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.

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.

Are the N-demethylated metabolites of U-47700 more active than their parent compound? In vitro µ-opioid receptor activation of N-desmethyl-U-47700 and N,N-bisdesmethyl-U-47700.

Studies on the tissue distribution of the new synthetic opioid U-47700 and its main metabolite N-desmethyl-U-47700 revealed about sixfold higher metabolite concentrations in pig brain as compared with the parent compound. To better assess the toxic potential of this drug, the aim of this study was to assess the in vitro µ-opioid receptor (MOR) activation potential of the main metabolites of U-47700, N-desmethyl-U-47700, and N,N-bisdesmethyl-U-47700, using a live cell-based reporter assay based on NanoLuc Binary Technology®. Cells stably expressing human MOR and ß-arrestin 2 (ßarr2), each fused via a flexible linker to two complementary inactive subunits of the nanoluciferase, were seeded on poly-d-lysine-coated 96-well plates and treated with N-desmethyl-U-47700, N,N-bisdesmethyl-U-47700, U-47700, or hydromorphone as reference standard. MOR activation results in functional complementation of the nanoluciferase, which can be assessed via luminescence monitoring. The potency of the metabolites is lower than that of U-47700 (EC50 of 186 nM for U-47700, 3770 nM for N-desmethyl-U-47700, and >5 µM for N,N-bisdesmethyl-U-47700). The maximal efficacy (Emax ) observed (relative to hydromorphone, set arbitrarily at 100%) decreased from 183% to 127% and 39.2% for U-47700, N-desmethyl-U-47700, and N,N-bisdesmethyl-U-47700, respectively. Thus, the loss of one or two methyl groups reduced the MOR activation potential, which was more pronounced if both methyl groups were removed. It is thus anticipated that the impact on MOR exerted by the higher metabolite concentration in brain has only little-if any relevance for the strong toxic effects of U-47700.

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.

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.

Is adipose tissue suitable for detection of (synthetic) cannabinoids? A comparative study analyzing antemortem and postmortem specimens following pulmonary administration of JWH-210, RCS-4, as well as ∆9-tetrahydrocannabinol to pigs.

Examining fatal poisonings, chronic exposure may be reflected by the concentration in tissues known for long-term storage of drugs. Δ9-tetrahydrocannabinol (THC) persists in adipose tissue (AT), but sparse data on synthetic cannabinoids (SC) are available. Thus, a controlled pig study evaluating antemortem (AM) disposition and postmortem (PM) concentration changes of the SC 4-ethylnaphthalene-1-yl-(1-pentylindole-3-yl)methanone (JWH-210) and 2-(4-methoxyphenyl)-1-(1-pentyl-indole-3-yl)methanone (RCS-4) as well as THC in AT was performed. The drugs were administered pulmonarily (200 µg/kg body weight) to twelve pigs. Subcutaneous (s.c.) AT specimens were collected after 15 and 30 min and then hourly up to 8 h. At the end, pigs were sacrificed and s.c., perirenal, and dorsal AT specimens were collected. The carcasses were stored at room temperature (RT; n = 6) or 4 °C (n = 6) and specimens were collected after 24, 48, and 72 h. After homogenization in acetonitrile and standard addition, LC-MS/MS was performed. Maximum concentrations were reached 0.5-2 h after administration amounting to 21 ± 13 ng/g (JWH-210), 24 ± 13 ng/g (RCS-4), and 22 ± 20 ng/g (THC) and stayed at a plateau level. Regarding the metabolites, very low concentrations of N-hydroxypentyl-RCS-4 (HO-RCS-4) were detected from 0.5 to 8 h. PM concentrations of parent compounds did not change significantly (p > 0.05) over time under both storage conditions. Concentrations of HO-RCS-4 significantly (p < 0.05) increased in perirenal AT during storage at RT. These results suggest a rapid distribution and persistence in s.c. AT. Furthermore, AT might be resistant to PM redistribution of parent compounds. However, significant PM increases of metabolite concentrations might be considered in perirenal AT.

Are pigs a suitable animal model for in vivo metabolism studies of new psychoactive substances? A comparison study using different in vitro/in vivo tools and U-47700 as model drug.

Being highly potent, New Synthetic Opioids (NSO) have become a public health concern. Little is known though about the metabolism and toxicokinetics (TK) of many of the non fentanyl NSO such as U-47700. Obtaining such data in humans is challenging and so we investigated if pigs were a suitable model species as TK model for U-47700. The metabolic fate of U-47700 was elucidated after intravenous administration to one pig in vivo and results were compared to metabolic patterns formed by different other in vitro systems (human and pig liver microsomes, human liver S9 fraction) and compared to rat and human in vivo data. Furthermore, monooxygenase isozymes responsible for the major metabolic steps were elucidated. In total, 12 phase I and 8 phase II metabolites of U-47700 could be identified. The predominant reactions were N-demethylation, hydroxylation, and combination of them followed by glucuronidation or sulfation. The most predominant monooxygenase catalyzed conversions were N-demethylation, and hydroxylation by CYP3A4 and 2B6, and FMO3 catalyzed N-oxidation. Similar main phase I metabolites were found in vitro as compared to in vivo (pig/human). The metabolic pattern elucidated in the pig was comparable to human in vivo data. Thus, pigs seem to be a suitable animal model for metabolism and further TK of U-47700.

Studies on the in vitro and in vivo metabolism of the synthetic opioids U-51754, U-47931E, and methoxyacetylfentanyl using hyphenated high-resolution mass spectrometry.

New Synthetic Opioids (NSOs) are one class of New Psychoactive Substances (NPS) enjoying increasing popularity in Europe. Data on their toxicological or metabolic properties have not yet been published for most of them. In this context, the metabolic fate of three NSOs, namely, trans-3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methyl-benzenacetamide (U-51754), trans-4-bromo-N-[2-(dimethylamino)cyclohexyl]-N-methyl-benzamide (U-47931E), and 2-methoxy-N-phenyl-N-[1-(2-phenylethyl)piperidin-4-yl] acetamide (methoxyacetylfentanyl), was elucidated by liquid chromatography high-resolution mass spectrometry after pooled human S9 fraction (phS9) incubations and in rat urine after oral administration. The following major reactions were observed: demethylation of the amine moiety for U-51754 and U-47931E, N-hydroxylation of the hexyl ring, and combinations thereof. N-dealkylation, O-demethylation, and hydroxylation at the alkyl part for methoxyacetylfentanyl. Except for U-47931E, parent compounds could only be found in trace amounts in rat urine. Therefore, urinary markers should preferably be metabolites, namely, the N-demethyl-hydroxy and the hydroxy metabolite for U-51754, the N-demethylated metabolite for U-47931E, and the N-dealkylated metabolite as well as the O-demethylated one for methoxyacetylfentanyl. In general, metabolite formation was comparable in vitro and in vivo, but fewer metabolites, particularly those after multiple reaction steps and phase II conjugates, were found in phS9. These results were consistent with those of comparable compounds obtained from human liver microsomes, human hepatocytes, and/or human case studies.