Disposition of Hexahydrocannabinol Epimers and Their Metabolites in Biological Matrices following a Single Administration of Smoked Hexahydrocannabinol: A Preliminary Study.

In 2023, hexahydrocannabinol (HHC) attracted the attention of international agencies due to its rapid spread in the illegal market. Although it was discovered in 1940, less is known about the pharmacology of its two naturally occurring epimers, 9(R)-HHC and 9(S)-HHC. Thus, we aimed to investigate the disposition of hexahydrocannabinol epimers and their metabolites in whole blood, urine and oral fluid following a single controlled administration of a 50:50 mixture of 9(R)-HHC and 9(S)-HHC smoked with tobacco. To this end, six non-user volunteers smoked 25 mg of the HHC mixture in 500 mg of tobacco. Blood and oral fluid were sampled at different time points up to 3 h after the intake, while urine was collected between 0 and 2 h and between 2 and 6 h. The samples were analyzed with a validated HPLC-MS/MS method to quantify 9(R)-HHC, 9(S)-HHC and eight metabolites. 9(R)-HHC showed the highest Cmax and AUC0-3h in all the investigated matrices, with an average concentration 3-fold higher than that of 9(S)-HHC. In oral fluid, no metabolites were detected, while they were observed as glucuronides in urine and blood, but with different profiles. Indeed, 11nor-9(R)-HHC was the most abundant metabolite in blood, while 8(R)OH-9(R) HHC was the most prevalent in urine. Interestingly, 11nor 9(S) COOH HHC was detected only in blood, whereas 8(S)OH-9(S) HHC was detected only in urine.

The first LC-MS/MS stereoselective bioanalytical methods to quantitatively detect 9R- and 9S-hexahydrocannabinols and their metabolites in human blood, oral fluid and urine.

A sensitive LC-MS/MS method for the simultaneous quantification of the (9 R)- and (9 S)- hexahydrocannabinols (HHCs), and their metabolites, in human urine, oral fluid (OF) and blood samples were developed, validated and used to the biological samples of volunteers. The analytes were extracted from 100 µL human samples. An isocratic elution mode with methanol was used for chromatographic separation of (9 R)- and (9 S)-HHC on an immobilized amylose tris(3-chloro-5-methylphenylcarbamate)-based chiral column Lux i-Amylose-3. The flow-rate of the mobile phase was 0.5 mL/min. An isocratic elution mode of methanol and water (80/20, v/v) was used for chromatographic separation of metabolites of (9 R)- and (9 S)-HHC on a Lux AMP chiral column (with a proprietary chiral selector) at a flow rate of 0.5 mL/min. MS/MS analysis was performed in positive ionization mode for HHC epimers, while in negative ionization mode was used for metabolites of HHCs. The calibration curves for HHCs and their metabolites in human samples ranged from 0.25- 240 ng mL-1 and 1 - 100 ng mL-1, respectively, with determination coefficients (r2) of ≥ 0.99. All analytes were stable at room temperature, 4 °C, in the autosampler (+10 °C) and -20 °C for 24 h, after three freeze/thaw cycles, and when stored at -20 °C up to one week after quality control (QC) sample preparation (concentration differences less than 20% with respect to time zero response), in blood, urine and OF.

Synthetic Cathinones and Neurotoxicity Risks: A Systematic Review.

According to the EU Early Warning System (EWS), synthetic cathinones (SCs) are the second largest new psychoactive substances (NPS) class, with 162 synthetic cathinones monitored by the EU EWS. They have a similar structure to cathinone, principally found in Catha Edulis; they have a phenethylamine related structure but also exhibit amphetamine-like stimulant effects. Illegal laboratories regularly develop new substances and place them on the market. For this reason, during the last decade this class of substances has presented a great challenge for public health and forensic toxicologists. Acting on different systems and with various mechanisms of action, the spectrum of side effects caused by the intake of these drugs of abuse is very broad. To date, most studies have focused on the substances' cardiac effects, and very few on their associated neurotoxicity. Specifically, synthetic cathinones appear to be involved in different neurological events, including increased alertness, mild agitation, severe psychosis, hyperthermia and death. A systematic literature search in PubMed and Scopus databases according to PRISMA guidelines was performed. A total of 515 studies published from 2005 to 2022 (350 articles from PubMed and 165 from Scopus) were initially screened for eligibility. The papers excluded, according to the criteria described in the Method Section (n = 401) and after full text analyses (n = 82), were 483 in total. The remaining 76 were included in the present review, as they met fully the inclusion criteria. The present work provides a comprehensive review on neurotoxic mechanisms of synthetic cathinones highlighting intoxication cases and fatalities in humans, as well as the toxic effects on animals (in particular rats, mice and zebrafish larvae). The reviewed studies showed brain-related adverse effects, including encephalopathy, coma and convulsions, and sympathomimetic and hallucinogenic toxidromes, together with the risk of developing excited/agitated delirium syndrome and serotonin syndrome.

New Psychoactive Substances Intoxications and Fatalities during the COVID-19 Epidemic.

In January 2020, the World Health Organization (WHO) issued a Public Health Emergency of International Concern, declaring the COVID-19 outbreak a pandemic in March 2020. Stringent measures decreased consumption of some drugs, moving the illicit market to alternative substances, such as New Psychoactive Substances (NPS). A systematic literature search was performed, using scientific databases such as PubMed, Scopus, Web of Science and institutional and government websites, to identify reported intoxications and fatalities from NPS during the COVID-19 pandemic. The search terms were: COVID-19, SARS-CoV-2, severe acute respiratory syndrome coronavirus 2, coronavirus disease 2019, intox*, fatal*, new psychoactive substance, novel psychoactive substance, smart drugs, new psychoactive substance, novel synthetic opioid, synthetic opioid, synthetic cathinone, bath salts, legal highs, nitazene, bath salt, legal high, synthetic cannabinoid, phenethylamine, phencyclidine, piperazine, novel benzodiazepine, benzodiazepine analogue, designer benzodiazepines, tryptamine and psychostimulant. From January 2020 to March 2022, 215 NPS exposures were reported in Europe, UK, Japan and USA. Single NPS class intoxications accounted for 25, while mixed NPS class intoxications represented only 3 cases. A total of 130 NPS single class fatalities and 56 fatalities involving mixed NPS classes were published during the pandemic. Synthetic opioids were the NPS class most abused, followed by synthetic cathinones and synthetic cannabinoids. Notably, designer benzodiazepines were frequently found in combination with fentalogues. Considering the stress to communities and healthcare systems generated by the pandemic, NPS-related information may be underestimated. However, we could not define the exact impacts of COVID-19 on processing of toxicological data, autopsy and death investigations.

Neuronal nitric oxide synthase positive neurons in the human corpus callosum: a possible link with the callosal blood-oxygen-level dependent (BOLD) effect.

Brain functions have been investigated in the past decades via the blood-oxygen-level dependent (BOLD) effect using functional magnetic resonance imaging. One hypothesis explaining the BOLD effect involves the Nitric Oxide (NO) gaseous neurotransmitter, possibly released also by cells in the corpus callosum (CC). The eventual presence of NO releasing neurons and/or glial cells in the CC can be assessed by immunohistochemistry. Serial sections both from paraffin-embedded and frozen samples of CC obtained from adult human brains autopsy were studied with immunohistochemistry and immunofluorescence analysis, using an antibody against the neuronal isoform of Nitric Oxide Synthase (nNOS), the enzyme synthetizing the NO. The staining revealed the presence of many nNOS-immunopositive cells in the CC, shown to be neurons with immunofluorescence. Neuronal NOS-positive neurons presented different morphologies, were more numerous 4 mm apart from the midline, and displayed a peak in the body of the CC. In some cases, they were located at the upper boundary of the CC, more densely packed in the proximity of the callosal arterioles. The significant presence of nNOS-immunopositive neurons within the commissure suggests their probable role in the CC neurovascular regulation in the adult brain and could explain the BOLD effect detected in human CC.

Molecular Insights and Clinical Outcomes of Drugs of Abuse Adulteration: New Trends and New Psychoactive Substances.

Adulteration is a well-known practice of drug manufacturers at different stages of drug production. The intentional addition of active ingredients to adulterate the primary drug may enhance or mask pharmacological effects or may produce more potent drugs to increase the number of available doses and the dealer's profit. Adulterants found in different drugs change over time in response to different factors. A systematic literature search in PubMed and Scopus databases and official international organizations' websites according to PRISMA guidelines was performed. A total of 724 studies were initially screened, with 145 articles from PubMed and 462 from Scopus excluded according to the criteria described in the Method Section. The remaining 117 records were further assessed for eligibility to exclude articles without sufficient data. Finally, 79 studies were classified as "non-biological" (n = 35) or "biological" (n = 35 case reports; n = 9 case series) according to the samples investigated. Although the seized samples analyses revealed the presence of well-established adulterants such as levamisole for cocaine or paracetamol/acetaminophen for heroin, the reported data disclosed new adulteration practices, such as the use of NPS as cutting agents for classic drugs of abuse and other NPS. For example, heroin adulterated with synthetic cannabinoids or cocaine adulterated with fentanyl/fentalogues raised particular concern. Notably, adulterants play a role in some adverse effects commonly associated with the primary drug, such as levamisole-adulterated cocaine that may induce vasculitis via an autoimmune process. It is essential to constantly monitor adulterants due to their changing availability that may threaten drug consumers' health.

Role of NRF2 in Ovarian Cancer.

Among gynaecologic malignancies, ovarian cancer is one of the most dangerous, with a high fatality rate and relapse due to the occurrence of chemoresistance. Many researchers demonstrated that oxidative stress is involved in tumour occurrence, growth and development. Nuclear factor erythroid 2-related factor 2 (NRF2) is an important transcription factor, playing an important role in protecting against oxidative damage. Increased levels of Reactive Oxygen Species (ROS) activate NRF2 signalling, inducing the expression of antioxidant enzymes, such as haem oxygenase (HO-1), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD), that protect cells against oxidative stress. However, NRF2 activation in cancer cells is responsible for the development of chemoresistance, inactivating drug-mediated oxidative stress that normally leads to cancer cells' death. In this review, we report evidence from the literature describing the effect of NRF2 on ovarian cancer, with a focus on its function in drug resistance, NRF2 natural and synthetic modulators and its protective function in normal ovarian preservation.

Neuronal nitric oxyde synthase positive neurons in human indusium griseum.

The study was designed to analyze the nNOS positive neurons present in the indusium griseum by describing their distribution and morphology. To this purpose, sagittal serial sections from paraffin or frozen autopsy specimens of corpus callosum including the overlying indusium griseum were processed by immunohistochemistry and immunofluorescence, using an antibody against the neuronal form of the enzyme nitric oxyde synthase. To test the specificity of the antibody used, Western Blot was performed in the indusium griseum of the same specimens. The stainings revealed the presence of many neuronal nitric oxyde synthase-immunopositive neurons in human indusium griseum, located along both rostral-caudal and medio-lateral directions. In particular, they were more numerous 1 mm apart from the midline, and their number peaked over the body of the corpus callosum. They showed different morphologies; in some cases, they were located at the boundary between indusium griseum and corpus callosum, more densely packed in proximity to the pial arteries penetrating into the corpus callosum. The significant presence and distribution of neuronal nitric oxyde synthase-immunopositive neurons suggests that indusium griseum likely plays a functional role in the neurovascular regulation within the corpus callosum. Schematic representation of human adult IG and the neurovascular unit originating from sopracallosal artery (Sca) that branches into smaller arterioles (Br) (created in PowerPoint). The arterioles cross the three layers of IG (layers I, II and III) and penetrate into the CC separated from IG by the Virchow-Robin space (VRs). As the arterioles go deeper, this space disappears and the vascular basement membrane comes into direct contact with the astrocytic end-feets (intracallosal arterioles and capillaries). nNOS-immunopositive neurons (nNOSIP N) surround the arterioles and control the vasomotore tone secreting nitric oxyde (NO). Two morphological types of nNOSIP N can be appreciated by the use of different colors: fusiform (blue) and ovoidal (pink). Also NeuN-immunopositive neurons (N) and many astrocytes (As) are present, more numerous in IG than in CC.

3F-α-pyrrolydinovalerophenone (3F-α-PVP) in vitro human metabolism: Multiple in silico predictions to assist in LC-HRMS/MS analysis and targeted/untargeted data mining.

Synthetic cathinones (SCs) constitute a heterogenous class of new psychoactive substances (NPS), structurally related to cathinone. SCs represent the widest NPS class, second to synthetic cannabinoids, accounting for approximately 160 different analogues with substitution at the phenyl group, the amine group, or the alkyl chain. In 2020, α-pyrrolidonophenone analogues were the most trafficked SCs, and were involved in many fatalities and intoxication cases. In particular, 3F-α-pyrrolidinovalerophenone (3F-α-PVP) was the cause of the highest number of SC-related fatal intoxications in Sweden in 2018. Minor structural modifications are used to avoid legal controls and analytical detection, but may also induce different toxicological profile. Therefore, the identification of specific markers of consumption is essential to discriminate SCs in clinical and forensic toxicology. In this study, we assessed 3F-α-PVP metabolic profile. 3F-α-PVP was incubated with 10-donor-pooled human hepatocytes, LC-HRMS/MS analysis, and software-assisted data mining. This well-established workflow was completed by in silico metabolite predictions using three different freeware. Ten metabolites were identified after 3 h incubation, including hydrogenated, hydroxylated, oxidated, and N-dealkylated metabolites. A total of 51 phase I and II metabolites were predicted, among which 7 were detected in the incubations. We suggest 3F-α-PVP N-butanoic acid, 3F-α-PVP pentanol, and 3F-α-PVP 2-ketopyrrolidinyl-pentanol as specific biomarkers of 3F-α-PVP consumption. This is the first time that an N-ethanoic acid is detected in the metabolic pathway of a pyrrolidine SC, demonstrating the importance of a dual targeted/untargeted data mining strategy.

Acute Intoxications and Fatalities Associated With Benzimidazole Opioid (Nitazene Analog) Use: A Systematic Review.

BACKGROUND: Synthetic benzimidazole opioids (BOs) are highly potent µ-opioid receptor agonists with heroin-like effects. Isotonitazene was first available in 2019 in the drug market, although new analogs have multiplied recently. The authors aimed to identify BO use trends and gather toxicological data from BO-related cases to assist in clinical and forensic investigations. METHODS: A systematic literature search was conducted according to the PRISMA guidelines. PubMed and Scopus databases were accessed in October 2021 to identify scientific reports of BO-related intoxication and fatalities. Publication dates, case descriptions, symptoms, autopsy findings, and concentrations of BOs and metabolites in biological matrices were compiled. RESULTS: Data from 8 case reports with 93 fatalities involving isotonitazene ( n = 65), metonitazene ( n = 20), etonitazepyne ( N -pyrrolidino etonitazene) ( n = 8), flunitazene ( n = 4), and/or butonitazene ( n = 1), and 1 acute intoxication involving etonitazepyne were collected. Autopsy findings included pulmonary congestion/high lung weight (66%), cardiomegaly/high cardiac weight (39%), cerebral edema (22%), gastric contents in the airways (22%), and organ congestion (22%). Median peripheral blood concentrations were 1.7 ng/mL for isotonitazene (0.4-9.5 ng/mL, n = 13), 5.4 ng/mL for metonitazene (0.52-33 ng/mL, n = 17), 5.4 ng/mL for etonitazepyne (2.4-8.3 ng/mL, n = 2), 1.3 ng/mL for flunitazene (0.58-2.1 ng/mL, n = 2), and 3.2 ng/mL for butonitazene ( n = 1). Central nervous system depressants were almost always coadministered. CONCLUSIONS: Isotonitazene was predominant in cases from 2019 to mid-2020 and was replaced by metonitazene after scheduling in the United States. Typical findings on opioid overdoses have been reported. Peripheral blood concentrations were consistent with a potency similar to that of fentanyl. These results must be interpreted carefully, considering the scarcity of reports on BO-related cases and drug co-exposures.

Hypoxia-induced expression of neuronal nitric oxide synthase in astrocytes of human corpus callosum.

Nitric oxide (NO) is a gaseous neurotransmitter largely diffused in the brain; among other functions, it regulates the cerebral blood flow in response to hypoxia. NO can be synthetized by three different isoforms of the enzyme NO synthase: neuronal (nNOS), typical of neurons, endothelial and inducible. The aim of this study was to assess nNOS expression in human corpus callosum (CC) astrocytes, and its relationship with the hypoxia duration. Autoptic samples of CC from adult human subjects have been processed with immunohistochemistry and immunofluorescence using antibodies anti-nNOS and anti-glial fibrillary acidic protein (GFAP), the astrocyte marker. Results demonstrated for the first time the presence of nNOS-immunopositive astrocytes in the human CC. In particular, nNOS-positive astrocytes were absent in subjects deceased after a short hypoxia; their number and labeling intensity, however, increased with hypoxia prolongation. Neuronal NOS immunopositivity of CC astrocytes seems thus related to the hypoxia duration and the consequent brain damage.

Is etizolam a safe medication? Effects on psychomotor perfomance at therapeutic dosages of a newly abused psychoactive substance.

Etizolam is a drug from the thienotriazoldiazepine class, widely prescribed as anxiolytic due to its apparently secure toxicological profile. Nevertheless, some recent cases of etizolam dependence, intoxications and fatalities associated to its abuse have been reported in the international literature. For this reason, the drug listed as new psychoactive substance (NPS) by the World Health Organization (WHO) since 2015. Euphoric effect at high dosage is the first cause of its recreational use that has determined a wider distribution in the illicit market. An experimental study was performed to obtain evidence that etizolam at low therapeutic dosages is a drug with negligible influence on the psychomotor performances involved in driving. The psychomotor performance was assessed by performing different tests, such as critical tracking task (CTT), critical flicker fusion (CFF), choice reaction time (CRT), visual vigilance task (VVT), response competition test (RCT) in a group of 16 healthy volunteers after a single administration of etizolam at two different dosages (0.25 mg or 1.00 mg) in comparison to placebo. The test results showed that etizolam at 0.25 mg and 1.00 mg had no significant effect on vigilance, short term memory, psychomotor coordination or speed in decision making. Differently, abuse of etizolam to obtain the euphoric effects at presumably high dosages or in combination with other psychoactive substances could be fatal. The negligible side effects on mental and behavioral function demonstrated by this study, could represent an incitement to abuse, which can be strongly discouraged with correct information on differences between its correct use and its misuse.

Delayed Intracerebral Hemorrhage After Pseudoaneurysm of Middle Meningeal Artery Rupture: Case Report, Literature Review, and Forensic Issues.

BACKGROUND: Traumatic pseudoaneurysm of the middle meningeal artery (PMMA) is rare. Its rupture is associated with high mortality, so an early diagnosis is recommended for this risky condition. In the absence of a specific guideline, computed tomography (CT), digital subtraction angiography, and CT angiography (CTA) are proposed for its diagnosis. CTA is the technique of choice even if it is almost never performed, especially in mild head injury. We report a rare case of a delayed rupture of PMMA, analyzed from a forensic point of view. METHODS: Fifteen days after mild blunt head trauma, characterized by temporal fracture and a small hemorrhage near the rim, a wide intraparenchymal hemorrhage (IPH) occurred. The onset of IPH was marked by neurologic deterioration and arm paralysis. Immediate head CT showed IPH, and CTA showed PMMA. Prompt surgery could not help patient survival. The goal of autopsy was to formulate the cause of death and to individuate potential medical negligence. RESULTS: In the literature, 16 cases of 54 are related to PMMA (26%) and are associated with IPH. IPH can be acute or delayed. Eight cases of acute IPH and 8 cases of delayed IPH (including our case), both coexisting with PMMA, are described. The literature review showed that the association of temporal rim fracture and a small hemorrhage nearby is highly predictive of PMMA formation. CONCLUSIONS: Therefore, in the presence of these 2 risk factors after heat trauma, CTA is strongly suggested.

Prophylaxis for venous thromboembolism after resection of hepatocellular carcinoma on cirrhosis: is it necessary?

AIM: To assess the safety and effectiveness of prophylaxis for venous thromboembolism (VTE) in a large population of patients with hepatocellular carcinoma (HCC) on cirrhosis. METHODS: Two hundred and twenty nine consecutive cirrhotic patients with HCC who underwent hepatic resection were retrospectively evaluated to assess whether there was any difference in the incidence of thrombotic or hemorrhagic complications between those who received and those who did not receive prophylaxis with low-molecular weight heparin. Differences and possible effects of the following parameters were investigated: age, sex, Child-Pugh and model for end-stage liver disease (MELD) score, platelet count, presence of esophageal varices, type of hepatic resection, duration of surgery, intraoperative transfusion of blood and fresh frozen plasma (FFP), body mass index, diabetes and previous cardiovascular disease. RESULTS: One hundred and fifty seven of 229 (68.5%) patients received antithromboembolic prophylaxis (group A) while the remaining 72 (31.5%) patients did not (group B). Patients in group B had higher Child-Pugh and MELD scores, lower platelet counts, a higher prevalence of esophageal varices and higher requirements for intraoperative transfusion of FFP. The incidence of VTE and postoperative hemorrhage was 0.63% and 3.18% in group A and 1.38% and 1.38% in group B, respectively; these differences were not significant. None of the variables analyzed including prophylaxis proved to be risk factors for VTE, and only the presence of esophageal varices was associated with an increased risk of bleeding. CONCLUSION: Prophylaxis is safe in cirrhotic patients without esophageal varices; the real need for prophylaxis should be better assessed.