Does a postmortem redistribution affect the concentrations of the 7 azaindole-derived synthetic cannabinoid 5F-MDMB-P7AICA in tissues and body fluids following pulmonary administration to pigs?

Many fatal intoxications have been reported in connection with the consumption of newer, highly potent synthetic cannabinoids. Yet, a possible postmortem redistribution (PMR) might complicate reliable interpretation of analytical results. Thus, it is necessary to investigate the PMR-potential of new synthetic cannabinoids. The pig model has already proven to be suitable for this purpose. Hence, the aim of this study was to study the PMR of the synthetic cannabinoid 5F-MDMB-P7AICA and its main metabolite 5F-MDMB-P7AICA-dimethylbutanoic acid (DBA). 5F-MDMB-P7AICA (200 µg/kg body weight) was administered by inhalation to anesthetized and ventilated pigs. At the end of the experiment, the animals were euthanized and stored at room temperature for 3 days. Tissue and body fluid samples were taken daily. Specimens were analyzed after solid phase extraction using a standard addition method and LC-MS/MS, blood was quantified after protein precipitation using a validated method. In perimortem samples, 5F-MDMB-P7AICA was found mainly in adipose tissue, bile fluid, and duodenum contents. Small amounts of 5F-MDMB-P7AICA were found in blood, muscle, brain, liver, and lung. High concentrations of DBA were found primarily in bile fluid, duodenum contents, urine, and kidney/perirenal fat tissue. In the remaining tissues, rather low amounts could be found. In comparison to older synthetic cannabinoids, PMR of 5F-MDMB-P7AICA was less pronounced. Concentrations in blood also appear to remain relatively stable at a low level postmortem. Muscle, kidney, fat, and duodenum content are suitable alternative matrices for the detection of 5F-MDMB-P7AICA and DBA, if blood specimens are not available. In conclusion, concentrations of 5F-MDMB-P7AICA and its main metabolite DBA are not relevantly affected by PMR.

"Scalping" in the context of criminal dismemberment and mutilation-a case report and review of the literature.

We report on a case of criminal dismemberment and attempted scalping of a homicide victim with a "Mohawk" haircut. Case findings are presented. A review of the literature was performed for scalping in its historical and cultural context and particularly in criminal dismemberment and mutilation: Historically, scalping was prevalent in many ancient cultures around the world, where scalps were taken as trophies or "proof of kill", much like shrunken heads, trophy skulls, and other artefacts. Scalping was particularly widespread in Northern America in the context of tribal warfare, both before and after colonization. The iconic "Mohawk" haircut is closely linked with scalping, as it was meant to taunt the enemy. In the modern forensic context, scalping constitutes a form of criminal mutilation. However, cases of criminal dismemberment and mutilation are rare in forensic casework. Our literature review revealed a low number of scalping in criminal dismemberment and mutilation cases. The documentation was overall poor. Positioning scalping within the classification of criminal mutilation and dismemberment was difficult. In literature, even though case numbers were small, the majority of "textbook scalping" cases were German. The presented case, to our best knowledge, is the first modern-day photo-documented case of (attempted) scalping, even more so of a person wearing a "Mohawk".

Toxicokinetics of U-47700, tramadol, and their main metabolites in pigs following intravenous administration: is a multiple species allometric scaling approach useful for the extrapolation of toxicokinetic parameters to humans?

New synthetic opioids (NSOs) pose a public health concern since their emergence on the illicit drug market and are gaining increasing importance in forensic toxicology. Like many other new psychoactive substances, NSOs are consumed without any preclinical safety data or any knowledge on toxicokinetic (TK) data. Due to ethical reasons, controlled human TK studies cannot be performed for the assessment of these relevant data. As an alternative animal experimental approach, six pigs per drug received a single intravenous dose of 100 µg/kg body weight (BW) of U-47700 or 1000 µg/kg BW of tramadol to evaluate whether this species is suitable to assess the TK of NSOs. The drugs were determined in serum and whole blood using a fully validated method based on solid-phase extraction and LC-MS/MS. The concentration-time profiles and a population (pop) TK analysis revealed that a three-compartment model best described the TK data of both opioids. Central volumes of distribution were 0.94 L/kg for U-47700 and 1.25 L/kg for tramadol and central (metabolic) clearances were estimated at 1.57 L/h/kg and 1.85 L/h/kg for U-47700 and tramadol, respectively. The final popTK model parameters for pigs were upscaled via allometric scaling techniques. In comparison to published human data, concentration-time profiles for tramadol could successfully be predicted with single species allometric scaling. Furthermore, possible profiles for U-47700 in humans were simulated. The findings of this study indicate that unlike a multiple species scaling approach, pigs in conjunction with TK modeling are a suitable tool for the assessment of TK data of NSOs and the prediction of human TK data.

Are the postmortem concentration changes of the synthetic cannabinoid cumyl-5F-P7AICA and its N-pentanoic acid metabolite dependent on the environmental conditions? - A systematic study following pulmonary administration to pigs.

The number of fatal cases involving synthetic cannabinoids (SCs) is increasing. However, interpretation of postmortem (PM) toxicological findings is challenging, due to unknown PM intervals and possible redistribution processes or instabilities. Only sparse data on SCs are available. Therefore, a controlled pig study was performed to determine the PM stability of cumyl-5F-P7AICA under different environmental conditions. Ten pigs inhalatively received 200 µg/kg body weight cumyl-5F-P7AICA each. Six hours later, they were euthanized and biopsies of the main tissues and body fluids were taken. Animals were stored in air or water (n=5 each) and samples were repeatedly taken for three days. Quantification of cumyl-5F-P7AICA and its N-pentanoic acid metabolite (NPA) was performed using standard addition or a fully validated method (blood) followed by LC-MS/MS. Time-dependent concentration changes of cumyl-5F-P7AICA were observed in liver, bile fluid and muscle specimens at both conditions. Concentrations of NPA only changed considerably in duodenum content of animals stored in air. Environment-dependent concentrations changes were only observed for cumyl-5F-P7AICA in kidney and the NPA metabolite in duodenum content. Overall, cumyl-5F-P7AICA and its metabolite seem to be quite stable in PM specimens. Hence, also central blood might be analyzed, if no peripheral blood is available.

Development and method validation of a sampling technique for a reproducible detection of synthetic cannabinoids in exhaled breath using an in vitro pig lung model.

Alternative matrices, especially exhaled breath (EB), have gained increasing attention for a few years. To interpret toxicological findings, knowledge on the toxicokinetic (TK) properties of a substance in EB is indispensable. Whilst such data are already accessible for various drugs (e.g. Δ9-tetrahydrocannabinol), they are still not available for new psychoactive substances, particularly synthetic cannabinoids (SCs). As SCs raise a high public health concern, the aim of this study was to assess these data in future TK studies in pigs. For this purpose, an in vitro sampling technique of EB was initially developed, being prospectively applied to anesthetized and ventilated pigs for the detection of SCs in a controlled and reproducible manner as exemplified by cumyl-5F-P7AICA. Furthermore, a method for the qualitative and quantitative detection of cumyl-5F-P7AICA in EB using glass fiber filters (GFF) was established und fully validated. Therefore, cumyl-5F-P7AICA (0.5 mg/mL in ethanol abs.) was initially nebulized using a ventilation machine and a breathing tube, as they are also used in surgeries. The aerosol was delivered into a simulated pig lung. To collect EB, a pump was connected to that part of the breathing tube, that contains EB (expiratory limb), and sampling was performed repeatedly (n=6) for 15 min (2 L EB/min) each using GFF. For extraction of the substance, the GFF were macerated with acetone and the remaining experimental components were rinsed with ethanol. After sample preparation, the extracts were analyzed by LC-MS/MS. In the complete experimental setup, about 40% of the initially nebulized cumyl-5F-P7AICA dose was found with 3.6 ± 1.3% being detected in the GFF. Regarding the comparably high loss of substance, the open ventilation system and a conceivable adsorption of the SC in the ventilator have to be considered. However, the herein introduced approach is promising to determine the TK properties of cumyl-5F-P7AICA in EB.

'Flying high?'-Jump from a height in a 'Spice' high?: A case report on the synthetic cannabinoid 5F-MDMB-P7AICA.

Regarding the high potency of synthetic cannabinoids (SC), many intoxications and fatal cases are reported in literature. Here, we report on a fatality with 5F-MDMB-P7AICA contributing to the occurrence of death. A 31-year-old man died 10 h after he fell from the rooftop of a house. Police investigations revealed that he had consumed a 'legal high' herbal blend some hours earlier. An initial toxicological screening for new psychoactive substances (NPS) was negative. One year after, the analysis of confiscated drug samples revealed the SC 5F-MDMB-P7AICA being unknown at the time of the first investigations. Hence, post-mortem specimens were retrospectively analysed for 5F-MDMB-P7AICA and its dimethylbutanoic acid (DBA) metabolite. Lung, liver, kidney and bile fluid (BF) of the decedent were analysed following solid-phase extraction and standard addition, heart blood (HB) and peripheral blood (PB) by fully validated liquid-liquid extraction and protein precipitation methods. Additionally, hair specimens were analysed to examine a possible chronic consumption of the SC. All specimens were analysed by liquid-chromatography tandem mass spectrometry. 5F-MDMB-P7AICA was detected in HB (0.69 ng/ml), PB (1.2 ng/ml) and hair. DBA was found in HB (46 ng/ml) and PB (5.7 ng/ml) and could additionally be identified in liver and kidney (approximately 4-5 ng/g), lung (approximately 12 ng/g) and BF (approximately 60 ng/g). Compared with the parent compound, much higher concentrations of DBA were quantified. This case shows that drugs found at the scene can provide helpful initial information for further toxicological screenings in biological samples, especially when there is evidence of NPS consumption.

Are the (New) Synthetic Opioids U-47700, Tramadol and Their Main Metabolites Prone to Time-Dependent Postmortem Redistribution?-A Systematic Study Using an In Vivo Pig Model.

The interpretation of analytical results in forensic postmortem (PM) cases often poses a great challenge, in particular, due to possible PM redistribution (PMR) phenomena. In terms of new synthetic opioids, such data are usually not available and, if so, they are from case reports without the exact knowledge of dose, user habits, time of consumption or PM interval (PMI). Hence, a controlled toxicokinetic pig study was performed allowing the examination of PM tissue distribution and possible PMR of U-47700, tramadol and the main metabolites N-desmethyl-U-47700 and O-desmethyltramadol (ODT). For this purpose, 12 domestic pigs received an intravenous dose of 100 µg/kg body weight (BW) U-47700 or 1,000 µg/kg BW tramadol, respectively. The animals were put to death with T61 8 h after administration, and relevant organs, tissues and body fluids were sampled. Subsequently, the animals were stored at room temperature (RT), and the samples were taken again after 24, 48, and 72 h PM. Following homogenization and solid-phase extraction, quantification was performed applying a standard addition approach and liquid chromatography-tandem mass spectrometry. Only low-to-moderate concentration changes of U-47700, tramadol and their main metabolites were found in the analyzed tissue specimens and body fluids during storage at RT depending on the chosen PMI. On the contrary, a remarkable concentration increase of tramadol was observed in the liver tissue. These findings indicate that both synthetic opioids and their main metabolites are only slightly prone to PMR and central blood might be the matrix of choice for quantification of these substances.

Systematic Studies on Temperature-Dependent In Vitro Stability during Storage and Smoking of the Synthetic Cannabinoid 5F-MDMB-P7AICA.

Metabolism studies have shown that the synthetic cannabinoid (SC) 5F-MDMB-P7AICA is predominantly degraded by ester hydrolysis to 5F-MDMB-P7AICA dimethyl butanoic acid. To investigate the stability of 5F-MDMB-P7AICA during storage for a certain period of time or smoking, in vitro stability tests were performed. Blood and serum samples were collected repeatedly during a toxicokinetic study using a pig model and were retested after a 5- and 12-month storage at different temperatures (-20°C, 4°C or room temperature (RT)). Analysis was performed using fully validated liquid chromatography tandem mass spectrometry methods following liquid-liquid extraction and protein precipitation. One set of samples was analyzed immediately following the experiment (without storage (WS)). In the WS samples, 5F-MDMB-P7AICA and 5F-MDMB-P7AICA dimethyl butanoic acid were present in every sample collected throughout the whole experiment. Analysis of the blood and serum samples stored for 5 and 12 months at -20°C and 4°C revealed relatively stable concentrations of the parent substance and the dimethyl butanoic acid metabolite. Regarding the samples stored at RT, the concentrations of 5F-MDMB-P7AICA decreased, while the concentrations of the hydrolysis product increased. This change could particularly be observed in samples with a high initial concentration of the analytes. A further screening of the samples stored at RT revealed no other degradation products. In conclusion, the SC 5F-MDMB-P7AICA could be detected even after 12 months of storage at RT and therefore seems to be more stable than its isomer, 5F-ADB. Regarding the smoke condensate, besides the parent compound, only trace amounts of dimethyl butanoic acid were found.

Perimortem Distribution of U-47700, Tramadol and Their Main Metabolites in Pigs Following Intravenous Administration.

In spite of a decreasing number of new releases, new synthetic opioids (NSOs) are gaining increasing importance in postmortem (PM) forensic toxicology. For the interpretation of analytical results, toxicokinetic (TK) data, e.g., on tissue distribution, are helpful. Concerning NSOs, such data are usually not available due to the lack of controlled human studies. Hence, a controlled TK study using pigs was carried out, and the tissue distribution of U-47700 and tramadol as reference was examined. Twelve pigs received an intravenous dose of 100 µg/kg body weight (BW) U-47700 or 1,000 µg/kg BW tramadol. Eight hours after administration, the animals were put to death with T61. Relevant organs, body fluids and tissues were sampled. After homogenization and solid-phase extraction, quantification was performed applying standard addition and liquid chromatography--tandem mass spectrometry. At the time of death, the two parent compounds were determined in all analyzed specimens. Regarding U-47700, concentrations were highest in duodenum content, bile fluid and adipose tissue (AT). Concerning tramadol, next to bile fluid and duodenum content, highest concentrations were determined in the lung. Regarding the metabolites, N-desmethyl-U-47700 and O-desmethyltramadol (ODT) were detected in all analyzed specimens except for AT (ODT). Higher metabolite concentrations were found in specimens involved in metabolism. N-desmethyl-U-47700 showed much higher concentrations in routinely analyzed organs (lung, liver and kidney) than U-47700. To conclude, besides the routinely analyzed specimens in PM toxicology such as blood, urine or standard specimens like kidney or liver, AT, bile fluid and duodenum content could serve as alternative matrices. In case of U-47700, quantification of the main metabolite N-desmethyl-U-47700 is highly recommendable.

Can a Recently Developed Pig Model Be Used for In Vivo Metabolism Studies of 7-Azaindole-Derived Synthetic Cannabinoids? A Study Using 5F-MDMB-P7AICA.

New psychoactive substances (NPS), especially synthetic cannabinoids (SC) remain a public health concern. Due to ethical reasons, systematic controlled human studies to elucidate their toxicodynamics and/or toxicokinetics are usually not possible. However, such knowledge is necessary, for example, for determination of screening targets and interpretation of clinical and forensic toxicological data. In the present study, the feasibility of the pig model as an alternative for human in vivo metabolism studies of SC was investigated. For this purpose, the metabolic pattern of the SC methyl-2-{[1-(5-fluoropentyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl]amino}-3,3-dimethylbutanoate (5F-MDMB-P7AICA) was elucidated in pig urine following inhalative administration (dosage: 200 µg/kg of body weight). The results were compared with human and pig liver microsomal assays and literature. In addition, different incubations with isolated cytochrome-P450 (CYP) monooxygenases were conducted to identify the involved isozymes. In total, nine phase I and three phase II metabolites were identified in pig urine. The most abundant reactions were ester hydrolysis, ester hydrolysis combined with glucuronidation and ester hydrolysis combined with hydroxylation at the tert-butyl moiety. The parent compound was only found up to 1 h after administration in pig urine. The metabolite formed after hydroxylation and glucuronidation was detectable for 2 h, the one formed after ester hydrolyzation and defluorination for 4 h after administration. All other metabolites were detected during the whole sampling time. The most abundant metabolites were also detected using both microsomal incubations and monooxygenase screenings revealed that CYP3A4 catalyzed most reactions. Finally, pig data showed to be in line with published human data. To conclude, the main metabolites recommended in previous studies as urinary targets were confirmed by using pig urine. The used pig model seems therefore to be a suitable alternative for in vivo metabolism studies of 7-azaindole-derived SC.

Comparison of in vitro and in vivo models for the elucidation of metabolic patterns of 7-azaindole-derived synthetic cannabinoids exemplified using cumyl-5F-P7AICA.

Due to the dynamic market involving synthetic cannabinoids (SCs), the determination of analytical targets is challenging in clinical and forensic toxicology. SCs usually undergo extensive metabolism, and therefore their main metabolites must be identified for the detection in biological matrices, particularly in urine. Controlled human studies are usually not possible for ethical reasons; thus, alternative models must be used. The aim of this work was to predict the in vitro and in vivo metabolic patterns of 7-azaindole-derived SCs using 1-(5-fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-pyrollo[2,3-b]pyridin-3-carboxamide (cumyl-5F-P7AICA) as an example. Different in vitro (pooled human liver S9 fraction, pooled human liver microsomes, and pig liver microsomes) and in vivo (rat and pig) systems were compared. Monooxygenase isoenzymes responsible for the most abundant phase I steps, namely oxidative defluorination (OF) followed by carboxylation, monohydroxylation, and ketone formation, were identified. In both in vivo models, OF/carboxylation and N-dealkylation/monohydroxylation/sulfation could be detected. Regarding pHS9 and pig urine, monohydroxylation/sulfation or glucuronidation was also abundant. Furthermore, the parent compound could still be detected in all models. Initial monooxygenase activity screening revealed the involvement of CYP2C19, CYP3A4, and CYP3A5. Therefore, in addition to the parent compound, the OF/carboxylated and monohydroxylated (and sulfated or glucuronidated) metabolites can be recommended as urinary targets. In comparison to literature, the pig model predicts best the human metabolic pattern of cumyl-5F-P7AICA. Furthermore, the pig model should be suitable to mirror the time-dependent excretion pattern of parent compounds and metabolites.