Peter H. Schmidt

Genetic–epigenetic interaction modulates µ-opioid receptor regulation

Genetic and epigenetic mechanisms play important roles in protein expression, although at different levels. Genetic variations can alter CpG sites and thus influence the epigenetic regulation of mRNA expression, providing an increasingly recognized mechanism of functional consequences of genetic polymorphisms. One of those genetic effects is the association of reduced μ-opioid receptor expression with the functional genetic variant N40D (OPRM1 118A>G, rs1799971) that causes an amino acid exchange in the extracellular terminal of the μ-opioid receptor. We report that the nucleotide exchange at gene position +118 introduces a new CpG-methylation site into the OPRM1 DNA at position +117. This leads to an enhanced methylation of the OPRM1 DNA at this site and downstream. This epigenetic mechanism impedes μ-opioid receptor upregulation in brain tissue of Caucasian chronic opiate addicts, assessed postmortem. While in wild-type subjects, a reduced signalling efficiency associated with chronic heroin exposure was compensated by an increased receptor density, this upregulation was absent in carriers of the 118G receptor variant due to a diminished OPRM1 mRNA transcription. Thus, the OPRM1 118A>G SNP variant not only reduces µ-opioid receptor signalling efficiency, but, by a genetic-epigenetic interaction, reduces opioid receptor expression and therefore, depletes the opioid system of a compensatory reaction to chronic exposure. This demonstrates that a change in the genotype can cause a change in the epigenotype with major functional consequences.

A simple extraction and LC-MS/MS approach for the screening and identification of over 100 analytes in eight different matrices.

In the present study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) multi-analyte approach using one single work-up approach in whole blood, plasma, serum, post-mortem blood, liver tissue, gastric content, hair, and urine was developed for fast target screening and reliable identification of 130 analytes often requested in clinical and forensic toxicology. Samples (500 μL each) of whole blood, plasma, serum, post-mortem blood, tissue (homogenized 1 + 4 with water), as well as 3 g of distilled gastric contents, 1 mL of urine, or 20 mg of pulverized hair were extracted at different pH values with an diethyl ether-ethyl acetate mixture (1:1). Separation and identification were performed using LC-QTRAP with electrospray ionization in positive mode. For identification 1 scheduled multi-reaction-mode (sMRM) method with 390 transitions was developed covering benzodiazepines, Z-drugs, antidepressants, neuroleptics, opioids, new synthetic drugs, and phosphodiesterase type 5 inhibitors. For positive sMRM transitions with intensities exceeding 5000 cps, dependent scans (EPI scan collision energy, 35 eV, collision energy spread, 15 eV) were performed for library search using our in-house library. The method was developed with respect to selectivity, matrix effects, recovery, process efficiency, limit of detection, and applicability. The simple work-up procedure was suitable for all biosamples with exception of urine in respect to low concentrated analytes, which showed median recovery values of 59%. The method was selective for 130 analytes in all 8 biosamples. For 106 analytes, the limit of detection in whole blood, plasma, and serum was lower than the lowest therapeutic concentration listed in blood level lists.

Pharmacokinetics of (synthetic) cannabinoids in pigs and their relevance for clinical and forensic toxicology

Synthetic cannabinoids (SCs) are gaining increasing importance in clinical and forensic toxicology. They are consumed without any preclinical safety studies. Thus, controlled human pharmacokinetic (PK) studies are not allowed, although being relevant for interpretation of analytical results in cases of misuse or poisoning. As alternative, in a controlled animal experiment, six pigs per drug received a single intravenous dose of 200μg/kg BW each of Δ(9)-tetrahydrocannabinol (THC), 4-ethylnaphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-210), or 2-(4-methoxyphenyl)-1-(1-pentyl-indol-3-yl)methanone (RCS-4). In addition, six pigs received a combination of the three drugs with the identical dose each. The drugs were determined in serum using LC-MS/MS. A population (pop) PK analysis revealed that a three-compartment model described best the PK data of all three cannabinoids. Central volumes of distribution were estimated at 0.29L/kg, 0.20L/kg, and 0.67L/kg for THC, JWH-210, and RCS-4, respectively. Clearances were 0.042L/min/kg, 0.048L/min/kg, and 0.093L/min/kg for THC, JWH-210, and RCS-4, respectively. The popPK THC pig model was upscaled to humans using allometric techniques. Comparison with published human data revealed that the concentration-time profiles could successfully be predicted. These findings indicate that pigs in conjunction with PK modeling technique may serve as a tool for prediction of human PK of SCs.

Analysis of laryngeal fractures in decomposed bodies using microfocus computed tomography (mfCT)

Postmortem computed tomography (CT) is now routinely performed in forensic autopsies. Microfocus computed tomography (mfCT) has attracted recent attention because it can provide more detailed information than routine postmortem CT can. This feasibility study evaluated the usefulness of mfCT for examination of the hyoid bone and thyroid cartilage in cases of suspected strangulation, where advanced decomposition precludes detection of petechial hemorrhages and hemorrhages adjacent to fractures. The results show that mfCT was useful for identification of thin fracture lines in the fragile laryngeal structures. We suggest that mfCT should be considered for forensic autopsies in cases of suspected strangulation with advanced decomposition.

Can JWH‐210 and JWH‐122 be detected in adipose tissue four weeks after single oral drug administration to rats?

Synthetic cannabinoids such as alkylindole derivatives entered the illicit drug market worldwide a few years ago. Only a few data are available concerning their pharmacokinetics, in particular their distribution and persistence in adipose tissue. For the present study, rats were administered a single 20 mg/kg oral dose of JWH-210 or JWH-122. After one month, they were dissected and adipose tissue was taken in order to study whether JWH-210 and JWH-122 persisted in this body sample. After extraction, the samples were analyzed by liquid chromatographic-mass spectrometry (LC-QTrap-MS). Validation of the analytical method for adipose tissue is also presented. The results of the matrix effects determination ranged between 30.6 and 43.8%. The limits of detection for JWH-210 and JWH-122 were 0.8 and 1.0 ng/g and lower limits of quantification were 3.7 and 2.1 ng/g, respectively. Calibration curves ranged from 10 to 75 ng/g for JWH-210 and from 5 to 50 ng/g for JWH-122. Intra- and interday precision values were lower than 15% and bias values within ±15%. Applying this method, in adipose tissue specimens obtained 4 weeks after single drug administration, JWH-210 and JWH-122 were detected in concentrations of 116 and 9 ng/g, respectively.

Metabolic patterns of JWH-210, RCS-4, and THC in pig urine elucidated using LC-HR-MS/MS: Do they reflect patterns in humans?

The knowledge of pharmacokinetic (PK) properties of synthetic cannabinoids (SCs) is important for interpretation of analytical results found for example in intoxicated individuals. In the absence of human data from controlled studies, animal models elucidating SC PK have to be established. Pigs providing large biofluid sample volumes were tested for prediction of human PK data. In this context, the metabolic fate of two model SCs, namely 4-ethylnaphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-210) and 2-(4-methoxyphenyl)-1-(1-pentyl-indol-3-yl)methanone (RCS-4), was elucidated in addition to Δ9 -tetrahydrocannabinol (THC). After intravenous administration of the compounds, hourly collected pig urine was analyzed by liquid chromatography-high resolution mass spectrometry. The following pathways were observed: for JWH-210, hydroxylation at the ethyl side chain or pentyl chain and combinations of them followed by glucuronidation; for RCS-4, hydroxylation at the methoxyphenyl moiety or pentyl chain followed by glucuronidation as well as O-demethylation followed by glucuronidation or sulfation; for THC, THC glucuronidation, 11-hydroxylation, followed by carboxylation and glucuronidation. For both SCs, parent compounds could not be detected in urine in contrast to THC. These results were consistent with those obtained from human hepatocyte and/or human case studies. Urinary markers for the consumption of JWH-210 were the glucuronide of the N-hydroxypentyl metabolite (detectable for 3-4 h) and of RCS-4 the glucuronides of the N-hydroxypentyl, hydroxy-methoxyphenyl (detectable for at least 6 h), and the O-demethyl-hydroxy metabolites (detectable for 4 h). Copyright

Helicopter induced propeller injuries

Following routine maintenance a military helicopter was returned to service. Due to negligence some hoses had not been connected correctly. The wrongly connected hoses resulted in a malfunction, causing the rotor blades of the helicopter to smash through the windows of the helicopter cockpit. Both the pilot and the co-pilot were hit by the rotor blades and decapitated. Other passengers in the helicopter were injured. Due to vibrations the helicopter fell to the ground and its tail partly broke away (Figs. 1, 2). The bodies and body parts of the pilot and co-pilot were found on the ground outside the helicopter cockpit.

Development of an In-vitro Drug Delivery Efficiency Test for a Pulmonary Toxicokinetic Pig Study

BACKGROUND Since their appearance on the drugs of abuse market, synthetic cannabinoids (SCs) are gaining increasing toxicological relevance. They are consumed without knowledge of their toxicokinetic (TK) and toxicodynamic properties and human studies are not allowed due to ethical reasons. A controlled animal TK study following nebulization of 4-ethylnaphthalene-1-yl-(1-pentylindole- 3-yl)methanone (JWH-210), 2-(4-methoxyphenyl)-1-(1-pentyl-indole-3-yl)methanone (RCS-4) as well as Δ9-tetrahydrocannabinol (THC) to pigs should be helpful for better interpretation of analytical results in cases of misuse or poisoning. As a prerequisite, an in-vitro test system mimicking a ventilated pig had to be developed to determine the quantity and reproducibility of which drug dose is delivered to the pig lung. METHODS JWH-210, RCS-4, and THC (1 mg in 2 mL ethanol each) were nebulized during ventilation using an ultrasonic nebulizer. The drug aerosol was delivered via the inspiratory limb and the endotracheal tube passing through a glass fiber filter (n = 6). The drugs were extracted from the filters using ethanol and ultrasonication. After several dilution steps and adding an internal standard solution, the extracts were analyzed by LC-MS/MS. RESULTS Extraction of the nebulized drugs revealed delivery efficiencies of 78.8 ± 5.0% for JWH-210, 70.5 ± 6.9% for RCS-4, and 70.8 ± 7.9% for THC. The loss of about 20-30% of the administered dose might be attributable to retention in the nebulizer device or adhesion of the aerosol particles to the tube wall. CONCLUSION Nevertheless, regarding delivery efficiencies, the minor standard deviations indicate an acceptable reproducibility, suggesting that this administration system is suitable for application in TK studies.

Can toxicokinetics of (synthetic) cannabinoids in pigs after pulmonary administration be upscaled to humans by allometric techniques

Graphical abstract Figure. No caption available. &NA; Being advertised and distributed as attractive substitutes of cannabis, synthetic cannabinoids (SC) are gaining increasing relevance in forensic and clinical toxicology. As no data from controlled human studies are available, SC are sold and consumed without the knowledge of their toxicokinetic (TK) and toxicodynamic properties. Hence, animal models coupled with mathematical approaches should be used to ascertain those properties. Therefore, a controlled pig TK study allowing for extrapolation to human data was performed. For this purpose, eleven pigs received a single pulmonary dose of 200 &mgr;g/kg body weight each of &Dgr;9‐tetrahydrocannabinol (THC), 4‐ethylnaphthalene‐1‐yl‐(1‐pentylindole‐3‐yl)methanone (JWH‐210) as well as 2‐(4‐methoxyphenyl)‐1‐(1‐pentyl‐indole‐3‐yl)methanone (RCS‐4) via an ultrasonic nebulizer. Blood and urine samples were repeatedly drawn over 8 h. Serum‐concentration‐time profiles of the parent compounds were determined using LC‐MS/MS. Urine specimens were analyzed by LC‐HR‐MS/MS in order to elucidate the main metabolites. Maximum serum concentrations were reached 10–15 min after beginning of nebulization and amounted to 66 ± 36 ng/mL for THC, 41 ± 11 ng/mL for JWH‐210, and 34 ± 8.9 ng/mL for RCS‐4. The serum‐concentration‐time profiles of THC, JWH‐210, and RCS‐4 were best described by three‐compartment models with first order absorption and elimination processes. Absorption from the lungs to serum was modeled by first‐order processes. The determination of the bioavailability yielded 23.0%, 24.2%, and 45.7% for THC, JWH‐210, and RCS‐4, respectively. Furthermore, the developed THC model was upscaled to humans using allometric scaling techniques. A successful prediction of human concentration‐time profiles could be done. Also the metabolic patterns were in good agreement with human data. In conclusion, these findings are the first reported regarding the TK properties of SC after pulmonary administration to pigs. The presented method of TK serves as an appropriate predictor of human TK of cannabinoids.