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).

Detection, chemical analysis, and pharmacological characterization of dipyanone and other new synthetic opioids related to prescription drugs.

The emergence of structurally diverse new synthetic opioids (NSOs) has caused the opioid crisis to spiral to new depths. Little information is available about the pharmacology of most novel opioids when they first emerge. Here, using a ß-arrestin 2 recruitment assay, we investigated the in vitro µ-opioid receptor (MOR) activation potential of dipyanone, desmethylmoramide, and acetoxymethylketobemidone (O-AMKD) - recent NSOs that are structurally related to the prescription opioids methadone and ketobemidone. Our findings indicate that dipyanone (EC50=39.9 nM; Emax=155% vs. hydromorphone) is about equally active as methadone (EC50=50.3 nM; Emax=152%), whereas desmethylmoramide (EC50=1335 nM; Emax=126%) is considerably less active. A close structural analogue of ketobemidone (EC50=134 nM; Emax=156%) and methylketobemidone (EC50=335 nM; Emax=117%), O-AMKD showed a lower potency (EC50=1262 nM) and efficacy (Emax=109%). Evaluation of the opioid substitution product buprenorphine and its metabolite norbuprenorphine confirmed the increased in vitro efficacy of the latter. In addition to in vitro characterization, this report details the first identification and full chemical analysis of dipyanone in a seized powder, as well as a postmortem toxicology case from the USA involving the drug. Dipyanone was quantified in blood (370 ng/mL), in which it was detected alongside other NSOs (e.g., 2-methyl AP-237) and novel benzodiazepines (e.g., flualprazolam). While dipyanone is currently not commonly encountered in forensic samples worldwide, its emergence is worrisome and representative of the dynamic NSO market. Graphical Abstract.

Opioid overdoses involving xylazine in emergency department patients: a multicenter study.

INTRODUCTION: Illicit opioids, consisting largely of fentanyl, novel synthetic opioids, and adulterants, are the primary cause of drug overdose fatality in the United States. Xylazine, an alpha-2 adrenergic agonist and veterinary tranquilizer, is being increasingly detected among decedents following illicit opioid overdose. Clinical outcomes in non-fatal overdose involving xylazine are unexplored. Therefore, among emergency department patients with illicit opioid overdose, we evaluated clinical outcome differences for patients with and without xylazine exposures. METHODS: This multicenter, prospective cohort study enrolled adult patients with opioid overdose who presented to one of nine United States emergency departments between 21 September 2020, and 17 August 2021. Patients with opioid overdose were screened and included if they tested positive for an illicit opioid (heroin, fentanyl, fentanyl analog, or novel synthetic opioid) or xylazine. Patient serum was analyzed via liquid chromatography quadrupole time-of-flight mass spectroscopy to detect current illicit opioids, novel synthetic opioids, xylazine and adulterants. Overdose severity surrogate outcomes were: (a) cardiac arrest requiring cardiopulmonary resuscitation (primary); and (b) coma within 4 h of arrival (secondary). RESULTS: Three hundred and twenty-one patients met inclusion criteria: 90 tested positive for xylazine and 231 were negative. The primary outcome occurred in 37 patients, and the secondary outcome occurred in 111 patients. Using multivariable regression analysis, patients positive for xylazine had significantly lower adjusted odds of cardiac arrest (adjusted OR 0.30, 95% CI 0.10-0.92) and coma (adjusted OR 0.52, 95% CI 0.29-0.94). CONCLUSIONS: In this large multicenter cohort, cardiac arrest and coma in emergency department patients with illicit opioid overdose were significantly less severe in those testing positive for xylazine.

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.

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.

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.

A Forward-Thinking Approach to Addressing the New Synthetic Opioid 2-Benzylbenzimidazole Nitazene Analogs by Liquid Chromatography-Tandem Quadrupole Mass Spectrometry (LC-QQQ-MS).

Novel psychoactive substances (NPS) continue to represent a threat to public health and safety. The number of new drugs in the latest emergent synthetic opioid class-the 2-benzylbenzimidazole analogs-also called the nitazenes-has begun to dominate the current new synthetic opioid (NSO) subclass of NPS. We describe a liquid chromatography-tandem quadrupole mass spectrometry method for the quantification of nine analogs and/or metabolites of drugs in this series: isotonitazene, metonitazene, protonitazene, etonitazene, clonitazene, flunitazene, N-desethyl isotonitazene, 5-amino isotonitazene and 4'-hydroxy nitazene in human whole blood, urine, and tissue. Samples were prepared for analysis using a basic liquid-liquid extraction. Chromatographic separation was achieved using a C-18 analytical column. Multiple reaction monitoring mode was used for detection. The calibration range for the analytes was 0.5-50 ng/mL (except for 5-amino isotonitazene, which was 1.0-50 ng/mL). The limit of detection was 0.1 ng/mL, and the limit of quantitation was 0.5 ng/mL. The method had no carryover or interferences. Ionization enhancement was observed but did not affect quantitation. All analytes passed the method validation assessment. Authentic human samples suspected of containing NSOs were obtained from a medical examiner and coroner offices, as well as partnering forensic toxicology laboratories. Isotonitazene was confirmed in 92 blood samples, and its metabolites were confirmed across various matrices. Metonitazene (n = 35), flunitazene (n = 5), protonitazene (n = 3), etodesnitazene (n = 2) and butonitazene (n = 1) were also detected in cases. These newly emerging 2-benzylbenzimidazole analogs were commonly found in combination with NPS benzodiazepines and opioids (e.g., flualprazolam, fentanyl). Nitazene analogs are potent esoteric drugs that may not be identified during routine toxicological screening, and specialized assays based on sensitive instrumentation are needed to accurately characterize these NSOs.

Pharmacokinetics and pharmacodynamics of the synthetic cannabinoid, 5F-MDMB-PICA, in male rats.

5F-MDMB-PICA is a popular synthetic cannabinoid associated with analytically confirmed intoxications. In vitro studies show 5F-MDMB-PICA is a potent cannabinoid-1 receptor (CB1) agonist, but little information is available about in vivo pharmacokinetics and pharmacodynamics. To this end, the present study had three aims: 1) to develop a validated method for detection of 5F-MDMB-PICA and its metabolites in rat plasma, 2) to utilize the method for investigating pharmacokinetics of 5F-MDMB-PICA in rats, and 3) to relate 5F-MDMB-PICA pharmacokinetics to pharmacodynamic effects. 5F-MDMB-PICA and its metabolites were quantified using liquid chromatography tandem mass spectrometry (LC-MS/MS) and method validation followed forensic standards. Male Sprague-Dawley rats bearing surgically implanted jugular catheters and subcutaneous (s.c.) temperature transponders received 5F-MDMB-PICA (50, 100, or 200 µg/kg, s.c.) or its vehicle. Blood samples were drawn at 15, 30, 60, 120, 240, and 480 min post-injection, and plasma was assayed using LC-MS/MS. At each blood draw, body temperature, and catalepsy scores were recorded. Maximum plasma concentrations (Cmax) of 5F-MDMB-PICA rose linearly with increasing dose (1.72-6.20 ng/mL), and plasma half-life (t1/2) ranged from 400 to 1000 min 5F-MDMB-PICA-3,3-dimethylbutanoic acid and 5OH-MDMB-PICA were the only metabolites detected, and plasma concentrations were much lower than the parent drug. 5F-MDMB-PICA induced robust hypothermia and catalepsy-like symptoms that were significantly correlated with concentrations of 5F-MDMB-PICA. Radioligand binding in rat brain membranes revealed 5F-MDMB-PICA displays high affinity for CB1 (IC50 = 2 nM) while metabolites do not. In summary, 5F-MDMB-PICA is a potent CB1 agonist in rats whose pharmacodynamic effects are related to circulating concentrations of the parent drug and not its metabolites.

Metonitazene in the United States-Forensic toxicology assessment of a potent new synthetic opioid using liquid chromatography mass spectrometry.

Metonitazene is considered a new psychoactive substance (NPS) and emerging potent synthetic opioid, causing increased public health concern beginning in 2020. Metonitazene joins a growing list of new synthetic opioids (NSOs) contributing to deaths among people who use drugs in the United States and other parts of the world. Metonitazene (a 2-benzylbenzimidazole analogue) first appeared in mid-2020 in the recreational drug supply and subsequently began proliferating in death investigation casework towards the end of 2020. Screening and metabolite discovery were performed by liquid chromatography quadrupole time-of-flight mass spectrometry. Quantitative confirmation was performed by liquid chromatography tandem quadrupole mass spectrometry. Metonitazene was confirmed in 20 authentic forensic postmortem cases with an average concentration in blood at 6.3 ± 7.5 ng/ml (median: 3.8 ng/ml, range: 0.5-33 ng/ml, n = 18) and in urine at 15 ± 13 ng/ml (median: 11 ng/ml, range: 0.6-46 ng/ml, n = 14). Metonitazene was the only opioid identified in 30% of cases but was also found in combination with fentanyl (55%) and NPS benzodiazepines, opioids, and hallucinogens (45%). Medical examiners included metonitazene as a drug responsible for the cause of death, and the manner of death was always ruled to be an accident. The metabolism of metonitazene was found to be similar to that of isotonitazene, a closely related analogue. Toxicology laboratories and death investigators should ensure that metonitazene is included in forensic testing protocols, all while remaining vigilant for subsequent NSOs to emerge.