In vitro structure-activity relationships and forensic case series of emerging 2-benzylbenzimidazole 'nitazene' opioids.

2-Benzylbenzimidazole 'nitazene' opioids are presenting a growing threat to public health. Although various nitazenes were previously studied, systematic comparisons of the effects of different structural modifications to the 2-benzylbenzimidazole core structure on µ-opioid receptor (MOR) activity are limited. Here, we assessed in vitro structure-activity relationships of 9 previously uncharacterized nitazenes alongside known structural analogues. Specifically, we focused on MOR activation by 'ring' substituted analogues (i.e., N-pyrrolidino and N-piperidinyl modifications), 'desnitazene' analogues (lacking the 5-nitro group), and N-desethyl analogues. The results from two in vitro MOR activation assays (ß-arrestin 2 recruitment and inhibition of cAMP accumulation) showed that 'ring' modifications overall yield highly active drugs. With the exception of 4'-OH analogues (which are metabolites), N-pyrrolidino substitutions were generally more favorable for MOR activation than N-piperidine substitutions. Furthermore, removal of the 5-nitro group on the benzimidazole ring consistently caused a pronounced decrease in potency. The N-desethyl modifications showed important MOR activity, and generally resulted in a slightly lowered potency than comparator nitazenes. Intriguingly, N-desethyl isotonitazene was the exception and was consistently more potent than isotonitazene. Complementing the in vitro findings and demonstrating the high harm potential associated with many of these compounds, we describe 85 forensic cases from North America and the United Kingdom involving etodesnitazene, N-desethyl etonitazene, N-desethyl isotonitazene, N-pyrrolidino metonitazene, and N-pyrrolidino protonitazene. The low-to-sub ng/mL blood concentrations observed in most cases underscore the drugs' high potencies. Taken together, by bridging pharmacology and case data, this study may aid to increase awareness and guide legislative and public health efforts.

MDMB-4en-PINACA-Related Deaths in Cook County Jail: Awareness and Preventive Measures.

ABSTRACT: Since January 2023, a series of fatalities has occurred in the Cook County Jail. Upon reviewing surveillance videos, in some cases, the inmates shared paper strips, and it was followed by the onset of labored breathing and loss of consciousness. Scene investigation revealed burnt paper strips near the body in 3 cases. No trauma was observed during autopsy. The expanded toxicology panels did not detect any illegal drugs in the postmortem blood samples. However, additional analysis specifically targeting synthetic cannabinoids revealed the presence of MDMB-4en-PINACA [methyl 3,3-dimethyl-2-{[1-(pent-4-en-1-yl)-1H-indazole-3-carbonyl]amino}butanoate], a synthetic cannabinoid associated with a wide range of adverse effects, including cardiovascular complications such as tachycardia and hypertension, respiratory depression, and acute kidney injury. There is limited research on the lethality of MDMB-4en-PINACA. This case series suggests that even isolated use can potentially lead to death. This study aims to raise public awareness regarding MDMB-4en-PINACA, highlighting its unpredictable effects and potential for severe adverse reactions, and to facilitate the development of effective prevention and harm reduction strategies. Implementing screening methods in correctional facilities is crucial to prevent the circulation of potentially fatal substances.

Toxicological and pharmacological characterization of novel cinnamylpiperazine synthetic opioids in humans and in vitro including 2-methyl AP-237 and AP-238.

The recent scheduling actions of fentanyl-related substances in both the United States and China have sparked the emergence and proliferation of other generations of "legal" opioids that are structurally distinct from fentanyl, including the recently emerged class of cinnamylpiperazines. In contrast to fentanyl, which contains a piperidine core and a phenethyl moiety, the primary structural components of cinnamylpiperazines are the piperazine core and a cinnamyl moiety. This manuscript reports on the toxicological profile for antemortem and postmortem cases where a cinnamylpiperazine was detected. Samples were quantitatively confirmed using liquid chromatography tandem mass spectrometry. The cases were received between February 2020 and April 2021. Concentrations of 2-methyl AP-237 from four postmortem cases ranged from 820 to 5800 ng/mL, and concentrations of AP-238 from two postmortem cases were 87 and 120 ng/mL. µ-Opioid receptor (MOR) activation potential for 2-methyl AP-237, AP-237, para-methyl AP-237, and AP-238 were studied using a ßarr2 recruitment assay. Efficacies (Emax, relative to hydromorphone) and potencies (EC50) were derived and of the compounds tested AP-238 was the most potent compound in the panel with an EC50 of 248 nM. 2-Methyl AP-237 was found to be the most efficacious drug (Emax = 125%) of the tested cinnamylpiperazines; however, it had substantially less efficacy than fentanyl. The in vitro MOR activation potential of the studied cinnamylpiperazines was lower than that of fentanyl and other novel synthetic opioids (NSOs), in line with the relatively higher concentrations observed in postmortem toxicology samples-an important observational link between in vitro pharmacology and in vivo toxicology.

Pharmacological evaluation and forensic case series of N-pyrrolidino etonitazene (etonitazepyne), a newly emerging 2-benzylbenzimidazole 'nitazene' synthetic opioid.

Novel synthetic opioids continue to emerge on recreational drug markets worldwide. In response to legislative bans on fentanyl analogues, non-fentanyl structural templates, such as 2-benzylbenzimidazoles ('nitazenes'), are being exploited to create new µ-opioid receptor (MOR) agonists. Here, we pharmacologically characterize an emerging cyclic analogue of etonitazene, called N-pyrrolidino etonitazene (etonitazepyne), using in vitro and in vivo methods. A series of analytically confirmed fatalities is described to complement preclinical findings. Radioligand binding assays in rat brain tissue revealed that N-pyrrolidino etonitazene has high affinity for MOR (Ki = 4.09 nM) over δ-opioid (Ki = 959 nM) and κ-opioid (Ki = 980 nM) receptors. In a MOR-ß-arrestin2 activation assay, N-pyrrolidino etonitazene displayed high potency (EC50 = 0.348 nM), similar to etonitazene (EC50 = 0.360 nM), and largely exceeding the potencies of fentanyl (EC50 = 14.9 nM) and morphine (EC50 = 290 nM). When administered s.c. to male Sprague Dawley rats, N-pyrrolidino etonitazene induced opioid-like antinociceptive, cataleptic, and thermic effects. Its potency in the hot plate test (ED50 = 0.0017 mg/kg) was tenfold and 2,000-fold greater than fentanyl (ED50 = 0.0209 mg/kg) and morphine (ED50 = 3.940 mg/kg), respectively. Twenty-one overdose fatalities associated with N-pyrrolidino etonitazene were found to contain low blood concentrations of the drug (median = 2.2 ng/mL), commonly in the context of polysubstance use. N-Pyrrolidino etonitazene was reported as a cause of death in at least two cases, demonstrating toxicity in humans. We demonstrate that N-pyrrolidino etonitazene is an extremely potent MOR agonist that is likely to present high risk to users. Continued vigilance is required to identify and characterize emergent 2-benzylbenzimidazoles, and other non-fentanyl opioids, as they appear in the marketplace.

Proliferation of Novel Synthetic Opioids in Postmortem Investigations After Core-Structure Scheduling for Fentanyl-Related Substances.

ABSTRACT: New generations of novel synthetic opioids (NSOs) have emerged to fill a void in the illicit drug markets left by the decline in popularity of fentanyl analogs subsequent to core-structure scheduling of fentanyl-related substances in the United States and China. These new opioids include members of the 2-benzyl benzimidazole (eg, isotonitazene, metonitazene, N -pyrrolidino etonitazene, protonitazene, etodesnitazene), benzimidazolone (eg, brorphine), and cinnamylpiperazine (eg, AP-238, 2-methyl AP-237) subclasses. Novel synthetic opioids continue to be detected in opioid-related fatal overdoses, demonstrating the harms associated with exposure to these drugs. Between January 2020 and December 2021, 384 casework blood samples were reported by our laboratory to contain 1 or more of the prior listed 8 NSOs. Isotonitazene (n = 144), metonitazene (n = 122), and brorphine (n = 91) were the 3 most prevalent substances, with positivity for isotonitazene and brorphine peaking just before the announcement of emergency scheduling. These NSOs have been documented as significant drivers of drug mortality, and this case series described here highlights the challenges medical examiners and coroners face in staying current with emerging drugs. Challenges include regional differences, rapid turnover, short lifecycles, variable toxicology testing, and difficulty in assessing individual drug toxicity in polydrug cases.

Levamisole-a Toxic Adulterant in Illicit Drug Preparations: a Review.

ABSTRACT: Discovered in the 1960s, the common anthelminthic levamisole has seen widespread use in veterinary applications. Its use rapidly expanded thereafter to include human medical treatments for a variety of acute and chronic disorders. Because of reports of severe adverse effects, the US Food and Drug Administration withdrew levamisole's approval for human use in 2000; however, medical options outside the United States and illicit options worldwide allow continued accessibility to levamisole. The compound is rapidly metabolized in the body, with at least 2 known active metabolites. Levamisole has a broad range of immunomodulatory effects, including both stimulatory and inhibitory effects on immune responses. It is generally well tolerated at therapeutic concentrations, although a variety of autoimmune-related adverse effects have been reported, including agranulocytosis, leukopenia, purpura, and visible necrotized skin tissue. Individuals with levamisole-compromised immune systems are more susceptible to infections, including COVID-19. Since the early 2000's, levamisole has been frequently used as an adulterating agent in illicit street drugs, especially cocaine, fentanyl, and heroin. Although its prevalence has varied over time and geographically, levamisole has been detected in up to 79% of the street supply of cocaine at levels up to 74% by weight. Its presence in illicit drug markets also raises concern over the potential for exposure of children and neonates, although this is supported by only limited anecdotal evidence. Levamisole is not currently included in routine drug testing panels, although a variety of confirmatory testing techniques exist across a range of antemortem and postmortem specimen options. Because of its varying presence in illicit drug markets, both the medical and forensic communities need to be aware of levamisole and its potential impact on toxicological investigations.

A Case of Unintentional Opioid (U-47700) Overdose in a Young Adult After Counterfeit Xanax Use.

ABSTRACT: We report the case of a young adult who became unresponsive after insufflating what he believed to be "crushed Xanax." Naloxone was administered, reversing his altered mental status and respiratory depression. Clinicians suspected opioid toxicity; however, the patient adamantly denied opioid use. Because of unclear etiology of his symptoms, blood and urine specimens were obtained. A urine specimen was split and then submitted for a clinical comprehensive drug screen using gas chromatography-mass spectrometry. The blood specimen and the remaining urine specimen were sent to a reference laboratory for analysis using liquid chromatography quadrupole time-of-flight mass spectrometry and liquid chromatography tandem mass spectrometry. The standard, clinical gas chromatography-mass spectrometry urine drug testing procedure only detected caffeine; however, analysis by liquid chromatography quadrupole time-of-flight mass spectrometry and liquid chromatography tandem mass spectrometry confirmed the presence of U-47700 (a high-potency clandestine opioid) and its metabolites in the urine and blood. These findings implicate U-47700 as the agent responsible for the patient's signs of opioid toxicity. In this case, a young adult intending to use alprazolam encountered U-47700 with life-threatening effect. Clinicians must remain vigilant for symptoms consistent with opioid overdose, especially with increasing prevalence of counterfeit drugs containing clandestine opioids. Clinicians must also consider obtaining specimens for appropriate analytical testing to improve surveillance and facilitate public health interventions.

Metabolic profiling of synthetic cannabinoid 5F-ADB and identification of metabolites in authentic human blood samples via human liver microsome incubation and ultra-high-performance liquid chromatography/high-resolution mass spectrometry.

RATIONALE: Indazole carboxamide synthetic cannabinoids, a prevalent class of recreational drugs, are a major clinical, forensic and public health challenge. One such compound, 5F-ADB, has been implicated in fatalities worldwide. Understanding its metabolism and distribution facilitates the development of laboratory assays to substantiate its consumption. Synthetic cannabinoid metabolites have been extensively studied in urine; studies identifying metabolites in blood are limited and no data on the metabolic stability (half-life, clearance and extraction ratio) of 5F-ADB have been published prior to this report. METHODS: The in vitro metabolism of 5F-ADB was elucidated via incubation with human liver microsomes for 2 h at 37°C. Samples were collected at multiple time points to determine its metabolic stability. Upon identification of metabolites, authentic forensic human blood samples underwent liquid-liquid extraction and were screened for metabolites. Extracts were analyzed via ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOFMS) operated in positive electrospray ionization mode. RESULTS: Seven metabolites were identified including oxidative defluorination (M1); carboxypentyl (M2); monohydroxylation of the fluoropentyl chain (M3.1/M3.2) and indazole ring system (M4); ester hydrolysis (M5); and ester hydrolysis with oxidative defluorination (M6). The half-life (3.1 min), intrinsic clearance (256.2 mL min-1 kg-1 ), hepatic clearance (18.6 mL min-1 kg-1 ) and extraction ratio (0.93) were determined for the first time. In blood, M1 was present in each sample as the most abundant substance; two samples contained M5; one contained 5F-ADB, M1 and M5. CONCLUSIONS: 5F-ADB is rapidly metabolized in HLM. 5F-ADB, M1 and M5 are pharmacologically active at the cannabinoid receptors (CB1 /CB2 ) and M1 and M5 may contribute to a user's impairment profile. The results demonstrate that it is imperative that synthetic cannabinoid assays screen for pharmacologically active metabolites, especially for drugs with short half-lives. The authors propose that M1 and M5 are appropriate markers to include in laboratory blood tests screening for 5F-ADB.

Phase I metabolism of synthetic cannabinoid receptor agonist PX-1 (5F-APP-PICA) via incubation with human liver microsomes and UHPLC-HRMS.

Studies of the metabolic and pharmacological profiles of indole carboxamide synthetic cannabinoids (a prevalent class of new psychoactive substances) are critical in ensuring that their use can be detected through bioanalytical testing. We have determined the in vitro Phase I metabolism of one such compound, PX-1 (5F-APP-PICA), and appropriate markers to demonstrate human consumption. PX-1 was incubated with human liver microsomes, followed by analysis of the extracts via high-resolution mass spectrometry. A total of 10 metabolites were identified, with simultaneous defluorination and monohydroxylation of the pentyl side chain as the primary biotransformation product (M1). Additional metabolites formed were hydroxylation products of the indole and benzyl moieties, distal amide hydrolysis, N-desfluoropentyl, and carboxypentyl metabolites. Three monohydroxylated metabolites specific to PX-1 were identified and are reported for the first time in this study. The primary metabolite, M1, was further oxidized to M5, a carboxypentyl metabolite. M8 is PX-1 specific, possessing an intact fluoropentyl side chain. These three metabolites are the most suitable for implementation into bioanalytical assays for demonstrating PX-1 consumption. The findings of this study can be used by analytical scientists and medical professionals to determine PX-1 ingestion and predict the metabolites of synthetic cannabinoids sharing structural elements.

Assessment of synthetic cannabinoid FUB-AMB and its ester hydrolysis metabolite in human liver microsomes and human blood samples using UHPLC-MS/MS.

FUB-AMB, an indazole carboxamide synthetic cannabinoid recreational drug, was one of the compounds most frequently reported to governmental agencies worldwide between 2016 and 2019. It has been implicated in intoxications and fatalities, posing a risk to public health. In the current study, FUB-AMB was incubated with human liver microsomes (HLM) to assess its metabolic fate and stability and to determine if its major ester hydrolysis metabolite (M1) was present in 12 authentic forensic human blood samples from driving under the influence of drug cases and postmortem investigations using UHPLC-MS/MS. FUB-AMB was rapidly metabolized in HLM, generating M1 that was stable through a 120-min incubation period, a finding that indicates a potential long detection window in human biological samples. M1 was identified in all blood samples, and no parent drug was detected. The authors propose that M1 is a reliable marker for inclusion in laboratory blood screens for FUB-AMB; this metabolite may be pharmacologically active like its precursor FUB-AMB. M1 frequently appears in samples in which the parent drug is undetectable and can point to the causative agent. The results suggest that it is imperative that synthetic cannabinoid laboratory assay panels include metabolites, especially known or potential pharmacologically active metabolites, particularly for compounds with short half-lives.

Bromazolam in Impaired Driving Investigations.

The designer benzodiazepine bromazolam is increasingly encountered in forensic casework, including impaired driving investigations. A series of suspected impaired driving cases that tested positive for bromazolam are described herein along with information about driving performance, driver appearance and observed behavior. Bromazolam was indicated in casework either through screening by liquid chromatography time of flight mass spectrometry (LC-TOF/MS) and/or a positive benzodiazepine immunoassay screen. Blood samples were forwarded for quantitative confirmatory analysis using a liquid chromatography tandem mass spectrometry (LC-MS/MS) method with a reporting limit of 2.0 ng/mL. Bromazolam was reported in 98 impaired driving cases from samples reported between January 2021 and December 2023, with the earliest detection from September 2020. Mean and median blood concentrations were 125 ± 145 ng/mL and 84 ng/mL respectively, with a range of 4.2 - 990 ng/mL. Additional positive findings were reported in almost all cases, with the highest result (990 ng/mL) being the only case in which bromazolam was the only finding. Fentanyl was the most frequent drug found in combination with bromazolam. Driving behaviors reported in these cases included erratic driving, errors in Standardized Field Sobriety Tests (SFSTs), and symptoms consistent with Central Nervous System (CNS) depressants, including slurred speech, incoordination, and lethargic behavior. Based on its prevalence and demonstrated impairing effects, bromazolam should be included in the scope of impaired driving testing as long as it continues to be prevalent in the drug supply.

Determination of fentanyl contamination on United States paper currency by LC-QQQ-MS.

Previous research has evaluated the extent to which cocaine and other drugs were detectable on currency in the USA. The literature was in agreement that the majority of bills exhibited some degree of contamination. With the increase of fentanyl in the illicit drug supply, this study was designed to evaluate the extent that fentanyl, cocaine, methamphetamine and other substances were present on circulating currency in 2022. A quantitative assay using liquid chromatography-triple quadrupole mass spectrometry was developed and validated to detect six analytes: fentanyl, 4-anilino-N-phenethylpiperidine, acetylfentanyl, benzylfentanyl, cocaine and methamphetamine. One-dollar bills were collected from 13 cities across the country. Sample preparation consisted of soaking the bills in methanol followed by liquid-liquid extraction. Chromatographic separation was achieved using a C18 analytical column and gradient elution with ammonium formate in water (5 mM, pH 3) and 0.1% formic acid in acetonitrile. The quantitative working range for this assay was 0.1 µg to 1.0 µg per bill (equivalent to 1 ng/mL to 100 ng/mL of extract). Fentanyl was detected on the majority (63%) of samples, with 61% of samples having ≥0.1 µg of fentanyl and 4% of samples having ≥1.0 µg. Cocaine and methamphetamine were detected on 100% and 98% of bills, respectively, typically in amounts >1.0 µg. The remaining fentanyl-related substances were detected in 15% of samples in amounts no >0.69 µg per bill and exclusively in the presence of fentanyl. Unsurprisingly, areas of the country with higher incidence of fentanyl use yielded higher frequency of contaminated bills and higher concentrations. Human exposure to drugs on currency is unlikely to have any significant impacts toxicologically or pharmacologically; however, our research findings suggest that paper currency could serve as a useful substrate for surveillance of drug trends regionally, nationally and/or internationally.

Evaluating cross-reactivity of new psychoactive substances (NPS) in human whole blood by enzyme-linked immunosorbent assay (ELISA).

Due to the increase in the use of novel psychoactive substances (NPS) and their overall prevalence, it is important to have effective and reliable screening technologies to detect NPS in biological matrices. Enzyme-linked immunosorbent assays (ELISA) are among the most popular screening methods. To evaluate the effectiveness of ELISA for NPS detection, five subclasses of NPS (novel synthetic opioids, fentanyl analogs, stimulants, benzodiazepines and hallucinogens) were evaluated in whole blood for their cross-reactivity on commercially available ELISA kits. A variety of novel synthetic opioids were tested at concentrations of 1-80 ng/mL and 50-2000 ng/mL and demonstrated no cross-reactivity to a morphine ELISA plate at either concentration range. Fentanyl analogs were tested at concentrations ranging from 0.01 to 1 ng/mL and had cross-reactivities ranging from 8% to 178% on the fentanyl ELISA kit used. Both para-chloro fentanyl (178%) and acryl fentanyl (164%) showed cross-reactivities well above that of fentanyl. Novel stimulants were tested at concentrations of 0.5-40 ng/mL and 20-2,000 ng/mL. 4-Fluoroamphetamine was the only novel stimulant with cross-reactivity (3,354%) to the amphetamine ELISA plate. Novel benzodiazepines were tested at concentrations of 1-40 ng/mL on a benzodiazepine plate. Cross-reactivities ranged from 36.1% to 263%, with desalkylflurazepam having the highest cross-reactivity. Finally, novel hallucinogens were tested at concentrations of 0.5-10 ng/mL on a phencyclidine (PCP) ELISA plate, which produced no cross-reactivity and then with 10-1,000 ng/mL, which gave results from 56.6% to 151%. Both hydroxy-PCP (151%) and chloro-PCP (137%) showed cross-reactivities above that of PCP. This research has demonstrated the utility of using ELISA-based screening for novel benzodiazepines, hallucinogens and for fentanyl analogs; however, there is limited application and risk of false-negative results for the other drug classes due to low or non-existent cross-reactivities.

Development and validation of a quantitative method for the analysis of delta-9-tetrahydrocannabinol (delta-9-THC), delta-8-tetrahydrocannabinol (delta-8-THC), delta-9-tetrahydrocannabinolic acid (THCA), and cannabidiol (CBD) in botanicals, edibles, liquids, oils, waxes, and bath products by gas chromatography mass spectrometry (GC/MS).

A quantitative gas chromatography mass spectrometry (GC/MS) method was developed for delta-9-tetrahydrocannabinol (delta-9-THC), delta-8-tetrahydrocannabinol (delta-8-THC), tetrahydrocannabinolic acid (THCA), and cannabidiol (CBD) in matrices including plant material, liquids and oils, waxes, edibles, and bath and body products. Samples were prepared by homogenization, extraction of the cannabinoids into solvent, liquid/liquid extraction, and derivatization. The GC/MS method was validated from 0.15% to 5.00% (weight basis) to encompass the 0.3% legal distinction between hemp and marijuana. Validation was performed assessing imprecision/bias, calibration model, recovery, interferences, limit of detection, matrix matching, carryover, accuracy, and an assessment of CBD conversion to delta-9-THC. The calibration curves were quadratic weighted 1/x with r2 > 0.990. The method had a detection limit of 0.075% in plant material for each analyte. Analyte recovery was greater than 70% in plant material. Carryover was not observed up to concentrations equivalent to 100% analyte, and no forensically significant conversion of CBD to delta-9-THC was observed. One cannabinoid isomer, 9(R)-delta-7-tetrahydrocannabinol (9(R)-delta-7-THC), was determined to interfere with the quantitation of delta-9-THC, but could be differentiated based on mass spectrum. The method was determined to be suitable for quantitation of delta-9-THC, delta-8-THC, delta-9-THCA, and CBD and was able to differentiate hemp samples from marijuana samples.

Four-year evaluation of drug-impaired driving drug concentrations.

Drug-impaired driving is a significant public health and safety concern in the USA. To help assess current patterns of drug use in drivers, we evaluated 4 years of drug positivity in a large cohort of suspected impaired drivers. Samples collected between January 2017 and December 2020 were tested via a method compliant with the National Safety Council's Alcohol, Drugs, and Impairment Division's Tier I scope of recommended drugs. In 2017, NMS Labs received 17 346 driving under the influence of drugs cases, 17 471 in 2018, 19 050 in 2019, and 16 539 in 2020. The most common drug class detected was cannabinoids in ∼50% of the cases each year. The most common drugs detected over the 4 years were delta-9 tetrahydrocannabinol (delta-9 THC), ethanol, amphetamine/methamphetamine, fentanyl, and alprazolam. Delta-9 THC increased in positivity over the study, having been identified in 45% of cases in 2017, 46% in 2018, 46% in 2019, and 49% in 2020. Ethanol was found in 59% of cases in 2017, 59% in 2018, 61% in 2019, and 53% in 2020. Delta-9 THC and ethanol were the most common drug combination, found together in ∼19% of the cases every year of the study. Statistically significant increases in the average concentration of the following drugs were observed: fentanyl (5.7 ng/mL in 2017 to 9.6 ng/mL in 2020), methamphetamine (301 ng/mL in 2017 to 381 ng/mL in 2020), and delta-9-THC (6.4 ng/mL in 2017 to 7.3 ng/mL in 2020). Other findings included increases in the maximum reported concentrations between 2017 and 2020 for amphetamine (1400 to 2700 ng/mL), methamphetamine (5550 to 13 000 ng/mL), and fentanyl (56 to 310 ng/mL). Statistically significant concentration decreases were noted for several central nervous system depressants, notably prescription benzodiazepines, and several prescription narcotic analgesics.

Prevalence and predictors of HIV among patients presenting to US emergency departments with opioid overdose.

INTRODUCTION: Opioid overdose deaths in the U.S. have risen dramatically in the past decade, largely due to the surge in illicitly manufactured fentanyl. Injection drug use is a known risk factor for HIV, further complicating the long-term consequences of opioid use. The baseline prevalence of HIV among adults in the US is 0.46 %. The primary purpose of this study was to determine the prevalence and risk factors of HIV among patients presenting to the emergency departments (ED) with an acute opioid overdose. METHODS: This study is a prospective observational cohort study from the ToxIC Fentalog Study group. Patients age 18 years of age or older are included if they present to one of 10 participating U.S. hospitals in 9 states between September 2020 and May 2023 with a suspected opioid overdose and had waste serum available after routine laboratory testing. Clinical data is collected from the medical record and patient serum is sent for comprehensive toxicologic analysis via liquid chromatography quadrupole time-of-flight mass spectroscopy to detect the presence of over 1200 substances including illicit opioids, novel synthetic opioids, medications, and adulterants. Logistic multivariable regression was performed to examine the association between demographic, behavioral, and serum toxicology data with risk factors and HIV status. RESULTS: Among the total cohort (n=1690), 1062 cases had known HIV status (62.8 % of total sample). Among patients with a known HIV status, 60 (5.6 % [95 % CI: 4.2 %, 7.0 %]) were HIV positive. Patients with HIV reported stimulant use more frequently (13.3 %) than those without HIV (6.8 %; p=0.003). After controlling for confounding, bipolar psychiatric history was a significant independent predictor of HIV positivity (aOR: 1.08; 95 % CI: 1.02, 1.13) in this population. CONCLUSIONS: In this large multicenter cohort, the prevalence of HIV for ED patients with illicit opioid overdose was 9 times higher than that expected by the general population. Bipolar disorder appears to be a novel risk factor for HIV positivity in this patient population.

Analysis of umbilical cord tissue as an indicator of in utero exposure to toxic adulterating substances.

In utero drug exposure is a significant public health threat to the well-being and normal development of the neonate. Recently, testing of umbilical cord tissue (UCT) has been employed to measure illicit drug exposure, as drugs used by the mother during the third trimester may be retained in the UCT. Focus has also been given to potential adverse health effects among drug users, resulting from exposure to pharmacologically active adulterants and cutting agents in the street drug supply. The in utero effects of these substances have not been well studied in humans, nor has their presence been demonstrated as a means for assessing adverse health effects in the neonate. Here, we describe the application of a novel test method to analyze UCT for the presence of more than 20 common adulterating/cutting substances via LC/Q-TOF. In total, 300 de-identified UCT samples were analyzed-all had previously tested positive for cocaine or opiates. Generally, the positivity rates of individual compounds were similar between the Cocaine and Opiates Subgroups, apart from levamisole, xylazine, dipyrone (metabolites), and promethazine. Many of the adulterants used in the street drug supply do have legitimate medicinal/therapeutic uses, including several of the compounds most frequently detected in this study. Caffeine and lidocaine were the most frequently identified compounds both individually (>70% each) and in combination with each other. Alternatively, levamisole, an adulterant with no legitimate therapeutic use, was present in 12% of cases. Importantly, this data demonstrates that the detection of traditional drugs of abuse may serve as indicators of potential in utero exposure to toxic adulterating substances during gestation. While there is cause for concern with respect to any unintentional drug exposure, illicit drug use during pregnancy, including uncontrolled dosing, poly-adulterant consumption, and the interactions of these drug mixtures, produces a significant public health threat to the neonate which warrants further study.

Plasma pharmacokinetics and pharmacodynamic effects of the 2-benzylbenzimidazole synthetic opioid, isotonitazene, in male rats.

RATIONALE: Isotonitazene is an illicit synthetic opioid associated with many intoxications and fatalities. Recent studies show that isotonitazene is a potent µ-opioid receptor (MOR) agonist in vitro, but little information is available about its in vivo effects. OBJECTIVES: The aims of the present study were to investigate the pharmacokinetics of isotonitazene in rats, and relate pharmacokinetic parameters to pharmacodynamic effects. METHODS: Isotonitazene and its metabolites were identified and quantified by liquid chromatography tandem quadrupole mass spectrometry (LC-QQQ-MS). Male Sprague-Dawley rats with jugular catheters and subcutaneous (s.c.) temperature transponders received isotonitazene (3, 10, 30 µg/kg, s.c.) or its vehicle. Blood samples were drawn at 15, 30, 60, 120, and 240 min post-injection, and plasma was assayed using LC-QQQ-MS. At each blood draw, body temperature, catalepsy scores, and hot plate latencies were recorded. RESULTS: Maximum plasma concentrations of isotonitazene rose in parallel with increasing dose (range 0.2-9.8 ng/mL) and half-life ranged from 23.4 to 63.3 min. The metabolites 4'-hydroxy nitazene and N-desethyl isotonitazene were detected, and plasma concentrations were below the limit of quantitation (0.5 ng/mL) but above the limit of detection (0.1 ng/mL). Isotonitazene produced antinociception (ED50 = 4.22 µg/kg), catalepsy-like symptoms (ED50 = 8.68 µg/kg), and hypothermia (only at 30 µg/kg) that were significantly correlated with concentrations of isotonitazene. Radioligand binding in rat brain tissue revealed that isotonitazene displays nM affinity for MOR (Ki = 15.8 nM), while the N-desethyl metabolite shows even greater affinity (Ki = 2.2 nM). CONCLUSIONS: In summary, isotonitazene is a potent MOR agonist whose pharmacodynamic effects are related to circulating concentrations of the parent drug. The high potency of isotonitazene portends substantial risk to users who are exposed to the drug.

N,N-Dimethylpentylone (dipentylone)-A new synthetic cathinone identified in a postmortem forensic toxicology case series.

Synthetic cathinones emerged on the novel psychoactive substance (NPS) drug market as alternatives to controlled stimulants and entactogens such as methamphetamine and 3,4-methylenedioxymethamphetamine. The majority of synthetic cathinones can be subclassified into two groups: beta-keto amphetamines (i.e., NPS with the suffix "drone") and beta-keto methylenedioxyamphetamines (i.e., NPS with the suffix "lone"). Although a significant number of beta-keto amphetamines have been identified, beta-keto methylenedioxyamphetamines have dominated the NPS market, including notable drugs like methylone, butylone, N-ethyl pentylone (ephylone), eutylone and now N,N-dimethylpentylone. N,N-Dimethylpentylone, also known as dipentylone or beta-keto-dimethylbenzodioxolylpentanamine, emerged into the illicit drug supply <2 months of the international control of eutylone (September 2021). A novel standard addition method was developed and validated for N,N-dimethylpentylone, pentylone and eutylone, and 18 postmortem cases were quantitated using the method described in this manuscript. The resulting blood concentration range for N,N-dimethylpentylone in this case series was 3.3 to 970 ng/mL (median: 145 ng/mL, mean: 277 ± 283 ng/mL). Pentylone, a metabolite of N,N-dimethylpentylone, was detected in all cases (range: 1.3-420 ng/mL, median: 31 ng/mL and mean: 88 ± 127 ng/mL). Due to the rise in identifications of N,N-dimethylpentylone in postmortem investigations as well as the potential misidentification of N,N-dimethylpentylone as N-ethyl pentylone, samples testing positive for pentylone should be additionally confirmed for the presence of N,N-dimethylpentylone. Based on prior trends of new synthetic cathinones, it can be theorized that N,N-dimethylpentylone may predominate the US synthetic stimulant market for the next 1-2 years; however, given the emergence of additional closely related isomeric compounds, it is important to utilize methodology capable of differentiating N,N-dimethylpentylone from its isomers (N-isopropylbutylone, N-ethyl pentylone, N-ethyl N-methyl butylone, hexylone, N-propylbutylone, diethylone and tertylone).