Cytochrome P450 2D6 Isozyme Research Articles

Nanohybrids of quantum dots and cytochrome P450 for light-driven drug metabolism

In this work, nanohybrids of CdTe quantum dots (QDs) and cytochrome P450 2D6 (CYP2D6) prepared by covalent binding of CYP2D6 to QDs were used as photocatalysts for drug metabolism. As a proof-of-concept of the light-driven drug metabolism, tramadol was chosen as the substrate. As control, the high performance liquid chromatography coupled with mass spectrometry (HPLC–MS) was used to confirm the production of metabolites. Under irradiation with a white-light, the catalytic cycle of P450-mediated oxygenation reactions occurred with the formation of radical species on QDs, which immediately activated the proximity of the bound P450 enzyme and transferred electrons from the conduction band (CB) of CdTe QDs to CYP2D6. This light-driven catalytic reaction via nanohybrids was comparable to the native biological metabolism by CYP2D6 isozyme microsomes with CYP reductase (CPR) using the enzyme cofactor nicotinamide-adenine dinucleotide phosphate (NADPH) as electron donor. Furthermore, a photoelectrochemical platform was designed for in-situ monitoring the metabolic reactions. Upon addition of tramadol, the photocurrent increased due to the electron transfer from the CB of CdTe QDs to CYP2D6. The apparent Michaelis–Menten constant was thus easily measured to be 1.35μM. This enzyme-based photoelectrochemical platform showed accepted stability and great potential for studies on drug metabolism in vitro.

Unsuspected poor metabolizer phenotypes of fluoxetine in bioavailability/bioequivalence studies from an Indian population perspective

Fluoxetine, belonging to the class of selective serotonin uptake inhibitors, has been extensively used for the treatment of depression and other psychiatry related disorders. Fluoxetine (CAS 59333-67-4) is a substrate of polymorphic cytochrome P450 2D6 isozyme (CYP2D6) leading to the formation of norfluoxetine (CAS 83891-03-06), which is not only active but long lived than the parent in the systemic circulation. Since the parent and metabolite levels are important from a therapeutic perspective, knowledge of phenotypic distribution of the population may be an important consideration. The aim of the work was to retrospectively evaluate the pharmacokinetic data of fluoxetine and norfluoxetine from several bioavailability/bioequivalence (BA/BE) studies to identify the poor metabolizer (PM) phenotypes from the unsuspected healthy subjects across varied protocol designs, dose sizes and differing formulations. The pharmacokinetic data of fluoxetine and norfluoxetine were gathered from several BA/BE studies conducted at clinical facilities located at Bangalore and Chennai, India. The BA/BE studies involved open label, two-way randomized crossover designs with two periods and two treatments (reference and test). Blood samples were collected for at least 672 h after fluoxetine dosing and the plasma was analyzed using validated tandem liquid chromatography mass spectrometric assay to determine fluoxetine and norfluoxetine levels. Standard pharmacokinetic parameters were computed using noncompartmental methods. For the purpose of this paper, retrospective evaluation of pharmnacokinetic data from only the reference formulation was considered. The AUC ratio of fluoxetine/norfluoxetine was computed. The individual fluoxetine/norfluoxetine AUC(0-infinity), ratios were plotted in increasing rank order and using outlier test ('T' procedure at 5% level of significance) the subjects were categorized as extensive metabolizer (EM) and PM phenotypes. The unequivocal confirmation of PM phenotypes was obtained by performing linear regression analysis of fluoxetine vs norfluoxetine AUC(0-infinity) values. Each study was evaluated for the distribution of EM and PM phenotypes of fluoxetine. There were 144 subjects evaluable from four studies, 89.6% (129 out of 144) of which could be categorised as EMs and 10.4% (15 out of 144) as PMs of fluoxetine. The pharmacokinetic parameters were quite distinct between the two phenotypes: (1) PM phenotypes showed much higher exposure (approximately 2.3-fold increase in AUC(0-infinity) and much slower elimination (almost 2-fold increase in elimination half-life) for fluoxetine as compared to EM phenotypes; (2) PM phenotypes showed approximately 0.5-fold lower exposure of norfluoxetine as compared to the EM counter parts; (3) There was no change (approximately 1.2-fold) in the elimination half life of norfluoxetine in EM and PM phenotypes. Retrospective evaluation of fluoxetine and norfluoxetine pharmacokinetic data demonstrated existence of both PM and EM phenotypes in the Indian population. Based on the overall data (n=144 subjects) there appeared to be 10.4% of PM phenotypes for fluoxetine and/or possibly for other polymorphic CYP2D6 substrates commonly used in this region.

First-in-Human Assays: Excitement and Introspection

First-in-Human Assays: Excitement and Introspection

Development and validation of a chemical hydrolysis method for dextromethorphan and dextrophan determination in urine samples: Application to the assessment of CYP2D6 activity in fibromyalgia patients

Dextromethorphan (DEM) is a widely used probe drug for human cytochrome P450 2D6 isozyme activity assessment by measuring the ratio between DEM and its N-demethylated metabolite dextrorphan (DOR). DOR is excreted in urine mainly conjugated to glucuronic acid. Prior to quantification, DOR must be deconjugated to avoid variability caused by the polymorphic glucuronosyltransferase enzyme. A chemical hydrolysis method was optimized using a chemometric approach. Three factors (acid concentration, hydrolysis time and temperature) were selected and simultaneously varied to study their effect on conjugated DOR hydrolysis. Hydrolysis conditions that maximize DOR release without significant degradation of both DEM and DOR were chosen and results were compared to those obtained by enzymatic method using β-glucuronidase. An HPLC method with fluorescence detection was developed for the simultaneous quantitation of DEM, DOR and levallorphan, used as an internal standard. Separation was performed on a phenyl analytical column (150 mm × 4.6 mm i.d., 5 μm) with a mobile phase consisting of 18% acetonitrile and 50 mM phosphoric acid (pH 3). The overall analytical procedure was validated and showed good performances in terms of selectivity, linearity, sensitivity, precision and accuracy. Finally, this assay was used to determine DEM/DOR molar ratios in fibromyalgia patients for the purpose of determining phenotype status for the CYP2D6.

Drug disposition of chiral and achiral drug substrates metabolized by cytochrome P450 2D6 isozyme: case studies, analytical perspectives and developmental implications

The concepts of drug development have evolved over the last few decades. Although number of novel chemical entitities belonging to varied classes have made it to the market, the process of drug development is challenging, intertwined as it is with complexities and uncertainities. The intention of this article is to provide a comprehensive review of novel chemical entities (NCEs) that are substrates to cytochrome P450 (CYP) 2D6 isozyme. Topics covered in this review aim: (1) to provide a framework of the importance of CYP2D6 isozyme in the biotransformation of NCEs as stand-alones and/or in conjunction with other CYP isozymes; (2) to provide several case studies of drug disposition of important drug substrates, (3) to cover key analytical perspectives and key assay considerations to assess the role and involvement of CYP2D6, and (4) to elaborate some important considerations from the development point of view. Additionally, wherever applicable, special emphasis is provided on chiral drug substrates in the various subsections of the review.

Influence of CYP2D6 Genetics on Opioid Kinetics, Metabolism and Response

Pharmacogenetics does seem to play a key role in the use of so-called weak opioids. It has been shown for codeine, dihydrocodeine, oxycodone and hydrocodone, that their O-demethylation in the 3-position results in metabolites which have much stronger μ-receptor binding. These opioids may therefore exert their pharmacological actions predominantly through their O-demethylated metabolites. However, this metabolic step is under genetic control of the polymorphic cytochrome P450 2D6 isozyme (CYP2D6). Poor metabolisers of CYP2D6 (∼10% of the Caucasian population) do not express this enzyme and hence can only form trace amounts of the O-demethylated metabolites of these four opioids. This might put these persons on risk of reduced or even abolished analgesic effects when given these weak opioids. From this point of view there are two major issues why weak opioids cannot wholeheartedly be recommended: large interindividual variability of the analgesic effect due to CYP2D6 polymorphism and 10% of patients with no benefit from these drugs. On the other hand it might be advantageous to use the O-demethylated metabolites morphine, oxymorphone and hydromorphone which are all strong opioids and have a smaller interindividual variability of the opioid effects. Instead of using weak opioids, small doses and controlled release formulations of strong opioids might be the future way to in analgesic therapy despite the fear of addiction and bureaucratic efforts involved with these compounds.

Ketone reductase activity and reduced haloperidol/haloperidol ratios in haloperidol-treated schizophrenic patients

Twenty schizophrenic patients were treated with a fixed haloperidol (HL) dose of 20 mg/day for 4 weeks. The conversion of HL to its reduced metabolite (reduced haloperidol, RH) occurs via the ketone reductase enzyme. RH is also converted back to HL by the cytochrome P450 2D6 isozyme. Ketone reductase activity can be measured in red blood cells. Plasma HL and RH levels were assayed by high performance liquid chromatography. Blood samples were obtained at baseline and during weeks 2 and 4 of HL therapy. Seventeen of 20 patients had ketone reductase values < 3. A significant correlation between ketone reductase activity and RH/HL plasma ratios was observed at week 4 in these 17 patients. Patients with ketone reductase activity |>3 could represent a subgroup of patients that metabolize HL differently. The wide interpatient variability observed with HL and RH plasma levels in HL-treated patients could reflect differences in ketone reductase activity and the metabolic status of debrisoquin hydroxylase (cytochrome P450IID6) in psychiatric patients.

Primary and secondary oxidative metabolism of dextromethorphan: In vitro studies with female sprague-dawley and dark agouti rat liver microsomes

The O-demethylation of dextromethorphan (DM) to dextrorphan (DR) is catalysed by the polymorphic CYP2D6 (cytochrome P4502D6) isozyme in man. DM is commonly used as a probe for phenotyping subjects as either poor or extensive metabolizers for the debrisoquine/sparteine oxidative polymorphism via CYP2D6. The enzyme kinetics of DM O- and N-demethylation, and the N- and O-demethylations of the primary metabolites DR and 3-methoxymorphinan (3MM), respectively, were studied in liver microsomes from female Dark Agouti (DA) rats, the poor metabolizer counterpart, and female Sprague-Dawley (SD) rats, the extensive metabolizer counterpart. The formation of metabolites was quantified by HPLC with fluorescence detection and kinetic parameters were calculated. The intrinsic clearance ( V max/ K m ) of the O-demethylation of 3MM to 3-hydroxymorphinan (3OHM) was 180-fold lower in DA rats (0.11 vs 20.77 mL/hr/mg) due to a 60-fold higher K m (108.7 vs 1.76 μM) and 3-fold lower V max (11.5 vs 35.95 nmol/mg/hr). The kinetics for DR N-demethylation to 30HM did not differ between rat strains. The Michaelis-Menten constant ( K m ) for DM N-demethylation to 3MM was similar between SD and DA rats (85.04 vs 68.99 μM); however, SD rats displayed a 2-fold higher V max (83.37 vs 35.49 nmol/mg/hr) and intrinsic clearance (0.96 vs 0.51 mL/hr/mg). The O-demethylation of DM to DR in SD rats showed a high and low affinity enzyme component, with the high affinity intrinsic clearance contributing 98% of the total intrinsic clearance in these rats. DM O-demethylation in DA rats was characterized by a single enzyme system. The high affinity O-demethylating enzyme in SD rats showed a 20-fold lower K m (2.5 vs 55.6 μM) and a three-fold higher V max (51.04 vs 16.84 nmol/ mg/ hr) resulting in a 66-fold higher intrinsic clearance (20.04 vs 0.31 mL/hr/mg) compared to DA rats. Quinine; dextropropoxyphene, (±)methadone and (±)propafenone were shown to be potent inhibitors of 3MM and DM O-emethylation but did not inhibit DR or DM N-demethylation at similar concentrations. SD and DA rats showed a clear strain difference in 3MM O-demethylation and DM O-demethylation. In contrast, DR N-demethylation and DM N-demethylation do not appear to be under genetic control in the female SD-DA rat model. Kinetic parameters and inhibition studies suggest that 3MM and DM O-demethylation pathways in the rat may be mediated by the same cytochrome P450 isozyme.

Ethylmorphine O-deethylation cosegregates with the debrisoquin genetic metabolic polymorphism.

The single oral dose kinetics of ethylmorphine and its fractional metabolic clearance by O-dealkylation and N-dealkylation was investigated in five extensive and four poor metabolizers of dextromethorphan. In addition, the urinary metabolic ratios for these pathways (MRO and MRN, respectively) were investigated in a larger group of 27 extensive metabolizers and six poor metabolizers. The mean values for the fractional metabolic clearance by O-dealkylation and the MRO differed significantly between the poor metabolizers and extensive metabolizers without overlap between the values of either of these parameters in the two groups of subjects. In contrast, the corresponding parameters for the N-demethylation did not differ between poor metabolizers and extensive metabolizers. The area under the plasma concentration versus time curve was significantly higher (about three times higher) in the poor metabolizers compared with the extensive metabolizers (p = 0.004). Our data suggest that ethylmorphine is O-deethylated by the cytochrome P4502D6 isozyme inasmuch as both the fractional metabolic clearance by O-dealkylation and the MRO were found to cosegregate with the phenotype for the O-demethylation of dextromethorphan in our group of subjects.