Introduction

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Methadone is an opiate µ-receptor agonist widely used for perioperative and chronic pain control. It is growing in use for first-line therapy for cancer pain (Manfredi et al., 1997). Methadone is also used for treating opiate withdrawal (O'Connor and Fiellin, 2000) and the therapeutic success and cost-effectiveness in the treatment of opiate addiction are legion (Ling et al., 1994). For preventing opiate withdrawal, methadone is typically administered daily. l--Acetylmethadol (LAAM) is an analog of methadone, characterized by equieffective and longer duration of effect and effectiveness when administered every 2 or 3 days (Eissenberg et al., 1999). LAAM has recently been approved as an alternative to opiate maintenance therapy and is commonly used in narcotic treatment programs (O'Connor and Fiellin, 2000).

Numerous studies have been performed regarding the metabolic fate of methadone. Methadone is predominantly N-demethylated and undergoes spontaneous cyclization to produce 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), which is subsequently N-demethylated to 2-ethyl-5-methyl-3,3-diphenylpyraline (EMDP) (Fig. 1) (Plummer et al., 1988; Inturrisi et al., 1990). These two metabolites are devoid of opiate activity (Sullivan and Due, 1973). LAAM is sequentially N-demethylated to form l--acetyl-N-normethadol (nor-LAAM) and l--acetyl-N,N-dinormethadol (dinor-LAAM) (Fig. 1) (Billings et al., 1973). In contrast to the metabolites of methadone, nor-LAAM and dinor-LAAM are pharmacologically active and are more potent than LAAM. The elimination half-lives of LAAM, nor-LAAM, and dinor-LAAM are approximately 0.5, 1 to 1.5, and 3 to 4 days, respectively (Kaiko and Inturrisi, 1975; Vaupel and Jasinski, 1997; Walsh et al., 1998), which contributes to the longer duration of clinical effects of LAAM compared with methadone.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   N-Demethylation of methadone and LAAM.

During treatment of chronic pain or opioid maintenance therapy, maintaining therapeutic concentrations of methadone, or LAAM and its active metabolites, is crucially important for adequate analgesia and in preventing the occurrence of withdrawal or overdose. Even patients maintained on stable methadone doses experience withdrawal, which has been causally related to unexpected changes in plasma concentration (Dyer et al., 1999). In addition, methadone (and possibly LAAM) is susceptible to pharmacokinetic drug interactions. Decreased methadone clearance occurs in patients treated with cimetidine, erythromycin, ketoconazole, and fluvoxamine, while increased clearance occurs following enzyme induction by rifampin, barbiturates, and phenytoin (Kreek, 1990; O'Connor and Fiellin, 2000). Initiation of rifampin or phenytoin therapy in patients maintained on methadone can precipitate withdrawal, associated with decreased plasma methadone concentrations and increased urinary EDDP and EMDP excretion (Tong et al., 1981; Kreek, 1990). To predict pharmacokinetics and effects of drug interactions, in vitro metabolic profiles are often useful, specifically identification of P450 isoforms responsible for metabolism and the Michaelis-Menten kinetic parameters (Houston and Carlile, 1997; Obach et al., 1997). Several reports showed the principal involvement of CYP3A4 in the N-demethylation of methadone by human liver microsomes and the possible interaction of methadone with other CYP3A4 substrates (Iribarne et al., 1996, 1997; Moody et al., 1997; Foster et al., 1999). CYP3A4 is also involved in the metabolism of LAAM in human liver microsomes (Moody et al., 1997; Oda and Kharasch, 2001).

In clinical practice, wide interindividual variability in methadone disposition is the greatest impediment to predictable dosing and effect (Plummer et al., 1988; Inturrisi et al., 1990). Kinetic variability is exaggerated with oral dosing, and there is substantial variability in oral methadone bioavailability (84 ± 26%, range 41-99%) (Sawe, 1986), suggesting significant first-pass clearance (Inturrisi et al., 1987; Plummer et al., 1988; Inturrisi et al., 1990). A 2- to 3-fold greater metabolite/parent drug area under the curve ratio after oral compared with intravenous LAAM (Walsh et al., 1998) also suggests first-pass metabolism of LAAM in the intestine.

CYP3A4 substrates have been shown to undergo significant first-pass intestinal extraction in vivo. Moreover the magnitude of such first-pass intestinal metabolism can be substantial; for example, intestinal extraction ratios for midazolam equal or exceed those in liver (Gomez et al., 1995; Thummel et al., 1996). Thus, it appears that intestinal metabolism may account for a significant portion of first-pass metabolism of both methadone and LAAM. The purpose of the present study is to test the hypothesis that methadone and LAAM are metabolized by human intestinal microsomes, that CYP3A4 is a predominant isoform, and to scale the in vitro kinetic parameters to predict in vivo intestinal clearances.

	Materials and Methods

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Chemicals. EDDP·perchlorate (d₀), deuterated EDDP·perchlorate ([ethyl-2',2',2'-²H₃]-1,5-dimethyl-3,3-diphenyl-2-ethylpyrrolinium·perchlo-rate) (d₃), EMDP·HCl (d₀), deuterated EMDP·HCl ([ethyl-2',2',2'-²H₃]-3,3-diphenyl-2-ethyl-5-methyl-1-pyrroline·HCl) (d₃), LAAM·HCl (d₀), deuterated LAAM·HCl {()-[1,1,1,2,2,3-²H₆]--acetylmethadol·HCl} (d₆), nor-LAAM·HCl (d₀), deuterated nor-LAAM·HCl {()-[1,1,1,2,2,3-²H₆]--acetyl-N-normethadol·HCl} (d₆), dinor-LAAM·HCl (d₀), and deuterated dinor-LAAM·HCl {()-[acetyl-²H₃]--acetyl-N,N-dinormethadol·HCl) (d₃) were prepared at Research Triangle Institute (Research Triangle Park, NC) and provided by the National Institute on Drug Abuse (Rockville, MD). Methadone, EDDP, and EMDP are racemic mixtures. LAAM, nor-LAAM, and dinor-LAAM are single enantiomers. Methadone, glucose 6-phosphate, glucose-6-phosphate dehydrogenase (type VII), NADP, troleandomycin, ketoconazole, and trifluoroacetic acid were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Human intestinal tissue medically unsuitable for transplant was obtained from University of Washington Medical Center (Seattle, WA). cDNA-expressed CYP3A4 in microsomes was obtained from GENTEST (Woburn, MA). Acetonitrile (high-performance liquid chromatography grade) was purchased from J. T. Baker (Phillipsburg, NJ). Unless specified, all other reagents were purchased from Sigma-Aldrich Chemical Co. and were of the highest purity available. All buffers and reagents were prepared with high-purity (18.2 M · cm) water (Milli-Q; Millipore, Bedford, MA).

Incubation Conditions with Human Intestinal Microsomes and cDNA-Expressed CYP3A4. Microsomes were prepared from human intestine by differential centrifugation of homogenates as described by Paine et al. (1997) and stored at 80°C until used. Protein concentration of the microsomal fractions was measured using the method of Lowry et al. (1951), with bovine serum albumin as standard. P450 content was determined from the difference spectrum of carbon monoxide-reduced versus oxidized microsomes as described by Omura and Sato (1964). Incubations were conducted at a final volume of 0.5 ml in 100 mM potassium phosphate buffer (pH 7.4) containing human intestinal microsomes (0.2 mg of protein) and substrate, methadone, LAAM, or nor-LAAM (0.05-1500 µM). The incubation mixture was preincubated at 37°C for 3 min then the reaction was initiated by addition of the NADPH-generating system (final concentrations: 10 mM glucose 6-phosphate, 1.0 mM NADP, and 1.0 unit of glucose-6-phosphate dehydrogenase and 5 mM magnesium chloride, preincubated at 37°C for 10 min to preform NADPH) and lasted for 10 min. Reactions were terminated by addition of 0.1 ml of 20% trichloroacetic acid, and transferring the incubation vials to an ice-water bath. Experiments using CYP3A4 with coexpressed cytochrome b₅ were done as described above, except 5 pmol of cDNA-expressed CYP3A4 was used instead of microsomes and incubation was for 30 min.

Inhibition Study with CYP Isoform-Specific Inhibitors. Experiments with the CYP3A4-selective inhibitor troleandomycin (100 µM) and with ketoconazole (5 µM) were conducted with microsomes from two individuals [human intestinal microsome (HIM) 40 and 46]. Inhibitor concentrations were chose to inhibit >80% of CYP3A4 activity (Kharasch and Thummel, 1993). These inhibitors were diluted in methanol (final methanol concentration 1%). Concentration of methadone, LAAM, and nor-LAAM was 5 µM. Ketoconazole, a competitive inhibitor of CYP3A4, was added to the incubation mixture with substrate, preincubated at 37°C for 3 min, and the reaction was initiated by the addition of the NADPH-generating system. Troleandomycin, a mechanism-based inhibitor of CYP3A4, was first preincubated at 37°C for 15 min with microsomes and the NADPH-generating system then the substrate was added to start the reaction. Reactions were carried out at 37°C for 10 min and then terminated with trichloroacetic acid as described above.

Analytical Determinations. After termination of the incubation, deuterated internal standards of EDDP (d₃, 89.3 pmol) and EMDP (d₃, 9.4 pmol) were added to the reaction mixture of methadone; nor-LAAM (d₆, 72.5 pmol) and dinor-LAAM (d₃, 7.6 pmol) were added to the mixture of LAAM and nor-LAAM. LAAM (d₆, 696 pmol) was also added to measure the remaining LAAM following incubation. Solid phase extraction of the samples was performed as described previously (Oda and Kharasch, 2001). Analytes were quantified by liquid chromatography-mass spectrometry. For EDDP and EMDP measurement, an isocratic mobile phase of 55% methanol in 0.05% trifluoroacetic acid (pH 3.6) was used at 0.25 ml/min. Detection was performed at m/z 264.1, 267.1, 278.1, and 281.1 for EMDP (d₀), EMDP (d₃), EDDP (d₀), and EDDP (d₃), respectively, with 80-V fragmentation. Retention times of EMDP (d₀), EMDP (d₃), EDDP (d₀), and EDDP (d₃) were 2.36, 2.36, 3.10, and 3.00 min, respectively. EDDP and EMDP were quantified from the peak area ratios, d₀/d₃ of EDDP and d₀/d₃ of EMDP, based on least-square regression of calibrators (2.5-25000 pmol for EDDP and 0.25-2500 pmol for EMDP). LAAM, nor-LAAM, and dinor-LAAM were measured as reported previously (Oda and Kharasch, 2001). The lower limit of quantitation was 0.5 pmol for EDDP, EMDP, nor-LAAM and dinor-LAAM. The intra-assay and interassay variations were less than 7% throughout the range. Minor contamination of EDDP, EMDP in methadone, and dinor-LAAM in nor-LAAM quantified at each substrate concentration was subtracted from the formation of metabolites. No contamination was detected in LAAM. Amount of the contamination was measured from the mixtures containing the substrate, microsomes, and NADPH without incubation.

Data Analysis. Microsomal velocity versus substrate concentration data were analyzed using a dual-enzyme Michaelis-Menten model. This model was selected because hyperbolic Eadie-Hofstee curves most often indicate multiple CYP isoforms participation in microsomal reactions. In the following equations, S is the substrate concentration; K_m1 and K_m2, high- and low-affinity Michaelis-Menten constants, respectively; V_max, maximum metabolic velocity; V_max1 and V_max2, high- and low-affinity maximum metabolic velocity, respectively; K', binding constant; and n, number of binding sites.

(1)

CYP3A4 data were analyzed using several models in addition to the above-described model, based on the recognition that this isoform contains at least two binding sites (Korzekwa et al., 1998; Shou et al., 1999; Hosea et al., 2000). If the binding sites are nonindependent and exhibit cooperativity then the general allosteric model (Hill equation) can be used, with known limitations (Shou et al., 1999).

(2)

A cooperative single-enzyme model with two binding sites in which product can be formed either from the single-substrate-bound form (ES) or from the two-substrate-bound form (ESS) of the enzyme was described by Korzekwa et al. (1998).

(3)

If K_m2 K_m1 and S K_m2 this reduces to the following:

(4)

where V_max2/K_m2 is modeled as a single parameter.

(5)

Predicted Intestinal Clearance and Extraction Ratio of Methadone, LAAM, and nor-LAAM. Predicted in vivo intestinal formation clearance (CL) and extraction ratio (ER) of EDDP and EMDP from methadone, nor-LAAM and dinor-LAAM from LAAM, and dinor-LAAM from nor-LAAM were scaled from the CL_int (V_max/K_m) of intestinal microsomes and CYP3A4 using the following scaling factors (Thummel et al., 1997): 71 nmol of total intestinal CYP3A4 and 23 pmol of CYP3A4/mg of intestinal microsomal protein. Intestinal extraction ratio was calculated as CL/(CL + blood flow to the small intestine) using the well stirred model. Assuming that drug distributes in red blood cells (ERb), blood flow to the small intestine was assumed to be 248 ml/min (Thummel et al., 1997). Assuming that drug distribution is restricted to plasma (ERp), plasma flow to the small intestine was assumed to be 52% of blood flow, 129 ml/min. Methadone (38%) and LAAM (50%) do partition into red cells (Toro-Goyco, 1980; Boulton and Devane, 2000); hence, both models were evaluated. Protein binding was not considered in the predictions since it does not affect the steady-state clearance of methadone (Foster et al., 2000), and the effect of protein binding on the pharmacokinetics of LAAM is unknown.

	Results

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Metabolism of Methadone by Human Intestinal Microsomes. Formation of both EDDP and EMDP from methadone was linear with microsomal protein content up to 0.2 mg and incubation time up to 20 min under the substrate concentrations tested (Fig. 2A). In addition, N-demethylation (sum of EDDP and EMDP) consumed less than 10% of the substrate. Relationships between formation rates of metabolites and substrate concentration showed hyperbolic saturation kinetics (Fig. 1). EMDP concentrations were approximately 10% of those of EDDP. Eadie-Hofstee plots were biphasic and concave hyperbolic (versus parabolic) (Fig. 3, insets), indicating apparent multienzyme kinetics. Kinetic parameters for the formation of EDDP and EMDP were obtained by nonlinear regression analysis of metabolite versus substrate data, using a dual-enzyme Michaelis-Menten model. K_m1 for the formation of EDDP and EMDP from methadone were comparable between intestinal microsomes from three individuals (HIM 21, 40, and 46) (Table 1). K_m2 was more than 10 times higher than K_m1 for EDDP and EMDP formation. The in vitro clearance estimate (CL_int = V_max/K_m) for EMDP from methadone was approximately 3% of that for EDDP from methadone (0.3 × 10³ and 11 × 10³ ml/min/mg of protein, respectively). Both troleandomycin and ketoconazole inhibited the formation of EDDP and EMDP from methadone by more than 70% (Fig. 4).

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Incubation time and formation of EDDP () and EMDP () from methadone by human microsomes (A) and CYP3A4 (B); nor-LAAM () and dinor-LAAM () from LAAM; and dinor-LAAM from nor-LAAM () by human microsomes (C) and by CYP3A4 (D). Substrate concentration was 1 µM. Microsomal protein and CYP3A4 contents were 200 µg and 5 pmol, respectively.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Formation of EDDP (A) and EMDP (B) from methadone (0.05-1500 µM) by human intestinal microsomes (HIM 40). Lines represent rates predicted using Michaelis-Menten kinetic parameters derived from regression analysis of the observed results (symbols). The insets show Eadie-Hofstee plots. Each plot is the mean of duplicate experiments.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Effects of CYP3A4 inhibitors on methadone demethylation by HIM 40 () and HIM 46 (). Rates of metabolite formation were expressed as a percentage of control values without inhibitors. Incubations contained methadone (5 µM), 0.1 mg protein, inhibitors, and NADPH-generating system. Inhibitor concentrations are described under Materials and Methods. Uninhibited rates of formation of EDDP and EMDP by HIM 40 were 5.1 and 0.35 pmol/min/mg, respectively; by HIM 46 they were 6.7 and 0.34 pmol/min/mg. Data are the mean ± S.D. of three experiments.

Metabolism of Methadone by cDNA-Expressed CYP3A4. Formation of both EDDP and EMDP was linear with CYP content up to 10 pmol and incubation time up to 30 min under substrate concentrations tested (Fig. 2B). Plots of metabolite formation versus substrate concentration for N-demethylation of methadone by CYP3A4 showed saturable hyperbolic kinetics with respect to methadone concentration (Fig. 5). Eadie-Hofstee plots were biphasic and hyperbolic, indicating apparent multisite kinetics with this single isoform.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Relationships between methadone concentration (0.05-1500 µM) and formation of EDDP (A) and EMDP (B) by expressed CYP3A4. Incubations (30 min) contained methadone, CYP3A4 (5 pmol), and NADPH-generating system. Lines, data points, and insets are as described for Fig. 3. Each plot is the mean of duplicates.

Metabolism of LAAM and nor-LAAM by Human Intestinal Microsomes. Formation of nor-LAAM and dinor-LAAM from LAAM and dinor-LAAM from nor-LAAM was linear with microsomal protein content up to 0.2 mg and incubation time up to 20 min with the substrate concentrations tested (Fig. 2C). Residual substrate concentrations were more than 90% of the starting value. Relationships between metabolite formation rates and substrate concentration showed hyperbolic kinetics (Fig. 6). Dinor-LAAM concentrations were approximately 1% those of nor-LAAM, when LAAM was the substrate. Eadie-Hofstee plots were biphasic, indicating multienzyme kinetics (Fig. 6, insets). Kinetic parameters for the formation of nor-LAAM and dinor-LAAM were obtained by nonlinear regression analysis of metabolite versus substrate data, using a dual-enzyme Michaelis-Menten model. K_m1 for the formation of nor-LAAM from LAAM and dinor-LAAM from nor-LAAM was comparable between intestinal microsomes from three individuals (HIM 28, 31, and 40) (Table 3). V_max1 for dinor-LAAM from nor-LAAM was lower than that for nor-LAAM from LAAM in each of these microsomes. K_m2 for nor-LAAM from LAAM and dinor-LAAM from nor-LAAM was 10 times higher than that of K_m1. The in vitro clearance estimate (CL_int = V_max/K_m) for the low-affinity enzyme was <10% of that for the high-affinity enzyme. For the high-affinity enzyme, CL_int for nor-LAAM from LAAM and dinor-LAAM from nor-LAAM was comparable. Both troleandomycin and ketoconazole inhibited the formation of nor-LAAM and dinor-LAAM from LAAM by more than 70% (Fig. 7). The same profiles of inhibition were observed for the formation of dinor-LAAM from nor-LAAM.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Relationships between LAAM concentration and formation of nor-LAAM (A) and dinor-LAAM (B), and between nor-LAAM concentration and dinor-LAAM formation (C), by human intestinal microsomes (HIM 40). Substrate concentrations were 0.05 to 1500 µM. Lines, data points, and insets are as described for Fig. 3. Each plot is the mean of duplicates.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Effects of CYP3A4 inhibitors on formation of nor-LAAM from LAAM, dinor-LAAM from LAAM, and dinor-LAAM from nor-LAAM by HIM 40 () and HLM 46 (). Rates are expressed as a percentage of control values. Incubations contained substrate (5 µM), microsomes (0.1 mg), inhibitors, and NADPH-generating system. Inhibitor concentrations are described under Materials and Methods. Uninhibited formation of nor-LAAM and dinor-LAAM from LAAM and dinor-LAAM from nor-LAAM by HIM 40 were 230, 31, and 195 pmol/min/mg, respectively. Formation of nor-LAAM and dinor-LAAM from LAAM and dinor-LAAM from nor-LAAM by HIM 46 were 254, 21, and 264 pmol/min/mg of protein. Data are the mean ± S.D. of three experiments.

Metabolism of LAAM and nor-LAAM by cDNA-Expressed CYP3A4. Metabolite formation was linear with CYP contents up to 10 pmol and incubation times up to 30 min (Fig. 2D). Residual amount of the substrates was more than 90% of the initial value. Metabolite formation versus substrate concentration plots for N-demethylation of LAAM by CYP3A4 were hyperbolic with respect to LAAM concentration, but were not saturable, even at 1.5 mM substrate (Fig. 8). Eadie-Hofstee plots for expressed CYP3A4 were biphasic and concave, indicating apparent multisite kinetics.

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Relationships between LAAM concentration and formation of nor-LAAM (A) and dinor-LAAM (B), and between nor-LAAM concentration and dinor-LAAM formation (C) by expressed CYP3A4. Incubations (30 min) contained substrate (0.05-1500 µM), CYP3A4 (5 pmol), and NADPH-generating system. Lines, data points, and insets are as described for Fig. 3. Each plot is the mean of duplicates.

View larger version (16K): [in this window] [in a new window]   Fig. 9.   Prediction of dinor-LAAM formation from LAAM by human intestinal microsomes (HIM 28) (A) and expressed CYP3A4 (B). Data points depict the observed rates (mean of duplicates). Lines represent the formation rates predicted by applying the Km and Vmax values for LAAM and nor-LAAM N-demethylation shown in Table 4 (Michaelis-Menten model) at each concentration of LAAM.

Predicted Intestinal Clearance and Extraction Ratio of Methadone, LAAM, and nor-LAAM. In vivo intestinal clearances and extraction ratios for the opioids were predicted from the in vitro clearances (CL_int = V_max1/K_m1, using only kinetic parameters for the high-affinity component) and appropriate scaling factors and blood flow estimates as described under Materials and Methods. Predicted in vivo clearances for EDDP and EMDP from methadone, nor-LAAM from LAAM, and dinor-LAAM from nor-LAAM were of the same order of magnitude between microsomes and CYP3A4 (Tables 5 and 6). Predicted intestinal first-pass extraction for methadone was 21 and 17%, respectively, for intestinal microsomes and CYP3A4. Inclusion of the low-affinity enzymatic component would yield a predicted extraction approximately 10% higher. Predicted intestinal first-pass metabolism of LAAM to nor-LAAM, and LAAM to both bioactivated metabolites, was 33 and 34%, respectively, using intestinal microsomes.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Results of the present study demonstrate that human intestinal microsomes catalyze the N-demethylation of methadone, LAAM, and nor-LAAM and suggest that CYP3A4 is a principal CYP isoform involved in the metabolism. This conclusion is based on the following: 1) metabolism of methadone, LAAM, and nor-LAAM by intestinal microsomes was significantly inhibited by CYP3A4 chemical inhibitors; 2) K_m values for N-demethylation of methadone, LAAM, and nor-LAAM by intestinal microsomes were comparable with those by CYP3A4; and 3) cDNA-expressed CYP3A4 had considerable catalytic activity toward methadone, LAAM, and nor-LAAM and CYP3A is the predominant intestinal CYP. In addition, N-demethylation of LAAM and nor-LAAM by intestinal microsomes showed multienzyme kinetics that were similar to the multisite kinetics shown by CYP3A4, consistent with the above-mentioned conclusion.

In addition to CYP3A4, a limited number of other CYP isoforms has been detected in human intestinal microsomes (Gervot et al., 1999; Zhang et al., 1999). Of those CYP isoforms, CYP2C and particularly 2B have some metabolic activity toward methadone and LAAM N-demethylation (Iribarne et al., 1996; Moody et al., 1997; Oda and Kharasch, 2001; E. D. Kharasch and C. Jubert, unpublished data). However, contribution of CYP2B and 2C to the metabolism of methadone in human intestinal microsomes would be expected to be small since the content of these isoforms is considerably lower than that of CYP3A (Zhang et al., 1999), although we cannot completely eliminate the possibility of minor contribution of these CYP isoforms to the metabolism of methadone in human liver microsomes.

Previous investigations of hepatic microsomal methadone metabolism reported an inability to detect EMDP formation (Iribarne et al., 1996; Foster et al., 1999). In contrast, in the present study, EMDP formation was detected with intestinal microsomes and CYP3A4. The V_max1 for EMDP from methadone was approximately 5% of that of EDDP with both intestinal microsomes and CYP3A4, accounting for the low formation rates of EMDP. In this study, improved analytical methods using liquid chromatography-mass spectrometry instead of high-performance liquid chromatography with ultraviolet detection (Iribarne et al., 1996; Foster et al., 1999) or gas chromatography with chemical ionization (Moody et al., 1997) significantly improved the assay sensitivity and permitted EMDP quantification.

There have been several studies evaluating the metabolism of methadone in human liver microsomes. Irbarne et al. (1996) identified the role of CYP3A4 in N-demethylation of methadone. In their studies, however, double-reciprocal plots for microsomal N-demethylation were linear over the substrate concentrations studied (10-5000 µM), no evidence for biphasic kinetics was observed, and the mean K_m obtained with microsomes from three individuals was 545 µM. A subsequent investigation reported a liver microsomal K_m of 755 µM (Iribarne et al., 1997). Correlation and inhibition experiments in these studies used relatively high substrate concentrations (2500 and 250-500 µM, respectively). Moody et al. (1997) also showed the involvement of CYP3A4 in the hepatic microsomal metabolism of methadone, at a single substrate concentration (10 µM, which was lower than that used previously) although kinetic parameters were not determined (Moody et al., 1997). Foster et al. (1999) also showed the predominant role of CYP3A4 in the hepatic microsomal metabolism of racemic, (R)-, and (S)-methadone to EDDP (Foster et al., 1999). Substrate concentrations were 1 to 1500 µM and the average K_m values were 165, 198, and 182 µM, respectively. Eadie-Hofstee plots were generally linear and data were fit to a single-enzyme model. K_m values were obtained only with microsomes, not with CYP3A4. Clinical methadone concentrations in plasma are typically 0.1 to 0.2 and 1 to 2 µM, respectively, after single and daily doses (Inturrisi and Verebely, 1972; Dyer et al., 1999). Intestinal concentrations are unknown, but presumably greater, and localized concentrations could be quite high. Therefore, in the present study, substrate concentrations of 0.05 to 1500 µM were evaluated. We clearly observed biphasic kinetics, both with intestinal microsomes and CYP3A4, and both K_m1 and K_m2 were comparable between microsomes and CYP3A4. Compared with the previous results obtained with liver microsomes and CYP3A4, the use of lower methadone concentrations might account for the biphasic kinetics detected presently with intestinal microsomes and CYP3A4. Alternatively, there may be intrinsic differences between hepatic and intestinal methadone metabolism. A preliminary experiment with human liver microsomes, however, also showed biphasic kinetics over this extended concentration range (E. D. Kharasch, unpublished results). Further investigation is warranted to understand these differences.

Racemic methadone used in the present study is a mixture of (R)- and (S)-enantiomeric forms, and there is a 10-fold higher affinity of (R)-methadone for opiate µ-receptors than (S)-methadone (Kristensen et al., 1995). We did not evaluate intestinal metabolism of methadone enantiomers. There were no differences, however, between enantiomers in the CL_int (V_max/K_m) for demethylation by human liver microsomes or rates of CYP3A4-catalyzed demethylation (Foster et al., 1999). Since CYP3A4 is predominantly involved in the metabolism of methadone in both liver and intestinal microsomes and there appear to be no enantiomeric differences in hepatic or CYP3A4-catalyzed demethylation, we would also not expect a remarkable enantiomeric difference in human intestinal microsomal methadone N-demethylation.

In the present study, methadone showed apparent multienzyme kinetics with intestinal microsomes, and apparent multisite kinetics with expressed CYP3A4, which is consistent with our previous results with human liver microsomes (E. D. Kharasch, unpublished results). It is now well established that the CYP3A4 active site contains two or more binding sites, and can accommodate the simultaneous presence of at least two substrate molecules, or one or more substrates and an allosteric effector (which may be a second substrate molecule) (Shou et al., 1994, 1999; Ueng et al., 1997; Korzekwa et al., 1998; Hosea et al., 2000; Houston and Kenworthy, 2000). Positive, negative, and noncooperativity are reported for multisite kinetics by CYP3A4. In the present study, no evidence for positive cooperativity with methadone was observed, as velocity curves and Eadie-Hofstee plots were both hyperbolic. Results were well fit to a negative cooperativity model, and a dual-enzyme model yielded similar parameters to the two-site model. Compared with positive cooperativity, apparent negative cooperativity in CYP3A4-catalyzed metabolism is relatively rare. Methadone may be a fourth example, in addition to naphthalene, the antiarrhythmic agent BRL32872, and LAAM (Clarke, 1998; Korzekwa et al., 1998; Oda and Kharasch, 2001). Further investigation is required to elucidate the mechanism of CYP3A4-catalyzed methadone metabolism.

Of the two methadone binding sites on CYP3A4, only the high-affinity binding site would appear relevant for methadone metabolism in the liver, since the K_m for this site was higher than plasma concentrations of methadone following intravenous and oral administration (Verebely et al., 1975), and K_m2 was substantially higher than in vivo concentrations. However, the low-affinity as well as high-affinity binding sites might be responsible for intestinal metabolism of methadone in vivo, since the concentration of methadone in the intestine would be much higher than in the peripheral blood.

In contrast to methadone, there are fewer studies evaluating the CYP isoforms contributing to the metabolism of LAAM (Moody et al., 1997; Oda and Kharasch, 2001). Both reports showed the principal role of CYP3A4 in LAAM and nor-LAAM N-demethylation. Unlike the pharmacologically inactive metabolites of methadone, both nor-LAAM and dinor-LAAM are more potent and longer acting than LAAM and contribute significantly to the pharmacological effect of LAAM (Billings et al., 1973; Sullivan and Due, 1973), suggesting that the formation and disposition of nor-LAAM and dinor-LAAM as well as LAAM are important in determining the clinical effect of LAAM. Results of the present investigation, showing intestinal microsomal LAAM N-demethylation and the role of CYP3A4, suggest that intestinal metabolism may contribute to LAAM bioactivation.

With both intestinal microsomes and CYP3A4, saturation curves were hyperbolic (without apparent sigmoidicity) and Eadie-Hofstee plots were hyperbolic concave. CYP3A4 clearly exhibited non-Michaelis-Menten kinetics, which paralleled the microsomal results, suggesting that the biphasic intestinal microsomal kinetics may derive from the non-Michaelis-Menten behavior of CYP3A4 and its predominant role in intestinal microsomal LAAM metabolism. These results are consistent with our previous study using human liver microsomes (Oda and Kharasch, 2001), and further support our conclusion that CYP3A4 is the predominant CYP isoform involved in the metabolism of LAAM.

K_m and V_max for LAAM metabolism by intestinal microsomes in the present study are comparable with our previous results for human liver microsomes (Oda and Kharasch, 2001). Kinetic parameters for CYP3A4-catalyzed metabolism are somewhat different. In the previous experiments, K_m1 for formation of dinor-LAAM from LAAM and nor-LAAM was lower than presently and K_m2 was greater. Previously, the highest substrate concentration was 1000 µM and the calculated K_m2 for dinor-LAAM from LAAM by CYP3A4 was 2800 µM. Since the predicted K_m was beyond the actual substrate concentration range, there was inherent inaccuracy in the parameters for the low-affinity site (which would also affect the estimates for the high-affinity site). In the present study, substrate concentrations were somewhat higher, thus the parameter estimates should be more accurate.

Recently, intrinsic clearance and extraction ratios scaled from in vitro kinetic parameters have been found useful for predicting intestinal drug disposition and are good indicators of the relative contribution of intestine and liver to presystemic clearance (Thummel et al., 1996, 1997). The intrinsic clearance of methadone by human intestinal microsomes obtained in the present study is comparable with that calculated from hepatic blood flow and hepatic clearance following intravenous administration (Inturrisi et al., 1987), suggesting the comparable contribution of intestinal metabolism with hepatic metabolism. The predicted intestinal extraction of methadone and LAAM (20-35%) is comparable with those of other CYP3A4 substrates, which are known to be susceptible to intestinal drug interactions (Thummel et al., 1997).

Inhibition and induction of intestinal CYP3A4 induce and diminish oral availability of CYP3A4 substrates, respectively (Gomez et al., 1995; Ameer and Weintraub, 1997). Intestinal interactions between oral CYP3A4 substrates may add to interindividual variability in intestinal presystemic metabolism and hepatic CYP3A4 drug interactions. Intestinal metabolism and predicted first-pass extraction of LAAM was even greater than that of methadone. Thus, intestinal metabolism may contribute significantly to the bioactivation and clinical effect of LAAM and methadone. The role of intestinal presystemic metabolism and intestinal drug interactions in methadone inactivation and LAAM bioactivation, and the clinical effects of these drugs, merits further investigation.

In summary, we have shown that methadone, LAAM, and nor-LAAM are metabolized in human intestinal microsomes, and predominantly by CYP3A4. Furthermore, CYP3A4-catalyzed methadone and LAAM metabolism exhibits unusual multisite kinetics.