Rate of onset of dopamine transporter inhibitors assessed with intracranial self-stimulation and in vivo dopamine photometry in rats.

Drug self-administration and intracranial self-stimulation (ICSS) are two preclinical behavioral procedures used to predict abuse potential of drugs, and abuse-related drug effects in both procedures are thought to depend on increased mesolimbic dopamine (DA) signaling. Drug self-administration and ICSS yield concordant metrics of abuse potential across a diverse range of drug mechanisms of action. The "rate of onset," defined as the velocity with which a drug produces its effect once administered, has also been implicated as a determinant of abuse-related drug effects in self-administration procedures, but this variable has not been systematically examined in ICSS. Accordingly, this study compared ICSS effects produced in rats by three DA transporter inhibitors that have different rates of onset (fastest to slowest: cocaine, WIN-35428, RTI-31) and that produced progressively weaker metrics of abuse potential in a drug self-administration procedure in rhesus monkeys. Additionally, in vivo photometry using the fluorescent DA sensor dLight1.1 targeted to the nucleus accumbens (NAc) was used to assess the time course of extracellular DA levels as a neurochemical correlate of behavioral effects. All three compounds produced ICSS facilitation and increased DA levels assessed by dLight. In both procedures, the rank order of onset rate was cocaine > WIN-35428 > RTI-31; however, in contrast to monkey drug self-administration results, maximum effects did not differ across compounds. These results provide additional evidence that drug-induced increases in DA drive ICSS facilitation in rats and illustrate the utility of both ICSS and photometry to evaluate the time course and magnitude of abuse-related drug effects in rats.

A Strategy to Prioritize Emerging Drugs of Abuse for Analysis: Abuse Liability Testing Using Intracranial Self-Stimulation (ICSS) in Rats and Validation with α-Pyrrolidinohexanophenone (α-PHP).

Novel psychoactive substances (NPS) threaten public health and safety while also straining the limited resources of forensic laboratories. To efficiently allocate the finite resources available, we propose a new strategy for prioritizing NPS with abuse liability testing using a preclinical behavioral procedure in rats known as intracranial self-stimulation (ICSS). To validate this assay, the recently-scheduled synthetic cathinone α-PHP was compared to cocaine, a mechanistically similar drug of abuse, as a positive control and saline as a negative control. Male Sprague-Dawley rats (n=6) were implanted with electrodes targeting the medial forebrain bundle and trained to respond by lever-press for electrical brain stimulation. The rats were tested with doses of 0.32, 1.0, and 3.2 mg/kg α-PHP as well as 10 mg/kg of cocaine and saline administered by intraperitoneal injection. Neither saline nor 0.32 mg/kg α-PHP altered ICSS response rates compared to baseline levels of responding; however, doses of 1.0 and 3.2 mg/kg α-PHP and 10 mg/kg cocaine facilitated ICSS responding. This ICSS profile suggests that α-PHP has high abuse potential, with a rapid onset of effects and a long duration of action, and supports the decision to schedule this compound. This study demonstrates the ability of ICSS to distinguish between compounds of low and high potential for abuse. A strategy is proposed here to screen NPS using ICSS and classify emerging drugs into four priority categories for further analysis.

Driving Impairment Cases Involving Etizolam and Flubromazolam.

This study describes 12 cases of drivers stopped for impaired driving, where a designer benzodiazepine was detected, specifically etizolam or flubromazolam. Etizolam was detected in three cases, with blood concentrations ranging from 40 to 330 ng/mL. Two of these cases had low concentrations of methamphetamine and/or amphetamine, and in the third case tetrahydrocannabinol (THC) was detected. Flubromazolam was detected in nine cases; in all cases, at least one other drug was detected, with THC being the most prevalent. The mean blood concentration of flubromazolam was 16.3 ng/mL and had a median concentration of 17.0 ng/mL, ranging from 7.0 to 31 ng/mL. The low concentrations of designer benzodiazepines that produce pharmacological effects may allow many of these drugs to go undetected using routine testing in laboratories; therefore, it is necessary to include these novel compounds within validated analytical methods to reduce the chance of reporting false negative results. The prevalence in which laboratories are detecting the presence of novel benzodiazepines in impaired drivers illustrates the increased threat to public safety. These case studies demonstrate the importance of investigating agencies and forensic laboratories to be vigilant in monitoring the emerging novel psychoactive substances in their region.

Investigation of the Optical Isomers of Methcathinone, and Two Achiral Analogs, at Monoamine Transporters and in Intracranial Self-Stimulation Studies in Rats.

Methcathinone (MCAT; 1), the progenitor of numerous and widely abused "synthetic cathinone" central stimulants, exists as a pair of optical isomers. Although S(-)MCAT is several-fold more potent than R(+)MCAT in rodent locomotor stimulation and in stimulus generalization studies in rat drug discrimination assays, the individual optical isomers of MCAT have never been directly compared for their actions at monoamine transporters that seem to underlie their actions and have never been examined for their relative abuse potential. Here, we found that the isomers of MCAT are nearly equieffective at dopamine and norepinephrine transporters (DAT and NET, respectively) as transporter substrates (i.e., as releasing agents) and are ≥63-fold less potent at the serotonin transporter (SERT). In intracranial self-stimulation (ICSS) studies to evaluate abuse-related drug effects in rats, S(-)MCAT was approximately twice as potent as its R-enantiomer. Achiral analogs, α-methyl MCAT (3) and α-des-methyl MCAT (4), also were DAT/NET substrates and also produced abuse-related ICSS effects, indicating that they retain abuse potential and that they might be useful for the further study of the stereochemistry of synthetic cathinone analogs with chiral ß- (or other) substituents.

Evaluation of Nicotine and the Components of e-Liquids Generated from e-Cigarette Aerosols.

Electronic cigarettes (e-cigs) deliver nicotine in an aerosol to the user that simulates the smoke of traditional cigarettes purportedly without the pathology of inhaling tobacco smoke due to the absence of combustion. Advanced versions of e-cigs enable the user to potentially moderate the concentration of drug in the aerosol by selecting from a range of voltages on the power supply. A method was developed to trap the aerosol produced by a KangerTech AeroTank, 1.8 Ω preassembled atomizer in order to analyze the concentration of nicotine and to evaluate the constituents of the aerosol at various voltages on the power supply. A 12-mg/mL formulation of nicotine in 50:50 propylene glycol (PG):vegetable glycerin (VG) was used to produce aerosol at 3.9, 4.3 and 4.7 V. The aerosol was trapped in a simple glass assemblage and analyzed by a 3200 Q Trap HPLC-MS-MS. The dose of nicotine delivered in the aerosol at 3.9, 4.3 and 4.7 V was determined to be 88 ± 12 µg, 91 ± 15 µg and 125 ± 22 µg. The average recovery of nicotine in the trap across the voltages was 99.8%. The glass trap system was an effective device for collecting the aerosol for analysis and an increase in drug yield was observed with increasing voltage from the power supply on the e-cig. The glass trap system was also used in combination with a 100-µm solid-phase microextraction fiber to capture the aerosol and analyze it via DART-MS and GC-MS. Four commercial e-liquids labeled to contain nicotine were aerosolized at 4.3 V. The pharmacologically active ingredient, nicotine, as well as PG, VG and a number of flavoring agents found in these formulations were identified.

U-47700: A Not So New Opioid.

U-47700 was developed by the Upjohn Co. in the 1970s as part of their search for a selective µ-opioid agonist with similar potency as morphine. U-47700 has re-emerged recently in the illicit drug market and is easily and cheaply obtained via the internet as well as on the street, many times falsely sold as another drug. Several fatalities from U-47700 have been reported in scientific literature, often in combination with other intoxicants. This case report describes the first death in south-central Kansas resulting solely from U-47700 intoxication: a 26-year-old white male found dead in his bedroom with apparent drug paraphernalia. Autopsy findings were consistent with opioid overdose, but toxicological examination, utilizing immunoassay and instrumental techniques, was negative for opioids. U-47700 was detected in a comprehensive alkaloid screen by GC/MS and GC-NPD, and quantitation was performed using GC-NPD on a variety of specimens to provide a full tissue distribution. Quantitation of U-47700 in this individual revealed the following: heart blood 0.26 mg/L, femoral blood 0.40 mg/L, vitreous fluid 0.09 mg/L, brain 0.38 mg/kg, liver 0.28 mg/kg and urine 4.6 mg/L.

Concentration of Nicotine and Glycols in 27 Electronic Cigarette Formulations.

Personal battery-powered vaporizers or electronic cigarettes were developed to deliver a nicotine vapor such that smokers could simulate smoking tobacco without the inherent pathology of inhaled tobacco smoke. Electronic cigarettes and their e-cigarette liquid formulations are virtually unregulated. These formulations are typically composed of propylene glycol and/or glycerin, flavoring components and an active drug, such as nicotine. Twenty-seven e-cigarette liquid formulations that contain nicotine between 6 and 22 mg/L were acquired within the USA and analyzed by various methods to determine their contents. They were screened by Direct Analysis in Real Time™ Mass Spectrometry (DART-MS). Nicotine was confirmed and quantitated by high-performance liquid chromatography-tandem mass spectrometry, and the glycol composition was confirmed and quantitated by gas chromatography-mass spectrometry. The DART-MS screening method was able to consistently identify the exact mass peaks resulting from the protonated molecular ion of nicotine, glycol and a number of flavor additives within 5 mmu. Nicotine concentrations were determined to range from 45 to 131% of the stated label concentration, with 18 of the 27 have >10% variance. Glycol composition was generally accurate to the product description, with only one exception where the propylene glycol to glycerin percentage ratio was stated as 50:50 and the determined concentration of propylene glycol to glycerin was 81:19 (% v/v). No unlabeled glycols were detected in these formulations.