A discordance between cytochrome P450 2D6 genotype and phenotype in patients undergoing methadone maintenance treatment

Abstract

Aims

To assess CYP2D6 activity and genotype in a group of patients undergoing methadone maintenance treatment (MMT).

Methods

Blood samples from 34 MMT patients were genotyped by a polymerase chain reaction-based method, and results were compared with CYP2D6 phenotype (n = 28), as measured by the molar metabolic ratio (MR) of dextromethorphan (DEX)/dextrorphan (DOR) in plasma.

Results

Whereas 9% of patients (3/34) were poor metabolizers (PM) by genotype, 57% (16/28) were PM by phenotype (P < 0.005). Eight patients, who were genotypically extensive metabolizers (EM), were assigned as PM by their phenotype. The number of CYP2D6*4 alleles and sex were significant determinants of CYP2D6 activity in MMT patients, whereas other covariates (methadone dose, age, weight) did not contribute to variation in CYP2D6 activity.

Conclusions

There was a discordance between genotype and in vivo CYP2D6 activity in MMT patients. This finding is consistent with inhibition of CYP2D6 activity by methadone and may have implications for the safety and efficacy of other CYP2D6 substrates taken by MMT patients.

Introduction

It has been shown that the liver expression and enzyme activity of most human drug metabolizing cytochromes P450 (CYPs) are highly variable for both genetic and environmental reasons [1–3]. Genetic polymorphism has been identified in several CYPs and CYP2D6 is the best characterized in this respect. It catalyses the biotransformation of about 25% of commonly prescribed drugs [4] including tricyclic antidepressants, antipsychotic agents, opioids, as well as amphetamines [1, 5–7]. The CYP2D6 polymorphism is due to multiple mutations of the gene, which result in absent, functionally deficient, under-expressed or over-expressed protein. The CYP2D6*3, CYP2D6*4, and CYP2D6*6 alleles are the major variants associated with loss of activity in poor metabolizers (PM) [8], who represent about 8% of Caucasians. The remainder of the population are termed extensive metabolizers (EM).

The activity of CYP2D6 is also subject to potent inhibition, which can lead to a discordance between phenotype and genotype. Thus, administration of drugs such as quinidine can cause phenocopying, the apparent conversion of an EM to a PM [5, 9–13].

Methadone has become one of the most widely used drugs for the treatment of opiate dependency. In vitro studies using human liver microsomes have shown that methadone competitively inhibits the O-demethylation of dextromethorphan (DEX), a marker substrate for measuring CYP2D6 activity [14]. However, the effect of methadone maintenance treatment (MMT) on CYP2D6 activity has not been investigated systematically in vivo. Any change could have a significant impact on the efficacy and toxicity of other drugs taken concomitantly with methadone. The aim of this study was to characterize CYP2D6 activity in a group of patients undergoing MMT and to investigate the concordance of CYP2D6 phenotype and genotype.

Subjects

Fifty-two Caucasian patients undergoing MMT (2–120 months (35 ± 32) (mean ± SD); 35 male; age range 2–55 years (33 ± 7); methadone daily dose range 20–130 mg day⁻¹ (49 ± 22)) and attending clinic at the Substance Misuse Service, Community Health Sheffield (CHS) participated in the study after giving written informed consent. In addition to methadone, 40 (77%) of patients were taking other medications (e.g. benzodiazepines). Eight patients were taking known CYP2D6 inhibitors (fluoxetine and paroxetine) [15] and the results of phenotyping these patients were not included in any analysis. The study was approved by the Research Ethics Committee of North Sheffield.

Biological samples

Blood samples were taken using a butterfly cannula and were transferred into sterile heparinized glass tubes. After centrifugation at 400 g for 10 min, the plasma and blood cells were transferred into sterile polypropylene tubes. Urine samples (20 ml) were collected for screening for drugs of abuse. As a preventative measure, all biological samples were HIV-deactivated by incubation for 30 min in a 58 °C water bath and exposed to UV light for 1 h. These treatments did not influence the assay for DEX and its metabolite dextrorphan (DOR). The samples were stored at − 25 °C pending assay.

Determination of CYP2D6 phenotype

Following a 12-h abstinence from any DEX-containing medicine, MMT patients received 15-mg capsules of DEX by mouth. The ratio of DEX/DOR concentrations in plasma samples taken at 3 h after dosing was used as the index of CYP2D6 activity [16, 17], and a value of 0.1 was used as the antimode separating EM and PM phenotypes [18]. DEX and DOR were assayed by the high-performance liquid chromatography method of Chen et al.[19]. The limit of quantification was 0.3 ng ml⁻¹ and 0.1 ng ml⁻¹ for DEX and DOR, respectively, and the intra- and interday assay coefficients of variation of the assays were < 15%.

Determination of CYP2D6 genotype by polymerase chain reaction

Lymphocytic genomic DNA was isolated from 5–10 ml whole blood [20, 21]. Allele-specific polymerase chain reaction (PCR) was carried out to detect the CYP2D6*3, CYP2D6*4, and CYP2D6*6 alleles, which are the major mutations leading to loss of CYP2D6 activity (over 90% of PM alleles) [8, 22]. The method of Smith et al.[23] was used to detect the 2D6*3 and 2D6*4 alleles and that of Sachse et al.[24] was used for 2D6*6.

Detection of drugs of abuse

Urinalyses were carried out for common drugs of abuse as a part of the routine clinical procedure at the Toxicology Department of the Royal Hallamshire Hospital, Sheffield.

Statistical analysis

Statistical analysis was performed using SPSS for Windows (ver.10; SPSS Inc., Chicago, IL, USA). Multiple linear regression analysis was used to investigate the effects of all available covariables (sex, weight, age, methadone dose, and number of defective alleles) on the DEX/DOR log molar metabolic ratio (MR).

Results

Urinalysis was positive for trazodone, dihydrocodeine, 6-monoacetylmorphine (6MAM), codeine, morphine, amphetamines, benzodiazepines, canabinoides, barbiturates and cocaine in three (7%), two (5%), three (7%), 17 (39%), 28 (64%), one (2%), 29 (66%), 12 (27%), two (5%), and 17 (39%) cases, respectively.

Of 52 subjects, 34 were genotyped and 36 were phenotyped. Since eight patients were taking fluoxetine or paroxetine, the data from these individuals were excluded from the statistical analysis for phenotyping. In 16 patients both genotype and phenotype were determined.

Whereas 9% of patients (3/34) were PM (all homozygous for the CYP2D6*4 allele) by genotype, 57% (16/28) were PM by phenotype. Thus, there was a significant difference between the proportion of genotypic and phenotypic PMs (P < 0.005; Z-test). In 10 (29%) subjects, a heterozygous EM genotype was identified (CYP2D6*1/CYP2D6*4). The other 21 (62%) subjects were found to be homozygous for the wild-type allele (CYP2D6*1/CYP2D6*1). A summary of the relationship between genotype and phenotype is shown in Figure 1. Assuming a value of 0.1 (i.e. − 1 on a log scale) as the antimode separating EM and PM phenotypes [18], genotype and phenotype were at variance in eight patients.

Figure 1.

CYP2D6 genotype-phenotype correlation in MMT patients (n = 16). The dash line represents the cut-off point separating EM (below dashed line) from PM phenotypes. Female patients are represented by solid circles and the thick lines indicate the mean MR value for each group. The two middle groups were ‘apparent PM’ as a result of CYP2D6 inhibition by methadone.

Multiple regression analysis indicated that CYP2D6*4 alleles and being female were associated with lower CYP2D6 activity as measured by the log (MR) (P < 0.001; r² = 0.75). There was no significant contribution from other potential covariates (methadone dose, weight, age) to CYP2D6 activity.

Discussion

In a previous report we showed that the log transformed DEX/DOR ratio was significantly higher in MMT patients compared with normal healthy EM subjects (P < 0.001) [25]. The present study indicates that the proportion of CYP2D6 PM genotypes is similar in MMT patients (9%) and normal healthy Caucasian populations (7–10%) [26, 27]. Our value is also consistent with the proportion of the PM genotype (7%) reported by Eap et al. in patients undergoing methadone maintenance treatment [28]. However, the proportion of apparent phenotypic PMs in our patients (57%) was much higher than that reported by Wu et al. (10%) [14].

In the present study the frequencies of the most common defective alleles (*3, *4, *6) [8] were analysed. These account for about 90% of PM genotypes [8]. Thus, we do not consider that the lack of data on other defective alleles (e.g. *5) contributes significantly to the observed discordance of genotype and phenotype, given the low frequency of such alleles (e.g. 0.02 for the *5 allele in Caucasians) [24].

As expected, the presence of the CYP2D6*4 allele was associated with a lower activity of CYP2D6 (P < 0.001; Figure 1). Regression analysis did not indicate any significant effect of methadone dose on CYP2D6 activity as measured by log (MR) (P = 0.53). The relationship between methadone dose and its plasma concentration is highly variable due to a large inter-individual variation in methadone metabolism as indicated in a recent meta-analysis [29]. This may explain the apparent lack of a dose–effect relationship between methadone and CYP2D6 inhibition in the present study. With an increased number of subjects or by using plasma methadone concentration instead of dose, a correlation might be expected. We observed a lower activity of CYP2D6 in female MMT patients (2.2-fold higher DEX/DOR ratio). This could be related to differences in plasma methadone concentrations in female compared with male patients at a given dose level. There have been reports of sex difference in combined CYP3A4 and P-glycoprotein activity [30] and methadone is known to be a substrate for both CYP3A4 [31] and P-glycoprotein [32]. However, to our knowledge there have been no reports of sex differences in methadone clearance [33–35]; the reported slower elimination of methadone in females [36] could be related to a larger volume of distribution of this lipophilic compound in female patients [34].

The high proportion of phenotypic PMs in MMT patients is consistent with inhibition of CYP2D6 activity by methadone reported in vitro[14, 37]. A greater degree of CYP2D6 inhibition observed in our study compared with that of Wu et al.[14] may be related to the time of day that phenotyping was performed in relation to methadone dosage. In our study DEX was given in the morning at the same time as methadone administration. In the study of Wu et al.[14] DEX was given at bed-time and urine was collected overnight. Thus, methadone concentrations during phenotyping are likely to have been substantially higher in our study. Another explanation for the higher proportion of PMs found in this study compared with that of Wu et al.[14] is the higher methadone dosage in our study (20–130 mg day⁻¹ as opposed to 20–50 mg day⁻¹ in Wu et al.). The probe drug used both by Wu et al. and in the current study was DEX. It is worth mentioning that DEX has a higher affinity for CYP2D6 (K_m approximately 5 µM) [38, 39] than other commonly used CYP2D6 probes such as debrisoquine (K_m approximately 15 µM) [40]. Thus, the phenocopying observed with this substrate indicates a relatively potent inhibition of CYP2D6 by methadone.

In conclusion, we report a marked discordance between CYP2D6 genotype and phenotype after methadone intake in patients undergoing MMT. Since CYP2D6 is involved in the metabolism of many clinically important drugs [4], the observed decrease in metabolic activity during methadone treatment may influence the efficacy and toxicity of these agents. Although the change in CYP2D6, as measured by the DEX/DOR ratio, is only around seven-fold (compared with a 50-fold difference between EM and true PM genotypes), clinicians should be aware of the potential for an increased risk of CYP2D6-related drug interactions in MMT EM patients.