Human Cytochrome P450 2D6 Research Articles

Ligand- and Protein-Based Modeling Studies of the Inhibitors of Human Cytochrome P450 2D6 and a Virtual Screening for Potential Inhibitors from the Chinese Herbal Medicine, Scutellaria baicalensis (Huangqin,Baikal Skullcap)

We have previously examined the binding patterns of various substrates to human cytochrome P450 2D6 (CYP2D6) using a series of molecular modeling methods. In this study, we further explored the binding modes of various types of inhibitors to CYP2D6 using a combination of ligand- and protein-based modeling approaches. Firstly, we developed and validated a pharmacophore model for CYP2D6 inhibitors, which consisted of two hydrophobic features and one hydrogen bond acceptor feature. Secondly, we constructed and validated a quantitative structure-activity relationship (QSAR) model for CYP2D6 inhibitors which gave a poor to moderate prediction accuracy. Thirdly, a panel of CYP2D6 inhibitors were subject to molecular docking into the active site of wild-type and mutated CYP2D6 enzyme. We demonstrated that 8 residues in the active site (Leu213, Glu216, Ser217, Gln244, Asp301, Ser304, Ala305, and Phe483) played an important role in the binding to the inhibitors via hydrogen bond formation and/or π-π stacking interaction. Apparent changes in the binding modes of the inhibitors have been observed with Phe120Ile, Glu216Asp, Asp301Glu mutations in CYP2D6. Finally, we screened for potential binders/inhibitors from the Chinese herbal medicine Scutellaria baicalensis (Huangqin, Baikal Skullcap) using the established pharmacophore model for CYP2D6 inhibitors and molecular docking approach. Overall, 18 out of 40 compounds from S. baicalensis were mapped to the pharmacophore model of CYP2D6 inhibitors and most herbal compounds from S. baicalensis could be docked into the active site of CYP2D6. Our study has provided insights into the molecular mechanisms of interaction of synthetic and herbal compounds with human CYP2D6 and further benchmarking studies are needed to validate our modeling and virtual screening results.

Heterologous Expression of Human Cytochromes P450 2D6 and CYP3A4 in Escherichia coli and Their Functional Characterization

This study aimed to express two major drug-metabolizing human hepatic cytochromes P450 (CYPs), CYP2D6 and CYP3A4, together with NADPH-cytochrome P450 oxidoreductase (OxR) in Escherichia coli and to evaluate their catalytic activities. Full length cDNA clones of both isoforms in which the N-terminus was modified to incorporate bovine CYP17α sequence were inserted into a pCWori(+) vector. The modified CYP cDNAs were subsequently expressed individually, each together with OxR by means of separate, compatible plasmids with different antibiotic selection markers. The expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. Enzyme activities were examined using high performance liquid chromatography (HPLC) assays with probe substrates dextromethorphan and testosterone for CYP2D6 and CYP3A4, respectively. Results from immunoblotting demonstrated the presence of both CYP proteins in bacterial membranes and reduced CO difference spectra of the cell preparations exhibited the characteristic absorbance peak at 450nm. Co-expressed OxR also demonstrated an activity level comparable to literature values. Kinetic parameters, K(m) and V(max) values determined from the HPLC assays also agreed well with literature values. As a conclusion, the procedures described in this study provide a relatively convenient and reliable means of producing catalytically active CYP isoforms suitable for drug metabolism and interaction studies.

Modeling Human Cytochrome P450 2D6 Metabolism and Drug-Drug Interaction by a Novel Panel of Knockout and Humanized Mouse Lines

The highly polymorphic human cytochrome P450 2D6 enzyme is involved in the metabolism of up to 25% of all marketed drugs and accounts for significant individual differences in response to CYP2D6 substrates. Because of the differences in the multiplicity and substrate specificity of CYP2D family members among species, it is difficult to predict pathways of human CYP2D6-dependent drug metabolism on the basis of animal studies. To create animal models that reflect the human situation more closely and that allow an in vivo assessment of the consequences of differential CYP2D6 drug metabolism, we have developed a novel straightforward approach to delete the entire murine Cyp2d gene cluster and replace it with allelic variants of human CYP2D6. By using this approach, we have generated mouse lines expressing the two frequent human protein isoforms CYP2D6.1 and CYP2D6.2 and an as yet undescribed variant of this enzyme, as well as a Cyp2d cluster knockout mouse. We demonstrate that the various transgenic mouse lines cover a wide spectrum of different human CYP2D6 metabolizer phenotypes. The novel humanization strategy described here provides a robust approach for the expression of different CYP2D6 allelic variants in transgenic mice and thus can help to evaluate potential CYP2D6-dependent interindividual differences in drug response in the context of personalized medicine.

Comparison of Cytochrome P450 2D6 and Variants in Terms of Drug Oxidation Rates and Substrate Inhibition

This review focuses on identification of the important active site residues of CYP2D6 in terms of CYP2D6 polymorphism. A meta-analysis was performed on the reported literature regarding (1) values of the Michaelis-Menten constant (K(m)), maximal velocity (V(max)), and intrinsic clearance (V(max)/K(m)) for 41 metabolic reactions of 31 substrates mediated by human cytochrome P450 2D6 and its variants and mutants and (2) inhibition constants (K(i)) for 15 inhibitors. The mean ratios of V(max)/K(m) values with respect to the wild type (CYP2D6.1) for CYP2D6.2 (R296C/S486T), CYP2D6.10 (P34S/S486T), CYP2D6.17 (T107I/R296C/S486T), CYP2D6.31 (R296C/R440H/S486T), CYP2D6.34 (R296C), CYP2D6.36 (P34S/S486T and 6 other amino acids substitutions), CYP2D6.49 (P34S /F120I/S486T), and P34S and G42R mutants but not CYP2D6.39 (S486T) were in the range 0.03-0.61, and the median ratios were in the range 0.03-0.57. More than 90% of V(max)/K(m) values for CYP2D6.10, CYP2D6.17, and CYP2D6.36 were less than half of those for CYP2D6.1. In addition, 20-59% of V(max)/K(m) values for these variants were less than one-tenth those of the wild type. These results suggest that the CYP2D6 polymorphism may affect the metabolic activities of many compounds. However, the kinetic behaviors of these variants and mutants depended on the metabolic reaction. The K(i) values of many of the inhibitors of CYP2D6.10 and CYP2D6.17 were comparable with or higher than those for CYP2D6.1. Collectively, these findings provide insights into the contributions of CYP2D6 polymorphisms to drug metabolism and adverse drug interactions.

Abstract 2283: Endoxifen exhibits potent in vitro and in vivo antitumor activity in ER+/HER2+ breast cancer and tamoxifen refractory tumors

Abstract Background: In estrogen receptor positive (ER+) breast cancer human epidermal growth factor receptor 2 (HER2) expression is associated with resistance to tamoxifen (Tam). Endoxifen is the most abundant active metabolite of Tam, resulting in part from cytochrome P450 2D6 (CYP2D6) metabolism. While Tam has been extensively used to model proliferative ER/HER2 cross-talk, there are no published data regarding endoxifen's effect on ER/HER2 nongenomic nor ER genomic signaling. Methods: Using ER+ cells (MCF7) and ER+/HER2+ cells (MCF7-HER2, BT474) we compared the effects of Tam and endoxifen on proliferation using crystal violet assays, Erk1/2 phosphorylation by western blotting, and E2-stimulated ER-regulated gene expression of amphiregulin (AREG), c-Myc (MYC) and progesterone receptor (PGR) by real-time RT-PCR assays. To examine the anti-proliferative affects of endoxifen compared to Tam in vivo we performed E2-stimulated MCF7-HER2 xenograft studies in athymic nude mice. Endoxifen doses of 25 (low) and 75 mg/kg (high) were used based on our prior pharmacokinetic studies showing achievable steady state levels of 100 nM and 1 uM, respectively. Z-Endoxifen HCl was synthesized by NCI. Results: Endoxifen, unlike Tam, potently inhibits growth in both ER+ and ER+/HER2+ breast cancer cell lines at concentrations observed in human CYP2D6 intermediate and extensive metabolizers (>40 nM). Tam (10-1000 nM) stimulates phosphorylation of Erk1/2 in stark contrast to endoxifen at similar doses. Endoxifen (100-1000 nM) significantly decreases E2-stimulated transcription of ER-regulated genes in MCF7-HER2 cells; AREG 2-3 fold, MYC 1-2 fold, and PGR 2-4 fold. Conversely, Tam (10-1000 nM) maintains or stimulates transcription; AREG 2-3 fold, MYC 0.5-2.5 fold, and PGR 0-3 fold. After 7 weeks of treatment of MCF7-HER2 xenografts, both high (p=0.001) and low dose endoxifen (p=0.01) resulted in greater antitumor activity than Tam, with high dose superior to low dose endoxifen (p=0.024). MCF7-HER2 xenografts that progressed on Tam to 200% growth at 7 weeks were randomized to either continued Tam or endoxifen. Four weeks after randomization endoxifen was superior to Tam (p<0.0001) with tumor size decreased by ≥ 20%. No significant toxicity was observed.Conclusions: We have demonstrated in both ER+ and ER+/HER2+ breast cancer cell lines that in contrast to Tam, endoxifen potently inhibits growth (both in vitro and in vivo), does not activate Erk1/2 signaling, and inhibits E2-stimulated transcription of ER-regulated genes. Endoxifen exhibited potent antitumor activity in MCF7-HER2 xenografts refractory to Tam. These data support the ongoing NCI sponsored phase I studies of Z-endoxifen at Mayo Clinic and NCI. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2283. doi:10.1158/1538-7445.AM2011-2283

Inhibition of Human Cytochrome P450 Enzymes 3A4 and 2D6 by β‐Carboline Alkaloids, Harmine Derivatives

β-Carboline alkaloids are the main chemical constituents of the plant Peganum harmala, while they also could be formed endogenously and found in coffee, alcoholic beverages and tobacco. Considering the fact that the possibility of herb-drug interactions has recently received great attention worldwide, the aim of the current study was to assess the potential for the metabolism-based drug-drug interactions arising from five β-carboline alkaloids (harmine, harmaline, harmalol, harmol and harmane) from P. harmala in vitro. With microsome incubation assays and UPLC/HPLC methods, the inhibitions on human liver CYP3A4 and CYP2D6 enzymes by those β-carboline alkaloids were studied kinetically. Harmine, harmol and harmane exhibited noncompetitive inhibition on the activity of CYP3A4 with K(i) values of 16.76, 5.13 and 1.66 μM, respectively. These β-carboline alkaloids were also found to be both substrates and inhibitors for CYP2D6. Harmaline, harmine and harmol showed typical competitive inhibition on the activity of CYP2D6 with K(i) values of 20.69, 36.48 and 47.11 μM, respectively. The inhibition of the two major CYP enzymes by those β-carboline alkaloids suggested that changes in the pharmacokinetics of co-administered drugs were likely to have occurred. Therefore, caution should be exercised for possible drug interactions of medicinal plants containing those β-carboline alkaloids and CYP substrates.

Substituted Imidazole of 5-Fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine Inactivates Cytochrome P450 2D6 by Protein Adduction

5-Fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine (SCH 66712) is a potent mechanism-based inactivator of human cytochrome P450 2D6 that displays type I binding spectra with a K(s) of 0.39 ± 0.10 μM. The partition ratio is ~3, indicating potent inactivation that addition of exogenous nucleophiles does not prevent. Within 15 min of incubation with SCH 66712 and NADPH, ∼90% of CYP2D6 activity is lost with only ~20% loss in ability to bind CO and ~25% loss of native heme over the same time. The stoichiometry of binding to the protein was 1.2:1. SDS-polyacrylamide gel electrophoresis with Western blotting and autoradiography analyses of CYP2D6 after incubations with radiolabeled SCH 66712 further support the presence of a protein adduct. Metabolites of SCH 66712 detected by mass spectrometry indicate that the phenyl group on the imidazole ring of SCH 66712 is one site of oxidation by CYP2D6 and could lead to methylene quinone formation. Three other metabolites were also observed. For understanding the metabolic pathway that leads to CYP2D6 inactivation, metabolism studies with CYP2C9 and CYP2C19 were performed because neither of these enzymes is significantly inhibited by SCH 66712. The metabolites formed by CYP2C9 and CYP2C19 are the same as those seen with CYP2D6, although in different abundance. Modeling studies with CYP2D6 revealed potential roles of various active site residues in the oxidation of SCH 66712 and inactivation of CYP2D6 and showed that the phenyl group of SCH 66712 is positioned at 2.2 Å from the heme iron.

Evolution of the CYP2D gene cluster in humans and four non-human primates

The human cytochrome P450 2D6 (CYP2D6) is a primary enzyme involved in the metabolism of about 25% of commonly used therapeutic drugs. CYP2D6 belongs to the CYP2D subfamily, a gene cluster located on chromosome 22, which comprises the CYP2D6 gene and pseudogenes CYP2D7P and CYP2D8P. Although the chemical and physiological properties of CYP2D6 have been extensively studied, there has been no study to date on molecular evolution of the CYP2D subfamily in the human genome. Such knowledge could greatly contribute to the understanding of drug metabolism in humans because it makes us to know when and how the current metabolic system has been constructed. The knowledge moreover can be useful to find differences in exogenous substrates in a particular metabolism between human and other animals such as experimental animals. Here, we conducted a preliminary study to investigate the evolution and gene organization of the CYP2D subfamily, focused on humans and four non-human primates (chimpanzees, orangutans, rhesus monkeys, and common marmosets). Our results indicate that CYP2D7P has been duplicated from CYP2D6 before the divergence between humans and great apes, whereas CYP2D6 and CYP2D8P have been already present in the stem lineages of New World monkeys and Catarrhini. Furthermore, the origin of the CYP2D subfamily in the human genome can be traced back to before the divergence between amniotes and amphibians. Our analyses also show that reported chimeric sequences of the CYP2D6 and CYP2D7 genes in the chimpanzee genome appear to be exchanged in its genome database.

Studies on the metabolism of the α‐pyrrolidinophenone designer drug methylenedioxy‐pyrovalerone (MDPV) in rat and human urine and human liver microsomes using GC–MS and LC–high‐resolution MS and its detectability in urine by GC–MS

Since the late 1990s, many derivatives of the α-pyrrolidinophenone (PPP) drug class appeared on the drugs of abuse market. The latest compound was described in 2009 to be a classic PPP carrying a methylenedioxy moiety remembering the classic entactogens (ecstasy). Besides Germany, 3,4-methylene-dioxypyrovalerone (MDPV) has appeared in many countries in Europe and Asia, indicating its worldwide importance for forensic and clinical toxicology. The aim of the presented work was to identify the phase I and II metabolites of MDPV and the human cytochrome-P450 (CYP) isoenzymes responsible for its main metabolic step(s). Finally, the detectability of MDPV in urine by the authors' systematic toxicological analysis (STA) should be studied. The urine samples were extracted after and without enzymatic cleavage of conjugates. The metabolites were separated and identified after work-up by GC-MS and liquid chromatography (LC)-high-resolution MS (LC-HR-MS). The studies revealed the following phase I main metabolic steps in rat and human: demethylenation followed by methylation, aromatic and side chain hydroxylation and oxidation of the pyrrolidine ring to the corresponding lactam as well as ring opening to the corresponding carboxylic acid. Using LC-HR-MS, most metabolite structures postulated according to GC-MS fragmentation could be confirmed and the phase II metabolites were identified. Finally, the formation of the initial metabolite demethylenyl-MDPV could be confirmed using incubation of human liver microsomes. Using recombinant human CYPs, CYP 2C19, CYP 2D6 and CYP 1A2 were found to catalyze this initial step. Finally, the STA allowed the detection of MDPV metabolites in the human urine samples.

Characterization of hybrid bilayer membranes on silver electrodes as biocompatible SERS substrates to study membrane–protein interactions

Hybrid bilayer lipid membranes (HBMs) were built on roughened silver electrodes exhibiting surface-enhanced Raman scattering (SERS) activity. The HBM consisted of a first layer of octadecanethiol (ODT) directly bound to the electrode surface, on which a second layer of 1,2-diphytanoyl- sn-glycero-3-phosphocholine (DPhPC) was obtained by self-assembled phospholipid vesicle fusion. The electrochemical properties of the HBM were investigated in situ by cyclic voltammetry (CV), AC voltammetry and electrochemical impedance spectroscopy (EIS). The results indicate that our HBMs are well-formed, and their insulating properties are comparable to those observed for HBM supported by smooth metal substrates. The interaction between the bilayer and the human enzyme cytochrome P450 2D6 (CYP2D6) was investigated. Surface-enhanced resonance Raman scattering (SERRS) measurements in combination with AC and EIS, performed on the same electrode sample, proved that the CYP2D6 is immobilized on the HBM without evident alterations of its active site and without significant perturbations of the bilayer architecture. This study yields novel insights into the properties of HBMs built on roughened surfaces, providing in situ electrochemical characterization of a substrate which is suitable for studying peripheral membrane proteins with SERRS spectroscopy.

The Effects of Azole-Based Heme Oxygenase Inhibitors on Rat Cytochromes P450 2E1 and 3A1/2 and Human Cytochromes P450 3A4 and 2D6

Heme oxygenases (HOs) catalyze the degradation of heme to biliverdin, carbon monoxide (CO), and free iron. The two major isoforms, HO-1 (inducible) and HO-2 (constitutive), are involved in a variety of physiological functions, including inflammation, apoptosis, neuromodulation, and vascular regulation. Major tools used in exploring these actions have been metalloporphyrin analogs of heme that inhibit the HOs. However, these tools are limited by their lack of selectivity; they affect other heme-dependent enzymes, such as cytochromes P450 (P450s), soluble guanylyl cyclase (sGC), and nitric-oxide synthase (NOS). Our laboratory has successfully synthesized a number of nonporphyrin azole-based HO inhibitors (QC-xx) that had little or no effect on sGC and NOS activity. However, their effects on various P450 isoforms have yet to be fully elucidated. To determine the effects of the QC-xx inhibitors on P450 enzyme activity, microsomal preparations of two rat P450 isoforms (2E1 and 3A1/3A2) and two human P450 supersome isoforms (3A4 and 2D6) were incubated with varying concentrations of HO inhibitor, and the activity was determined by spectrophotometric or fluorometric analysis. Results indicated that some QC compounds demonstrated little to no inhibition of the P450s, whereas others did inhibit these P450 isoforms. Four structural regions of QC-xx were analyzed, leading to the identification of structures that confer a decreased effect on both rat and human P450 isoforms studied while maintaining an inhibitory effect on the HOs.

Cytochrome P450 2D6 as a Model Antigen

Cytochrome P450 2D6 (CYP2D6) has been identified as the major autoantigen in type 2 autoimmune hepatitis (AIH). However, because of a lack of appropriate animal models, the etiology of AIH is still poorly understood. We generated a mouse model for AIH using the human CYP2D6 as a triggering molecule for autoimmunity. We infected wild-type FVB mice with an adenovirus expressing human CYP2D6 (Ad-2D6) to break self-tolerance to the mouse CYP2D6 homologues. Ad-2D6-infected mice showed persistent features of liver damage including hepatic fibrosis, cellular infiltrations, focal-to-confluent necrosis and generation of anti-CYP2D6 antibodies, which predominantly recognized the identical immunodominant epitope recognized by LKM-1 antibodies from AIH patients. Interestingly, Ad-2D6 infection of transgenic mice expressing the human CYP2D6 (CYP2D6 mice) resulted in delayed kinetics and reduced severity of liver damage. However, the quantity and quality of anti-CYP2D6 antibodies was only moderately reduced in CYP2D6 mice. In contrast, the frequency of CYP2D6-specific CD4 and CD8 T cells was dramatically decreased in CYP2D6 mice, indicating the presence of a strong T cell tolerance to human CYP2D6 established in CYP2D6 mice, but not in wild-type mice. CYP2D6-specific T cells reacted to human CYP2D6 peptides with intermediate homology to the mouse homologues, but not to those with high homology, indicating that molecular mimicry rather than molecular identity breaks tolerance and subsequently causes severe persistent autoimmune liver damage. The CYP2D6 model provides a platform to investigate mechanisms involved in the immunopathogenesis of autoimmune-mediated chronic hepatic injury and evaluate possible ways of therapeutic interference.

Mechanism‐Based Inhibition of Human Cytochrome P450 2D6 by Schering 66712

Schering 66712 (5‐fluoro‐2‐[4‐[(2‐phenyl‐1H‐imidazol5‐yl)methyl]‐1‐piperzainyl]pyrimidine) is a mechanism‐based inactivator of human cytochrome P450 2D6 (P450 2D6) that displays Type I binding with KS of 0.39 ± 0.10 μM. The partition ratio is approximately 300. Inactivation was not prevented by addition of an exogenous nucleophiles including potassium cyanide. Within 15 minutes of incubation with Schering 66712 and NADPH, ~100% of P450 2D6 activity was lost with ~40% loss in ability to binding CO. These findings support inactivation primarily by adduction of protein. ESI‐LC‐MS analysis of whole protein also indicates the presence of a protein adduct. Modeling of Schering 66712 in the active site of P450 2D6 suggests interaction between the phenyl group of Schering 66712 and the heme iron. (Supported by: NIH GM086767‐01 and RGM086767Z, a grant from the Howard Hughes Medical Institute to Kalamazoo College, and the Hutchcroft Fund of Kalamazoo College).

Effects of monoamine oxidase inhibitor and cytochrome P450 2D6 status on 5-methoxy- N, N-dimethyltryptamine metabolism and pharmacokinetics

5-Methoxy- N, N-dimethyltryptamine (5-MeO-DMT) is a natural psychoactive indolealkylamine drug that has been used for recreational purpose. Our previous study revealed that polymorphic cytochrome P450 2D6 (CYP2D6) catalyzed 5-MeO-DMT O-demethylation to produce active metabolite bufotenine, while 5-MeO-DMT is mainly inactivated through deamination pathway mediated by monoamine oxidase (MAO). This study, therefore, aimed to investigate the impact of CYP2D6 genotype/phenotype status and MAO inhibitor (MAOI) on 5-MeO-DMT metabolism and pharmacokinetics. Enzyme kinetic studies using recombinant CYP2D6 allelic isozymes showed that CYP2D6.2 and CYP2D6.10 exhibited 2.6- and 40-fold lower catalytic efficiency ( V max/ K m), respectively, in producing bufotenine from 5-MeO-DMT, compared with wild-type CYP2D6.1. When co-incubated with MAOI pargyline, 5-MeO-DMT O-demethylation in 10 human liver microsomes showed significantly strong correlation with bufuralol 1′-hydroxylase activities ( R 2 = 0.98; P < 0.0001) and CYP2D6 contents ( R 2 = 0.77; P = 0.0007), whereas no appreciable correlations with enzymatic activities of other P450 enzymes. Furthermore, concurrent MAOI harmaline sharply reduced 5-MeO-DMT depletion and increased bufotenine formation in human CYP2D6 extensive metabolizer hepatocytes. In vivo studies in wild-type and CYP2D6-humanized (Tg- CYP2D6) mouse models showed that Tg- CYP2D6 mice receiving the same dose of 5-MeO-DMT (20 mg/kg, i.p.) had 60% higher systemic exposure to metabolite bufotenine. In addition, pretreatment of harmaline (5 mg/kg, i.p.) led to 3.6- and 4.4-fold higher systemic exposure to 5-MeO-DMT (2 mg/kg, i.p.), and 9.9- and 6.1-fold higher systemic exposure to bufotenine in Tg- CYP2D6 and wild-type mice, respectively. These findings indicate that MAOI largely affects 5-MeO-DMT metabolism and pharmacokinetics, as well as bufotenine formation that is mediated by CYP2D6.

Polymorphism of Human Cytochrome P450 2D6 and Its Clinical Significance

Part I of this article discussed the potential functional importance of genetic mutations and alleles of the human cytochrome P450 2D6 (CYP2D6) gene. The impact of CYP2D6 polymorphisms on the clearance of and response to a series of cardiovascular drugs was addressed. Since CYP2D6 plays a major role in the metabolism of a large number of other drugs, Part II of the article highlights the impact of CYP2D6 polymorphisms on the response to other groups of clinically used drugs. Although clinical studies have observed a gene-dose effect for some tricyclic antidepressants, it is difficult to establish clear relationships of their pharmacokinetics and pharmacodynamic parameters to genetic variations of CYP2D6; therefore, dosage adjustment based on the CYP2D6 phenotype cannot be recommended at present. There is initial evidence for a gene-dose effect on commonly used selective serotonin reuptake inhibitors (SSRIs), but data on the effect of the CYP2D6 genotype/phenotype on the response to SSRIs and their adverse effects are scanty. Therefore, recommendations for dose adjustment of prescribed SSRIs based on the CYP2D6 genotype/phenotype may be premature. A number of clinical studies have indicated that there are significant relationships between the CYP2D6 genotype and steady-state concentrations of perphenazine, zuclopenthixol, risperidone and haloperidol. However, findings on the relationships between the CYP2D6 genotype and parkinsonism or tardive dyskinesia treatment with traditional antipsychotics are conflicting, probably because of small sample size, inclusion of antipsychotics with variable CYP2D6 metabolism, and co-medication. CYP2D6 phenotyping and genotyping appear to be useful in predicting steady-state concentrations of some classical antipsychotic drugs, but their usefulness in predicting clinical effects must be explored. Therapeutic drug monitoring has been strongly recommended for many antipsychotics, including haloperidol, chlorpromazine, fluphenazine, perphenazine, risperidone and thioridazine, which are all metabolized by CYP2D6. It is possible to merge therapeutic drug monitoring and pharmacogenetic testing for CYP2D6 into clinical practice. There is a clear gene-dose effect on the formation of O-demethylated metabolites from multiple opioids, but the clinical significance of this may be minimal, as the analgesic effect is not altered in poor metabolizers (PMs). Genetically caused inactivity of CYP2D6 renders codeine ineffective owing to lack of morphine formation, decreases the efficacy of tramadol owing to reduced formation of the active O-desmethyl-tramadol and reduces the clearance of methadone. Genetically precipitated drug interactions might render a standard opioid dose toxic. Because of the important role of CYP2D6 in tamoxifen metabolism and activation, PMs are likely to exhibit therapeutic failure, and ultrarapid metabolizers (UMs) are likely to experience adverse effects and toxicities. There is a clear gene-concentration effect for the formation of endoxifen and 4-OH-tamoxifen. Tamoxifen-treated cancer patients carrying CYP2D6*4, *5, *10, or *41 associated with significantly decreased formation of antiestrogenic metabolites had significantly more recurrences of breast cancer and shorter relapse-free periods. Many studies have identified the genetic CYP2D6 status as an independent predictor of the outcome of tamoxifen treatment in women with breast cancer, but others have not observed this relationship. Thus, more favourable tamoxifen treatment seems to be feasible through a priori genetic assessment of CYP2D6, and proper dose adjustment may be needed when the CYP2D6 genotype is determined in a patient. Dolasetron, ondansetron and tropisetron, all in part metabolized by CYP2D6, are less effective in UMs than in other patients. Overall, there is a strong gene-concentration relationship only for tropisetron. CYP2D6 genotype screening prior to antiemetic treatment may allow for modification of antiemetic dosing. An alternative is to use a serotonin agent that is metabolized independently of CYP2D6, such as granisetron, which would obviate the need for genotyping and may lead to an improved drug response. To date, the functional impact of most CYP2D6 alleles has not been systematically assessed for most clinically important drugs that are mainly metabolized by CYP2D6, though some initial evidence has been identified for a very limited number of drugs. The majority of reported in vivo pharmacogenetic data on CYP2D6 are from single-dose and steady-state pharmacokinetic studies of a small number of drugs. Pharmacodynamic data on CYP2D6 polymorphisms are scanty for most drug studies. Given that genotype testing for CYP2D6 is not routinely performed in clinical practice and there is uncertainty regarding genotype-phenotype, gene-concentration and gene-dose relationships, further prospective studies on the clinical impact of CYP2D6-dependent metabolism of drugs are warranted in large cohorts.

Probabilistic Prediction of the Human CYP3A4 and CYP2D6 Metabolism Sites

This article briefly introduces the results of in silico prediction of the most probable metabolism sites for the human cytochrome P450 3A4 and 2D6 isoforms. Ligand-based QSAR models have been developed using a novel GALAS modeling approach, and provide probabilities of being a target of CYP3A4 or CYP2D6 for any atom in a molecule. The GALAS-model development methodology allows evaluation of the reliability of predictions in the form of estimated prediction Reliability Indices (RIs). For all the models considered in this study, the number of misclassifications and inconclusive results was reduced significantly when only predictions of high quality (RI>0.5) were taken into account, demonstrating that RI reflects accuracy of prediction. The applicability domain of regioselectivity models is shown to be easily expandable to cover compound classes of interest to the user. The results obtained so far show promising perspectives for the utilization of the GALAS modeling in the analysis of regioselectivity for other important biotransformation enzymes--a work currently in progress.

Polymorphism of Human Cytochrome P450 2D6 and Its Clinical Significance

Cytochrome P450 (CYP) 2D6 is one of the most investigated CYPs in relation to genetic polymorphism, but accounts for only a small percentage of all hepatic CYPs (approximately 2-4%). There is a large interindividual variation in the enzyme activity of CYP2D6. The enzyme is largely non-inducible and metabolizes approximately 25% of current drugs. Typical substrates for CYP2D6 are largely lipophilic bases and include some antidepressants, antipsychotics, antiarrhythmics, antiemetics, beta-adrenoceptor antagonists (beta-blockers) and opioids. The CYP2D6 activity ranges considerably within a population and includes ultrarapid metabolizers (UMs), extensive metabolizers (EMs), intermediate metabolizers (IMs) and poor metabolizers (PMs). There is a considerable variability in the CYP2D6 allele distribution among different ethnic groups, resulting in variable percentages of PMs, IMs, EMs and UMs in a given population. To date, 74 allelic variants and a series of subvariants of the CYP2D6 gene have been reported and the number of alleles is still growing. Among these are fully functional alleles, alleles with reduced function and null (non-functional) alleles, which convey a wide range of enzyme activity, from no activity to ultrarapid metabolism of substrates. As a consequence, drug adverse effects or lack of drug effect may occur if standard doses are applied. The alleles *10, *17, *36 and *41 give rise to substrate-dependent decreased activity. Null alleles of CYP2D6 do not encode a functional protein and there is no detectable residual enzymatic activity. It is clear that alleles *3, *4, *5, *6, *7, *8, *11, *12, *13, *14, *15, *16, *18, *19, *20, *21, *38, *40, *42, *44, *56 and *62 have no enzyme activity. They are responsible for the PM phenotype when present in homozygous or compound heterozygous constellations. These alleles are of clinical significance as they often cause altered drug clearance and drug response. Among the most important variants are CYP2D6*2, *3, *4, *5, *10, *17 and *41. On the other hand, the CYP2D6 gene is subject to copy number variations that are often associated with the UM phenotype. Marked decreases in drug concentrations have been observed in UMs with tramadol, venlafaxine, morphine, mirtazapine and metoprolol. The functional impact of CYP2D6 alleles may be substrate-dependent. For example, CYP2D6*17 is generally considered as an allele with reduced function, but it displays remarkable variability in its activity towards substrates such as dextromethorphan, risperidone, codeine and haloperidol. The clinical consequence of the CYP2D6 polymorphism can be either occurrence of adverse drug reactions or altered drug response. Drugs that are most affected by CYP2D6 polymorphisms are commonly those in which CYP2D6 represents a substantial metabolic pathway either in the activation to form active metabolites or clearance of the agent. For example, encainide metabolites are more potent than the parent drug and thus QRS prolongation is more apparent in EMs than in PMs. In contrast, propafenone is a more potent beta-blocker than its metabolites and the beta-blocking activity during propafenone therapy is more prominent in PMs than EMs, as the parent drug accumulates in PMs. Since flecainide is mainly eliminated through renal excretion, and both R- and S-flecainide possess equivalent potency for sodium channel inhibition, the CYP2D6 phenotype has a minor impact on the response to flecainide. Since the contribution of CYP2D6 is greater for metoprolol than for carvedilol, propranolol and timolol, a stronger gene-dose effect is seen with this beta-blocker, while such an effect is lesser or marginal in other beta-blockers. Concordant genotype-phenotype correlation provides a basis for predicting the phenotype based on genetic testing, which has the potential to achieve optimal pharmacotherapy. However, genotype testing for CYP2D6 is not routinely performed in clinical practice and there is uncertainty regarding genotype-phenotype, gene-concentration and gene-dose relationships. Further prospective studies on the clinical impact of CYP2D6-dependent metabolism of drugs are warranted in large cohorts of subjects.

Identification of genetic variants of human cytochrome P450 2D6 with impaired mitochondrial targeting

Human cytochrome P450 2D6 (CYP2D6) is responsible for the metabolism of approximately 20% of drugs in common clinical use. The CYP2D6 gene locus is highly polymorphic. Many of the polymorphisms have been shown to be clinically relevant and can account for inter-individual differences in the metabolism of specific drugs. In addition to the established sources of variability in CYP2D6-dependent drug metabolism, a recent study in our laboratory identified CYP2D6 in the mitochondria of human liver samples and found that it is metabolically active in this novel location. In the present study we show that mutations are present in the targeting signal region of CYP2D6 that may help to account for the inter-individual variability that was observed previously in the level of the mitochondrial enzyme in human liver samples. These mutations were identified within the ER targeting domain, the proline-rich domain as well as the putative protein kinase A (PKA) and protein kinase C (PKC)-specific phosphorylation sites. In vitro studies demonstrate that the mutations identified in the targeting signals affect the efficiency of mitochondrial targeting of CYP2D6. Since the mitochondrial enzyme has been shown to be active in drug metabolism, this pharmacogenetic variation could play a role in modulating the response of an individual to drug therapy.

Substrate Specificity, Inhibitors and Regulation of Human Cytochrome P450 2D6 and Implications in Drug Development

CYP2D6 accounts for only a small percentage of total hepatic CYPs (<2%), but it metabolizes approximately 25% of clinically used drugs (>100) with significant polymorphisms. A number of drugs acting on the central nervous system and cardiovascular system are substantially metabolized by CYP2D6. The enzyme also utilizes hydroxytryptamines and neurosteroids as endogenous substrates. In addition, CYP2D6 metabolizes procarcinogens and neurotoxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 1,2,3,4-tetrahydroquinoline, and indolealkylamines. Typical CYP2D6 substrates are usually lipophilic bases with a planar hydrophobic aromatic ring and a nitrogen atom which can be protonated at physiological pH, but several atypical substrates such as spirosulfonamide and pactimibe do not contain a basic nitrogen atom. The structure of human CYP2D6 has been recently determined and shows the characteristic CYP fold as observed in other members of the CYP superfamily, with a well-defined active site cavity above the heme group with a volume of approximately 540 A(3). CYP2D6 is largely uninducible by prototypical CYP inducers such as phenobarbital, rifampin and dexamethasone, but it is regulated by hepatocyte nuclear factor-4alpha, a nuclear receptor. CYP2D6 is subject to inhibition by a number of drugs and this may provide an explanation for numerous clinical drug interactions. CYP2D6 has an important role in drug development and it is a common practice for pharmaceutical industry nowadays to a great extent screen drug candidates early in development as possible CYP2D6 substrates and/or inhibitors and drop such candidates where they have alternatives. This candidate selection might eventually lead to a less prominent role of this enzyme in the future for drug metabolism and less interindividual variability in drug exposure and minimize potentially adverse drug interactions. Further studies are warranted to delineate the molecular mechanisms involved in the function and regulation of CYP2D6.

Human liver mitochondrial cytochrome P450 2D6 – individual variations and implications in drug metabolism

Constitutively expressed human cytochrome P450 2D6 (CYP2D6; EC 1.14.14.1) is responsible for the metabolism of approximately 25% of drugs in common clinical use. It is widely accepted that CYP2D6 is localized in the endoplasmic reticulum of cells; however, we have identified this enzyme in the mitochondria of human liver samples and found that extensive inter-individual variability exists with respect to the level of the mitochondrial enzyme. Metabolic assays using 7-methoxy-4-aminomethylcoumarin as a substrate show that the human liver mitochondrial enzyme is capable of oxidizing this substrate and that the catalytic activity is supported by mitochondrial electron transfer proteins. In the present study, we show that CYP2D6 contains an N-terminal chimeric signal that mediates its bimodal targeting to the endoplasmic reticulum and mitochondria. In vitro mitochondrial import studies using both N-terminal deletions and point mutations suggest that the mitochondrial targeting signal is localized between residues 23-33 and that the positively-charged residues at positions 24, 25, 26, 28 and 32 are required for mitochondrial targeting. The importance of the positively-charged residues was confirmed by transient transfection of a CYP2D6 mitochondrial targeting signal mutant in COS-7 cells. Both the mitochondria and the microsomes from a CYP2D6 stable expression cell line contain the enzyme and both fractions exhibit bufuralol 1'-hydroxylation activity, which is completely inhibited by CYP2D6 inhibitory antibody. Overall, these results suggest that the targeting of CYP2D6 to mitochondria could be an important physiological process that has significance in xenobiotic metabolism.