Role of human placental efflux transporter P-glycoprotein in the transfer of buprenorphine, levo-alpha-acetylmethadol, and paclitaxel.

This study examines the role of placental P-glycoprotein (P-gp) in the transfer of buprenorphine (BUP) and L-alpha-acetylmethadol (LAAM) across the dually perfused human placental lobule. BUP (10 ng/mL) and LAAM (35 ng/mL) were perfused in the maternal-to-fetal direction. The following kinetic parameters were determined: fetal transfer rate (TR (f)), maternal clearance (Cl (m)), and clearance index (Cl (index)). The opiates were perfused in the presence of P-gp inhibitor GF120918 (experimental group) and in its absence (control group). The kinetic parameters for the control group were set at 100% and were as follows for LAAM in the experimental group: TR (f), 123 +/- 20%, Cl (m) 116 +/- 23%, and Cl (index) 123 +/- 22% ( P < 0.05). The corresponding parameters for BUP were not different from controls. The data indicate that LAAM, but not BUP, is extruded by the efflux transporter P-gp. Therefore, it is reasonable to assume that the activity of P-gp could be one of the factors affecting the extent of fetal exposure to LAAM during pregnancy.

N-demethylation of levo-alpha-acetylmethadol by human placental aromatase.

Levo-alpa-acetylmethadol (LAAM) is a methadone derivative used to treat the opiate addict. We previously reported on the kinetics for transplacental transfer of LAAM and its levels in the fetal circuit using the technique of dual perfusion of the placental lobule. The aim of this investigation was to identify the enzyme responsible for the biotransformation of LAAM and norLAAM and the metabolites formed in the term human placenta. Placental microsomes exhibited higher activities than the mitochondrial and cytosolic fractions in metabolizing LAAM to norLAAM. None of these subcellular fractions catalyzed the formation of dinorLAAM from either LAAM or norLAAM as determined by HPLC/UV. Evidence obtained from the effects of cytochrome P450 (CYP) inhibitors on the demethylation of LAAM to norLAAM by placental microsomes suggested that CYP 19/aromatase is the major enzyme involved. Out of 10 monoclonal antibodies raised against various CYP isoforms, only that for aromatase caused over 80% inhibition of norLAAM formation. The biotransformation of LAAM to norLAAM exhibited monophasic kinetics with apparent Km and Vmax values of 105 +/- 57 microM and 86.8 +/- 15.6 pmol mg(-1) protein min(-1), respectively. The kinetic profile determined for a cDNA-expressed CYP 19 metabolism of LAAM to norLAAM was similar to that determined for placental microsomes. Taken together, the above data indicate that CYP 19/aromatase is the enzyme responsible for the N-demethylation of LAAM to norLAAM in term human placentas obtained from healthy pregnant women.

Transfer of L-alpha-acetylmethadol (LAAM) and L-alpha-acetyl-N-normethadol (norLAAM) by the perfused human placental lobule.

The agonists buprenorphine and l-alpha-acetylmethadol (LAAM) were introduced as alternatives to methadone for treatment of the adult opiate addict. The direct and indirect effects of these drugs on normal fetal growth and development are currently under investigation in our laboratory. The goal of this report is to provide part of the data necessary to assess the safety of LAAM in treatment of the pregnant opiate addict. To achieve this goal, the technique of dual perfusion of placental lobule was utilized to determine the kinetics for transplacental transfer of LAAM and its effects on the viability and functional parameters of the tissue. LAAM is rapidly metabolized to the pharmacologically active norLAAM that was also included in this investigation. The two opiates were transfused at their plasma levels in patients under treatment, a concentration of 35 ng/ml. The drugs exhibited similar pharmacokinetic profiles, characterized by an initial phase of distribution into placental tissue followed by their low transfer to the fetal circuit. During the 4-h experimental period, the transfused tissue retained significant amounts of LAAM and norLAAM, and neither drug was metabolized. LAAM did not affect placental tissue viability and functional parameters. However, norLAAM caused a significant decrease in the release of human chorionic gonadotropin. At this time, it is unclear whether a similar effect for norLAAM may occur in vivo and, if so, what the consequences would be on its role in implantation and normal fetal growth and development.

Metabolism of methadone and levo-alpha-acetylmethadol (LAAM) by human intestinal cytochrome P450 3A4 (CYP3A4): potential contribution of intestinal metabolism to presystemic clearance and bioactivation.

Methadone and levo-alpha-acetylmethadol (LAAM) are opioid agonists used for analgesia and preventing opiate withdrawal. Methadone is sequentially N-demethylated to the inactive metabolites 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyraline (EMDP). LAAM is essentially a prodrug that undergoes bioactivation via sequential N-demethylation to levo-alpha-acetyl-N-normethadol (nor-LAAM) and levo-alpha-acetyl-N,N-dinormethadol (dinor-LAAM). Methadone and LAAM are metabolized by CYP3A4 in human liver. Since they are administered orally, and CYP3A4 is expressed in human intestine, we tested the hypotheses that human intestine can metabolize methadone and LAAM, and evaluated the participation of CYP3A4. Intestinal microsomal methadone N-demethylation exhibited hyperbolic noncooperative kinetics and biphasic Eadie-Hofstee plots. Using a dual-enzyme Michaelis-Menten model, K(m) values were 11 and 1200 microM for EDDP and 23 and 930 microM for EMDP formation, respectively. CYP3A4 inhibitors (troleandomycin and ketoconazole) inhibited EDDP and EMDP formation by >70%. Methadone N-demethylation by CYP3A4 showed biphasic Eadie-Hofstee plots without evidence of positive cooperativity; K(m) values were 10 and 1100 microM for EDDP and 20 and 1000 microM for EMDP formation. Intestinal microsomal LAAM and nor-LAAM N-demethylation also exhibited hyperbolic kinetics and biphasic Eadie-Hofstee plots. K(m) values were 21 and 980 microM for nor-LAAM from LAAM and 18 and 1200 microM for dinor-LAAM from nor-LAAM. Troleandomycin and ketoconazole inhibited N-demethylation by >70%. LAAM and nor-LAAM metabolism by CYP3A4 showed biphasic Eadie-Hofstee plots without evidence of positive cooperativity; K(m) values were 8 and 1300 microM, 6 and 950 microM, respectively. Predicted in vivo intestinal extraction of methadone and LAAM is 21 and 33%, respectively. We conclude that methadone, LAAM, and nor-LAAM are metabolized by human intestinal microsomes; CYP3A4 is the predominant cytochrome P450 isoform; CYP3A4-catalyzed methadone, LAAM, and nor-LAAM metabolism is characterized by noncooperative, multisite kinetics; and intestinal metabolism may contribute to presystemic methadone inactivation and LAAM bioactivation.

Differential N-demethylation of l-alpha-acetylmethadol (LAAM) and norLAAM by cytochrome P450s 2B6, 2C18, and 3A4.

Incubation of l-alpha-acetylmethadol (LAAM) or norLAAM with cDNA-expressed P450s 3A4, 2B6, and 2C18 produced significant N-demethylation products. P450s 2C19, 2C8, 3A5, 2C9, 3A7, 1A1, and 2D6 (norLAAM only), also produced detectable product. Coexpression of cytochrome b(5) enhanced LAAM N-demethylation, most dramatically for 3A4, but had marginal effects on norLAAM N-demethylation. Modeling total liver metabolism using immunoquantification and relative activity factors of P450s suggests contributions of P450 3A4 > 2B6 > 2C18, with the importance of 2B6 to 2C isozymes enhanced by relative activity factors. The ratio of dinorLAAM to norLAAM plus dinorLAAM formed from LAAM did not exceed 20%, and was isozyme and cytochrome b(5) coexpression dependent. This ratio decreased with concentration with 3A4, but was relatively constant for 2B6 and 2C18. The human liver microsomes substrate-concentration response was similar to cDNA-expressed 3A4, but the ratio was higher. Changes in the environment of cDNA-expressed 3A4 also effected the magnitude of the ratio, but not the concentration-dependent decrease. These studies show that the N-demethylation of LAAM and norLAAM is not restricted to P450 3A4, particularly P450s 2B6 and 2C18, and suggest that the mechanism of sequential metabolism for 3A4 differs from that of 2B6 and 2C18.

Metabolism of levo-alpha-Acetylmethadol (LAAM) by human liver cytochrome P450: involvement of CYP3A4 characterized by atypical kinetics with two binding sites.

levo-alpha-Acetylmethadol (LAAM) is a long-acting opioid agonist prodrug used for preventing opiate withdrawal. LAAM undergoes bioactivation via sequential N-demethylation to nor-LAAM and dinor-LAAM, which are more potent and longer-acting than LAAM. This study examined LAAM and nor-LAAM metabolism using human liver microsomes, cDNA-expressed CYP, CYP isoform-selective chemical inhibitors, and monoclonal antibody to determine kinetic parameters for predicting in vivo drug interactions, involvement of constitutive CYP isoforms, and mechanistic aspects of sequential N-demethylation. N-Demethylation of LAAM and nor-LAAM by human liver microsomes exhibited biphasic Eadie-Hofstee plots. Using a dual-enzyme Michaelis-Menten model, K(m) values were 19 and 600 microM for nor-LAAM and 4 and 450 microM for dinor-LAAM formation, respectively. LAAM and nor-LAAM metabolism was inhibited by the CYP3A4-selective inhibitors troleandomycin, erythromycin, ketoconazole, and midazolam. Of the cDNA-expressed isoforms examined, CYP2B6 and 3A4 had the highest activity toward LAAM and nor-LAAM at both low (2 microM) and high (250 microM) substrate concentrations. N-Demethylation of LAAM and nor-LAAM by expressed CYP3A4 was unusual, with hyperbolic velocity curves and Eadie-Hofstee plots and without evidence of positive cooperativity. Using a two-site model, K(m) values were 6 and 0.2 microM, 1250 and 530 microM, respectively. Monoclonal antibody against CYP2B6 inhibited CYP2B6-catalyzed but not microsomal LAAM or nor-LAAM metabolism, whereas troleandomycin inhibited metabolism in all microsomes studied. The ratio [dinor-LAAM/(nor-LAAM plus dinor-LAAM)] with microsomes and CYP3A4 decreased with increasing LAAM concentration, suggesting most dinor-LAAM is formed from released nor-LAAM that subsequently reassociates with CYP3A4. Based on these results, we conclude that LAAM and nor-LAAM are predominantly metabolized by CYP3A4 in human liver microsomes, and CYP3A4 exhibits unusual multisite kinetics.

Variables in human liver microsome preparation: impact on the kinetics of l-alpha-acetylmethadol (LAAM) n-demethylation and dextromethorphan O-demethylation.

We examined three primary variables in the preparation of human liver microsomes. In three experiments, each using three livers, we manipulated 1) the force of the first centrifugation (9,000, 10,500, or 12,000g); 2) the presence of sucrose in the homogenization buffer; and 3) the number of homogenizing strokes (6, 8, or 10). Sedimentation plots for the marker enzymes succinate dehydrogenase, NADPH cytochrome P450 reductase (reductase), and glutathione S-transferase in the resulting premicrosomal, microsomal, and cytosolic fractions suggest that enhanced purity of microsomes can be obtained by reducing force of centrifugation, including sucrose, and increasing the number of homogenization strokes. Each microsomal fraction was also assayed for protein content, cytochrome P450, NADH cytochrome b(5) reductase, cytochrome b(5), absorbance at 420, p-nitrophenol hydroxylation, tolbutamide hydroxylation, dextromethorphan N- and O-demethylation, glucuronidation of morphine and 1-naphthol, and ester cleavage of p-nitrophenolacetate. These microsomal indicators were ranked and tested for statistical differences. The use of 9000g statistically increased optimal recovery (per gram of liver) and specific activity (per milligram of protein). The inclusion of sucrose improved activity specific to reductase activity. Ten homogenization strokes improved activity specific to reductase activity. Substrate-dependent activities of dextromethorphan O-demethylation to dextrorphan and the N-demethylation of l-alpha-acetylmethadol (LAAM) to norLAAM and dinorLAAM were compared in microsomes prepared with or without sucrose and microsomes prepared using 9,000 or 12,000g force, respectively. No significant differences were found in the concentration-dependent activities. Variation of the methods used to prepare human liver microsomes can significantly affect the recovery and specific activity of microsomal components; however, they do not appear to affect enzyme kinetics.

Detection of methadone, LAAM, and their metabolites by methadone immunoassays.

l-Alpha-acetylmethadol (LAAM) was recently approved as a substitute for methadone. LAAM, methadone, and their common metabolite, methadol, are extensively N-demethylated. The structural similarities of LAAM and its metabolites to methadone suggest that they may cross-react in methadone immunoassays. To test this hypothesis, drug-free urine was fortified with LAAM, norLAAM, dinorLAAM, methadol, normethadol, dinormethadol, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), or 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP) at 12 concentrations (0.03 to 100 microg/mL). Samples were analyzed using two enzyme immunoassays (Behring Diagnostics, EIA-b; Diagnostic Reagents, EIA-d); a fluorescent polarization immunoassay (Abbott, FPIA); two enzyme-linked immunosorbant immunoassays (Diagnostix, ELISA-d; STC Technologies, ELISA-s); a kinetic microparticles in solution immunoassay (Roche Diagnostic Systems, KIMS); and a radioimmunoassay (Diagnostic Products, RIA). LAAM had high cross-reactivity with ELISA-d (318.3%), RIA (249.5%), EIA-d (100.8%), KIMS (91.1%), and ELISA-s (75.3%). Methadol also displayed relatively high cross-reactivity as follows: EIA-d (97.8%), KIMS (85.4%), ELISA-d (70.3%), and FPIA (37.7%). Successive N-demethylations of LAAM and methadol were associated with loss of cross-reactivity. The methadone metabolites EDDP and EMDP showed little cross-reactivity. These findings suggest that LAAM use could result in positive immunoassay test results when using many of the commercially available methadone immunoassay kits and that confirmation of LAAM and its metabolites should be considered.

The involvement of cytochrome P450 3A4 in the N-demethylation of L-alpha-acetylmethadol (LAAM), norLAAM, and methadone.

The N-demethylation of LAAM, norLAAM, and methadone has been investigated in human liver microsomes and microsomes containing cDNA-expressed human P450s. Gas chromatography/mass spectrometry methods allowed detection of norLAAM and dinorLAAM formation from LAAM, dinorLAAM formation from norLAAM, and EDDP and EMDP formation from methadone. The rates of N-demethylation varied 4- to 7-fold in microsomes from four different donors with activities for LAAM and norLAAM consistently greater (5- to 14-fold) than for methadone. The N-demethylation of LAAM, norLAAM, and methadone were significantly inhibited by ketoconazole. IC50s could be determined for ketoconazole inhibition of LAAM and norLAAM N-demethylation of 1.6 and 1.1 microM, respectively. The ability of ketoconazole to reduce methadone N-demethylation below 40% varied in regard to liver donor. No other P450-selective inhibitors reduced the average activities more than 43%. cDNA-expressed P450 3A4 N-demethylated LAAM, norLAAM, and methadone at greater rates than the other cDNA-expressed P450s studied (1A2, 2C9, 2D6, or 2E1). P450 3A N-demethylation of LAAM, norLAAM, and methadone exceeded the next most active P450, respectively, by at least 2.5, 9.6, and 13.4 times when expressed per milligram protein and by 18.2, 6.0, and 6.1 times when expressed per nanomole P450. These results suggest that P450 3A4 is the primary site of N-demethylation of LAAM, norLAAM, and methadone in human liver. Although other enzymes may also be capable of N-demethylating these compounds, identification of specific enzymes, except P450 3A4, has yet to be established. Knowledge of these enzymatic pathways is essential for assessment of the impact of metabolic drug-drug interactions on therapeutic success and/or adverse events.

A gas chromatographic-positive ion chemical ionization-mass spectrometric method for the determination of I-alpha-acetylmethadol (LAAM), norLAAM, and dinorLAAM in plasma, urine, and tissue.

l-alpha-Acetylmethadol (LAAM) is approved as a substitute for methadone for the treatment of opiate addiction. Analytical methods are needed to quantitate LAAM and its two psychoactive metabolites, norLAAM and dinorLAAM, to support pharmacokinetic and other studies. We developed a gas chromatographic-positive ion chemical ionization-mass spectrometric method for these analyses. The method uses 0.5 mL urine or 1.0 mL plasma or tissue homogenate, deuterated (d3) isotopomers as internal standards, methanolic denaturation of protein (for plasma and tissue), and extraction of the buffered sample with n-butyl chloride. For tissue homogenates, an acidic back extraction is included. norLAAM and dinorLAAM were derivatized with trifluoroacetic anhydride. Chromatographic separation of LAAM and derivatized norLAAM and dinorLAAM is achieved with a 5% phenyl methylsilicone capillary column. Positive ion chemical ionization detection using methane-ammonia as the reagent gas produces abundant protonated ions (MH+) for LAAM (m/z 354) and LAAM-d3 (m/z 357) and ammonia adduct ions (MNH4+) for the derivatized norLAAM (m/z 453), norLAAM-d3 (m/z 45 6), dinorLAAM (m/z 439), and dinorLAAM-d3 (m/z 442). The linear range of the calibration curves were matrix dependent: 5-300 ng/mL for plasma, 10-1000 ng/mL for urine, and 10-600 ng/g for tissue homogenates. The low calibrator was the validated limit of quantitation for that matrix. The method is precise and accurate with percent coefficients of variation and percent of targets within 13%. The method was applied to the analysis of human urine and plasma samples; rat plasma, liver, and brain samples; and human liver microsomes following incubation with LAAM.

Clinical toxicology of drugs used in the treatment of opiate dependency.

Many aspects of the pharmacokinetics of methadone have been evaluated since 1970. Analytic techniques used to monitor urine and serum or plasma concentrations of methadone and its metabolites have improved with advances in chromatography and development of immunoassay techniques. On reviewing the literature on methadone since 1970, however, there were several recurring limitations of the experimental design in a large number of the studies reported. These include: 1. No appreciation for the effect of urinary pH on excretion of methadone 2. Small number of patients enrolled with inadequate control subjects 3. The effect of smoking cigarettes was not evaluated or adequately controlled 4. Urine collections were often obtained without supervision and correcting results to creatinine excretion 5. Blood specimens were generally not collected from 0-15 minutes after intravenous dosing 6. Incomplete excretion data (nonhydrolysis of glucuronide conjugates) in the urine 7. Most studies did not evaluate protein binding of methadone when attempting to correlate therapeutic control or failure with serum concentrations of methadone. In general, the literature supporting the use of naltrexone was more favorable than for methadone, buprenorphine, LAAM, and clonidine. The major limitation on the use of naltrexone, however, is the lack of incentive for the patient to keep taking the medication. If the use of naltrexone, LAAM, buprenorphine, or clonidine becomes widely available, robust analytic techniques must be developed for monitoring of these drugs, their metabolites, or both in the urine to verify patient compliance.

In vivo carcinogenesis assay of L-alpha-acetylmethadol.HCl in rodents.

With increasing clinical use of L-alpha-acetylmethadol.HCl (LAAM), findings of a carcinogenic bioassay could be useful in risk assessment. Initial studies provided a sex-related oral LD50 in B6C3F1 mice, 126 mg/kg for males and 71 mg/kg for females, and changes after treatment with mean doses of 8, 18, and 33 mg/kg for 90 days which included hyperactivity and unchanged growth rate, food intake, and morphology. Twenty-four-month oral doses were 7.6 and 30.1 mg/kg for mice, 3.1 and 9.7 mg/kg for male rats, and 5.7 and 16.6 mg/kg for female rats. LAAM was lethal in high-dose male mice (40% survival) whereas survival rates of 74-78% were similar in other treated and control groups. An 80-90% survival rate was seen for rats. Deaths related to aging and concomitant morphologic changes occurred randomly in all groups. The causes of LAAM-induced deaths were not established. Central nervous system stimulation and fighting were more common to male rodents. A dose-related inhibition of growth was also common but food intake was stimulated in male mice and both sexes of rats. An increase in liver neoplastic nodules was dose related and drug related in rats. Some nonneoplastic lesions may have been drug related. Terminal plasma LAAM and metabolite levels were generally dose related.

Quantitation of l-alpha-acetylmethadol and its metabolites in human serum by capillary gas-liquid chromatography and nitrogen detection.

A procedure is described for the simultaneous measurement of l-alpha-acetylmethadol and its two pharmacologically active metabolites: noracetylmethadol and dinoracetylmethadol. In the method an intramolecular conversion reaction of the two metabolites to their amide configuration is utilized. The reaction is performed while the metabolites are still in the serum. Following solvent extraction the samples are analyzed by capillary gas-liquid chromatography coupled with nitrogen detection. Quantitation is achieved by internal standardization. The lower limit of sensitivity is 5 ng/ml in serum. Absolute sensitivity is 0.1 ng for all three compounds. The advantages over other procedures are: speed due to the single extraction step; increased recovery of noracetylmethadol and dinoracetylmethadol due to decreased polarity of the amides; greater stability of the metabolites in the amide configuration; better chromatographic quantitation and separation because detector response for the amides is greater than it is for the original configuration of the metabolites and the area of the chromatographic tracing is free of interfering substances.

Effect of phenobarbital pretreatment on the plasma and urinary levels of (-)-alpha-acetylmethadol and its metabolites.

The effect of phenobarbital (PB), an inducer of the hepatic microsomal enzyme system, on the plasma levels and urinary elimination of (-)-alpha-acetylmethadol 1 and its metabolites have been examined in the rat. [3H]1 was administered to saline control and PB-pretreated rats at doses of 5 mg/kg ip (55 muCi/kg). The concentration of 1 and its metabolites noracetylmethadol 2, dinoracetylmethadol 3, methadol 4, normethadol 5, and N-acetylnormethadol 6 were quantitated in plasma and urine over 48 h by TLC and liquid scintillation counting. PB pretreatment significantly decreased the plasma total radioactivity and the levels of 1 and its five metabolites over the 48-h period investigated. Urinary total radioactivity and elimination of 1 and its five metabolites were also reduced in PB-pretreated rats. The results indicated that PB pretreatment markedly affects the in vivo transformation and elimination of 1 and its metabolites. The decrease in the levels observed for 1 and its metabolites in the plasma and urine can be due either to an increase in the metabolism of 1 via a different pathway than the formation of the biologically active metabolites 2, 3, 4, and 5, or it may be that PB is enhancing the further metabolism of these compounds to more polar water-soluble products which are mainly excreted through the bile.

Postnatal abstinence or acute toxicity can account for morbidity in developmental studies with opiates.

The incidence of neonatal morbidity and mortality in rats exposed to opiates in utero is generally high. To determine the extent to which neonatal opioid intoxication and/or withdrawal contribute to this effect, addicted pups from dams treated chronically with the long-acting opioid levo-alpha-acetylmethadol (LAAM) and appropriate controls were injected within 12 h of birth with saline, an opioid agonist (LAAM and metabolites) or an antagonist (naloxone). The incidence of neonatal mortality for pups born to dams maintained on a high dose of LAAM was 52%. A single injection of agonist on the first day of life reduced mortality in this group to 29% while a single injection of the antagonist increased mortality to 88%. In contrast, administration of the agonist to control pups and pups born to dams maintained on lower doses of LAAM resulted in increased mortality. Naloxone was apparently innocuous in non-dependent neonates. These data show that, despite LAAM's long duration of action in the mature rat, newborn rats experience withdrawal soon after drug exposure is terminated. These data also indicate that continued opioid exposure is a highly effective means of treating/preventing severe spontaneous withdrawal in the newborn.

Uptake of l-alpha-acetylmethadol (LAAM) and its analgesically active metabolites, nor-LAAM and dinor-LAAM, in the isolated perfused rat lung.

The pulmonary uptake of l-alpha-acetylmethadol (LAAM) and its major analgesically active metabolites, nor-LAAM and dinor-LAAM, was studied during a single pass through the isolated perfused rat lung (IPL). The radiolabeled drugs were infused into the IPL for 10 min followed by a 30-min drug-free perfusion. All three drugs were extensively taken up into the IPL; however, dinor-LAAM, the least lipophilic, accumulated to the greatest extent. Their rates of efflux from the IPL with time could be described by the sum of three exponentials and 20-25% of each compound accumulated in a "slowly effluxable pool," suggesting a highly sequestered pool of drug in the lung. These findings suggest that tissue sequestration of the active metabolites is equal to or greater than LAAM itself. Such tissue sequestration could limit the sequential metabolic activation or inactivation, and serve as a reservoir of the active compounds. These factors favor the persistence of LAAM and its active metabolites in the body, thus prolonging the opiate-like effects after LAAM administration. The data also indicated that the relationship between drug basicity or lipophilicity and extent of uptake is complex and it is difficult to associate a particular physicochemical property with the extent or character of pulmonary drug uptake.

Uptake and efflux of 1-alpha-acetylmethadol (LAAM) and methadone in the isolated perfused rat lung.

The pulmonary uptake of 1-alpha-acetylmethadol (LAAM) and methadone was studied during a single pass through the isolated perfused rat lung (IPL). The IPL was perfused for 10 min with perfusate containing various concentrations of one of the radiolabeled drugs, followed by a 30-min drug-free perfusion and the amount of radioactivity in the pulmonary venous effluent was used to estimate uptake and efflux of the drugs with time. Both drugs were extensively taken up by the IPL; however, LAAM uptake was significantly greater than that of methadone. The amount of each drug taken up was a nonlinear function of concentration in the perfusate, suggesting saturability of the major uptake processes. During drug-free perfusion LAAM and methadone previously accumulated were released from the lung at two different efflux rates; however, a third, more rapid efflux process for methadone was also evident. The total venous effluent collected during the drug-free perfusion contained a significantly greater fraction of the accumulated methadone as compared to LAAM. Calculation of the total amount of each drug effluxable at the observed rates indicated that another pool for both drugs existed in the lung that did not efflux at an observable rate. The accumulated LAAM remaining in this "slowly effluxable pool" was significantly greater than methadone. These data demonstrate a greater pulmonary uptake and tissue sequestration of LAAM as compared to methadone, and may be one of the contributing factors in the persistence of LAAM and its active metabolites in the body.

Plasma and urine disposition of 1-alpha-acetylmethadol and its principal metabolites in man.

The disposition of 1-alpha-acetylmethadol (LAAM) in plasma and urine was monitored by GC/CIMS following oral administration of 10 doses (0.73-1.5 mg/kg) over 42 days, to twelve human subjects. Plasma concentration-time course profiles fitted a two-compartment, first order kinetic model. Mean plasma t1/2 alpha for LAAM was 2.4 hours; t1/2 beta was 37.5 hours for the first dose and 46.8 hours for the last dose. The mean terminal half-life for nor-LAAM was 38.2 hours for first and 64.6 for last dose; for dinor-LAAM t1/2 beta was 168 hours, last dose. Drug accumulation occurred in some subjects, but within the study range, dosage was not related to maximum plasma levels nor to accumulation. In urine, the sum of LAAM, nor-LAAM, and dinor-LAAM represented 25% of the dose, and unconjugated methadol metabolites, 1.6-1.7%.

Cumulation of active metabolites of levo-alpha-acetylmethadol in the rat fetus and neonate.

Levo-alpha-acetylmethadol (LAAM, 0.2 or 2.0 mg/kg/day) was orally administered to female Sprague-Dawley rats for one month prior to and throughout pregnancy. The rats were killed on the 18th day of gestation along with a group of 18-day pregnant females given a single oral 2.0 mg/kg dose of LAAM 24 hours earlier. Although cumulation of LAAM or its active metabolites was not seen in plasma or brain of pregnant rats given drug chronically, significant cumulation was observed in whole fetus and in fetal brain. In addition, a 2-3 fold elevation in the concentrations, and an even greater elevation of total content, was noted in the newborn pup. These data suggest that opiate intoxication soon after birth may be a factor responsible for the increased morbidity and mortality of rat pups prenatally exposed to LAAM.

Acute effects of ethanol on hepatic uptake and distribution of narcotics in the isolated perfused rabbit liver.

This study was performed as an initial step in systematically defining the hepatic interactions between ethanol and opioids using a controlled in vitro system. The acute effects of ethanol on the initial uptake and distribution of long- and short-acting narcotics were studied using isolated rabbit liver perfused with rabbit blood without or with ethanol. A pulse injection of 1.5 mg of 14C-labeled narcotic [methadone, 1-alpha-acetylmethadol (LAAM), morphine, or meperidine] was made into the portal vein cannula followed by perfusion for 2 min. Radioactivity was determined in liver homogenates and subcellular fractions; methadone and its metabolites were measured by thin-layer chromatography with zonal scanning in each fraction. Ethanol preperfusion and concomitant ethanol perfusion did not effect hepatic uptake of methadone, LAAM, morphine, or meperidine. Although subcellular localization of morphine and meperidine differed from that of methadone and LAAM, perfusion with ethanol did not alter the acute hepatic uptake and distribution of any of the narcotics. These findings suggest that acute exposure to ethanol does not alter the acute hepatic disposition of narcotics.