Mecamylamine elicits withdrawal-like signs in rats following a single dose of nicotine

Rationale

The ability of nicotine to induce dependence (result in a withdrawal syndrome) is typically thought to require long-term, daily smoking. Emerging evidence suggests that symptoms of nicotine withdrawal may occur following only a few cigarettes. Whether acute exposure to nicotine can induce dependence in animals has not been well established.

Objective

The objective of this paper is to examine whether the nicotinic acetylcholine receptor antagonist mecamylamine elicits withdrawal-like signs in rats following acute nicotine exposure.

Methods and Results

Mecamylamine (3.0 mg/kg, s.c.) administered ≈2 h after a single dose of nicotine (0.5 mg/kg, s.c.) elicited increases in intracranial self-stimulation (ICSS) thresholds and somatic signs, two well-established effects of withdrawal from long-term (chronic) nicotine exposure. The magnitude of these effects remained constant across five daily test sessions. A lower dose of mecamylamine (1.5 mg/kg, s.c.) had little or no effect on ICSS thresholds or somatic signs following acute nicotine exposure, but precipitated robust increases in these measures during a chronic nicotine infusion. Finally, rats exhibited a small increase in ICSS thresholds over time following a single nicotine injection (0.5 mg/kg, s.c.), possibly reflecting a modest spontaneous withdrawal-like effect.

Conclusions

Mecamylamine elicited withdrawal-like signs in rats following a single dose of nicotine. The different effects of mecamylamine 1.5 mg/kg following acute versus chronic nicotine exposure supports the notion that these models simulate the early and more advanced stages of nicotine dependence, respectively. While further optimization and validation of these models is necessary, they may provide a novel approach for studying the earliest stages of nicotine dependence.     

The incorporation of cocaine and metabolites into hair: effects of dose and hair pigmentation.

The relationship between xenobiotic concentrations in hair and the degree of systemic xenobiotic exposure is poorly defined. The purpose of this study was to evaluate the effect of dose, time, and pigment on the hair incorporation of cocaine (COC) and its metabolites, benzoylecgonine (BE), ecgonine methyl ester (EME), and norcocaine (NCOC). COC was administered by the i.p. route to male Long-Evans (LE) rats at three doses (5, 10, and 20 mg/kg) once daily for 5 days. Fourteen days after the initial injection, the hair was collected and analyzed by gas chromatography/mass spectrometry for the compounds of interest. COC, EME, and NCOC were preferentially incorporated into pigmented hair in a dose-dependent manner. None of the analytes were detected in nonpigmented hair. The plasma pharmacokinetic profile of each analyte was determined at each dose. After normalizing for the plasma concentrations, the incorporation of COC into pigmented hair was 2 orders of magnitude greater than BE. The time course of COC and metabolite distribution into hair was also investigated from 1 h to 14 days after a single dose. After COC disappears from plasma, there is a 3-day delay before maximal hair concentrations are reached in pigmented hair. In nonpigmented hair, concentrations of BE and COC did not exceed 0.25 ng/mg and were undetectable after 4 h and 2 days, respectively. This study demonstrates that the pigment-mediated differences in the incorporation of COC and its metabolites noted at 14 days after dosing are also evident a few hours after drug administration.     

Deposition of 7-aminoclonazepam and clonazepam in hair following a single dose of Klonopin

The objective of this paper was to determine whether benzodiazepine clonazepam (CLO) and its major metabolite 7-aminoclonazepam (7-ACLO) could be detected in hair collected from healthy volunteers after receiving a single 3-mg dose of Klonopin (clonazepam). Such data would be of great importance to law enforcement agencies trying to determine the best time interval for hair collection from a victim of drug-facilitated sexual assault (DFSA) in order to reveal drug use. Ten healthy volunteers (6 women and 4 men, 23-49 years old) participated in the study. The following hair samples were collected from each volunteer: one before CLO administration, and 1, 3, 5, 14, 21, and 28 days after. All hair samples were pulverized and 50-mg aliquots were sonicated in methanol and digested with 0.1 N HCl at 55 degrees C for 18-24 h. Internal standard, diazepam-d5 (DIAZ-d5) was used. Both extracts were combined and extracted using HCX solid-phase extraction columns. After derivatization with HFBA all extracts were analyzed using highly sensitive negative chemical ionization gas chrometography-mass spectrometry. Standard curves for CLO (20-100 pg/mg) and 7-ACLO (1-20 pg/mg) were prepared by spiking aliquots (50 mg) of negative hair and had correlation coefficients of 0.985 and 0.989, respectively. In addition, two levels of control hair were prepared for CLO and 7-ACLO. All method validation parameters were within acceptable limits. 7-ACLO was detected in hair of 6 out of 10 volunteers. In two cases 7-ACLO appeared in hair three days after CLO intake and remained detectable for the entire 28-day study period (3.6-8.4 pg/mg and 2.7-3.0 pg/mg), and in two subjects it was detectable 21 days later (4.9 and 2.7 pg/mg and 1.2 and 23 pg/mg). In two volunteers 7-ACLO was detected only on day 28 (1.8 and 3.3 pg/mg). CLO was not detected in any of the samples.     

Accelerated solvent extraction for gas chromatographic analysis of nicotine and cotinine in meconium samples

Adverse effects associated with smoking during pregnancy are well documented. Although self-report surveys on drug consumption during pregnancy have been improved with new interviewing techniques, underreporting is still a concern. Therefore, a series of biological markers and specimens to diagnose fetal exposure to tobacco have been studied. In the present study, an analytical method was developed to detect nicotine and cotinine (the main nicotine metabolite) in meconium samples. Accelerated solvent extraction (ASE) followed by solid-phase extraction (SPE) were used as sample preparation techniques. The analytes were detected by gas-chromatography with nitrogen-phosphorus detection. The limits of detection were 3.0 and 30 ng/g for cotinine and nicotine, respectively. The method showed good linearity (r(2) > 0.98) in the concentration range studied (LOQ-500 ng/g). The intraday precision, given by the RSD of the method, was less than 15% for cotinine and nicotine. The method proved to be fast, practical, and sensitive. Smaller volumes of organic solvents are necessary compared to other chromatographic methods published in the scientific literature. This is the first report in which ASE was used as sample preparation technique in methods to detect xenobiotics in meconium.     

High dose transdermal nicotine therapy for heavy smokers: safety,tolerability and measurement of nicotine and cotinine levels

Transdermal nicotine has been shown to relieve nicotine withdrawal and to double smoking cessation rates compared to placebo in clinical trials. A 21 or 22 mg/day dose provides a steady state serum nicotine that is less than obtained from smoking. Limited information is available about higher nicotine patch doses. To define better the optimal dosing of nicotine patch therapy, we undertook an open-label study to determine the safety and tolerability of 44 mg/day dose for smoking cessation in subjects smoking 20 cigarettes per day. Forty smokers received 44 mg/day of transdermal nicotine for 4 weeks followed by 4 weeks of 22 mg/day. Of the 40 subjects enrolled, 38 (95%) completed the 4 weeks of 44 mg patch therapy and 36 (90%) completed the entire 8 weeks of patch therapy. Non-smokers at week 4 had a mean serum nicotine level of 23.4±11.7 ng/ml and cotinine of 152.2±87.3 ng/ml. Percent replacement was calculated by dividing the steady state level at week 4 by the baseline level while the subjects were smoking their usual number of cigarettes. Percent nicotine replacement for non-smokers at week 4 (while on 44 mg nicotine patch) averaged 158%±108.4, and for cotinine was 112.0±73.8. For nicotine, 33% of non-smokers at week 4 had 100% nicotine replacement and for cotinine 63% 100% replacement. Biochemically confirmed point prevalence smoking cessation rates were 65% and 55% at weeks 4 and 8 of patch therapy, respectively, and self-reported smoking cessation at 3 months was 50%. The most common effect was skin irritation at the patch site. A single subject was admitted for myocardial infarction following step-down from 44 to 22 mg of replacement nicotine. The subject was not smoking and the adverse event was deemed to be not related to the patch therapy. Sleep complaints were reported in 33% of subjects during the 44 mg phase. Other complaints were infrequent. We conclude that 44 mg per 24-h nicotine patch therapy in heavy smokers is safe, tolerable, and without significant adverse events.     

Cefodizime penetration into skin suction blister fluid following a single intravenous dose

Summary Cefodizime pharmacokinetics was investigated, evaluating drug concentrations in serum, skin suction blister fluid (SBF), saliva and urine in six healthy male subjects who were administered a 1-g dose intravenously. Serum levels in five subjects can be described according to a two-compartment open model; terminal half-life is 181±14 min. Volume of distribution (V_d) amounts to 15.3±1.61, serum clearance to 59±6 ml/min, renal clearance to 33±3 ml/min. Of the administered dose, 54% is renally excreted unchanged within 27 h. Unbound drug fraction in serum is 19.0% and in SBF 38.4%. Thus renal clearance of free cefodizime amounts to 172 ml/min, V_dss to 68.91 (free drug). Whereas cefodizime has not been detected in saliva samples, SBF concentration 3–9 h post administration parallel serum levels, amounting to 40% of the respective serum concentration. At 9 h, unbound cefodizime concentrations in SBF amount to 1.4±0.4 µg/ml, this value being well above the MIC_90% values of many clinically relevant bacteria.     

Time course of the locomotor stimulant and depressant effects of a single low dose of ethanol in mice

The acute effects of alcohol on spontaneous locomotor activity in male Swiss mice were studied at various times after an IP injection of 2 g/kg ethanol. Subjects were placed alone in a novel arena and videotape recordings were made of behaviour: trials were of 500-s duration and commenced at either 30, 60, 120, or 180 min after alcohol administration. Measures of behaviour included various indices of ambulation and immobility, together with a more detailed ethological analysis of the frequencies of all other acts and postures shown by test animals. Ambulation showed a biphasic response to alcohol treatment, consisting of an initial stimulation followed by a suppression after 3 h. Immobility was also increased by alcohol, and showed peak stimulation in trials commencing 30 min after administration: thereafter there was a progressive return to baseline levels. Many behavioural elements were suppressed including rearing, digging, shaking, and abbreviated grooming. Ethanol thus appeared to produce two distinct types of depression, in terms of increased immobility (and suppression of other behaviour) and in terms of decreased ambulation, the latter occurring when immobility had returned to baseline levels.     

Transdermal delivery of nicotine in normal human volunteers: a single dose and multiple dose study

The absorption of nicotine delivered by a transdermal delivery system (TDS) was investigated in two separate studies, (A) a dose proportionality study and (B) a multiple dose study. In the dose range of 15-60 mg nicotine, the AUC and Cmax values were proportional to the dose. The levels achieved were in the same range as reported in smokers, following absorption from nicotine chewing gum. The TDS used in the present study produced sustained levels of nicotine for 24 h. No significant accumulation of nicotine was evident as a result of multiple dose administration using a 30-mg nicotine patch. Absorption of nicotine from the TDS was 80-90% and the rate of delivery was similar during both studies.     

A comparison of nicotine dose estimates in smokers between filter analysis, salivary cotinine, and urinary excretion of nicotine metabolites

Rationale Nicotine uptake during smoking was estimated by either analyzing the metabolites of nicotine in various body fluids or by analyzing filters from smoked cigarettes. However, no comparison of the filter analysis method with body fluid analysis methods has been published.Objectives Correlate nicotine uptake estimates between filter analysis, salivary cotinine, and urinary excretion of selected nicotine metabolites to determine the suitability of these methods in estimating nicotine absorption in smokers of filtered cigarettes.Materials and methods A 5-day clinical study was conducted with 74 smokers who smoked 1–19 mg Federal Trade Commission tar cigarettes, using their own brands ad libitum. Filters were analyzed to estimate the daily mouth exposure of nicotine. Twenty-four-hour urine samples were collected and analyzed for nicotine, cotinine, and 3′-hydroxycotinine plus their glucuronide conjugates. Saliva samples were collected daily for cotinine analysis.Results Each method correlated significantly (p < 0.01) with the other two. The best correlation was between the mouth exposure of nicotine, as estimated by filter analysis, and urinary nicotine plus metabolites. Multiple regression analysis implies that saliva cotinine and urinary output are dependent on nicotine mouth exposure for multiple days. Creatinine normalization of the urinary metabolites degrades the correlation with mouth exposure.Conclusions The filter analysis method was shown to correlate with more traditional methods of estimating nicotine uptake. However, because filter analysis is less complicated and intrusive, subjects can collect samples easily and unsupervised. This should enable improvements in study compliance and future study designs.F. K. St.Charles and G. R. Krautter were formerly with Brown & Williamson Tobacco Company, Macon, GA 31202, USA.     

Distribution of dieldrin following a single oral dose

Two groups of investigators have used the classical model for the distribution of thiopental following iv injection to predict the distribution of dieldrin following accidental ingestion. They concluded that the concentration of dieldrin in the brain is reduced first by redistribution to muscle and only later by redistribution to fat. The model as originally designed for thiopental had been evaluated by analysis of samples from persons undergoing surgery under thiopental anesthesia. Since it is not appropriate to give large doses of dieldrin to humans or to collect samples of muscle and various vital organs from them, the distribution of dieldrin was studied in rats. Dieldrin dissolved in corn oil was administered to these animals by stomach tube. The highest concentration of dieldrin in the brain was reached in 4 hr, and the concentration decreased gradually thereafter. The concentration in muscle remained essentially steady during the interval from 4 to 48 hr. There was no peak for muscle that could be interpreted as replacing a peak for brain. The concentration of dieldrin in fat was already slightly higher than that in the brain at 1 hr, very much higher at 4 hr when the concentration in the brain was maximal, and the concentration in the fat continued to increase during the first 24 hr. Either on the basis of concentration or on the basis of the total amount in each organ, no reason was found to assign any special importance to muscle as a sink into which dieldrin is redistributed from the brain. The fat appears to be far more important in this regard. The results indicate the danger of applying a mathematical model to a new situation without checking the results experimentally.     

Pharmacokinetics of methyprylon following a single oral dose

Single oral doses of 300 mg of methyprylon were administered to 10 healthy volunteers. Plasma concentrations of methyprylon and its dehydro metabolite were measured using a recently developed HPLC assay. Plasma concentration-time data were fitted to a two-compartment model with either first-order absorption, zero-order absorption, or two consecutive, discontinuous, first-order absorption rate constants. Based on the criteria of visual inspection, the correlation coefficient, standard deviations of the parameter estimates, and the residual sum of squares, it was concluded that the zero-order absorption model fit the data best. Mean (+/- SD) values for the half-life (9.2 +/- 2.2 h), apparent clearance, (11.91 +/- 4.42 mL/h/kg) and apparent steady-state volume of distribution, (0.97 +/- 0.33 L/kg) were found.     

Time course of smoking withdrawal symptoms as a function of nicotine replacement

Subjects (N = 32) provided morning, afternoon, and evening data for week-1 withdrawal from smoking. Withdrawal symptoms were measured using Schneider's Smoker Complaint Scale. Twenty subjects received nicotine gum and 12 subjects received placebo gum. Carbon monoxide levels verified smoking abstinence. Results showed significantly less withdrawal for nicotine gum subjects compared to the placebo group. A significant treatment- x -time of day interaction was also observed: Placebo subjects reported increased withdrawal in the evenings compared to their morning and afternoon scores, and in contrast to nicotine-group responses. The results provide evidence for nicotine withdrawal and its alleviation by nicotine gum.     

Time course of oxipurinol levels in plasma following single dose and chronic administration of allopurinol in different pharmaceutical preparations (author's transl)

Oxipurinol plasma levels and plasma elimination half-life were investigated in five healthy volunteers after oral administration of 300 mg allopurinol in customary (A 300) and in slow-release preparation (A ret) in a double blind cross-over study. After a single oral dose of 300 mg allopurinol in customary preparation maximum oxipurinol plasma levels were 5.24 microgram/ml. After allopurinol in slow-release preparation maximum oxipurinol levels were 2.22 microgram/ml. 24-h oxipurinol plasma levels were 3.78 microgram/ml after A 300 and 2.08 microgram/ml after A ret, respectively. Chronic oral administration of a daily dose of 300 mg allopurinol resulted in accumulation of oxipurinol until day 8. 24-h steady-state levels of oxipurinol were found to be 9.98 microgram/ml after A 300 and 7.14 microgram/ml after A ret. All oxipurinol plasma levels on A 300 exceeded those on A ret. The time course of oxipurinol plasma levels after a single oral dose of allopurinol suggests that oxipurinol elimination from plasma follows an exponential function (first-order elimination). Plasma elimination half-life of oxipurinol was 42.65 h after administration of A 300 and 49.91 h after administration of A ret. The difference between the elimination half-lives is statistically not significant.     

Correlates of individual differences in compensatory nicotine self-administration in rats following a decrease in nicotine unit dose

Rationale  The ability of tobacco harm reduction strategies to produce significant reductions in toxin exposure is limited by compensatory increases in smoking behavior. Characterizing factors contributing to the marked individual variability in compensation may be useful for understanding this phenomenon and assessing the feasibility of harm reduction interventions. Objective  The objective of the study was to use an animal model of human compensatory smoking that involves a decrease in unit dose supporting nicotine self-administration (NSA) to examine potential contributors to individual differences in compensation. Methods  Rats were trained for NSA during daily 23-h sessions at a unit dose of 0.06 mg/kg/inf until responding was stable. The unit dose was then reduced to 0.03 mg/kg/inf for at least 10 sessions. Following reacquisition of NSA at the training dose and extinction, single-dose nicotine pharmacokinetic parameters were determined. Results  Decreases in nicotine intake following dose reduction were proportionally less than the decrease in unit dose, indicating partial compensation. Compensatory increases in infusion rates were observed across the course of the 23-h sessions. The magnitude of compensation differed considerably between rats. Rats exhibiting the highest baseline infusion rates exhibited the lowest levels of compensation. Nicotine pharmacokinetic parameters were not significantly correlated with compensation. Infusion rates immediately returned to pre-reduction levels when baseline conditions were restored. Conclusions  These findings provide initial insights into correlates of individual differences in compensation following a reduction in nicotine unit dose. The present assay may be useful for characterizing mechanisms and potential consequences of the marked individual differences in compensatory smoking observed in humans.     

Urinary excretion of alpha-hydroxytriazolam following a single dose of halcion

As an approved medicinal product and reportedly an abused substance that have been associated with death and "considered to be a factor...of impaired driving, sexual assault, and other violent crimes", triazolam is controlled at the same level (Level III) as flunitrazepam in Taiwan. Alleged misuses of this substance have been associated with case specimens submitted to this laboratory. A sample preparation (with and without enzymatic hydrolysis) and gas chromatography-mass spectrometry protocols were evaluated and applied to the analysis of free and total alpha-hydroxytriazolam (the main metabolite of triazolam) in urine. Ions designated for TMS-derivatized alpha-hydroxytriazolam and alpha-hydroxytriazolam-d4 are m/z 415, 417, and 430 and 419, 421, and 434, respectively. The overall protocol achieved the following results when applied to the analysis of 2-mL drug-free urine specimens fortified with 10-200 ng/mL alpha-hydroxytriazolam: recovery, 95%; interday and intraday precision ranges, 1.50-3.52% and 0.93-4.71%, respectively; linearity, r2 > 0.99; and limits of detection and quantitation, 0.05 and 0.1 ng/mL, respectively. This protocol was applied to the analysis of case specimens and urine samples collected from two patients (A and B) taking one oral dose of Halcion (0.25 mg triazolam). Excretion profiles of free and total alpha-hydroxytriazolam show that free alpha-hydroxytriazolam is detectable, but at very low levels (< 5 ng/mL). Peak excretion of total alpha-hydroxytriazolam occurs at approximately 5-10 h following the drug intake. Total alpha-hydroxytriazolam is excreted at detectable levels approximately 2-35 h following an oral dose of 0.25 mg triazolam. Total free and conjugated alpha-hydroxytriazolam excreted by A and B are 0.61% and 31.6%; and 0.36% and 57.2% of the dose, respectively.     

Effects on memory following a single oral dose of diazepam

The effects of diazepam on learning and memory were investigated in a double-blind study of 28 Uruguayan university women who were randomly assigned to four treatments, seven subjects each: placebo, 0.1, 0.2, and 0.3 mg/kg diazepam orally. Subjects were presented with five uncategorized and categorized word lists, a 12-digit number list, and a tonal discrimination test, before and up to 200 min after treatment. Immediate and delayed recall were tested. Performance of the 0.1 mg/kg diazepam group did not show significant differences from placebo in any of the tests. For the 0.2 dose group, marginal impairments were noted at 60 min followed by full recovery at 190 min. On the contrary, 0.3 mg/kg diazepam produced significant impairments on immediate and delayed recall, with marginal impairments still evident at 190 min. The finidng that diazepam did not impair performance on a tonal discrimination test provided some evidence that memory impairments did not result from failures in general alertness.     

Enzyme induction following a single dose of amobarbital in dogs

The elimination of amobarbital in dogs was investigated by injecting various doses of amobarbital into a given animal. At low doses (3mg/kg) serum levels declined in a first-order fashion. Superficially, at high doses (20 mg/kg) the relationship between serum concentration and time could be quantitatively characterized by simple one-compartment saturable kinetics. Indeed, qualitatively, saturation of the amobarbital-metabolizing enzymes was indicated by a shallower initial slope of the semilogarithmic concentration-time profile at the high than at the low dose. However, in addition, an acute enzyme induction phenomenon was observed which was indicated by a shorter terminal half-life of amobarbital at the high dose than after the low dose and also by a shortening in antipyrine half-life.This work was supported by Medical Research Council of Canada, Ottawa Grant No. MA-4763.