Toxicokinetic Studies on (New) Synthetic Opioids

Toxicokinetic (TK) data are essential for an enhanced interpretation of clinical and forensic cases. In the presented work, the metabolic fate of three different model new synthetic opioids (NSO), namely U 47931E, U-51754, and methoxyacetylfentanyl, was investigated using different in vitro and in vivo approaches. The results revealed a good agreement between the various applied models. Furthermore, the TK of one of the most popular NSO, U-47700, was elucidated in a pig model following intravenous administration. Tramadol was assessed as reference for the examination of the comparability of porcine and human TK. Main metabolites of all U-substances were formed by N-demethylation, hydroxylation and combinations thereof. Methoxyacetylfentanyl was metabolized by dealkylation and hydroxylation. Phase II metabolites were formed to a limited extent. The major urinary metabolic pathways for U 47700 in pigs were comparable to those of humans. A three-compartment model best described the TK properties as modeled in a single species population TK approach. By usage of this TK model, published human data could successfully be predicted in terms of tramadol. Investigations of perimortem and postmortem (PM) distribution patterns of the drugs revealed that lung, liver, kidney, duodenum content, and bile are suitable matrices for qualitative PM analysis. Moreover, U-47700, tramadol and their metabolites show only low to moderate tendency for PM redistribution in most of the studied organs.Für die Erhebung toxikokinetischer (TK) Daten wurde in dieser Arbeit der Metabolismus ausgewählter neuer synthetischer Opioide (NSO) (U-47931E, U-51754 und Methoxyacetylfentanyl) in verschiedenen in vitro/in vivo Modellen untersucht. Hierbei zeigte sich eine gute Übereinstimmung der verschiedenen angewandten Modelle. Zusätzlich wurde eine Schweinestudie mit dem populären NSO U-47700 nach intravenöser Verabreichung durchgeführt. Tramadol diente dabei als Referenzsubstanz, um die Kompatibilität des Schweinemodells mit humanen Daten zu überprüfen. Alle untersuchten U Substanzen wurden hauptsächlich durch N-Demethylierung, Hydroxylierung und Kombinationen beider Wege verstoffwechselt und Methoxyacetylfentanyl durch Dealkylierung und Hydroxylierung. Phase II Metabolite wurden nur in geringem Ausmaß gebildet. Das Schweinemodell zeigte eine gute Übereinstimmung mit dem humanen Metabolismus. Eine populationskinetische Modellierung ergab, dass ein Drei-Kompartiment-Modell die TK Parameter beider Substanzen am besten beschrieben hat. Das Modell konnte in Zusammenhang mit einer allometrischen Einzelspezies-Skalierung erfolgreich für die Vorhersage humaner Tramadol-Expositionen genutzt werden. Die Ermittlung der Gewebeverteilung der Substanzen zeigte, dass Lunge, Leber, Niere, Duodenalinhalt und Gallenflüssigkeit geeignete Matrizes für qualitative postmortale Analysen sind. Die Substanzen zeigten zudem in den meisten Organen nur geringe Tendenzen für postmortale Umverteilungsprozesse

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

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, Ndesmethyl-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 β-arrestin 2 (βarr2), 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-bisdesmethylU-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

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 refected 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-indole3-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 sacrifced 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 signifcantly (p>0.05) over time under both storage conditions. Concentrations of HO-RCS-4 signifcantly (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, signifcant PM increases of metabolite concentrations might be considered in perirenal AT

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 N-demethylated metabolites of U-47700 more active than their parent compound? In vitro mu-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 mu-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 (R). Cells stably expressing human MOR and beta-arrestin 2 (beta arr2), 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 mu M for N,N-bisdesmethyl-U-47700). The maximal efficacy (E-max) 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

Betrunken oder doch nur verwest? Lassen sich ante mortem erfolgte Aufnahme und postmortale Neubildung von Alkohol anhand eines Begleitstoffmusters unterscheiden?

Background: Several pitfalls arise in the assessment of postmortem blood alcohol concentrations (BAC). The purpose of the present study was to evaluate in a controlled and systematic manner in a porcine model whether a postmortem congener pattern can provide evidence for antemortem or postmortem ethanol neogenesis. Methods: Ethanol was administered intravenously to six pigs, whereas six control pigs remained sober. The animals were euthanized 1h after the start of administration, and peripheral and heart blood (HB) as well as muscle tissue were collected. The animals were stored at room temperature and the aforementioned range of samples was collected daily for 3 days. Samples were analyzed for ethanol and congener substances by headspace gas chromatography-mass spectrometry. Results: Over the course of storage, ethanol formation was observed in the sober pigs, resulting in a median BAC of 0.24 g/kg body weight. The BAC in the pigs with alcohol remained comparatively stable. In addition, a distinct increase in n-propanol, n-butanol, and acetaldehyde was observed. The median blood concentration of n-propanol in sober animals was higher after storage than that of pigs with alcohol, but no significant differences could be substantiated between the two groups (p> 0.05). Acetaldehyde and n-butanol concentrations in HB of the sober pigs increased to the level of the pigs with alcohol at death after 3 days. Until the end of the experiment (3 days postmortem), no significant differences in concentrations were detected. Concentrations in muscle tissue did not increase to the same extent. Discussion: In the present study, no marker could be identified that could reliably discriminate antemortem ethanol ingestion from postmortem genesis.Hintergrund: Bei der Beurteilung postmortaler Blutalkoholkonzentrationen (BAK) ergeben sich mehrere Fallstricke. In der vorliegenden Studie sollte im Schweinemodell kontrolliert und systematisch evaluiert werden, ob ein postmortales Begleitstoffmuster Anhaltspunkte für eine ante mortem oder post mortem erfolgte Ethanolgenese liefern kann. Methodik: Sechs Schweinen wurde Ethanol intravenös verabreicht, während 6 Kontrollschweine nüchtern blieben. Eine Stunde nach Applikationsbeginn wurden die Tiere getötet und peripheres und zentrales Blut sowie Muskelgewebe entnommen. Die Kadaver wurden bei Raumtemperatur gelagert und das genannte Probenspektrum jeweils täglich über 3 Tage entnommen. Die Proben wurden mittels HeadspaceGaschromatographie-Massenspektrometrie auf Ethanol und Begleitstoffe untersucht. Ergebnisse: Über den Verlauf der Lagerung wurde eine Ethanolneubildung in den nüchternen Schweinen beobachtet, die zu einer medianen BAK von 0,24 g/kg Körpergewicht führte. Die BAK in den alkoholisierten Schweinen blieb vergleichsweise stabil. Zudem konnte ein deutlicher Anstieg an n-Propanol, n-Butanol und Acetaldehyd beobachtet werden. Die mediane Blutkonzentration von n-Propanol lag bei ursprünglich nüchternen Tieren nach Lagerung über der der alkoholisierten Schweine, es ergaben sich jedoch keine signifikanten Unterschiede (p> 0,05). Die Acetaldehyd- und n-Butanol-Konzentrationen im Herzblut der nüchternen Schweine stiegen innerhalb vom 3 Tagen auf das Niveau der bei Todeseintritt alkoholisierten Schweine an. Bis zum Versuchsende (3 Tage post mortem) konnten keine signifikanten Unterschiede in den Konzentrationen mehr festgestellt werden. Die Konzentrationen im Muskelgewebe stiegen nicht in demselben Maße an. Diskussion: In der vorliegenden Studie konnte kein Marker ermittelt werden, mit dem sich eine ante mortem erfolgte Ethanolaufnahme von einer postmortalen Genese zuverlässig diskriminieren ließe