Change from the CYP2D6 extensive metabolizer to the poor metabolizer phenotype during treatment With bupropion

Some data indicate that bupropion inhibits the cytochrome P-450 enzyme CYP2D6, but very little published data is available on the extent of this inhibition. The objective of the present study was to quantify this inhibition in a subject treated with bupropion for smoking cessation. Genotypically, the patient was a CYP2D6 homozygous extensive metabolizer (EM). His CYP2D6 phenotype was assessed using the test drug dextromethorphan before, during, and after treatment with bupropion. During treatment with bupropion, he clearly changed from the EM to the poor metabolizer (PM) phenotype. Although the results from a single patient should be interpreted with great caution, the extent of the interaction indicates that bupropion might be a CYP2D6 inhibitor as potent as the most powerful CYP2D6 inhibitors known, such as quinidine and paroxetine.     

Effects of CYP2D6 status on harmaline metabolism, pharmacokinetics and pharmacodynamics, and a pharmacogenetics-based pharmacokinetic model

Harmaline is a β-carboline alkaloid showing neuroprotective and neurotoxic properties. Our recent studies have revealed an important role for cytochrome P450 2D6 (CYP2D6) in harmaline O-demethylation. This study, therefore, aimed to delineate the effects of CYP2D6 phenotype/genotype on harmaline metabolism, pharmacokinetics (PK) and pharmacodynamics (PD), and to develop a pharmacogenetics mechanism-based compartmental PK model. In vitro kinetic studies on metabolite formation in human CYP2D6 extensive metabolizer (EM) and poor metabolizer (PM) hepatocytes indicated that harmaline O-demethylase activity (V_max/K_m) was about 9-fold higher in EM hepatocytes. Substrate depletion showed mono-exponential decay trait, and estimated in vitro harmaline clearance (CL_int, μL/min/10⁶ cells) was significantly lower in PM hepatocytes (28.5) than EM hepatocytes (71.1). In vivo studies in CYP2D6-humanized and wild-type mouse models showed that wild-type mice were subjected to higher and longer exposure to harmaline (5 and 15 mg/kg; i.v. and i.p.), and more severe hypothermic responses. The PK/PD data were nicely described by our pharmacogenetics-based PK model involving the clearance of drug by CYP2D6 (CL_CYP2D6) and other mechanisms (CL_other), and an indirect response PD model, respectively. Wild-type mice were also more sensitive to harmaline in marble-burying tests, as manifested by significantly lower ED₅₀ and steeper Hill slope. These findings suggest that distinct CYP2D6 status may cause considerable variations in harmaline metabolism, PK and PD. In addition, the pharmacogenetics-based PK model may be extended to define PK difference caused by other polymorphic drug-metabolizing enzyme in different populations.     

Minimizing polymorphic metabolism in drug discovery: evaluation of the utility of in vitro methods for predicting pharmacokinetic consequences associated with CYP2D6 metabolism.

Minimizing interindividual variability in drug exposure is an important goal for drug discovery. The reliability of the selective CYP2D6 inhibitor quinidine was evaluated in a retrospective analysis using a standardized approach that avoids laboratory-to-laboratory variation. The goal was to evaluate the reliability of in vitro metabolism studies for predicting extensive metabolizer (EM)/poor metabolizer (PM) exposure differences. Using available literature, 18 CYP2D6 substrates were selected for further analysis. In vitro microsomal studies were conducted at 1 microM substrate and 0.5 microM P450 to monitor substrate depletion. An estimate of the fraction metabolized by CYP2D6 in microsomes was derived from the rate constant determined with and without 1 microM quinidine for 11 substrates. Clearance in EM and PM subjects and fractional recovery of metabolites were taken from the literature. A nonlinear relationship between the contribution of CYP2D6 and decreased oral clearance for PMs relative to EMs was evident. For drugs having <60% CYP2D6 involvement in vivo, a modest difference between EM and PM exposure was observed (<2.5-fold). For major CYP2D6 substrates (>60%), more dramatic exposure differences were observed (3.5- to 53-fold). For compounds primarily eliminated by hepatic P450 and with sufficient turnover to be evaluated in vitro, the fraction metabolized by CYP2D6 in vitro compared favorably with the in vivo data. The in vitro estimation of fraction metabolized using quinidine as a specific inhibitor provided an excellent predictive tool. Results from microsomal substrate depletion experiments can be used with confidence to select compounds in drug discovery using a cutoff of >60% metabolism by CYP2D6.     

Genotype and phenotype of cytochrome P450 2D6 in human immunodeficiency virus-positive patients and patients with acquired immunodeficiency syndrome

Objective: To examine the distribution of the cytochrome P ₄₅₀ (CYP) CYP2D6 phenotype and its relation to genotype, concomitant medication, and disease state in human immunodeficiency virus (HIV)-positive patients. Design: A cross sectional study with a longitudinal component compared individual genotypes for CYP2D6 to the CYP2D6 phenotype. Methods: Sixty-one predominately male Caucasian, HIV-positive patients were recruited and CYP2D6 genotypes [extensive metabolizer (EM) or poor metabolizer (PM)] determined by polymerase chain reaction (PCR)-based amplification, followed by restriction fragment-length analysis. The patients were also phenotyped using dextromethorphan (DM) to determine their respective enzyme activity and assigned either a CYP2D6 EM or PM phenotype. Complete medical and treatment histories were compiled. A total of 44 patients were tested longitudinally. Results: Fifty-nine patients (97%) possessed an EM genotype, consistent with previously observed distributions in demographically similar populations. In healthy seronegative populations, genotype and phenotype have been shown to be essentially interchangeable measures of CYP2D6 activity. In this cohort, 2 of the 59 patients with an EM genotype expressed a PM phenotype. In addition, 4 EM patients were less extensive DM metabolizers than any of the patients receiving medication known to inhibit CYP2D6. This apparent shift toward the PM phenotype from the EM genotype was associated with the presence of active illness. Conclusion: Changes may occur in HIV-positive patients such that their CYP2D6 activity approaches that of PMs, despite having an EM genotype. Neither active disease nor drug interactions alone explain the shift. Received: 1 September 1999 / Accepted in revised form: 10 February 2000     

Disposition and metabolic fate of atomoxetine hydrochloride: the role of CYP2D6 in human disposition and metabolism.

The role of the polymorphic cytochrome p450 2D6 (CYP2D6) in the pharmacokinetics of atomoxetine hydrochloride [(-)-N-methyl-gamma-(2-methylphenoxy)benzenepropanamine hydrochloride; LY139603] has been documented following both single and multiple doses of the drug. In this study, the influence of the CYP2D6 polymorphism on the overall disposition and metabolism of a 20-mg dose of (14)C-atomoxetine was evaluated in CYP2D6 extensive metabolizer (EM; n = 4) and poor metabolizer (PM; n = 3) subjects under steady-state conditions. Atomoxetine was well absorbed from the gastrointestinal tract and cleared primarily by metabolism with the preponderance of radioactivity being excreted into the urine. In EM subjects, the majority of the radioactive dose was excreted within 24 h, whereas in PM subjects the majority of the dose was excreted by 72 h. The biotransformation of atomoxetine was similar in all subjects undergoing aromatic ring hydroxylation, benzylic oxidation, and N-demethylation with no CYP2D6 phenotype-specific metabolites. The primary oxidative metabolite of atomoxetine was 4-hydroxyatomoxetine, which was subsequently conjugated forming 4-hydroxyatomoxetine-O-glucuronide. Due to the absence of CYP2D6 activity, the systemic exposure to radioactivity was prolonged in PM subjects (t(1/2) = 62 h) compared with EM subjects (t(1/2) = 18 h). In EM subjects, atomoxetine (t(1/2) = 5 h) and 4-hydroxyatomoxetine-O-glucuronide (t(1/2) = 7 h) were the principle circulating species, whereas atomoxetine (t(1/2) = 20 h) and N-desmethylatomoxetine (t(1/2) = 33 h) were the principle circulating species in PM subjects. Although differences were observed in the excretion and relative amounts of metabolites formed, the primary difference observed between EM and PM subjects was the rate at which atomoxetine was biotransformed to 4-hydroxyatomoxetine.     

CYP2D6 and CYP3A4 involvement in the primary oxidative metabolism of hydrocodone by human liver microsomes

AIM: To determine the Michaelis-Menten kinetics of hydrocodone metabolism to its O- and N-demethylated products, hydromorphone and norhydrocodone, to determine the individual cytochrome p450 enzymes involved, and to predict the in vivo hepatic intrinsic clearance of hydrocodone via these pathways. METHODS: Liver microsomes from six CYP2D6 extensive metabolizers (EM) and one CYP2D6 poor metabolizer (PM) were used to determine the kinetics of hydromorphone and norhydrocodone formation. Chemical and antibody inhibitors were used to identify the cytochrome p450 isoforms catalyzing these pathways. Expressed recombinant cytochrome p450 enzymes were used to characterize further the metabolism of hydrocodone. RESULTS: Hydromorphone formation in liver microsomes from CYP2D6 EMs was dependent on a high affinity enzyme (Km = 26 microm) contributing 95%, and to a lesser degree a low affinity enzyme (Km = 3.4 mm). In contrast, only a low affinity enzyme (Km = 8.5 mm) formed this metabolite in the liver from the CYP2D6 PM, with significantly decreased hydromorphone formation compared with the livers from the EMs. Norhydrocodone was formed by a single low affinity enzyme (Km = 5.1 mm) in livers from both CYP2D6 EM and PM. Recombinant CYP2D6 and CYP3A4 formed only hydromorphone and only norhydrocodone, respectively. Hydromorphone formation was inhibited by quinidine (a selective inhibitor of CYP2D6 activity), and monoclonal antibodies specific to CYP2D6. Troleandomycin, ketoconazole (both CYP3A4 inhibitors) and monoclonal antibodies specific for CYP3A4 inhibited norhydrocodone formation. Extrapolation of in vitro to in vivo data resulted in a predicted total hepatic clearance of 227 ml x h-1 x kg-1 and 124 ml x h-1 x kg-1 for CYP2D6 EM and PM, respectively. CONCLUSIONS: The O-demethylation of hydrocodone is predominantly catalyzed by CYP2D6 and to a lesser extent by an unknown low affinity cytochrome p450 enzyme. Norhydrocodone formation was attributed to CYP3A4. Comparison of recalculated published clearance data for hydrocodone, with those predicted in the present work, indicate that about 40% of the clearance of hydrocodone is via non-CYP pathways. Our data also suggest that the genetic polymorphisms of CYP2D6 may influence hydrocodone metabolism and its therapeutic efficacy.     

CYP2D6 and CYP2C19 activity in a large population of Dutch healthy volunteers: indications for oral contraceptive-related gender differences

OBJECTIVE: We examined a large database containing results on CYP2D6 and CYP2C19 activity of 4301 Dutch volunteers phenotyped in the context of various clinical pharmacology studies. METHODS: The subjects were given 22 mg dextromethorphan, 100 mg mephenytoin and 200 mg caffeine. For CYP2D6, the dextromethorphan/dextrorphan metabolic ratios in urine samples taken for a subsequent 8 h were used. Dextromethorphan and dextrorphan were quantified by reversed-phase high performance liquid chromatography. For CYP2C19 similarly obtained (R)-mephenytoin and (S)-mephenytoin ratios were used. (S)-mephenytoin and (R)-mephenytoin were analysed and quantified by enantioselective capillary gas chromatography. In addition, CYP2C19 poor metabolizer (PM) subjects were reanalysed after acidic pre-treatment of urine samples to confirm the PM status. RESULTS: The investigated population mainly comprised Caucasian (98.9%) males (68%). The age ranged from 18 to 82 years. For CYP2D6, it was found that 8.0% of the subjects were PMs. The average metabolic ratio was 0.014 (0.033) for subjects who showed extensive metabolizing activity (EM) and 5.4 (7.6) for PM subjects. For CYP2C19, it was found that 1.8% of the subjects were PMs. The metabolic ratio was 0.162 (0.124) for EM subjects and 1.076 (0.040) for PM subjects. Within the EM group the metabolic ratio in females was significantly lower for CYP2D6 (-20%) and significantly higher for CYP2C19 (+40%) compared with males. For PMs there was no such difference for CYP2D6 (P = 0.79) or CYP2C19 (P = 0.20). Oral contraceptive (OC) use significantly decreased the CYP2C19 activity by 68% for mephenytoin as compared to non-OC using females. CONCLUSIONS: For CYP2D6, the PM incidence (8.0%) is in accordance with literature data. The CYP2C19, PM incidence (1.8%) is low compared to reports from other European countries. For mephenytoin, the acidification procedure has been shown to be very important for the confirmation of CYP2C19 PMs. In EM females compared to EM males, CYP2D6 activity is increased and CYP2C19 activity is reduced. For CYP2C19 in particular this reduction is substantial and most pronounced in the age range from 18 to 40 years. For CYP2C19, the reduced activity is associated with the use of oral contraceptives.     

CYP3A4 mediates dextropropoxyphene N-demethylation to nordextropropoxyphene: human in vitro and in vivo studies and lack of CYP2D6 involvement

The individual cytochrome P450 isoforms in dextropropoxyphene N-demethylation to nordextropropoxyphene were determined and the pharmacokinetics of dextropropoxyphene and nordextropropoxyphene in cytochrome P4502D6 (CYP2D6) extensive (EM) and poor (PM) subjects were characterized. Microsomes from six CYP2D6 extensive metabolizers and one CYP2D6 poor metabolizer were used with isoform specific chemical and antibody inhibitors and expressed recombinant CYP enzymes. Groups of three CYP2D6 EM and PM subjects received a single 65-mg oral dose of dextropropoxyphene, and blood and urine were collected for 168 and 96 h, respectively. Nordextropropoxyphene formation in vitro was not different between the CYP2D6 extensive metabolizers (Km = 179 +/- 74 microM, Cl(int) = 0.41 +/- 0.26 ml mg(-1)h(-1)) and the PM subject (K = 225 microM, Cl(int) = 0.19 ml mg(-1) h(-1)) and was catalysed predominantly by CYP3A4. There was no apparent difference in the pharmacokinetics of dextropropoxyphene and nordextropropoxyphene in CYP2D6 EM and PM subjects. CYP3A4 is the major CYP enzyme catalysing the major metabolic pathway of dextropropoxyphene metabolism. Hence variability in the pharmacodynamic effects of dextropropoxyphene are likely due to intersubject variability in hepatic CYP3A4 expression and/or drug-drug interactions. Reported CYP2D6 phenocopying is not due to dextropropoxyphene being a CYP2D6 substrate.     

CYP3A4 mediates dextropropoxyphene N-demethylation to nordextropropoxyphene: human in vitro and in vivo studies and lack of CYP2D6 involvement

1.?The individual cytochrome P450 isoforms in dextropropoxyphene N-demethylation to nordextropropoxyphene were determined and the pharmacokinetics of dextropropoxyphene and nordextropropoxyphene in cytochrome P4502D6 (CYP2D6) extensive (EM) and poor (PM) subjects were characterized.

2.?Microsomes from six CYP2D6 extensive metabolizers and one CYP2D6 poor metabolizer were used with isoform specific chemical and antibody inhibitors and expressed recombinant CYP enzymes. Groups of three CYP2D6 EM and PM subjects received a single 65-mg oral dose of dextropropoxyphene, and blood and urine were collected for 168 and 96 h, respectively.

3.?Nordextropropoxyphene formation in vitro was not different between the CYP2D6 extensive metabolizers (K_m = 179 ± 74 μM, Cl_int = 0.41 ± 0.26 ml mg^?1 h^?1) and the PM subject (K_m = 225 μM, Cl_int = 0.19 ml mg^?1 h^?1) and was catalysed predominantly by CYP3A4. There was no apparent difference in the pharmacokinetics of dextropropoxyphene and nordextropropoxyphene in CYP2D6 EM and PM subjects.

4.?CYP3A4 is the major CYP enzyme catalysing the major metabolic pathway of dextropropoxyphene metabolism. Hence variability in the pharmacodynamic effects of dextropropoxyphene are likely due to intersubject variability in hepatic CYP3A4 expression and/or drug–drug interactions. Reported CYP2D6 phenocopying is not due to dextropropoxyphene being a CYP2D6 substrate.     

No effect of the new antidepressant reboxetine on CYP2D6 activity in healthy volunteers.

The effect of the new antidepressant reboxetine on the activity of the cytochrome P450 (CYP) 2D6 isoenzyme was investigated in 10 healthy volunteers using dextromethorphan as a model CYP2D6 substrate. Each volunteer received a single 30 mg oral dose of dextromethorphan on three different occasions separated by an interval of at least 4 weeks: a) in a control session; b) after 1 week of treatment with reboxetine, 8 mg/day; and c) after 1 week of treatment with paroxetine (an inhibitor of CYP2D6 activity) 20 mg/day. Urine was collected over the next 8 hours for the determination of the dextromethorphan/dextrorphan metabolic ratio. All subjects were classified as extensive metabolizers (EM) with a dextromethorphan/dextrorphan ratio < 0.3. There were no notable changes in the urinary dextromethorphan/dextrorphan ratio in the reboxetine phase as compared to the control session. By contrast, there was a statistically significant increase in the metabolic ratio in the paroxetine phase (p < 0.001), with 4 subjects switching to poor metabolizer (PM) phenotype. These results suggest that reboxetine is unlikely to cause clinically significant interactions with substrates of CYP2D6.     

Concordance of P450 2D6 (debrisoquine hydroxylase) phenotype and genotype: inability of dextromethorphan metabolic ratio to discriminate reliably heterozygous and homozygous extensive metabolizers.

Debrisoquine-hydroxylase (P450 2D6) not equal to phenotype was determined in 116 individuals using dextromethorphan as the substrate probe. Polymerase chain reaction and restriction fragment length polymorphism analyses were used to detect inactivating mutations in the CYP2D6 gene and assign genotype in all 116 individuals. Using a urinary metabolic ratio (DM/DT) of > or = 0.3 to define poor metabolizer (PM) phenotypes, 96 subjects were extensive metabolizers (EM) and 20 were PMs. The CYP2D6(B) mutation was the most common mutation, present in 18% of phenotypic EM alleles and 66% of the alleles in PM phenotypes. The CYP2D6(A) mutation (8% of PM alleles) and the CYP2D6 gene deletion (2.6% of PM alleles) were found less frequently. Seven different variants of the CYP2D6 gene were found. In subjects with two mutant alleles, genotype correctly predicted the PM phenotype in 100% (n = 13). Overall, genotype agreed with phenotype assignments in 109 of 116 (94%) subjects. Seven subjects with a wild-type allele at the CYP2D6(A) and CYP2D6(B) loci were phenotypic PMs, representing the only discrepant results. These discrepancies could be due to the imprecision of phenotype assignment or to as yet unknown mutations in CYP2D6. Although the median urinary metabolic ratio was significantly lower in homozygous EMs compared with heterozygous EMs, there was extensive overlap in metabolic ratios in these two groups, indicating that the DM/DT metabolic ratio cannot reliably discriminate homozygous EMs from heterozygous EMs.     

Lack of interaction of milnacipran with the cytochrome p450 isoenzymes frequently involved in the metabolism of antidepressants

OBJECTIVE: To compare the pharmacokinetics of milnacipran in extensive metabolisers (EMs) and poor metabolisers (PMs) of sparteine and mephenytoin, and to assess the influence of multiple administrations of milnacipran on the activity of cytochrome P450 (CYP) isoenzymes through its own metabolism and through various probes, namely CYP2D6 (sparteine/dextromethorphan), CYP2C19 (mephenytoin), CYP1A2 (caffeine) and CYP3A4 (endogenous 6-beta-hydroxy-cortisol excretion). METHODS: Twenty-five healthy subjects, 12 EMs for both sparteine/dextromethorphan and mephenytoin, nine EMs for mephenytoin and PMs for sparteine/dextromethorphan (PM(2D6)) and four PMs for mephenytoin and EMs for sparteine/dextromethorphan (PM(2C19)) were administered milnacipran as a single 50 mg capsule on day 1 followed by a 50 mg capsule twice daily for 7 days. The pharmacokinetics of milnacipran and its oxidative metabolites were assessed after the first dose (day 1) and after multiple administration (day 8), and were compared for differences between CYP2D6 and CYP2C19 PMs and EMs. Metabolic tests were performed before (day -2), during (days 1 and 8) and after (day 20) milnacipran administration. RESULTS: Milnacipran steady state was rapidly achieved. Metabolism was limited: approximately 50% unchanged drug, 30% as glucuronide and 20% as oxidative metabolite (mainly F2800 the N-dealkyl metabolite). Milnacipran administration to PM2D6 and PM2C19 subjects did not increase parent drug exposure or decrease metabolite exposure. Milnacipran oxidative metabolism is not mediated through CYP2D6 or CYP2C19 polymorphic pathways nor does it significantly interact with CYP1A2, CYP2C19, CYP2D6 or CYP3A4 activities. CONCLUSION: Limited reciprocal pharmacokinetic interaction between milnacipran and CYP isoenzymes would confer flexibility in the therapeutic use of the drug when combined with antidepressants. Drug-drug interaction risk would be low, even if the combined treatments were likely to inhibit CYP2D6 and CYP2C19 isoenzyme activities.     

Evaluation of analytical and clinical performance of a dual-probe phenotyping method for CYP2D6 polymorphism and CYP3A4 activity screening

A bioanalytical method for the determination of dextromethorphan (DEX) and its metabolites dextrorphan (DTX), 3-methoxymorphinan (3MM), and 3-hydroxymorphinan (3HM) in human urine was developed for CYP2D6 phenotyping and CYP3A4 activity measurements in clinical pharmacology studies using dextromethorphan administered in a drinking solution as substrate. The method was evaluated by thorough conventional validation and by a cross-validation of the method with a previously applied method for dextromethorphan and dextrorphan only (CYP2D6 phenotyping). Cross-validation with the former method showed no significant differences in measured concentrations of volunteer samples. This guaranteed the consistency of epidemiologic data in the database collected from two methods. For the CYP2D6 and CYP3A4 evaluations, the clinical parameters are ratios of concentrations. It appeared that severe variance in individual concentrations generally did not influence the variance of ratios significantly, because experimental errors in concentrations of two analytes proved to correlate considerably. For CYP2D6 values around the antimodes, the chance of a misclassification is very small. The chance of classifying an extensive metabolizer as a poor metabolizer or vice versa is negligible. For CYP3A4 activity determinations it was concluded that in general a change in dextromethorphan/3-methoxymorphinan (DEX/3MM) ratios of 10% or more as detected with the current method, is a significant increase or decrease in the activity of CYP3A4. The authors concluded that they had obtained an analytically valid and clinically reliable bioanalytical method for the determination of dextromethorphan and its metabolites dextrorphan, 3-methoxymorphinan, and 3-hydroxymorphinan in human urine for CYP2D6 phenotyping and CYP3A4 activity measurements for clinical pharmacology studies.     

Evidence for a role of cytochrome P450 2D6 and 3A4 in ethylmorphine metabolism.

Ethylmorphine is metabolised by N-demethylation (to norethylmorphine) and by O-deethylation (to morphine). The O-deethylation reaction was previously shown in vivo to co-segregate with the O-demethylation of dextromethorphan indicating that ethylmorphine is a substrate of polymorphic cytochrome P450(CYP)2D6. To study further the features of ethylmorphine metabolism we investigated its N-demethylation and O-deethylation in human liver microsomes from eight extensive (EM) and one poor metaboliser (PM) of dextromethorphan. Whereas N-demethylation varied only two-fold there was a 4.3-fold variation in the O-deethylation of ethylmorphine, the lowest rate being observed in the PM. Quinidine, at a concentration of 1 microM, inhibited O-deethylation in microsomes from an EM, but was unable to do so in microsomes from the PM. The immunoidentified CYP2D6 and CYP3A4 correlated with the rates of O-deethylation (r = 0.972) and N-demethylation (r = 0.969), respectively. We conclude that the O-deethylation of ethylmorphine is catalysed by the CYP2D6 in human liver microsomes consistent with previous findings in healthy volunteers.     

Assessment of in vivo CYP2D6 activity: differential sensitivity of commonly used probes to urine pH

Drug/metabolite ratios (MRs) are used as in vivo markers of enzyme activity. The ratios are potentially confounded by the renal clearance of the drug (urine-based MRs) or metabolite (plasma-based MRs). The authors have investigated the relative sensitivity of urinary MR of 3 in vivo probe substrates of CYP2D6 debrisoquine (DB), dextromethorphan (DM), and metoprolol (MP) to changes in urine pH. Three groups of healthy volunteers each comprising 12 individuals were given DB (10 mg), DM (25 mg), or MP (100 mg) on 3 occasions. In 1 study arm, urine was acidified by the oral intake of ammonium chloride; in another, it was alkalinized by intake of sodium bicarbonate; and in the third, urine pH was uncontrolled. Urinary MP/alpha-hydroxy-MP, DM/dextrorphan, and DB/4-hydroxy-DB ratios were calculated. The mean(geo) MR for DB was not significantly different in any of the study arms, whereas those for MP and DM were significantly different under acidified and alkalinized urine conditions compared to uncontrolled urine pH (P < .01) and were correlated with urine pH (P < .001). Without control of urine pH, in vivo estimates of CYP2D6 metabolic activity are likely to be less precise using DM or MP as probe substrates compared to DB. Although this is unlikely to cause any problem in distinguishing the large functional differences in CYP2D6 in poor metabolizer (PM) and extensive metabolizer (EM) phenotypes, this may contribute to difficulties in differentiating in vivo metabolic activity among allelic variants within the overall CYP2D6 EM phenotype using MP or DM. However, because DB is not available in many countries (eg, United States), alternative in vivo markers of CYP2D6 with low sensitivity to urine pH should be sought.     

Steady state plasma levels of the enantiomers of trimipramine and of its metabolites in CYP2D6-, CYP2C19- and CYP3A4/5-phenotyped patients

Steady state plasma concentrations of the (L)- and (D)-enantiomers of trimipramine (TRI), desmethyltrimipramine (DTRI), 2-hydroxytrimipramine (TRIOH) and 2-hydroxydesmethyl-trimipramine (DTRIOH) were measured in 27 patients receiving between 300 and 400 mg/day racemic TRI. The patients were phenotyped with dextromethorphan and mephenytoin, and the 8-hour urinary ratios of dextromethorphan/dextrorphan, dextromethorphan/3-methoxymorphinan, and (S)-mephenytoin/(R)mephenytoin were used as markers of cytochrome P-450IID6 (CYP2D6), CYP3A4/5 and CYP2C19 activities, respectively. One patient was a CYP2D6 and one was a CYP2C19 poor metabolizer. A stereoselectivity in the metabolism of TRI has been found, with a preferential N-demethylation of (D)-TRI and a preferential hydroxylation of (L)-TRI. CYP2D6 appears to be involved in the 2-hydroxylation of (L)-TRI, (L)DTRI and (D)-DTRI, but not of (D)-TRI, as significant correlations were measured between the dextromethorphan/dextrorphan ratios and the (L)-TRI/(L)-TRIOH (r = 0.45, p = 0.019), the (L)-DTRI/(L)-DTRIOH (r = 0.47, p = 0.014), and the (D)-DTRI/(D)-DTRIOH (r = 0.51, p = 0.006), but not with the (D)-TRI/(D)-TRIOH ratios (r = 0.29, NS). CYP2C19, but not CYP2D6, appears to be involved in the demethylation pathway, with a stereoselectivity toward the (D)-enantiomer of TRI, as a significant positive correlation was calculated between the mephenytoin (S)/(R) ratios and the concentrations to dose-to-weight ratios of (D)-TRI (r = 0.69, p = 0.00006). CYP3A4/5 appears to be involved in the metabolism of (L)-TRI to a presently not determined metabolite. The CYP2D6 poor metabolizer had the highest (L)-DTRI and (D)-DTRI concentrations to dose-to-weight ratios, and the CYP2C19 poor metabolizer had the highest (L)-TRI and (D)-TRI concentrations to dose-to-weight ratios of the group.     

Polymorphism in diazepam metabolism in Wistar rats

We observed variations in the metabolism of diazepam in Wistar rats. We studied these variations carefully, and found that the variations are dimorphic and about 17% of male rats of Wistar strain we examined showed two times higher diazepam metabolic activities in their liver microsomes than the rest of animals at the substrate concentrations less than 5 microM. We classified them as extensive metabolizer (EM) and poor metabolizer (PM) of diazepam. No sex difference was observed in the frequency of appearance of EM. Activities of the primary metabolic pathways of diazepam were examined to elucidate the cause of this polymorphism in male Wistar rats. No significant differences were observed in activities of neither diazepam 3-hydroxylation or N-desmethylation between EM and PM rats, while activity of diazepam p-hydroxylation was markedly (more than 200 times) higher in EM rats, indicating that this reaction is responsible for the polymorphism of diazepam metabolism in Wistar rats. We examined the expression levels of CYP2D1, which was reported to catalyze diazepam p-hydroxylation in Wistar rats to find no differences in the expression levels of CYP2D1 between EM and PM rats. The kinetic study on diazepam metabolism in male Wistar rats revealed that EM rats had markedly higher V(max) and smaller K(m) in diazepam p-hydroxylation than those of PM rats, indicating the presence of high affinity high capacity p-hydroxylase enzyme in EM rats. As a consequence, at low concentrations of diazepam, major pathways of diazepam metabolism were p-hydroxylation and 3-hydroxylation in male EM rats, while in male PM rats, 3-hydroxylation followed by N-desmethylation. Due to this kinetic nature of p-hydroxylase activity, EM rats had markedly higher total CL(int) of diazepam than that of PM rats. Polymorphism in diazepam metabolism in humans is well documented, but this is the first report revealing the presence of the polymorphism in diazepam metabolism in rats. The current results infer polymorphic expression of new diazepam p-hydroxylating enzyme with lower K(m) than CYP2D1 in EM Wistar rats.     

Extension of a pilot study: impact from the cytochrome P450 2D6 polymorphism on outcome and costs associated with severe mental illness

The influence of cytochrome P450 2D6 (CYP2D6) genetic variability was examined in psychiatric inpatients by evaluating adverse drug events (ADEs), hospital stays, and total costs over a 1-year period in an extension of a previously published brief report. One hundred consecutive psychiatric patients from Eastern State Hospital in Lexington, Kentucky, were genotyped for CYP2D6 expression. ADEs were evaluated by a neurologic rating scale, modified Udvalg for Kliniske Undersogelser Side Effect Rating Scale, or chart review. Information on total hospitalization days and total costs were gathered for a 1-year period. Forty-five percent of the patients received medications that were primarily dependent on the CYP2D6 enzyme for their elimination. When the analysis was restricted to just those patients in each group receiving medication heavily dependent on the CYP2D6 enzyme, the following were observed: (1) a trend toward greater numbers of ADEs from medications as one moved from the group with ultrarapid CYP2D6 activity (UM) to the group with absent CYP2D6 activity (PM); (2) the cost of treating patients with extremes in CYP2D6 activity (UM and PM) was on average $4,000 to $6,000 per year greater than the cost of treating patients in the efficient metabolizer (EM) and intermediate metabolizer (IM) groups; and (3) total duration of hospital stay was more pronounced for those in CYP2D6 PM group. Variance of hospital stays and costs calculated from these preliminary data suggests that 1,500 to 2,000 patients must be evaluated over at least a 1-year period to determine whether the CYP2D6 genetic variation significantly alters the duration of hospital stay and costs.     

Cytochrome P450 dependent metabolism of the new designer drug 1-(3-trifluoromethylphenyl)piperazine (TFMPP). In vivo studies in Wistar and Dark Agouti rats as well as in vitro studies in human liver microsomes

1-(3-Trifluoromethylphenyl)piperazine (TFMPP) is a designer drug with serotonergic properties. Previous studies with male Wistar rats (WI) had shown, that TFMPP was metabolized mainly by aromatic hydroxylation. In the current study, it was examined whether this reaction may be catalyzed by cytochrome P450 (CYP)2D6 by comparing TFMPP vs. hydroxy TFMPP ratios in urine from female Dark Agouti rats, a model of the human CYP2D6 poor metabolizer phenotype (PM), male Dark Agouti rats, an intermediate model, and WI, a model of the human CYP2D6 extensive metabolizer phenotype. Furthermore, the human hepatic CYPs involved in TFMPP hydroxylation were identified using cDNA-expressed CYPs and human liver microsomes. Finally, TFMPP plasma levels in the above mentioned rats were compared. The urine studies suggested that TFMPP hydroxylation might be catalyzed by CYP2D6 in humans. Studies using human CYPs showed that CYP1A2, CYP2D6 and CYP3A4 catalyzed TFMPP hydroxylation, with CYP2D6 being the most important enzyme accounting for about 81% of the net intrinsic clearance, calculated using the relative activity factor approach. The hydroxylation was significantly inhibited by quinidine (77%) and metabolite formation in poor metabolizer genotype human liver microsomes was significantly lower (63%) compared to pooled human liver microsomes. Analysis of the plasma samples showed that female Dark Agouti rats exhibited significantly higher TFMPP plasma levels compared to those of male Dark Agouti rats and WI. Furthermore, pretreatment of WI with the CYP2D inhibitor quinine resulted in significantly higher TFMPP plasma levels. In conclusion, the presented data give hints for possible differences in pharmacokinetics in human PM and human CYP2D6 extensive metabolizer phenotype subjects relevant for risk assessment.     

CYP2D6 Genotyping as an alternative to phenotyping for determination of metabolic status in a clinical trial setting

The emerging application of pharmacogenomics in the clinical trial setting requires careful comparison with more traditional phenotyping methodologies, particularly in the drug metabolism area where phenotyping is used extensively. The research objectives of this study were 1) to assess the utility of cytochrome P450 2D6 (CYP2D6) genotyping as an alternative to traditional phenotyping as a predictor of poor metabolizer status; 2) to identify issues for consideration when implementing CYP2D6 genotyping in clinical trials; and 3) to outline the advantages and disadvantages of CYP2D6 genotyping compared with phenotyping. DNA samples obtained from 558 previously phenotyped individuals were blindly genotyped at the CYP2D6 locus, and the genotype-phenotype correlation was then determined. The CYP2D6 genotyping methodology successfully predicted all but 1 of the 46 poor metabolizer subjects, and it was determined that this 1 individual had a novel (presumably inactive) mutation within the coding region. In addition, we identified 2 subjects with CYP2D6 genotypes indicative of poor metabolizers who had extensive metabolizer phenotypes as determined by dextromethorphan/dextrorphan ratios. This finding suggests that traditional phenotyping methods do not always offer 100% specificity. Our results suggest that CYP2D6 genotyping is a valid alternative to traditional phenotyping in a clinical trial setting, and in some cases may be better. We also discuss some of the issues and considerations related to the use of genotyping in clinical trials and medical practice.