Plasma Methadone Research Articles

Evaluation of methadone concentrations in bitches and in umbilical cords after epidural or systemic administration for caesarean section: A randomized trial

ObjectiveTo measure plasma methadone concentrations in bitches and the umbilical cords of their puppies after systemic or epidural administration. Study designProspective, randomized, clinical study. AnimalsA total of 27 healthy pregnant female dogs undergoing caesarean section, 4.3 ± 2.3 years of age and weighing 19.9 ± 13.2 kg. MethodsThe dogs were randomly divided into three groups: 1) intramuscular methadone (0.3 mg kg–1) (group MET; n = 9); 2) epidural methadone (0.1 mg kg–1) (group METEPI; n = 9); and 3) epidural lidocaine (4.4 mg kg–1) [group CON (control group); n = 9]. Ten minutes before induction, methadone was administered intramuscularly to the group MET dogs. Anaesthesia was induced with propofol and maintained with isoflurane. Cardiovascular and respiratory parameters were monitored throughout the anaesthesia. After induction, epidural anaesthesia was administered to dogs in groups METEPI and CON. Before any treatment (T0) and, as soon as the last foetus was removed from the uterus (T1), venous blood samples were collected from each dog into heparinized tubes; the umbilical cords were collected and stored at –80 °C until pharmacological analysis was carried out. The samples were analysed using ultra performance liquid chromatography. ResultsThe cardiorespiratory parameters of the bitches and of the puppies at birth, and the Apgar scores did not differ significantly between groups. At T1 both the median maternal methadone plasma concentration and the median methadone umbilical cord concentration were higher in group MET compared to group METEPI (p = 0.0018 and p = 0.004, respectively). The maternal plasma concentration was higher than the concentration in the umbilical cords (p = 0.05) in group METEPI but not in group MET (p = 0.25). Conclusions and clinical relevanceEpidural methadone (0.1 mg kg–1) administered to bitches undergoing caesarean section is associated with lower umbilical cord methadone concentrations as compared with intramuscularly administered methadone at higher dosages (0.3 mg kg–1).

Methadone Dosage and Plasma Levels, SNPs of OPRM1 Gene and Age of First Drug Use Were Associated With Outcomes of Methadone Maintenance Treatment.

Objective: To explore the association between methadone dosage, plasma drug concentration, SNPs of μ-opioid receptor gene (OPRM1), ATP-binding cassette subfamily B member 1 gene (ABCB1), and methadone maintenance treatment (MMT) response.Method: A total of 240 Chinese Han participants receiving MMT were recruited from Shanghai. Nine single nucleotide polymorphisms (SNPs) of the OPRM1 gene and three SNPs of the ABCB1 gene were genotyped, plasma methadone concentration was detected, and a morphine urine test was taken from all subjects.Results: Methadone dosage, plasma methadone concentration, and negative rate of morphine urine test of retention participants were significantly higher, although the addiction severity index (ASI) was not significantly different between the two groups. A allele and AA genotype carriers of rs562859 (OPRM1 gene) had better compliance of MMT, and AA genotype carriers had a higher negative rate of morphine urine test. However, the difference was not significant after adjusting influence factors (age, sex, and methadone dosage). GG genotype carriers of rs3192723 (OPRM1 gene) had a significantly lower negative rate of morphine urine test, and the difference was still significant after adjusting influence factors. Logistic regression analysis showed that methadone-free trough concentration (OR = 0.910, p = 0.023) and AA genotype of rs526859 (OR = 0.580, p = 0.037) were associated with better compliance of MMT. After Bonferroni correction, only free trough concentration of methadone was negatively correlated with MMT compliance. The SNPs rs6912029 (OR = 0.021, p = 0.066) and rs6902403 (OR = 0.910, p = 0.007) of the OPRM1 gene, age at first use (OR = 1.118, p = 0.005), and average methadone dosage (OR = 1.033, p = 0.045) were associated with MMT effect. After Bonferroni correction, average methadone dosage was no longer correlated with MMT effect.Conclusion: Dosage of methadone, plasma methadone concentration, several SNPs (rs3192723, rs6912029, rs6902403) of the OPRM1 gene, and age of first drug use were associated with better MMT outcomes.

Ultrasonically formation of supramolecular based ultrasound energy assisted solidification of floating organic drop microextraction for preconcentration of methadone in human plasma and saliva samples prior to gas chromatography–mass spectrometry

In this work, an ultrasonic-assisted supramolecular based on solidification of floating organic drop microextraction (UA-SM-SFO-ME) was developed as a green method for preconcentration of methadone prior to gas chromatography–mass spectrometry (GC–MS). The supramolecular solvent aggregates containing reverse micelles of 1-dodecanol in tetrahydrofuran (THF) were formed by ultrasonication that subsequently dispersed in the sample solution. Ultrasonic waves caused the fast formation of supramolecular solvent aggregates. In this work, ultrasonication was used in two phases: First phase, the formation of reverse micelles and the second phase, the dispersion of supramolecular solvent in the sample solution. Actually, ultrasonication was basic of this presented work. In order to provide the highest extraction efficiency, the influence of various parameters on the method performance (supramolecular solvent type and volume, disperser solvent condition, pH, extraction time and salt concentration) was investigated. Based on the obtained optimum conditions, the limits of detection (LODs) and the limits of quantitation (LOQs) were obtained 0.5–1.2 µg L−1 and 1.2–2.5 µg L−1 with preconcentration factors in the range of 182–191, in water and biological samples, respectively. Subsequently, the method was assessed for preconcentration of the methadone in human plasma and saliva samples.

Simultaneous chiral separation of tramadol and methadone in tablets, human urine, and plasma by capillary electrophoresis using maltodextrin as the chiral selector

The stereoselective analysis and separation of racemic drugs play an important role in pharmaceutical industry to eliminate the unwanted isomer and find the right therapeutic control for the patient. Present study suggests a maltodextrin-modified capillary electrophoresis method for a single-run chiral separation of two closely similar opiate pain relief drugs: tramadol (TRA) and methadone (MET). The best separation method possible for the both enantiomers was achieved on an uncoated fused-silica capillary at 25°C using 100 mM phosphate buffer (pH8.0) containing 20% (wv-1 ) maltodextrin with dextrose equivalent of 4-7 and an applied voltage of 16kV. Under optimal conditions, the baseline resolution of TRA and MET enantiomers was obtained in less than 12minutes. The relative standard deviations (n=3) of 20μgmL-1 TRA and MET were 2.28% and 3.77%, respectively. The detection limits were found to be 2μgmL-1 for TRA and 1.5μgmL-1 for MET. This method was successfully applied to the measurement of drugs concentration in their tablets, urine, and plasma samples.

Methadone Dose Adjustments, Plasma R-Methadone Levels and Therapeutic Outcome of Heroin Users: A Randomized Clinical Trial

Aims: We aimed to improve the retention in treatment and therapeutic outcome of methadone maintenance treatment (MMT) patients by adjusting the oral methadone dose in order to reach a “target” plasma R-methadone level (80–250 ng/mL). Methods: A multicenter randomized controlled trial was organized. Results: The intention-to-treat statistical analysis showed that repeated dose adjustments performed in order to obtain therapeutic plasma R-methadone levels did not improve retention in treatment of heroin-dependent patients. However, patients having plasma methadone levels in the “target range” at the beginning of the study had a better retention in treatment than controls. Furthermore, patients succeeding in keeping plasma R-methadone target levels (per protocol analysis) remained in treatment and improved their social scores better than controls. ­Conclusion: Although the primary endpoint of this study was not demonstrated, a post hoc and a per protocol analysis suggested that patients in MMT with plasma R-methadone concentrations in the target range have a better therapeutic outcome than controls.

Corrected QT Interval and Methadone Dose and Concentrations in Pregnant and Postpartum Women

Methadone is a standard treatment for opioid dependence in pregnancy; however, its impact on maternal corrected QT interval (QTc) has not been evaluated. We studied the association between methadone dose and enantiomer-specific plasma concentrations and QTc among pregnant and postpartum women and newborns. We assessed the relevance of QTc screening guidelines for pregnant women and infants. From 2006 to 2008, plasma methadone concentrations were measured during pregnancy, postpartum, and in cord blood in women treated for opioid dependence at a single treatment program. Electrocardiograms (ECGs) were obtained at peak methadone concentrations in mothers and within 48 hours of birth for infants. Pearson correlations were performed at each time point for QTc and R-methadone, S-methadone, and total methadone concentrations and ratio of R-methadone/S-methadone concentrations. Mean (SD) daily methadone dose for the 25 women was 94.2 (39.1) mg during pregnancy and 112.5 (46.6) mg postpartum. During the third trimester, higher methadone dose and R-methadone concentration correlated with longer QTc (Pearson r = 0.67, P < .001 and Pearson r = 0.49, P = .02, respectively), while S-methadone concentration, R-methadone/S-methadone concentration ratio, and total methadone concentration did not. Postpartum, QTc did not significantly correlate with dose or enantiomer concentrations. Infant QTc did not correlate with maternal dose at delivery or enantiomer-specific cord methadone concentrations. In pregnant and postpartum women, 13% and 17%, respectively, had QTc ≥ 450 ms, as did 19% of infants. QTc correlated with dose and R-methadone concentration during the third trimester. However, longer QTc was common among women during and after pregnancy. Given the relatively high rate of QTc > 450 ms, an ECG before and after methadone initiation is advisable for pregnant and postpartum women.

Increased DNA Methylation of ABCB1, CYP2D6, and OPRM1 Genes in Newborn Infants of Methadone-Maintained Opioid-Dependent Mothers

To investigate whether in utero opioid exposure, which has been linked to adverse neurodevelopmental and social outcomes, is associated with altered DNA methylation of opioid-related genes at birth. Observational cohort study of 21 healthy methadone-maintained opioid-dependent mother-infant dyads consecutively delivered at >36 weeks of gestation, and 2 comparator groups: smoking, "deprived" opioid-naïve mother-infant dyads (n = 17) and nonsmoking, "affluent" opioid-naïve mother-infant dyads (n = 15). DNA methylation of ABCB1, CYP2D6, and OPRM1 genes for mothers and babies was determined from buccal swabs. Plasma methadone concentrations were additionally measured for methadone-maintained opioid-dependent mothers. DNA methylation for ABCB1 and CYP2D6 was similar in opioid-naïve infants compared with their mothers, but was less for OPRM1 (3 ± 1.6% vs 8 ± 1%, P < .0005). Opioid-exposed newborns had similar DNA methylation to their mothers for all genes studied and greater methylation of ABCB1 (18 ± 4.8% vs 3 ± 0.5%), CYP2D6 (92 ± 1.2% vs 89 ± 2.4%), and OPRM1 (8 ± 0.3% vs 3 ± 1.6%) compared with opioid-naïve newborns (P < .0005 for all 3 genes). Infant DNA methylation was not related to birth weight, length of hospital stay, maternal smoking, dose or plasma concentration of methadone at delivery, or postcode of residence. In utero exposure to opioids is associated with increased methylation of opioid-related genes in the newborn infant. It is not clear whether these findings are due to opioid exposure per se or other associated lifestyle factors.

Can Saliva and Plasma Methadone Concentrations Be Used for Enantioselective Pharmacokinetic and Pharmacodynamic Studies in Patients With Advanced Cancer?

PurposeMethadone is a potent analgesic used to treat refractory cancer pain. It is administered as a racemic mixture, with the l-enantiomer being primarily a μ-receptor agonist, whereas the d-enantiomer is an N-methyl-d-aspartate antagonist and inhibits serotonin and norepinephrine reuptake. Dose requirements vary greatly among patients to achieve optimal pain control and to avoid the risk of adverse effects. The relationship between plasma and saliva methadone enantiomer concentrations was investigated to determine if saliva could be a substitute for plasma in pharmacodynamic and pharmacokinetic studies for clinical monitoring and dose optimization of methadone in patients with advanced cancer. MethodsPatients with advanced cancer who were prescribed varying doses of oral methadone for pain management were recruited to obtain paired plasma and saliva samples. Pain scores were recorded at the time of sampling. The total and unbound plasma and saliva concentrations of the l- and d-enantiomers of methadone were quantified by using an HPLC-MS/MS method. The relationship between plasma (total and unbound) and saliva concentrations were compared. The saliva-to-plasma concentration ratio was compared versus the dose administered and the time after dosing for both enantiomers. The association of methadone concentrations with reported pain scores was compared by using a Mann-Whitney U test for significance. FindingsFifty patients receiving a mean dose of 11mg/d of methadone provided 151 paired plasma and saliva samples. The median age of the population was 61 years with an interquartile range of 53-71 years with total body weight ranging from 59-88 kg. Median (interquartile) total plasma concentrations for l- and d-methadone were 50.78 ng/mL (30.6–113.0 ng/mL) and 62.0 ng/mL (28.7–116.0 ng/mL), respectively. Median (interquartile range) saliva concentrations for l- and d-methadone were 81.5 ng/mL (28.0–203.2 ng/mL) and 44.2 (16.2–149.7 ng/mL). No relationship could be established between plasma and saliva concentrations for l- and d-methadone (r2 = 0.35 and 0.25). The saliva-to-plasma concentration analyzed with the methadone dose showed higher saliva concentrations at lower doses. Dose-normalized saliva concentrations followed a similar pattern over time compared with plasma concentrations. No correlation was found between l-methadone plasma, d-methadone plasma, l-methadone saliva, d-methadone saliva concentrations, and pain score. ImplicationsSaliva concentration was not a better predictor of pain control than plasma concentration for dose optimization and monitoring studies of methadone in patients with cancer. Although the saliva-to-plasma ratio of the concentration of methadone enantiomers was stable across the dosing range, due to the variability in individual saliva-to-plasma ratios, saliva sampling may not be a valid substitute in pharmacokinetic studies of methadone in cancer.

Variations in Infant CYP2B6 Genotype Associated with the Need for Pharmacological Treatment for Neonatal Abstinence Syndrome in Infants of Methadone-Maintained Opioid-Dependent Mothers.

Background Neonatal abstinence syndrome (NAS) in infants of methadone-maintained opioid-dependent (MMOD) mothers cannot be predicted in individual cases. We investigated whether variation in infant genotype is associated with severity of NAS. Methods This is a pilot observational cohort study of 21 MMOD mothers and their newborns. Infant buccal swabs were obtained soon after delivery, together with a maternal blood sample for the determination of maternal plasma methadone concentration. Genomic variation in five opioid-related genes (ABCB1, COMT, CYP2B6, CYP2D6, and OPRM1) was ascertained from infant buccal swabs and related to need for pharmacological treatment of NAS. Results Out of 21 infants, 11 (52%) required treatment for NAS. Mothers of treated infants tended to have been prescribed higher doses of methadone, but plasma methadone concentrations did not differ between mothers of treated or untreated babies. Treated and untreated babies did not differ in terms of method of feeding. Treated infants were more likely to carry the normal (homozygous) allele at 516 and 785 regions of CYP2B6 gene (p = 0.015 and 0.023, respectively). There were no differences in any other genes between infants who did or did not require treatment for NAS. Conclusion Genomic variation in CYP2B6 may explain, at least in part, severity of NAS.

Current Concepts in Methadone Metabolism and Transport.

Methadone is a cornerstone therapy for opioid addiction and a public health strategy for HIV/AIDS and hepatitis C reduction. Methadone is also used for acute and chronic pain. As use for chronic pain has grown, so too have adverse events. Constitutive and acquired (drug interactions) inter- and intraindividual variability in methadone pharmacokinetics and pharmacodynamics confounds reliable clinical use. Identification of enzymes and transporters responsible for methadone disposition has been a long-sought ideal. Initial in vitro studies identified CYP3A4 as metabolizing methadone. Subsequently, by extrapolation, CYP3A4 was long assumed to be responsible for clinical methadone disposition. However, CYP2B6 is also a major catalyst of methadone metabolism in vitro. It has now been unequivocally established that CYP2B6, not CYP3A4, is the principal determinant of methadone metabolism, clearance, elimination, and plasma concentrations in humans. Methadone disposition is susceptible to inductive and inhibitory drug interactions. CYP2B6 genetics also influences methadone metabolism and clearance, which were diminished in CYP2B6*6 carriers and increased in CYP2B6*4 carriers. CYP2B6 genetics can explain, in part, interindividual variability in methadone metabolism and clearance. Thus, both constitutive variability due to CYP2B6 genetics, and CYP2B6-mediated drug interactions, can alter methadone disposition, clinical effect, and drug safety. Methadone is not a substrate for major influx or efflux transporters.

Dose, Plasma Level, and Treatment Outcome Among Methadone Patients in Shanghai, China

The purpose of this study was to investigate the blood levels of methadone in participants receiving methadone for the treatment of opioid dependence. After stabilization on methadone for four weeks, blood samples from 95 participants were collected between treatment weeks 4 and 12, before and after receiving doses of methadone, and its blood levels were measured. A multiple linear regression model was used to examine the association between methadone blood levels and the outcomes of methadone maintenance treatment (MMT). Outcome differences between participants who had high (≥2) or low (<2) peak-to-trough ratios were also compared using an independent sample t-test. The blood level of methadone was not correlated with the clinical outcome of MMT with the moderate range of doses given. However, the retention of patients who had a free peak-to-trough ratio >2 was significantly poorer than those whose ratio was <2. Thus, monitoring plasma methadone levels is unlikely to be effective for guiding dosing decisions in situations where compliance with MMT is already very high or when the methadone dose is no longer the dominant factor in determining the clinical outcome. However, monitoring plasma methadone levels is still helpful for guiding the dosage for patients with a rapid metabolism.

Effect of intramuscular methadone on pharmacokinetic data and thermal and mechanical nociceptive thresholds in the cat.

Objectives The aim of the study was to assess simultaneous pharmacokinetics and thermal and mechanical antinociception after intramuscular methadone (0.6 mg/kg) in 10 cats. Methods Thermal and mechanical threshold (TT and MT, respectively) testing and blood collection were conducted at baseline and up to 24 h after administration. Methadone plasma concentrations were determined by liquid chromatography-tandem mass spectrometry and pharmacokinetic parameters were estimated by a non-compartmental method. TT and MT were analysed using ANOVA ( P <0.05). Time of maximum plasma concentration (Tmax), time of onset of antinociception and time of reaching cut-out threshold (TT 55°C; MT 30 Newtons [N]) were determined. Results TT and MT increased above baseline from 20-240 mins and 5-40 mins, respectively, after intramuscular (IM) administration ( P <0.005). Mean maximum delta T (measured as TT minus baseline threshold) was 7.9°C (95% confidence interval [CI] 4.3-11.6) at 60 mins and mean maximum delta F (measured as MT minus baseline threshold) was 4.2 (95% CI 1.6-6.7) N at 45 mins. IM methadone concentration-time data decreased curvilinearly, and gave a clearance estimate of mean 9.1 ml/kg/min (range 5.2-15.7) with median Tmax at 20 mins (range 5-360 mins). Conclusions and relevance IM data followed classical disposition and elimination in all cats. Plasma concentrations after IM administration were associated with an antinociceptive effect, including negative hysteresis. These data can be used for devising dosing schedules for methadone in clinical feline practice.

Molecularly imprinted polymer-sol-gel tablet toward micro-solid phase extraction: I. Determination of methadone in human plasma utilizing liquid chromatography–tandem mass spectrometry

In the present work molecularly imprinted sol-gel tablet (MIP-Tablet) was prepared. The MIP-sol-gel was prepared as a thin layer on polyethylene material in a tablet form. Methadone-d9 was selected as the template and 3-(propylmethacrylate)-trimethoxysilane was used as precursor. MIP-Tablet was applied for micro-solid phase extraction (μ-SPE). The MIP-Tablet was used for the determination of methadone in human plasma samples utilizing liquid chromatography-tandem mass spectrometry; and each tablet could be used twenty times. The extraction time was 10 min while desorption time was 6 min. Factors affecting the extraction efficiency such as desorption solvents, sample pH, salt addition, extraction time, desorption time and adsorption capacity were investigated. The calibration curves were obtained within the range of 5–5000 ng/mL using methadone in human plasma samples. The coefficients of determination (r2) values were ≥0.999 for all runs and the extraction recovery was >80%. The accuracy values for quality control samples varied from +3.6 to +9.7% and the inter-day precision (RSD %) values were ranged from 5.0 to 8.0%. The limit of detection was 1.0 ng/mL and the lower limit of quantification was 5 ng/mL utilizing methadone in human plasma samples.

Methadone Pharmacogenetics

Background: Interindividual variability in methadone disposition remains unexplained, and methadone accidental overdose in pain therapy is a significant public health problem. Cytochrome P4502B6 (CYP2B6) is the principle determinant of clinical methadone elimination. The CYP2B6 gene is highly polymorphic, with several variant alleles. CYP2B6.6, the protein encoded by the CYP2B6*6 polymorphism, deficiently catalyzes methadone metabolism in vitro. This investigation determined the influence of CYP2B6*6, and other allelic variants encountered, on methadone concentrations, clearance, and metabolism. Methods: Healthy volunteers in genotype cohorts CYP2B6*1/*1 (n = 21), CYP2B6*1/*6 (n = 20), and CYP2B6*6/*6 (n = 17), and also CYP2B6*1/*4 (n = 1), CYP2B6*4/*6 (n = 3), and CYP2B6*5/*5 (n = 2) subjects, received single doses of IV and oral methadone. Plasma and urine methadone and metabolite concentrations were determined by tandem mass spectrometry. Results: Average S-methadone apparent oral clearance was 35 and 45% lower in CYP2B6*1/*6 and CYP2B6*6/*6 genotypes, respectively, compared with CYP2B6*1/*1. R-methadone apparent oral clearance was 25 and 35% lower in CYP2B6*1/*6 and CYP2B6*6/*6 genotypes, respectively, compared with CYP2B6*1/*1. R- and S-methadone apparent oral clearance was threefold and fourfold greater in CYP2B6*4 carriers. IV and oral R- and S-methadone metabolism was significantly lower in CYP2B6*6 carriers compared with that of CYP2B6*1 homozygotes and greater in CYP2B6*4 carriers. Methadone metabolism and clearance were lower in African Americans in part because of the CYP2B6*6 genetic polymorphism. Conclusions: CYP2B6 polymorphisms influence methadone plasma concentrations, because of altered methadone metabolism and thus clearance. Genetic influence is greater for oral than IV methadone and S- than R-methadone. CYP2B6 pharmacogenetics explains, in part, interindividual variability in methadone elimination. CYP2B6 genetic effects on methadone metabolism and clearance may identify subjects at risk for methadone toxicity and drug interactions.

Simultaneous Determination of Methadone, Fluoxetine, Venlafaxine and Their Metabolites in Rat Plasma by UPLC–MS/MS for Drug Interaction Study

A sensitive and rapid ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed to determine methadone, EDDP, fluoxetine, norfluoxetine, venlafaxine and O-desmethylvenlafaxine in rat plasma using diazepam as the internal standard (IS). Sample preparation was accomplished through a simple one-step protein precipitation with 200 µL acetonitrile added to 100 µL rat plasma. The analytes were separated on an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm) and the mobile phase was acetonitrile and 0.1 % formic acid in water in gradient elution at a flow rate of 0.40 mL min−1. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions. The linearity of this method was found to be within the concentration range of 0.5–250.0 ng mL−1 for methadone, EDDP, fluoxetine and norfluoxetine and 0.4–200.0 ng mL−1 for venlafaxine and O-desmethylvenlafaxine, respectively. The limit of quantification (LOQ) was 0.5 ng mL−1 for methadone, EDDP, fluoxetine and norfluoxetine and 0.4 ng mL−1 for venlafaxine and O-desmethylvenlafaxine, respectively. A time period of 3 min was needed for an analytical run. The method described above was successfully applied to the subsequent pharmacokinetic study.

Quantitative determination of the enantiomers of methadone in human plasma and saliva by chiral column chromatography coupled with mass spectrometric detection

Methadone is a potent lipophilic synthetic opioid that is effective in the treatment of cancer pain and perceived benefit in difficult pain control scenarios (especially in cases of neuropathic pain). The use of methadone in clinical practice is challenging however, due to the narrow therapeutic window and large inter- and intra-individual variability in therapeutic response. Quantitation of the enantiomers d- and l-methadone (d- and l-MTD) in plasma and saliva provides a basis for studying its pharmacokinetics in patients with cancer and for monitoring efficacy, toxicity and side-effects. This assay involves quantitation of the enantiomers of methadone using their respective deuterated internal standards, in plasma and saliva matrices with no impact of ion suppression in either matrix. The analytical recoveries of d- and l-MTD from the saliva collection devices (Salivette®) are optimised in this novel method with an accurate and simple extraction method employing dichloromethane. Optimal enantioselective separations were achieved using an α1-acid glycoprotein chiral stationary phase and triple quadrupole tandem mass spectrometer. Linearity was demonstrated over 0.05–1000µg/L for both enantiomers in plasma and in saliva with correlation coefficients greater than 0.998. The lower limit of quantitation (LLOQ) was determined to be 0.1µg/L in plasma and saliva for d- and l-MTD. Accuracy of the method ranges from 100% to 106% even at the LLOQ and total precision, expressed as the coefficient of variation, was between 0.2% and 4.4% for both analytes in both matrices. A simple one step extraction procedure resulted in recoveries greater than 95% for both analytes, at concentrations as low as 0.5µg/L, from the Salivette®. The validated method was applied successfully in 14 paired plasma and saliva samples obtained from adult patients with cancer pain receiving methadone.

Pharmacokinetics of Oral Methadone in the Treatment of Neonatal Abstinence Syndrome: A Pilot Study

To characterize the population pharmacokinetics of oral methadone in neonates requiring pharmacologic treatment of neonatal abstinence syndrome and to develop a pharmacokinetic (PK) model toward an evidence-based treatment protocol. Based on a methadone dosing protocol, serum concentrations of methadone and its metabolites were assessed by high performance liquid chromatography-tandem mass spectrometry from dried blood spots. Population PK analysis was performed to determine the volume of distribution and clearance of oral methadone. Methadone plasma concentration-time profiles were simulated from the deduced PK model to optimize the dosing regimen. There was substantial interindividual variability in methadone concentrations. Blood concentrations of methadone were best described by a 1-compartment model with first-order absorption. The population mean estimates (coefficient of variation percentage) for oral clearance and volume of distribution were 8.94 (103%) L/h/70 kg and 177 (133%) L/70 kg, respectively. Optimized dosing strategies were developed based on the simulated PK profiles. We suggest a starting dose of 0.1 mg/kg per dose every 6 hours for most patients requiring pharmacologic treatment of neonatal abstinence syndrome followed by an expedited weaning phase. The proposed dosing regimen may reduce the cumulative dose of opioid and shorten the length of hospitalization. Future studies should aim to validate the simulated dosing schemes with clinical data and expand our understanding of the between-patient PK variability. ClinicalTrials.gov: NCT01754324.

Differences in Methadone Metabolism by CYP2B6 Variants.

Methadone is a long-acting opioid with considerable unexplained interindividual variability in clearance. Cytochrome P450 2B6 (CYP2B6) mediates clinical methadone clearance and metabolic inactivation via N-demethylation to 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). Retrospective studies suggest that individuals with the CYP2B6*6 allelic variant have higher methadone plasma concentrations. Catalytic activities of CYP2B6 variants are highly substrate- and expression-system dependent. This investigation evaluated methadone N-demethylation by expressed human CYP2B6 allelic variants in an insect cell coexpression system containing P450 reductase. Additionally, the influence of coexpressing cytochrome b5, whose role in metabolism can be inhibitory or stimulatory depending on the P450 isoform and substrate, on methadone metabolism, was evaluated. EDDP formation from therapeutic (0.25-1 μM) R- and S-methadone concentrations was CYP2B6.4 ≥ CYP2B6.1 ≥ CYP2B6.5 >> CYP2B6.9 ≈ CYP2B6.6, and undetectable from CYP2B6.18. Coexpression of b5 had small and variant-specific effects at therapeutic methadone concentrations but at higher concentrations stimulated EDDP formation by CYP2B6.1, CYP2B6.4, CYP2B6.5, and CYP2B6.9 but not CYP2B6.6. In vitro intrinsic clearances were generally CYP2B6.4 ≥ CYP2B6.1 > CYP2B6.5 > CYP2B6.9 ≥ CYP2B6.6. Stereoselective methadone metabolism (S>R) was maintained with all CYP2B6 variants. These results show that methadone N-demethylation by CYP2B6.4 is greater compared with CYP2B6.1, whereas CYP2B6.9 and CYP2B6.6 (which both contain the 516G>T, Q172H polymorphism), are catalytically deficient. The presence or absence of b5 in expression systems may explain previously reported disparate catalytic activities of CYP2B6 variants for specific substrates. Differences in methadone metabolism by CYP2B6 allelic variants provide a mechanistic understanding of pharmacogenetic variability in clinical methadone metabolism and clearance.

Relationship between plasma concentrations of the l-enantiomer of methadone and response to methadone maintenance treatment

This study evaluated the relationship between the plasma concentration of l-methadone and response to methadone in real-world patients, in order to identify a minimum plasma concentration above which methadone treatment is effective.Ninety-four patients with opioid dependence under maintenance methadone treatment were consecutively recruited. Response was defined as negative urine analyses in the three weeks prior to the blood sampling. The percentage of participants with a plasma l-methadone concentration between 100 and 250ng/ml was 54.2% among those with a methadone dosage ≥60mg/day. Plasma l-methadone concentrations were significantly higher in patients with negative urine analyses compared with those with positive urine analyses (median 93 vs. 77ng/ml, Mann–Whitney test, P<0.05). Above plasma l-methadone concentrations of 200ng/ml no heroin use was reported and urine analyses were negative. Moreover, above concentrations of 250ng/ml craving was absent. Examination of demographic correlates of treatment outcome indicated that older age, a stable job and being married were protective against the use of heroin. Mean plasma l-methadone concentration was significantly lower in patients who used cannabis compared with those who did not use cannabis, after adjusting for methadone dosage.In conclusion our results identify specific cut-offs for plasma l-methadone concentrations about which therapeutic response is observed and provide new evidence that therapeutic response is associated with patient׳s demographic characteristics. This underscores the need to monitor plasma methadone concentrations as part of Drug Addiction Services routine practice, in order to provide an objective framework for changing the methadone dosage.

A Possible Mechanistic Link Between the CYP2C19 Genotype, the Methadone Metabolite Ethylidene-1,5-Dimethyl-3,3-Diphenylpyrrolidene (EDDP), and Methadone-Induced Corrected QT Interval Prolongation in a Pilot Study.

Methadone use and methadone-associated sudden cardiac death have increased dramatically. Prolongation of the QT interval of the cardiac cycle predisposes to arrhythmia and is common among methadone users. We studied the relationship between pharmacogenetic variables and methadone metabolites and QT prolongation. Blood was obtained on days 1, 7, and 21 from consenting individuals initiating methadone treatment. Plasma methadone and ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidene (EDDP) were measured using liquid chromatographic-electrospray ionization-tandem mass spectrometry. The corrected QT interval (QTc) from 12-lead electrocardiograms (ECGs) was obtained at baseline and at 21 days. Total plasma EDDP, (S)-EDDP, and (R)-EDDP concentrations were significantly higher for individuals carrying the CYP2C19*2 variant (n=8) than in 17 subjects carrying the CYP2C19 wild-type allele (p<0.004). Seventeen (68%) of 25 subjects experienced a QTc (Bazett) of 39.9±28.4 ms (mean±standard deviation). The methadone dose and the plasma EDDP concentration corrected for dose were both significantly associated with QTc at study termination and with change in QTc interval from baseline (∆QTc) (p<0.03 to p<0.0003). Based on a QTc increase, five subjects had a potentially increased arrhythmia risk. Compared with other participants, the mean dose for those individuals was higher (50.8 vs. 42.5 mg/day; p<0.04) as was the total plasma EDDP (36.8 vs. 21.0 ng/mL; p<0.002) and dose-corrected EDDP (0.27 vs. 0.16 ng/mL/mg; p<0.003). These results suggest that a notable change in the QTc interval was associated with both oral dose and increased methadone metabolism, as indicated by the higher plasma concentration of the principal methadone metabolite. The oral dose and plasma EDDP concentration may be useful in identifying individuals at risk for methadone-related arrhythmia.