological improvements in gene scanning and gene variant identification. The number of variant alleles identified for genes coding for drug metabolizing enzymes (DMEs) considerably increased in the early 2000s, and continues to increase. The clinical consequences – or at least genotyping-phenotyping relationships – of DME polymorphisms have not been demonstrated for all variants. Only those DME allele variants will be mentioned for which significant changes in enzyme activity have been found using probe drugs.
We systematically characterized the levels and substrate specificity of P450s from humans and rats to extrapolate drug metabolism data from experimental animals to humans. Human P450s (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2E1, and 3A4) were expressed in Saccharomyces cerevisiae and purified. Rat P450s were purified from hepatic microsomes of rats. We investigated the catalytic activities of purified P450s in a reconstituted system. Human CYP2B6 and rat CYP2B1 had high lidocaine N-deethylation activity. Human and rat CYP2D forms had high debrisoquine 4-hydroxylation activity. Human CYP3A4 and rat CYP3A2 had high testosterone 2β- and 6β-hydroxylation activities in a modified reconstituted system with a lipid mixture. The hydroxylation site of testosterone by CYP2B6 (16α- and 16β-positions) agreed with that by rat CYP2B1. Human CYP2E1 had the highest lauric acid (ω-1)-hydroxylation activity and also had catalytic properties similar to those of rat CYP2E1. Human CYP2A and 2C forms had catalytic properties in testosterone metabolism different from those of rats. Antibodies raised against purified P450s were used to measure the levels of hepatic P450s. The level of CYP3A4 was the highest in human hepatic microsomes, comprising 30–40% of the total P450. CYP2C9 comprised 10–20% of the total. The levels of CYP1A2, 2A6, 2C8, 2D6, and 2E1 were moderate (5–15% of total P450). CYP2B6 content was very low. The information of this study is useful for drug metabolism and toxicological studies.
The pharmacokinetics of bupropion (BUP) and its three major basic metabolites (the erythroamino alcohol [EB], the threoamino alcohol [TB], and the hydroxy [HB] metabolites) were characterized after a single, oral, 200 mg dose of BUP in six healthy men. Twenty-one sequential plasma samples for analysis by HPLC were drawn from each subject over the 56-hour period after dosing. Pharmacokinetic analyses were by noncompartmental methods. The mean elimination t1/2 values of BUP, TB, EB, and HB were 9.8, 19.8, 26.8, and 22.2 hours, respectively. The mean plasma AUCs of TB and HB were 2.4 and 10.3 times greater, respectively, than that for BUP. Because of the substantial presence of these metabolites in systemic circulation, further studies are recommended to understand further their roles in the clinical profile of this new antidepressant.
To study cytochrome P-450 (CYP) 2B6 contribution to methoxychlor metabolism within human liver microsomes and to initiate an investigation of CYP2B6 protein expression, we developed a polyclonal antibody targeted to a 20-residue peptide within that protein. The antibody was found to be highly sensitive and monospecific for CYP2B6 on immunoblots. Although many immunological studies have described the absence or low expression of CYP2B6 in human livers, in the present investigation, we have found this not to be the case. We immunoquantified CYP2B6 apoprotein expression in a panel of 28 livers and found concentrations ranging from 2 to 82 pmol/mg protein, with a mean value of 25 pmol/mg protein. Five livers ( approximately 18%) displayed relatively high levels of CYP2B6 (>40 pmol/mg protein). There were no sex-related differences, although the highest level was observed in a 1-week postpartum donor given several medications. A marked diminution in variability was found in individuals aged 56 or older (n = 12), but there were no age-related trends in mean CYP2B6 content. We suggest that CYP2B6 represents a significant portion of total CYP in human liver. The exquisite sensitivity of this antibody (fmol quantities are detected easily on immunoblots) may explain our detection of CYP2B6 in 100% of livers versus its detection in a limited number of livers by certain other investigators. The antibody also was found to immunoinhibit CYP2B6-catalyzed N-demethylation of (S)-mephenytoin in human liver microsomes by 68 to 79%. The utility of this antibody for determining human liver microsomal CYP2B6 contribution to the ortho-hydroxylation of methoxychlor was demonstrated.
Fifteen xanthates with carbon chains of different lengths or substitutions, including the antiviral compound D609 (O-tricyclo[5.2. 1.0(2,6)]dec-9-yl-dithiocarbonate), were tested for their ability to inactivate cytochromes P-450 (P-450s) 2B1 and 2B6. All of the xanthates tested were found to inactivate P-450 2B1 in a time- and concentration-dependent manner. The rates of inactivation at 30 degrees C ranged from 0.22 min-1 to 0.02 min-1. The concentrations required for half-maximal inactivation were between 2.4 and 69 microM. A general trend in the inactivation kinetics could be observed with an increasing chain length of the xanthates. Longer carbon chains resulted in slower rates of inactivation with longer half-times of inactivation and higher partition ratios. For P-450 2B1, the most effective inactivators were xanthates with substitutions of intermediate length. The best inactivator for P-450 2B1 was the C8 xanthate, with an inactivation potency (KI) of 2.4 microM, a rate of inactivation of 0.07 min-1, and a partition ratio of 4. Four xanthates were further examined for their effect on the 7-ethoxy-4-(trifluoromethyl)coumarin activity of P-450 2B6. The C8 xanthate was again the most effective inactivator, with a KI of 1 microM. Although the KI values were generally lower than those found with P-450 2B1, the rates of inactivation for P-450 2B6 with the various xanthates were 3- to 5-fold slower. In addition, the isozyme selectivity of xanthates was tested with P-450s 2E1, 1A1, 3A2, 3A4, 2C9, and 2D6. P-450 2E1 was inactivated by xanthates at concentrations 15- to 100-fold higher than those required to inactivate either P-450 2B1 or 2B6. P-450 1A1 was not inactivated by xanthates. However, all of the xanthates tested were able to inhibit the enzymatic activity of P-450 1A1 to a different extent, depending on the length of the xanthate carbon chain. Virtually no inactivation of P-450s 2D6 or 2C9 was seen, except that C8 and D609 were inhibitory at high concentrations (0.2-0.6 mM). None of the xanthates studied had any effect on the activities of P-450s 3A2 or 3A4.
Expression of human cytochrome P450 (P450) 2B6 in Escherichia coli was achieved following supplementation of the expression medium with chloramphenicol. The recombinant protein was purified using Ni(2+)-nitrilotriacetate chromatography and was characterized with regard to its spectral properties and catalytic activities toward typical P450 substrates. The purified recombinant protein was also used to raise polyclonal antibodies in rabbits. Examination of a panel of human liver microsomal preparations revealed expression of P450 2B6 in most samples, with levels of <1 to 30 pmol 2B6/mg microsomal protein. Examination of purified P450 2B6 preparations revealed the presence of a protease-sensitive site located 126 residues away from the N-terminus. The identity of the cleavage boundary was verified by protein sequence analysis. Cleavage of P450 2B6 at that site results in the presence of a lower molecular weight fragment of approximately 35 kDa in purified preparations. An immunoreactive peptide of a similar molecular weight was consistently observed in some but not all human liver microsomal preparations suggesting cleavage at the same site. Examination of catalytic activities of the purified reconstituted protein indicated the potential utility of (S)-mephenytoin N-demethylation and testosterone 16beta-hydroxylation as markers for P450 2B6.
Members of the nuclear-receptor superfamily mediate crucial physiological functions by regulating the synthesis of their target genes. Nuclear receptors are usually activated by ligand binding. Cytochrome P450 (CYP) isoforms often catalyse both formation and degradation of these ligands. CYPs also metabolize many exogenous compounds, some of which may act as activators of nuclear receptors and disruptors of endocrine and cellular homoeostasis. This review summarizes recent findings that indicate that major classes of CYP genes are selectively regulated by certain ligand-activated nuclear receptors, thus creating tightly controlled networks.
Inhibition of human cytochrome P4502D6 (CYP2D6)-catalysed metabolism can lead to clinically significant alterations in pharmacokinetics. Since there is evidence that the selective serotonin reuptake inhibitor (SSRI) class of antidepressant drugs might inhibit CYP2D6, the effects of five SSRIs on human liver microsomal CYP2D6 activity were compared with each other and with three tricyclic antidepressant drugs. On a molar basis, paroxetine was the most potent of the SSRIs at inhibiting the CYP2D6-catalysed oxidation of sparteine (Ki = 0.15 microM), although fluoxetine (0.60 microM) and sertaline (0.70 microM) had Ki values in the same range. Fluvoxamine (8.2 microM) and citalopram (5.1 microM) also inhibited CYP2D6 activity. The major circulating metabolites of paroxetine in man produced negligible inhibition. In contrast, norfluoxetine the active metabolite of fluoxetine, was a potent CYP2D6 inhibitor (0.43 microM). CYP2D6 activity was also diminished by the tricyclic antidepressant drugs clomipramine (2.2 microM), desipramine (2.3 microM) and amitriptyline (4.0 microM). These findings suggest that compounds with SSRI activity are likely to interact with human CYP2D6 in vivo with the potential of causing drug interactions.
The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite,trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPH-cytochrome P450 reductase. Bacterial membranes were harvested and incubated with tamoxifen ortrans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis. CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 ± 20 pmol/40 min/0.2 nmol P450 using 18 μM tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 ± 0.8 and 3.1 ± 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 μM tamoxifen). These two forms also catalyzed 4′-hydroxylation (13.0 ± 1.9 and 1.4 ± 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 μM tamoxifen; 0.51 ± 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 μM tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations. CYP1B1 was the principal catalyst of 4-hydroxytamoxifentrans-cis isomerization but CYP2B6 and CYP2C19 also contributed.
Background and objective:
Recent in vitro studies have suggested an important role of cytochrome P450 (CYP) 2B6 and CYP2C19 in methadone metabolism. We aimed to determine the influence of CYP2B6, CYP2C9, and CYP2C19 genetic polymorphism on methadone pharmacokinetics and on the response to treatment.
Methods:
We included 209 patients in methadone maintenance treatment on the basis of their response to treatment and their daily methadone dose. Patients were genotyped for CYP2B6, CYP2C9, and CYP2C19. Steady-state trough and peak (R)-, (S)-, and (R,S)-plasma levels and peak-to-trough plasma level ratios were measured.
Results:
CYP2B6 genotype influences (S)-methadone and, to a lesser extent, (R)-methadone plasma levels, with the median trough (S)-methadone plasma levels being 105, 122, and 209 ng . kg/mL . mg for the noncarriers of allele *6, heterozygous carriers, and homozygous carriers (*6/*6), respectively (P = .0004). CYP2C9 and CYP2C19 genotypes do not influence methadone plasma levels. Lower peak and trough plasma levels of methadone and higher peak-to-trough ratios were measured in patients considered as nonresponders [median (R,S)-methadone trough plasma levels of 183 and 249 ng . kg/mL . mg (P = .0004) and median peak-to-trough ratios of 1.82 and 1.58 for high-dose nonresponders and high-dose responders, respectively (P = .0003)].
Conclusion:
Although CYP2B6 influences (S)-methadone plasma levels, given that only (R)-methadone contributes to the opioid effect of this drug, a major influence of CYP2B6 genotype on response to treatment is unlikely and has not been shown in this study. Lower plasma levels of methadone in nonresponders, suggesting a higher clearance, and higher peak-to-trough ratios, suggesting a shorter elimination half-life, are in agreement with the usual clinical measures taken for such patients, which are to increase methadone dosages and to split the daily dose into several intakes.
The pharmacokinetics of bupropion and 3 of its basic metabolites were determined in 8 young, healthy, male volunteers after single and multiple oral doses of bupropion. Plasma profiles were obtained: 1) after a single 100 mg oral dose of bupropion hydrochloride, 2) following administration of 100 mg 8-hourly for 14 days and 3) again after a single 100 mg dose 14 days later. Plasma concentrations of the parent drug and metabolites were determined by high-performance liquid chromatography. Saliva secretion and pupil diameters were measured, subjective assessments of sleep made using visual analogue scales and side effects, blood counts and biochemistry were monitored. After the first dose mean elimination half lives (t1/2) of bupropion, and metabolites I and II were 8, 19 and 19 h respectively. On repeated administration there was little accumulation of the parent drug and no evidence for induction of its own metabolism. Accumulation of I was consistent with its rate of elimination after single doses while that of II was greater than predicted with prolongation of t1/2 to 35 h. Metabolite III was barely detectable after single doses but its accumulation on multiple dosing was consistent with its long half life (35 h) determined on occasion 2. Saliva secretion was significantly reduced during the multiple dosing period but there were no complaints of dry mouth. Subjective assessments of sleep were not significantly altered though one subject reported vivid dreams. There were no other adverse reactions.
1. A novel modelling alignment for P450s, utilizing NADPH-P450 reductase for electron transfer, is proposed on the basis of analysis of their amino acid sequences. 2. Information used to facilitate the alignment process includes: the recent X-ray crystal structure of P450102 (P450bm3), site-directed mutagenesis experiments, chemical modification of specific residues, and antibody recognition studies. 3. The alignment has been used to construct a number of microsomal P450s of relevance to xenobiotic and endogenous metabolism.
Ropivacaine is a local anesthetic with a long duration of action. Although it is less toxic than bupivacaine, local anesthetic toxicity is possible when the plasma concentration is increased. Because ropivacaine is an amide-type local anesthetic, it is metabolized by cytochrome P450 (P450) in the liver, and its elimination and plasma concentration can be dependent on the level of P450. The purpose of this investigation was to elucidate the metabolism of ropivacaine by human hepatic P450.
The metabolism of ropivacaine was compared using recombinant human and purified rat hepatic P450 isozymes. An inhibition study using antibodies against rat P450 was performed using hepatic microsomes from human and rat to identify which P450s are involved in ropivacaine metabolism.
Ropivacaine was metabolized to 2',6'-pipecoloxylidide (PPX), 3'-hydroxyropivacaine (3'-OH Rop), and 4'-hydroxyropivacaine (4'-OH Rop) by hepatic microsomes from human and rat. PPX was a major metabolite of both human and rat hepatic microsomes. In a reconstituted system with rat P450. PPX was produced by CYP2C11 and 3A2, 4'-OH Rop by CYP1A2, and 3'-OH Rop by CYP1A2 and 2D1. Formation of PPX in rat hepatic microsomes was inhibited by anti CYP3A2, but not by CYP2C11 antibody, and formation of 3'-OH Rop was inhibited by CYP1A2 and 2D1 antibodies. Anti CYP3A2 and 1A2 antibodies inhibited the formation of PPX and 3'-OH Rop in human hepatic microsomes, respectively. Recombinant human P450s expressed in lymphoblast cells were used for further study. CYP3A4 and 1A2 formed the most PPX and 3'-OH Rop, respectively. Ropivacaine N-dealkylation and 3'-hydroxylation activities correlated well with the level of CYP3A4 and 1A2 in human hepatic microsomes, respectively.
Ropivacaine was metabolized to PPX, 3'-OH Rop, and 4'-OH Rop by hepatic P450. PPX was a major metabolite in human hepatic microsomes. CYP3A4 was involved in producing PPX. CYP1A2 was involved in the formation of 3'-OH Rop in human hepatic microsomes.
Methoxyflurane nephrotoxicity is mediated by cytochrome P450-catalyzed metabolism to toxic metabolites. It is historically accepted that one of the metabolites, fluoride, is the nephrotoxin, and that methoxyflurane nephrotoxicity is caused by plasma fluoride concentrations in excess of 50 microM. Sevoflurane also is metabolized to fluoride ion, and plasma concentrations may exceed 50 microM, yet sevoflurane nephrotoxicity has not been observed. It is possible that in situ renal metabolism of methoxyflurane, rather than hepatic metabolism, is a critical event leading to nephrotoxicity. We tested whether there was a metabolic basis for this hypothesis by examining the relative rates of methoxyflurane and sevoflurane defluorination by human kidney microsomes.
Microsomes and cytosol were prepared from kidneys of organ donors. Methoxyflurane and sevoflurane metabolism were measured with a fluoride-selective electrode. Human cytochrome P450 isoforms contributing to renal anesthetic metabolism were identified by using isoform-selective inhibitors and by Western blot analysis of renal P450s in conjunction with metabolism by individual P450s expressed from a human hepatic complementary deoxyribonucleic acid library.
Sevoflurane and methoxyflurane did undergo defluorination by human kidney microsomes. Fluoride production was dependent on time, reduced nicotinamide adenine dinucleotide phosphate, protein concentration, and anesthetic concentration. In seven human kidneys studied, enzymatic sevoflurane defluorination was minima, whereas methoxyflurane defluorination rates were substantially greater and exhibited large interindividual variability. Kidney cytosol did not catalyze anesthetic defluorination. Chemical inhibitors of the P450 isoforms 2E1, 2A6, and 3A diminished methoxyflurane and sevoflurane defluorination. Complementary deoxyribonucleic acid-expressed P450s 2E1, 2A6, and 3A4 catalyzed methoxyflurane and sevoflurane metabolism, in diminishing order of activity. These three P450s catalyzed the defluorination of methoxyflurane three to ten times faster than they did that of sevoflurane. Expressed P450 2B6 also catalyzed methoxyflurane defluorination, but 2B6 appeared not to contribute to renal microsomal methoxyflurane defluorination because the P450 2B6-selective inhibitor had no effect.
Human kidney microsomes metabolize methoxyflurane, and to a much lesser extent sevoflurane, to fluoride ion. P450s 2E1 and/or 2A6 and P450 3A are implicated in the defluorination. If intrarenally generated fluoride or other metabolites are nephrotoxic, then renal metabolism may contribute to methoxyflurane nephrotoxicity. The relative paucity of renal sevoflurane defluorination may explain the absence of clinical sevoflurane nephrotoxicity to date, despite plasma fluoride concentrations that may exceed 50 microM.
Previous studies demonstrated that methoxychlor [1,1,1-trichloro-2,2-bis-(4-methoxyphenyl)ethane] is a proestrogen and is toxic to mammalian reproductive processes. Mammalian liver microsomes sequentially demethylate methoxychlor (I), yielding two estrogenic metabolites, mono-OH-M (II) and bis-OH-M (III). Liver microsomes from phenobarbital (PB)-treated rats (PB microsomes) additionally formed a catechol product, tris-OH-M (VII) (Kupfer et al., Chem. Res. Toxicol. 3, 8-16, 1990). This study shows that, in addition to compounds II, III and VII, male and female rat PB microsomes catalyze the formation of a novel ring-hydroxylated methoxychlor metabolite, ring-OH-M (IV). Liver microsomes from male rats treated with pregnenolone-16 alpha-carbonitrile formed the same metabolites as PB microsomes, but the ring-OH-M was formed only in minute amounts, and there was no tris-OH-M. Liver microsomes from methylchlolanthrene-treated and control male rats demethylated methoxychlor, but did not form ring-hydroxylated products. Similarly, human liver microsomes exhibited demethylation but not ring-hydroxylation. Incubation of mono-OH-M (II) with control rat liver microsomes yielded only bis-OH-M (III), whereas incubation of ring-OH-M (IV) resulted in monodemethylated (dihydroxy) compounds V/VI and didemethylated ring-hydroxylated compound, tris-OH-M (VII). Incubation of (IV) with PB microsomes led to compounds V and/or VI and tris-OH-M (VII), whereas incubation of mono-OH-M (II) yielded bis-OH-M (III) and tris-OH-M (VII). The evidence indicates that ring-hydroxylation is catalyzed by CYP2B: a) induction of CYP2B was required for ring-hydroxylation; b) antibodies against CYP2B1/2 strongly inhibited the formation of the ring-hydroxylated products by PB microsomes; c) incubation of methoxychlor with reconstituted CYP2B1 yielded both the hydroxylated (IV and VII) and the demethylated (II and III) metabolites; and d) reconstituted CYP2B1 converted mono-OH-M into bis-OH-M and tris-OH-M, whereas bis-OH-M was converted into tris-OH-M. Human CYP2B6 exhibits ring-hydroxylation, indicating that this reaction is catalyzed by several CYP2B isozymes. In addition, this study demonstrates that the formation of the catechol tris-OH-M involves two metabolic pathways: via O-demethylation followed by ring-hydroxylation and via ring-hydroxylation and subsequent O-demethylation.
Antipyrine has been widely used as a probe drug for human oxidative drug metabolism. To evaluate the role of antipyrine as a model drug, we have identified the cytochrome P450 enzymes involved in 4-hydroxyantipyrine, 3-hydroxymethylantipyrine, and norantipyrine formation.
We used the following methods for this study: (1) determination of enzyme kinetics for antipyrine metabolite formation in human liver microsomes, (2) inhibition studies with antibodies and inhibitors, and (3) formation of metabolites by stable expressed human P450 enzymes.
Antipyrine biotransformation could be described by Michaelis-Menten kinetics: norantipyrine: maximum rate of metabolite formation (Vmax), 0.91 +/- 0.04 nmol . mg-1 . min-1; Michaelis-Menten constant (Km), 19.0 +/- 0.8 mmol/L; 4-hydroxyantipyrine: Vmax, 1.54 +/- 0.08 nmol . mg-1 . min-1;Km,39.6 +/- 2.5 mmol/L. Antibodies against CYP3A4 inhibited the formation of 4-hydroxyantipyrine by 25% to 65%. LKM-2 antibodies (anti-CYP2C) caused a 75% to 100% inhibition of norantipyrine and a 58% to 80% inhibition of 3-hydroxymethylantipyrine formation. Sulfaphenazole inhibited the formation of 3-hydroxymethylantipyrine and norantipyrine by about 50%. Furafylline and fluvoxamine inhibited norantipyrine, 4-hydroxyantipyrine, and 3-hydroxymethylantipyrine formation by about 30%, 30%, and 50%, respectively. Ketoconazole reduced formation of norantipyrine, 3-hydroxymethylantipyrine, and 4-hydroxyantipyrine by up to 80%. Formation in stable expressed enzymes indicated involvement of CYP1A2, CYP2B6, CYP2C, and CYP3A4 in metabolite formation.
Antipyrine metabolites are formed by at least six hepatic cytochrome P450 enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C18, and CYP3A4). 4-Hydroxylation is mainly catalyzed by CYP3A4 and, to a lesser extent, by CYP1A2. The CYP2C subfamily contains the predominant enzymes for norantipyrine formation, and CYP1A2 is also involved. Formation of 3-hydroxymethylantipyrine is mediated by CYP1A2 and CYP2C9. Because several cytochrome P450 enzymes are involved in the formation of each metabolite, antipyrine is not well suited as a probe for distinct human cytochrome P450 enzymes.
Anin vitroradiometric assay selective for inducible CYP2B activity is described. The assay is based on the quantification of3H2O release that occurs duringo-ring hydroxylation of [o-3H]methoxychlor by liver microsomes in the presence of NADPH.3H2O is isolated by removing >99.9% of the parent compound and organic metabolites by facile charcoal extraction and filtration. There was no evidence for an NIH shift during ring hydroxylation, and there was little or no isotope effect. Selectivity for CYP2B was demonstrated using liver microsomes prepared from rats and mice treated with inducers of different CYP isoforms. Ring hydroxylation of [o-3H]methoxychlor was elevated 11.4-fold over control values in liver microsomes from male rats treated with phenobarbital. With mice, phenobarbital treatment elevated liver microsomal ring hydroxylation 7.1-fold. Clofibrate, 3-methylcholanthrene, or β-naphthoflavone treatment of male rats or pyridine treatment of female rats did not elevate liver microsomal ring-hydroxylase activity, indicating that CYP4A, 1A, and 2E1 do not support this reaction. In female rats, dexamethasone and pregnenolone-16 α-carbonitrile treatment elevated ring hydroxylation up to 5.5- and 3.2-fold, respectively, an activity that may be attributed to CYP2B induction in those animals. Incubation of liver microsomes from phenobarbital-treated males with monospecific anti-CYP2B monoclonal antibodies (Mab) inhibited ring-hydroxylase activity up to 86%, demonstrating predominantly CYP2B-mediated catalysis. An 86% inhibition by these Mabs was also observed using liver microsomes from male mice treated with phenobarbital, indicating the assay is not limited to rats. The CYP2B mechanism-based inhibitor orphenadrine caused a 76% decline in activity, providing further evidence for CYP2B involvement. Unlike other CYP2B-selective assays, this method may be readily adapted toin vivostudies, by measuring urinary excretion of3H2O as an indication of total body CYP2B activity.
In vitro methods were used to identify the cytochrome P450 (CYP) enzyme(s) involved in S-mephenytoin N-demethylation. S-Mephenytoin (200 microM) was incubated with human liver microsomes, and nirvanol formation was quantitated by reversed-phase HPLC. S-Mephenytoin N-demethylase activity in a panel of human liver microsomes ranged 35-fold from 9 to 319 pmol/min/mg protein and correlated strongly with microsomal CYP2B6 activity (r = 0.91). Additional correlations were found with microsomal CYP2A6 and CYP3A4 activity (r = 0.88 and 0.74, respectively). Microsomes prepared from human beta-lymphoblastoid cells transformed with individual P450 cDNAs were assayed for S-mephenytoin N-demethylase activity. Of 11 P450 isoforms (P450s 1A1, 1A2, 2A6, 2B6, 2E1, 2D6, 2C8, 2C9, 2C19, 3A4, and 3A5) tested, only CYP2B6 catalyzed the N-demethylation of S-mephenytoin with an apparent K(m) of 564 microM. Experiments with P450 form-selective chemical inhibitors, competitive substrates, and anti-P450 antibodies were also performed. Troleandomycin, a mechanism-based CYP3A selective inhibitor, and coumarin, a substrate for CYP2A6 and therefore a potential competitive inhibitor, failed to inhibit human liver microsomal S-mephenytoin N-demethylation. In contrast, orphenadrine, an inhibitor of CYP2B forms, produced a 51 +/- 4% decrease in S-mephenytoin N-demethylase activity in human liver microsomes and a 45% decrease in recombinant microsomes expressing CYP2B6. Also, both CYP2B6-marker 7-ethoxytrifluoromethylcoumarin O-deethylase and S-mephenytoin N-demethylase activities were inhibited by approximately 65% by 5 mg anti-CYP2B1 IgG/mg microsomal protein. Finally, polyclonal antibody inhibitory to CYP3A1 failed to inhibit S-mephenytoin N-demethylase activity. Taken together, these studies indicate that the N-demethylation of S-mephenytoin by human liver microsomes is catalyzed primarily by CYP2B6.
1. We have examined the metabolism of diazepam by ten human cytochrome P450 forms (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5) expressed in HepG2 cells using a recombinant vaccinia virus system. 2. Among the P450 forms tested, diazepam was significantly demethylated by CYP2B6, 2C9, 2C19, 3A4 and 3A5, with 2C19 exhibiting the highest rate at concentrations < 0.1 mM, and hydroxylated only by the latter three enzymes, with 3A5 being the most active. The N-demethylation activity of diazepam by 2C19 at a concentration of 20 microM was six times of that by 3A4. However, that by 2C9 was detected at only a trace level. 3. CYP2C19, 3A4 and 3A5 of the ten human P450s catalysed the 3-hydroxylation of nordiazepam, and 2B6, the 2C subfamily and the 3A subfamily catalysed the N-demethylation of temazepam. CYP3A4 exhibited the highest activity of nordiazepam 3-hydroxylation and temazepam N-demethylation. 4. Diazepam N-demethylation by human liver microsomes correlated with diazepam 3-hydroxylation, but not S-mephenytoin 4'-hydroxylation. 5. Our results suggest that in the human liver, the metabolism of diazepam to nordiazepam is mediated by CYP3A4, which has been reported as the most abundant P450 form in human liver as well as 2C19, which has been reported as a polymorphic enzyme.
The specificities of orphenadrine and methimazole on eight human liver P450 enzyme activities were evaluated by studying the extent of inhibition at different concentrations in two protocols: competitive inhibition and preincubation. In the competitive inhibition protocol, orphenadrine decreased CYP2B6 marker activity up to 45-57% in human liver microsomes and up to 80-97% in cell microsomes containing cDNA-expressed CYP2B6. Orphenadrine strongly decreased CYP2D6 marker activity by 80-90%. Orphenadrine also partially decreased the CYP1A2, CYP2A6, CYP3A4, and CYP2C19 marker activities. In the preincubation protocol, orphenadrine decreased the CYP2B6 activity in cDNA-expressed cell microsomes to completion. In human liver microsomes, orphenadrine strongly decreased the marker activities of CYP2B6, CYP2D6, as well as CYP2C9; and partially decreased the marker activities of CYP1A2, CYP2A6, CYP3A4, and CYP2C19. In the competitive inhibition protocol, methimazole had no effect on the marker activities of CYP2E1 and CYP2A6; slightly decreased CYP2D6 marker activity; partially decreased the marker activities of CYP2C19, CYP2C9, and CYP2B6; and dramatically decreased CYP3A4 marker activity. Methimazole decreased CYP1A2 marker activity at lower concentrations, but not at the highest concentration studied (1 mM). In the preincubation protocol, methimazole was shown to be a potent and nonspecific inhibitor of all the enzyme activities. Marker activities of CYP2C9, CYP2C19, and CYP3A4 were completely inhibited at relatively low concentrations. This study indicates orphenadrine cannot be used as a selective inhibitor of CYP2B6 in human liver microsomes and that methimazole is not a selective inhibitor of the flavin-containing monooxygenase in human liver microsomes.
1. The construction of three-dimensional models of CYP2B isozymes from rat (CYP2B1), rabbit (CYP2B4) and man (CYP2B6), based on a multiple sequence alignment with CYP102, a unique eukaryotic-like bacterial P450 (in terms of possessing an NADPH-dependent FAD- and FMN-containing oxidoreductase redox partner) of known crystal structure, is reported. 2. The enzyme models described are shown to be consistent with experimental evidence from site-directed mutagenesis studies, antibody recognition sites and amino acid residues identified as being associated with redox partner interactions, together with the location of a key serine residue (Ser-128) likely to be involved in protein kinaseA-mediated phosphorylation. 3. A substantial number of known substrates and inhibitors of CYP2B isozymes are shown to fit the putative active sites of the enzyme models in agreement with their reported position of metabolism or mode of inhibition respectively. In particular, there is complementarity between the characteristic non-planar geometries of CYP2B substrates and key groups in the enzymes' active sites. 4. Molecular modelling of CYP2B isozymes appears to rationalize a number of the reported findings from quantitative structure-activity relationship investigations on series of CYP2B substrates and inhibitors.
7-Ethoxy-4-trifluoromethylcoumarin (7-EFC) was examined as a substrate for cytochrome P450 (P450) in microsomes from human livers and expressed in B-lymphoblastoid cells. The O-deethylation of 7-EFC to 7-hydroxy-4-trifluoromethylcoumarin (7-HFC) varied over a liver bank (n = 19) by a factor of 13 (40-507 pmol min-1 mg-1 protein). When compared with the ability of the bank of human liver samples to metabolize form-selective substrates of the P450, 7-HFC formation correlated strongly with the formation of the S-mephenytoin metabolite, nirvanol (r2 = 0.86, p < 0.0001). alpha-Napthoflavone (ANF), diethyldithiocarbamate (DDC) and chloramphenicol (CAP) inhibited the O-deethylation of 7-EFC by microsomes from human livers by greater than 60%. Orphenadrine (ORP), a reported specific CYP2B6 inhibitor, was a less potent inhibitor of 7-HFC formation by microsomes from human liver than DDC or ANF. Using microsomes from B-lymphoblastoid cells expressing specific P450s, CYP2B6 and CYP1A2 were found to produce substantial levels of 7-HFC whereas CYP2E1 and CYP2C19 produced detectable amounts of this metabolite. ORP inhibited expressed CYP2E1 and CYP2B6 mediated 7-HFC formation to a greater extent than the inhibition observed for CYP1A2. Methoxychlor and S-mephenytoin inhibited expressed CYP2B6 but not CYP1A2 mediated 7-EFC O-deethylation. Livers (n = 5) with high relative rates of 7-HFC formation displayed biphasic enzyme kinetics with the low K(m) site (average K(m) = 3.3 microM) demonstrating allosteric activation. Five livers with low relative rates of 7-HFC formation also exhibited biphasic kinetics but lacked evidence of an allosteric mechanism being involved in the low K(m) component (average K(m) = 2.4 microM). Furthermore, expressed CYP2B6 and CYP2E1 converted 7-EFC to 7-HFC with allosteric activation indicated, while CYP1A2 mediated metabolism of 7-EFC to 7-HFC best fit the classic Michaelis-Menten model. A commercially available antibody to rat CYP2B, suggested to be specific for CYP2B6, was found to cross react with all members to the CYP2 family examined including CYP2C19, which possessed a nearly identical electrophoretic mobility to that of CYP2B6 in the system examined. In total, the evidence presented indicates that multiple P450s are involved in the formation of 7-HFC from 7-EFC, therefore this does not appear to be a useful or a selective probe of CYP2B6 catalytic activity. Furthermore, the specificity of both antibody and chemical inhibitor (ORP) probes previously suggested to be specific for CYP2B6 is also questioned.
Biotransformation of the selective serotonin reuptake inhibitor antidepressant, fluoxetine, to its principal metabolite, norfluoxetine, was evaluated in human liver microsomes and in microsomes from transfected cell lines expressing pure human cytochromes. In human liver microsomes, formation of norfluoxetine from R,S-fluoxetine was consistent with Michaelis-Menten kinetics (mean K(m) = 33 microM), with evidence of substrate inhibition at high substrate concentrations in a number of cases. The reaction was minimally inhibited by coincubation with chemical probes inhibitory for P450-2D6 (quinidine), -1A2 (furafylline, alpha-naphthoflavone), and -2E1 (diethyldithiocarbamate). Substantial inhibition was produced by coincubation with sulfaphenazole (Ki = 2.8 microM), an inhibitory probe for P450-2C9, and by ketoconazole (Ki = 2.5 microM) and fluvoxamine (Ki = 5.2 microM). However, ketoconazole, relatively specific for P450-3A isoforms only at low concentrations, reduced norfluoxetine formation by only 20% at 1 microM, and triacetyloleandomycin (> or = 5 microM) reduced the velocity by only 20-25%. Microsomes from cDNA-transfected human lymphoblastoid cells containing human P450-2C9 produced substantial quantities of norfluoxetine when incubated with 100 microM fluoxetine. Smaller amounts of product were produced by P450-2C19 and -2D6, but no product was produced by P450-1A2, -2E1, or 3A4. Cytochrome P450-2C9 appears to be the principal human cytochrome mediating fluoxetine N-demethylation. P450-2C19 and -3A may make a further small contribution, but P450-2D6 is unlikely to make an important contribution.
RP 73401 is a potent inhibitor of cyclic nucleotide phosphodiesterase type IV. RP 73401 is metabolized by human liver microsomes almost exclusively by transhydroxylation of the cyclopentyl group to RPR 113406. Liquid chromatography/mass spectrometry/mass spectrometry analysis of plasma from patients given RP 73401 also revealed a molecular ion and fragmentation consistent with RPR 113406. Thus, the objective was to determine the oxidative enzyme(s) responsible for RP 73401 hydroxylation. Kinetic constants of RP 113406 formation ranged from 8 to 26 MM and 0.83 to 5.99 nmol/min/mg protein for K(m) and V(max), respectively (n = 3). Enzyme activity varied 23-fold among 15 human liver microsome samples and correlated with CYP2A6-catalyzed coumarin hydroxylase (r2 = 0.85, P < .01) and CYP2B6-catalyzed 7-ethoxytrifluoromethylcoumarin O-deethylase (r2 = 0.82, P < .01) activities. Chemical inhibition studies showed a 63% decrease in RP 73401 hydroxylation by 500 microM orphenadrine. Coumarin (10 microM), however, did not inhibit RP 73401 hydroxylation. Also, anti-CYP2B1 IgG produced 85% inhibition of RP 73401 hydroxylation, but only a negligible decline in coumarin hydroxylase activity. Of the 10 expressed P450 forms studied, only CYP2B6 catalyzed RP 73401 hydroxylation. Finally, expressed CYP2B6 showed a high affinity (K(m) = 22.5 microM) for RP 73401 hydroxylation, similar to the human liver microsome studies.
We tested the ability of human liver microsomes (HLMs) and recombinant human cytochrome P450 (CYP or P450) isoforms to catalyze the N-demethylation of nirvanol-free (S)-mephenytoin [(S)-MP] in vitro. In mixed HLMs, the kinetics of (S)-MP N-demethylation suggested two contributing activities. A high-affinity/low-capacity component exhibited a KM of 174.1 microM and a Vmax of 170.5 pmol/mg protein/min, whereas a low-affinity/high-capacity component exhibited a KM of 1911 microM and a Vmax of 3984 pmol/mg protein/min. The activity of the high-affinity component was completely abolished by sulfaphenazole, with little effect on the low-affinity component. Of the recombinant P450 isoforms tested, only CYP2B6 and CYP2C9 formed nirvanol from (S)-MP. The KM value (150 +/- 42 microM) derived for recombinant CYP2C9 was close to that obtained for the high-affinity/low-capacity component in mixed HLMs (KM = 174.1 microM). The predicted contribution of this activity at concentrations (1-25 microM) achieved after a single 100-mg dose of racemic MP is approximately 30% of the rate of nirvanol formation. At concentrations of >1000 microM, we estimate that >90% of the rate can be explained by the low-affinity activity (CYP2B6). Therefore, the N-demethylation of (S)-MP to nirvanol may be a useful means of probing the activity of CYP2B6 in vitro when concentrations of >1000 microM are used, but it is unlikely to be a suitable phenotyping tool for this isoform in vivo, where concentrations of >1000 microM are rarely encountered.
Previous studies in this laboratory have determined the lack of specificity of several antibody and substrate probes of CYP2B6. The goals of the current study were to examine the expression of CYP2B6 in a bank of human liver microsome (HLM) samples using a new specific monoclonal antibody (MAb 49-10-20) and to further characterize the substrate specificity of CYP2B6. A 100-fold variability in expression of immunodetectable CYP2B6 was demonstrated in a bank of 19 HLM samples (0.7 pmol/mg protein to 71. 1 pmol/mg protein) using MAb 49-10-20. CYP2B6 levels were found to significantly (P < .0001) correlate with S-mephenytoin N-demethylation to nirvanol (r2 = 0.89), 7-hydroxy-4-trifluoromethylcoumarin formation (r2 = 0.81) and several markers of CYP3A levels and activity. The relationships between nirvanol formation and CYP3A levels or activity were found to depend on two HLM samples. Km (apparent) values were generated for benzyloxyresorufin O-deethylation (1.3 microM), benzphetamine N-demethylation (93.4 microM), 3-cyano 7-ethoxycoumarin O-deethylation (71.3 microM), midazolam 1'-hydroxylation (46.1 microM) and 4-chloromethyl-7-ethoxycoumarin O-deethylation (33.7 microM) using expressed CYP2B6. Testosterone 16beta-hydroxylation by expressed CYP2B6 resulted in atypical kinetics characteristic of substrate activation. The data best fit the Hill equation with a Km (apparent) of 50.5 microM and an n of 1.3 (n = number of sites bound by activator). In conclusion, the highly specific MAb 49-10-20 was used to provide further confirmation that S-mephenytoin N-demethylation to nirvanol is a CYP2B6 selective probe. Finally, some, but not all substrates of CYP2B6 demonstrate autoactivation.
To determine the possible impact of CYP2D6 polymorphism on the pharmacokinetics and pharmacodynamics of selegiline.
Five poor metabolizers and 8 extensive metabolizers of debrisoquin (INN, debrisoquine) were given 10 mg selegiline hydrochloride. The concentrations of selegiline and its main metabolites in serum were determined for 4 days. The pharmacodynamics were quantitated by measuring platelet monoamine oxidase type B activity for 3 weeks. In addition, the effect of selegiline and its main metabolites on the CYP2D6-catalyzed dextromethorphan O-demethylase activity and the effect of quinidine on the metabolism of selegiline were studied in human liver microsomes.
Peak serum concentrations of selegiline were reached rapidly and ranged from 1 to 32 nmol/L. The metabolite concentrations were considerably higher and remained so for a longer period. There were no significant differences in the pharmacokinetic parameters of selegiline, desmethylselegiline, and l-amphetamine between poor metabolizers and extensive metabolizers. However, the area under the serum concentration-time curve (AUC) values of l-methamphetamine were, on average, 46% higher (P = .01) in poor metabolizers than in extensive metabolizers. No significant correlations were found between debrisoquin metabolic ratio and AUC values of selegiline or its metabolites, except for l-methamphetamine (rs = 0.90; P < .001). The maximum monoamine oxidase type B inhibition was 97% in both groups. The inhibitory potency of selegiline, desmethylselegiline, and l-methamphetamine toward dextromethorphan O-demethylase was very low (50% inhibitory concentration values from 160 to 580 mumol/L). Quinidine (< or = 100 mumol/L) did not inhibit the formation of desmethylselegiline or l-methamphetamine from selegiline.
CYP2D6 is not important in the primary elimination of selegiline, and the biological effect of selegiline seems to be similar in poor metabolizers and extensive metabolizers of debrisoquin. The inhibitory effect of selegiline and its main metabolites on CYP2D6 activity seems to be negligible.
To begin to build an understanding of the interactions of CYP2B6 with substrates, two different 3-dimensional quantitative structure activity relationship (3D-QSAR) models were constructed using 16 substrates of B-lymphoblastoid expressed CYP2B6. A pharmacophore model was built using the program Catalyst, which was compared with a partial least-squares (PLS) model using molecular surface-weighted holistic invariant molecular (MS-WHIM) descriptors. The Catalyst model yielded a 3-dimensional model of the common structural features of CYP2B6 substrates, whereas PLS MS-WHIM generated a model based on statistical analyses of molecular descriptors for size and shape of the substrate. The pharmacophore model obtained with Catalyst consisted of three hydrophobes and one hydrogen bond acceptor region. The cross-validated PLS MS-WHIM model gave a good q2 value of 0.607. Size, positive electrostatic potential, hydrogen bonding acceptor capacity, and hydrophobicity were found to be the most relevant descriptors for the model. These models were then used to predict the Km (apparent) values of a test set of structurally diverse substrates for CYP2B6 not included in the model building, specifically lidocaine, amitriptyline, bupropion, arteether, and verapamil. Overall, both 3D-QSAR methods yielded satisfactory Km (apparent) value predictions for the majority of the molecules in the test set. However, PLS MS-WHIM was unable to reliably predict the Km (apparent) value for verapamil, whereas Catalyst did not predict the Km (apparent) value for lidocaine. In both of these cases the residual of the Km (apparent) value was greater than one log unit. The strengths and limitations of both of these 3D-QSAR approaches are discussed.
The central nervous system toxicity of ifosfamide (IFF), a chiral antineoplastic agent, is thought to be dependent on its N-dechloroethylation by hepatic cytochrome P-450 (CYP) enzymes. The purpose of this study was to identify the human CYPs responsible for IFF-N-dechloroethylation and their corresponding regio- and enantioselectivities. IFF exists in two enantiomeric forms, (R) - and (S)-IFF, which can be dechloroethylated at either the N2 or N3 positions, producing the corresponding (R,S)-2-dechloroethyl-IFF [(R, S)-2-DCE-IFF] and (R,S)-3-dechloroethyl-IFF [(R,S)-3-DCE-IFF]. The results of the present study suggest that the production of (R)-2-DCE-IFF and (S)-3-DCE-IFF from (R)-IFF is catalyzed by different CYPs as is the production of (S)-2-DCE-IFF and (R)-3-DCE-IFF from (S)-IFF. In vitro studies with a bank of human liver microsomes revealed that the sample-to-sample variation in the production of (S)-3-DCE-IFF from (R)-IFF and (S)-2-DCE-IFF from (S)-IFF was highly correlated with the levels of (S)-mephenytoin N-demethylation (CYP2B6), whereas (R)-2-DCE-IFF production from (R)-IFF and (R)-3-DCE-IFF production from (S)-IFF were both correlated with the activity of testosterone 6beta-hydroxylation (CYP3A4/5). Experiments with cDNA-expressed P-450 and antibody and chemical inhibition studies supported the conclusion that the formation of (S)-3-DCE-IFF and (S)-2-DCE-IFF is catalyzed primarily by CYP2B6, whereas (R)-2-DCE-IFF and (R)-3-DCE-IFF are primarily the result of CYP3A4/5 activity.
1. Aminopyrine N-demethylase activity was determined for 11 forms of human hepatic cytochrome P450s (P450s) expressed in yeast Saccharomyces cerevisiae and for human steroidogenic CYP17 expressed in Escherichia coli. 2. Among the hepatic P450s, the N-demethylation of aminopyrine was catalysed most efficiently by CYP2C19, followed by CYP2C8, 2D6, 2C18 and 1A2, whereas the activity with CYP2E1 was negligible. The kinetics of the N-demethylation process by CYP1A2, 2C8, 2C19 and 2D6 were studied by fitting to Michaelis-Menten kinetics by Lineweaver-Burk plots. CYP2C19 exhibited the highest affinity and a high capacity for the aminopyrine N-demethylation. CYP2C8 showed the highest Vmax, followed by CYP2C19, 2D6 and 1A2, whereas the Km for CYP2C8, 2D6 and 1A2 were 10-17 times higher than that for CYP2C19. Accordingly, the Vmax/Km for CYP2C19 was more than nine times higher than that of other P450s. 3. Human steroidogenic CYP17 also catalysed aminopyrine N-demethylation and the activity was comparable with that for CYP3A4 which is a dominant P450 in human liver. The activity was increased 1.5-fold by the addition of cytochrome b5, whereas the activity was not affected by the addition of Mg2+. 4. These results suggest that several human hepatic P450s, especially CYP2C19, and steroidogenic CYP17 exhibit aminopyrine N-demethylase activity.
The contributions of specific human liver cytochrome P-450 (CYP) enzymes to the activation, via 4-hydroxylation, of the oxazaphosphorine anticancer prodrugs cyclophosphamide (CPA) and ifosfamide (IFA) were investigated. Analysis of a panel of 15 human P-450 cDNAs expressed in human lymphoblasts and/or baculovirus-infected insect cells (Supersomes) demonstrated that CYPs 2A6, 2B6, 3A4, 3A5, and three CYP2C enzymes (2C9, 2C18, 2C19) exhibited significant oxazaphosphorine 4-hydroxylase activity, with 2B6 and 3A4 displaying the highest activity toward CPA and IFA, respectively. CYP2B6 metabolized CPA at a approximately 16-fold higher in vitro intrinsic clearance (apparent Vmax/Km) than IFA, whereas 3A4 demonstrated approximately 2-fold higher Vmax/Km toward IFA. A relative substrate-activity factor (RSF)-based method was developed to calculate the contributions of individual P-450s to total human liver microsomal metabolism based on cDNA-expressed P-450 activity data and measurements of the liver microsomal activity of each P-450 form. Using this method, excellent correlations were obtained when comparing measured versus predicted (calculated) microsomal 4-hydroxylase activities for both CPA (r = 0. 96, p <.001) and IFA (r = 0.90, p <.001) in a panel of 17 livers. The RSF method identified CYP2B6 as a major CPA 4-hydroxylase and CYP3A4 as the dominant IFA 4-hydroxylase in the majority of livers, with CYPs 2C9 and 2A6 making more minor contributions. These predicted P-450 enzyme contributions were verified using an inhibitory monoclonal antibody for 2B6 and the P-450 form-specific chemical inhibitors troleandomycin for 3A4 and sulfaphenazole for 2C9, thus validating the RSF approach. Finally, Western blot analysis using anti-2B6 monoclonal antibody demonstrated the presence of 2B6 protein at a readily detectable level in all but one of 17 livers. These data further establish the significance of human liver CYP2B6 for the activation of the clinically important cancer chemotherapeutic prodrug CPA.
1. Molecular modelling studies of CYP2B isoforms from rat (CYP2B1), rabbit (CYP2B4) and man (CYP2B6) are reported, with particular emphasis on substrate interactions with the human CYP2B isoform, CYP2B6. 2. The findings represent an advance on our previous study that focused primarily on the rat CYP2B isoform, CYP2B1, and involved homology modelling with substrate-free CYP102. 3. The current work utilizes the recently published substrate-bound CYP102 crystal structure as a template for construction of the CYP2B subfamily isoforms and shows, in particular, that known CYP2B6 substrate specificity and regioselectivity can be rationalized by putative active site interactions.
Sertraline, a new antidepressant of the selective serotonin reuptake inhibitor class, is extensively metabolized to desmethylsertraline in humans. We identified the cytochrome P-450 (CYP) isoforms involved in sertraline N-demethylation using pooled human liver microsomes and cDNA-expressed CYP isoforms. Eadie-Hofstee plots for the sertraline N-demethylation in human liver microsomes were monophasic. The estimated Michaelis-Menten kinetic parameters were: KM = 18.1 +/- 2.0 microM, Vmax = 0.45 +/- 0.03 nmol/min/mg of protein, and Vmax/KM = 25.2 +/- 4.3 microl/min/mg of protein. At the substrate concentration of 20 microM, which approximated the apparent KM value, sulfaphenazole (CYP2C9 inhibitor) and triazolam (CYP3A substrate) reduced the N-demethylation activities by 20 to 35% in human liver microsomes, whereas the inhibition induced by mephenytoin (CYP2C19 substrate) or quinidine (CYP2D6 inhibitor) was marginal. The anti-CYP2B6 antibody inhibited the sertraline N-demethylation activities by 35%. Sertraline N-demethylation activities were detected in all cDNA-expressed CYP isoforms studied. In particular, CYP2C19, CYP2B6, CYP2C9-Arg, CYP2D6-Val, and CYP3A4 all showed relatively high activity. When the contributions of CYP2D6, CYP2C9, CYP2B6, CYP2C19, and CYP3A4 were estimated from the Vmax/KM of cDNA-expressed CYP isoforms and from their contents in pooled human liver microsomes, the values were found to be 35, 29, 14, 13, and 9%, respectively. The results suggest that at least five isoforms of CYP (CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4) are involved in the sertraline N-demethylation in human liver microsomes and that the contribution of any individual isoform does not exceed 40% of overall metabolism. Therefore, concurrent administration of a drug that inhibits a specific CYP isoform is unlikely to cause a marked increase in the plasma concentration of sertraline.
The pharmacokinetics and biotransformation of the antiretroviral agent nevirapine (NVP) after autoinduction were characterized in eight healthy male volunteers. Subjects received 200-mg NVP tablets once daily for 2 weeks, followed by 200 mg twice daily for 2 weeks. Then they received a single oral dose (solution) of 50 mg containing 100 microCi of [(14)C]NVP. Biological fluids were analyzed for total radioactivity, parent compound (HPLC/UV), and metabolites (electrospray liquid chromatography/mass spectroscopy and liquid chromatography/tandem mass spectroscopy). Mean recovery of radioactivity was 91.4%, with 81.3% excreted in urine and 10.1% recovered in the feces over a period of 10 days. Circulating radioactivity was evenly distributed between whole blood and plasma. At maximum plasma concentration, parent compound accounted for approximately 75% of the circulating radioactivity. Mean plasma elimination half-lives for total radioactivity and NVP were 21.3 and 20.0 h, respectively. Several metabolites were identified in urine including 2-hydroxynevirapine glucuronide (18.6%), 3-hydroxynevirapine glucuronide (25.7%), 12-hydroxynevirapine glucuronide (23.7%), 8-hydroxynevirapine glucuronide (1.3%), 3-hydroxynevirapine (1.2%), 12-hydroxynevirapine (0.6%), and 4-carboxynevirapine (2.4%). Greater than 80% of the radioactivity in urine was made up of glucuronidated conjugates of hydroxylated metabolites of NVP. Thus, cytochrome P-450 metabolism, glucuronide conjugation, and urinary excretion of glucuronidated metabolites represent the primary route of NVP biotransformation and elimination in humans. Only a small fraction of the dose (2.7%) was excreted in urine as parent compound.
The aim of this study was to re-examine the human hepatic metabolism of diclofenac, with special focus on the generation of minor hydroxylated metabolites implicated in the idiosyncratic hepatotoxicity of the drug. Different experimental approaches were used: human hepatocytes, human microsomes, and engineered cells expressing single human CYP (cytochromes P450). Human hepatocytes formed 3'-hydroxy-, 4'-hydroxy-, 5-hydroxy- 4',5-dihydroxy-, and N,5-dihydroxydiclofenac, as well as several lactams. Formation of 4'- and 5-hydroxydiclofenac by human liver microsomes followed a Michaelis-Menten kinetics (Km 9 +/- 1 microM; Vmax 432 +/- 15 pmol/min/mg and Km 43 +/- 5 microM; and Vmax 15.4 +/- 0.6 pmol/min/mg, respectively). Secondary metabolites were detected after incubation of 5-hydroxydiclofenac with human liver microsomes, yielding 4',5-dihydroxydiclofenac (Km 15 +/- 1 microM; Vmax 96 +/- 3 pmol/min/mg) and small amounts of N,5-dihydroxydiclofenac (non-Michaelis-Menten kinetics). Based on microsome studies and the incubations with human hepatocytes and engineered cells, we estimated that in vivo CYP2C9 would be exclusively responsible for the 4' hydroxylation of diclofenac (>99.5%) as well as 5-hydroxydiclofenac (>97%). CYP2C9 was exclusively responsible for the formation of 3'-hydroxydiclofenac. Multiple regression analysis evidenced that the rate of production of 5-hydroxydiclofenac in human microsomes followed the algorithm: 0.040 x S-mephenytoin 4'-hydroxylation + 0.083 x tolbutamide methylhydroxylation, (multiple correlation coefficient = 0.969). However, the incubation of diclofenac with cell lines expressing different human CYP suggested that 7 isoforms could be involved. Comparison of data obtained with CYP-expressing cells and human hepatocytes suggests that CYP2C8 > CYP2C19 approximately CYP2C18 > CYP2B6 are the isoforms implicated in the 5-hydroxylation of diclofenac in vivo.
The structural basis for functional differences between human cytochrome P-450 2B6 and rat 2B1 was investigated. An amino acid sequence alignment predicted the location of 2B6 substrate recognition site (SRS) residues. Ten residues within these SRSs unique to 2B6 compared with 2B1, 2B4, and 2B11 were chosen for mutagenesis. Two additional sites that differ between 2B6 and 2B1 and are known to have a role in 2B1 substrate specificity were also mutated. The 2B6 mutants were expressed in Spodoptera frugiperda cells and characterized using the 2B6-specific substrate RP 73401 [3-cyclopentyloxy-N-(3,5-dichloro-4-pyridyl)-4-methoxybenzamide], the 2B1-selective substrate androstenedione, and the common substrate 7-ethoxy-4-trifluoromethylcoumarin. Mutants F107I and L363V exhibited decreased RP 73401 hydroxylation but retained most of the wild-type level of 2B6 7-ethoxy-4-trifluoromethylcoumarin O-deethylase activity. In addition, SRS exchanges were studied in which the amino acid sequence of 2B6 SRSs was converted to the sequence of 2B1. Each of these constructs, having two to seven substitutions, expressed at levels similar to 2B6 but did not acquire significant androstenedione hydroxylase activity. Docking of RP 73401 into the active site of a 2B6 homology model suggested a direct interaction with residue L363 but not with F107. Findings from this study suggest that 1) residues F107 and L363 are necessary for 2B6 RP 73401 hydroxylase activity, 2) 2B6 is able to tolerate multiple SRS substitutions without compromising protein expression levels or protein stability, and 3) conferring androstenedione hydroxylase function to cytochrome P-450 2B6 is more complex than altering a single SRS.
Human cytochrome (CYP)2B6 cDNA was cloned and expressed in bacteria and in yeast. Its expression in Saccharomyces cerevisiae enabled us to obtain, at a high level, an active yeast-expressed CYP2B6 protein, so as to assess its role in the metabolism of ethoxyresorufin, pentoxyresorufin, benzyloxyresorufin, ethoxycoumarin, testosterone and cyclophosphamide. Kinetic analysis showed that human CYP2B6 preferentially metabolized benzyloxyresorufin and pentoxyresorufin, although other CYPs also metabolized these substrates in human liver microsomes. CYP2B6 also manifested a strong 4-hydroxycyclophosphamide activity. Its expression in Escherichia coli enabled us to produce a very specific anti-human CYP2B6 antibody. No cross reactivity of this antibody was observed with CYPs1A1, 1A2, 3A4, 3A5, 2C8, 2C9, 2C18, 2C19, 2D6 or 2E1. This antibody enabled us to study the hepatic and extrahepatic expression of CYP2B6 in man, as well as its expression and inducibility in primary cultured human hepatocytes and in different human cell lines. Immunoblot analysis revealed that the CYP2B6 protein was expressed in 43 of the 48 human liver samples tested, with levels ranging from 0.4 to 8 pmol/mg of microsomal protein with a mean of 1.7 pmol/mg protein. CYP2B was also expressed in human brain, intestine and kidney, and at a lower level in the lung. CYP2B mRNA was detected in human liver, kidney, lung, trachea and intestine. We also found that CYP2B6 is induced at protein and mRNA levels by phenobarbital (2 mM) and cyclophosphamide (1 mM), an anticancer drug known to be metabolized by CYP2B6. No expression or inducibility of CYP2B6 was observed in any of the human cell lines tested.
The anticancer prodrug ifosfamide (IFA) contains a chiral phosphorous atom and is administered clinically as a racemic mixture of R and S enantiomers. Animal model studies and clinical data indicate enantioselective differences in cytochrome P-450 (CYP) metabolism, pharmacokinetics, and therapeutic efficacy between the two enantiomers; however, the metabolism of individual IFA enantiomers has not been fully characterized. The role of CYP enzymes in the stereoselective metabolism of R-IFA and S-IFA was investigated by monitoring the formation of both 4-hydroxy (activated) and N-dechloroethyl (DCl) (inactive, neurotoxic) metabolites. In the 4-hydroxylation reaction, cDNA-expressed CYPs 3A4 and 3A5 preferentially metabolized R-IFA, whereas CYP2B6 was more active toward S-IFA. Enantioselective IFA 4-hydroxylation (R > S) was observed with six of eight human liver samples. In the N-dechloroethylation reaction, CYPs 3A4 and 2B6 both catalyzed a significantly higher intrinsic metabolic clearance (V(max)/K(m)) of S-IFA compared with R-IFA. Striking P-450 form specificity in the formation of individual DCl metabolites was evident. CYPs 3A4 and 3A5 preferentially produced (R)N2-DCl-IFA and (R)N3-DCl-IFA (derived from R-IFA and S-IFA, respectively), whereas CYP2B6 correspondingly formed (S)N3-DCl-IFA and (S)N2-DCl-IFA. In human liver microsomes, the CYP3A-specific inhibitor troleandomycin suppressed (R)N2- and (R)N3-DCl-IFA formation by >/=80%, whereas (S)N2- and (S)N3-DCl-IFA formation were selectively inhibited (>/=85%) by a CYP2B6-specific monoclonal antibody. The overall extent of IFA N-dechloroethylation varied with the CYP3A4 and CYP2B6 content of each liver, but was significantly lower for R-IFA (32 +/- 13%) than for S-IFA (62 +/- 17%, n = 8; p <.001) in all livers examined. R-IFA thus has more favorable liver metabolic properties than S-IFA with respect to less extensive N-dechloroethylation and more rapid 4-hydroxylation, indicating that R-IFA may have a distinct clinical advantage over racemic IFA.
The role of cytochrome P-450s (CYPs) in S-mephobarbital N-demethylation was investigated by using human liver microsomes and cDNA-expressed CYPs. Among the 10 cDNA-expressed CYPs studied (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4), only CYP2B6 could catalyze S-mephobarbital N-demethylation. The apparent K(m) values of human liver microsomes for S-mephobarbital N-demethylation were close to that of cDNA-expressed CYP2B6 (about 250 microM). The N-demethylase activity of S-mephobarbital in 10 human liver microsomes was strongly correlated with immunodetectable CYP2B6 levels (r = 0.920, p<.001). Orphenadrine (300 microM), a CYP2B6 inhibitor, inhibited the N-demethylase activity of S-mephobarbital in human liver microsomes to 29% of control activity. Therefore, it appears that CYP2B6 mainly catalyzes S-mephobarbital N-demethylation in human liver microsomes.
The study aimed to identify the specific human cytochrome P450 (CYP450) enzymes involved in the metabolism of artemisinin.
Microsomes from human B-lymphoblastoid cell lines transformed with individual CYP450 cDNAs were investigated for their capacity to metabolize artemisinin. The effect on artemisinin metabolism in human liver microsomes by chemical inhibitors selective for individual forms of CYP450 was investigated. The relative contribution of individual CYP450 isoenzymes to artemisinin metabolism in human liver microsomes was evaluated with a tree-based regression model of artemisinin disappearance rate and specific CYP450 activities.
The involvement of CYP2B6 in artemisinin metabolism was demonstrated by metabolism of artemisinin by recombinant CYP2B6, inhibition of artemisinin disappearance in human liver microsomes by orphenadrine (76%) and primary inclusion of CYP2B6 in the tree-based regression model. Recombinant CYP3A4 was catalytically competent in metabolizing artemisinin, although the rate was 10% of that for recombinant CYP2B6. The tree-based regression model suggested CYP3A4 to be of importance in individuals with low CYP2B6 expression. Even though ketoconazole inhibited artemisinin metabolism in human liver microsomes by 46%, incubation with ketoconazole together with orphenadrine did not increase the inhibition of artemisinin metabolism compared to orphenadrine alone. Troleandomycin failed to inhibit artemisinin metabolism. The rate of artemisinin metabolism in recombinant CYP2A6 was 15% of that for recombinant CYP2B6. The inhibition of artemisinin metabolism in human liver microsomes by 8-methoxypsoralen (a CYP2A6 inhibitor) was 82% but CYP2A6 activity was not included in the regression tree.
Artemisinin metabolism in human liver microsomes is mediated primarily by CYP2B6 with probable secondary contribution of CYP3A4 in individuals with low CYP2B6 expression. The contribution of CYP2A6 to artemisinin metabolism is likely of minor importance.
The anticancer alkylating agents cyclophosphamide (CPA) and ifosfamide (IFA) are prodrugs that undergo extensive P450-catalyzed metabolism to yield both active (4-hydroxylated) and therapeutically inactive but neurotoxic (N-dechloroethylated) metabolites. Whereas the human liver microsomal P450 catalysts of CPA and IFA 4-hydroxylation are well characterized, the P450 enzyme catalysts of the alternative N-dechloroethylation pathway are poorly defined. Analysis of a panel of fifteen human P450 cDNAs in the baculovirus expression system ('Supersomes') demonstrated that CYP3A4 exhibited the highest N-dechloroethylation activity toward both CPA and IFA, whereas CYP2B6 displayed high N-dechloroethylation activity toward IFA, but not CPA. The contributions of each human P450 to overall liver microsomal N-dechloroethylation were calculated using a recently described relative substrate-activity factor method, and were found to be in excellent agreement with the results of inhibition studies using the CYP3A inhibitor troleandomycin and an inhibitory monoclonal antibody to CYP2B6. With CPA as substrate, CYP3A4 was shown to catalyze >/=95% of liver microsomal N-dechloroethylation, whereas with IFA as substrate, CYP3A4 catalyzed an average of approximately 70% of liver microsomal N-dechloroethylation (range = 40-90%), with the balance of this activity catalyzed by CYP2B6 (range = 10-70%, dependent on the CYP2B6 content of the liver). Because CYP2B6 can make a significant contribution to human liver microsomal IFA N-dechloroethylation, but only a minor contribution to IFA 4-hydroxylation, the selective inhibition of hepatic CYP2B6 activity in individuals with a high hepatic CYP2B6 content may provide a useful approach to minimize the formation of therapeutically inactive but toxic N-dechloroethylated IFA metabolites.
Effect of bisphenol A on drug-metabolizing enzyme activities by human hepatic cytochrome P450s (CYP) was investigated. We measured aminopyrine N-demethylation by eleven kinds of cDNA-expressed CYPs. CYP2C19 and CYP2B6 catalyzed most efficiently the aminopyrine N-demethylation, followed by CYP2C8 and CYP2D6. Bisphenol A (1 mM) most efficiently inhibited aminopyrine N-demethylation by CYP2C8 and CYP2C19 by 82% and 85%, respectively, whereas inhibition of the activities by CYP 2B6 and 2D6 was less than 40%. Bisphenol A exhibited a noncompetitive-type inhibition of aminopyrine N-demethylase activity by CYP2C8 with Ki value of 97 microM. Additionally, we investigated the inhibitory effect of bisphenol A on CYP2C19-mediated S-mephenytoin 4-hydroxylation. Bisphenol A exhibited a mixed-type inhibition with Ki value of 113 microM. These results suggest that bisphenol A inhibits human hepatic CYP activities, especially CYP2C8 and CYP2C19.
The characteristics of mammalian microsomal P450 xenobiotic substrates are described, particularly with reference to the major P450 isoforms associated with drug metabolism in humans. It is further reported that a relatively small number of molecular, electronic, and physico-chemical properties are required to discriminate between chemicals that exhibit specificity for human P450 isoforms: CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. Molecular templates of superimposed substrates are shown to be complementary with the putative active sites of the relevant enzymes, thus enabling a possible prediction of P450 specificity from structure. Factors contributing to metabolic clearance and binding affinity are also discussed, and methods for their calculation are described.
2-Phenyl-2-(1-piperidinyl)propane (PPP), an analog of phencyclidine, was tested for its ability to inactivate cytochrome P450s (P450s) 2B1 and 2B6. PPP inactivated the 7-(benzyloxy)resorufin O-dealkylation activity of liver microsomes obtained from phenobarbital-induced rats with a K(I) of 11 microM. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of purified rat liver P450 2B1 and expressed human P450 2B6 was inactivated by PPP in a reconstituted system containing NADPH-cytochrome P450 reductase and lipid. In the presence of NADPH, the loss of activity was time- and concentration-dependent, and followed pseudo first order kinetics. The rate of inactivation for P450 2B1 was 0.3 min(-1), and the concentration of PPP required to achieve half-maximal inactivation was 12 microM. The time for 50% of the P450 2B1 to become inactivated at saturating concentrations of PPP was 2.5 min. P450 2B6 was inactivated with a k(inact) of 0.07 min(-1), a K(I) of 1.2 microM, and a t(1/2) of 9.5 min. The inactivated P450s 2B1 and 2B6 lost about 25 and 15%, respectively, of their ability to form a CO-reduced complex, suggesting that the loss of activity was caused by a PPP modification of the apoprotein rather than the heme. The estimated partition ratio for P450s 2B1 and 2B6 with PPP was 31 and 15, respectively. The inactivation was not reversible and reductase activity was not affected. Coincubation of P450 2B1 and 2B6 with PPP and NADPH in the presence of an alternate substrate protected both enzymes from inactivation. The exogenous nucleophile GSH did not affect the rate of inactivation. PPP-inactivated P450s 2B1 and 2B6 were recognized on Western blots by an antibody generated to phencyclidine that had been conjugated to BSA. Stoichiometries of 1.4:1 and 0.7:1 were determined for the binding of a [3H]PPP metabolite to P450 2B1 and 2B6, respectively.
The purpose of this study was to establish bupropion (BUP) hydroxylation as a selective in vitro marker of cytochrome P450 (CYP) 2B6 catalytic activity. Among a panel of 16 human liver microsomes (HLMs), BUP hydroxylase activity varied 80-fold when assayed at 500 microM substrate and significantly correlated with CYP2B6 blotting density (r(2) = 0.99) and S-mephenytoin N-demethylase activity (r(2) = 0.98). Kinetic analysis of BUP hydroxylation was performed in a subset of seven HLMs representative of the 80-fold range in activity. Sigmoidal kinetics suggestive of allosteric activation was observed in five HLMs exhibiting low or high activity; the mean apparent K(m) for BUP hydroxylation in these HLMs (130 microM) was similar to the K(m) for cDNA-expressed CYP2B6 (156 microM). Nonsaturable, biphasic kinetics was observed in two HLMs exhibiting low activity. Among a panel of cDNA-expressed P450 isoforms, CYP2B6 and CYP2E1 demonstrated the highest rates of BUP hydroxylation at 12 mM BUP (7.0 and 2.4 pmol/min/pmol of P450, respectively). The relative contributions of CYP2B6 and CYP2E1 to BUP hydroxylation were estimated by using immunoinhibitory monoclonal antibodies (MAB) to these enzymes. MAB-2B6 produced 88% maximum inhibition of BUP hydroxylation when assayed at 12 mM BUP in a high activity HLM, whereas MAB-2E1 produced 81% maximum inhibition in a low activity HLM. However, negligible inhibition by MAB-2E1 was observed when low and high activity HLMs were assayed at 500 microM BUP. These results demonstrate selectivity of BUP hydroxylation for CYP2B6 at 500 microM BUP, thereby validating its use as a diagnostic in vitro marker of CYP2B6 catalytic activity.
Fluoxetine is one of the most widely prescribed selective serotonin reuptake inhibitors (SSRIs) that is marketed worldwide. However, details of its human hepatic metabolism have been speculative and incomplete, possibly due to the sensitivity of analytical techniques and selectivity of specific in vitro probes and reagents used. Studies with (R)-, (S)-, and racemic fluoxetine were undertaken to determine the stereospecific nature of its metabolism and estimate intrinsic clearance contributions of each CYP for fluoxetine N-demethylation. Measurable fluoxetine N-demethylase activity was catalyzed by CYP1A2, -2B6, -2C9, -2C19, -2D6, -3A4, and -3A5. All enzymes catalyzed this reaction for both enantiomers and the racemate, and intrinsic clearance values were similar for the enantiomers for all CYP enzymes except CYP2C9, which demonstrated stereoselectivity for R- over the S-enantiomer. Scaling the intrinsic clearance values for the individual CYP enzymes to estimate contributions of each in human liver microsomes suggested that CYP2D6, CYP2C9, and CYP3A4 contribute the greatest amount of fluoxetine N-demethylation in human liver microsomes. These data were corroborated with the examination of the effects of CYP-specific inhibitors quinidine (CYP2D6), sulfaphenazole (CYP2C9), and ketoconazole (CYP3A4) on fluoxetine N-demethylation in pooled human liver microsomes. Together, these findings suggest a significant role for the polymorphically expressed CYP2D6 in fluoxetine clearance and are consistent with reports on the clinical pharmacokinetics of fluoxetine.
The in vitro biotransformation of bupropion to hydroxybupropion was studied in human liver microsomes and microsomes containing heterologously expressed human cytochromes P450 (CYP). The mean (+/-S.E.) K(m) in four human liver microsomes was 89 (+/-14) microM. In microsomes containing cDNA-expressed CYPs, hydroxybupropion formation was mediated only by CYP2B6 at 50 microM bupropion (K(m) 85 microM). A CYP2B6 inhibitory antibody produced more than 95% inhibition of bupropion hydroxylation in four human livers. Bupropion hydroxylation activity at 250 microM was highly correlated with S-mephenytoin N-demethylation activity (yielding nirvanol), another CYP2B6-mediated reaction, in a panel of 32 human livers (r = 0.94). The CYP2B6 content of 12 human livers highly correlated with bupropion hydroxylation activity (r = 0.96). Thus bupropion hydroxylation is mediated almost exclusively by CYP2B6 and can serve as an index reaction reflecting activity of this isoform. IC(50) values for inhibition of a CYP2D6 index reaction (dextromethorphan O-demethylation) by bupropion and hydroxybupropion were 58 and 74 microM, respectively. This suggests a low inhibitory potency versus CYP2D6, the clinical importance of which is not established. Since bupropion is frequently coadministered with other antidepressants, IC(50) values (microM) for inhibition of bupropion hydroxylation were determined as follows: paroxetine (1.6), fluvoxamine (6.1), sertraline (3.2), desmethylsertraline (19.9), fluoxetine (59.5), norfluoxetine (4.2), and nefazodone (25.4). Bupropion hydroxylation was only weakly inhibited by venlafaxine, O-desmethylvenlafaxine, citalopram, and desmethylcitalopram. The inhibition of bupropion hydroxylation in vitro by a number of newer antidepressants suggests the potential for clinical drug interactions.
Oxidation of propofol to 4-hydroxypropofol represents a significant pathway in the metabolism of this anesthetic agent in humans. The aim of this study was to identify the principal cytochrome P-450 (CYP) isoforms mediating this biotransformation.
Propofol hydroxylation activities and enzyme kinetics were determined using human liver microsomes and cDNA-expressed CYPs. CYP-specific marker activities and CYP2B6 protein content were also quantified in hepatic microsomes for correlational analyses. Finally, inhibitory antibodies were used to ascertain the relative contribution of CYPs to propofol hydroxylation by hepatic microsomes.
Propofol hydroxylation by hepatic microsomes showed more than 19-fold variability and was most closely correlated to CYP2B6 protein content (r = 0.904), and the CYP2B6 marker activities, S-mephenytoin N-demethylation (r = 0.919) and bupropion hydroxylation (r = 0.854). High- and intermediate-activity livers demonstrated high-affinity enzyme kinetics (K(m) < 8 microm), whereas low-activity livers displayed low-affinity kinetics (K(m) > 80 microm). All of the CYPs evaluated were capable of hydroxylating propofol; however, CYP2B6 and CYP2C9 were most active. Kinetic analysis indicated that CYP2B6 is a high-affinity (K(m) = 10 +/- 2 microm; mean +/- SE of the estimate), high-capacity enzyme, whereas CYP2C9 is a low-affinity (K(m) = 41 +/- 8 microm), high-capacity enzyme. Furthermore, immunoinhibition showed a greater contribution of CYP2B6 (56 +/- 22% inhibition; mean +/- SD) compared with CYP2C isoforms (16 +/- 7% inhibition) to hepatic microsomal activity.
Cytochrome P-450 2B6, and to a lesser extent CYP2C9, contribute to the oxidative metabolism of propofol. However, CYP2B6 is the principal determinant of interindividual variability in the hydroxylation of this drug by human liver microsomes.