Systematic web monitoring of drug test subversion strategies in the United States.

As negative drug tests are frequently a condition for employment, some people who use drugs will try to subvert the testing. In this study, systematic web monitoring was used to investigate how drug test subversion is discussed online. Posts pertaining to drug test subversion were obtained from public websites and the dark web (n = 634, July-December 2021). Most information from public websites came from Twitter (65%), and 94% of dark web posts were from Reddit. The posts were manually coded to extract quantitative and qualitative information about drug test subversion tactics. Most posts discussed urine drug tests (85%), followed by hair (11%) and oral fluid (2%), and the most discussed drugs were marijuana (72%) and cocaine (7.3%). Urine drug test subversion mainly pertained to specimen substitution, with synthetic urine or urine from another person. Another strategy was to mask diluted urine by ingesting creatine. Urine adulteration was rarely discussed. Hair test subversion involved harsh treatments with products such as bleach, baking soda, and/or detergent. Hair removal was also discussed. Oral fluid test subversion focused on removing drugs from the oral cavity through vigorous brushing of teeth and tongue as well as the use of mouthwash, hydrogen peroxide, gum, and commercial detox products. This study highlights subversion strategies used by donors. Although little evidence was provided as to the effectiveness of these strategies, this information may help guide future studies and development of specimen validity testing to minimize the impact of drug test subversion attempts.

Evaluation of the long-term stability of select phenylacetylindole, cycloalkylindole, quinolinyl, and carboxamide synthetic cannabinoids using LC-MS-MS.

Forensic toxicology laboratories often encounter casework backlogs, which raise concerns for drug stability that can be affected by long storage times, temperature and preservatives, or the lack thereof. The focus of this research was to evaluate the impact of these factors on the stability of 17 synthetic cannabinoids (SCs) in human whole blood and 10 associated metabolites in human urine. The fortified biological specimens were stored under room temperature (20°C), refrigerator (4°C) and freezer (-20°C) conditions for a period of 52 weeks. Preservatives included potassium oxalate, sodium ethylenediaminetetraacetic acid and sodium fluoride. Extraction of analytes was conducted using supported liquid extraction and analyzed using a liquid chromatograph-tandem mass spectrometer. Under all three storage conditions, the majority of urine metabolites were stable up to 9 weeks. All analytes in frozen sodium fluoride-preserved blood were stable at 21-52 weeks with the exception of APP-PICA. Analytes in the blood that were stable up to 52 weeks in the freezer generally had a core structure of a carbonyl substituent on a pyrazole or pyrrole with surrounding nonpolar groups. In contrast, compounds with two adjacent polar carbonyl functional groups experienced degradation at ≤1 week under ambient temperature and refrigeration. 5-Fluoropentyl analogs, XLR11 and 5-fluoro ADB-PINACA, in comparison to their counterpart analytes, UR144 and ADB-PINACA, were unstable at earlier time points under all temperatures. Based on these data, forensic blood evidence suggesting the presence of SC compounds is recommended to be frozen with sodium fluoride and potassium oxalate preservatives for optimal quantitative results.

Associations between opioid overdose deaths and drugs confiscated by law enforcement and submitted to crime laboratories for analysis, United States, 2014-2019: an observational study.

Background: The overdose epidemic in the United States (US) continues to generate unprecedented levels of mortality. There is urgent need for a national data system capable of yielding high-quality, timely, and actionable information on existing and emerging drugs. Public health researchers have started using law enforcement forensic laboratory data to obtain surveillance information on illicit drugs. This study is the first to use drug reports from the entire US to examine correlations between a changing drug supply and increasing opioid-involved overdose deaths (OOD) on a national scale. Methods: This study is observational and investigates associations between law enforcement drug reports and OOD for the US from 2014 to 2019. OOD data are from the Centers for Disease Control and Prevention's National Vital Statistics System restricted-use multiple cause of death files. The US Drug Enforcement Administration's National Forensic Laboratory Information System (NFLIS) contains forensic laboratory-tested drug exhibit information for the entire US (NFLIS-Drug). Counts of forensic laboratory reports and OOD were aggregated for each state by month, quarter, and year. A difference-in-differences framework was used to estimate contemporaneous and lagged associations. Findings: Between 2014 and 2019 in the US, 249,522 OOD were reported, with the annual number nearly doubling from 28,723 to 50,179. OOD involving illicitly manufactured fentanyls (IMF) also increased substantially during this period, from 19.4% to 72.9%. In addition, 3,817,438 forensic laboratory reports in the US that were reported to NFLIS-Drug contained an opioid, stimulant, or benzodiazepine. Reports of fentanyl and fentanyl-related compounds (FFRC) had the strongest association with OOD. Each additional FFRC exhibit was associated with a 2.97% (95% CI: 1.7%, 4.1%) increase in OOD per 100,000 persons per quarter. Interpretation: Adding to the emerging consensus, protracted growth in IMF supply was more strongly associated with OOD than all other illicit drugs reported to NFLIS-Drug over the study time period. Findings demonstrate NFLIS-Drug data usefulness for research that require proxy indicators for the illicit drugs supply. A concerted effort between public health and public safety to make NFLIS-Drug more timely could strengthen its utility as a national, public health, drug surveillance system. Funding: Sangeetha Arctic Slope Mission Services, LLC, ASMS Contract No. ASM5-00017.

Understanding research methods, limitations, and applications of drug data collected by the National Forensic Laboratory Information System (NFLIS-Drug).

The National Forensic Laboratory Information System (NFLIS) is a drug surveillance program of the US Drug Enforcement Administration that systematically collects data on drugs that are seized by law enforcement and submitted to and analyzed by the Nation's forensic laboratories (NFLIS-Drug). NFLIS-Drug data are increasingly used in predictive modeling and drug surveillance to examine drug availability patterns. Given the complexity of the data and data collection, there are some common methodological pitfalls that we highlight with the aim of helping researchers avoid these concerns. The analysis done for this Technical Note is based on a review of the scientific literature that includes 428 unique, refereed article citations in 182 distinct journals published between January 1, 2005, and April 30, 2021. Each article was analyzed according to how NFLIS-Drug data were mentioned and whether NFLIS-Drug data were included. A sample of 37 articles was studied in-depth, and data issues were summarized. Using examples from the literature, this Technical Note highlights eight broad concerns that have important implications for the proper applications, interpretations, and limitations of NFLIS-Drug data with suggestions for improving research methods and accurate reporting of forensic drug data. NFLIS-Drug data are timely and provide key information to inform drug use trends across the United States; however, our present analysis shows that NFLIS-Drug data are misunderstood and represented in the literature. In addition to highlighting these issues, DEA has created several resources to assist NFLIS data users and researchers, which are summarized in the discussion.

Urinary Excretion Profile of Cannabinoid Analytes Following Acute Administration of Oral and Vaporized Cannabis in Infrequent Cannabis Users.

Traditionally, smoking has been the predominant method for administering cannabis, but alternative routes of administration have become more prevalent. Additionally, research examining urinary cannabinoid excretion profiles has primarily focused on 11-nor-9-carboxy-∆9-tetrahydrocannabinol (∆9-THC-COOH), a metabolite of ∆9-tetrahydrocannabinol (∆9-THC), as the primary analyte. The aim of the current study was to characterize the urinary excretion profile of ∆9-THC-COOH, ∆9-THC, ∆8-tetrahydrocannabinol (∆8-THC), 11-hydroxy-∆9-tetrahydrocannabinol (11-OH-∆9-THC), ∆9-tetrahydrocannabivarin (THCV), 11-nor-∆9-tetrahydrocannabivarin-9-carboxlic acid (THCV-COOH), cannabidiol (CBD), cannabinol (CBN) and 8,11-dihydroxytetrahydrocannabinol (8,11-diOH-∆9-THC) following controlled administration of both oral and vaporized cannabis. Participants (n = 21, 11 men/10 women) who were infrequent cannabis users ingested cannabis-containing brownies (0, 10 and 25 mg ∆9-THC) and inhaled vaporized cannabis (0, 5 and 20 mg ∆9-THC) across six double-blind outpatient sessions. Urinary concentrations of ∆9-THC analytes were measured at baseline and for 8 h after cannabis administration. Sensitivity, specificity and agreement between the three immunoassays (IAs) for ∆9-THC-COOH (cutoffs of 20, 50 and 100 ng/mL) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analyses (confirmatory cutoff concentrations of 15 ng/mL) were assessed. Urinary concentrations for ∆9-THC-COOH, ∆9-THC, 11-OH-∆9-THC, THCV, CBN and 8,11-diOH-∆9-THC all peaked at 5-6 h and 4 h following oral and vaporized cannabis administration, respectively. At each active dose, median maximum concentrations (Cmax) for detected analytes were quantitatively higher after oral cannabis administration compared to vaporized. Using current recommended federal workplace drug-testing criteria (screening via IA with a cutoff of ≥50 ng/mL and confirmation via LC-MS-MS at a cutoff of ≥15 ng/mL), urine specimens tested positive for ∆9-THC-COOH in 97.6% of oral sessions and 59.5% of vaporized sessions with active ∆9-THC doses. These data indicate that while ∆9-THC-COOH may serve as the most consistent confirmatory analyte under the current drug-testing guidelines, future work examining 11-OH-∆9-THC under similar parameters could yield an alternative analyte that may be helpful in distinguishing between licit and illicit cannabis products.

Assessment of cognitive and psychomotor impairment, subjective effects, and blood THC concentrations following acute administration of oral and vaporized cannabis.

BACKGROUND: Cannabis legalization is expanding, but there are no established methods for detecting cannabis impairment. AIM: Characterize the acute impairing effects of oral and vaporized cannabis using various performance tests. METHODS: Participants (N = 20, 10 men/10 women) who were infrequent cannabis users ingested cannabis brownies (0, 10, and 25 mg Δ-9-tetrahydrocannabinol, THC) and inhaled vaporized cannabis (0, 5, and 20 mg THC) in six double-blind outpatient sessions. Cognitive/psychomotor impairment was assessed with a battery of computerized tasks sensitive to cannabis effects, a novel test (the DRiving Under the Influence of Drugs, DRUID®), and field sobriety tests. Blood THC concentrations and subjective drug effects were evaluated. RESULTS: Low oral/vaporized doses did not impair cognitive/psychomotor performance relative to placebo but produced positive subjective effects. High oral/vaporized doses impaired cognitive/psychomotor performance and increased positive and negative subjective effects. The DRUID® was the most sensitive test to cannabis impairment, as it detected significant differences between placebo and active doses within both routes of administration. Women displayed more impairment on the DRUID® than men at the high vaporized dose only. Field sobriety tests showed little sensitivity to cannabis-induced impairment. Blood THC concentrations were far lower after cannabis ingestion versus inhalation. After inhalation, blood THC concentrations typically returned to baseline well before pharmacodynamic effects subsided. CONCLUSIONS: Standard approaches for identifying impairment due to cannabis exposure (i.e. blood THC and field sobriety tests) have severe limitations. There is a need to identify novel biomarkers of cannabis exposure and/or behavioral tests like the DRUID® that can reliably and accurately detect cannabis impairment at the roadside and in the workplace.

Benzodiazepines reported in NFLIS-Drug, 2015 to 2018.

The National Forensic Laboratory Information System (NFLIS) is a program of the U.S. Drug Enforcement Administration, Diversion Control Division. The NFLIS-Drug component collects drug identification results and associated information from drug cases submitted to and analyzed by federal, state, and local forensic laboratories. This paper presents national annual estimates and national and regional yearly trend differences for clonazepam, diazepam, flubromazolam, clonazolam, and etizolam using annual report rates per 100,000 persons aged 15 or older between 2015 and 2018. An estimated 263,538 benzodiazepine reports were identified by state and local laboratories between 2015 and 2018. Methamphetamine, cocaine, and heroin accounted for 32% of the drugs reported in the same item as alprazolam. Depressants and tranquilizers and narcotic analgesics were the drug classes most frequently identified in the same item as etizolam. A timeline of some benzodiazepines' emergence in NFLIS-Drug is shown, as well as state- and county-level data for selected benzodiazepines.

In vitro and in vivo pharmacological evaluation of the synthetic cannabinoid receptor agonist EG-018.

Synthetic cannabinoid receptor agonists (SCRAs) possess high abuse liability and complex toxicological profiles, making them serious threats to public health. EG-018 is a SCRA that has been detected in both illicit products and human samples, but it has received little attention to date. The current studies investigated EG-018 at human CB1 and CB2 receptors expressed in HEK293 cells in [3H]CP55,940 competition binding, [35S]GTPγS binding and forskolin-stimulated cAMP production. EG-018 was also tested in vivo for its ability to produce cannabimimetic and abuse-related effects in the cannabinoid tetrad and THC drug discrimination, respectively. EG-018 exhibited high affinity at CB1 (21 nM) and at CB2 (7 nM), but in contrast to typical SCRAs, behaved as a weak partial agonist in [35S]GTPγS binding, exhibiting lower efficacy but greater potency, than that of THC at CB1 and similar potency and efficacy at CB2. EG-018 inhibited forskolin-stimulated cAMP with similar efficacy but lower potency, compared to THC, which was likely due to high receptor density facilitating saturation of this signaling pathway. In mice, EG-018 (100 mg/kg, 30 min) administered intraperitoneally (i.p.) did not produce effects in the tetrad or drug discrimination nor did it shift THC's ED50 value in drug discrimination when administered before THC, suggesting EG-018 has negligible occupancy of brain CB1 receptors following i.p. administration. Following intravenous (i.v.) administration, EG-018 (56 mg/kg) produced hypomotility, catalepsy, and hypothermia, but only catalepsy was blocked by the selective CB1 antagonist rimonabant (3 mg/kg, i.v.). Additional studies of EG-018 and its structural analogues could provide further insight into how cannabinoids exert efficacy through the cannabinoid receptors.

Development of a Quantitative LC-MS-MS Assay for Codeine, Morphine, 6-Acetylmorphine, Hydrocodone, Hydromorphone, Oxycodone and Oxymorphone in Neat Oral Fluid.

Recent advances in analytical capabilities allowing for the identification and quantification of drugs and metabolites in small volumes at low concentrations have made oral fluid a viable matrix for drug testing. Oral fluid is an attractive matrix option due to its relative ease of collection, reduced privacy concerns for observed collections and difficulty to adulterate. The work presented here details the development and validation of a liquid chromatography tandem mass spectrometry (LC-MS-MS) method for the quantification of codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone and oxymorphone in neat oral fluid. The calibration range is 0.4-150 ng/mL for 6-acetylmorphine and 1.5-350 ng/mL for all other analytes. Within-run and between-run precision were <5% for all analytes except for hydrocodone, which had 6.2 %CV between runs. Matrix effects, while evident, could be controlled using matrix-matched controls and calibrators with deuterated internal standards. The assay was developed in accordance with the proposed mandatory guidelines for opioid confirmation in federally regulated workplace drug testing. The use of neat oral fluid, as opposed to a collection device, enables collection of a single sample that can be split into separate specimens.

Detection and quantification of codeine-6-glucuronide, hydromorphone-3-glucuronide, oxymorphone-3-glucuronide, morphine 3-glucuronide and morphine-6-glucuronide in human hair from opioid users by LC-MS-MS.

Current hair testing methods that rely solely on quantification of parent drug compounds are unable to definitively distinguish between drug use and external contamination. One possible solution to this problem is to confirm the presence of unique drug metabolites that cannot be present through contamination, such as phase II glucuronide conjugates. This work demonstrates for the first time that codeine-6-glucuronide, hydromorphone-3-glucuronide, oxymorphone-3-glucuronide, morphine-3-glucuronide and morphine-6-glucuronide are present at sufficient concentrations to be quantifiable in hair of opioid users and that their concentrations generally increase as the concentrations of the corresponding parent compounds increase. Here, we present a validated liquid chromatography tandem mass spectrometry method to quantify codeine-6-glucuronide, dihydrocodeine-6-glucuronide, hydromorphone-3-glucuronide, morphine-3-glucuronide, morphine-6-glucuronide, oxymorphone-3-glucuronide, codeine, dihydrocodeine, dihydromorphine, hydrocodone, hydromorphone, morphine, oxycodone, oxymorphone and 6-acetylmorphine in human hair. The method was used to analyze 46 human hair samples from known drug users that were confirmed positive for opioids by an independent laboratory. Glucuronide concentrations in samples positive for parent analytes ranged from ~1 to 25 pg/mg, and most samples had glucuronide concentrations in the range of ~1 to 5 pg/mg. Relative to the parent concentrations, the average concentrations of the four detected glucuronides were as follows: codeine-6-glucuronide, 2.33%; hydromorphone-3-glucuronide, 0.94%; oxymorphone-3-glucuronide, 0.77%; morphine 3-glucuronide, 0.59%; and morphine-6-glucuronide, 0.93%.

Identification of Eight Synthetic Cannabinoids, Including 5F-AKB48 in Seized Herbal Products Using DART-TOF-MS and LC-QTOF-MS as Nontargeted Screening Methods.

Synthetic cannabinoids are sprayed onto plant material and smoked for their marijuana-like effects. Clandestine manufacturers modify synthetic cannabinoid structures by creating closely related analogs. Forensic laboratories are tasked with detection of these analog compounds, but targeted analytical methods are often thwarted by the structural modifications. Here, direct analysis in real time coupled to accurate mass time-of-flight mass spectrometry (DART-TOF-MS) in combination with liquid chromatography quadruple time-of-flight mass spectrometry (LC-QTOF-MS) are presented as a screening and nontargeted confirmation method, respectively. Methanol extracts of herbal material were run using both methods. Spectral data from four different herbal products were evaluated by comparing fragmentation pattern, accurate mass and retention time to available reference standards. JWH-018, JWH-019, AM2201, JWH-122, 5F-AKB48, AKB48-N-(4-pentenyl) analog, UR144, and XLR11 were identified in the products. Results demonstrate that DART-TOF-MS affords a useful approach for rapid screening of herbal products for the presence and identification of synthetic cannabinoids.

Thermolytic Degradation of Synthetic Cannabinoids: Chemical Exposures and Pharmacological Consequences.

Synthetic cannabinoids are manufactured clandestinely with little quality control and are distributed as herbal "spice" for smoking or as bulk compound for mixing with a solvent and inhalation via electronic vaporizers. Intoxication with synthetic cannabinoids has been associated with seizure, excited delirium, coma, kidney damage, and other disorders. The chemical alterations produced by heating these structurally novel compounds for consumption are largely unknown. Here, we show that heating synthetic cannabinoids containing tetramethylcyclopropyl-ring substituents produced thermal degradants with pharmacological activity that varied considerably from their parent compounds. Moreover, these degradants were formed under conditions simulating smoking. Some products of combustion retained high affinity at the cannabinoid 1 (CB1) and CB2 receptors, were more efficacious than (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55,940) in stimulating CB1 receptor-mediated guanosine 5'-O-(3-thiotriphosphate) (GTPγS) binding, and were potent in producing Δ9-tetrahydrocannabinol-like effects in laboratory animals, whereas other compounds had low affinity and efficacy and were devoid of cannabimimetic activity. Degradants that retained affinity and efficacy also substituted in drug discrimination tests for the prototypical synthetic cannabinoid 1-pentyl-3-(1-naphthoyl)indole (JWH-018), and are likely to produce psychotropic effects in humans. Hence, it is important to take into consideration the actual chemical exposures that occur during use of synthetic cannabinoid formulations to better comprehend the relationships between dose and effect.

Medullary Endocannabinoids Contribute to the Differential Resting Baroreflex Sensitivity in Rats with Altered Brain Renin-Angiotensin System Expression.

CB1 cannabinoid receptors are expressed on vagal afferent fibers and neurons within the solitary tract nucleus (NTS), providing anatomical evidence for their role in arterial baroreflex modulation. To better understand the relationship between the brain renin-angiotensin system (RAS) and endocannabinoid expression within the NTS, we measured dorsal medullary endocannabinoid tissue content and the effects of CB1 receptor blockade at this brain site on cardiac baroreflex sensitivity (BRS) in ASrAOGEN rats with low glial angiotensinogen, normal Sprague-Dawley rats and (mRen2)27 rats with upregulated brain RAS expression. Mass spectrometry revealed higher levels of the endocannabinoid 2-arachidonoylglycerol in (mRen2)27 compared to ASrAOGEN rats (2.70 ± 0.28 vs. 1.17 ± 0.09 ng/mg tissue; P < 0.01), while Sprague-Dawley rats had intermediate content (1.85 ± 0.27 ng/mg tissue). Microinjection of the CB1receptor antagonist SR141716A (36 pmol) into the NTS did not change cardiac BRS in anesthetized Sprague-Dawley rats (1.04 ± 0.05 ms/mmHg baseline vs. 1.17 ± 0.11 ms/mmHg after 10 min). However, SR141716A in (mRen2)27 rats dose-dependently improved BRS in this strain: 0.36 pmol of SR141716A increased BRS from 0.43 ± 0.03 to 0.71 ± 0.04 ms/mmHg (P < 0.001), and 36 pmol of SR141716A increased BRS from 0.47 ± 0.02 to 0.94 ± 0.10 ms/mmHg (P < 0.01). In contrast, 0.36 pmol (1.50 ± 0.12 vs. 0.86 ± 0.08 ms/mmHg; P < 0.05) and 36 pmol (1.38 ± 0.16 vs. 0.46 ± 0.003 ms/mmHg; P < 0.01) of SR141716A significantly reduced BRS in ASrAOGEN rats. These observations reveal differential dose-related effects of the brain endocannabinoid system that influence cardiovagal BRS in animals with genetic alterations in the brain RAS.

Evaluation of first generation synthetic cannabinoids on binding at non-cannabinoid receptors and in a battery of in vivo assays in mice.

Anecdotal reports suggest that abused synthetic cannabinoids produce cannabis-like "highs," but some of their effects may also differ from traditional cannabinoids such as Δ(9)-tetrahydrocannabinol (THC). This study examined the binding affinities of first-generation indole-derived synthetic cannabinoids at cannabinoid and noncannabinoid receptors and their effects in a functional observational battery (FOB) and drug discrimination in mice. All seven compounds, except JWH-391, had favorable affinity (≤159 nM) for both cannabinoid receptors. In contrast, binding at noncannabinoid receptors was absent or weak. In the FOB, THC and the six active compounds disrupted behaviors in CNS activation and muscle tone/equilibrium domains. Unlike THC, however, synthetic cannabinoids impaired behavior across a wider dose and domain range, producing autonomic effects and signs of CNS excitability and sensorimotor reactivity. In addition, mice acquired JWH-018 discrimination, and THC and JWH-073 produced full substitution whereas the 5-HT2B antagonist mianserin did not substitute in mice trained to discriminate JWH-018 or THC. Urinary metabolite analysis showed that the compounds were extensively metabolized, with metabolites that could contribute to their in vivo effects. Together, these results show that, while first-generation synthetic cannabinoids shared some effects that were similar to those of THC, they also possessed effects that differed from traditional cannabinoids. The high nanomolar (or absent) affinities of these compounds at receptors for most major neurotransmitters suggests that these divergent effects may be related to the greater potencies and/or efficacies at CB1 receptors; however, action(s) at noncannabinoid receptors yet to be assessed or via different signaling pathways cannot be ruled out.

AB-CHMINACA, AB-PINACA, and FUBIMINA: Affinity and Potency of Novel Synthetic Cannabinoids in Producing Δ9-Tetrahydrocannabinol-Like Effects in Mice.

Diversion of synthetic cannabinoids for abuse began in the early 2000s. Despite legislation banning compounds currently on the drug market, illicit manufacturers continue to release new compounds for recreational use. This study examined new synthetic cannabinoids, AB-CHMINACA (N-[1-amino-3-methyl-oxobutan-2-yl]-1-[cyclohexylmethyl]-1H-indazole-3-carboxamide), AB-PINACA [N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide], and FUBIMINA [(1-(5-fluoropentyl)-1H-benzo[d]imadazol-2-yl)(naphthalen-1-yl)methanone], with the hypothesis that these compounds, like those before them, would be highly susceptible to abuse. Cannabinoids were examined in vitro for binding and activation of CB1 receptors, and in vivo for pharmacological effects in mice and in Δ(9)-tetrahydrocannabinol (Δ(9)-THC) discrimination. AB-CHMINACA, AB-PINACA, and FUBIMINA bound to and activated CB1 and CB2 receptors, and produced locomotor suppression, antinociception, hypothermia, and catalepsy. Furthermore, these compounds, along with JWH-018 [1-pentyl-3-(1-naphthoyl)indole], CP47,497 [rel-5-(1,1-dimethylheptyl)-2-[(1R,3S)-3-hydroxycyclohexyl]-phenol], and WIN55,212-2 ([(3R)-2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone, monomethanesulfonate), substituted for Δ(9)-THC in Δ(9)-THC discrimination. Rank order of potency correlated with CB1 receptor-binding affinity, and all three compounds were full agonists in [(35)S]GTPγS binding, as compared with the partial agonist Δ(9)-THC. Indeed, AB-CHMINACA and AB-PINACA exhibited higher efficacy than most known full agonists of the CB1 receptor. Preliminary analysis of urinary metabolites of the compounds revealed the expected hydroxylation. AB-PINACA and AB-CHMINACA are of potential interest as research tools due to their unique chemical structures and high CB1 receptor efficacies. Further studies on these chemicals are likely to include research on understanding cannabinoid receptors and other components of the endocannabinoid system that underlie the abuse of synthetic cannabinoids.

Alterations in the Medullary Endocannabinoid System Contribute to Age-related Impairment of Baroreflex Sensitivity.

As they age, Sprague-Dawley (SD) rats develop elevated systolic blood pressure associated with impaired baroreflex sensitivity (BRS) for control of heart rate. We previously demonstrated in young hypertensive (mRen2)27 rats that impaired BRS is restored by CB1 cannabinoid receptor blockade in the solitary tract nucleus (NTS), consistent with elevated content of the endocannabinoid 2-arachidonoylglycerol (2-AG) in dorsal medulla relative to normotensive SD rats. There is no effect of CB1 receptor blockade in young SD rats. We now report in older SD rats that dorsal medullary 2-AG levels are 2-fold higher at 70 versus 15 weeks of age (4.22 ± 0.61 vs. 1.93 ± 0.22 ng/mg tissue; P < 0.05). Furthermore, relative expression of CB1 receptor messenger RNA is significantly lower in aged rats, whereas CB2 receptor messenger RNA is significantly higher. In contrast to young adult SD rats, microinjection of the CB1 receptor antagonist SR141716A (36 pmole) into the NTS of older SD rats normalized BRS in animals exhibiting impaired baseline BRS (0.56 ± 0.06 baseline vs. 1.06 ± 0.05 ms/mm Hg after 60 minutes; P < 0.05). Therefore, this study provides evidence for alterations in the endocannabinoid system within the NTS of older SD rats that contribute to age-related impairment of BRS.

Effects of α-pyrrolidinopentiophenone and 4-methyl-N-ethylcathinone, two synthetic cathinones commonly found in second-generation "bath salts," on intracranial self-stimulation thresholds in rats.

BACKGROUND: Use of synthetic cathinones, which are designer stimulants found in "bath salts," has increased dramatically in recent years. Following governmental bans of methylenedioxypyrovalerone, mephedrone, and methylone, a second generation of synthetic cathinones with unknown abuse liability has emerged as replacements. METHODS: Using a discrete trials current intensity threshold intracranial self-stimulation procedure, the present study assessed the effects of 2 common second-generation synthetic cathinones, α-pyrrolidinopentiophenone (0.1-5 mg/kg) and 4-methyl-N-ethcathinone (1-100 mg/kg) on brain reward function. Methamphetamine (0.1-3 mg/kg) was also tested for comparison purposes. RESULTS: Results revealed both α-pyrrolidinopentiophenone and 4-methyl-N-ethcathinone produced significant intracranial self-stimulation threshold reductions similar to that of methamphetamine. α-Pyrrolidinopentiophenone (1 mg/kg) produced a significant maximal reduction in intracranial self-stimulation thresholds (~19%) most similar to maximal reductions produced by methamphetamine (1 mg/kg, ~20%). Maximal reductions in intracranial self-stimulation thresholds produced by 4-methyl-N-ethcathinone were observed at 30 mg/kg (~15%) and were comparable with those observed with methamphetamine and α-pyrrolidinopentiophenone tested at the 0.3-mg/kg dose (~14%). Additional analysis of the ED50 values from log-transformed data revealed the rank order potency of these drugs as methamphetamine ≈ α-pyrrolidinopentiophenone>4-methyl-N-ethcathinone. CONCLUSIONS: These data suggest that the newer second-generation synthetic cathinones activate the brain reward circuitry and thus may possess a similar degree of abuse potential as prototypical illicit psychostimulants such as methamphetamine as well as the first generation synthetic cathinone methylenedioxypyrovalerone, as previously reported.

Evaluation of laser diode thermal desorption-tandem mass spectrometry (LDTD-MS-MS) in forensic toxicology.

Many forensic laboratories experience backlogs due to increased drug-related cases. Laser diode thermal desorption (LDTD) has demonstrated its applicability in other scientific areas by providing data comparable with instrumentation, such as liquid chromatography-tandem mass spectrometry, in less time. LDTD-MS-MS was used to validate 48 compounds in drug-free human urine and blood for screening or quantitative analysis. Carryover, interference, limit of detection, limit of quantitation, matrix effect, linearity, precision and accuracy and stability were evaluated. Quantitative analysis indicated that LDTD-MS-MS produced precise and accurate results with the average overall within-run precision in urine and blood represented by a %CV <14.0 and <7.0, respectively. The accuracy for all drugs in urine ranged from 88.9 to 104.5% and 91.9 to 107.1% in blood. Overall, LDTD has the potential for use in forensic toxicology but before it can be successfully implemented that there are some challenges that must be addressed. Although the advantages of the LDTD system include minimal maintenance and rapid analysis (∼10 s per sample) which makes it ideal for high-throughput forensic laboratories, a major disadvantage is its inability or difficulty analyzing isomers and isobars due to the lack of chromatography without the use of high-resolution MS; therefore, it would be best implemented as a screening technique.

Potent rewarding and reinforcing effects of the synthetic cathinone 3,4-methylenedioxypyrovalerone (MDPV).

Reports of abuse and toxic effects of synthetic cathinones, frequently sold as 'bath salts' or 'legal highs', have increased dramatically in recent years. One of the most widely used synthetic cathinones is 3,4-methylenedioxypyrovalerone (MDPV). The current study evaluated the abuse potential of MDPV by assessing its ability to support intravenous self-administration and to lower thresholds for intracranial self-stimulation (ICSS) in rats. In the first experiment, the rats were trained to intravenously self-administer MDPV in daily 2-hour sessions for 10 days at doses of 0.05, 0.1 or 0.2 mg/kg per infusion. The rats were then allowed to self-administer MDPV under a progressive ratio (PR) schedule of reinforcement. Next, the rats self-administered MDPV for an additional 10 days under short access (ShA; 2 hours/day) or long access (LgA; 6 hours/day) conditions to assess escalation of intake. A separate group of rats underwent the same procedures, with the exception of self-administering methamphetamine (0.05 mg/kg per infusion) instead of MDPV. In the second experiment, the effects of MDPV on ICSS thresholds following acute administration (0.1, 0.5, 1 and 2 mg/kg, i.p.) were assessed. MDPV maintained self-administration across all doses tested. A positive relationship between MDPV dose and breakpoints for reinforcement under PR conditions was observed. LgA conditions led to escalation of drug intake at 0.1 and 0.2 mg/kg doses, and rats self-administering methamphetamine showed similar patterns of escalation. Finally, MDPV significantly lowered ICSS thresholds at all doses tested. Together, these findings indicate that MDPV has reinforcing properties and activates brain reward circuitry, suggesting a potential for abuse and addiction in humans.