Methadone is a synthetic opioid used for detoxification and maintenance treatment of patients who are dependent on opiates, mainly heroin. In the liver it is metabolised to 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) through
Methadone kinetics is also slightly affected by
In addition,
The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the University Hospital Split, Croatia (No. 530-01/12-01/164). All patients signed informed consent to participation in the study.
Our study participants were 35 adult men aged ≥21 years, who were in the MMT programme conducted by the Institute for Public Health of the Split-Dalmatia County, Croatia. To arrive to this number we first assessed for eligibility 74 adult male heroin addicts in the programme according to the following inclusion criteria: male, Caucasian, at least 9 months on MMT with regular attendance, no HIV or HBV co-infection or infection, negative urine tests for the presence of heroin or other pharmacological substances that could interfere with methadone metabolism, and no liver cirrhosis or history of significant alcohol abuse. HCV-positive patients were receiving interferon therapy that does not interfere with methadone metabolism (9). Thirty-nine candidates who did not meet these criteria were excluded.
The remaining 35 participants were divided in groups according to their HCV status: HCV-negative (HCV-) (N=12), HCV positive (HCV+) (N=16), and those in clinical remission (HCV CR) (N=7). Their liver damage was assessed according to the fibrosis-4 (FIB-4) index as described elsewhere (10). They were receiving different recommended doses of oral methadone based on their clinical presentation, and these doses were not modified for at least six months before the study (Table 1).
Average FIB-4 index, methadone-to-EDDP urine concentration ratio, and methadone dose received by study groups (N=35)
Parameters | HCV-negative (n=12) | HCV-positive (n=16) | HCV clinical remission (n=7) |
---|---|---|---|
FIB-4 index | 0.81 | 2.61 | 2.08 |
Methadone-to-EDDP urine concentration ratio | 0.87 | 2.04 | 0.79 |
Methadone dose (mg) | 83.2 | 85.0 | 62.5 |
For this study, methadone and EDDP concentrations were tested only in urine during three regular check-ups 15 days apart. We took two urine samples per check-up; one immediately before and the other 90 min after oral methadone administration, which totalled six samples per participant. The samples were stored at 4 °C and analysed within 1–4 days as described in detail in our previous reports (8, 11). Methadone and EDDP concentrations were used to calculate their ratio (Table 1).
Blood samples for DNA analysis were collected at regular check-ups and stored in EDTA tubes. DNA was isolated with a commercial genomic DNA isolation kit (High Pure PCR Template Preparation Kit, Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. Extracted DNA was quantified using a Qubit 4 fluorimeter (Thermo Fischer Scientific, Waltham, MA, USA).
Using the TaqMan® SNP genotyping assay (Thermo Fischer Scientific) and an Applied Biosystems 7500 real-time polymerase chain reaction (RT-PCR) system (Applied Biosystems, Foster City, CA, USA) we genotyped for the following single-nucleotide polymorphisms (SNPs):
Statistics and differences between the samples and groups were tested by using GraphPad Prism version 8.0.0 for Mac (GraphPad Software, San Diego, CA, USA). Hardy-Weinberg equilibrium, chi-square, and the
Methadone is converted to its inactive metabolites EDDP and EMDP by hepatic CYP450 enzymes. Its pharmacokinetics is individual, not only because of differences in sex, age, body weight, and use of other drugs, but also because of different allelic frequencies of genetic polymorphisms (2). Some earlier studies have already shown that allelic variations in the
Table 2 shows the genotypes of the four investigated SNP loci in DNA obtained from blood samples. Genotype frequencies were consistent with the Hardy-Weinberg equilibrium and did not significantly differ for any of the studied SNPs (the
Genotype and allele frequencies (%) by loci for the
Gene | SNP | Genotype | n (%) | Minor allele | MAF (%) | Hardy-Weinberg equilibrium | |
---|---|---|---|---|---|---|---|
χ2 p-value | |||||||
12 (34.3) | |||||||
rs1045642 | 16 (45.7) | 42.9 | 0.57 | 0.15 0.69 | |||
7 (20) | |||||||
15 (42.9) | |||||||
rs3745274 | 17 (48.5) | 32.9 | 0.67 | 0.36 0.55 | |||
3 (8.6) | |||||||
30 (85.7) | |||||||
rs2242480 | 5 (14.3) | 7.1 | 0.93 | 0.21 0.65 | |||
0 (0) | |||||||
34 (97.1) | |||||||
rs2740574 | 1 (2.9) | 1.4 | 0.99 | 0.007 0.93 | |||
0 (0) |
In contrast to
We observed a small difference in wild and mutant genotypes between the
Although several studies have suggested an association between genetic variability and methadone metabolism, our results showed no significant difference in methadone-to-EDDP ratio between the SNP genotypes. For
Even within the groups divided by HCV status this difference was not significant (
The liver is the primary target organ of HCV infection and is the main organ responsible for metabolism of drugs. Wu et al. (16) reported that HCV affected methadone metabolism in MMT patients. Our results seem to confirm this finding, as the severity of liver damage (median FIB-4 index) was the highest in HCV patients, who also showed the slowest methadone metabolism (the highest methadone-to-EDDP ratio; Table 1).
Figure 3 shows FIB-4 indices relative to the
In conclusion, our study found no significant influence of the established genetic polymorphisms in our patients with methadone metabolism. Similar has been reported by Fonseca et. al. (17), who found negligible impact of the
The only major influence on the methadone-to-EDDP ratio in HCV+ patients we found was the stage of liver damage. HCV- and HCV+ patients were taking similar methadone doses. The FIB-4 index and the methadone-to-EDDP ratio in HCV- patients were lower, regardless of the genotype.
However, our conclusion should be taken with reserve, as this study had a small sample size. Future research should therefore involve more MMT patients.