Pharmacology and toxicology of xylazine: quid novum?

The current opioid overdose crisis is characterized by the presence of unknown psychoactive adulterants. Xylazine is an alpha-2 receptor agonist that is not approved for human use but is commonly used in veterinary medicine due to its sedative and muscle-relaxant properties. Cases of human intoxication due to accidental or voluntary use have been reported since the 1980s. However, reports of adulteration of illicit opioids (heroin and illicit fentanyl) with xylazine have been increasing all over Western countries. In humans, xylazine causes respiratory depression, bradycardia, and hypotension-posing individuals, using xylazine-adulterated opioids. We present a narrative review of the latest intoxication cases related to xylazine, to bring awareness to readers and also to help pathologists to detect and deal with xylazine cases.

Targeted screening and quantification of synthetic cathinones and metabolites in hair by UHPLC-HRMS.

OBJECTIVE: Synthetic cathinones (SCs) are new psychoactive substances with sympathomimetic effects, which emerged into the illegal drug market to replace controlled stimulants. Since every year more powerful and toxic substances enter the illicit market, there is the need for analytical methodologies able to detect these new compounds in conventional and non-conventional biological matrices. We sought to develop and validate a targeted screening and quantification method for thirty-two parent SCs and two metabolites in hair samples by ultra-high-performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC-HRMS). MATERIALS AND METHODS: 20 mg hair samples were soaked in 250 µL of 2 mM ammonium formate, methanol and acetonitrile mixture (50/25/25, v/v/v) and incubated overnight at 40°C. After incubation, the samples were evaporated to dryness under nitrogen stream and reconstituted with 100 µL of mobile phase mix (A:B, 80:20) and 10 µL were injected into UHPLC-HRMS. A Q ExactiveTM Focus Orbitrap Mass spectrometer with full scan and targeted data-dependent MS/MS scan acquisition was used for the screening and quantitation analysis. RESULTS: The assay was linear from 5 to 500 pg/mg hair for all the analytes under investigation. Intra-day and inter-day precision were always < 15% and matrix effect and analytical recovery were always within acceptable criteria (±25% and >50%, respectively). The developed method was applied to authentic hair samples from SCs consumers. The most prevalent found SCs were 3,4-Methylenedioxy-α-Pyrrolidinohexanophenone with a concentration range of 6.0-1,000.0 pg/mg along with α-Pyrrolidinohexiophenone (54.0 and 554.0 pg/mg, respectively), 3-Methylmetcathinone (556.0 and 5,000.0 pg/mg) and 4-Methylethcathinone (11.5 and 448.0 pg/mg) CONCLUSIONS: The developed method showed good selectivity, specificity, an easy and low-cost sample preparation and an analysis time compatible with a high throughput laboratory.

Determination of nine new fentanyl analogues and metabolites in consumers' urine by ultra-high-performance liquid chromatography-tandem mass spectrometry.

OBJECTIVE: New fentanyl analogues have been constantly emerging into the illegal drug market as cheap substitutes of heroin posing a serious health threat for consumers because of their high toxicity. Analytical methods to disclose the presence of these compounds in biological fluids of intoxicated individuals need to be updated to keep up with the new trends. In this study, we updated an ultra-high-performance liquid chromatography-tandem mass spectrometry method previously developed, for detecting some new fentanyl analogues and metabolites (sufentanil and norsufentanil, cis-3-methylnorfentanyl, trans-3-methylnorfentanyl, metabolites of cis and transmethylfentanyl, beta-phenylfentanyl, phenylfentanyl, para-fluoro furanyl fentanyl, isobutyryl fentanyl and ocfentanil) in urine sample. MATERIALS AND METHODS: Urine samples were simply diluted before injection in the chromatograph equipped with a reversed phase microcolumn. Detection was achieved with a triple quadrupole mass spectrometer with an electrospray ionization source in positive ion mode and operated in multiple reaction monitoring. RESULTS: The chromatographic separation was short (5 min) and the method was fully validated with a high sensitivity being limits of quantifications from 0.003 to 0.006 µg/L urine for the analytes under investigation. CONCLUSIONS: The suitability of the method was tested with urine specimens from former heroin addicts, which resulted positive by immunological screening to the class of fentanyl analogues. This method represents a valid tool to document recent exposure to the above-reported compounds for clinical and forensic purposes.

Suitability of miRNA assessment in postmortem interval estimation.

OBJECTIVE: The aim of this review was to explore recent pieces of evidence focused on the use of miRNAs for PMI estimation both in humans and animal experiments, with particular interest on the best miRNAs to use as reference/target markers in different tissues or biological fluids. MiRNAs are innovative biomarkers used in clinical and research field; they appear very attractive, being introduced in forensic research scenarios even for PMI estimation. MATERIALS AND METHODS: Data from PubMed and Scopus were analyzed from January 2013 to August 2020. Based on inclusion/exclusion criteria, high-quality articles have been selected to become the subject of this review. RESULTS: A total of 737 papers were found but, after titles/abstracts screening for inclusion criteria and a full-text careful selection, 33 papers were deeply studied. After the exclusion of 19 papers, 15 articles remained. Eight papers dealt with animals (mice/rats), two both with animals and humans (for method validation previously built), while 5 exclusively with humans. Myocardium (6/15) and brain (6/15) were the most studied tissues, respectively in mice/rats and humans. PMI considered was up to 7.5 days in mouse studies and less than 3 days in human models. CONCLUSIONS: Because of their significant stability in both early and long PMI, miRNAs are the cleverest reference markers to be used. Temperature and environmental conditions influence mostly mRNA, while miRNAs are less susceptible to them. The best miRNA to choose depends on its tissue specificity, i.e., miR-9 and miR-125 in brain or miR-1 and miR-133 in skeletal muscle/heart.

SARS-CoV-2 identification in lungs, heart and kidney specimens by transmission and scanning electron microscopy.

From two COVID-19-related deaths, samples of lung, heart and kidney were collected and processed for Transmission and Scanning Electron Microscopy (TEM and SEM) with the aim of identifying the virus. Virions of SARS-CoV-2 were found in all tissues by TEM and SEM, corroborating the hypothesis that the virus enters the cells of different organs. This is the first report identifying SARS-CoV-2 in different human tissues by TEM and SEM.

Commentary - Heroin purity and adulteration: an updated snapshot from the Italian Early Warning System.

Not Available.

Drug-facilitated sexual assaults (DFSA): a serious underestimated issue.

OBJECTIVE: Drug-facilitated sexual assault (DFSA) is a nonconsensual sexual act in which the victim is incapacitated or unconscious due to the effects of alcohol, a drug and/or other intoxicating substances. Dozens of drugs (including ethanol) can potentially be used to commit sexual assaults, but γ-Hydroxybutyric acid (GHB) and flunitrazepam are the most common "date rape drugs". MATERIALS AND METHODS: Multidisciplinary databases were browsed using the following search terms: "drug-facilitated sexual assault", "chemical submission", "date rape", "rape drugs", and "drink-spiking". Moreover, a search for reports was conducted on Institutional websites to identify documentation published by international agencies or institutions. Articles and reports were independently evaluated by each author. RESULTS: There are no accurate estimates of the number of DFSA occurring each year, although assaults are increasingly reported. Many DFSA, however, are still not reported. Victims are reluctant to report incidents because of embarrassment, guilt or perceived responsibility, or because they do not clearly remember the assault. Moreover, most of the drugs typically used in sexual assaults are rapidly metabolized, making them undetectable in routine drug screenings. CONCLUSIONS: Most of the substances involved in DFSA, with the exception of alcohol, are under international control and scheduled under the United Nations Single Convention on Narcotic Drugs of 1961 and the Convention on Psychotropic Substances of 1971. However, several psychotropic substances and antihistamines used in sexual assaults are still not under international control, allowing for trafficking, often via the Internet and courier. The absence of international control makes it difficult to obtain accurate data on the nature and the extent of the problem.

Post-mortem toxicology in the diagnosis of sudden death in young and middle-aged victims.

OBJECTIVE: We aimed to investigate the impact of the toxicological results found in cases of sudden death (SD) and to correlate the clinical, autopsy and genetic findings with the toxicology results. MATERIALS AND METHODS: Consecutive SD in people aged between 16 and 50 years with medico-legal autopsies and toxicology studies were included over a 3-year period. The comparison between the toxicological data and demographic characteristics, clinical circumstances, autopsy, and genetic results were taken into account. RESULTS: 101 cases were finally included. They were predominately males (84%) and the mean age was 39.8 years. 52 (51.5%) cases had positive toxicological findings and in 25 cases (24.8%), toxic compounds were considered the first cause of death. Ethanol was the most frequently identified agent (69%), following by licit drugs (56%) and drugs of abuse (39%). Cases with positive toxicology were younger than those with negative results (37.9±9.1 vs. 41.9±7.8; p=0.02). Patients with more than 3 comorbidities showed an association with positive toxicological results (n=14 vs. n=3; p=0.017). The genetic study was performed in 70 (69.3%) SD cases. We identified pathogenic or likely pathogenic variants in 17.1% cases and uncertain significance variants in 42.8% cases. 58% of these variants were probably related to the cause of death. CONCLUSIONS: A large fraction of SD victims had positive toxicological findings and a quarter of deaths were directly caused by toxic substances. The identification of the factors that trigger SD provides a good approach to contribute in avoiding future episodes.

Use of cognitive enhancers: methylphenidate and analogs.

OBJECTIVE: In the last decades, several cognitive-enhancing drugs have been sold onto the drug market. Methylphenidate and analogs represent a sub-class of these new psychoactive substances (NPS). We aimed to review the use and misuse of methylphenidate and analogs, and the risk associated. Moreover, we exhaustively reviewed the scientific data on the most recent methylphenidate analogs (methylphenidate and ethylphenidate excluded). MATERIALS AND METHODS: Literature search was performed on methylphenidate and analogs, using specialized search engines accessing scientific databases. Additional reports were retrieved from international agencies, institutional websites, and drug user forums. RESULTS: Methylphenidate/Ritalin has been used for decades to treat attention deficit disorders and narcolepsy. More recently, it has been used as a cognitive enhancer and a recreational drug. Acute intoxications and fatalities involving methylphenidate were reported. Methylphenidate was scheduled as an illegal drug in many countries, but NPS circumventing the ban and mimicking the psychostimulant effects of methylphenidate started being available: ethylphenidate, 3,4-dichloromethylphenidate, 3,4-dichloroethylphenidate, 4-fluoromethylphenidate, 4-fluoroethylphenidate, methylnaphthidate, ethylnaphthidate, isopropylphenidate, propylphenidate, 4-methylmethylphenidate, and N-benzylethylphenidate have been available in the past few years. Only little data is currently available for these substances. Many intoxications involving methylphenidate analogs were reported. To date, ethylphenidate was involved in 28 fatalities, although it was reportedly directly related to the cause of death in only 7 cases; 3,4-dichloroethylphenidate was involved in 1 death. CONCLUSIONS: The rapid expansion of methylphenidate analogs onto the drug market in the past few years makes likely the occurrence of intoxications and fatalities in the next years. Careful monitoring and systematic control of methylphenidate analogs should be undertaken to reduce the uprising threat, and education efforts should be made among high-risk populations.

Relationship Between Postmortem Urine and Blood Concentrations of GHB Furnishes Useful Information to Help Interpret Drug Intoxication Deaths.

This article reports the concentrations of gamma-hydroxybutyrate (GHB) in femoral blood and bladder urine in a case series of drug intoxication deaths (N = 37). GHB was determined in blood (B-GHB) and urine (U-GHB) by a GC-FID-GBL method and 30 mg/L was used as a cut-off concentration for reporting positive results. The mean (median) and range of GHB concentrations in bladder urine were 2,818 mg/L (1,900 mg/L) and 120-13,000 mg/L, respectively. These concentrations were appreciably higher than those in femoral blood, 637 mg/L (260 mg/L) and 30-9,200 mg/L, respectively. Urine/blood ratios of GHB were highly variable (mean 8.99, median 5.33 and range 0.16-29.3). GHB is rapidly metabolized and cleared from the bloodstream, whereas there is no metabolism occurring in the urinary bladder. In five autopsy cases, U-GHB was lower than B-GHB, which suggests that these individuals died before equilibration of the drug in all body fluids and tissues. In the other 32 deaths, U-GHB was higher than B-GHB, sometimes appreciably higher, which points towards a longer survival time after intake or administration of GHB. The analysis of urine extends the window of detection of GHB by several hours compared with blood samples, depending in part on when the bladder was last voided before death. Furthermore, the urinary concentration of GHB gives a hint about the concentration in blood during the time that the urine was produced in the kidney and stored in the bladder since the previous void.

Synthetic cathinones related fatalities: an update.

OBJECTIVE: Synthetic cathinones, more commonly known as "bath salts", are synthetic drugs chemically related to cathinone, a psychostimulant found in the khat plant. They are the first most consumed products among new psychoactive substances, which cause psychostimulant and hallucinogenic effects determining a number of fatalities worldwide.  In this paper, we have systematically reviewed cases of synthetic cathinones-related fatalities analytically confirmed, which have occurred in the last few years. MATERIALS AND METHODS: Relevant scientific articles were identified in Medline, Cochrane Central, Scopus, Web of Science and Institutional/government websites up to November 2017 using the following keywords: synthetic cathinones, mephedrone, methylenedioxypyrovalerone, MDPV, methylone, ethylone, buthylone, fatal intoxication, fatalities and death. RESULTS: In total, 20 citations met the criteria for inclusion, representing several fatal cases with analytically confirmed synthetic cathinones in biological sample/s of the deceased. The death was attributed to hyperthermia, hypertension, cardiac arrest and more in general to the classic serotonin syndrome. Only rarely did the concentration of the parent drug causing fatality overcome the value of 1 mg/L in post-mortem biological fluids. CONCLUSIONS: Abuse of synthetic cathinones still represents a serious public health issue. Systematic clinical studies on both the animal and human model are lacking; therefore, the only available data are from the users who experience the possible hazardous consequences. Analytical methodologies for the identification of parent compounds and eventual metabolites both in ante-mortem and post-mortem cases need to be developed and validated. Analytical data should be shared through different communication platforms with the aim of stopping this serious health threat for drug users.

Hepatotoxicity of paracetamol and related fatalities.

Paracetamol, also known as acetaminophen, is the most commonly used antipyretic and pain reliever and since 1955 it is available over-the-counter as a single formulation or in combination with other substances and, as indicated by the World Health Organization, it can be used in all the three steps of pain intensity. Paracetamol toxicity is one of the most common causes of poisoning worldwide. While paracetamol is described as relatively nontoxic when administered in therapeutic doses, it is known to cause toxicity when taken in a single or repeated high dose, or after chronic ingestion. Repeated supratherapeutic misuse, non-intentional misuse, and intentional ingestion may all result in hepatic toxicity, the main cause of acute liver failure (ALF) in the United States and Europe. Since paracetamol is responsible for nearly half of the cases in the US of acute liver failure and remains the leading cause of liver transplantation, continued awareness promotion, education and research should be constantly undertaken. We herein review the literature on paracetamol toxicity with particular attention to aspects of liver damage and related fatalities.

Hepatotoxicity induced by greater celandine (Chelidonium majus L.): a review of the literature.

The available literature assessing Chelidonium majus L. (CM) hepatotoxicity potential, and its risk to benefit assessment has been reviewed in this paper. Identification of significant scientific literature was performed via the following research databases: Cochrane Central, Google Scholar, EMBASE, Medline, Science Direct, Scopus, Web of Science, using the following keywords: "Chelidonium majus", "greater celandine", "Hepatotoxicity", "Liver" "Injury", "Toxicity" individually investigated and then again in association. CM named also greater celandine, swallow-wort, or bai-qu-cai (Chinese), has been used for a long time in traditional Chinese medicine and phytotherapy. Its extracts have been claimed to display a wide variety of biological activities: antimicrobial, anti-inflammatory, spasmolytic, antineoplastic, hepatoprotective, and analgesic. Moreover, herbal medicine suggests this plant have numerous additional effects which have not yet been scientifically evaluated, such as antitussive, diuretic, and eye-regenerative. However, despite its claimed hepatoprotective effects, several hepatotoxicity cases have been reported to be probably or highly probably connected with CM exposure, after their evaluation through liver-targeted causality assessment methods. CM hepatotoxicity has been defined as a distinct form of herb-induced liver injury (HILI), due to an idiosyncratic reaction of the metabolic type. This evidence has to be considered in relationship with the absence of considerable benefits of CM therapy. Therefore, the risk to benefit ratio of the use of herbal products containing greater celandine can actually be considered as negative.