Cora Wunder

Resistant hypertension? Assessment of adherence by toxicological urine analysis.

Objective: Uncontrolled hypertension under antihypertensive multidrug regimen is not necessarily always true resistance. Incomplete adherence is one of several possible causes of uncontrolled hypertension. Nonadherence remains largely unrecognized and is falsely interpreted as treatment resistance, as it is difficult to confirm or exclude objectively. This is the first study assessing adherence in patients with apparent resistant hypertension systematically via toxicological urine screening. Methods: All patients referred from primary care physicians because of uncontrolled hypertension between 2004 and 2011 were analysed. Adherence was assessed in all patients with uncontrolled hypertension despite the concurrent use of at least four antihypertensive agents by using liquid chromatography-mass spectrometry analysis for antihypertensive drugs or their corresponding metabolites in urine. Results: A total of 375 patients with uncontrolled hypertension were referred. After optimization of drug therapy and exclusion of white coat hypertension, 108 patients met criteria for resistant hypertension. Of those, 15 patients had secondary causes of hypertension and 17 achieved goal blood pressure with quadruple antihypertensive therapy. Of the remaining 76 patients, 40 patients (53%) were found to be nonadherent. Among nonadherent patients, 30% had complete and 70% had incomplete adherence; 85% of the latter had taken less than 50% of drugs prescribed. Lack of adherence was almost evenly distributed between different classes of antihypertensive drugs. Conclusion: Low adherence was the most common cause of poor blood pressure control in patients with apparent resistant hypertension, being twice as frequent as secondary causes of hypertension. Incomplete adherence was far more common than complete nonadherence; thus, assessment of adherence in patients on multiple drug regime is only reliable when all drugs are included in assessment. Assessing adherence by toxicological urine screening is a useful tool in detecting low adherence, especially in the setting of multidrug regimen as a cause of apparently resistant hypertension.

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.

Venomic study on cone snails (Conus spp.) from South Africa

From six Conus species (Conus coronatus, Conus lividus, Conus mozambicus f. lautus, Conus pictus, Conus sazanka, Conus tinianus) collected off the eastern coast of South Africa the venoms were analyzed using MALDI-TOF mass spectrometry. Between 56 and 151 molecular masses most in a range of 1000 to 2500 Da, were identified. Among the six venoms, between 0 and 27% (C. coronatus versus C. sazanka) of the peptide masses were found to be similar. In a study on venoms from 6 Conus species collected in the Philippines, the percentage of identical masses was between none and 9% only. The venoms from the South African Conus species antagonized the rat neuronal nicotinic acetylcholine receptors (nAChRs) α3β2, α4β2, and α7, except for C. coronatus venom that blocked the α4β2 and α7 nAChRs only. HPLC-fractionation of C. tinianus venom led to the isolation of a peptide that is active on all three receptor subtypes. It consists of 16 amino acid residues cross-linked by two disulfide bridges as revealed by de novo sequencing using tandem mass spectrometry: GGCCSHPACQNNPDYC. Posttranslational modifications include C-terminal amidation and tyrosine sulfation. The new peptide is a member of the α-conotoxin family that are competitive antagonists of nAChRs. Phylogenetic analysis of the 16S RNA from numerous Conus species has clarified the evolutionary position of endemic South African Conus species and provided the first evidence for their close genetic relationships.

Variable adverse effects in subjects after ingestion of equal doses of Argyreia nervosa seeds.

As the new drug Spice hit the market in 2006 and was a hot topic in the media, the general issue of legal highs has been brought to the attention of a large number of (young) people. One of these so called legal highs are the seeds of Argyreia nervosa, also known as Hawaiian Baby Woodrose, which contains the psychotropic alkaloid lysergic acid amide (LSA). A study was designed to assess how driving ability is affected by Argyreia nervosa. However, the study could not be continued due to severe adverse effects in 3 of 4 subjects, such as cardiovascular dysregulation in two and a psychosis like state in one subject. All of the participants recovered completely within 9h after ingestion. Despite body normalized doses interindividually highly differing reactions in type and intensity were observed. Furthermore, fluctuating alkaloid contents in seeds and multi-drug intoxications make the use of this legal high far more dangerous than commonly believed.

Altered gene expression in the prefrontal cortex of young rats induced by the ADHD drug atomoxetine

Atomoxetine (ATX), a selective norepinephrine reuptake inhibitor, is a non-stimulant approved for the treatment of attention deficit/hyperactivity disorder (ADHD). Little is known about the molecular basis for its therapeutic effect. The objective of this animal study was to determine alterations in gene expression patterns in the prefrontal cortex after long-term administration of atomoxetine. Rats were treated for 21 days during childhood and early adolescent stages of development with a once-daily oral application of 0.05 g/kg atomoxetine, which resulted in plasma levels similar to those described in children. A whole genome RNA-microarray of rat prefrontal cortical gene expression after administration of atomoxetine versus sterile water revealed an mRNA increase in 114 genes (≥2-fold) while 11 genes were down-regulated (≤0.5-fold). By applying quantitative real-time PCR (qRT-PCR) and Western Blot we confirmed a significant increase in the expression of GABA A receptor subunits as well as ubiquinol-cytochrome c reductase complex core protein 2 (Uqcrc2). SNAP-25 (synaptosomal-associated protein of 25 kDa), which is an ADHD candidate gene and an important vesicle protein involved in axonal growth, synaptic plasticity and regulation of neurotransmitter release was also significantly upregulated on RNA- and protein level after atomoxetine treatment. In summary, we could show that long-term treatment with the ADHD drug atomoxetine induces the regulation of several genes in the prefrontal cortex of young rats. Especially the increased expression of SNAP-25 and GABA-A receptor subunits may indicate additional active therapeutic mechanisms for atomoxetine.

Retrospective analysis of synthetic cannabinoids in serum samples – epidemiology and consumption patterns

Herbal mixtures contain synthetic cannabinoids, which can cause severe intoxications. Due to the great variety and the changing spectrum of substances on the drug market, prevalence data are limited, and data on prevalence rates of synthetic cannabinoids in forensic cases are not available. The present study was performed to survey the prevalence of synthetic cannabinoids in cases of traffic and criminal offences in the German state Hesse in 2010. The applied analytical method covered all synthetic cannabinoids on the drug market at that time, and with 20% of the blood samples (422 out of 2201) a representative number was reanalyzed. In twelve samples synthetic cannabinoids were identified and a prevalence of 2.8% was estimated. Consumption patterns showed predominantly cases of multi-drug consumption (10 cases); the combination with cannabis or alcohol was frequent (four cases each). The observed deficits were moderate with the exception of aggravation of paranoia in one case. The symptoms were either compatible with the effects of cannabinoid agonists or attributable to alcohol or other drugs found in the blood samples. Our current analytical strategy is to perform such analyses only in cases where use is suspected or where symptoms are not explained by routine toxicological analyses. Hence, the positive rate is rather low highlighting the need to keep up with the developments on the drug market and to establish sensitive screening methods covering a broad range of substances that can be updated fast, e.g., relying on collections of mass spectrometric reference data.

Pharmacokinetic properties of the synthetic cannabinoid JWH-018 and of its metabolites in serum after inhalation

&NA; Each year, synthetic cannabinoids are occurring in high numbers in the illicit drug market, but data on their pharmacology and toxicology are scarcely available. Therefore, a pilot study was performed to assess adverse effects of JWH‐018, which is one of the oldest and best known synthetic cannabinoids. Six subjects inhaled smoke from 2 and 3 mg JWH‐018. The drug and nine of its metabolites were analyzed in their blood samples taken during the following 12 h by liquid chromatography–mass spectrometry (LC–MSMS). The maximum concentration of JWH‐018 reached 2.9–9.9 ng/ml after inhalation and markedly decreased during the next 1.5 h, followed by a multiexponential decline (t1/2 in median 1.3 h and 5.7 h). The concentration of the pentanoic acid metabolite was slightly higher than that of the 3‐, 4‐ and 5‐hydroxypentyl metabolites and of the 6‐hydroxyindol metabolite. The data also suggest a multiexponential decline and slow terminal elimination of JWH‐018 and all metabolites. The detection of JWH‐018 and of its metabolites in serum requires high analytical sensitivity. The pharmacokinetic properties of inhaled JWH‐018 are similar to that of THC. A slow terminal elimination of drug and metabolites may lead to accumulation in chronic users. HighlightsAfter inhalation of JWH‐018, serum concentrations are maximal within minutes.During the 12 h period a multicompartmental distribution/elimination is obvious.Pentanoic acid, 3‐, 4‐ and 5‐hydroxypentyl and 6‐hydroxyindol met. are detected.Results indicate a novel metabolite, an isomer of the pentanoic acid metabolite.A slow terminal elimination may lead to accumulation in chronic users.

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.

Studies on the alkaloid composition of the Hawaiian Baby Woodrose Argyreia nervosa, a common legal high

Seeds from the Hawaiian Baby Woodrose Argyreia nervosa of different origin and labelling and with allegedly high levels of ergot alkaloids were analysed using high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS/MS) technique. Lysergic acid amide (LSA), ergometrine, lysergol/elymoclavine/setoclavine, chanoclavine, lysergic acid and their respective stereoisomers were identified as well as penniclavine and lysergic acid α-hydroxyethylamide. In addition, methylergometrine, methysergide, and lysergylalanine were detected, some high molecular weight ergot alkaloid derivatives and hydroxyalanine derived ergopeptide fragments were detected indicating the presence of ergopeptides in the seeds. The results of the study demonstrate that the content of ergot alkaloids in Argyreia nervosa seeds depends on the quality of the material. For a consumer the quality of the seeds is unforeseeable. For the toxicological expert it is essential to investigate not only the identity of such a confiscated seed material, but also the various ergot alkaloid constituents to assess the hazardous nature and the toxic potential of the material.

Selective sequestration of cardenolide isomers by two species of Danaus butterflies (Lepidoptera: Nymphalidae: Danainae)

Several species of milkweed butterflies (Danaini) are known to sequester cardenolides from their milkweed host plants. In adults of Danaus plexippus and D. gilippus, jointly raised on Asclepias curassavica (Asclepiadaceae), two host-plant cardenolides (calotropin and calactin) were found in significantly different ratios: in D. plexippus and the plant, they occurred in roughly equal ratios, but in D. gilippus, calotropin had a 10–12 times lower concentration, suggesting a selective sequestration of calactin. The two Danaus species belong to different subgenera and the results may be relevant to a better understanding of the evolution of cardenolide sequestration in Danaini.