Substrate specificity of CYP2D6 genetic variants.

Genetic variation in the gene encoding CYP2D6 is used to guide drug prescribing in clinical practice. However, genetic variants in CYP2D6 show substrate-specific effects that are currently not accounted for. With a systematic literature, we retrieved 22 original studies describing in vitro experiments focusing on CYP2D6 alleles (CYP2D6*1, *2, *10 and *17) and substrates. Allele activity (clearance of the allele of interest divided by the clearance of the wildtype) was extracted. The results support the hypothesis of the existence of substrate specificity of the CYP2D6*17-allele (higher debrisoquine clearance), a subtle effect of the CYP2D6*10-allele (lower dextromethorphan clearance) but no substrate-specific effect of the CYP2D6*2-allele. Although our results support substrate specificity, for most substrates data are too sparse and require further studies.

Cytochrome b5 is a major determinant of human cytochrome P450 CYP2D6 and CYP3A4 activity in vivo.

The cytochrome P450-dependent mono-oxygenase system is responsible for the metabolism and disposition of chemopreventive agents, chemical toxins and carcinogens, and >80% of therapeutic drugs. Cytochrome P450 (P450) activity is regulated transcriptionally and by the rate of electron transfer from P450 reductase. In vitro studies have demonstrated that cytochrome b5 (Cyb5) also modulates P450 function. We recently showed that hepatic deletion of Cyb5 in the mouse (HBN) markedly alters in vivo drug pharmacokinetics; a key outstanding question is whether Cyb5 modulates the activity of the major human P450s in drug disposition in vivo. To address this, we crossed mice humanized for CYP2D6 or CYP3A4 with mice carrying a hepatic Cyb5 deletion. In vitro triazolam 4-hydroxylation (probe reaction for CYP3A4) was reduced by >50% in hepatic microsomes from CYP3A4-HBN mice compared with controls. Similar reductions in debrisoquine 4-hydroxylation and metoprolol α-hydroxylation were observed using CYP2D6-HBN microsomes, indicating a significant role for Cyb5 in the activity of both enzymes. This effect was confirmed by the concentration-dependent restoration of CYP3A4-mediated triazolam turnover and CYP2D6-mediated bufuralol and debrisoquine turnover on addition of Escherichia coli membranes containing recombinant Cyb5. In vivo, the peak plasma concentration and area under the concentration time curve from 0 to 8 hours (AUC0-8 h) of triazolam were increased 4- and 5.7-fold, respectively, in CYP3A4-HBN mice. Similarly, the pharmacokinetics of bufuralol and debrisoquine were significantly altered in CYP2D6-HBN mice, the AUC0-8 h being increased ∼1.5-fold and clearance decreased by 40-60%. These data demonstrate that Cyb5 can be a major determinant of CYP3A4 and CYP2D6 activity in vivo, with a potential impact on the metabolism, efficacy, and side effects of numerous therapeutic drugs.

Extrapolating in vitro metabolic interactions to isolated perfused liver: predictions of metabolic interactions between R-bufuralol, bunitrolol, and debrisoquine.

Drug-drug interactions (DDIs) are a great concern to the selection of new drug candidates. While in vitro screening assays for DDI are a routine procedure in preclinical research, their interpretation and relevance for the in vivo situation still represent a major challenge. The objective of the present study was to develop a novel mechanistic modeling approach to quantitatively predict DDI solely based upon in vitro data. The overall strategy consisted of developing a model of the liver with physiological details on three subcompartments: the sinusoidal space, the space of Disse, and the cellular matrix. The substrate and inhibitor concentrations available to the metabolizing enzyme were modeled with respect to time and were used to relate the in vitro inhibition constant (K(i)) to the in vivo situation. The development of the liver model was supported by experimental studies in a stepwise fashion: (i) characterizing the interactions between the three selected drugs (R-bufuralol (BUF), bunitrolol (BUN), and debrisoquine (DBQ)) in microsomal incubations, (ii) modeling DDI based on binary mixtures model for all the possible pairs of interactions (BUF-BUN, BUF-DBQ, BUN-DBQ) describing a mutual competitive inhibition between the compounds, (iii) incorporating in the binary mixtures model the related constants determined in vitro for the inhibition, metabolism, transport, and partition coefficients of each compound, and (iv) validating the overall liver model for the prediction of the perfusate kinetics of each drug determined in isolated perfused rat liver (IPRL) for the single and paired compounds. Results from microsomal coincubations showed that competitive inhibition was the mechanism of interactions between all three compounds, as expected since those compounds are all substrates of rat CYP2D2. For each drug, the K(i) values estimated were similar to their K(m) values for CYP2D2 indicative of a competition for the same substrate-binding site. Comparison of the performance between the novel liver physiologically based pharmacokinetic (PBPK) model and published empirical models in simulating the perfusate concentration-time profile was based on the area under the curve (AUC) and the shape of the curve of the perfusate time course. The present liver PBPK model was able to quantitatively predict the metabolic interactions determined during the perfusions of mixtures of BUF-DBQ and BUN-DBQ. However, a lower degree of accuracy was obtained for the mixtures of BUF-BUN, potentially due to some interindividual variability in the relative proportion of CYP2D1 and CYP2D2 isoenzymes, both involved in BUF metabolism. Overall, in this metabolic interaction prediction exercise, the PBPK model clearly showed to be the best predictor of perfusate kinetics compared to more empirical models. The present study demonstrated the potential of the mechanistic liver model to enable predictions of metabolic DDI under in vivo condition solely from in vitro information.

Relation between CYP2D6 phenotype and genotype and personality in healthy volunteers.

OBJECTIVES: Our group has previously show that interindividual variability in CYP2D6 hydroxylation capacity was related to personality differences in cognitive social anxiety. Thus, we aimed to analyze whether this relationship between personality and CYP2D6 phenotype and genotype was found in a similar population of healthy volunteers from a different latitude and culture by using the same methodology. METHODS: A total of 253 university students and staff from Havana Psychiatric Hospital and Calixto García Medical School in Cuba completed the Karolinska Scales of Personality (KSP), and were evaluated on debrisoquine hydroxylation capacity and CYP2D6 genotypes. KSP scores were compared between four groups, divided according to their CYP2D6 metabolic capacity: one of poor and three of extensive metabolizers. Furthermore, KSP scores were compared between another four different groups divided according to their number of CYP2D6 active genes: zero, one, two, and more than two. RESULTS: In Cubans, the differences in cognitive social anxiety-related personality traits across the four CYP2D6 hydroxylation capacity groups were strikingly similar to those found in Spaniards. These differences also came out to be significant for psychic anxiety (p = 0.02) and socialization (p = 0.02). The same pattern of results obtained for the subscales of psychic anxiety, socialization, psychasthenia and inhibition of aggression with regard to phenotype in both the Cuban and Spanish studies were seen with regard to CYP2D6 genotypes. CONCLUSIONS: Corroborating these results further strengthens evidence of the relationship between CYP2D6 metabolic capacity and personality. In this population of healthy Cuban volunteers, the CYP2D6 hydroxylation capacity was related to the degree of anxiety and socialization. These results support the postulated reduction of serotonin in CYP2D6 poor metabolizers, which may be associated with a cluster of behavioral traits (e.g., anxiety, impulsivity). Thus, research is warranted to determine CYP2D6 functional implications for interindividual differences in vulnerability to neuropsychiatric diseases and drug response.

Chimeric mice with humanized liver.

Recently, chimeric mice with humanized liver were established by transplanting human hepatocytes into an urokinase-type plasminogen activator(+/+)/severe combined immunodeficient transgenic mouse line. The replacement with human hepatocytes is more than 80-90% and is higher than any other chimeric mouse reported previously. In drug development, the liver is one of the most important organs because it is mainly involved in the pharmacokinetics of drugs and is frequently damaged by many drugs due to the accumulation of drugs and/or metabolites. The pharmacokinetics could affect the efficacy and toxicity of a drug, and thus prediction of the human pharmacokinetics is important for developing new drugs without adverse reactions and toxicity. Extrapolation from experimental animals or in vitro studies to the human in vivo pharmacokinetics is still difficult. To date, human hepatocytes and liver microsomes are recognized as better tools and are frequently used to estimate the human pharmacokinetics. We thought that chimeric mice with humanized liver could become a new tool for estimating the human toxicity and pharmacokinetics. At first, metabolism, which plays an essential role in pharmacokinetics, was investigated in the chimeric mice. In the liver of the chimeric mice, human drug metabolizing enzymes were found to be expressed and to reflect the capacities and genetic polymorphism of the donor. In an in vivo study on metabolism, human specific metabolites could be detected in the serum of the chimeric mice indicating that the chimeric mice could be used as an in vivo model to address human metabolism. These results suggested that the chimeric mice could overcome the species differences in drug metabolism and be used to evaluate drug toxicity due to genetic polymorphism. The reasons for drug interaction are often enzyme induction and inhibition. By the treatment with a typical inducer of cytochrome P450 (P450), which is the central drug-metabolizing enzyme, P450s expressed in the liver of the chimeric mice were found to possess induction potencies. After the treatment with a specific inhibitor of human P450, the area under the curve of the P450 metabolite was significantly decreased in the chimeric mice but not in the control mice. Therefore, it was indicated that the chimeric mice could be useful for assessing drug interactions in vivo. Moreover, drug excretion was determined to be humanized because cefmetazole was mainly excreted in urine both in the chimeric mice and humans but in the feces in control uPA(-/-)/SCID mice. Drug transporters expressed in the liver of the chimeric mice were also humanized. In this review, studies of the chimeric mice with humanized liver, particularly on metabolism and excretion, are summarized and the possibility of using the chimeric mice is proposed for the advanced prediction of human pharmacokinetics and toxicity.

The effect of a long term epidural infusion of ropivacaine on CYP2D6 activity.

BACKGROUND: Ropivacaine and one of its metabolites, pipecoloxylidide, inhibit CYP2D6 in. human liver microsomes in vitro with K(i) values of 5 microM (1.4 mg/L) and 13 microM (3.6 mg/L), respectively. We investigated the effect of a 50 h continuous epidural infusion of ropivacaine 2 mg/mL at a rate of 14 mL/h on CYP2D6 activity. METHODS: Nineteen patients (41-85 yr) undergoing hip or knee replacement, all extensive metabolizers with respect to CYP2D6 activity, were included. Medications known to inhibit or be metabolized by CYP2D6, or known to be strong inhibitors/inducers of CYP1A2 or CYP3A4 were not allowed. Patients received 10 mg debrisoquine (a marker for CYP2D6 activity) before surgery and after 40 h epidural infusion. The metabolic ratio (MR) for debrisoquine hydroxylation was calculated as the amount of debrisoquine/amount of 4-OH-debrisoquine excreted in 0-10 h urine. RESULTS: The median (range) of MR before and after ropivacaine were 0.54 (0.1-3.4) and 1.79 (0.3-6.7), respectively. The Hodges Lehman estimate of the ratio MR after/MR before ropivacaine was 2.2 with a 95% confidence interval 1.9-2.7 (P < 0.001). CONCLUSION: A continuous epidural infusion of ropivacaine inhibits CYP2D6 activity in patients who are extensive metabolizers resulting in a twofold increase in the MR for debrisoquine hydroxylation. However, since none of the patients was converted into a functional poor metabolizer (MR >12.6), the effect on the metabolism of other drugs metabolized by CYP2D6 is unlikely to be of major clinical importance.

Comparative metabolic capabilities and inhibitory profiles of CYP2D6.1, CYP2D6.10, and CYP2D6.17.

Polymorphisms in the cytochrome P450 2D6 (CYP2D6) gene are a major cause of pharmacokinetic variability in human. Although the poor metabolizer phenotype is known to be caused by two null alleles leading to absence of functional CYP2D6 protein, the large variability among individuals with functional alleles remains mostly unexplained. Thus, the goal of this study was to examine the intrinsic enzymatic differences that exist among the several active CYP2D6 allelic variants. The relative catalytic activities (enzyme kinetics) of three functionally active human CYP2D6 allelic variants, CYP2D6.1, CYP2D6.10, and CYP2D6.17, were systematically investigated for their ability to metabolize a structurally diverse set of clinically important CYP2D6-metabolized drugs [atomoxetine, bufuralol, codeine, debrisoquine, dextromethorphan, (S)-fluoxetine, nortriptyline, and tramadol] and the effects of various CYP2D6-inhibitors [cocaine, (S)-fluoxetine, (S)-norfluoxetine, imipramine, quinidine, and thioridazine] on these three variants. The most significant difference observed was a consistent but substrate-dependent decease in the catalytic efficiencies of cDNA-expressed CYP2D6.10 and CYP2D6.17 compared with CYP2D6.1, yielding 1.32 to 27.9 and 7.33 to 80.4% of the efficiency of CYP2D6.1, respectively. The most important finding from this study is that there are mixed effects on the functionally reduced allelic variants in enzyme-substrate affinity or enzyme-inhibitor affinity, which is lower, higher, or comparable to that for CYP2D6.1. Considering the rather high frequencies of CYP2D6*10 and CYP2D6*17 alleles for Asians and African Americans, respectively, these data provide further insight into ethnic differences in CYP2D6-mediated drug metabolism. However, as with all in vitro to in vivo extrapolations, caution should be applied to the clinical consequences.

Genotyping of the cytochrome P450 2D6 4469 C>T polymorphism using SimpleProbes.

BACKGROUND: Genotyping of human cytochrome P450s is a pharmacogenetic approach to diagnosing inherited deficiencies in drug metabolizing enzymes that influence therapeutic responses. The P450 CYP2D6 (debrisoquine hydroxylase) metabolizes numerous antidepressants and neuroleptic agents and there is evidence of a relationship between gene polymorphism and variant therapeutic response. Polymorphism in CYP2D6 causes poor, intermediate, efficient or ultrarapid metabolization of substrate drugs affecting pharmacokinetic parameters and requiring dose adjustments. Predictive genotyping for broader clinical application is reliant on fast, technically simple analyses. A new genotyping method was explored. It identifies the single nucleotide polymorphism (SNP) 4469 C>T (NCBI access no. M33388) with one fluorescent hybridization probe (SimpleProbes; SP) using the LightCycler (LC). This SNP is found in 21 alleles, comprising 30% in Caucasian populations and encoding enzymes with poor, intermediate or efficient activity. The remaining 65 known alleles either harbour a C in position 4469 or are deletion mutants. METHODS: Comparative detection of C>T polymorphism was done using a well-established polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique and PCR followed by melting-point (T(m)) analysis with an SP covering the SNP position in 144 samples encompassing alleles *2 and *41 with a T, alleles *1,*3, *4, *6, *9, *10, *15 with a C and the deletion mutant allele *5. RESULTS: C>T polymorphism was detected with complete concordance. T(m) of SP/target heteroduplex complexes for C was: T(m) 67, 89 degrees C to 68, 62 degrees C and for T: T(m) 60, 70 degrees C to 61, 51 degrees C. CONCLUSION: By one-step SP methodology it proved possible within 2 h to identify an SNP in genotypes comprising >90% in Caucasian populations.

Appropriate phenotyping procedures for drug metabolizing enzymes and transporters in humans and their simultaneous use in the "cocktail" approach.

Phenotyping for drug metabolizing enzymes and transporters is used to assess quantitatively the effect of an intervention (e.g., drug therapy, diet) or a condition (e.g., genetic polymorphism, disease) on their activity. Appropriate selection of test drug and metric is essential to obtain results applicable for other substrates of the respective enzyme/transporter. The following phenotyping metrics are recommended based on the level of validation and on practicability: CYP1A2, paraxanthine/caffeine in plasma 6 h after 150 mg caffeine; CYP2C9, tolbutamide plasma concentration 24 h after 125 mg tolbutamide; CYP2C19, urinary excretion of 4'-OH-mephenytoin 0-12 h after 50 mg mephenytoin; CYP2D6, urinary molar ratio debrisoquine/4-OH-debrisoquine 0-8 h after 10 mg debrisoquine; and CYP3A4, plasma clearance of midazolam after 2 mg midazolam (all drugs given orally).

In vivo drug metabolism model for human cytochrome P450 enzyme using chimeric mice with humanized liver.

We previously clarified that major human drug metabolizing enzymes were expressed in a chimeric urokinase-type plasminogen activator (uPA)+/+/severe combined immunodeficient (SCID) mouse line established recently, in which the liver could be replaced by more than 80% with human hepatocytes. In the present study, we investigated the in vivo drug metabolism of a CYP2D6 substrate, debrisoquin (DB), in chimeric mice with high (High) or low (Low) human albumin (hAlb) concentrations and in control uPA-/-/SCID mice. The hAlb in the mouse blood is one of the indices of humanized liver because the chimeric mice produce hAlb. After oral administration of DB at 2.0 mg/kg, the AUC0-8 value of a major CYP2D6 metabolite of DB, 4'-hydroxydebrisoquin (4-OH DB), in High was 3.6-fold higher than those of Low and uPA-/-/SCID mice. By pre-treatment with a typical CYP2D6 inhibitor, quinidine, the AUC0-8 value of 4-OH DB in High was decreased although such values in Low and uPA-/-/SCID mice did not change. The in vitro kinetic analyses and the Ki values of quinidine on the DB 4'-hydroxylase activity in liver microsomes also supported the humanization of the chimeric mice. In conclusion, the chimeric mice exhibited a humanized profile of drug metabolism and the inhibition of P450.

Liver disease selectively modulates cytochrome P450--mediated metabolism.

BACKGROUND: The liver plays a significant role in drug metabolism; thus it would be expected that liver disease may have a detrimental effect on the activity of cytochrome P450 (CYP) enzymes. The extent to which the presence and severity of liver disease affect the activity of different individual drug-metabolizing enzymes is still not well characterized. The purpose of this study was to assess the effect of liver disease on multiple CYP enzymes by use of a validated cocktail approach. METHODS: The participants in this investigation were 20 patients with different etiologies and severity of liver disease and 20 age-, sex-, and weight-matched healthy volunteers. Liver disease severity was categorized by use of the Child-Pugh score. All participants received a cocktail of 4 oral drugs simultaneously, caffeine, mephenytoin, debrisoquin (INN, debrisoquine), and chlorzoxazone, as in vivo probes of the drug-metabolizing enzymes CYP1A2, CYP2C19, CYP2D6, and CYP2E1, respectively. The primary end points were measurements of specific CYP metabolism indexes for each enzyme. RESULTS: Mephenytoin metabolism was significantly decreased in both patients with mild liver disease (Child-Pugh score of 5/6) (-63% [95% confidence interval (CI), -86% to -40%]; P = .0003) and patients with moderate to severe liver disease (Child-Pugh score >6) (-80% [95% CI, -95% to -64%]; P = .0003). In comparison with control subjects, the caffeine metabolic ratio was 69% lower (95% CI, -85% to -54%; median, 0.14 versus 0.62; P = .0003), the debrisoquin recovery ratio was 71% lower (95% CI, -96% to -47%; median, 0.10 versus 0.65; P = .012), and the chlorzoxazone metabolic ratio was 60% lower (95% CI, -91% to -29%; median, 0.21 versus 0.83; P = .0111) in patients with moderate to severe liver disease. All 4 drugs showed significant negative relationships with the Child-Pugh score. CONCLUSIONS: CYP enzyme activity is differentially affected by the presence of liver disease. We propose that the data can be explained by the "sequential progressive model of hepatic dysfunction," whereby liver disease severity has a differential effect on the metabolic activity of specific CYP enzymes.

3,4-Dehydrodebrisoquine, a novel debrisoquine metabolite formed from 4-hydroxydebrisoquine that affects the CYP2D6 metabolic ratio.

Considerable unexplained intersubject variability in the debrisoquine metabolic ratio (urinary debrisoquine/4-hydroxydebrisoquine) exists within individual CYP2D6 genotypes. We speculated that debrisoquine was converted to as yet undisclosed metabolites. Thirteen healthy young volunteers, nine CYP2D6*1 homozygotes [extensive metabolizers (EMs)] and four CYP2D6*4 homozygotes [poor metabolizers (PMs)] took 12.8 mg of debrisoquine hemisulfate by mouth and collected 0- to 8- and 8- to 24-h urines, which were analyzed by gas chromatography-mass spectrometry (GCMS) before and after treatment with beta-glucuronidase. Authentic 3,4-dehydrodebrisoquine was synthesized and characterized by GCMS, liquid chromatography-tandem mass spectrometry, and (1)H NMR. 3,4-Dehydrodebrisoquine is a novel metabolite of debrisoquine excreted variably in 0- to 24-h urine, both in EMs (3.1-27.6% of dose) and PMs (0-2.1% of dose). This metabolite is produced from 4-hydroxydebrisoquine in vitro by human and rat liver microsomes. A previously unstudied CYP2D6*1 homozygote was administered 10.2 mg of 4-hydroxydebrisoquine orally and also excreted 3,4-dehydrodebrisoquine. EMs excreted 6-hydroxydebrisoquine (0-4.8%) and 8-hydroxydebrisoquine (0-1.3%), but these phenolic metabolites were not detected in PM urine. Debrisoquine and 4-hydroxydebrisoquine glucuronides were excreted in a highly genotype-dependent manner. A microsomal activity that probably does not involve cytochrome P450 participates in the further metabolism of 4-hydroxydebrisoquine, which we speculate may also lead to the formation of 1- and 3-hydroxydebrisoquine and their ring-opened products. In conclusion, this study suggests that the traditional metabolic ratio is not a true measure of the debrisoquine 4-hydroxylation capacity of an individual and thus may, in part, explain the wide intragenotype variation in metabolic ratio.

Amodiaquine, its desethylated metabolite, or both, inhibit the metabolism of debrisoquine (CYP2D6) and losartan (CYP2C9) in vivo.

OBJECTIVE: To study the extent of in vivo inhibition by the antimalarial drug amodiaquine, its active metabolite N-desethylamodiaquine, or both, of the metabolism of four probe drugs of the enzymes CYP2D6, CYP2C19, CYP2C9 and CYP1A2. METHODS: Twelve healthy Swedish volunteers received a cocktail of four probe drugs (debrisoquine, omeprazole, losartan and caffeine) to determine their baseline metabolic capacities. After a washout period, they received a 600 mg oral dose of amodiaquine hydrochloride; and 2-3 h later the cocktail was administered again. One week after the intake of amodiaquine, the subjects received the cocktail a third time. The levels of probe drugs and their metabolites as well as amodiaquine and its metabolite were determined by HPLC. RESULTS: Plasma levels of amodiaquine and N-desethylamodiaquine could be followed in all subjects for 6 h and 28 days, respectively. Among the 12 subjects, a 3-fold variation in amodiaquine AUC and a 2-fold variation in N-desethylamodiaquine AUC, were observed. The CYP2D6 and CYP2C9 activities of the subjects were measured by debrisoquine and losartan phenotyping tests, respectively. There were significant mean increases in debrisoquine metabolic ratio (MR) between baseline and the second cocktail [MR(2 h)-MR(baseline) 1.426 (95% confidence interval 1.159, 1.755), P=0.002; ANOVA, Fisher LSD test] and in mean losartan MR between baseline and the second cocktail [MR(2 h)-MR(baseline) 1.724 (95% confidence interval 1.076, 2.762), P=0.026; ANOVA, Fisher LSD test]. The effects on CYP2D6 and CYP2C9 activities subsided within a week after intake of amodiaquine as tested by the phenotyping cocktail. The changes in omeprazole MRs and caffeine MRs were not statistically significant between any of the study phases. CONCLUSION: A single dose of amodiaquine decreased CYP2D6 and CYP2C9 activities significantly compared to baseline values. Amodiaquine has the potential to cause drug-drug interactions and should be further investigated in malarial patients treated with drug combinations containing amodiaquine.

Influence of chronic renal failure on stereoselective metoprolol metabolism in hypertensive patients.

The influence of chronic renal failure on the stereoselective metabolism of rac-metoprolol was investigated in 15 hypertensive patients, 7 of them with chronic renal failure and 8 with normal renal function. They were treated with rac-metoprolol (200 mg) for 7 days. The patients of both groups presented stereoselectivity in metoprolol metabolism, favoring the formation of 1'R-alpha-hydroxymetoprolol (AUC(1(')R/1(')S)(0-24) approximately 2.5) and (R)-metoprolol acidic metabolite (AUC((S)/(R))(0-24) = 0.8), the latter resulting in the plasma accumulation of (S)-metoprolol (AUC((S)/(R))(0-24) = 1.2). Patients with chronic renal failure presented plasma accumulation of the 4 alpha-hydroxymetoprolol isomers and of both metoprolol acidic metabolite enantiomers. A 50% reduction in Cl(R) does not explain the 3- to 4-fold plasma accumulation of metoprolol acidic metabolite in this group, suggesting that other pathways of metoprolol elimination are affected in chronic renal failure in addition to renal excretion. Chronic renal failure does not change the stereoselective kinetic disposition of metoprolol but modifies its stereoselective metabolism, inducing some of the CYP enzymes involved in the formation of the metoprolol acid metabolite.

Cytochrome P450 mRNA expression in peripheral blood lymphocytes as a predictor of enzyme induction.

OBJECTIVE: Previous reports have supported the concept that messenger ribonucleic acid (mRNA) concentrations for cytochrome P450 (CYP) enzymes in peripheral blood mononuclear cells may be predictive of systemic enzyme activity. We investigated whether changes in mRNA expression for CYP1A2,CYP2C19, CYP2D6 and CYP3A4 in peripheral blood lymphocytes (PBLs) may serve as surrogate markers for changes in CYP enzyme activity following the administration of rifampin. METHODS: On day 1 and day 9 of the study, 12 healthy volunteers were administered caffeine 100 mg, debrisoquine 10 mg and omeprazole 40 mg orally, along with midazolam 0.025 mg/kg intravenously. Blood samples and urine were collected for 8 h after drug administration. The subjects took rifampin 300 mg (n = 6) or 600 mg (n = 6) daily on days 2-8. Total RNA was isolated from PBLs on day 1 and day 9, and mRNA expression for the CYP enzymes and hGAPDH were determined by means of quantitative, real-time, reverse-transcriptase polymerase chain reaction. CYP1A2 activity was estimated by calculating the plasma paraxanthine to caffeine AUC ratio (caffeine metabolic ratio; CMR), CYP2C19 activity by the 2-h omeprazole hydroxylation index (HI), CYP2D6 activity by the urinary debrisoquine recovery ratio (DBRR) and CYP3A4 activity by midazolam clearance. RESULTS: Median midazolam clearance (0.362 to 0.740 l/kg/h), omeprazole HI (0.752 to 0.214), CMR (0.365 to 0.450) and DBRR (0.406 to 0.479) all changed significantly following rifampin, consistent with the expected enzyme induction. CYP1A2,CYP2D6 and CYP3A4 mRNA content were measurable in all samples. CYP2C19 mRNA was inconsistently detectable. There were no significant correlations between changes in enzyme activity and mRNA expression by Spearman's rank order correlation. CONCLUSION: The results do not support the use of mRNA expression assays for CYP1A2, CYP2C19, CYP2D6 and CYP3A4 enzymes in PBLs as surrogates for quantifying changes in systemic enzyme activity in the setting of enzyme induction.

Determination of debrisoquine and 4-hydroxydebrisoquine by high-performance liquid chromatography: application to the evaluation of CYP2D6 genotype and debrisoquine metabolic ratio relationship.

The drug-metabolizing cytochrome P450 (CYP) enzyme CYP2D6 is involved in the metabolism of several clinically important drugs. So far more than 50 different CYP2D6 allelic variants have been described, and thus there is an increased need for routine high-performance liquid chromatography (HPLC) methods for the evaluation of the functional implication of CYP2D6 polymorphism. Debrisoquine is metabolized to 4-hydroxydebrisoquine by CYP2D6, and therefore it has been used widely to determine the hydroxylation capacity of the enzyme. The aim of the present study was to develop a simple, accurate HPLC method with ultraviolet detection for the measurement of debrisoquine and 4-hydroxydebrisoquine in urine for evaluation of the relationship between CYP2D6 enzyme activity and genotypes. For the HPLC determination, a C18 extraction column was used with a flow rate of 0.8 mL/min and detection at 210 nm. The compounds were eluted from the column in less than 10 min. Coefficients of variation at all concentrations were less than 4% for both compounds. The debrisoquine/4-hydroxydebrisoquine ratio (debrisoquine metabolic ratio) was determined in a panel of 16 Caucasian healthy volunteers with zero (poor metabolizers), one, two or more than two (ultrarapid metabolizers) CYP2D6 active genes. Significant correlation (p<0.05) between the number of CYP2D6 active genes and the hydroxylation capacity of the enzyme was found. The present HPLC method was simple, fast and accurate, and thus will be useful for the evaluation of CYP2D6 hydroxylation capacity in pharmacogenetic studies.

In vivo assessment of botanical supplementation on human cytochrome P450 phenotypes: Citrus aurantium, Echinacea purpurea, milk thistle, and saw palmetto.

OBJECTIVES: Phytochemical-mediated modulation of cytochrome P450 (CYP) activity may underlie many herb-drug interactions. Single-time point phenotypic metabolic ratios were used to determine whether long-term supplementation of Citrus aurantium , Echinacea purpurea , milk thistle (Silybum marianum), or saw palmetto (Serenoa repens) extracts affected CYP1A2, CYP2D6, CYP2E1, or CYP3A4 activity. METHODS: Twelve healthy volunteers (6 women, 6 men) were randomly assigned to receive C aurantium , E purpurea , milk thistle, or saw palmetto for 28 days. For each subject, a 30-day washout period was interposed between each supplementation phase. Probe drug cocktails of midazolam and caffeine, followed 24 hours later by chlorzoxazone and debrisoquin (INN, debrisoquine), were administered before (baseline) and at the end of supplementation. Presupplementation and postsupplementation phenotypic trait measurements were determined for CYP3A4, CYP1A2, CYP2E1, and CYP2D6 by use of 1-hydroxymidazolam/midazolam serum ratios (1-hour sample), paraxanthine/caffeine serum ratios (6-hour sample), 6-hydroxychlorzoxazone/chlorzoxazone serum ratios (2-hour sample), and debrisoquin urinary recovery ratios (8-hour collection), respectively. The content of purported "active" phytochemicals was determined for each supplement. RESULTS: Comparisons of presupplementation and postsupplementation phenotypic ratios suggested that these particular supplements had no significant effect on CYP1A2, CYP2D6, CYP2E1, or CYP3A4 activity. Phytochemical profiles indicated that C aurantium was devoid of the CYP3A4 inhibitor 6',7'-dihydroxybergamottin. Quantities of fatty acids, flavonolignans, and cichoric acid were consistent with label claims for saw palmetto, milk thistle, and E purpurea , respectively. CONCLUSIONS: Botanical supplements containing C aurantium , milk thistle, or saw palmetto extracts appear to pose a minimal risk for CYP-mediated herb-drug interactions in humans. Although the effects of E purpurea on CYP activity were minor, further study into the interaction potential of this botanical is merited.

Pharmacokinetic and pharmacodynamic assessment of a five-probe metabolic cocktail for CYPs 1A2, 3A4, 2C9, 2D6 and 2E1.

AIMS: The primary objectives of the present study were to establish whether there was a pharmacokinetic or pharmacodynamic interaction between the probe drugs caffeine (CYP1A2), tolbutamide (CYP2C9), debrisoquine (CYP2D6), chlorzoxazone (CYP2E1) and midazolam (CYP3A4), when administered in combination as a cocktail. Furthermore, the tolerability of these probe drugs, both alone and in combination as a cocktail was assessed. METHODS: Twelve healthy volunteer subjects (age range 22-48 years) were entered into an open, fixed sequence, 6-limb, single centre study. The randomization was such that all drugs were given individually followed by the full "cocktail" as the last treatment limb. The phenotypic index used to assess the intrinsic activity of the CYP isoforms included metabolite/parent ratios in plasma and urine (CYPs 1A2, 2E1 & 2C9), parent/metabolite ratios in urine (CYP2D6) and plasma AUClast (CYP3A4). Blood pressure and blood glucose measurements were used to assess pharmacodynamic interactions. Tolerability was assessed through reporting of adverse events RESULTS: Overall, there was little evidence that the probe drugs interacted metabolically when co-administered as the cocktail. The ratio of the geometric mean (and 90% confidence interval) of the phenotypic index, obtained after administration of the probe as part of the cocktail and when given alone were: caffeine, 0.86 (0.67-1.10), midazolam, 0.96 (0.74-1.24), tolbutamide, 0.86 (0.72-1.03), debrisoquine 1.04 (0.97-1.12) and chlorzoxazone, 0.95 (0.86-1.05). There was no difference in blood pressure and blood glucose concentrations following the cocktail and dosing of the individual probes. There was no effect on ECG recordings at any time-point. The adverse events reported for individual drug administrations were mild, transient and expected. Overall no more adverse events were reported on the cocktail study days than on the days when the drugs were administered alone. CONCLUSIONS: The five probe drugs when coadministered, in this dosing regimen, demonstrated no evidence of either a metabolic or pharmacodynamic interaction that might confound the conclusions drawn during a cocktail study. The present cocktail methodology has the potential to become a useful tool to aid the detection of clinically important drug-drug interactions during drug development.

Disposition of debrisoquine and nortriptyline in Korean subjects in relation to CYP2D6 genotypes, and comparison with Caucasians.

AIMS: To study the influence of the CYP2D6*10 allele on the disposition of debrisoquine and nortriptyline. METHODS: The pharmacokinetics of debrisoquine and nortriptyline and their main metabolites were determined in ten Koreans with the CYP2D6*1/*1 (n = 5) and CYP2D6*1/*10 (n = 5) genotypes after single oral doses of 20 mg debrisoquine and 25 mg nortriptyline, respectively. The data were compared with previously published findings from 21 Caucasians with 0, one, two, three, four or 13 functional CYP2D6 genes. RESULTS: The AUC0-8 of 4-hydroxydebrisoquine was significantly lower in Koreans with CYP2D6*1/*10 genotype compared with CYP2D6*1/*1[95% confidence interval (CI) for the ratio between means 1.17, 1.85]. No other genotype-related differences were found in the plasma kinetics of nortriptyline and debrisoquine, or their hydroxy metabolites. The AUCnortriptyline/AUC10-hydroxynortriptyline ratio did not differ between the *1/*1 and *1/*10 genotype groups (95% CI for the ratio of means 0.60, 1.26). Similarly, there was no difference between these genotypes with respect to the AUCdebrisoquine/AUC4-hydroxydebrisoquine ratio (95% CI for the ratio of mean values 0.38, 1.46). Both Korean genotype groups had similar AUCs and parent compound/metabolite AUC ratios of debrisoquine and nortriptyline to Caucasians with two functional CYP2D6 genes. CONCLUSIONS: Heterozygosity for CYP2D6*10 decreases the CYP2D6-dependent elimination of nortriptyline and debrisoquine to only a limited degree. Further studies in subjects homozygous for CYP2D6*10 are required to elucidate fully the pharmacokinetic consequences of this CYP2D6 genotype in Orientals.

[Prevalence of ultraextensive drug metabolizers in Croatian population--long-PCR based detection of amplified CYP2D6 gene]. / Prevalencija vrlo brzih metabolizatora lijekova u hrvatskoj populaciji--detekcija amplificiranog gena CYP2D6 metodom long-PCR.

Cytochrome P450 enzyme debrisoquine 4-hydroxylase, responsible for the metabolism of different classes of drugs and other chemical substances, exhibits genetic polymorphism with great interindividual and interethnic differences in metabolic capacity. The activity of enzyme ranges from very expressed, rapid, to total absence of activity. Up to 7% of Caucasians may demonstrate ultrarapid metabolism--UEM of debrisoquine and other drugs, substrates of debrisoquin hydroxylase, due to inheritance of multiplicate functional CYP2D6 gene, causing an increased amount of enzyme to be expressed. Identification of subjects with ultrarapid metabolism is of potential clinical value for optimization of therapy and avoidance of therapeutic failure due to inadequate dosage. In our study we wanted to determine the prevalence of UEM genotype in Croatian population applying long-PCR method. We found a 4% prevalence of ultrarapid metabolizers with multiplicated CYP2D6 gene.