RESUMO
OBJECTIVE: Develop a cytochrome P450 (CYP) phenotyping cocktail for dogs using specific substrates for hepatic P450 enzymes CYP2B11, CYP2D15, and CYP3A12 and determine whether alternative sampling methods (saliva and urine) or single time point samples could be used instead of multiple blood sampling. ANIMALS: 12 healthy client-owned dogs (8 females and 4 males) from February 2019 to May 2019. METHODS: In a randomized crossover study, dogs received oral administration of the probe drug bupropion (75 mg), dextromethorphan (30 mg), or omeprazole (40 mg) alone or as a 3-drug combination (Program in Individualized Medicine [PrIMe] cocktail) to evaluate simultaneous phenotyping of CYP2B11, CYP2D15, and CYP3A12. Pharmacokinetic profiles for the probe drugs and metabolites were determined using plasma, saliva, and urine. Dogs received probe drugs alone or combined. Pharmacokinetic profiles up to 6 hours postdose for the probe drugs and metabolites were determined using plasma, saliva, and urine. RESULTS: The PrIMe cocktail was well tolerated. There was no statistically significant interaction between the probe drugs when administered together. Single time point plasma metabolic ratios at 4 hours postdose for all probe drugs strongly correlated with the corresponding area under the plasma concentration-versus-time curve (AUC) ratios. Saliva AUC metabolic ratios for CYP3A12 and CYP2D15 and 6-hour urine for CYP2B11 and CYP2D15 were correlated with plasma AUC ratios. CONCLUSIONS: The PrIMe cocktail can be used for simultaneous CYP phenotyping using plasma 4-hour single time point sample metabolic ratios. Saliva and urine sampling are suitable for specific CYPs. CLINICAL RELEVANCE: The PrIMe cocktail has potential as a useful tool in dogs to detect clinically important CYP-mediated drug-drug interactions, identify novel pharmacogenes, determine the drug-metabolizing phenotype of individual dogs, aid in individualized dose selection, and evaluate the effects of various physiological states on drug metabolism.
Assuntos
Bupropiona , Estudos Cross-Over , Dextrometorfano , Omeprazol , Animais , Cães , Dextrometorfano/farmacocinética , Dextrometorfano/urina , Dextrometorfano/metabolismo , Bupropiona/farmacocinética , Bupropiona/metabolismo , Bupropiona/sangue , Omeprazol/farmacocinética , Feminino , Masculino , Sistema Enzimático do Citocromo P-450/metabolismo , Fenótipo , Hidrocarboneto de Aril Hidroxilases/metabolismoRESUMO
Although great progress has been made in the fine-tuning of diplotypes, there is still a need to further improve the predictability of individual phenotypes of pharmacogenetically relevant enzymes. The aim of this study was to analyze the additional contribution of sex and variants identified by exome chip analysis to the metabolic ratio of five probe drugs. A cocktail study applying dextromethorphan, losartan, omeprazole, midazolam, and caffeine was conducted on 200 healthy volunteers. CYP2D6, 2C9, 2C19, 3A4/5, and 1A2 genotypes were analyzed and correlated with metabolic ratios. In addition, an exome chip analysis was performed. These SNPs correlating with metabolic ratios were confirmed by individual genotyping. The contribution of various factors to metabolic ratios was assessed by multiple regression analysis. Genotypically predicted phenotypes defined by CPIC discriminated very well the log metabolic ratios with the exception of caffeine. There were minor sex differences in the activity of CYP2C9, 2C19, 1A2, and CYP3A4/5. For dextromethorphan (CYP2D6), IP6K2 (rs61740999) and TCF20 (rs5758651) affected metabolic ratios, but only IP6K2 remained significant after multiple regression analysis. For losartan (CYP2C9), FBXW12 (rs17080138), ZNF703 (rs79707182), and SLC17A4 (rs11754288) together with CYP diplotypes, and sex explained 50% of interindividual variability. For omeprazole (CYP2C19), no significant influence of CYP2C:TG haplotypes was observed, but CYP2C19 rs12777823 improved the predictability. The comprehensive genetic analysis and inclusion of sex in a multiple regression model significantly improved the explanation of variability of metabolic ratios, resulting in further improvement of algorithms for the prediction of individual phenotypes of drug-metabolizing enzymes.
Assuntos
Genótipo , Fenótipo , Polimorfismo de Nucleotídeo Único , Humanos , Masculino , Feminino , Adulto , Exoma/genética , Cafeína/farmacocinética , Cafeína/metabolismo , Dextrometorfano/farmacocinética , Dextrometorfano/metabolismo , Losartan/farmacocinética , Preparações Farmacêuticas/metabolismo , Adulto Jovem , Omeprazol/farmacocinética , Fatores Sexuais , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Estudos de Associação Genética/métodos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Tree shrews are a nonprimate species used in a range of biomedical studies. Recent genome analysis of tree shrews found that the sequence identities and the numbers of genes of cytochrome P450 (CYP or P450), an important family of drug-metabolizing enzymes, are similar to those of humans. However, tree shrew P450s have not yet been sufficiently identified and analyzed. In this study, novel CYP2D8a and CYP2D8b cDNAs were isolated from tree shrew liver and were characterized, along with human CYP2D6, dog CYP2D15, and pig CYP2D25. The amino acid sequences of these tree shrew CYP2Ds were 75%-78% identical to human CYP2D6, and phylogenetic analysis showed that they were more closely related to human CYP2D6 than rat CYP2Ds, similar to dog and pig CYP2Ds. For tree shrew CYP2D8b, two additional transcripts were isolated that contained different patterns of deletion. The gene and genome structures of CYP2Ds are generally similar in dogs, humans, pigs, and tree shrews. Tree shrew CYP2D8a mRNA was most abundantly expressed in liver, among the tissue types analyzed, similar to dog CYP2D15 and pig CYP2D25 mRNAs. Tree shrew CYP2D8b mRNA was also expressed in liver, but at a level 7.3-fold lower than CYP2D8a mRNA. Liver microsomes and recombinant protein of both tree shrew CYP2Ds metabolized bufuralol and dextromethorphan, selective substrates of human CYP2D6, but the activity level of CYP2D8a greatly exceeded that of CYP2D8b. These results suggest that tree shrew CYP2D8a and CYP2D8b are functional drug-metabolizing enzymes, of which CYP2D8a is the major CYP2D in liver. SIGNIFICANCE STATEMENT: Novel tree shrew CYP2D8a and CYP2D8b cDNAs were isolated from liver. Their amino acid sequences were 75%-78% identical to human CYP2D6. For CYP2D8b, two additional transcripts contained different patterns of deletion. Tree shrew CYP2D8a mRNA was abundantly expressed in liver, similar to dog CYP2D15 and pig CYP2D25 mRNAs. Recombinant tree shrew CYP2Ds catalyzed the oxidation of bufuralol and dextromethorphan. Tree shrew CYP2D8a and CYP2D8b are functional drug-metabolizing enzymes, of which CYP2D8a is the major CYP2D in liver.
Assuntos
Citocromo P-450 CYP2D6 , Dextrometorfano , Etanolaminas , Humanos , Ratos , Suínos , Animais , Cães , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Dextrometorfano/metabolismo , Tupaia/genética , Tupaia/metabolismo , Tupaiidae/genética , Tupaiidae/metabolismo , Filogenia , Musaranhos/genética , Musaranhos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos Hepáticos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Cytochrome P450 2D6 (CYP2D6) is involved in the metabolism of >20% of marketed drugs. CYP2D6 expression and activity exhibit high interindividual variability and is induced during pregnancy. The farnesoid X receptor (FXR) is a transcriptional regulator of CYP2D6 that is activated by bile acids. In pregnancy, elevated plasma bile acid concentrations are associated with maternal and fetal risks. However, modest changes in bile acid concentrations may occur during healthy pregnancy, thereby altering FXR signaling. A previous study demonstrated that hepatic tissue concentrations of bile acids positively correlated with the hepatic mRNA expression of CYP2D6. This study sought to characterize the plasma bile acid metabolome in healthy women (n = 47) during midpregnancy (25-28 weeks gestation) and ≥3 months postpartum and to determine if plasma bile acids correlate with CYP2D6 activity. It is hypothesized that during pregnancy, plasma bile acids would favor less hydrophobic bile acids (cholic acid vs. chenodeoxycholic acid) and that plasma concentrations of cholic acid and its conjugates would positively correlate with the urinary ratio of dextrorphan/dextromethorphan. At 25-28 weeks gestation, taurine-conjugated bile acids comprised 23% of the quantified serum bile acids compared with 7% ≥3 months postpartum. Taurocholic acid positively associated with the urinary ratio of dextrorphan/dextromethorphan, a biomarker of CYP2D6 activity. Collectively, these results confirm that the bile acid plasma metabolome differs between pregnancy and postpartum and provide evidence that taurocholic acid may impact CYP2D6 activity during pregnancy. SIGNIFICANCE STATEMENT: Bile acid homeostasis is altered in pregnancy, and plasma concentrations of taurocholic acid positively correlate with CYP2D6 activity. Differences between plasma and/or tissue concentrations of farnesoid X receptor ligands such as bile acids may contribute to the high interindividual variability in CYP2D6 expression and activity.
Assuntos
Citocromo P-450 CYP2D6 , Dextrometorfano , Humanos , Feminino , Gravidez , Citocromo P-450 CYP2D6/metabolismo , Dextrometorfano/metabolismo , Dextrorfano , Ácido Taurocólico , Período Pós-PartoRESUMO
CYP2D15 is a major drug metabolizing P450 in canine liver. Like the human orthologue (CYP2D6), this enzyme is highly polymorphic with at least five common nonsynonymous variants reported that result in amino acid changes, including p.Ile109Val, p.Leu115Phe, p.Gly186Ser, p.Ile250Phe and p.Ile307Val. Furthermore, a mRNA splice variant of CYP2D15 has been found in canine liver that lacks the exon 3 gene region resulting in an inactive enzyme. The objective of this study was to evaluate whether any of these amino acid variants or the exon 3 deletion mRNA variant (exon3-delta) was associated with differences in CYP2D15-selective activities or protein content in a bank of canine livers. Livers were obtained from 25 Beagles and 34 dogs of various other breeds. CYP2D15-selective activities measured included dextromethorphan o-demethylation and tramadol o-demethylation. Reverse transcription PCR showed that 76% of livers (44/58) expressed both exon3-delta and normally spliced CYP2D15 RNA, while the remaining 24% (14/58) expressed only normally spliced RNA. The presence of exon3-delta was not correlated with CYP2D15 activities or protein content. Compared with wild-type livers, Beagle dog livers heterozygous for the p.Ile109Val and p.Gly186Ser variants showed from 40 to 50% reductions in median enzyme activities, while heterozygous p.Gly186Ser livers were associated with a 41% reduction in median CYP2D15 protein content (p < .05; Dunn's test). In the entire liver bank, livers homozygous for p.Ile109Val were also associated with a 40% reduction in median dextromethorphan O-demethylation activities versus wild-type livers (p < .05). These results identify several nonsynonymous CYP2D15 gene variants associated with variable CYP2D15 metabolism in canine liver.
Assuntos
Microssomos Hepáticos , RNA , Cães , Animais , Humanos , Microssomos Hepáticos/metabolismo , RNA/metabolismo , Dextrometorfano/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Éxons , RNA Mensageiro/metabolismoRESUMO
INTRODUCTION: Tobacco products are addictive, with nicotine serving as the major addictive ingredient. Chronic tobacco use or chronic administration of nicotine alone results in both physiological and psychological dependence. Our previous studies indicated that dextromethorphan (DM) could effectively attenuate the dependence of morphine and methamphetamine. Thus, we further investigated the possible effects of DM on nicotine dependence. AIMS AND METHODS: Conditioned place preference (CPP) test was used to examine nicotine-induced rewarding effects as well as the drug-seeking-related behavior in rats. Nicotine dependence was induced by continuous subcutaneous infusion of nicotine via an osmotic minipump for 7 days and abstinence was initiated by removal of the pump. Withdrawal signs were observed and quantified. Locomotor activity was measured to determine the behavioral sensitization induced by nicotine. To investigate the activity of mesolimbic dopaminergic neuronal activity in correlation with the effects of nicotine, the animals were sacrificed and the nucleus accumbens (NAc), dorsal striatum (DS), and medial prefrontal cortex (mPFC) were dissected and used to determine the contents of dopamine (DA) and its metabolites using high-performance liquid chromatography (HPLC). RESULTS: Our results showed that DM could suppress nicotine-induced rewarding effect and drug-seeking-related behavior. In addition, co-administration and post-treatment of DM could both attenuate nicotine withdrawal signs. Moreover, DM could suppress nicotine-induced behavioral sensitization. Neurochemical experiments show that co-administration and post-treatment of DM abolished nicotine-induced increase of the DA turnover rate in the mPFC, but not in the NAc and DS. CONCLUSIONS: The results suggest that DM has a great therapeutic potential in the treatment of nicotine dependence. IMPLICATIONS: Our results showed that DM could suppress nicotine-induced rewarding effect and drug-seeking-related behavior. In addition, co-administration and post-treatment of DM could both attenuate nicotine withdrawal signs. Moreover, DM could suppress nicotine-induced behavioral sensitization. Neurochemical experiments show that co-administration and post-treatment of DM abolished nicotine-induced increase of the DA turnover rate in the mPFC, but not in the NAc and DS. These results suggest that DM has a great therapeutic potential in the treatment of nicotine dependence.
Assuntos
Síndrome de Abstinência a Substâncias , Tabagismo , Ratos , Animais , Nicotina/efeitos adversos , Nicotina/metabolismo , Dextrometorfano/farmacologia , Dextrometorfano/metabolismo , Ratos Sprague-Dawley , Tabagismo/tratamento farmacológico , Tabagismo/metabolismo , Recompensa , Núcleo Accumbens/metabolismo , Dopamina/metabolismo , Síndrome de Abstinência a Substâncias/tratamento farmacológico , Síndrome de Abstinência a Substâncias/metabolismoRESUMO
Inter-individual variability in pharmacokinetics and drug response is heavily influenced by single-nucleotide variants (SNVs) and copy-number variations (CNVs) in genes with importance for drug disposition. Nowadays, a plethora of studies implement next generation sequencing to capture rare and novel pharmacogenomic (PGx) variants that influence drug response. To address these issues, we present a comprehensive end-to-end analysis workflow, beginning from targeted PGx panel re-sequencing to in silico analysis pipelines and in vitro validation assays. Specifically, we show that novel pharmacogenetic missense variants that are predicted or putatively predicted to be functionally deleterious, significantly alter protein activity levels of CYP2D6 and CYP2C19 proteins. We further demonstrate that variant priorization pipelines tailored with functional in vitro validation assays provide supporting evidence for the deleterious effect of novel PGx variants. The proposed workflow could provide the basis for integrating next-generation sequencing for PGx testing into routine clinical practice.
Assuntos
Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2D6/genética , Sequenciamento de Nucleotídeos em Larga Escala , Variantes Farmacogenômicos , Algoritmos , Linhagem Celular , Simulação por Computador , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Citocromos b5/genética , Dextrometorfano/metabolismo , Humanos , Mefenitoína/metabolismo , Microssomos/metabolismo , Mutação de Sentido Incorreto , Reprodutibilidade dos TestesRESUMO
CYP2D6 is an important enzyme of the cytochrome P450 superfamily, and catalyzes nearly 25% of the drugs sold in the market. For decades, the interactions and metabolism between CYP2D6 and substrates have been a hot topic. However, the key factors of the catalytic regioselectivity for CYP2D6 still remain controversial. Here, we construct four systems to explore the interaction between dextromethorphan (DM) and CYP2D6. A new binding mode of CYP2D6 is defined, and two key residues (residue Asp301 and residue Glu216) are discovered working simultaneously to stabilize the DM at the reactive site by forming water bridge hydrogen bonds when CYP2D6 binds DM. Our results also indicate that the substrate concentration could mediate the binding mode between the substrate and CYP2D6 by decreasing the volume of the catalytic pocket, which is not conducive to the O-demethylation of DM but benefits the N-demethylation of DM. These results could shed light on the process of CYP2D6 binding to the substrate, and help to better understand the regioselectivity of CYP2D6 catalyzing the substrates.
Assuntos
Citocromo P-450 CYP2D6/metabolismo , Dextrometorfano/metabolismo , Domínio Catalítico , Citocromo P-450 CYP2D6/química , Dextrometorfano/química , Humanos , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Ligação ProteicaRESUMO
Dextromethorphan (DXM) acts as cough suppressant via its central action. Cell-protective effects of this drug have been reported in peripheral tissues, making DXM potentially useful for treatment of several common human diseases, such as type 2 diabetes mellitus (T2DM). Pancreatic islets are among the peripheral tissues that positively respond to DXM, and anti-diabetic effects of DXM were observed in two placebo-controlled, randomized clinical trials in humans with T2DM. Since these effects were associated with central side effects, we here developed chemical derivatives of DXM that pass the blood-brain barrier to a significantly lower extent than the original drug. We show that basic nitrogen-containing residues block central adverse events of DXM without reducing its anti-diabetic effects, including the protection of human pancreatic islets from cell death. These results show how to chemically modify DXM, and possibly other morphinans, as to exclude central side effects, while targeting peripheral tissues, such as pancreatic islets.
Assuntos
Glicemia/análise , Dextrometorfano/farmacologia , Hipoglicemiantes/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Cálcio/metabolismo , Dextrometorfano/análogos & derivados , Dextrometorfano/metabolismo , Dextrometorfano/uso terapêutico , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/patologia , Desenho de Fármacos , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/metabolismo , Hipoglicemiantes/uso terapêutico , Insulina/sangue , Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos Endogâmicos C57BLRESUMO
Interest in developing NMDA receptor antagonists with reduced side-effects for neurological and psychiatric disorders has been re-energized by the recent introduction of esketamine into clinical practice for treatment-resistant depression. Structural analogs of dextromethorphan bind with low affinity to the NMDA receptor ion channel, have functional effects in vivo, and generally display a lower propensity for side-effects than that of ketamine and other higher affinity antagonists. As such, the aim of the present study was to determine whether a series of N-substituted-3-alkoxy-substituted dextromethorphan analogs produce their anticonvulsant effects through NMDA receptor blockade. Compounds were studied against NMDA-induced seizures in rats. Compounds were administered intracerebroventricularly in order to mitigate confounds of drug metabolism that arise from systemic administration. Comparison of the anticonvulsant potencies to their affinities for NMDA, σ1, and σ2 binding sites were made in order to evaluate the contribution of these receptors to anticonvulsant efficacy. The potencies to block convulsions were positively associated with their affinities to bind to the NMDA receptor ion channel ([3H]-TCP binding) (r = 0.71, p < 0.05) but not to σ1 receptors ([3H]-SKF 10047 binding) (r = -0.31, p = 0.46) or to σ2 receptors ([3H]-DTG binding) (p = -0.38, p = 0.36). This is the first report demonstrating that these dextromethorphan analogs are functional NMDA receptor antagonists in vivo. Given their potential therapeutic utility and favorable side-effect profiles, such low affinity NMDA receptor antagonists could be considered for further development in neurological (e.g., anticonvulsant) and psychiatric (e.g., antidepressant) disorders.
Assuntos
Anticonvulsivantes/administração & dosagem , Dextrometorfano/análogos & derivados , Dextrometorfano/administração & dosagem , Dextrorfano/administração & dosagem , Agonistas de Aminoácidos Excitatórios/efeitos adversos , Antagonistas de Aminoácidos Excitatórios/administração & dosagem , N-Metilaspartato/efeitos adversos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Convulsões/induzido quimicamente , Convulsões/tratamento farmacológico , Álcoois/química , Animais , Anticonvulsivantes/metabolismo , Sítios de Ligação , Dextrometorfano/metabolismo , Dextrorfano/metabolismo , Modelos Animais de Doenças , Antagonistas de Aminoácidos Excitatórios/metabolismo , Infusões Intraventriculares , Ligantes , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores sigma/metabolismo , Resultado do Tratamento , Receptor Sigma-1RESUMO
Polyphenolic compounds (including flavonoids, chalcones, phenolic acids, and furanocoumarins) represent a common part of our diet, but are also the active ingredients of several dietary supplements and/or medications. These compounds undergo extensive metabolism by human biotransformation enzymes and the microbial flora of the colon. CYP2D6 enzyme metabolizes approximately 25% of the drugs, some of which has narrow therapeutic window. Therefore, its inhibition can lead to the development of pharmacokinetic interactions and the disruption of drug therapy. In this study, the inhibitory effects of 17 plant-derived compounds and 19 colonic flavonoid metabolites on CYP2D6 were examined, employing two assays with different test substrates. The O-demethylation of dextromethorphan was tested employing CypExpress 2D6 kit coupled to HPLC analysis; while the O-demethylation of another CYP2D6 specific substrate (AMMC) was investigated in a plate reader assay with BioVision Fluorometric CYP2D6 kit. Interestingly, some compounds (e.g., bergamottin) inhibited both dextromethorphan and AMMC demethylation; however, certain substances proved to be inhibitors only in one of the assays applied. Our results demonstrate that some polyphenols and colonic metabolites are inhibitors of CYP2D6-catalyzed reactions. Nevertheless, the inhibitory effects showed strong substrate dependence.
Assuntos
Colo/metabolismo , Inibidores do Citocromo P-450 CYP2D6/farmacologia , Citocromo P-450 CYP2D6/metabolismo , Polifenóis/farmacologia , Acetamidas/metabolismo , Dextrometorfano/metabolismo , Flavonoides/farmacologia , Humanos , Polifenóis/metabolismo , Piridazinas/metabolismoRESUMO
OBJECTIVES: Cytochrome P450 2D6 (CYP2D6) enzyme metabolizes a quarter of prescription drugs. Polymorphisms of CYP2D6 gene and resultant phenotypic variations in metabolic activity have been described in various populations. We assessed the prevalence of CYP2D6 activity phenotypes, employing dextromethorphan (DXM) as probe drug in subjects with at least two parental generations residing in eastern India. MATERIALS AND METHODS: Unrelated healthy subjects took 60 mg DXM after fasting overnight. Blood samples were collected 3 h after dosing and plasma separated. DXM and its primary metabolite dextrorphan (DXT) were measured by liquid chromatography with tandem mass spectrometry. The DXM-to-DXT metabolic ratio (MR) was obtained for each subject. Histogram of MR values suggested bimodal distribution. A polynomial regression equation derived through probit analysis was solved to identify the antimode of the MR values. Subjects with log(MR) < antimode were extensive metabolizers (EMs). Log(MR) ≥ antimode indicated poor metabolizers (PMs). RESULTS: We evaluated the results from 97 participants. The median MR was 0.209 (interquartile range: 0.090-0.609), while the antimode for MR was 3.055. From these, it was inferred that three subjects were PMs, while the rest were EMs. CYP2D6 polymorphism prevalence is low (3.09%; 95% confidence interval: 0.35%-6.54%) in the population of eastern India and matches the prevalence in other zones of the country. CONCLUSIONS: Differences in CYP2D6 activity has treatment implications and may lead to adverse events or therapeutic failure. Phenotyping of subjects receiving CYP2D6 metabolized drugs may help clinicians personalize treatment and avert adverse drug-drug interactions. However, the frequency of the PM phenotype is low in India, and routinely phenotyping for CYP2D6 activity will not be cost-effective. We cannot recommend it at this stage.
Assuntos
Citocromo P-450 CYP2D6/genética , Variantes Farmacogenômicos , Polimorfismo Genético , Adulto , Citocromo P-450 CYP2D6/metabolismo , Dextrometorfano/metabolismo , Interações Medicamentosas , Feminino , Humanos , Índia , Masculino , Farmacogenética , Fenótipo , Adulto JovemRESUMO
Measuring in vivo changes in the drug metabolizing activity of cytochrome P450 (CYP) enzymes is critical to understanding and assessing drug-drug, drug-diet and drug-disease interactions. The sensitivity and specificity of ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) makes it an ideal tool for analyzing drugs and their metabolites in biological matrices, and has demonstrated utility in CYP phenotyping across varied applications. Published CYP phenotyping cocktail assays often require large plasma sample volumes (0.5-1 mL), have relatively low sensitivity and multi-step complex sample preparation and extraction procedures. Further, variability exists in the way that recovery and matrix effects are investigated and reported, and some studies fail to report these data altogether. Therefore, the aim of this study was to develop, validate and optimize a simplified assay for the probe drugs caffeine (metabolized by CYP1A2), omeprazole (CYP2C19), losartan (CYP2C9), dextromethorphan (CYP2D6), midazolam (CYP3A4) and their respective enzyme-specific metabolites in small volumes (100 µL) of human plasma, that addresses the issues noted. Analyte extraction involved protein precipitation with acetonitrile and solid-phase extraction (SPE). Samples were analyzed using an Agilent 1290 infinity LC system in tandem with 6460A triple quadrupole mass spectrometers. The assay met FDA guideline-recommended requirements for specificity, sensitivity (analyte LLOQs 0.78-23.4 ng/mL), accuracy (intra-day RE% nominal concentration 90.7-110.2%; inter-day RE% 87.0-110.5%) and precision (intra-day analyte RSD% 0.46-11.4%; inter-day RSD% 1.36-11.2%). Recovery and matrix effects were thoroughly investigated and excluded as potential interferers with assay performance. This assay has been used successfully to phenotype CYP activity in a human clinical trial participant. Importantly, the authors provide a contemporary commentary on commonly found issues in the CYP phenotyping cocktail assay literature, and make recommendations concerning best-practice approaches and the standardization of data reporting in this area.
Assuntos
Sistema Enzimático do Citocromo P-450/sangue , Sistema Enzimático do Citocromo P-450/classificação , Técnicas Biossensoriais/métodos , Cafeína/metabolismo , Cromatografia Líquida de Alta Pressão , Dextrometorfano/metabolismo , Interações Medicamentosas , Humanos , Limite de Detecção , Losartan/metabolismo , Midazolam/metabolismo , Omeprazol/metabolismo , Fenótipo , Sensibilidade e Especificidade , Espectrometria de Massas em TandemRESUMO
While CYP2D6 allele and phenotype frequencies have been extensively studied, currently, very little ethnically specific data is available regarding the East African and South Pacific region, including Kenya and Vanuatu. The absence of information regarding gene polymorphisms and their resulting clinical effects in these populations may hinder treatment strategies and patient outcome. Given the scarceness of CYP2D6 related data in these populations, the purpose of this study was to perform a pharmacogenomic analysis of the Kenyan and Ni-Vanuatu population and ultimately characterize the enzymatic properties of eight novel CYP2D6 variant proteins expressed in 293FT cells in vitro using dextromethorphan as a substrate. Our study revealed a prevalence of functional alleles in both populations a low frequency for decreased function defining genotypes in the Ni-Vanuatu population, with approximately 36% of our Kenyan subjects presenting substrate-dependent decreased function alleles. Additionally, 6 variants (P171L, G306R, V402L, K1, K2, and K3) showed significantly reduced intrinsic clearance compared to wild-type CYP2D6.1. Our findings aid in efforts to bridge the gap between pharmacogenomic analysis and clinical application, by providing useful information in the development of ethnic-specific strategies as well as stressing the importance of population-specific genotyping when conducting multi-regional clinical trials and designing therapeutic strategies.
Assuntos
Alelos , Citocromo P-450 CYP2D6/genética , Dextrometorfano/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Desmetilação , Variação Genética/genética , Genótipo , Células HEK293 , Humanos , Japão , Polimorfismo Genético/genéticaRESUMO
There is evidence that the antipsychotic drug perazine is an inhibitor of CYP2D6. This study aimed at evaluating its effect on CYP2D6 and CYP2C19 activities in submitting psychiatric patients to phenotyping with dextromethorphan and mephenytoin, respectively, substrates of these enzymes, before and during a treatment with perazine. A total of 31 patients were phenotyped with dextromethorphan (CYP2D6) and mephenytoin (CYP2C19) before and after a 2-week treatment with 450 ± 51 mg/day (mean ± sd) perazine. At baseline, five patients appeared to be poor metabolizers (PM) of dextromethorphan and two patients of mephenytoin. The metabolic ratio (MR) of dextromethorphan/dextrorphan as determined in collected urine increased significantly (Wilcoxon; P < .0001) from baseline (0.39 ± 1.38 [mean ± sd]) till day 14 (1.46 ± 2.22). In 19 out of 26 extensive metabolizers (EM) of dextromethorphan, the phenotype changed from EM to PM. This suggests an almost complete inhibition of CYP2D6 by perazine and/or its metabolites. On the other hand, perazine (or some of its metabolites) did seemingly not inhibit CYP2C19. In conclusion, this study suggests that in patients treated with perazine and co-medicated with CYP2D6 substrates, there could be an increased risk of adverse effects as a consequence of a pharmacokinetic interaction.
Assuntos
Antipsicóticos/farmacologia , Citocromo P-450 CYP2C19/metabolismo , Inibidores do Citocromo P-450 CYP2D6/farmacologia , Citocromo P-450 CYP2D6/metabolismo , Dextrometorfano/metabolismo , Mefenitoína/metabolismo , Perazina/farmacologia , Esquizofrenia/tratamento farmacológico , Adulto , Interações Medicamentosas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Esquizofrenia/enzimologia , Adulto JovemRESUMO
The aim of the present study was to determine the involvement of the VTA 5-HT1A receptors in analgesia induced by the co-administration of morphine and dextromethorphan (DM). Male Wistar rats were bilaterally cannulated in the VTA by the stereotaxic instrument. The tail-flick and formalin tests were performed to assess nociception in the acute and tonic pain conditions respectively. The present data indicated that intraperitoneal (i.p.) administration of morphine increased the tail-flick latency (1-4â¯mg/kg) and decreased the pain score of formalin test (2-8â¯mg/kg), showing an analgesic effect. Co-administration of ineffective doses of morphine (1 or 2â¯mg/kg) with DM (30â¯mg/kg, i.p.) induced analgesia in both animal models. Interestingly, intra-VTA microinjection of 5HT1A receptors antagonist, S-WAY100-135 (0.5 and 1⯵g/kg), inhibited the analgesic effect of morphine plus DM in both acute and tonic pain models. It should be considered that the same doses of DM or S-WAY100-135 by itself had no effects on antinociception in the animal models. Overall, these results indicated that systemic blockade of NMDA receptors via DM administration potentiated the response of a low dose of morphine to induce analgesic effect. Additionally, it seems that the VTA serotonergic system via 5HT1A receptors mediates the potentiative effect of DM on morphine-induced analgesia.
Assuntos
Dor/fisiopatologia , Receptor 5-HT1A de Serotonina/metabolismo , Área Tegmentar Ventral/metabolismo , Analgesia/métodos , Analgésicos Opioides/farmacologia , Animais , Dextrometorfano/metabolismo , Dextrometorfano/farmacologia , Masculino , Modelos Animais , Morfina/metabolismo , Morfina/farmacologia , Nociceptividade/efeitos dos fármacos , Núcleo Accumbens/efeitos dos fármacos , Manejo da Dor/métodos , Ratos , Ratos Wistar , Receptor 5-HT1A de Serotonina/fisiologia , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Serotonina/metabolismo , Área Tegmentar Ventral/fisiologiaAssuntos
Antitussígenos/efeitos adversos , Citocromo P-450 CYP2D6/genética , Dextrometorfano/efeitos adversos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/genética , Transtornos Mentais/induzido quimicamente , Antitussígenos/metabolismo , Antitussígenos/uso terapêutico , Criança , Citocromo P-450 CYP2D6/metabolismo , Dextrometorfano/metabolismo , Dextrometorfano/uso terapêutico , Feminino , Genótipo , Humanos , FenótipoRESUMO
Cytochrome P450 (CYP) 2D6 metabolizes a wide range of xenobiotics and is characterized by a huge interindividual variability. A recent clinical study highlighted differential magnitude of CYP inhibition as a function of CYP2D6 genotype. The aim of this study was to investigate the effect of CYP2D6 genotype on the inhibition of dextromethorphan O-demethylation by duloxetine and paroxetine in human liver microsomes (HLMs). The study focused on genotypes defined by the combination of two fully functional alleles (activity score 2, AS 2, n = 6), of one fully functional and one reduced allele (activity score 1.5, AS 1.5, n = 4) and of one fully functional and one non-functional allele (activity score 1, AS 1, n = 6), which all predict extensive metabolizer phenotype. Kinetic experiments showed that maximal reaction velocity was affected by CYP2D6 genotype, with a decrease in 33% of Vmax in AS 1 HLMs compared to AS 2 (P = 0.06). No difference in inhibition parameters Ki , KI and kinact was observed neither with the competitive inhibitor duloxetine nor with the time-dependent inhibitor paroxetine. Among the genotypes tested, we found no difference in absolute CYP2D6 microsomal levels with ELISA immunoquantification. Therefore, our results suggest that genotype-sensitive magnitude of drug-drug interactions recently observed in vivo is likely to be due to differential amounts of functional enzymes at the microsomal level rather than to a difference in inhibition potencies across genotypes, which motivates for further quantitative proteomic investigations of functional and variant CYP2D6 alleles.
Assuntos
Inibidores do Citocromo P-450 CYP2D6/farmacologia , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Microssomos Hepáticos/enzimologia , Alelos , Inibidores do Citocromo P-450 CYP2D6/farmacocinética , Desmetilação , Dextrometorfano/antagonistas & inibidores , Dextrometorfano/metabolismo , Cloridrato de Duloxetina/farmacologia , Genótipo , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Paroxetina/farmacologiaRESUMO
Formation of dextrorphan (DXT) from dextromethorphan (DXM) has been widely used to assess cytochrome P450 2D (CYP2D) activity. Additionally, the kinetics of CYP2D activity have been well characterized in the liver microsomes. However, studies in brain microsomes are limited due to the lower microsomal content and abundance of CYP2D in the brain relative to the liver. In the present study, we developed a micro-scale enzymatic incubation method, coupled with a sensitive UPLC-MS/MS assay for the quantitation of the rate of DXT formation from DXM in brain microsomes. Rat brain microsomes were incubated with different concentrations of DXM for various times. The reaction was stopped, and the proteins were precipitated by the addition of acetonitrile, containing internal standard (d3-DXT). After centrifugation, supernatant (2⯵L) was injected onto a UPLC, C18 column with gradient elution. Analytes were quantitated using triple-quadrupole MS/MS with electrospray ionization in positive ion mode. The assay, which was validated for accuracy and precision in the linear range of 0.25â¯nM to 100â¯nM DXT, has a lower limit of quantitation of 0.125â¯fmol on the column. Using our optimized incubation and quantitation methods, we were able to reduce the incubation volume (25⯵L), microsomal protein amount (5⯵g), and incubation time (20â¯min), compared with reported methods. The method was successfully applied to estimation of the Michaelis-Menten (MM) kinetic parameters of dextromethorphan-O-demethylase activity in the rat brain microsomes (mean⯱â¯SD, nâ¯=â¯4), which showed a maximum velocity of 2.24⯱â¯0.42â¯pmol/min/mg and a MM constant of 282⯱â¯62⯵M. It is concluded that by requiring far less biological material and time, our method represents a significant improvement over the existing techniques for investigation of CYP2D activity in rat brain microsomes.
Assuntos
Encéfalo/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Sistema Enzimático do Citocromo P-450/metabolismo , Dextrometorfano/metabolismo , Microssomos/metabolismo , Oxirredutases O-Desmetilantes/metabolismo , Animais , Encéfalo/citologia , Desmetilação , Dextrometorfano/análise , Dextrorfano/análise , Dextrorfano/metabolismo , Cinética , Modelos Lineares , Masculino , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/métodosRESUMO
We investigate the mechanism of time-dependent inhibition (TDI) of human cytochrome P450 2D6 (CYP2D6) by 3,4-methylenedioxymethamphetamine (MDMA, ecstasy), one of the most widespread recreational drugs of abuse. In an effort to unravel the kinetic mechanism of the formation of metabolic inhibitory complex (MIC) of CYP2D6 with MDMA-derived carbene we carried out a series of spectrophotometric studies paralleled with registration of the kinetics of time-dependent inhibition (TDI) in CYP2D6-incorporated proteoliposomes. The high amplitude of spectral signal in this system allowed us to characterize the spectral properties of the formed MIC in details and obtain an accurate spectral signature of MIC formation. This information was then used in the studies with CYP2D6-containing microsomes of insect cells (CYP2D6 Supersomes™). Our results demonstrate that in both systems the formation of the ferrous carbene-derived MIC is relatively slow, reversible and is not associated with the accumulation of the ferric carbene intermediate, as takes place in the case of CYP3A4 and podophylotoxin. Furthermore, the limited amplitude of MIC formation suggests that only a fraction (â¼50%) of spectrally detectable CYP2D6 in both proteoliposomes and Supersomes participates in the formation of MIC and is therefore involved in the MDMA metabolism. This observation reveals yet another example of a cytochrome P450 that exhibits persistent functional heterogeneity of its population in microsomal membranes. Our study provides a solid methodological background for further mechanistic studies of MIC formation in human liver microsomes and demonstrates that the potency and physiological relevance of MDMA-dependent TDI of CYP2D6 may be overestimated.