Comparison of novel and existing methods for detection of linkage disequilibrium using parent-child trios in the GAW12 genetic isolate simulated data.

A novel method for joint detection of association caused by linkage disequilibrium (LD) and estimation of both recombination fraction and linkage disequilibrium parameters was compared to several existing implementations of the transmission/disequilibrium test (TDT) and modifications of the TDT in the simulated genetic isolate data from Genetic Analysis Workshop 12. The first completely genotyped trio of affected child and parents was selected from each family in each replicate so that the TDT tests are valid tests of linkage and association, rather than being only valid as tests for linkage. In general, power to detect LD using the genome-wide scan markers was inadequate in the individual replicate samples, but the power was better when analyzing several SNP markers in candidate gene 1.

Elimination of cocaine and metabolites in plasma, saliva, and urine following repeated oral administration to human volunteers.

Chronic administration of lipophilic drugs can result in accumulation and prolonged elimination during abstinence. It has been suggested that cocaine and/or metabolites can be detected in saliva and urine for an extended period following long-term, high-dose administration. The effects of chronic oral cocaine administration in healthy volunteer subjects with a history of cocaine abuse were investigated. Subjects were housed on a closed clinical ward and were administered oral cocaine in up to 16 daily sessions. In each session, volunteers received five equal doses of oral cocaine with 1 h between doses. Across sessions, cocaine was administered in ascending doses from an initial dose of 100 mg (500 mg/day) up to 400 mg (2 g/day), increasing by 25 mg/dose/session (125 mg/session). Participation in the study was terminated if cardiovascular safety parameters were exceeded. Plasma and saliva specimens were collected periodically during the dosing sessions and during the one-week withdrawal phase at the end of the study. All urine specimens were collected throughout the entire study. Specimens were analyzed for cocaine and metabolites by solid-phase extraction followed by gas chromatographic-mass spectrometric analysis in the SIM mode. The limit of detection for each analyte was approximately 1 ng/mL. The analytes measured included benzoylecgonine (BZE), ecgonine methyl ester, cocaine, benzoylnorecgonine, norcocaine, m- and p-hydroxycocaine, and m- and p-hydroxybenzoylecgonine. Noncompartmental analysis was employed for the determination of plasma and saliva pharmacokinetic parameters. Urinary elimination half-lives for cocaine and metabolites were determined by constructing ARE (amount remaining to be excreted) plots. Two phases of urinary elimination of cocaine and metabolites were observed. An initial elimination phase was observed during withdrawal that was similar to the elimination pattern observed after acute dosing. The mean (N = 6) plasma, saliva, and urine cocaine elimination half-lives were 1.5 +/- 0.1 h, 1.2 +/- 0.2 h, and 4.1 +/- 0.9 h, respectively. For three subjects, the mean cocaine urinary elimination half-life for the terminal phase was 19.0 +/- 4.2 h. There was some difficulty in determining if a terminal elimination phase for cocaine was present for the remaining three subjects because of interference by high concentrations of BZE. A terminal elimination phase was also observed for cocaine metabolites with half-life estimates ranging from 14.6 to 52.4 h. These terminal elimination half-lives greatly exceeded previous estimates from studies of acute cocaine administration. These data suggest that cocaine accumulates in the body with chronic use resulting in a prolonged terminal elimination phase for cocaine and metabolites.

Lamotrigine distribution in two postmortem cases.

Lamotrigine (Lamictal) is a new anticonvulsant drug recently approved for use in the United States. Although a therapeutic range for lamotrigine has not been definitively established, a range of between 2 and 14 mg/L has been reported. Two cases are presented in which lamotrigine was identified in cases investigated by the Office of the Chief Medical Examiner, State of Maryland. Lamotrigine was identified by gas chromatography-nitrogen-phosphorus detection following an alkaline extraction. A DB-5 column provided analytical separation; no derivatization was required. Confirmation was achieved by full scan electron ionization gas chromatography-mass spectrometry. In Case 1, primidone (11 mg/L) and phenobarbital (5.5 mg/L) were found in the heart blood in addition to lamotrigine (8.3 mg/L); in Case 2, no drugs other than lamotrigine (52 mg/L) were detected in the heart blood. The peripheral blood concentration in Case 2 was 54 mg/L. The liver lamotrigine concentrations in the two cases were 41 and 220 mg/kg. The medical examiner ruled that the cause of death in Case 1 was seizure disorder and the manner of death was natural. In Case 2, the medical examiner ruled that the cause of death was lamotrigine intoxication and the manner of death was undetermined.

Butyrylcholinesterase accelerates cocaine metabolism: in vitro and in vivo effects in nonhuman primates and humans.

Butyrylcholinesterase (BChE) is known to metabolize cocaine in humans. In the present study, three different experiments were performed to determine whether the addition of horse serum-derived BChE would accelerate the metabolism of cocaine. In the first experiment, the addition of BChE to squirrel monkey plasma in vitro reduced the half-life of cocaine by over 80%, decreased the production of the metabolic product benzoylecgonine, and increased ecgonine methyl ester formation. The effect of BChE on cocaine metabolism was reversed by a specific BChE inhibitor. In the second, in vivo, experiment, exogenously administered BChE reduced peak cocaine concentrations when given to anesthetized squirrel monkeys. Finally, incubation of cocaine with added BChE in human plasma in vitro resulted in a decrease in cocaine half-life similar to that observed with squirrel monkey plasma. The magnitude of the decrease in cocaine half-life was proportional to the amount of added BChE. Together, these results indicate that exogenously administered BChE can accelerate cocaine metabolism in such a way as to potentially lessen the behavioral and toxic effects of cocaine. Therefore, BChE may be useful as a treatment for cocaine addiction and toxicity.

Cocaine and metabolite concentrations in plasma during repeated oral administration: development of a human laboratory model of chronic cocaine use.

Long-term use of cocaine may produce neurophysiological and metabolic alterations that differ from acute drug use. A laboratory model that was capable of evaluating the effects of chronic cocaine administration in human subjects was needed. Chronic oral administration of cocaine was considered a feasible route because of the ease of administration, control of dosing patterns, and possible reduction in medical risks compared with the intravenous and smoked routes. This clinical study was conducted to evaluate chronically administered oral cocaine as a means of studying cocaine addiction and withdrawal in humans. Cocaine-abusing volunteers were given multiple doses of oral cocaine each day in up to 16 daily sessions (including three placebo sessions). In each daily session, volunteers received five equal doses separated by hourly intervals. Across sessions, the dose was increased from an initial dose of 100 mg (500 mg/day) to 400 mg (2000 mg/day) in the last session. The dose for each consecutive cocaine session was increased by 25 mg/dose/session (125-mg total increase per session). Twelve subjects were enrolled in the study; however, three subjects dropped out prior to completion of at least three sessions. Two subjects completed all 13 cocaine sessions. The remaining seven subjects completed from 3 to 11 sessions; their participation was terminated early for safety and behavioral reasons. Plasma was collected during all sessions and analyzed for cocaine and metabolites by solid-phase extraction followed by gas chromatography-mass spectrometry. Oral cocaine administration resulted in peak plasma concentrations of cocaine approximately 1 h after administration. Accumulation of cocaine was evident between hourly doses and there was evidence of dose-proportional increases in area under the curve (AUC) measures across sessions. A variety of cocaine metabolites was measured in plasma including benzoylecgonine, ecgonine methyl ester, norcocaine, benzoylnorecgonine, and p and m-hydroxy metabolites of cocaine and benzoylecgonine. During chronic oral dosing, there appeared to be a trend for AUC ratios (AUCmetabolite/AUCcocaine) of benzoylecgonine and ecgonine methyl ester to decrease and norcocaine to increase, indicating the possibility of dose-, time-, or route-dependent changes in the absorption and/or metabolism of cocaine. Overall, this study demonstrated that chronic oral dosing of cocaine produced dose-related increases in plasma cocaine concentration, and this model could be useful for studying the effects of chronic cocaine use in human subjects.

Attenuation of cocaine-induced locomotor activity by butyrylcholinesterase.

A primary enzyme for the metabolism of cocaine is butyrylcholinesterase (BChE). To determine whether the systemic administration of BChE can increase the metabolism of cocaine sufficiently to alter a behavioral effect, rats were tested in a locomotor activity chamber after receiving 17 mg of cocaine per kg intraperitoneally. In rats pretreated intravenously with 5,000 IU of horse serum-derived BChE, the locomotor activity effect was significantly attenuated. BChE pretreatment increased plasma BChE levels approximately 400-fold. When added to rat plasma, this amount of BChE reduced the cocaine half-life from over 5 hr to less than 5 min. BChE altered the cocaine metabolic pattern such that the relatively nontoxic metabolite ecgonine methyl ester was produced, rather than benzoylecgonine. These results suggest that systemic administration of BChE can increase the metabolism of cocaine sufficiently to alter a behavioral effect of cocaine and thus should be investigated as a potential treatment for cocaine abuse.

Effects of delivery rate and non-contingent infusion of cocaine on cocaine self-administration in rhesus monkeys.

The goal of this study was to determine whether slowly infused, response-independent cocaine would reduce cocaine self-administration in an animal model of drug abuse. Seven male rhesus monkeys self-administered i.v. cocaine on a fixed-ratio 30 schedule (5-min time-out). With unit dose (0.056 mg/kg per infusion for one monkey and 0.032 mg/kg per infusion for the rest) and infusion volume (0.5 ml) held constant, the rate of delivery was manipulated (0.125, 0.1875, 0.375, 0.75 and 3 ml/min, with infusions lasting 240, 160, 80, 40, and 10 s, respectively). Response rates increased monotonically as a function of delivery rate. Responding for cocaine at the slowest delivery rate did not differ from saline. The effects of infusing additional cocaine (starting 30 min prior to the session) at this non-reinforcing rate (0.125 ml/min) were then determined. Delivery rate of the self-administered infusion was manipulated as before. Non-contingent cocaine significantly increased responding for cocaine (at the fastest delivery rate) and for saline. While non-contingent cocaine reduced responding for cocaine in two of the seven monkeys, it also significantly reduced responding in three monkeys that responded for food on the same schedule. Plasma levels of cocaine delivered at rates of 0.125 and 3 ml/min were compared in five other monkeys. While a higher peak was reached with the faster infusion, levels did not differ after 5 min. Thus, when an infusion became available (after the 5-min time-out) in the self-administration experiments, plasma levels should not have differed regardless of the delivery rate. These results suggest that a low-dose, slow-delivery treatment with cocaine might prime or reinstate drug seeking rather than decrease it.

Forensic drug testing for opiates. VII. Urinary excretion profile of intranasal (snorted) heroin.

The urinary excretion profile of free and conjugated morphine and 6-acetylmorphine was determined by gas chromatography-mass spectrometry (GC-MS) and immunoassay for six healthy male subjects after intranasal administration of 6 and 12 mg of heroin HCI. Results were compared with heroin administration (6 mg) by the intramuscular route. Heroin metabolites were rapidly excreted with peak concentrations appearing in the first or second specimen collection after drug administration. Concentrations of total morphine and 6-acetylmorphine after intranasal heroin were similar to those after intramuscular administration, but free morphine concentrations after the lower intranasal dose were significantly lower than the same dose given intramuscularly. Detection times for total morphine by GC-MS and immunoassay (300-ng/mL cutoff concentration) were generally 24-36 h, but were reduced to less than 12 h at the higher cutoff concentration of 2000 ng/mL. 6-Acetylmorphine concentrations were highly variable and short-lived; detection times (10 ng/mL) were approximately 2-3 h for most subjects, but some failed to produce positive specimens. Of 14 specimens with 6-acetylmorphine concentrations of 10 ng/mL or more, ten were associated with total morphine concentrations greater than 2000 ng/mL, and four specimens had total morphine concentrations less than 2000 ng/mL. Overall, intranasal administration of heroin produced a similar profile of excretion of heroin metabolites to intramuscular administration.

Distribution of venlafaxine in three postmortem cases.

Venlafaxine (V) is a second-generation antidepressant approved for use in the United States in 1993. It is a derivative of phenethylamine and is structurally unrelated to first- and other second-generation antidepressants. Nevertheless, its mechanism of action is similar to other antidepressants; it inhibits the reuptake of presynaptic norepinephrine and serotonin. Its major routes of elimination involve O and N demethylation. O-Desmethylvenlafaxine (ODV) is biologically active. Therapeutic concentrations of V and ODV are approximately 0.2 and 0.4 mg/L, respectively. Three cases of drug intoxication involving V are presented. V and ODV were identified by gas chromatography-nitrogen-phosphorus detection after alkaline extraction of the biological specimen. On an HP-5 column, V and ODV elute after bupropion and fluoxetine, but prior to the first-generation antidepressants, sertraline, amoxapine, and trazodone. V and ODV were confirmed by full scan electron impact gas chromatography-mass spectrometry. The heart-blood V and ODV concentrations (mg/L) in the three cases were 6.6 and 31; 84 and 15; and 44 and 50, respectively. In Case 1, acetaminophen and diphenhydramine were found in the heart blood at 140 and 2.6 mg/L respectively. In Case 2, amitriptyline, nortriptyline, and chlordiazepoxide were found in the blood at 2.8, 0.5 and 3.3 mg/L, respectively. In each case, the manner of death was suicide.

Plasma butyrylcholinesterase activity and cocaine half-life differ significantly in rhesus and squirrel monkeys.

In vitro studies have implicated butyrylcholinesterase (BChE, E.C.3.1.1.8) as the major enzyme for metabolizing cocaine in humans, but little is known about endogenous BChE activity in monkeys and other animals often used in preclinical studies of cocaine. We compared BChE activity in 18 rhesus and 11 squirrel monkeys, using the colorimetric method of Ellman with butyrylthiocholine as substrate, and in vitro cocaine half-life in pooled plasma samples measuring cocaine concentrations over 60 minutes by GC-MS. Rhesus monkeys had a significantly higher plasma BChE activity than squirrel monkeys (8.2 +/- 0.5 U/L vs. 2.8 +/- 0.5 U/L), and a three-fold shorter in vitro cocaine half-life (20.1 min vs. 60.2 min). BChE activity in rhesus monkeys was comparable to the activity reported in humans. There was no significant influence of age, weight, or prior cocaine exposure. These results indicate that BChE level can vary between species of non-human primates, a factor that should be taken into account when studying drugs such as cocaine which are metabolized by BChE.