Rapid clinical induction of hepatic cytochrome P4502B6 activity by ritonavir

Ritonavir is the most potent and efficacious inhibitor of cytochrome P4503A (CYP3A), and it is used accordingly for the pharmacoenhancement of other antiretrovirals. Paradoxically, ritonavir induces the clinical metabolism and clearance of many drugs. The mechanism by which ritonavir inhibits and induces clinical drug metabolism is unknown. Ritonavir induces CYP2B6 in human hepatocytes. This investigation tested the hypothesis that ritonavir induces human CYP2B6 in vivo. Thirteen healthy human immunodeficiency virus-negative volunteers underwent a three-way sequential crossover protocol, receiving racemic bupropion after nothing (control), 3 days of treatment with ritonavir, and 2.5 weeks of treatment with ritonavir (400 mg twice a day). Stereoselective bupropion hydroxylation was used as an in vivo probe for CYP2B6 activity. Plasma and urine (R)- and (S)-bupropion and (R,R)- and (S,S)-hydroxybupropion concentrations were measured by liquid chromatography-mass spectrometry. Racemic, (R)-, and (S)-bupropion plasma ratios of the area under the concentration-time curve from 0 h to infinity (AUC(0-infinity)) (ritonavir/control) were significantly reduced to 0.84, 0.86, and 0.80, respectively, after 3 days of ritonavir treatment and to 0.67, 0.69, and 0.60 after steady-state ritonavir treatment. Apparent oral clearances for racemic, (R)-, and (S)-bupropion all were significantly increased by 1.2-fold after 3 days of ritonavir treatment and by 1.4-, 1.7-, and 1.5-fold after steady-state ritonavir treatment. The plasma (S,S)-hydroxybupropion/(S)-bupropion AUC(0-72) ratio was significantly increased by ritonavir. Formation clearances of both (R,R)- and (S,S)-hydroxybupropion were increased 1.8-fold after 3 days of ritonavir treatment and 2.1-fold after steady-state ritonavir treatment. These results show that ritonavir induces human CYP2B6 activity. Induction is rapid, occurring after only 3 days of ritonavir, and is sustained for at least 2 weeks. The ritonavir induction of CYP2B6 activity may have significant implications for drug interactions and clarify previously unexplained interactions.     

Duloxetine is both an inhibitor and a substrate of cytochrome P4502D6 in healthy volunteers

BACKGROUND AND OBJECTIVES: Duloxetine, a potent dual reuptake inhibitor of serotonin and norepinephrine currently undergoing clinical investigation for treatment of depression and stress urinary incontinence, has the potential to act as both a substrate and an inhibitor of cytochrome P4502D6 (CYP2D6). Our objectives were to determine the effect of duloxetine on the pharmacokinetics of desipramine, a tricyclic antidepressant metabolized by CYP2D6 (study 1), and the effect of paroxetine, a potent CYP2D6 inhibitor, on duloxetine pharmacokinetics (study 2). METHODS: Subjects were healthy men and women between 21 and 63 years old. All subjects were genotypically CYP2D6 extensive metabolizers. In study 1, 50 mg of desipramine was administered as a single dose alone and in the presence of steady-state duloxetine 60 mg twice daily. In study 2, steady-state pharmacokinetics of duloxetine 40 mg once daily were determined in the presence and absence of steady-state paroxetine 20 mg once daily. RESULTS: Duloxetine increased the maximum plasma concentration of desipramine 1.7-fold and the area under the concentration-time curve 2.9-fold. Paroxetine increased the maximum plasma concentration of duloxetine and the area under the concentration-time curve at steady state 1.6-fold. Reports of adverse events were similar whether duloxetine was administered alone or in combination with desipramine or paroxetine. CONCLUSION: Duloxetine 60 mg twice daily is a moderately potent CYP2D6 inhibitor, intermediate between paroxetine and sertraline. The potent CYP2D6 inhibitor paroxetine has a moderate effect on duloxetine concentrations. The results of these 2 studies suggest that caution should be used when CYP2D6 substrates and inhibitors are coadministered with duloxetine.     

Inhibition of cytochrome P4502D6 activity with paroxetine normalizes the ultrarapid metabolizer phenotype as measured by nortriptyline pharmacokinetics and the debrisoquin test.

BACKGROUND: The ultrarapid metabolizer phenotype of the cytochrome P4502D6 (CYP2D6) enzyme has been considered a relevant cause of nonresponse to antidepressant drug therapy. Prescribing high doses of antidepressants to such patients leads to high concentrations of potentially toxic metabolites and an increased risk for adverse reactions. Normalization of the metabolic status of ultrarapid metabolizers by inhibition of CYP2D6 activity could offer a clinically acceptable method to successfully treat such patients with antidepressants. METHODS: Five ultrarapid metabolizers with a CYP2D6 gene duplication or triplication were treated with 25 mg nortriptyline twice a day for 3 consecutive weeks, alone during the first week and concomitantly with the CYP2D6 inhibitor paroxetine 10 mg or 20 mg twice a day, respectively, during the second and third weeks. After the third week, nortriptyline was discontinued and the subjects were treated with paroxetine 20 mg twice a day during the fourth study week. At the end of each study week, the steady-state pharmacokinetic parameters of nortriptyline or paroxetine were determined within the dose interval. In addition, the CYP2D6 phenotype was determined by debrisoquin (INN, debrisoquine) test at baseline and at the end of each study phase. Treatment-related adverse events were recorded during drug administration and for 1 week thereafter. RESULTS: All 5 subjects had very low (subtherapeutic) nortriptyline concentrations after 7 days' treatment with nortriptyline only. Addition of paroxetine 10 mg twice a day to the nortriptyline regimen resulted in a change in all individuals to the "normal" extensive debrisoquine metabolizer phenotype, and therapeutic plasma nortriptyline concentrations were achieved in 4 of 5 subjects after a 3 times mean increase in nortriptyline trough concentration (P =.0011). Doubling the paroxetine dose caused a 15 times mean increase in paroxetine trough concentration (P <.001), indicating strong inhibition by paroxetine of its own metabolism. The high paroxetine concentrations in 2 subjects caused them to have the poor debrisoquine metabolizer phenotype and resulted in a further increase in plasma nortriptyline trough concentration (P =.0099). A strong correlation (rank correlation coefficient [r(s)] = 0.89; P <.0001) was observed between paroxetine and nortriptyline trough concentrations. Paroxetine also significantly decreased the fluctuation of nortriptyline concentrations within the dose interval. One subject discontinued the study after the second study week because of adverse effects; otherwise, the study drugs were well tolerated. CONCLUSIONS: Paroxetine, with a daily dosage from 20 to 40 mg, is an effective tool in normalizing the metabolic status of CYP2D6 ultrarapid metabolizers.     

Cytochrome P4502D6 (CYP2D6) gene locus heterogeneity: characterization of gene duplication events

Duplications and multiplications of active CYP2D6 genes can cause ultrarapid drug metabolism and lead to therapeutic failure. Multiple functional and non-functional duplication alleles have been further characterized. Duplications were detected by long-range polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism, and sequence analysis. A PCR fragment encompassing the entire duplicated gene was utilized for detailed characterization. Duplications occurred at 1.3, 5.75, and 2.0% in Caucasian, African American, and racially mixed populations, respectively (n=887 total). Of those 28, 47, and 17% were non-functional CYP2D6*4 x N. Twelve unique duplication alleles were detected: *1 x N, *2 x N, *4 x N, *6 x N, *10 x N, *17 x N, *17 x N[spacer], *29 x N, *35 x N, *43 x N, *45 x N, and a novel non-functional tandem arrangement of a chimeric 2D7/2D6 and *1 gene. All novel duplications except *35 x N were found in African Americans. Accurate identification of gene duplication events is essential to avoid false-positive ultrarapid metabolism assignments and thus, overestimation of predicted activity and increased risk for unwanted adverse events.     

Mechanism-based inactivation of human cytochrome P4502C8 by drugs in vitro

Studies were conducted to evaluate the potential mechanism-based inactivation of recombinant and human liver microsomal CYP2C8 by clinically used drugs. Several tricyclic antidepressants, calcium channel blockers, monoamine oxidase inhibitors, and various other known CYP3A4 inhibitors exhibited greater inhibition of CYP2C8 (paclitaxel 6alpha-hydroxylation) following preincubation, consistent with mechanism-based inactivation. Inactivation of recombinant CYP2C8 by phenelzine, amiodarone, verapamil, nortriptyline, fluoxetine, and isoniazid was of the pseudo-first order type and was characterized by respective inactivation kinetic constants (KI and kinact) of 1.2 microM and 0.243 min(-1), 1.5 microM and 0.079 min(-1), 17.5 microM and 0.065 min(-1), 49.9 microM and 0.036 min(-1), 294 microM and 0.083 min(-1), and 374 microM and 0.042 min(-1). Spectral scanning of recombinant CYP2C8 demonstrated the formation of metabolite-intermediate complexes with verapamil, nortriptyline, fluoxetine, and isoniazid, but not amiodarone. In contrast, inactivation by phenelzine resulted from heme destruction by free radicals. Studies with human liver microsomes (HLMs) revealed that nortriptyline, verapamil, and fluoxetine were not mechanism-based inactivators (MBIs) of CYP2C8. Simultaneous inactivation of CYP2C8 and CYP3A4 (paclitaxel 3'-phenyl-hydroxylation) was observed using amiodarone, isoniazid, and phenelzine with the efficiency of inactivation greater for the CYP3A4 pathway. With the exception of phenelzine, glutathione and superoxide dismutase failed to protect CYP2C8 (recombinant and HLMs) or CYP3A4 from inactivation by MBIs. However, the alternate CYP2C8 substrate, torsemide, prevented CYP2C8 inactivation in all cases. These data are consistent with mechanism-based inactivation of CYP2C8 by a range of commonly prescribed drugs, several of which have been implicated in clinically important drug-drug interactions.     

Effect of low-dose ritonavir (100 mg twice daily) on the activity of cytochrome P450 2D6 in healthy volunteers

OBJECTIVE: In the treatment of human immunodeficiency virus infection, the protease inhibitor ritonavir is used in a low dose (100 mg twice daily) to inhibit cytochrome P450 (CYP) 3A4 and thereby increase plasma concentrations of coadministered protease inhibitors. When applied in a therapeutic dose (600 mg twice daily), ritonavir also inhibits CYP2D6. The effect of low-dose ritonavir on CYP2D6 is unknown and was investigated in this study. METHODS: This was a 1-arm, 2-period, fixed-order study in 13 healthy male volunteers who were extensive metabolizers of CYP2D6. The first period examined baseline CYP2D6 activity by evaluating the pharmacokinetics of a single dose of desipramine and by metabolic phenotyping with dextromethorphan. During the second period, participants took ritonavir, 100 mg twice daily, for 2 weeks, followed by repeat assessment of desipramine pharmacokinetics and the dextromethorphan metabolic phenotype in the presence of ritonavir. RESULTS: Low-dose ritonavir (100 mg twice daily) significantly increased the exposure to single-dose desipramine, as reflected in a geometric mean ratio (with ritonavir/without ritonavir) of 1.26 (95% confidence interval, 1.13-1.40) for the desipramine area under the concentration versus time curve from time 0 to infinity (P < .001). Coadministration of low-dose ritonavir did not significantly affect the dextromethorphan/dextrorphan urinary metabolic ratio and did not convert any extensive metabolizer to a poor metabolizer. CONCLUSIONS: Low-dose ritonavir (100 mg twice daily) exerts a modest inhibitory effect on the activity of CYP2D6 in extensive metabolizers, as assessed with desipramine as the index substrate. This effect was not apparent with the dextromethorphan/dextrorphan metabolic ratio as an indicator for CYP2D6 activity. It is expected that the effect of low-dose ritonavir on CYP2D6 will not require standard dose reductions for CYP2D6 substrates.     

Dose dependency of dextromethorphan for cytochrome P450 2D6 (CYP2D6) phenotyping

Most dextromethorphan CYP2D6 phenotyping studies use a 30-mg dose, but data that show superiority of any particular dose are lacking. We compared metabolic ratios from six different dextromethorphan phenotyping doses to ascertain whether linearity existed over a dosage range. Forty subjects were enrolled in the study. Each subject received 0.05 mg/kg, 0.15 mg/kg, 0.3 mg/kg, 30 mg, 0.8 mg/kg, and 1.2 mg/kg dextromethorphan in a randomized crossover fashion. Urinary dextromethorphan to dextrorphan molar ratios were used to measure CYP2D6 activity. Single blood samples were obtained for CYP2D6 genotyping, which revealed one poor metabolizer and 39 extensive metabolizers. A statistical difference was found for the molar ratio between the 0.8 mg/kg and the 1.2 mg/kg dose compared with the other four doses. None of the 39 genotypic extensive metabolizers were incorrectly phenotyped with any of these doses. These data support the use of moderate doses of dextromethorphan for phenotyping to avoid dose dependency.     

基因芯片法测定中国人群细胞色素P4502D6的基因多态性

目的：根据细胞色素P4502D6基因序列的多态性分布特点，针对在中．国人群药物代谢中具有潜在作用的突变位点C188T和G4268C，设计寡核苷酸探针，制备寡核苷酸芯片，探讨基因芯片技术应用于CYP2D6基因分型的可行性，观察中国人群细胞色素P4502D6的基因多态性。 方法：实验于2005-02／08进行。以克隆并已测序的细胞色素P4502D6基因重组质粒作为标准样品，扩增产物与芯片进行杂交，制备基因芯片，测定312名中国健康志愿者（已签署知情同意书）细胞色素P4502D6基因分型，并使用直接测序法对结果进行验证。 结果：312名全部进入结果分析。①312名中国．健康志愿者中CYP2D6＊2和CYP2D6＊10的变异频率分别为CYP2D6＊2W＊10W 8．3％，＊2H＊10W13．5％．＊2M＊10W 10．6％．＊2M＊10H 17．0％，＊2M＊10M 34．6％，＊2H＊10H 9．6％，＊2H＊10m 6.4％。②对部分样本采用直接测序的方法进行进一步确证，结果完全吻合。 结论：①CYP2D6＊2和CYP2D6＊10突变型等位基因在中国人群中出现的频率较高。②基因芯片法简便、快捷，适用于中国人群细胞色素P4502D6基因分型研究。     

基因芯片法测定中国人群细胞色素P450 2D6的基因多态性

目的:根据细胞色素P4502D6基因序列的多态性分布特点,针对在中国人群药物代谢中具有潜在作用的突变位点C188T和G4268C,设计寡核苷酸探针,制备寡核苷酸芯片,探讨基因芯片技术应用于CYP2D6基因分型的可行性,观察中国人群细胞色素P4502D6的基因多态性。方法:实验于2005-02/08进行。以克隆并已测序的细胞色素P4502D6基因重组质粒作为标准样品,扩增产物与芯片进行杂交,制备基因芯片,测定312名中国健康志愿者(已签署知情同意书)细胞色素P4502D6基因分型,并使用直接测序法对结果进行验证。结果:312名全部进入结果分析。①312名中国健康志愿者中CYP2D62和CYP2D610的变异频率分别为CYP2D62W10W8.3%,2H10W13.5%,2M10W10.6%,2M10H17.0%,2M10M34.6%,2H10H9.6%,2H10M6.4%。②对部分样本采用直接测序的方法进行进一步确证,结果完全吻合。结论:①CYP2D62和CYP2D610突变型等位基因在中国人群中出现的频率较高。②基因芯片法简便、快捷,适用于中国人群细胞色素P4502D6基因分型研究。     

The antipsychotic clozapine is metabolized by the polymorphic human microsomal and recombinant cytochrome P450 2D6.

A large interindividual variability for clozapine bioavailability and plasma steady-state concentrations and clearance exists. The enzymatic system which is involved in clozapine metabolism has not been fully characterized, yet structurally related tricyclic drugs have been found to be metabolized by cytochrome P450 2D6 (CYP2D6), which is polymorphically expressed in humans. The involvement of CYP2D6 in clozapine and fluperlapine metabolism was studied with human liver microsomes and in recombinant RT2D6 7-8 (RT2D6) cells, which specifically express human CYP2D6. Clozapine and its structural analog fluperlapine both bind to the active site of CYP2D6, as demonstrated by the competitive inhibition of dextromethorphan metabolism at inhibitor concentrations up to 40 microM. The inhibition constants (Ki) for both clozapine and fluperlapine were about 4 microM in microsomes from human liver. Clozapine exhibited a higher inhibition constant of 18.7 microM in microsomes from RT2D6 cells, but the difference was not statistically significant (P less than .05). These concentrations are close to the plasma concentrations of 0.3 to 3 microM achieved during clozapine therapy. Both clozapine and fluperlapine are also metabolized by CYP2D6. RT2D6 cells produced a number of metabolites from clozapine, whereas only a single metabolite was obtained from fluperlapine. The clozapine metabolites were not identified; however, they were different from N-oxide and N-demethyl clozapine. The fluperlapine metabolite was found to be the 7-hydroxy fluperlapine, which is also a major metabolite in vivo. In conclusion, both drug-drug interactions on the binding site of CYP2D6 and polymorphic metabolism of clozapine by CYP2D6 could contribute to the observed variability in clozapine kinetics in humans.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of cytochrome P4502C19 genotypic differences on cure rates for gastroesophageal reflux disease by lansoprazole

BACKGROUND AND OBJECTIVES: The acid-inhibitory effect of lansoprazole depends on differences in cytochrome P450 (CYP) 2C19 genotypes. We assessed whether therapeutic effects of lansoprazole on gastroesophageal reflux disease (GERD) depended on the CYP2C19 genotype status in relation to the grade of GERD. METHODS: A total of 65 patients with GERD (grades A-D) completed treatment with lansoprazole, by taking 30 mg orally once a day for 8 weeks. The CYP2C19 genotype status of patients was determined by polymerase chain reaction-restriction fragment length polymorphism analysis. Before and after treatment, esophageal endoscopy was performed. GERD was considered to be cured on the basis of endoscopic findings at the end of treatment. Plasma lansoprazole levels could be determined at 3 hours after the last 30-mg dose of lansoprazole in the 27 genotyped patients. RESULTS: Cure rates for GERD depended significantly on the CYP2C19 genotype status, as well as the grade of GERD before treatment. Cure rates in the homozygous extensive, heterozygous extensive, and poor metabolizer groups were 45.8%, 67.9%, and 84.6%, respectively. Cure rates in the groups with GERD grade A, grade B, and grade C or D were 85.0%, 60.0%, and 45.0%, respectively. The cure rate in patients with the homozygous extensive metabolizer genotype of CYP2C19 with a GERD grade of C or D was very low (16.7%). Plasma lansoprazole levels in patients with the homozygous extensive metabolizer genotype were the lowest of the 3 groups. CONCLUSIONS: CYP2C19 genotype status, as well as the grade of GERD before treatment, is one of the determinants for the success or failure of treatment of GERD with lansoprazole. The low cure rate in patients with the homozygous extensive metabolizer genotype appeared to be a result of these patients having the lowest plasma lansoprazole levels among the 3 genotype groups.     

A longitudinal study of cytochrome P450 2D6 (CYP2D6) activity during adolescence

CYP2D6 substrates are among the most highly prescribed medications in teenagers and also commonly associated with serious adverse events. To investigate the relative contributions of genetic variation, growth, and development on CYP2D6 activity during puberty, healthy children and adolescents 7–15 years of age at enrollment participated in a longitudinal phenotyping study involving administration of 0.3 mg/kg dextromethorphan (DM) and 4‐h urine collection every 6 months for 3 years (7 total visits). At each visit, height, weight, and sexual maturity were recorded, and CYP2D6 activity was determined as the urinary molar ratio of DM to its metabolite dextrorphan (DX). A total of 188 participants completed at least one visit, and 102 completed all seven study visits. Following univariate analysis, only CYP2D6 activity score (p < 0.001), urinary pH (p < 0.001), weight (p = 0.018), and attention‐deficit/hyperactivity disorder (ADHD) diagnosis (p < 0.001) were significantly correlated with log(DM/DX). Results of linear mixed model analysis with random intercept, random slope covariance structure revealed that CYP2D6 activity score had the strongest effect on log(DM/DX), with model‐estimated average log(DM/DX) being 3.8 SDs higher for poor metabolizers than for patients with activity score 3. A moderate effect on log(DM/DX) was observed for sex, and smaller effects were observed for ADHD diagnosis and urinary pH. The log(DM/DX) did not change meaningfully with age or pubertal development. CYP2D6 genotype remains the single, largest determinant of variability in CYP2D6 activity during puberty. Incorporation of genotype‐based dosing guidelines should be considered for CYP2D6 substrates given the prevalent use of these agents in this pediatric age group.

Study Highlights

• WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

Several CYP2D6 substrates are used in the management of neurodevelopmental, behavioral, and psychiatric disorders in children and adolescents, often off‐label, for indications that often differ from the adult conditions for which the medications originally were developed. Therefore, little information is available to guide dosing in pediatric patients, and even less regarding the influence of growth and development on the processes influencing drug disposition and response.

• WHAT QUESTION DID THIS STUDY ADDRESS?

What is the relative contribution of genetic variation and the processes of growth and development on variability in CYP2D6 activity during puberty?

• WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

CYP2D6 genotype remains the single, largest biological determinant of variability of CYP2D6 activity during puberty. Effects of factors associated with growth and sexual maturation are small and have limited ability to explain variability in CYP2D6 activity.

• HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

Incorporation of genotype‐based dosing guidelines should be considered for CYP2D6 substrates, especially given the prevalent use of antipsychotic and antidepressant medications in children and adolescents. A challenge for the future remains to build on, and extend the accumulating database describing the relative contribution of ontogeny and genetic variation to observed variability in drug disposition and response across the continuum from birth to adulthood.     

Rapid detection of common cytochrome P450 2D6 alleles in Caucasians

CYP2D6 is a highly polymorphic enzyme that mediates the metabolism of around 20% of all currently prescribed drugs. Genetic variability within CYP2D6 results in poor (PM), intermediate (IM), extensive (EM) and ultra-rapid metabolisers (UM) of CYP2D6 substrates. Here we describe an assay which is able to detect the major PM (CYP2D6*3, *4, *5, *6), IM (CYP2D6*9, *10, *41) and UM (CYP2D6*nxn) alleles found in Caucasians. This assay is performed in two stages. The first stage is a multiplex long-range PCR which is used to simultaneously screen for whole gene deletions and duplications while isolating CYP2D6 from the CYP2D gene cluster to avoid pseudogene contamination. In the second stage, individuals with one or more copies of CYP2D6 are genotyped for PM and IM alleles using a two-tube multiplex Amplification Refractory Mutation System (ARMS). The specificity and reliability of the multiplex long-range PCR and subsequent ARMS were confirmed using a panel of positive controls that had been previously validated by PCR-RFLPs and DNA sequencing. This two-stage assay offers a robust and cheap alternative to many currently available CYP2D6 genotyping approaches. Our entire assay, once patient DNA has been extracted, can be run within 7 h using 10 microl PCRs.