Methadone administered to rat dams during pregnancy and lactation affects the circadian rhythms of their pups.

The circadian clock is one of the most important homeostatic systems regulating the majority of physiological functions. Its proper development contributes significantly to the maintenance of health in adulthood. Methadone is recommended for the treatment of opioid use disorders during pregnancy, increasing the number of children prenatally exposed to long-acting opioids. Although early-life opioid exposure has been studied for a number of behavioral and physiological changes observed later in life, information on the relationship between the effects of methadone exposure and circadian system development is lacking. Using a rat model, we investigated the effects of prenatal and early postnatal methadone administration on the maturation of the circadian clockwork in the suprachiasmatic nucleus (SCN) and liver, the rhythm of aralkylamine N-acetyltransferase (AA-NAT) activity in the pineal gland, and gene expression in the livers of 20-day-old rats. Our data show that repeated administration of methadone to pregnant and lactating mothers has significant effect on rhythmic gene expression in the SCN and livers and on the rhythm of AA-NAT in the offspring. Similar to previous studies with morphine, the rhythm amplitudes of the clock genes in the SCN and liver were unchanged or enhanced. However, six of seven specific genes in the liver showed significant downregulation of their expression, compared to the controls in at least one experimental group. Importantly, the amplitude of the AA-NAT rhythm was significantly reduced in all methadone-treated groups. As there is a strong correlation with melatonin levels, this result could be of importance for clinical practice.

Detection of Three Opioids (Morphine, Codeine and Methadone) and Their Metabolites (6-Monoacetylmorphine and 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine) in Larvae of Lucilia sericata Species by UHPLC-TF-MS and Validation.

Insects on corpses could be a useful tool for the detection of exogenous substances such as drugs of abuse. The identification of exogenous substances in carrion insects is critical for proper estimation of the postmortem interval. It also provides information about the deceased person that may prove useful for forensic purposes. High-performance liquid chromatography coupled with Fourier transform mass spectrometry is a highly sensitive analytical technique that can identify substances even at very low concentrations, such as in the case of searching for exogenous substances in larvae. In this paper, a method is proposed for the identification of morphine, codeine, methadone, 6-monoacetylmorphine (6-MAM) and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in the larvae of Lucilia sericata, a common carrion fly widely distributed in temperate areas of the world. The larvae, which were reared on a pig meat substrate, were killed once they reached their third stage by immersion in hot water at 80 °C and aliquoted into 400 mg samples. The samples were fortified with 5 ng of morphine, methadone and codeine. After solid-phase extraction, the samples were processed with a liquid chromatograph coupled to a Fourier transform mass spectrometer. This qualitative method has been validated and tested on larvae from a real case. The results lead to the correct identification of morphine, codeine, methadone and their metabolites. This method could prove useful in cases where toxicological analysis must be conducted on highly decomposed human remains, where biological matrices are very limited. Furthermore, it could help the forensic pathologist to better estimate the time of death, as the growth cycle of carrion insects can undergo changes if exogenous substances are taken.

Punica granatum Seed Essential Oil Suppressed Methadone-Induced Cell Death by Natural Antioxidant Activity.

OBJECTIVES: Methadone is an opioid used in treating chronic and acute pains as well as opioid dependence. It induces death in neural cells. This study investigates Punica granatum oil's effects as a natural antioxidant on methadone-induced cell death. MATERIALS AND METHODS: The cell death index indicating the apoptosis occurrence is calculated using the TUNEL test. Rhodamine123 evaluated mitochondrial membrane permeability. Griess reaction was used to detect nitric oxide production. Furthermore, IL-1ß, IL-6, INFγ, and TNFα inflammatory cytokines were measured using the Rat inflammatory cytokine assay kit, Rat Kit V-Plex, and the caspase-3 activity was calculated through the Caspase-3 Colorimetric Assay Kit. RESULTS: Different treatment processes of Punica granatum oil reduced cell cytotoxicity and cell death index and increased viability and proliferation in methadone-treated PC12 cells. NO production decreased in different treatment processes compared to methadone-induced PC12 cells and decreased IL-1ß, IL-6, INFγ, and TNFα inflammatory cytokines. In these treatment processes, mitochondrial membrane potential increased, and caspase-3 activity decreased compared to methadone-induced PC12 cells. CONCLUSION: Punica granatum essential oil declined methadone-induced cell death in PC12 cells in a dose-dependent manner through suppressing NO production, IL-1ß, IL-6, INF-γ, and TNF-α inflammatory cytokines production, mitochondrial membrane disruption, and caspase-3 activities.

Chronic exposure to methadone induces activated microglia and astrocyte and cell death in the cerebellum of adult male rats.

Methadone is a centrally-acting synthetic opioid analgesic widely used in the methadone maintenance therapy (MMT) programs throughout the world. Considering its neurotoxic effects particularly on the cerebellum, this study aims to address the behavioral and histological alterations in the cerebellar cortex associated with methadone administration. Twenty-four adult male albino rats were randomized into two groups of control and methadone treatment. Methadone was subcutaneously administered (2.5-10 mg/kg) once a day for two consecutive weeks. The functional and structural changes in the cerebellum were compared to the control group. Our data revealed that treating rats with methadone not only induced cerebellar atrophy, but also prompted the actuation of microgliosis, astrogliosis, and apoptotic biomarkers. We further demonstrated that treating rats with methadone increased complexity of astrocyte processes and decreased complexity of microglia processes. Our result showed that methadone impaired motor coordination and locomotor performance and neuromuscular activity. Additionally, relative gene expression of TNF-α, caspase-3 and RIPK3 increased significantly due to methadone. Our findings suggest that methadone administration has a neurodegenerative effect on the cerebellar cortex via dysregulation of microgliosis, astrogliosis, apoptosis, and neuro-inflammation.

GC-MS Analysis of Methadone and EDDP in Addicted Patients under Methadone Substitution Treatment: Comparison of Urine and Plasma as Biological Samples.

(1) Background: Methadone, along with buprenorphine, is the most commonly used drug for the treatment of opioid dependence. This study aimed to analyze methadone and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenyl pyrrolidine (EDDP), in the urine and plasma of opiate addicts. The study group consisted of drug users voluntarily admitted to the detoxification center C.E.T.T.T. "St. Stelian" of Bucharest. Secondly, the study aimed to identify whether urine or plasma provides better results for the proposed method. (2) Methods: A GC-MS method, using an internal standard (diphenylamine) in the FULL-SCAN and SIM modes of operation and using the m/z = 72 ion for methadone and the m/z = 277 ion for EDDP, combined with a liquid-liquid extraction procedure was performed. (3) Results: The applied procedure allows the detection and quantification of methadone in both urine and plasma samples. EDDP was identified in patients with higher levels of methadone. Higher levels of methadone were detected in urine than in plasma samples. (4) Conclusions: This procedure can be used in clinical laboratories for the rapid determination of methadone levels in urine rather than in plasma. The procedure can be applied for the monitoring of methadone substitution treatment.

Chronic exposure to methadone impairs memory, induces microgliosis, astrogliosis and neuroinflammation in the hippocampus of adult male rats.

Methadone is a centrally-acting synthetic opioid analgesic widely used in methadone maintenance therapy (MMT) programs throughout the world. Given its neurotoxic effects, particularly on the hippocampus, this study aims to address the behavioral and histological alterations in the hippocampus associated with methadone administration. To do so, twenty-four adult male albino rats were randomized into two groups, methadone treatment and control. Methadone was administered subcutaneously (2.5-10 mg/kg) once a day for two consecutive weeks. A comparison was drawn with behavioral and structural changes recorded in the control group. The results showed that methadone administration interrupted spatial learning and memory function. Accordingly, treating rats with methadone not only induced cell death but also prompted the actuation of microgliosis, astrogliosis, and apoptotic biomarkers. Furthermore, the results demonstrated that treating rats with methadone decreased the complexity of astrocyte processes and the complexity of microglia processes. These findings suggest that methadone altered the special distribution of neurons. Also, a substantial increase was observed in the expression of TNF-α due to methadone. According to the findings, methadone administration exerts a neurodegenerative effect on the hippocampus via dysregulation of microgliosis, astrogliosis, apoptosis, and neuro-inflammation.

Review of LC techniques for determination of methadone and its metabolite in the biological samples.

Methadone (MTD) is a synthetic analgesic drug used for treating opioid dependence and effectively used clinically for patients with severe pain. The abuse of MTD may lead to poisoning, disorder in the central nervous system and even death. The regular monitoring of MTD in biological matrices including serum, plasma and urine samples is an effective way to control abuse of MTD. In this manner, the selection of analytical monitoring of MTD in biological matrices is of paramount importance. This study was conducted to review high-performance liquid chromatography (HPLC) techniques carried out on MTD and its main metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in the biological samples during 2015-June 2021.

Novel associations between CYP2B6 polymorphisms, perioperative methadone metabolism and clinical outcomes in children.

Aim: Methadone exhibits significant variability in clinical response. This study explores the genetic influence of variable methadone pharmacokinetics. Methods: This is a prospective study of methadone in children undergoing major surgery. CYP2B6 genotyping, plasma methadone and metabolite levels were obtained. Clinical outcomes include pain scores and postoperative nausea and vomiting (PONV). Results:CYP2B6 poor metabolizers (*6/*6) had >twofold lower methadone metabolism compared with normal/rapid metabolizers. The incidence of PONV was 4.7× greater with CYP2B6 rs1038376 variant. AG/GG variants of rs2279343 SNP had 2.86-fold higher incidence of PONV compared with the wild variant (AA). Nominal associations between rs10500282, rs11882424, rs4803419 and pain scores were observed. Conclusion: We have described novel associations between CYP2B6 genetic variants and perioperative methadone metabolism, and associations with pain scores and PONV.

Screening for Opioid and Stimulant Exposure In Utero Through Targeted and Untargeted Metabolomics Analysis of Umbilical Cords.

BACKGROUND: Neonatal abstinence syndrome is an array of signs and symptoms experienced by a newborn due to abrupt discontinuation of intrauterine exposure to certain drugs, primarily opioids. In the United States, the incidence of neonatal abstinence syndrome has tripled over the past decade. The current standard of care for drug testing includes the analysis of infant urine and meconium. Sample collection is associated with several limitations, including diaper media interferences, limited sample amount, sample heterogeneity, and the need for professional staff for collection. Umbilical cord tissue has emerged as a convenient sample matrix for testing owing to its universal availability. The purpose of this study was to examine umbilical cords using an untargeted metabolomics approach to determine the detected drugs and validate an analytical method to confirm and quantify the identified drugs. METHODS: A metabolomics analysis was performed with 21 umbilical cords to screen for drugs and drug metabolites by liquid chromatography-mass spectrometry. Drugs were identified using the National Institute of Standards and Technology database, and an analytical method was developed and validated using secondary liquid chromatography-mass spectrometry instrument for positive confirmation and quantitative analysis. RESULTS: Twenty-one random umbilical cords from women were tested: 4 were positive for cocaine and the primary and secondary metabolites; one was positive for methadone, the primary metabolite; 3 were positive for cotinine, the metabolite of nicotine; and 5 were positive for acetyl norfentanyl. CONCLUSIONS: Our research is a prospective method development study using untargeted and targeted approaches to characterize steady-state drug metabolite levels in the umbilical cord matrix at the time of delivery. By characterizing drug type and concentration, this methodology can be used to develop a reliable complementary testing method for meconium toxicology screens.

A Sensitive LC-MS/MS Assay for the Quantification of Methadone and its Metabolites in Dried Blood Spots: Comparison With Plasma.

INTRODUCTION: Methadone, a synthetic narcotic, is widely used both in adults and children for pain control and as a replacement drug in opioid use disorder to prevent craving and withdrawal. To support clinical pharmacokinetic trials in neonates, infants, and children, the authors developed and validated a novel, automated, highly sensitive liquid chromatography-electrospray-tandem mass spectrometry ionization (LC-ESI-MS/MS) method for the quantification of methadone and its metabolites, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyraline (EMDP), in samples collected as dried blood spots. METHODS: Blood was spiked with different concentrations of methadone, EDDP, and EMDP, and blood drops were applied to filter paper cards. Punches of 6.4 mm were removed from the cards, and 600 µL of protein precipitation solution (methanol/0.2M ZnSO4, 7:3, vol/vol) containing the internal standards (methadone-d9 and EDDP-d5) at a concentration of 1 mcg/L was added. The extracts were analyzed using LC-ESI-MS/MS in combination with online extraction. The mass spectrometer was run in the positive multiple reaction monitoring mode, and the total run time was 3.2 minutes. RESULTS: For the dried blood spots, the assay has a lower limit of quantification of 0.1 mcg/L for methadone, EDDP, and EMDP. The range of reliable response for methadone for the ion transition m/z = 310.2→265.1 was 0.1-100 mcg/L and for the ion transition m/z = 310.2→223.1 5-1000 mcg/L. For EDDP, on the range of reliable response for the ion transition, m/z = 278.2→234.3 was 0.1-100 mcg/L and for the ion transition m/z = 278.2→186.1 5-1000 mcg/L. The calibration range for EMDP was 0.1-100 mcg/L. Accuracy (85%-115%) and imprecision (<15%) met predefined acceptance criteria. DISCUSSION: This assay allows for the measurement of small volume blood samples without the need for an intravenous blood draw, and thus, it is suitable for pharmacokinetics studies and therapeutic drug monitoring in pediatric patients.

Opioid Toxidrome Following Grapefruit Juice Consumption in the Setting of Methadone Maintenance.

BACKGROUND: Methadone is a synthetic mu-opioid receptor agonist used in the treatment of chronic pain and opioid dependence. Methadone is metabolized by several cytochrome P450 isoenzymes; primarily CYP3A4, CYP2B6, and CYP2D6 before renal and fecal elimination. Exposure to substances like grapefruit juice, that inhibit these isoenzymes may result in increased blood levels of methadone, and thus may manifest clinically as unexpected opioid toxicity. CASE: A 51-year-old male was found unresponsive. He was hypoxic and bradypneic with pinpoint pupils. Multiple boluses followed by infusion of naloxone were required before improvement of respiratory status. Upon awakening, the patient reported participating in an opioid treatment program where he is administered 90 mg of oral methadone daily and denied any other substance use. On further questioning, he admitted to drinking grapefruit juice (estimated to be approximately 500 mL/day) every day for 3 consecutive days before presentation. The patient was discharged home after being counseled to stop drinking grapefruit juice. DISCUSSION: Grapefruit juice is known to be an inhibitor of the CYP3A4 isoenzyme. Various studies demonstrate that through CYP3A4 inhibition, grapefruit juice increases serum levels of opioids, such as methadone, though no clinically significant effects have been reported. CONCLUSIONS: Grapefruit juice inhibits the metabolism of methadone, raising its serum levels. To our knowledge, this is the first reported case in which the interaction between grapefruit juice and methadone was significant enough to cause an opioid toxidrome. It is, therefore, recommended that opioid treatment programs (OTPs) advise patients about this interaction before administering methadone.

Nicotine oxidation by genetic variants of CYP2B6 and in human brain microsomes.

Common variation in the CYP2B6 gene, encoding the cytochrome P450 2B6 enzyme, is associated with substrate-specific altered clearance of multiple drugs. CYP2B6 is a minor contributor to hepatic nicotine metabolism, but the enzyme has been proposed as relevant to nicotine-related behaviors because of reported CYP2B6 mRNA expression in human brain tissue. Therefore, we hypothesized that CYP2B6 variants would be associated with altered nicotine oxidation, and that nicotine metabolism by CYP2B6 would be detected in human brain microsomes. We generated recombinant enzymes in insect cells corresponding to nine common CYP2B6 haplotypes and demonstrate genetically determined differences in nicotine oxidation to nicotine iminium ion and nornicotine for both (S) and (R)-nicotine. Notably, the CYP2B6.6 and CYP2B6.9 variants demonstrated lower intrinsic clearance relative to the reference enzyme, CYP2B6.1. In the presence of human brain microsomes, along with nicotine-N-oxidation, we also detect nicotine oxidation to nicotine iminium ion. However, unlike N-oxidation, this activity is NADPH independent, does not follow Michaelis-Menten kinetics, and is not inhibited by NADP or carbon monoxide. Furthermore, metabolism of common CYP2B6 probe substrates, methadone and ketamine, is not detected in the presence of brain microsomes. We conclude that CYP2B6 metabolizes nicotine stereoselectively and common CYP2B6 variants differ in nicotine metabolism activity, but did not find evidence of CYP2B6 activity in human brain.

Biodegradation study of methadone by adapted activated sludge: Elimination kinetics, transformation products and ecotoxicological evaluation.

The biotransformation study of difficult-to-degrade opioid analgesic methadone (MTHD) was performed by activated sludge culture adapted to high concentration of methadone (10 mg/L). The study included determination of elimination kinetics of the parent compound, taxonomic characterization of microbial culture, identification of biotransformation products (TPs) and assessment of ecotoxicological effects of biotransformation processes. The chemical analyses were performed by ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry, whereas the ecotoxicological assessment was made based on determinations of toxicity to freshwater algae. Changes of the adapted sludge culture during the experiment were followed using the 16S rRNA gene amplicon sequencing. Depending on the experimental conditions, the elimination efficiency of methadone (10 mg/L) varied from 9% to 93% with the corresponding half-lives from 11.4 days to 1.5 days. A significantly faster elimination (t1/2 from 1.5 days to 5.8 days) was achieved at cometabolic conditions, using glucose-containing media, as compared to the experiments with MTHD as a single organic carbon source (t1/2 = 11.4 days). Moreover, increased biotransformation rate following the additional supplementation of ammonia, revealed a possible importance of nitrogen availability for the transformation at cometabolic conditions. The elimination of parent compound was associated with the formation of 3 different TPs, two of which were identical to main human metabolites of MTHD, 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP). EDDP represented over 90% of the total TP concentration at the end of experiment. The biodegradation of MTHD was associated with a pronounced drop in algal toxicity, confirming a rather positive ecotoxicological outcome of the achieved biotransformation processes.

Methadone Metabolism and Drug-Drug Interactions: In Vitro and In Vivo Literature Review.

Methadone is utilized for the treatment of individuals with opiate dependence. Methadone undergoes N-demethylation by multiple cytochrome P450 (CYP) enzymes including CYP3A4, CYP2B6, CYP2C19, CYP2D6, CYP2C9, and CYP2C8. In vivo, polymorphism effects on methadone systemic exposure have been noted for CYP2B6, CYP3A4, and CYP2D6. Clinical drug interaction studies with antiviral drugs in methadone maintenance treatment patients yield varying results on methadone pharmacokinetics and pharmacodynamics. In general, CYP inhibitors altered methadone exposure with no adverse effects. CYP inducers generally decreased methadone exposure with some reports of withdrawal symptoms in the subjects. Interaction studies with antiviral drug combinations yielding differing results depend on the enzyme(s) affected. For certain antiviral medicines which are dual inhibitor(s) and inducer(s) for CYP enzymes, their effect on methadone pharmacokinetics can change with time since the effect of induction is usually delayed compared to the effect of inhibition.

Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor.

The intrinsic efficacy of orthosteric ligands acting at G-protein-coupled receptors (GPCRs) reflects their ability to stabilize active receptor states (R*) and is a major determinant of their physiological effects. Here, we present a direct way to quantify the efficacy of ligands by measuring the binding of a R*-specific biosensor to purified receptor employing interferometry. As an example, we use the mu-opioid receptor (µ-OR), a prototypic class A GPCR, and its active state sensor, nanobody-39 (Nb39). We demonstrate that ligands vary in their ability to recruit Nb39 to µ-OR and describe methadone, loperamide, and PZM21 as ligands that support unique R* conformation(s) of µ-OR. We further show that positive allosteric modulators of µ-OR promote formation of R* in addition to enhancing promotion by orthosteric agonists. Finally, we demonstrate that the technique can be utilized with heterotrimeric G protein. The method is cell-free, signal transduction-independent and is generally applicable to GPCRs.

Episodio maniaco con síntomas psicóticos tras desintoxicación de heroína: estudio de un caso / Manic episode with psychotic symptoms after heroin detoxification: a case study

El uso de fármacos agonistas opioides, como la metadona, en el tratamiento de la dependencia a la heroína es altamente frecuente. A su vez, la prevalencia de trastornos depresivos en esta población es elevada. Por otra parte, entre los síntomas de abstinencia aguda a opioides se incluyen alteraciones afectivas como la disforia. Se presenta el caso de una mujer de 43 años con diagnóstico de dependencia a la heroína de varios años de evolución y antecedentes de distimia sin tratamiento antidepresivo continuado. Ingresa en la unidad de desintoxicación hospitalaria para desintoxicación de la heroína con inducción de metadona. Tras la retirada de este fármaco, presentó un cuadro maniaco con síntomas psicóticos

The use of opioid agonist drugs, such as methadone, in the treatment of heroin dependence is very common. At the same time, the prevalence of depressive disorders in this population is high. On the other hand, symptoms of acute withdrawal from opioids include affective alterations such as dysphoria. The case is presented of a 43-year-old woman with a diagnosis of heroin dependence of several years onset, and a history of dysthymia with no continuous antidepressant treatment. She was admitted to the hospital detoxification unit, for heroin detox with methadone induction. After the withdrawal of this drug, the patient had a manic episode with psychotic symptoms

A Conformationally Gated Model of Methadone and Loperamide Transport by P-Glycoprotein.

P-glycoprotein (Pgp) is a multidrug resistance transporter that limits the penetration of a wide range of neurotherapeutics into the brain including opioids. The diphenylpropylamine opioids methadone and loperamide are structurally similar, but loperamide has about a 4-fold higher Pgp-mediated transport rate. In addition to these differences, they showed significant differences in their effects on Pgp-mediated adenosine triphosphate (ATP) hydrolysis. The activation of Pgp-mediated ATP hydrolysis by methadone was monophasic, whereas loperamide activation of ATP hydrolysis was biphasic implying methadone has a single binding site and loperamide has 2 binding sites on Pgp. Quenching of tryptophan fluorescence with these drugs and digoxin showed competition between the opioids and that loperamide does not compete for the digoxin-binding site. Acrylamide quenching of tryptophan fluorescence to probe Pgp conformational changes revealed that methadone- and loperamide-induced conformational changes were distinct. These results were used to develop a model for Pgp-mediated transport of methadone and loperamide where opioid binding and conformational changes are used to explain the differences in the opioid transport rates between methadone and loperamide.

Effects of cytochrome P450 single nucleotide polymorphisms on methadone metabolism and pharmacodynamics.

Methadone is a synthetic, long-acting opioid with a single chiral center forming two enantiomers, (R)-methadone and (S)-methadone, each having specific pharmacological actions. Concentrations of (R)- and (S)-methadone above therapeutic levels have the ability to cause serious, life-threatening, and fatal side effects. This toxicity can be due in part to the pharmacogenetics of an individual, which influences the pharmacokinetic and pharmacodynamic properties of the drug. Methadone is primarily metabolized in the liver by cytochrome P450 (CYP) enzymes, predominately by CYP2B6, followed by CYP3A4, 2C19, 2D6, and to a lesser extent, CYP2C18, 3A7, 2C8, 2C9, 3A5, and 1A2. Single nucleotide polymorphisms (SNPs) located within CYPs have the potential to play an important role in altering methadone metabolism and pharmacodynamics. Several SNPs in the CYP2B6, 3A4, 2C19, 2D6, and 3A5 genes result in increases in methadone plasma concentrations, decreased N-demethylation, and decreased methadone clearance. In particular, carriers of CYP2B6*6/*6 may have a greater risk for detrimental adverse effects, as methadone metabolism and clearance are diminished in these individuals. CYP2B6*4, on the other hand, has been observed to decrease plasma concentrations of methadone due to increased methadone clearance. The involvement, contribution, and understanding the role of SNPs in CYP2B6, and other CYP genes, in methadone metabolism can improve the therapeutic uses of methadone in patient outcome and the development of personalized medicine.

Liver damage indices as a tool for modifying methadone maintenance treatment: a cross-sectional study.

AIM: To assess the effect of liver damage on methadone metabolism in opiate addicts undergoing methadone maintenance treatment (MMT). METHODS: This cross-sectional study recruited 74 patients treated at the outpatient clinic of Public Health Institute of Split-Dalmatia County from 2013-2016. Concentrations of methadone and its main inactive metabolite were measured in participants' biological samples on regular check-ups. Urine samples obtained before oral methadone intake, and blood and urine samples obtained 90 minutes after methadone intake were analyzed using gas chromatography/mass spectrometry. Participants were divided into groups according to liver damage criteria: hepatitis C virus status (positive, negative, or clinical remission); aspartate aminotransferase to platelet ratio (APRI) index (<0.7 and ≥0.7); and fibrosis-4 score (<1.45, 1.45-3.25, >3.25). RESULTS: Metabolic ratio and methadone metabolite concentration in plasma decreased linearly with HCV infection status by the factor of 1.67 (P=0.001) and 2.2 (P=0.043), respectively. Metabolic ratio in plasma decreased in patients with APRI index ≥0.7 by the average factor of 2.12 (P=0.01) and methadone metabolite concentration in plasma decreased by the factor of 6.16 (P=0.009). Metabolic ratio in urine decreased with the severity of fibrosis-4 score by the average factor of 1.63 (P=0.008), whereas methadone metabolite concentration decreased by the factor of 3.53 (P=0.007). CONCLUSION: Liver damage decreases methadone metabolism. Indices of liver function should be calculated regularly during MMT for methadone dose titration.