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Free and bound enantiomers of methadone and its metabolite, EDDP in methadone maintenance treatment: Relationship to dosage?
Clinical Biochemistry 38 (2005) 1088 – 1094
Free and bound enantiomers of methadone and its metabolite, EDDP in methadone maintenance treatment: Relationship to dosage? D.C. Lehotay a,c,⁎, S. George a , M.L. Etter a , K. Graybiel a , J.C. Eichhorst a , B. Fern e , W. Wildenboer f , P. Selby d , B. Kapur b a
Provincial Laboratory, 3211 Albert Street, Regina, SK, Canada S4S 5W6 b St. Michael's Hospital, University of Toronto, Canada c University of Saskatchewan, Saskatchewan, Canada d Centre of Addiction and Mental Health, University of Toronto, Canada e College Park Medical Clinic 2600, 8th Street, Saskatoon, SK, Canada f Regina Community Clinic, 1106 Winnipeg St., Regina, SK, Canada
Received 7 April 2005; received in revised form 16 August 2005; accepted 17 September 2005 Available online 10 November 2005
Abstract Objectives: To determine the effects of metabolism and protein binding on the relationship between administered dose, blood levels of R methadone and biological response by measuring the free and protein-bound forms of the R and S enantiomers of methadone and EDDP, its metabolite. Design and methods: To measure free and total drug, trough levels were collected from 45 methadone clients. To measure free methadone, samples were filtered using ultrafiltration with a MW weight cut-off of 10,000 and extracted using liquid–liquid extraction. The solvent was evaporated and samples reconstituted in mobile phase for analysis by LC/MS/MS. Total analyte was determined by extracting unfiltered samples. Enantiomeric separation of methadone and EDDP was by chiral chromatography. Results: The presence of unmetabolized methadone suggested that none of the patients were very fast metabolizers. R and S forms were metabolized at the same rate at all administered doses. Free R methadone levels correlated both with methadone dose and with the total amount of R methadone. The free fraction of R methadone (%free R) was higher at lower doses than at high doses, varied from 5 to 25% and was inversely proportional to the total dose of administered drug in a relationship that was logarithmic and non-linear. Conclusions: By measuring the free, biologically active form of the drug, we were unable to account for the large variations in dose required between different patients to prevent the onset of withdrawal symptoms. The reason for the large range in dosage may be multifactorial. © 2005 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Methadone; EDDP; Enantiomer; Bound; Free; Tandem MS; LC/MS/MS; Dosage; Withdrawal symptoms
Introduction Methadone is commonly used in the treatment of opioid dependence. The drug is usually administered orally as a racemic mixture of (R) and (S) methadone. The (R) form accounts for most or all of the opioid effects, while the (S) form is devoid of biological activity. In addition, methadone is bound to plasma proteins: it has been estimated that about 85% of the drug is bound to α-1 acid glycoprotein and to a much lesser extent to ⁎ Corresponding author. Provincial Laboratory, 3211 Albert Street, Regina, SK, Canada S4S 5W6. E-mail address: [email protected] (D.C. Lehotay).
serum albumin [1,2]. Alpha-1 acid glycoprotein is an acute phase protein that may be elevated in inflammation and may thus alter the availability of free or unbound drug [3]. As proteinbound drug is not normally available to interact with drug receptors, biological activity is due primarily to the free fraction of the drug. Methadone is metabolized by cytochrome P450 enzymes in liver via N-demethylation followed by cyclization to 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), a biologically inactive metabolite [4]. Several additional minor metabolites have also been identified [5]. There is marked individual variability in the optimal dose of methadone required to maintain clients in a methadone maintenance treatment (MMT) program. In a recent review,
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D.C. Lehotay et al. / Clinical Biochemistry 38 (2005) 1088–1094
Eap et al. [6] commented that an adequate methadone dosage is at least 60 mg/day but may be closer to 80–100 mg/day. Providing adequate methadone to clients to reduce craving (to prevent the onset of withdrawal symptoms) is a major factor in the success of an MMT program. Many MMT programs prescribe low dosages of methadone for political, psychological, philosophical or moral reasons. One of the major concerns is that, to date, there has not been a scientific basis for determining adequate and/or optimal doses. Most physicians prescribing methadone follow the guidelines outlined by Eap [6] and others [7–9]. They also test their clients for other drugs of abuse and consult with their clients directly regarding the adequacy of the dose. Among prescribers, there is continuing disagreement regarding optimal methadone dosage for MMT. Most MMT programs monitor their clients for compliance by periodic urine drug testing. Measuring methadone, or its major metabolite EDDP, has not been very successful in determining optimal methadone dosage. Urine drug testing programs normally measure methadone by an immunoassay that quantifies the sum of both the R and S enantiomers of methadone. The concentrations of free drug in blood or the effects of metabolism on the bioavailability of R methadone are not reported. The experiments described below were designed to gain a better understanding of the factors that may influence optimal methadone dosage in a MMT program. We developed methods to quantify free and bound forms of the R and S enantiomers of methadone and its metabolite, EDDP in blood. These were measured in 45 patients on long-term methadone maintenance therapy who have been on the same dose of the drug for at least 3 months. The objective of these experiments was to (a)determine if there is a relationship between administered dose of methadone, blood levels of the active form of the drug and biological response; (b) to measure the free and protein-bound forms of the active R and inactive S enantiomers of methadone, and R and S forms of EDDP, its metabolite; and (c) to determine the nature of the relationship between methadone metabolism, protein binding and the large variations in maintenance dosage required by different methadone clients. Materials and methods Patients and sample collection Ambulatory patients on a stable maintenance dosage of methadone were selected for this study. All of the 45 patients received their daily dose of methadone once a day. Doses varied between 10 and 205 mg/day. All patients were on the same dose for at least 3 months prior to the beginning of the study. Trough level samples were collected from all patients just prior to the administration of their daily dose of the drug, which was given as an oral solution consisting of a racemic mixture of equal amounts of R and S methadone. Eight of the 45 patients in the study also had blood collected at 2 h after their daily dose (peak levels). Patients from whom extra samples were collected
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provided consent for the study and were paid an honorarium. The study was approved by the Ethics Review Board (ERB) of the University of Saskatchewan College of Medicine and the ERB of the University of Toronto. Sample processing Serum was collected in non-gel red top tubes, plasma was anti-coagulated with EDTA and separated within 30 min. 300 μL Table 1 Free and total pre-dose R and S Methadone and EDDP (μmol/L) Pt. Dose Total Total Free code R S R
Free S
% Free R
A01 A02 A03 A04 A05 A06 A07 A08 A09 A10 A11 A12 A13 A14 A15 A16 A17 A18 A19 A20 A21 A22 A23 A24 A25 A26 A27 A28 A29 A30 A31 A32 A33 A34 A35 A36 A37 A38 A39 A40 A41 A42 A43
0.003 0.027 0.003 0.024 0.003 0.008 0.034 0.006 0.007 0.006 0.014 0.011 0.044 0.060 0.008 0.032 0.042 0.019 0.059 0.034 0.049 0.047 0.031 0.032 0.061 0.033 0.034 0.046 0.051 0.055 0.037 0.035 0.031 0.062 0.043 0.036 0.047 0.043 0.053 0.127 0.042 0.039 0.043
9.01 13.37 5.77 17.04 3.49 10.51 12.85 5.71 7.26 8.33 7.41 5.43 8.21 7.06 6.41 4.60 5.50 4.38 4.81 10.17 5.89 8.26 22.72 18.10 5.21 22.77 6.55 9.92 5.63 9.99 14.83 5.93 18.60 6.04 7.24 7.79 13.11 3.84 8.18 9.70 5.21 15.70 11.85
20 24 25 25 45 55 60 70 70 70 80 105 110 115 120 130 155 160 170 40 140 125 30 30 190 32 65 85 160 25 150 9 205 110 94 70 125 100 150 190 50 60
0.12 0.20 0.15 0.16 0.19 0.22 0.69 0.37 0.26 0.16 0.37 0.48 0.80 1.65 0.58 1.58 1.34 1.09 2.02 0.38 1.22 0.85 0.15 0.19 2.04 0.15 0.59 0.65 1.64 1.23 0.28 0.97 0.17 1.54 0.77 0.61 0.48 1.78 1.06 1.81 1.27 0.30 0.54
0.06 0.18 0.13 0.14 0.18 0.16 0.54 0.29 0.25 0.13 0.33 0.30 0.64 1.75 0.28 1.23 1.04 0.81 2.40 0.36 1.27 0.89 0.12 0.16 1.87 0.15 0.65 0.72 1.49 0.83 0.15 0.44 0.16 1.97 0.82 0.54 0.50 1.67 0.90 2.84 1.01 0.30 0.46
0.010 0.026 0.009 0.027 0.006 0.023 0.088 0.021 0.019 0.013 0.028 0.026 0.066 0.116 0.037 0.073 0.073 0.048 0.097 0.038 0.072 0.071 0.034 0.035 0.106 0.035 0.038 0.065 0.092 0.123 0.042 0.058 0.031 0.093 0.056 0.048 0.063 0.069 0.087 0.176 0.066 0.047 0.064
A1AG fR/ fS
23.25 15.09 26.46 13.60 16.32 19.79 6.43 18.80 14.10 18.05 24.24 11.38 13.85 18.80 30.42 14.59 22.01 38.34 21.02 16.82 15.58 12.86 9.15
3.79 0.98 3.24 1.16 1.91 2.98 2.63 3.25 2.60 2.08 2.03 2.44 1.50 1.94 4.53 2.29 1.73 2.48 1.64 1.14 1.46 1.51 1.08 1.09 1.76 1.07 1.13 1.39 1.81 2.23 1.13 1.66 1.02 1.51 1.30 1.32 1.34 1.60 1.66 1.39 1.56 1.23 1.49
fR-EDDP/ fS-EDDP 1.02 1.00 1.05 1.02 0.90 0.97 0.88 1.36 1.09 0.90 0.80 1.12 0.91 0.77 1.21 1.00 1.21 0.99 0.89 0.77 0.87 1.14 1.00 0.82 0.84 1.12 1.17 1.19 1.26 1.08 1.24 0.73 1.12 0.77 1.11 0.98 1.37 0.99 0.92 1.22 0.83 1.09
All concentrations of methadone and/or EDDP are expressed as μmol/L of serum or plasma. Dose of drug administered to patients is in milligrams and is the total weight of the enantiomeric mixture given to the patient orally. Units of A1AG are in μmol/L. fR and fS represent free R methadone and free S methadone, respectively. Similarly, fR-EDDP and fS-EDDP represent free R EDDP and free S EDDP.
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Fig. 1. Relationship between ratio of free R- and S-EDDP and dose.
of serum or plasma was centrifuged at 9000 rpm (7437 × g) for 2 h using a selective ultrafiltration membrane with a molecular weight cut-off of 10,000 Da. Protein-bound methadone and EDDP are retained on the filter and all compounds with a molecular weight less than approximately 10,000 Da including free drugs and water pass through. 100 μL of ultrafiltrate or 250 μL of serum was extracted with 1-chlorobutane, evaporated to dryness under N2 and reconstituted in 200 μL HPLC mobile phase prior to further analysis of free and total drug concentrations by LC/MS/MS. Separation of R and S enantiomers of free and total methadone and free and total EDDP
was by HPLC on a Chiral-AGP 100 × 3.0 mm (0.5 μm) column followed by analysis on an Applied Biosystems API-4000 tandem mass spectrometer using a thermospray source in positive ion mode using MRM as described in the accompanying paper. Results Trough levels of total R and S methadone, and total R and S forms of EDDP were quantified as described in Materials and methods. Free R and S forms of the drug and metabolite were
Fig. 2. Relationship between free R and total R methadone.
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Fig. 3. Relationship between %free R methadone and dose.
also measured following separation of bound from free forms by ultrafiltration as described above. All of the data not shown. Table 1 shows the data used for the more important comparisons made in the Results and Discussion. Metabolism At steady state (trough level), all samples analyzed had measurable levels of R and S forms of methadone, suggesting that none of the patients were very fast metabolizers. On a small subset (eight) of the clients, we were able to collect two blood samples, one just prior to administration of their daily dose (trough level) and another one at about 2–3 h after their daily dose (peak level). The ratio of post-dose total R methadone to the pre-dose R methadone level in eight patients had an average value of 1.4 (range = 1.1–1.9), supporting our claim that in this population, there were no
fast or very fast metabolizers. The relationship between R and S forms of methadone and EDDP remained very close to one, indicating that both the biologically active R form and the inactive S form are metabolized at the same rate to EDDP as shown in Fig. 1. The correlation coefficients r2 between total R and S methadone and R and S EDDP were 0.94 and 0.98, respectively. The ratio between R and S forms of methadone converted to EDDP (R-EDDP/R methadone/S-EDDP/S methadone), as well as the relationship between total dose given and total R methadone, was also very close to one (correlation coefficient r2 = 0.88 and r2 = 0.97, respectively). We also determined the fraction of methadone metabolized at increasing administered doses. The results indicated that the ratio of R-EDDP to R methadone, which is a measure of the fraction of methadone metabolized at steady state, was the same regardless of the administered dose. The rate of metabolism remained unchanged at all doses.
Fig. 4. Relationship between A1AG and %free R methadone.
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Fig. 5. Relationship between %free R and total R methadone.
Free methadone Biological activity is exerted by the free (unbound) fraction of R methadone. Free R methadone levels were measured and found to be proportional to methadone dose with a correlation coefficient of r2 = 0.47, a statistically significant relationship (P b 0.05). Free R methadone correlated even better with the total amount of R methadone with a correlation of r2 = 0.55 (P b 0.05) (see Fig. 2). The free fraction of R methadone ((% free R = free R methadone/total R methadone) × 100) was higher at lower doses than at high doses of total administered drug. The correlation was statistically significant correlation of r2 = 0.47 (P b 0.05). The %free R methadone varied from 5 to 25% of total R methadone. As the total administered dose increased from low to high, %free methadone decreased from 25% to approximately 5% of the total R methadone detected. The data indicate that the relationship between %free R methadone and total R methadone is logarithmic and nonlinear as shown in Figs. 3 and 5. The binding of methadone is primarily to alpha-1 acid glycoprotein (A1AG) [1,2]. We measured the concentration of
A1AG and related it to %free R methadone present in each sample. The results are shown in Fig. 4. The relationship is very similar to that shown in Fig. 3 (which shows the correlation between %free R methadone and total R methadone). The relationship shown in Fig. 4 provides direct evidence that R methadone binds to A12AG. Fig. 4 shows that the correlation between A1AG and %free R methadone had a coefficient of r2 = 0.37 (P b 0.05) which is also statistically significant. The relationship between these two parameters was also logarithmic and non-linear. Fig. 6 depicts the relationship between free R methadone and A1AG. Free R methadone concentrations increased at higher concentrations of A1AG. To determine if the large variations in the dose of methadone required to prevent the onset of withdrawal symptoms could be accounted for by metabolism or by differences in protein binding of the drug, we compared the range of administered doses given to the different patients to the range of concentrations of free R methadone, the biologically active form that acts on the mu opioid receptor. The range of dosages from 9 to 205 mg represents a 23-fold increase in the amount of drug between the lowest and
Fig. 6. Relationship between free R Methadone and A1AG.
D.C. Lehotay et al. / Clinical Biochemistry 38 (2005) 1088–1094
highest amount given to the 45 patients in our data set. These doses and concentrations were not corrected for body weight. In a small subset (n = 11) of patients, the weights were used to calculate a dose/body weight value. Using this value instead of the uncorrected dose gave a range of values that varied from 3.8 to 21.5, an almost six-fold difference. However, when the range of steady-state concentrations of free R methadone is compared, it was found to vary between 6.5 and 175.9 nmol/L, a 27-fold increase in concentrations. Similarly, the range of total R methadone levels varied by about 20-fold between 110 and 2040 nmol/L in the same 45 patients. By measuring the free, biologically active form of the drug, we were unable to account for the large variations in dose required between different patients to prevent the onset of withdrawal symptoms. Discussion Biological response to methadone was not measured directly. However, all of the clients tested had been on stable maintenance therapy for 3 months or longer. The clients in this study were from methadone clinics in Regina, Saskatoon and from Toronto. All these are Harm Reduction Programs, where no attempt is made to force the clients to take less of the drug than is required to eliminate withdrawal symptoms. We therefore assumed that the amount of drug given was sufficient to prevent their onset. It has been recommended that trough levels of methadone need to be between 150 and 200 ng/ml (0.5–0.67 μmol/L), but preferably in the 400 ng/ml (1.35 μmol/L) range to be effective in reducing or eliminating withdrawal symptoms [10]. The total amount of methadone (sum of R and S methadone) was less than 0.5 μmol/L in 12 of a total of 45 of our patients. A total of 25/45 of the patients in our study had total methadone concentrations less than 1.35 μmol/L. The recommendation that dose of methadone needs to be sufficient to allow trough levels to be at or above 1.35 μmol/L is based on studies that show high rates of use of illegal opiates as the dose of the drug is reduced. While we support this argument, we also believe that it may be possible to prevent withdrawal symptoms in some clients with lower doses as also indicated in the present study. In fact, in a subset of the patients in this study who were on the lowest doses, they were less likely to use other illicit drugs that those on the highest doses (Table 2). The differences between patients on low and high maintenance doses have not been adequately described and are unclear. Further work is required for a better understanding of these differences. Eap found large individual variability in the R/S methadone ratios [11]. The average R/S methadone ratio of patients in this study was 1.19 ± 0.3 (mean ± SD), a value close to 1.0, with numbers ranging from 0.64 to 2.1. While the range of R/S values is only slightly less than that of Eap, the correlation between the two values was very high, suggesting that R and S methadone were metabolized at approximately the same rate. Methadone is metabolized by the cytochrome P450 enzymes in liver. A number of genetic variants of the P450CYP isoforms
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Table 2 Methadone dosage, free R methadone, and illegal drug use Dosage (mg)
Free R methadone (μmol/l)
Low dose patients 20 0.010 24 0.026 25 0.009 25 0.027 45 0.006 55 0.023 32 ± 14 0.017 ± 0.009 High dose patients 115 0.116 120 0.037 130 0.073 155 0.073 160 0.048 170 0.097 142 ± 23 P b 0.001
Documented illicit drug use None None THC THC, cocaine THC THC, cocaine, ritalin
Opiates, benzodiazepines Opiates, benzodiazepines None Opiates, benzodiazepines, ritalin THC, “anti-depressants”, Ritalin “Sleeping pills”, THC, cocaine, benzodiazepines
0.074 ± 0.029 P b 0.005
Results show individual administered doses of methadone in mg, free R methadone concentrations in blood in μmol/l, mean ± SD, and P values indicating levels of significance between low and high dose patients.
exist, some of which metabolize methadone much faster than others [12,13]. We were not able to analyze which of the various P450 enzymes were present in the patients in this study, future experiments are planned to accomplish this task. In the absence of such information, we relied on the concentration and ratio of the R and S methadone and its main metabolite, EDDP, to draw conclusions about the rate and specificity of metabolism of the drug. The presence of significant amounts of total and free R methadone and a ratio that was less than 2 between peak and trough levels of methadone suggested that there were no fast or very fast metabolizers of the drug in our patients, and that most if not all of the patients in this study are probably of the same genotype of the P450 isoenzyme, which may be the common P450 3A4 form. Others who have detected fast metabolism of methadone studied a different population of methadone clients from the one reported in this study [6]. The difference between our data and that of others most likely represents a geographical and possibly genetic difference in patient populations. In order to explain the large individual differences in the dose required to prevent withdrawal symptoms, we measured the free form of R methadone, the fraction that exerts the biological activity of this drug. Methadone binds mostly to alpha-1 acid glycoprotein (A1AG) and to serum albumin with lower affinity [2]. Free R methadone concentrations in serum varied between 0.007 and 0.176 μmol/L. Total R methadone concentrations were between 0.110 and 2.04 μmol/L. The binding affinity of A1AG for methadone is around 5 × 10− 5 M/L. The concentration of A1AG in samples varied between 6 and 38 μmol/l. Binding of a drug to serum proteins is determined by the concentration of the drug, the binding affinity, the total number of binding sites available for binding, and the presence of other compounds that may compete for binding. At a concentration of
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methadone equal to the binding affinity constant, half of the drug is bound, the remainder is free. At lower concentrations, less methadone is expected to be bound. Since there are other serum proteins that also bind methadone with differing affinities, and other drugs may also be present that compete for the available binding sites on A1AG, it is difficult to estimate the fraction of methadone that will be free at a given concentration of methadone, A1AG, and albumin. It appeared to be simpler to measure the free fraction of methadone directly. When this was done, a complex relationship between the amount of methadone and A1AG emerged. As a generalization, it may be stated that as A1AG increased, a higher percentage of methadone was bound. It was interesting, that as levels of A1AG increased, free R methadone levels increased also as shown in Fig. 6, indicating a complicated relationship between dose, binding to serum proteins, total R methadone levels, and biological response. Garrido et al. [2] recently showed that the mean of A1AG concentration was significantly increased in patients showing signs of withdrawal, while the albumin concentrations did not change. They also show that binding of methadone is related to A1AG (P b 0.05) levels and not to albumin. Patients in withdrawal had unbound methadone significantly decreased when compared to the control. A positive correlation (Pearson r = 0.48; P b 0.005) indicates that AGP levels rise during abstinence as the score of withdrawal symptoms increases. In our patient population, the %free fraction of R methadone decreased as the dose increased. This was probably because at higher doses, the concentration of methadone approached the binding constant; hence, more of the total drug was bound. At the lowest doses, methadone concentration is well below the affinity constant of A1AG for the drug, so less is bound. A comparison of the ratio of free methadone R to free methadone S revealed that on average, almost twice as much of the R form than of the S enantiomer was free. There was significant variability in the ratio, but the correlation between free R and free S was excellent: as one increased, so did the other (r2 = 0.76). These findings are consistent with and support those of others [14]. Attempts have been made in the past to predict the required methadone dose based on the 6-OH-cortisol/17-OHcorticosteroid ratio [15]. The rationale for this was that, since cortisol is metabolized via the same P450 enzymes as methadone, namely, cytochrome P450 3E4, an estimate of the flux through this pathway might predict the rate at which methadone is metabolized. The finding that this method is not predictive of the appropriate dose was confirmed by a more direct measurement of the ratio of trough levels of EDDP-R to methadone-R in this paper, a ratio which is indicative of the rate at which methadone is converted to its inactive metabolite, EDDP. In summary, we measured free and protein-bound forms of the R and S enantiomers of methadone and its major inactive metabolite, EDDP. Knowing the concentration of free, biologically active R methadone did not allow us to predict the dose of methadone required to reduce withdrawal symptoms. This suggested that while there may be differences in the
metabolism of methadone in different patients, there are also many other factors whose role will have to be understood better in order to predict the dose required to prevent withdrawal symptoms. These relate to the possible down-regulation of the mu opioid receptor in drug addicts, as well as variations in receptor number and affinity, all of which may change from patient to patient, and may be hard to predict without direct measurement of these variables in individual patients. A direct determination of the P450 methadone metabolizing enzyme genotype by molecular analysis, as well as a detailed study of the events taking place at the receptor, may be required to gain a better understanding of the quantitative relationships that may exist between dose of methadone, free R methadone, receptor activation, and the biological response, which is the prevention of withdrawal symptoms. References [1] Olsen GD. Methadone binding to human plasma proteins. Clin Pharmacol Ther 1973;14:338–43. [2] Garrido MJ, Aguirre C, Troconiz IF, Marot M, Valle M, Zamacona MK, et al. Alpha 1-acid glycoprotein (AAG) and serum protein binding of methadone in heroin addicts with abstinence syndrome. Int J Clin Pharmacol Ther 2000;38:35–40. [3] Romach MK, Piafsky KM, Abel JG, Khouw V, Sellers EM. Methadone binding to orosomucoid (alpha 1-acid glycoprotein): determinant of free fraction in plasma. Clin Pharmacol Ther 1981;29:211–7. [4] Sullivan HR, Due SL. Urinary metabolites of DL-methadone in maintenance subjects. J Med Chem 1973;16:909–13. [5] Anggard E, Gunne LM, Homstrand J, McMahon RE, Sandberg CG, Sullivan HR. Disposition of methadone in methadone maintenance. Clin Pharmacol Ther 1975;17:258–66. [6] Eap CB, Buclin T, Baumann P. Interindividual variability of the clinical pharmacokinetics of methadone: implications for the treatment of opioid dependence. Clin Pharmacokinet 2002;41:1153–93 [Review, 236 refs]. [7] Dole VP, Nyswander M. A medical treatment for diacetylmorphine (heroin) addiction: a clinical trial with methadone hydrochloride. JAMA 1965;193:80–4. [8] Strain EC, Bigelow GE, Liebson IA, Stitzer ML. Moderate- vs. high-dose methadone in the treatment of opioid dependence: a randomized trial. JAMA 1999;281:1000–5 [see comment]. [9] Caplehorn JR, Bell J. Methadone dosage and retention of patients in maintenance treatment. Med J Aust 1991;154:195–9 [see comment] [erratum appears in Med J Aust 1993; 159(9):640;]. [10] Maxwell S, Shinderman M. Optimizing response to methadone maintenance treatment: use of higher-dose methadone. J Psychoactive Drugs 1999;31:95–102. [11] Eap CB, Bourquin M, Martin J, Spagnoli J, Livoti S, Powell K, et al. Plasma concentrations of the enantiomers of methadone and therapeutic response in methadone maintenance treatment. Drug Alcohol Depend 2000;61:47–54. [12] Eap CB, Broly F, Mino A, Hammig R, Deglon JJ, Uehlinger C, et al. Cytochrome P450 2D6 genotype and methadone steady-state concentrations. J Clin Psychopharmacol 2001;21:229–34. [13] Shinderman M, Maxwell S, Brawand-Amey M, Golay KP, Baumann P, Eap CB. Cytochrome P4503A4 metabolic activity, methadone blood concentrations, and methadone doses. Drug Alcohol Depend 2003;69:205–11. [14] Boulton DW, Devane CL. Development and application of a chiral high performance liquid chromatography assay for pharmacokinetic studies of methadone. Chirality 2000;12:681–7. [15] Charlier C. Methadone maintenance treatment: is it possible to adapt the daily doses to the metabolic activity of the patient? Ther Drug Monit 2001;23:1–3.
Free and bound enantiomers of methadone and its metabolite, EDDP in methadone maintenance treatment: Relationship to dosage? D.C. Lehotay a,c,⁎, S. George a , M.L. Etter a , K. Graybiel a , J.C. Eichhorst a , B. Fern e , W. Wildenboer f , P. Selby d , B. Kapur b a
Provincial Laboratory, 3211 Albert Street, Regina, SK, Canada S4S 5W6 b St. Michael's Hospital, University of Toronto, Canada c University of Saskatchewan, Saskatchewan, Canada d Centre of Addiction and Mental Health, University of Toronto, Canada e College Park Medical Clinic 2600, 8th Street, Saskatoon, SK, Canada f Regina Community Clinic, 1106 Winnipeg St., Regina, SK, Canada
Received 7 April 2005; received in revised form 16 August 2005; accepted 17 September 2005 Available online 10 November 2005
Abstract Objectives: To determine the effects of metabolism and protein binding on the relationship between administered dose, blood levels of R methadone and biological response by measuring the free and protein-bound forms of the R and S enantiomers of methadone and EDDP, its metabolite. Design and methods: To measure free and total drug, trough levels were collected from 45 methadone clients. To measure free methadone, samples were filtered using ultrafiltration with a MW weight cut-off of 10,000 and extracted using liquid–liquid extraction. The solvent was evaporated and samples reconstituted in mobile phase for analysis by LC/MS/MS. Total analyte was determined by extracting unfiltered samples. Enantiomeric separation of methadone and EDDP was by chiral chromatography. Results: The presence of unmetabolized methadone suggested that none of the patients were very fast metabolizers. R and S forms were metabolized at the same rate at all administered doses. Free R methadone levels correlated both with methadone dose and with the total amount of R methadone. The free fraction of R methadone (%free R) was higher at lower doses than at high doses, varied from 5 to 25% and was inversely proportional to the total dose of administered drug in a relationship that was logarithmic and non-linear. Conclusions: By measuring the free, biologically active form of the drug, we were unable to account for the large variations in dose required between different patients to prevent the onset of withdrawal symptoms. The reason for the large range in dosage may be multifactorial. © 2005 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Methadone; EDDP; Enantiomer; Bound; Free; Tandem MS; LC/MS/MS; Dosage; Withdrawal symptoms
Introduction Methadone is commonly used in the treatment of opioid dependence. The drug is usually administered orally as a racemic mixture of (R) and (S) methadone. The (R) form accounts for most or all of the opioid effects, while the (S) form is devoid of biological activity. In addition, methadone is bound to plasma proteins: it has been estimated that about 85% of the drug is bound to α-1 acid glycoprotein and to a much lesser extent to ⁎ Corresponding author. Provincial Laboratory, 3211 Albert Street, Regina, SK, Canada S4S 5W6. E-mail address: [email protected] (D.C. Lehotay).
serum albumin [1,2]. Alpha-1 acid glycoprotein is an acute phase protein that may be elevated in inflammation and may thus alter the availability of free or unbound drug [3]. As proteinbound drug is not normally available to interact with drug receptors, biological activity is due primarily to the free fraction of the drug. Methadone is metabolized by cytochrome P450 enzymes in liver via N-demethylation followed by cyclization to 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), a biologically inactive metabolite [4]. Several additional minor metabolites have also been identified [5]. There is marked individual variability in the optimal dose of methadone required to maintain clients in a methadone maintenance treatment (MMT) program. In a recent review,
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Eap et al. [6] commented that an adequate methadone dosage is at least 60 mg/day but may be closer to 80–100 mg/day. Providing adequate methadone to clients to reduce craving (to prevent the onset of withdrawal symptoms) is a major factor in the success of an MMT program. Many MMT programs prescribe low dosages of methadone for political, psychological, philosophical or moral reasons. One of the major concerns is that, to date, there has not been a scientific basis for determining adequate and/or optimal doses. Most physicians prescribing methadone follow the guidelines outlined by Eap [6] and others [7–9]. They also test their clients for other drugs of abuse and consult with their clients directly regarding the adequacy of the dose. Among prescribers, there is continuing disagreement regarding optimal methadone dosage for MMT. Most MMT programs monitor their clients for compliance by periodic urine drug testing. Measuring methadone, or its major metabolite EDDP, has not been very successful in determining optimal methadone dosage. Urine drug testing programs normally measure methadone by an immunoassay that quantifies the sum of both the R and S enantiomers of methadone. The concentrations of free drug in blood or the effects of metabolism on the bioavailability of R methadone are not reported. The experiments described below were designed to gain a better understanding of the factors that may influence optimal methadone dosage in a MMT program. We developed methods to quantify free and bound forms of the R and S enantiomers of methadone and its metabolite, EDDP in blood. These were measured in 45 patients on long-term methadone maintenance therapy who have been on the same dose of the drug for at least 3 months. The objective of these experiments was to (a)determine if there is a relationship between administered dose of methadone, blood levels of the active form of the drug and biological response; (b) to measure the free and protein-bound forms of the active R and inactive S enantiomers of methadone, and R and S forms of EDDP, its metabolite; and (c) to determine the nature of the relationship between methadone metabolism, protein binding and the large variations in maintenance dosage required by different methadone clients. Materials and methods Patients and sample collection Ambulatory patients on a stable maintenance dosage of methadone were selected for this study. All of the 45 patients received their daily dose of methadone once a day. Doses varied between 10 and 205 mg/day. All patients were on the same dose for at least 3 months prior to the beginning of the study. Trough level samples were collected from all patients just prior to the administration of their daily dose of the drug, which was given as an oral solution consisting of a racemic mixture of equal amounts of R and S methadone. Eight of the 45 patients in the study also had blood collected at 2 h after their daily dose (peak levels). Patients from whom extra samples were collected
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provided consent for the study and were paid an honorarium. The study was approved by the Ethics Review Board (ERB) of the University of Saskatchewan College of Medicine and the ERB of the University of Toronto. Sample processing Serum was collected in non-gel red top tubes, plasma was anti-coagulated with EDTA and separated within 30 min. 300 μL Table 1 Free and total pre-dose R and S Methadone and EDDP (μmol/L) Pt. Dose Total Total Free code R S R
Free S
% Free R
A01 A02 A03 A04 A05 A06 A07 A08 A09 A10 A11 A12 A13 A14 A15 A16 A17 A18 A19 A20 A21 A22 A23 A24 A25 A26 A27 A28 A29 A30 A31 A32 A33 A34 A35 A36 A37 A38 A39 A40 A41 A42 A43
0.003 0.027 0.003 0.024 0.003 0.008 0.034 0.006 0.007 0.006 0.014 0.011 0.044 0.060 0.008 0.032 0.042 0.019 0.059 0.034 0.049 0.047 0.031 0.032 0.061 0.033 0.034 0.046 0.051 0.055 0.037 0.035 0.031 0.062 0.043 0.036 0.047 0.043 0.053 0.127 0.042 0.039 0.043
9.01 13.37 5.77 17.04 3.49 10.51 12.85 5.71 7.26 8.33 7.41 5.43 8.21 7.06 6.41 4.60 5.50 4.38 4.81 10.17 5.89 8.26 22.72 18.10 5.21 22.77 6.55 9.92 5.63 9.99 14.83 5.93 18.60 6.04 7.24 7.79 13.11 3.84 8.18 9.70 5.21 15.70 11.85
20 24 25 25 45 55 60 70 70 70 80 105 110 115 120 130 155 160 170 40 140 125 30 30 190 32 65 85 160 25 150 9 205 110 94 70 125 100 150 190 50 60
0.12 0.20 0.15 0.16 0.19 0.22 0.69 0.37 0.26 0.16 0.37 0.48 0.80 1.65 0.58 1.58 1.34 1.09 2.02 0.38 1.22 0.85 0.15 0.19 2.04 0.15 0.59 0.65 1.64 1.23 0.28 0.97 0.17 1.54 0.77 0.61 0.48 1.78 1.06 1.81 1.27 0.30 0.54
0.06 0.18 0.13 0.14 0.18 0.16 0.54 0.29 0.25 0.13 0.33 0.30 0.64 1.75 0.28 1.23 1.04 0.81 2.40 0.36 1.27 0.89 0.12 0.16 1.87 0.15 0.65 0.72 1.49 0.83 0.15 0.44 0.16 1.97 0.82 0.54 0.50 1.67 0.90 2.84 1.01 0.30 0.46
0.010 0.026 0.009 0.027 0.006 0.023 0.088 0.021 0.019 0.013 0.028 0.026 0.066 0.116 0.037 0.073 0.073 0.048 0.097 0.038 0.072 0.071 0.034 0.035 0.106 0.035 0.038 0.065 0.092 0.123 0.042 0.058 0.031 0.093 0.056 0.048 0.063 0.069 0.087 0.176 0.066 0.047 0.064
A1AG fR/ fS
23.25 15.09 26.46 13.60 16.32 19.79 6.43 18.80 14.10 18.05 24.24 11.38 13.85 18.80 30.42 14.59 22.01 38.34 21.02 16.82 15.58 12.86 9.15
3.79 0.98 3.24 1.16 1.91 2.98 2.63 3.25 2.60 2.08 2.03 2.44 1.50 1.94 4.53 2.29 1.73 2.48 1.64 1.14 1.46 1.51 1.08 1.09 1.76 1.07 1.13 1.39 1.81 2.23 1.13 1.66 1.02 1.51 1.30 1.32 1.34 1.60 1.66 1.39 1.56 1.23 1.49
fR-EDDP/ fS-EDDP 1.02 1.00 1.05 1.02 0.90 0.97 0.88 1.36 1.09 0.90 0.80 1.12 0.91 0.77 1.21 1.00 1.21 0.99 0.89 0.77 0.87 1.14 1.00 0.82 0.84 1.12 1.17 1.19 1.26 1.08 1.24 0.73 1.12 0.77 1.11 0.98 1.37 0.99 0.92 1.22 0.83 1.09
All concentrations of methadone and/or EDDP are expressed as μmol/L of serum or plasma. Dose of drug administered to patients is in milligrams and is the total weight of the enantiomeric mixture given to the patient orally. Units of A1AG are in μmol/L. fR and fS represent free R methadone and free S methadone, respectively. Similarly, fR-EDDP and fS-EDDP represent free R EDDP and free S EDDP.
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Fig. 1. Relationship between ratio of free R- and S-EDDP and dose.
of serum or plasma was centrifuged at 9000 rpm (7437 × g) for 2 h using a selective ultrafiltration membrane with a molecular weight cut-off of 10,000 Da. Protein-bound methadone and EDDP are retained on the filter and all compounds with a molecular weight less than approximately 10,000 Da including free drugs and water pass through. 100 μL of ultrafiltrate or 250 μL of serum was extracted with 1-chlorobutane, evaporated to dryness under N2 and reconstituted in 200 μL HPLC mobile phase prior to further analysis of free and total drug concentrations by LC/MS/MS. Separation of R and S enantiomers of free and total methadone and free and total EDDP
was by HPLC on a Chiral-AGP 100 × 3.0 mm (0.5 μm) column followed by analysis on an Applied Biosystems API-4000 tandem mass spectrometer using a thermospray source in positive ion mode using MRM as described in the accompanying paper. Results Trough levels of total R and S methadone, and total R and S forms of EDDP were quantified as described in Materials and methods. Free R and S forms of the drug and metabolite were
Fig. 2. Relationship between free R and total R methadone.
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Fig. 3. Relationship between %free R methadone and dose.
also measured following separation of bound from free forms by ultrafiltration as described above. All of the data not shown. Table 1 shows the data used for the more important comparisons made in the Results and Discussion. Metabolism At steady state (trough level), all samples analyzed had measurable levels of R and S forms of methadone, suggesting that none of the patients were very fast metabolizers. On a small subset (eight) of the clients, we were able to collect two blood samples, one just prior to administration of their daily dose (trough level) and another one at about 2–3 h after their daily dose (peak level). The ratio of post-dose total R methadone to the pre-dose R methadone level in eight patients had an average value of 1.4 (range = 1.1–1.9), supporting our claim that in this population, there were no
fast or very fast metabolizers. The relationship between R and S forms of methadone and EDDP remained very close to one, indicating that both the biologically active R form and the inactive S form are metabolized at the same rate to EDDP as shown in Fig. 1. The correlation coefficients r2 between total R and S methadone and R and S EDDP were 0.94 and 0.98, respectively. The ratio between R and S forms of methadone converted to EDDP (R-EDDP/R methadone/S-EDDP/S methadone), as well as the relationship between total dose given and total R methadone, was also very close to one (correlation coefficient r2 = 0.88 and r2 = 0.97, respectively). We also determined the fraction of methadone metabolized at increasing administered doses. The results indicated that the ratio of R-EDDP to R methadone, which is a measure of the fraction of methadone metabolized at steady state, was the same regardless of the administered dose. The rate of metabolism remained unchanged at all doses.
Fig. 4. Relationship between A1AG and %free R methadone.
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Fig. 5. Relationship between %free R and total R methadone.
Free methadone Biological activity is exerted by the free (unbound) fraction of R methadone. Free R methadone levels were measured and found to be proportional to methadone dose with a correlation coefficient of r2 = 0.47, a statistically significant relationship (P b 0.05). Free R methadone correlated even better with the total amount of R methadone with a correlation of r2 = 0.55 (P b 0.05) (see Fig. 2). The free fraction of R methadone ((% free R = free R methadone/total R methadone) × 100) was higher at lower doses than at high doses of total administered drug. The correlation was statistically significant correlation of r2 = 0.47 (P b 0.05). The %free R methadone varied from 5 to 25% of total R methadone. As the total administered dose increased from low to high, %free methadone decreased from 25% to approximately 5% of the total R methadone detected. The data indicate that the relationship between %free R methadone and total R methadone is logarithmic and nonlinear as shown in Figs. 3 and 5. The binding of methadone is primarily to alpha-1 acid glycoprotein (A1AG) [1,2]. We measured the concentration of
A1AG and related it to %free R methadone present in each sample. The results are shown in Fig. 4. The relationship is very similar to that shown in Fig. 3 (which shows the correlation between %free R methadone and total R methadone). The relationship shown in Fig. 4 provides direct evidence that R methadone binds to A12AG. Fig. 4 shows that the correlation between A1AG and %free R methadone had a coefficient of r2 = 0.37 (P b 0.05) which is also statistically significant. The relationship between these two parameters was also logarithmic and non-linear. Fig. 6 depicts the relationship between free R methadone and A1AG. Free R methadone concentrations increased at higher concentrations of A1AG. To determine if the large variations in the dose of methadone required to prevent the onset of withdrawal symptoms could be accounted for by metabolism or by differences in protein binding of the drug, we compared the range of administered doses given to the different patients to the range of concentrations of free R methadone, the biologically active form that acts on the mu opioid receptor. The range of dosages from 9 to 205 mg represents a 23-fold increase in the amount of drug between the lowest and
Fig. 6. Relationship between free R Methadone and A1AG.
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highest amount given to the 45 patients in our data set. These doses and concentrations were not corrected for body weight. In a small subset (n = 11) of patients, the weights were used to calculate a dose/body weight value. Using this value instead of the uncorrected dose gave a range of values that varied from 3.8 to 21.5, an almost six-fold difference. However, when the range of steady-state concentrations of free R methadone is compared, it was found to vary between 6.5 and 175.9 nmol/L, a 27-fold increase in concentrations. Similarly, the range of total R methadone levels varied by about 20-fold between 110 and 2040 nmol/L in the same 45 patients. By measuring the free, biologically active form of the drug, we were unable to account for the large variations in dose required between different patients to prevent the onset of withdrawal symptoms. Discussion Biological response to methadone was not measured directly. However, all of the clients tested had been on stable maintenance therapy for 3 months or longer. The clients in this study were from methadone clinics in Regina, Saskatoon and from Toronto. All these are Harm Reduction Programs, where no attempt is made to force the clients to take less of the drug than is required to eliminate withdrawal symptoms. We therefore assumed that the amount of drug given was sufficient to prevent their onset. It has been recommended that trough levels of methadone need to be between 150 and 200 ng/ml (0.5–0.67 μmol/L), but preferably in the 400 ng/ml (1.35 μmol/L) range to be effective in reducing or eliminating withdrawal symptoms [10]. The total amount of methadone (sum of R and S methadone) was less than 0.5 μmol/L in 12 of a total of 45 of our patients. A total of 25/45 of the patients in our study had total methadone concentrations less than 1.35 μmol/L. The recommendation that dose of methadone needs to be sufficient to allow trough levels to be at or above 1.35 μmol/L is based on studies that show high rates of use of illegal opiates as the dose of the drug is reduced. While we support this argument, we also believe that it may be possible to prevent withdrawal symptoms in some clients with lower doses as also indicated in the present study. In fact, in a subset of the patients in this study who were on the lowest doses, they were less likely to use other illicit drugs that those on the highest doses (Table 2). The differences between patients on low and high maintenance doses have not been adequately described and are unclear. Further work is required for a better understanding of these differences. Eap found large individual variability in the R/S methadone ratios [11]. The average R/S methadone ratio of patients in this study was 1.19 ± 0.3 (mean ± SD), a value close to 1.0, with numbers ranging from 0.64 to 2.1. While the range of R/S values is only slightly less than that of Eap, the correlation between the two values was very high, suggesting that R and S methadone were metabolized at approximately the same rate. Methadone is metabolized by the cytochrome P450 enzymes in liver. A number of genetic variants of the P450CYP isoforms
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Table 2 Methadone dosage, free R methadone, and illegal drug use Dosage (mg)
Free R methadone (μmol/l)
Low dose patients 20 0.010 24 0.026 25 0.009 25 0.027 45 0.006 55 0.023 32 ± 14 0.017 ± 0.009 High dose patients 115 0.116 120 0.037 130 0.073 155 0.073 160 0.048 170 0.097 142 ± 23 P b 0.001
Documented illicit drug use None None THC THC, cocaine THC THC, cocaine, ritalin
Opiates, benzodiazepines Opiates, benzodiazepines None Opiates, benzodiazepines, ritalin THC, “anti-depressants”, Ritalin “Sleeping pills”, THC, cocaine, benzodiazepines
0.074 ± 0.029 P b 0.005
Results show individual administered doses of methadone in mg, free R methadone concentrations in blood in μmol/l, mean ± SD, and P values indicating levels of significance between low and high dose patients.
exist, some of which metabolize methadone much faster than others [12,13]. We were not able to analyze which of the various P450 enzymes were present in the patients in this study, future experiments are planned to accomplish this task. In the absence of such information, we relied on the concentration and ratio of the R and S methadone and its main metabolite, EDDP, to draw conclusions about the rate and specificity of metabolism of the drug. The presence of significant amounts of total and free R methadone and a ratio that was less than 2 between peak and trough levels of methadone suggested that there were no fast or very fast metabolizers of the drug in our patients, and that most if not all of the patients in this study are probably of the same genotype of the P450 isoenzyme, which may be the common P450 3A4 form. Others who have detected fast metabolism of methadone studied a different population of methadone clients from the one reported in this study [6]. The difference between our data and that of others most likely represents a geographical and possibly genetic difference in patient populations. In order to explain the large individual differences in the dose required to prevent withdrawal symptoms, we measured the free form of R methadone, the fraction that exerts the biological activity of this drug. Methadone binds mostly to alpha-1 acid glycoprotein (A1AG) and to serum albumin with lower affinity [2]. Free R methadone concentrations in serum varied between 0.007 and 0.176 μmol/L. Total R methadone concentrations were between 0.110 and 2.04 μmol/L. The binding affinity of A1AG for methadone is around 5 × 10− 5 M/L. The concentration of A1AG in samples varied between 6 and 38 μmol/l. Binding of a drug to serum proteins is determined by the concentration of the drug, the binding affinity, the total number of binding sites available for binding, and the presence of other compounds that may compete for binding. At a concentration of
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methadone equal to the binding affinity constant, half of the drug is bound, the remainder is free. At lower concentrations, less methadone is expected to be bound. Since there are other serum proteins that also bind methadone with differing affinities, and other drugs may also be present that compete for the available binding sites on A1AG, it is difficult to estimate the fraction of methadone that will be free at a given concentration of methadone, A1AG, and albumin. It appeared to be simpler to measure the free fraction of methadone directly. When this was done, a complex relationship between the amount of methadone and A1AG emerged. As a generalization, it may be stated that as A1AG increased, a higher percentage of methadone was bound. It was interesting, that as levels of A1AG increased, free R methadone levels increased also as shown in Fig. 6, indicating a complicated relationship between dose, binding to serum proteins, total R methadone levels, and biological response. Garrido et al. [2] recently showed that the mean of A1AG concentration was significantly increased in patients showing signs of withdrawal, while the albumin concentrations did not change. They also show that binding of methadone is related to A1AG (P b 0.05) levels and not to albumin. Patients in withdrawal had unbound methadone significantly decreased when compared to the control. A positive correlation (Pearson r = 0.48; P b 0.005) indicates that AGP levels rise during abstinence as the score of withdrawal symptoms increases. In our patient population, the %free fraction of R methadone decreased as the dose increased. This was probably because at higher doses, the concentration of methadone approached the binding constant; hence, more of the total drug was bound. At the lowest doses, methadone concentration is well below the affinity constant of A1AG for the drug, so less is bound. A comparison of the ratio of free methadone R to free methadone S revealed that on average, almost twice as much of the R form than of the S enantiomer was free. There was significant variability in the ratio, but the correlation between free R and free S was excellent: as one increased, so did the other (r2 = 0.76). These findings are consistent with and support those of others [14]. Attempts have been made in the past to predict the required methadone dose based on the 6-OH-cortisol/17-OHcorticosteroid ratio [15]. The rationale for this was that, since cortisol is metabolized via the same P450 enzymes as methadone, namely, cytochrome P450 3E4, an estimate of the flux through this pathway might predict the rate at which methadone is metabolized. The finding that this method is not predictive of the appropriate dose was confirmed by a more direct measurement of the ratio of trough levels of EDDP-R to methadone-R in this paper, a ratio which is indicative of the rate at which methadone is converted to its inactive metabolite, EDDP. In summary, we measured free and protein-bound forms of the R and S enantiomers of methadone and its major inactive metabolite, EDDP. Knowing the concentration of free, biologically active R methadone did not allow us to predict the dose of methadone required to reduce withdrawal symptoms. This suggested that while there may be differences in the
metabolism of methadone in different patients, there are also many other factors whose role will have to be understood better in order to predict the dose required to prevent withdrawal symptoms. These relate to the possible down-regulation of the mu opioid receptor in drug addicts, as well as variations in receptor number and affinity, all of which may change from patient to patient, and may be hard to predict without direct measurement of these variables in individual patients. A direct determination of the P450 methadone metabolizing enzyme genotype by molecular analysis, as well as a detailed study of the events taking place at the receptor, may be required to gain a better understanding of the quantitative relationships that may exist between dose of methadone, free R methadone, receptor activation, and the biological response, which is the prevention of withdrawal symptoms. References [1] Olsen GD. Methadone binding to human plasma proteins. Clin Pharmacol Ther 1973;14:338–43. [2] Garrido MJ, Aguirre C, Troconiz IF, Marot M, Valle M, Zamacona MK, et al. Alpha 1-acid glycoprotein (AAG) and serum protein binding of methadone in heroin addicts with abstinence syndrome. Int J Clin Pharmacol Ther 2000;38:35–40. [3] Romach MK, Piafsky KM, Abel JG, Khouw V, Sellers EM. Methadone binding to orosomucoid (alpha 1-acid glycoprotein): determinant of free fraction in plasma. Clin Pharmacol Ther 1981;29:211–7. [4] Sullivan HR, Due SL. Urinary metabolites of DL-methadone in maintenance subjects. J Med Chem 1973;16:909–13. [5] Anggard E, Gunne LM, Homstrand J, McMahon RE, Sandberg CG, Sullivan HR. Disposition of methadone in methadone maintenance. Clin Pharmacol Ther 1975;17:258–66. [6] Eap CB, Buclin T, Baumann P. Interindividual variability of the clinical pharmacokinetics of methadone: implications for the treatment of opioid dependence. Clin Pharmacokinet 2002;41:1153–93 [Review, 236 refs]. [7] Dole VP, Nyswander M. A medical treatment for diacetylmorphine (heroin) addiction: a clinical trial with methadone hydrochloride. JAMA 1965;193:80–4. [8] Strain EC, Bigelow GE, Liebson IA, Stitzer ML. Moderate- vs. high-dose methadone in the treatment of opioid dependence: a randomized trial. JAMA 1999;281:1000–5 [see comment]. [9] Caplehorn JR, Bell J. Methadone dosage and retention of patients in maintenance treatment. Med J Aust 1991;154:195–9 [see comment] [erratum appears in Med J Aust 1993; 159(9):640;]. [10] Maxwell S, Shinderman M. Optimizing response to methadone maintenance treatment: use of higher-dose methadone. J Psychoactive Drugs 1999;31:95–102. [11] Eap CB, Bourquin M, Martin J, Spagnoli J, Livoti S, Powell K, et al. Plasma concentrations of the enantiomers of methadone and therapeutic response in methadone maintenance treatment. Drug Alcohol Depend 2000;61:47–54. [12] Eap CB, Broly F, Mino A, Hammig R, Deglon JJ, Uehlinger C, et al. Cytochrome P450 2D6 genotype and methadone steady-state concentrations. J Clin Psychopharmacol 2001;21:229–34. [13] Shinderman M, Maxwell S, Brawand-Amey M, Golay KP, Baumann P, Eap CB. Cytochrome P4503A4 metabolic activity, methadone blood concentrations, and methadone doses. Drug Alcohol Depend 2003;69:205–11. [14] Boulton DW, Devane CL. Development and application of a chiral high performance liquid chromatography assay for pharmacokinetic studies of methadone. Chirality 2000;12:681–7. [15] Charlier C. Methadone maintenance treatment: is it possible to adapt the daily doses to the metabolic activity of the patient? Ther Drug Monit 2001;23:1–3.