Impaired psychomotor function and plasma methadone and levo-alpha-acetylmethadol (LAAM) concentrations in opioid-substitution patients.

Tolerance to the psychomotor impairing effects of opioid drugs is expected to develop with repeated dosing, but may be incomplete. The relationship between plasma opioid concentration and psychomotor function in opioid-dependent patients was examined to determine whether impairment was more likely at the time of highest plasma drug concentration. Sixteen patients participating in a cross-over trial comparing methadone and LAAM completed a tracking task (OSPAT) 11 times over the dosing-interval for methadone (24-hrs) and LAAM (48-hrs). Venous blood was collected for the quantification of plasma (R)-(-)-methadone, LAAM, and nor-LAAM concentrations. The Digit Symbol Substitution Test (DSST) and Trail-Making Test were administered at the time of peak plasma concentration. Ten healthy controls (HCs) also participated. OSPAT scores (obtained for 15 patients) fluctuated significantly across the dosing-interval for both drugs and were lower in patients than HCs at the times of peak concentrations of (R)-(-)-methadone (1 hr: (mean difference; 95% CI) (2.13; 0.18-4.08); 2 hrs: (2.38; 0.48-4.28) postdosing) and LAAM (2 hrs: (1.81; 0.09-3.53), and 4 hrs (1.90: 0.9-3.71) postdosing). Within-participant analysis of the peak-change from baseline for OSPAT scores found that 10 of the 15 patients could be categorized as impaired on methadone and 9 on LAAM. No HCs were impaired. Patients performed worse on the DSST and Trails-A than HCs, but not on Trails-B. Results suggest that some patients receiving opioids long term may exhibit impairment at the time of highest plasma drug concentration. These patients should be made aware that their ability to undertake complex tasks may be affected. (PsycINFO Database Record

Paradoxical role of cytochrome P450 3A in the bioactivation and clinical effects of levo-alpha-acetylmethadol: importance of clinical investigations to validate in vitro drug metabolism studies.

OBJECTIVE: Levo-alpha-acetylmethadol (LAAM, levacetylmethadol) is a long-acting opioid agonist used for the prevention of opioid withdrawal. LAAM undergoes sequential N-demethylation to norLAAM and dinorLAAM, which are more potent and longer-acting than LAAM. Hepatic and intestinal microsomal N-demethylation in vitro is catalysed mainly by cytochrome P450 (CYP) 3A4; however, the role of CYP3A in LAAM disposition in humans in vivo is unknown. This investigation tested the hypothesis that CYP3A induction (or inhibition) would increase (or decrease) LAAM metabolism and bioactivation and, thus, clinical effects. It also related changes in LAAM disposition during enzyme inhibition or induction to any changes in pharmacological effect. METHODS: Healthy volunteers (n = 13) completed the three-way, randomised, balanced crossover study. Subjects received oral LAAM (0.25 mg/kg) after CYP3A induction (rifampicin [rifampin]), inhibition (troleandomycin) or nothing (controls). Plasma and urine LAAM, norLAAM and dinorLAAM were determined by electrospray high-performance liquid chromatography/mass spectrometry (HPLC/MS). Dark-adapted pupil diameter change from baseline (miosis) was the LAAM effect measure. Results were analysed by noncompartmental methods and by a combined pharmacokinetic/pharmacodynamic model. RESULTS: Compared with controls, CYP3A induction (or inhibition) decreased (or increased) plasma LAAM concentrations and mean area under the plasma concentration-time curve from time zero to infinity (AUC(infinity) 199 +/- 91 [control] versus 11.3 +/- 4.0 [rifampicin] and 731 +/- 229 ng . h/mL [troleandomycin]; p < 0.05), and increased (or decreased) median formation clearances of norLAAM (1740 versus 14 100 and 302 mL/h/kg; p < 0.05) and dinorLAAM (636 versus 7840 and 173 mL/h/kg; p < 0.05). Surprisingly, however, CYP3A induction (or inhibition) decreased (or increased) mean plasma metabolite AUC from 0 to 96 hours (AUC(96)) [norLAAM + dinorLAAM] (859 +/- 241 versus 107 +/- 48 and 1185 +/- 179 ng . h/mL; p < 0.05) and clinical effects (mean miosis AUC(96) 128 +/- 40 versus 22.5 +/- 14.9 and 178 +/- 81 mm . h; p < 0.05). Clinical effects were best correlated with plasma norLAAM concentrations. CONCLUSION: CYP3A mediates human LAAM N-demethylation and bioactivation to norLAAM and dinorLAAM in vivo. Paradoxically, however, CYP3A induction decreased and inhibition increased LAAM active metabolite concentrations and clinical effects. This suggests a CYP3A-mediated metabolic pathway leading to inactive metabolites, which predominates over CYP3A-dependent bioactivation. These results highlight the need for clinical investigations to validate in vitro drug metabolism studies.

Suppressed fever and hypersensitivity responses in chicks prenatally exposed to opiates.

We have established procedures to reliably induce opiate dependence in the chick embryo via in ovo injection, early in embryonic development, of the long-acting and potent opiate N-desmethyl-l-alpha-noracetylmethadol (NLAAM). Prior studies found that there is continual exposure to NLAAM throughout embryogenesis and shortly after hatching there are signs of spontaneous withdrawal. In the present study, we used three doses of NLAAM (2.5, 5, and 10 mg/kg egg weight) to determine if prenatal opiate exposure followed by postnatal withdrawal interfered with appropriate neural-endocrine-immune interactions in the young chick. To ensure that effects were not a consequence of inappropriately large doses, we first examined acute and chronic toxicity and additional characteristics of postnatal opiate withdrawal. We then measured the corticosterone and fever responses to LPS stimulation during the withdrawal period. After the conclusion of opiate withdrawal, we assessed the hypersensitivity response to phytohemagglutinin (PHA). The fever response to LPS and the hypersensitivity response to PHA were suppressed by prenatal opiate exposure and postnatal withdrawal. The corticosterone response to LPS was not affected, but there were exaggerated corticosterone responses to saline injection in chicks exposed in ovo to NLAAM. It was unlikely that the effects of prenatal NLAAM were the result of toxicity, as little chronic toxicity was seen with the lower two doses of NLAAM, doses that yielded significant suppressions of neural-endocrine-immune responses. However, effects found in the chicks treated with 10 mg NLAAM/kg may have been partly related to the greater toxicity and/or protracted postnatal withdrawal in this group.

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.

Interactions of the narcotic l-alpha-acetylmethadol with human cardiac K+ channels.

l-alpha-acetylmethadol is a long-acting narcotic analgesic that is used in the treatment of opiate addiction. However, the drug has been associated with cases of QT interval prolongation and ventricular arrhythmia. To understand the mechanism underlying these clinical findings, we examined the effects of l-alpha-acetylmethadol on the cloned human cardiac K(+) channels HERG (human ether-a-go-go-related gene), KvLQT1/minK and Kv4.3. Using patch clamp electrophysiology, we found that l-alpha-acetylmethadol inhibited HERG channel currents in a voltage-dependent manner displaying an IC(50) value of 3 microM. The major active metabolite of l-alpha-acetylmethadol, noracetylmethadol, inhibited HERG with an estimated IC(50) values of 12 microM. l-alpha-acetylmethadol had little or no effect on Kv4.3 or KvLQT1/minK K(+) channel currents at concentration up to 10 microM. We conclude that the proarrhythmic effects of l-alpha-acetylmethadol are due to specific blockade of the HERG cardiac K(+) channel and that its active metabolite noracetylmethadol may provide a safer alternative in the treatment of opiate addiction.

Determination of l-alpha-acetylmethadol (LAAM), norLAAM, and dinorLAAM in clinical and in vitro samples using liquid chromatography with electrospray ionization and tandem mass spectrometry.

l-Alpha-acetylmethadol (LAAM) is an alternative to methadone for the maintenance treatment of opioid dependence. LAAM has a longer therapeutic half-life than methadone, primarily because it is metabolized to more active metabolites, norLAAM and dinorLAAM. We have developed a liquid chromatography-tandem mass spectrometry method capable of measuring LAAM and its metabolites, norLAAM and dinorLAAM, at lower concentrations with 1.0-mL aliquots of plasma (range of 0.25 to 100 ng/mL) or higher concentration with 0.2-mL aliquots of plasma (range 1.25 to 500 ng/mL). It has acceptable precision and accuracy across both linear ranges, as well as in the urine matrix. Results from this assay correlate well with our previously validated gas chromatograghy-mass spectrometry method. All analytes had acceptable stability after three freeze-thaw cycles, room temperature storage for 20 h, or storage of extracts either at -20 degrees C for 6 days or on the autosampler (10 degrees C) for 4 days. The pharmacokinetics of LAAM, norLAAM, and dinorLAAM were determined for the first time in three male opioid-naive individuals receiving a single oral dose of 5 mg LAAM/70 kg. Using this method, we could monitor the in vitro N-demethylation of LAAM and norLAAM at substrate concentrations in the therapeutic range of 0.5 and 1.0 microM by cDNA-expressed cytochrome P450s. This confirmed the involvement of cytochrome P450s 3A4, 2B6, 2C8, and 2C18 at therapeutic concentrations of LAAM. An accurate and precise method for determination of LAAM and its metabolites, norLAAM and dinorLAAM, that is suitable for both in vivo and in vitro metabolism studies has been developed and validated.

Prenatal opiate withdrawal activates the chick embryo hypothalamic pituitary-adrenal axis and dilates vitelline blood vessels via serotonin(2) receptors.

Opiate withdrawal during pregnancy may occur because of voluntary or forced detoxification, or from rapid cycling associated with exposure to short-acting "street" opiates. Thus, animal modeling of prenatal withdrawal and development of potential therapeutic interventions is important. Direct developmental effects of opiates and/or withdrawal can be studied using a chick model. In ovo administration of the long-acting opiate N-desmethyl-l-alpha-noracetylmethadol (NLAAM) induces opiate dependence in the chick embryo. We examined activation of the hypothalamic-pituitary-adrenal (HPA) axis (assessed via serum corticosterone) and hemodynamic changes (assessed as changes in apparent diameter of vitelline (extraembryonic) blood vessels) after chronic NLAAM exposure and naloxone (Nx)-precipitated withdrawal during late stages of embryogenesis. Nx-precipitated withdrawal increased corticosterone 2- to 4.5-fold and diameters of vitelline blood vessels by 15 to 45%. NLAAM exposure itself did not effect these measures. In a second set of experiments, isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, was injected into eggs with embryos. IBMX similarly increased corticosterone and vitelline vessel diameter, with a similar time course and response magnitude. Previous studies found that serotonin(2) (5-HT(2)) receptors were involved in other withdrawal manifestations, so we determined whether they were likewise involved. Pretreatment with the 5-HT(2) antagonist ritanserin completely blocked HPA axis activation and vasodilation associated with both Nx-precipitated withdrawal and IBMX administration. This indicates that 5-HT(2) receptors, directly or indirectly, mediate these withdrawal manifestations in the chick embryo.

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.

Pharmacologic treatment of heroin-dependent patients.

Patients with heroin dependence frequently present to internists and other physicians for heroin-related medical, psychiatric, and behavioral health problems and often seek help with reducing their heroin use. Thus, physicians should be familiar with the identification and diagnosis of heroin dependence in their patients and be able to initiate treatment of heroin dependence both directly and by referral. Recent research has provided much information concerning effective pharmacologically based treatment approaches for managing opioid withdrawal and helping patients to remain abstinent Methadone maintenance and newer approaches using L-alpha acetylmethadol and buprenorphine seem to be particularly effective in promoting relapse prevention. Although these treatments are currently provided in special drug treatment settings, recent and ongoing research indicates that the physician's office may be an effective alternative site for these treatments.

l-alpha-Acetylmethadol, l-alpha-acetyl-N-normethadol and l-alpha-acetyl-N,N-dinormethadol: comparisons with morphine and methadone in suppression of the opioid withdrawal syndrome in the dog.

l-alpha-Acetyl-N-normethadol (nor-LAAM) and l-alpha-acetyl-N, N-dinormethadol (dinor-LAAM) are active metabolites of the opiate l-alpha-acetylmethadol (LAAM), and they contribute to the prolonged actions of the parent compound. Single doses of nor-LAAM, dinor-LAAM, LAAM, methadone and morphine were given intravenously to the chronic spinal dog to determine acute, single-dose effects and their ability to suppress withdrawal in morphine-dependent dogs. These opioids produced dose-dependent antinociception, decreases in body temperature and pupillary constriction. For these measures, dinor-LAAM was 1.5 to 3 times and nor-LAAM 6 to 12 times as potent as LAAM. Five hours after the acute administration of LAAM or either of the metabolites, a 1-mg/kg dose of naltrexone given intravenously produced withdrawal, indicating the presence of acute physical dependence. In dogs physically dependent on a daily dose of 125 mg of morphine, nor-LAAM was 9 times as potent as either LAAM or dinor-LAAM in suppressing spontaneous withdrawal 40 hr after the last dose of morphine. The efficacies of LAAM and its demethylated metabolites in the dog for producing acute opiate effects were comparable with those of morphine and methadone. There was a trend, however, for LAAM to suppress the expression of abstinence more fully than either metabolite. The usefulness of LAAM as a treatment for opiate addiction is likely due in part to the equivalent efficacies and higher potencies of its nor and dinor metabolites.

Quantitative analysis of l-alpha-acetylmethadol, l-alpha-acetyl-N-normethadol, and l-alpha-acetyl-N,N-dinormethadol in human hair by positive ion chemical ionization mass spectrometry.

A sensitive and specific method was developed for the quantitative analysis of l-alpha-acetylmethadol (LAAM), l-alpha-acetyl-N-normethadol (norLAAM), and l-alpha-acetyl-N,N-dinormethadol (dinorLAAM) in hair. In the development of this method, it was determined that sample pretreatment methods performed by the laboratory greatly affect the measured concentrations of drug and metabolite in hair. Deuterated internal standards were added to 20-mg hair samples and the samples digested overnight in a buffered solution of Protease Type VIII enzyme. Digests were extracted by modification of a liquid-liquid extraction procedure developed previously in our laboratory for the analysis of plasma and tissues. Derivatized extracts were analyzed on a Finnigan MAT 4500 mass spectrometer in positive ion chemical ionization mode using methane and ammonia reagent gases, helium carrier gas, and a DB-5MS (30 m, 0.25-micron film thickness) capillary column. The assay was linear to 50 ng/mg hair (r = 0.99) for all three compounds with a limit of quantitation experimentally determined to be 0.5 ng/mg for LAAM and 0.3 ng/mg for norLAAM and dinorLAAM. Intra-assay precision ranged from 1.0 to 10.5% for the three analytes at concentrations of 0.5, 5.0, and 25.0 ng/mg of hair. Interassay precision ranged from 4.7 to 12.9%. The performance of the method was also evaluated for its utility in detecting and quantitating LAAM, norLAAM, and dinorLAAM in hair from rats (n = 6) that had been administered 3 mg/kg LAAM intraperitoneally once daily for five days. LAAM, norLAAM and dinorLAAM were detectable in pigmented hair at concentrations of 1.27 ng/mg (+/-0.04), 1.28 ng/mg (+/-0.014), and 2.89 ng/mg (+/-0.014), respectively. Five laboratory wash solvents were then evaluated for their effect on the measured concentration of LAAM and metabolites in the rat hair. Phosphate buffer and 1% SDS washes substantially reduced the measured LAAM, norLAAM, and dinorLAAM concentrations by at least 30%, which suggests that drug incorporated into hair is removed (extracted) during the laboratory wash procedures. Wash procedures using methanol, methylene chloride, or water reduced the measured concentrations by no more than 20%. Because measured concentrations of LAAM, norLAAM, and dinorLAAM in hair appear to depend on the specific wash procedures used by a laboratory, quantitative data must be interpreted cautiously based on the sample pretreatment conditions.

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.

Toxicological evaluation of mu-agonists. Part II: Assessment of toxicity following 30 days of repeated oral dosing of male and female rats with levo-alpha-noracetylmethadol HCl (NorLAAM).

This study evaluated levo-alpha-noracetylmethadol (NorLAAM), the first N-demethylated metabolite of levo-alpha-acetylmethadol (LAAM), a long-acting morphine-like (mu) agonist, approved in 1993 to treat opiate dependence. After acute and 7-day pilot studies to define dose levels appropriate for use in longer term evaluations, Sprague-Dawley rats (20 of each sex per group) were gavaged with doses of 4.4-25.9 mg kg(-1) day(-1) for 30 days followed by a 14-day recovery period. Treatment-related effects included dose-dependent CNS depression paralleled by changes in food consumption, body weight gain and fecal output, as well as reddish urine and abdominal staining. Tolerance developed by day 7. The spectrum of activity observed differed from the parent compound primarily in its time course. Cage-biting and gnawing behavior were observed only with NorLAAM. Mortality was dose-dependent, with deaths occurring predominantly during the first week. At day 30, all male-treated groups exhibited statistically significant, dose-dependent decreases in body weight gain and increases in serum cholesterol that returned to the control range following recovery. Increases in brain/body weight and testes/body weight ratios and decreases in kidney/brain, liver/brain, spleen/brain and heart/brain ratios, as well as decreases in kidney, liver, spleen and heart absolute weights, achieved statistical significance only for males. At terminal sacrifice, histological findings in the kidneys included increased incidences of tubular mineral deposition in mid- and high-dose groups of both sexes and of corticomedullary mineral deposition in females. Hepatic centrilobular hypertrophy was evident in male and female mid- and high-dose groups. Histopathological changes abated following the recovery period. In summary, acute and repeated administration of NorLAAM produced a pharmacodynamic profile commensurate with its role as the primary N-demethylated metabolite of LAAM, which is more potent and less lipophilic than the parent compound; this was reflected in the toxicological outcomes observed. Like LAAM, NorLAAM's overall pattern of activity is consistent with its activity as a mu-agonist, which stimulates hepatic microsomal enzymes in rodents.

Determination of l-alpha-acetylmethadol, l-alpha-noracetylmethadol and l-alpha-dinoracetylmethadol in plasma by gas chromatography-mass spectrometry.

A method is described for the simultaneous determination of l-alpha-acetylmethadol (LAAM) and its N-demethylated metabolites, l-alpha-noracetylmethadol (norLAAM) and l-alpha-dinoracetylmethadol (dinorLAAM), in plasma by gas chromatography-chemical ionization mass spectrometry. Deuterated internal standards for each analyte serve as carriers and control for recovery during sample purification on a solid-phase extraction column (C18), and subsequent separation and analysis on a DB-17 capillary column. With this method, we have determined levels of LAAM, norLAAM, and dinorLAAM in small volumes of plasma (100 microliters). The limit of quantitation for all analytes was approximately 1.0 ng/g plasma and the limit of detection was approximately 0.5 ng/g plasma. An experimental application is also described where these analytes are quantitated in plasma obtained from rats before, during, and after chronic administration of LAAM-HCl. Since this technique affords a selective and sensitive means of detection of LAAM and its active, N-demethylated metabolites in small samples of blood, it may enable patient compliance to be more easily assessed by allowing samples to be collected by a simple finger-prick technique.

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.