Verena Angerer

Phase I metabolism of the highly potent synthetic cannabinoid MDMB-CHMICA and detection in human urine samples

Among the recently emerged synthetic cannabinoids, MDMB-CHMICA (methyl N-{[1-(cyclohexylmethyl)-1H-indol-3-yl]carbonyl}-3-methylvalinate) shows an extraordinarily high prevalence in intoxication cases, necessitating analytical methods capable of detecting drug uptake. In this study, the in vivo phase I metabolism of MDMB-CHMICA was investigated using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and liquid chromatography-electrospray ionization-quadrupole time-of-flight-mass spectrometry (LC-ESI-Q ToF-MS) techniques. The main metabolites are formed by hydrolysis of the methyl ester and oxidation of the cyclohexyl methyl side chain. One monohydroxylated metabolite, the ester hydrolysis product and two further hydroxylated metabolites of the ester hydrolysis product are suggested as suitable targets for a selective and sensitive detection in urine. All detected in vivo metabolites could be verified in vitro using a human liver microsome assay. Two of the postulated main metabolites were successfully included in a comprehensive LC-ESI-MS/MS screening method for synthetic cannabinoid metabolites. The screening of 5717 authentic urine samples resulted in 818 cases of confirmed MDMB-CHMICA consumption (14%). Since the most common route of administration is smoking, smoke condensates were analyzed to identify relevant thermal degradation products. Pyrolytic cleavage of the methyl ester and amide bond led to degradation products which were also formed metabolically. This is particularly important in hair analysis, where detection of metabolites is commonly considered a proof of consumption. In addition, intrinsic activity of MDMB-CHMICA at the CB1 receptor was determined applying a cAMP accumulation assay and showed that the compound is a potent full agonist. Based on the collected data, an enhanced interpretation of analytical findings in urine and hair is facilitated. Copyright

Metabolites of synthetic cannabinoids in hair—proof of consumption or false friends for interpretation?

AbstractThe detection of drug metabolites in hair is widely accepted as a proof for systemic uptake of the drug, unless the metabolites can be formed as artefacts. However, regarding synthetic cannabinoids, not much is known about mechanisms of incorporation into hair. For a correct interpretation concerning hair findings of these compounds and their metabolites, it is necessary to identify the different routes of incorporation and to assess their contribution to analytical findings. This study presents the results of the LC-ESI-MS/MS analysis of an authentic hair sample taken from a patient with a known history of heavy consumption of synthetic cannabinoids. In the authentic hair sample, 5F-PB-22 and AB-CHMINACA as well as their main metabolites 5F-PB-22 3-carboxyindole, PB-22 5-OH-pentyl, and AB-CHMINACA valine were detected in all segments, comprising segments grown in a time period where the substances had not been distributed on the ‘legal high’ market. To enable interpretation of the results regarding the distribution of the detected analytes along the hair shaft, the stability of 5F-PB-22 and AB-CHMINACA in hair matrix and under thermal stress was assessed. The stability tests revealed that the three ‘metabolites’ are also formed in externally contaminated hair after storage of the samples under different conditions. In addition, 5F-PB-22 3-carboxyindole and AB-CHMINACA valine were identified as degradation products in smoke condensate. Therefore, interpretation of ‘metabolite’ findings of compounds comprising chemically labile amide/ester bonds or 5-fluoro-pentyl side chains should be carried out with utmost care, taking into account the different mechanisms of formation and incorporation into hair. Graphical AbstractDegradation processes leading to artefacts identical with main metabolites of synthetic cannabinoids

Immunoassay screening in urine for synthetic cannabinoids – an evaluation of the diagnostic efficiency

Abstract Background: The abuse of synthetic cannabinoids (SCs) as presumed legal alternative to cannabis poses a great risk to public health. For economic reasons many laboratories use immunoassays (IAs) to screen for these substances in urine. However, the structural diversity and high potency of these designer drugs places high demands on IAs regarding cross-reactivity of the antibodies used and detection limits. Methods: Two retrospective studies were carried out in order to evaluate the capability of two homogenous enzyme IAs for the detection of currently prevalent SCs in authentic urine samples. Urine samples were analyzed utilizing a ‘JWH-018’ kit and a ‘UR-144’ kit. The IA results were confirmed by an up-to-date liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) screening method covering metabolites of 45 SCs. Results: The first study (n=549) showed an 8% prevalence of SCs use (LC-MS/MS analysis) among inpatients of forensic-psychiatric clinics, whereas all samples were tested negative by the IAs. In a second study (n=200) the combined application of both IAs led to a sensitivity of 2% and a diagnostic accuracy of 51% when applying the recommended IA cut-offs. Overall, 10 different currently prevalent SCs were detected in this population. The results can be explained by an insufficient cross-reactivity of the antibodies towards current SCs in combination with relatively high detection limits of the IAs. Conclusions: In light of the presented study data it is strongly recommended not to rely on the evaluated IA tests for SCs in clinical or forensic settings. For IA kits of other providers similar results can be expected.

Three fatalities associated with the synthetic cannabinoids 5F-ADB, 5F-PB-22, and AB-CHMINACA

The use of synthetic cannabinoids (SC) has been widespread in certain groups of drug users for many years. In the scientific literature many intoxication cases and a number of fatalities after the use of synthetic cannabinoids were reported. In this paper three death cases are described with involvement of the synthetic cannabinoids 5F-PB-22, AB-CHMINACA, and 5F-ADB. The three cases occurred in the eastern region of Germany, which is known as a region of high methamphetamine abuse. All decedents were male, between 25 and 41 years old, and had a known history of drug use. Femoral blood concentrations of the synthetic cannabinoids were measured using a validated LC-MS/MS method. The concentration of 5F-PB-22 in the first case was 0.37ng/mL, the concentration of AB-CHMINACA in the second case was approximately 4.1ng/mL (extrapolated) and the 5F-ADB concentration in the third case was 0.38ng/mL. Compared to other published cases the concentrations in the here presented cases seem to be in the lower range. However, taking into account the scene of death, the results of the forensic autopsy and the full toxicological analysis, the deaths can be explained as a direct consequence of consumption of synthetic cannabinoids, although in case one and two relevant amounts of ethanol were found, and in case three trimipramine and olanzapine were present in non-toxic concentrations. It has to be noted that concentrations of synthetic cannabinoids in femoral blood cannot directly be judged as toxic or lethal due to the possibility of postmortem redistribution and the development of tolerance after frequent use. Therefore, all available information has to be considered carefully before stating SC use as the cause of death.

Acute side effects after consumption of the new synthetic cannabinoids AB-CHMINACA and MDMB-CHMICA

Abstract Introduction: In 2014, the “European Monitoring Centre for Drugs and Drug Addiction” (EMCDDA) reported on 30 novel synthetic cannabinoids (SCs). Among these were indole- and indazole-based valine derivatives with a cyclohexylmethyl side chain (e.g., AB-CHMINACA and MDMB-CHMICA), which represent a new class of SCs. Methods: A prospective observational study of patients treated in emergency departments (EDs) after the intake of SCs was conducted. Clinical and laboratory data were combined and reported to a poison control centre. Serum and/or urine samples of ED patients were analyzed using LC–MS/MS. Results: Forty four patients (39 male, five female, 12–48 years) were included. AB-CHMINACA (MDMB-CHMICA) was identified in 20 (19) serum samples, and in 21 (25) urine samples, respectively. In 19 of the cases, more than one SC was present. Other psychoactive substances (mainly amfetamines) were identified in seven cases, but in five out of these in urine samples only. Based on the Poison Severity Score, severity of poisoning was minor (4), moderate (31) or severe (9). Most frequently reported neuropsychiatric symptoms were CNS-depression (n = 21, 61%), disorientation (n = 20, 45%), generalized seizures (n = 12, 27%), combativeness (n = 8, 18%) and extreme agitation (n = 7, 16%). Duration of symptoms lasting 24 hours or longer occurred in 15 cases (34%). Discussion: The prevalence of certain neuropsychiatric symptoms was higher in our study than in former reports after the intake of SCs of the aminoalkylindole-type (first generation) SCs. In addition, severe poisoning and duration of symptoms were also higher. Conclusions: In this study, the valine derivative AB-CHMINACA and the tert-leucine derivative MDMB-CHMICA (“third generation of SCs”) seem to be associated with more severe clinical toxicity than was previously reported in patients exposed to earlier generation SCs such as JWH-018. However, this observation needs to be confirmed with a larger cohort of patients with analytically confirmed abuse of third generation SCs. The rapid turnover of SCs on the drug market together with the occurrence of SCs such as AB-CHMINACA and MDMB-CHMICA is alarming, especially because of the unexpectedly high frequency of neuropsychiatric symptoms.

Bad trip due to 25I-NBOMe: a case report from the EU project SPICE II plus

Abstract Objective: The potent hallucinogenic drug 25I-NBOMe has recently emerged on the drug market. We present a case with analytically confirmed 25I-NBOMe intoxication from the prospective study “SPICE II Plus”. Case report: Because of a severe headache a 42-year-old man took one sip of a pediatric analgesic syrup, which had been refilled with a self-made solution of 25I-NBOMe in ethanol. Thirty minutes later restlessness occurred. On arrival in the emergency department mydriasis, strong sweating, disorientation, and agitation were noticed. Within short time the patient developed severe agitation, coenesthesia, and complex hallucinations. In blood serum samples obtained at admission revealed the presence of 25I-NBOMe (34 ng/mL), 2C-I (12 ng/mL) and 25I-NBOH (<1 ng/mL) (LC-ESI-MS/MS). The presumed analgesic syrup contained 25I-NBOMe (2800 μg/mL), and besides ethanol no other compounds were detected. After six hours, the symptoms resolved without further complications. Conclusions: This is a unique case of an analytically confirmed, accidental ingestion of 25I-NBOMe in a drug naïve adult. The finding of 2C-I in the serum sample 50 minutes after intake indicates a fast metabolic breakdown of 25I-NBOMe due to first-pass metabolism.

Genotoxic properties of XLR-11, a widely consumed synthetic cannabinoid, and of the benzoyl indole RCS-4

Abstract Aim of this study was the investigation of the genotoxic properties of XLR-11 [1-(5-fluoropentyl)-1H-indol-3-yl](2,2,3,3-tetramethylcyclopropyl)methanone, a widely consumed synthetic cannabinoid (SC), and of the benzoyl indole RCS-4 (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone). We characterized the DNA-damaging properties of these drugs in different experimental systems. No evidence for induction of gene mutations was detected in bacterial (Salmonella/microsome) tests, but clear dose-dependent effects were found in in vitro single cell gel electrophoresis (SCGE) assays with human lymphocytes and with buccal- and lung-derived human cell lines (TR-146 and A-549). These experiments are based on the determination of DNA migration in an electric field and enable the detection of single- and double-strand breaks and apurinic sites. Furthermore, we found that both drugs induce micronuclei which are formed as a consequence of chromosomal aberrations. The lack of effects in SCGE experiments with lesion-specific enzymes (FPG, Endo III) shows that the DNA damage is not caused by formation of oxidatively damaged bases; experiments with liver enzyme homogenates and bovine serum albumin indicate that the drugs are not converted enzymatically to DNA-reactive intermediates. Furthermore, results with buccal- and lung-derived human cells show that gaseous treatment of the cells under conditions which reflect the exposure situation in drug users may cause damage of the genetic material in epithelia of the respiratory tract. Since DNA instability is involved in the etiology of cancer, these findings can be taken as an indication that consumption of the SCs may cause tumors in the respiratory tract of consumers.