Gerold F. Kauert

Neurocognitive performance during acute THC intoxication in heavy and occasional cannabis users

Abstract Performance impairment during Δ9-tetrahydrocannabinol (THC) intoxication has been well described in occasional cannabis users. It is less clear whether tolerance develops to the impairing effects of THC in heavy users of cannabis. The aim of the present study was to assess neurocognitive performance during acute THC intoxication in occasional and heavy users. Twenty-four subjects (12 occasional cannabis users and 12 heavy cannabis users) participated in a double-blind, placebo-controlled, two-way mixed model design. Both groups received single doses of THC placebo and 500 μg/kg THC by smoking. Performance tests were conducted at regular intervals between 0 and 8 h after smoking, and included measures of perceptual motor control (critical tracking task), dual task processing (divided attention task), motor inhibition (stop signal task) and cognition (Tower of London). THC significantly impaired performance of occasional cannabis users on critical tracking, divided attention and the stop signal task. THC did not affect the performance of heavy cannabis users except in the stop signal task, i.e. stop reaction time increased, particularly at high THC concentrations. Group comparisons of overall performance in occasional and heavy users did not reveal any persistent performance differences due to residual THC in heavy users. These data indicate that cannabis use history strongly determines the behavioural response to single doses of THC.

Determination of amphetamine and methylenedioxy-amphetamine-derivatives in hair.

Two GC/MS-procedures for the detection of amphetamine and its methylenedioxy-derivatives (MDA, MDMA and MDE) in hair are presented. In these methods a methanol sonication extraction technique was applied. The extracted drugs were derivatized either with propionic acid anhydride (PSA) or trifluoroacetic acid anhydride (TFA). PSA-derivatives are more stable than TFA-derivatives, but the latter provide more specific mass-spectrometric information, and, therefore, seem to be preferably for amphetamine determination. The detection limit for all compounds was in a range of about 0.01 ng/mg, if at least 50-100 mg of hair were analyzed, independent of the derivatization used.

Determination of clopidogrel main metabolite in plasma: a useful tool for monitoring therapy?

This study was performed to determine whether analysis of clopidogrel and its main carboxylic acid metabolite in plasma provides additional information about the wide variability of platelet aggregation inhibition in clopidogrel-treated patients with peripheral arterial occlusive disease. Consecutive outpatients (n = 56) with stable peripheral arterial occlusive disease treated with 75 mg clopidogrel daily, without co-administration of aspirin, were investigated. With use of a standardized questionnaire, the time of drug intake was documented. Blood sampling was performed within 24 hours after the most recent drug intake. Platelet function was measured by optical aggregometry using adenosine diphosphate (ADP) (2 μmol/L) as the agonist. Plasma concentrations of clopidogrel and its main metabolite, clopidogrel carboxylic acid, were quantitated using high-performance liquid chromatography analysis coupled to mass spectrometry. In 95% (53/56) of patients, clopidogrel carboxylic acid was detected. In 40% (22/56) of patients, the ADP-induced aggregation response was within the normal range despite clopidogrel treatment. In 14% (3/22) of these patients, neither clopidogrel nor its main metabolite could be detected. Two of these patients agreed to ingest 75 mg/d clopidogrel under observation and to undergo blood sampling after 2, 12, and 24 hours. Clopidogrel carboxylic acid and a significant inhibition of platelet aggregation were detected even after 24 hours in both patients, confirming noncompliance as the reason for the lack of inhibition of ADP-induced platelet aggregation observed in the initial measurements. In the subgroup of patients who had taken clopidogrel within 4 hours before blood sampling, a large range of carboxylic acid concentrations was detected, indicating a high variability of drug metabolism among patients. In conclusion, determining clopidogrel metabolite plasma concentrations could be a useful tool for identifying poor compliance and variable metabolism in clopidogrel-treated patients. Nevertheless, in the majority of clopidogrel-treated patients, the variability of platelet response is not caused by noncompliance.

Gas chromatographic-mass spectrometric detection of anhydroecgonine methyl ester (methylecgonidine) in human serum as evidence of recent smoking of crack.

The discrimination between smoking of crack and other routes of cocaine application has forensic implications. The pyrolysis product anhydroecgonine methyl ester (AEME, methylecgonidine) has been found to be a marker for smoked cocaine. An improved method for the determination of AEME in serum was developed, consisting of mixed phase solid-phase extraction and GC-MS. Special care was taken for the volatility of AEME and tert.-butyldimethylsilylation was used for derivatization. Thus AEME could be determined for the first time in 13 serum samples from living subjects. The concentrations found were in a range of 3 to 34 ng/ml, a correlation with the storage time of the samples or with benzoylecgonine concentrations could not be found.

Artifact production in the assay of anhydroecgonine methyl ester in serum using gas chromatography-mass spectrometry

The detection of the pyrolysis product anhydroecgonine methyl ester (AEME, methylecgonidine) after cocaine smoking using gas chromatography-mass spectrometry is hampered by the artifactual production of AEME. The amount of AEME increases with the amount of cocaine used producing false positive values in authentic samples. A method for the correction of quantitative values was established using calibration of pyrolysis and estimation of the artifactual AEME. Authentic AEME in serum was differentiated from the artifact above 3.5 microg/l, 99% prediction limits of the quantitation were +/-3.1 microg/l. In 16 serum samples and five postmortem blood samples, cocaine and AEME were detected, but after application of the correction method only ten were truly positive for AEME.

New conopeptides of the D-superfamily selectively inhibiting neuronal nicotinic acetylcholine receptors.

The venom of cone snails (Conus spp.) is a rich source of peptides exhibiting a wide variety of biological activities. Several of these conopeptides are neuronal nicotinic acetylcholine receptor (nAChR) antagonists and belong to the A-, M-, S-, C and the recently described D-superfamily (alphaD-conopeptides). Here we describe the discovery and characterization of two alphaD-conopeptides isolated from the venom of Conus mustelinus and Conus capitaneus. Their primary structure was determined by Edman degradation, MS/MS analysis and by a PCR based approach. These peptides show close structural homology to the alphaD-VxXIIA, -B and -C conopeptides from the venom of Conus vexillum and are dimers (about 11kDa) of similar or identical peptides with 49 amino acid residues and a characteristic arrangement of ten conserved cysteine residues. These novel types of conopeptides specifically block neuronal nAChRs of the alpha7, alpha3beta2 and alpha4beta2 subtypes in nanomolar concentrations. Due to their high affinity, these new ligands may provide a tool to decipher the localisation and function of the various neuronal nAChRs.

Criminal poisoning of commuters in Bangladesh: Prospective and retrospective study

Travel-related poisoning is an emerging social and public health emergency in Bangladesh but its cause and significance have not been determined. To investigate this syndrome we performed a prospective clinical study and retrospective analysis of hospital records in a general medicine unit of a public tertiary care teaching hospital in Dhaka, Bangladesh, using toxicological analysis by fluorescence polarization immunoassay (FPIA) and liquid chromatography coupled to time-of-flight mass spectrometry (LC-TOF MS). The participants of the prospective study were 130 consecutive patients aged 16-80 years who were admitted with central nervous system depression (Glasgow Coma Score 3-14) after using public transportation, in the absence of other abnormalities, from January through June 2004, and a convenience sample of 15 such patients admitted during 3 days in May 2006. In 2004-2006, travel-related poisoning increased from 6.1 to 9.5% of all admissions (210-309 of 3266-3843 per year), representing 46.6-55.7% of all admitted poisoning cases. Incidents were associated with bus (76%), taxi, train, and air travel, or local markets; 98% of patients remembered buying or accepting food or drinks before losing consciousness. Direct financial damage (missing property) was diverse and frequently existential. Among 94 urine samples analyzed by FPIA, 74% tested positive for benzodiazepines. Among 15 urine samples analyzed by LC-TOF MS, lorazepam was detected in all; five also contained diazepam or metabolites; nitrazepam was present in three. FPIA results obtained for these 15 samples were below the recommended cut-off in eight (53%; lorazepam only). Our findings show that the massive medicosocial emergency of travel-related poisoning in Bangladesh is the result of drug-facilitated organized crime and that benzodiazepine drugs are used to commit these crimes, suggesting modifications to the local emergency management of the victims of this type of poisoning. They also highlight the need for more research in the neglected field of acute poisoning in Bangladesh, and for criminal investigations of the use of benzodiazepine drugs in this country.

Day–night expression patterns of clock genes in the human pineal gland

Abstract:  Rhythm generation within the mammalian circadian system is achieved by clock genes and their protein products. As an integral part of this system, the pineal gland serves the need to tune the body to the temporal environment by the rhythmic synthesis and release of melatonin. A number of human disorders and syndromes are associated with alterations in circadian rhythms of clock genes and their protein products and/or a dysfunction in melatonin synthesis. In the human, little is known about the molecular signature of time management. Pineal tissue from regular autopsies was allocated to asserted time‐of‐death groups (dawn, day, dusk, night), and analyzed by RT‐PCR, immunoblotting, immunohistochemistry, and confocal laser scanning microscopy for expression of clock genes. Despite the observed diurnal rhythms in activity of the arylalkylamine N‐acetyltransferase and in melatonin content, mRNA levels for the clock genes Period1, Cryptochrome1, Clock, and Bmal1, and also amounts of corresponding clock gene proteins showed no differences between time‐ of‐death groups. In contrast, a time‐of‐day‐dependent nucleocytoplasmic shuttling of clock gene proteins was detected. These data confirm the minor importance of a transcriptional regulation for dynamics in the human pineal gland, and offer a novel twist in the molecular competence of clock gene proteins.

Influence of Ethanol on the Pharmacokinetic Properties of Δ9-Tetrahydrocannabinol in Oral Fluid

Oral fluid (OF) tests aid in identifying drivers under the influence of drugs. In this study, 17 heavy cannabis users consumed alcohol to achieve steady blood alcohol concentrations of 0 to 0.7 g/L and smoked cannabis 3 h afterward. OF samples were obtained before and up to 4 h after smoking and on-site tests were performed (Dräger DrugTest 5000 and Securetec DrugWipe 5+). Maximum concentrations of tetrahydrocannabinol (THC) immediately after smoking (up to 44,412 ng/g) were below 4,300 (median 377) ng/g 1 h after smoking and less than 312 (median 88) ng/g 3 h later with 5 of 49 samples negative, suggesting that recent cannabis use might occasionally not be detectable. An influence of alcohol was not observed. Drinking 300 mL variably influenced THC concentrations (median only -29.6%), which suggests that drinking does not markedly affect on-site test performance. Many (92%) Dräger tests performed 4 h after smoking were still positive, indicating sufficient sensitivity for recent cannabis use. Differences in the results of a roadside study with DrugTest 5000 (sensitivity 84.8%, specificity 96.0%, accuracy 84.3%) could be explained by a higher number of true negatives, differences between OF and serum and differences between occasional and chronic users.

Up in Smoke: Comparability of THC Dosing across Performance Studies

SirWe thank Drs Nordstrom and Hart (2006) for theircomments, because it gives an opportunity to addressan issue of growing importance in experimental cannabisresearch: the comparability of THC dosing across experi-mental performance studies. In general, researchers haveemployed two types of units for reporting the mean amountof THC delivered to subjects when smoking an experimentalcannabis cigarette: (1) %THC contained in a cannabiscigarette and (2) absolute mass of THC (mg). The formerprovides only a relative measure of mass THC that cannotbe used for comparison between studies that usually employdifferent sizes/weights of cannabis cigarettes. The latterprovides an absolute measure of total amount of THCcontained in a cigarette and is much to be preferred overrelative units, because it does allow comparison of THCdose across studies. However, even comparison betweenabsolute units of THC doses can be misleading becauseof variations in smoking procedures that are beingemployed across studies. It has been amply shown thatvariations in puff volume, number of puffs, and breathholdduration produce dose-related changes in plasma levels ofTHC (Azorlosa et al, 1995). Therefore, the best approach forincreasing comparability between studies is to report THCdose as well as plasma THC concentrations. Unfortunately,assessment of plasma THC concentration is not standardpractice in experimental performance research. However, ifit were, it would have helped to evaluate the claims byDrs Nordstrom and Hart that THC doses in the studiesby Hart et al (2001) and Ramaekers et al (2006) werecomparable and that experienced cannabis users aretolerant to the impairing effects of THC on cognition.We will illustrate this point.In their original publication, Hart et al (2001) did notindicate the total amount of THC that was delivered to theirsubjects by smoking. They merely indicated that theirhigh concentration cigarettes contained 3.9% THC of whicheach subject smoked three standardized puffs. Each puffconsisted of 5s of inhalation, 10s of breathhold, and 40s ofexhalation and rest. They failed to report the total massof the cannabis cigarette. Nordstrom and Hart (2006)have now corrected this omission by stating that eachcigarette weighed 1g, on average of which subjects smokedthree-quarters, within a 3min period. This, they argue, isapproximately 30mg of THC which is close to the largestdose (35mg) in the study by Ramaekers et al (2006).It is difficult to see, however, how subjects in the study byHart et al (2001) could have smoked three-quarters of acigarette in only three standardized puffs, when subjectsin the study by Ramaekers et al (2006) needed about 25standardized puffs, that is, 4s of inhalation, 10s ofbreathhold, and 15s of exhalation and rest, to finishsmoking a cannabis cigarette completely. These compara-tive data on THC dosing regimens certainly do not supportthe claim by Drs Nordstrom and Hart that the totalamount of THC delivered to subjects were comparable inboth studies. Of course, the final confirmation of theirclaim can only come from comparative plasma THCdata. These however are not available, as Hart et al (2001)did not assess plasma THC. In addition, their claim is atodds with a previous conclusion by Hart et al (2002)that the behavioral effects of smoked cannabis containing3.1% THC is similar to the effects of 20mg oral THC.Again, no plasma THC data were provided but it is wellestablished that bioavailability of oral THC is only 10% andgenerally three times lower as compared to smoked THC(McGilveray, 2005; Ohlsson et al, 1980). This seems toindicate that THC concentrations during the smokedcondition in this and perhaps other experiments may havebeen very low as well. Of course, this is very speculative butit goes to show how data on plasma THC concentrationswould have come in very handy.