Postmortem Blood Concentrations of R - and S -Enantiomers of Methadone and EDDP in Drug Users: Influence of Co-Medication and P-glycoprotein Genotype (original) (raw)
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Methadone and methadone metabolites in postmortem specimens
Forensic Science, Medicine, and Pathology, 2008
We have determined drug/metabolite concentrations and ratios of methadone (METH) to two of its metabolites (EDDP, 2-ethylidene-1, 5-dimethyl-3, 3diphenylpyrrolidine; and EMDP, 2-ethyl-5-methyl-3, 3-diphenylpyrroline) in postmortem peripheral blood and liver tissue by liquid chromatography/tandem mass spectrometry. The assays employed deuterated internal standards and multiple reaction monitoring (MRM) techniques. The assay linear range was 0.01-2.0 mg/l for each analyte. METH, EDDP, and EMDP were determined in liver and peripheral blood from 46 methadone-positive cases. METH and EDDP were detected in all specimens, whether blood or liver. EMDP was detected, only in liver, and only 17 cases, at concentrations much lower than those of EDDP. Concentrations of METH and EDDP in blood and liver from EMDP-positive cases were in ranges higher than, but overlapping with, concentrations in blood and liver from EMDP-negative cases. These data suggest that although METH is readily demethylated and cyclized to EDDP, in vivo, conversion to EMDP may be less efficient and its accumulation in postmortem tissues may be highly individual.
Forensic Science International, 2013
Aim: Methadone (MTD) is frequently used for treatment of opiate addiction. S-MTD shows analgesic effects only in large doses, whereas R-MTD is mainly responsible for pharmacological effects. MTD clearance is primarily attributed to CYP3A4 and CYP2B6, and metabolism of the racemic compound is highly stereoselective. Aim of the present study was to investigate the enantiomeric ratios of MTD and 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in postmortem body fluids and tissues after administration of both racemic and enantiomerically pure R-MTD. It had to be established if R-MTD accumulates in body fluids and tissues after repeated dosing. Furthermore, mean femoral blood concentrations and R/Sratios of MTD and EDDP of cases participating in methadone maintenance therapy (MMT) and non-MMT cases have been compared. Methods: R-and S-MTD as well as R-and S-EDDP concentrations were determined by chiral LC-MS/MS following liquid-liquid extractions of body fluids and tissue homogenates of 16 MTD-related fatalities. Results and discussion: R/S ratios of MTD and EDDP in femoral blood ranged from 0.97 to 3.88 for MTD and from 0.68 to 1.35 for EDDP, respectively. R-MTD appeared to accumulate in all media after repeated dosing. Mean femoral blood concentrations of MTD and EDDP and mean femoral blood R/S ratio of MTD and in MMT cases were significantly higher than in non-MMT cases, whereas no difference of the mean femoral blood R/S ratios of EDDP could be observed in both groups. Conclusion: Accumulation of R-MTD might be due to repeated dosing and its longer mean half life compared to S-MTD. The enantiomeric ratios of MTD and EDDP are useful for the interpretation of postmortem concentrations and to differentiate between the consumption of racemic or enantiomerically pure R-MTD.
CYP2B6 Polymorphisms Influence the Plasma Concentration and Clearance of the Methadone S-Enantiomer
Journal of Clinical Psychopharmacology, 2011
Methadone is a racemic compound composed of the R-form and S-form enantiomers. The drug is usually used in maintenance therapy for the heroin-addicted patients. In our previous study, we found that the cytochrome P-450 (CYP) isozyme 2B6 preferentially metabolizes the S-methadone enantiomer. We thus tested whether CYP2B6 gene polymorphisms had any influence on the concentration or clearance of methadone. Ten single nucleotide polymorphisms within this gene region were evaluated in 366 patients undergoing methadone maintenance for at least 3 months. The plasma steady-state levels of racemic methadone and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine were then measured in these individuals. The rs10403955 (T allele in intron 1), rs3745274 (G allele in exon 4), rs2279345 (T allele in intron 5), and rs707265 (A allele in exon 9) CYP2B6 allele types were found to be significantly associated with a higher clearance, a lower plasma concentration, and a lower concentration-to-dosage (C/D) ratio of (S)-methadone (P G 0.0017). Two haplotype blocks of a trinucleotide haplotype (rs8100458-rs10500282-rs10403955 in intron 1) and a hexanucleotide haplotype (rs2279342-rs3745274-rs2279343-rs2279345-rs1038376-rs707265 from intron 2 to exon 9) were constructed within CYP2B6. The major combinations of T-T-T and A-G-AT A A of these particular haplotypes showed significant associations with the plasma concentrations of Smethadone and its C/D ratio (P G 0.0001, respectively). We conclude that genetic polymorphisms in the CYP2B6 gene may therefore be indicators of the clearance, plasma concentration and C/D ratio of S-methadone.
British journal of clinical pharmacology, 2014
Methadone is characterized by wide intersubject variability regarding the dose needed to obtain full therapeutic response. We assessed the influence of sociodemographic, ethnic, clinical, metabolic & genotype variables on methadone maintenance dose requirement in opioid-dependent responder patients. Eighty-one stable patients (60 men/21 women, 43.7±8.1 years, 63.1±50.9 mg/day methadone), divided into quartiles with respect to the median daily dose, were enrolled and underwent clinical examination, treatment history, liver/intestinal cytochrome P450 (CYP) 3A4 activity measured by the midazolam test, R,S-methadone trough concentration and clinically significant polymorphisms determination of the OPRM1, DRD2, COMT, ABCB1, CYP2B6, CYP3A5, CYP2C19 and CYP2D6 genes. Methadone maintenance dose was correlated to the highest dose ever used (r(2) =0.57, P<0.0001). Fractioned methadone intake (OR 4.87, 95%CI 1.27-18.6, P=0.02), body weight (OR 1.57, 95%CI 1.01-2.44, P=0.04), history of coca...
Revisiting Pharmacokinetics and Pharmacogenetics of Methadone in Healthy Volunteers
Drug Discovery and Development - New Advances, 2018
Methadone acts as a μ opioid agonist, a serotonin and norepinephrine reuptake inhibitor, and a noncompetitive N-methyl-D-aspartate receptor antagonist. These actions altogether are responsible for its efficacy in the management of chronic pain. It is available as a racemic mixture of (R)-and (S)-methadone, both being stereoisomers responsible for its analgesic effect. Methadone elimination occurs mainly through metabolism in the liver by CYP3A4, CYP2B6, and CY2C19 and to a lesser extent by CYP2D6 and in the intestine by CYP3A4. The relative intestinal content of CYP2B6 and CY2C19 is unknown but it seems that CYP2B6 is not present at the intestine. CYP3A4, CYP2B6, and CYP2C19 convert methadone mainly into 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine(EDDP). CYP2B6 and CYP2C19 are stereoselective to Sand R-enantiomer, respectively. The pharmacokinetic study carried out in healthy volunteers by our research group confirmed that MTD undergoes recirculation via gastric secretion and intestinal reabsorption and revealed that the drug is extensively metabolized in the liver but intestinal metabolism is not only relevant but also stereoselective. Polymorphisms of the CYP2B6 and CYP2C19 isoenzymes and their relationship with the pharmacokinetics of MTD were also assessed.
Fatal Methadone Toxicity: Potential Role of CYP3A4 Genetic Polymorphism
Journal of Analytical Toxicology, 2014
Methadone is difficult to administer as a therapeutic agent because of a wide range of interindividual pharmacokinetics, likely due to genetic variability of the CYP450 enzymes responsible for metabolism to its principal metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). CYP3A4 is one of the primary CYP450 isoforms responsible for the metabolism of methadone to EDDP in humans. The purpose of this study was to evaluate the role of CYP3A4 genetic polymorphisms in accidental methadone fatalities. A study cohort consisting of 136 methadone-only and 92 combined methadone/benzodiazepine fatalities was selected from cases investigated at the West Virginia and Kentucky Offices of the Chief Medical Examiner. Seven single nucleotide polymorphisms (SNPs) were genotyped within the CYP3A4 gene. Observed allelic and genotypic frequencies were compared with expected frequencies obtained from The National Center for Biotechnology Information dbSNP database. SNPs rs2242480 and rs2740574 demonstrated an apparent enrichment within the methadone-only overdose fatalities compared with the control group and the general population. This enrichment was not apparent in the methadone/benzodiazepine cases for these two SNPs. Our findings indicate that there may be two or more SNPs on the CYP3A4 gene that cause or contribute to the methadone poor metabolizer phenotype.
Journal of Analytical Toxicology, 2008
A chiral liquid chromatography-tandem mass spectrometry method was developed for the measurement of methadone and 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolinium (EDDP) enantiomers in postmortem blood. Blood (0.100 g) was mixed with water and aqueous methadone-d 3 and EDDP-d 3 solutions. After addition of NaOH, extraction was carried out with butyl acetate. Chromatographic separation was performed on a chiral-AGP analytical column (100 mm × 4.0-mm i.d., 5 µm) with a mobile phase of acetonitrile/ammonium acetate buffer (10 mM, pH 7.0, 22:78, v/v). A Quattro micro mass spectrometer was operated in the positive ion mode with an electrospray source. Multiple reaction monitoring was used with two transitions for each compound. The calibration curves were linear over the range from 0.001 to 2.5 mg/kg blood per enantiomer, and the limit of detection was 0.001 mg/kg, and the limit of quantitation was 0.003 mg/kg for all compounds. The precision (coefficient of variation%) was below 6.3%, and the trueness was 88-104% for all compounds. R-and S-methadone were measured in femoral blood from 10 postmortem cases. The R-methadone concentrations extended from 0.006 to 1.235 mg/kg with a median of 0.41 mg/kg, and the S-methadone concentrations ranged from 0 to 0.794 mg/kg (median 0.33 mg/kg). The median R/S-ratio was 1.46 (total range from 1.00 to 2.62), which tends to be higher than that reported in plasma of living subjects. Experimental R/S-methadone and R/S-EDDP (R,S-2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolinium) were purchased from Lipomed (Arlesheim, Switzerland) as HCl and HClO 4 salts, respectively. Purity according to the manufacturer's declaration was 99.99% (± 0.5%). The internal standards (IS) R/S-methadone-d 3 and R/S-EDDP-d 3 were from CIL (Andover, MA) and Lipomed, respectively. Stock solutions of methadone and EDDP were prepared in methanol (1 mg/mL). Dilutions were made in water, and calibrators were produced by adding dilutions to whole blood. The methanol amount accounted for less than 0.1% of Abstract Downloaded from https://academic.oup.com/jat/article/32/7/499/874261 by guest on 30 April 2021
Pharmacogenetics of Methadone Response
Molecular diagnosis & therapy, 2017
The efficacy of methadone maintenance treatment (MMT) in opioid use disorder is well established but responses vary. The influence of methadone pharmacodynamics and pharmacokinetics on dose requirements and program outcomes remains controversial despite the increasing number of studies evaluating genetic influences on response to methadone treatment. Furthermore, patients require different doses (usually between 60 and 100 mg/day), and there are no clear data on a plasma concentration associated with treatment success. We review the evidence regarding the influence of genetics on pharmacokinetic and pharmacodynamic factors in terms of MMT outcome. We also analyse the influence of genetics on the occurrence of severe adverse events such as respiratory depression and ventricular arrhythmia in methadone treatment. The outcomes of MMT may be influenced by a combination of environmental, drug-induced, and genetic factors. The influence of pharmacokinetic genetic variability can be clinic...
PLoS ONE, 2011
Although the efficacy of methadone maintenance treatment (MMT) in opioid dependence disorder has been well established, the influence of methadone pharmacokinetics in dose requirement and clinical outcome remains controversial. The aim of this study is to analyze methadone dosage in responder and nonresponder patients considering pharmacogenetic and pharmacokinetic factors that may contribute to dosage adequacy. Opioid dependence patients (meeting Diagnostic and Statistical Manual of Mental Disorders, [4 th Edition] criteria) from a MMT community program were recruited. Patients were clinically assessed and blood samples were obtained to determine plasma concentrations of (R,S)-, (R) and (S)-methadone and to study allelic variants of genes encoding CYP3A5, CYP2D6, CYP2B6, CYP2C9, CYP2C19, and Pglycoprotein. Responders and nonresponders were defined by illicit opioid consumption detected in random urinalysis. The final sample consisted in 105 opioid dependent patients of Caucasian origin. Responder patients received higher doses of methadone and have been included into treatment for a longer period. No differences were found in terms of genotype frequencies between groups. Only CYP2D6 metabolizing phenotype differences were found in outcome status, methadone dose requirements, and plasma concentrations, being higher in the ultrarapid metabolizers. No other differences were found between phenotype and responder status, methadone dose requirements, neither in methadone plasma concentrations. Pharmacokinetic factors could explain some but not all differences in MMT outcome and methadone dose requirements.
Clinical Pharmacology & Therapeutics, 2006
Background and Objective: The in vivo implication of various cytochrome P450 (CYP) isoforms and of P-glycoprotein on methadone kinetics is unclear. We aimed to thoroughly examine the genetic factors influencing methadone kinetics and response to treatment. Methods: Genotyping for CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, ABCB1, and UGT2B7 polymorphisms was performed in 245 patients undergoing methadone maintenance treatment. To assess CYP3A activity, the patients were phenotyped with midazolam. Results: The patients with lower CYP3A activity presented higher steady-state trough (R,S)-methadone plasma levels (4.3, 3.0, and 2.3 ng/mL • mg for low, medium, and high activity, respectively; P ؍ .0002). As previously reported, CYP2B6*6/*6 carriers had significantly higher trough (S)-methadone plasma levels (P ؍ .0001) and a trend toward higher (R)-methadone plasma levels (P ؍ .07). CYP2D6 ultrarapid metabolizers presented lower trough (R,S)-methadone plasma levels compared with the extensive or intermediate metabolizers (2.4 and 3.3 ng/mL • mg, respectively; P ؍ .04), whereas CYP2D6 poor metabolizer status showed no influence. ABCB1 3435TT carriers presented lower trough (R,S)-methadone plasma levels (2.7 and 3.4 ng/mL • mg for 3435TT and 3435CC carriers, respectively; P ؍ .01). The CYP1A2, CYP2C9, CYP2C19, CYP3A5, and UGT2B7 genotypes did not influence methadone plasma levels. Only CYP2B6 displayed a stereoselectivity in its activity. Conclusion: In vivo, CYP3A4 and CYP2B6 are the major CYP isoforms involved in methadone metabolism, with CYP2D6 contributing to a minor extent. ABCB1 genetic polymorphisms also contribute slightly to the interindividual variability of methadone kinetics. The genetic polymorphisms of these 4 proteins had no influence on the response to treatment and only a small influence on the dose requirement of methadone.