Association of CYP2B6 and OPRM1 Genotypes with Methadone Dose Requirements and Serum Concentrations in a Vietnamese Cohorts

The effectiveness and safety of methadone maintenance treatment (MMT) are challenged by considerable inter-individual variability in methadone pharmacokinetics and clinical response. Emerging evidence suggests that genetic factors significantly contribute to this variability. Among the genes studied, OPRM1 and CYP2B6 have been consistently identified as key determinants influencing methadone metabolism and treatment outcomes [1,2,3]. The OPRM1 gene encodes the mu-opioid receptor, the primary target of methadone. A common functional polymorphism, A118G (rs1799971), has been associated with altered receptor binding and downstream signaling, potentially influencing both the efficacy and tolerability of opioid therapy [1,4,5] In parallel, CYP2B6 encodes the cytochrome P450 2B6 enzyme, which is responsible for the major metabolic pathway of methadone. The CYP2B6 *6 allele, comprising rs3745274 and rs2279343, is linked to reduced enzymatic activity, slower methadone clearance, elevated plasma levels, and increased risk of toxicity [2,6,7,8,9].

The distribution of CYP2B6 and OPRM1 alleles varies widely across global populations, contributing to inter-individual differences in methadone metabolism and response. In East Asian countries such as China and Korea, the CYP2B6 4 allele is highly prevalent, often found in over 70% of individuals, while the 6 allele—associated with reduced enzyme activity and slower methadone metabolism—remains relatively uncommon [10,11]. In contrast, African and African-American populations exhibit much higher frequencies of the 6 allele, with some studies reporting prevalence rates up to 47% [6,12]. For OPRM1, the A118G polymorphism (rs1799971) shows significant ethnic variation as well. The G allele is most common in African populations, followed by Europeans, with reported frequencies ranging from 10% to over 20% in these groups [13,14]. However, few studies have investigated the prevalence or clinical implications of these polymorphisms specifically in methadone-maintained populations in Vietnam.

Given the lack of pharmacogenetic data in this context, a research gap exists in understanding how OPRM1 and CYP2B6 polymorphisms may affect methadone dosing and response among Vietnamese patients. The objective of this study is twofold: (1) to characterize the distribution of OPRM1 and CYP2B6 genotypes in a cohort of patients undergoing MMT in northern Vietnam; and (2) to assess their associations with key clinical outcomes, including serum methadone concentrations and maintenance dose requirements. Clarifying these relationships may support the development of genotype-informed strategies to optimize methadone therapy in Vietnam and similar resource-constrained settings.

Among the 200 participants, the vast majority were male (99.0%) and aged between 35 and 54 years. Nearly half had completed secondary school (47.5%), most were married (66.2%) and had children (70.1%). The most common occupation reported was housework or informal labor (55.2%). With respect to methadone treatment duration, over half (53.5%) had been on therapy for more than 3 years, while 26.3% had received treatment for less than 1 year, and 20.2% for 1 to 3 years. Longer treatment duration was significantly associated with older age (p = 0.029), Kinh ethnicity (p = 0.013), having children (p = 0.039), and higher mean age (p = 0.011).

Table 1 shows that participants with shorter treatment durations (<3 years) received higher initial methadone doses (approximately 21 mg/day), compared to those treated for over 3 years (18.8 mg/day; p = 0.021). However, maintenance doses and serum methadone concentrations were similar across groups, with mean serum levels ranging from 235.0 to 285.1 ng/mL (p = 0.124). The concentration-to-dose ratio (CDR), calculated as serum concentration (ng/mL) divided by maintenance dose (mg/day), had a mean value of 6.6 ± 3.7 ng/mL/mg and did not significantly differ across treatment duration groups (p = 0.801), indicating relatively consistent methadone pharmacokinetics across patients.

Table 2 presents the distribution of CYP2B6 and OPRM1 genotypes. The most prevalent CYP2B6 diplotypes were *1/*4 (33.3%) and *4/*6 (23.7%), with allele *4 observed in 81.8% of the sample. At the single nucleotide polymorphism (SNP) level, the most frequent genotypes were GG at rs3745274 (52.0%), AG at rs1799971 (47.5%), and CC at rs8192709 (88.4%). Hardy–Weinberg equilibrium (HWE) tests revealed that while most loci conformed to expected distributions, rs3745274 and rs2279343 deviated from equilibrium, a pattern consistent with findings in Iranian methadone-maintained populations reported by Sara Sadat Aghabozorg Afjeh and colleagues [15].

In the multivariable regression models, several significant associations were observed. The CYP2B6 *1/*6 genotype was associated with a significantly lower maintenance methadone dose (coefficient = −26.33; 95% confidence interval: −51.97 to −0.69). In addition, the *2/*6 genotype was significantly associated with lower log-transformed serum methadone concentrations (coefficient = −2.485; 95% CI: −3.024 to −1.947) and lower log CDR (coefficient = −2.595; 95% CI: −3.145 to −2.046). Higher final methadone dose also predicted increased serum methadone concentrations (coefficient = 0.019; 95% CI: 0.015 to 0.022). Other variables, including age, treatment duration, and OPRM1 genotype, were not significantly associated with methadone-related outcomes. The explanatory power of the regression models was modest, with R² values of 0.1062 for methadone dose, 0.4435 for serum methadone concentration, and 0.1114 for CDR (Table 3).

This study found that specific CYP2B6 genotypes, particularly *1/*2 and *1/*6, were associated with differences in methadone dose and serum concentration among patients on methadone maintenance. Most other genetic and clinical factors showed no significant association with methadone pharmacokinetics. These results suggest that certain genetic variants may be useful for individualizing methadone therapy, though much of the variability remains unexplained and warrants further research.

In terms of genotype distribution, the most frequent CYP2B6 diplotype in our Vietnamese cohort was *1/*4, accounting for approximately one-third of participants, followed by *4/*6. Less common genotypes included *1/*1, *1/*2, and *6/*9, each representing less than 10% of the sample. The predominance of the *1/*4 and *4/*6 diplotypes, as well as the high prevalence of the *4 allele (present in over 80% of participants), is broadly consistent with other studies conducted in East Asian populations, such as those in China and Korea ¹⁰,¹¹. In contrast, the *6 allele—which is associated with slower methadone metabolism and higher plasma concentrations—is much more prevalent in African and African-American populations, with frequencies reaching up to 47% in some cohorts, compared to the lower frequencies observed in both our Vietnamese sample and other Asian studies [6,12]. These ethnic differences in CYP2B6 allele and diplotype distribution may contribute to the variability in methadone pharmacokinetics and dosing requirements reported across global populations. For the OPRM1 gene, the AG genotype at rs1799971 (A118G) was most common in our cohort, with AA and GG genotypes being less prevalent. This distribution is consistent with published data from other Asian populations but differs from frequencies reported in European and especially African groups, where the G allele is more common [13,14]. The OPRM1 A118G polymorphism has clinical significance, as the G allele has been associated with reduced receptor function, higher methadone plasma levels, and increased risk of poor treatment response or toxicity in several studies [14,16]. Notably, while the prevalence of the A118G allele in European populations is typically 10–15%, it is higher in African populations and generally lower in East Asian groups—findings which align with our data and those of previous research [12,13]. Overall, the distribution of CYP2B6 and OPRM1 genotypes in our Vietnamese sample reflects expected patterns for East Asian populations but differs substantially from those observed in African and Western populations. This underscores the importance of considering ethnic and population-specific pharmacogenetic differences in the management of methadone maintenance therapy. As highlighted by recent studies, such as Twesigomwe et al. (2023) [12] and Chen et al. (2022) [11], the combination of CYP2B6 and OPRM1 genotype distributions, as well as additional genetic variants (e.g., ABCB1), may influence both methadone plasma concentrations and treatment outcomes, reinforcing the value of region-specific research and genotype-guided approaches to opioid dependence treatment.

Our study found that, among clinical and genetic factors, the CYP2B6*1/*6 genotype was significantly associated with a lower final methadone dose, consistent with previous research. Polymorphisms in the CYP2B6 gene, particularly *1/*6 and *2/*6, have been widely recognized as major determinants of methadone pharmacokinetic variability. Literature has demonstrated that individuals carrying these variants display reduced enzyme activity, resulting in decreased metabolic clearance of methadone and consequently higher plasma drug concentrations at standard doses [17] Surprisingly, our data showed that carriers of the CYP2B6*2/*6 genotype had significantly lower log serum methadone concentrations and lower log CDR, which is contrary to much of the published literature. Typically, *2/*6 is associated with reduced methadone metabolism, leading to higher serum concentrations and an increased CDR [10,15,18]. Most studies report that these alleles, by reducing enzyme activity, slow methadone clearance and result in higher steady-state levels and a higher CDR, particularly for the S-enantiomer of methadone [3,7,19]. The presence of the 2 allele, on the other hand, has been less extensively studied and its effect on methadone pharmacokinetics is not well defined. The unexpected finding of reduced serum concentrations in *2/6 carriers in our study could reflect unique genetic backgrounds or environmental influences in the Vietnamese population. Potential explanations include unmeasured gene–gene or gene–environment interactions, dietary or co-medication factors influencing enzyme expression (phenoconversion), or subtle differences in treatment practices such as formulation, absorption, or adherence variability. Given the inconsistencies with previously published pharmacogenetic data, our findings underscore the importance of conducting population-specific studies and caution against the universal application of pharmacogenetic interpretations across diverse ethnic groups. Future studies incorporating enantiomer-specific methadone measurement (e.g., LC-MS/MS) and broader genotype panels (e.g., ABCB1, CYP3A4) are needed to clarify these complex interactions and better guide genotype-informed methadone therapy.

The OPRM1 A118G polymorphism has been extensively studied for its role in modulating opioid response, particularly in pain management and addiction. While G allele carriers often require higher doses of opioids and report fewer side effects such as nausea [20], the specific impact of this variant on methadone pharmacokinetics remains unclear. Several studies have suggested that methadone, due to its unique dual mechanism of action (as both μ-opioid receptor agonist and NMDA receptor antagonist), may be less influenced by the A118G variant compared to other opioids [21]. A genome-wide association study in European-Americans found no significant association between A118G and methadone dose requirements, while other loci were implicated in African-American populations [22]. Additionally, recent research indicates that methadone may be particularly suitable for G allele carriers precisely because its efficacy is relatively unaffected by this polymorphism [21]. Although the A118G variant has been associated with altered brain connectivity and opioid-related behaviors, such neurobiological effects do not appear to directly influence serum methadone concentrations [23,24]. Taken together, these findings support our observation of no significant association between OPRM1 A118G and methadone serum levels, reinforcing the view that this polymorphism, while important in opioid response broadly, may have limited pharmacokinetic impact in the context of methadone maintenance treatment.

The implications of these findings are twofold. First, they reinforce the need for a precision medicine approach in MMT, particularly in ethnically distinct populations such as Vietnam, where CYP2B6 allele frequencies may differ significantly from those assumed in Western pharmacogenetic guidelines. Second, although our results suggest that genetic testing could help identify individuals with atypical dose requirements or suspected poor metabolizer status, the overall incremental explanatory power of genetic variables in multivariable models remains modest. Therefore, routine genotyping for all patients may not be currently justified. In Vietnam, the implementation of genotype-guided methadone dosing may face practical challenges, including limited access to pharmacogenetic testing, lack of clinical infrastructure, and cost constraints. However, as testing becomes more affordable and local evidence accumulates, targeted genotyping for high-risk subgroups—such as patients with poor treatment response or adverse effects—could be a feasible and impactful strategy to enhance treatment safety and effectiveness.

Several limitations must be acknowledged. Although the study sample reflects the demographic profile of real-world patients in Vietnamese methadone maintenance treatment (MMT) clinics, it was almost exclusively male (99%) and predominantly of Kinh ethnicity (96.9%). This homogeneity may limit the generalizability of our findings to female patients and ethnic minority groups, who may have different genetic backgrounds, methadone metabolism profiles, or treatment responses. Future research should aim to recruit more diverse and gender-balanced cohorts to ensure broader applicability of pharmacogenetic insights. Moreover, the cross-sectional design precludes causal inference and does not allow for assessment of temporal or dose-adjustment dynamics. Although methadone concentrations were measured at steady state and patients were scheduled for blood sampling before their next supervised dose, strict timing at exactly 24 hours was not always feasible due to behavioral unpredictability among this high-risk population. This could introduce some variability in serum concentration data. Additionally, we did not account for clinical and environmental factors such as, concomitant medications (e.g., antibiotics, antifungals), and smoking status, all of which may influence CYP2B6 enzyme activity regardless of genotype through phenoconversion. Lastly, our genetic analysis was limited to selected variants in CYP2B6 and OPRM1; future studies should focus on the combined analysis of multiple genes, which may also contribute to inter-individual variability in methadone pharmacokinetics and pharmacodynamics.

In conclusion, this study highlights the influence of specific CYP2B6 genotypes and SNPs on methadone pharmacokinetics in Vietnamese patients. Although genetic factors account for a portion of the variability in methadone dose requirements and serum concentrations, clinical variables such as actual daily dose remain the most important determinants. Personalized approaches to MMT dosing that integrate both genetic and clinical information may enhance the safety and effectiveness of therapy for opioid dependence.