Psychoactive Component Research Articles

Pharmacokinetics and central accumulation of delta-9-tetrahydrocannabinol (THC) and its bioactive metabolites are influenced by route of administration and sex in rats

Up to a third of North Americans report using cannabis in the prior month, most commonly through inhalation. Animal models that reflect human consumption are critical to study the impact of cannabis on brain and behaviour. Most animal studies to date utilize injection of delta-9-tetrahydrocannabinol (THC; primary psychoactive component of cannabis). THC injections produce markedly different physiological and behavioural effects than inhalation, likely due to distinctive pharmacokinetics. The current study directly examined if administration route (injection versus inhalation) alters metabolism and central accumulation of THC and metabolites over time. Adult male and female Sprague–Dawley rats received either an intraperitoneal injection or a 15-min session of inhaled exposure to THC. Blood and brains were collected at 15, 30, 60, 90 and 240-min post-exposure for analysis of THC and metabolites. Despite achieving comparable peak blood THC concentrations in both groups, our results indicate higher initial brain THC concentration following inhalation, whereas injection resulted in dramatically higher 11-OH-THC concentration, a potent THC metabolite, in blood and brain that increased over time. Our results provide evidence of different pharmacokinetic profiles following inhalation versus injection. Accordingly, administration route should be considered during data interpretation, and translational animal work should strongly consider using inhalation models.

Quantification of hordenine in a complex plant matrix by direct analysis in real time-high-resolution mass spectrometry: Application to the "plant of concern" Sceletium tortuosum.

Recently, there has been an increase in the recreational abuse of several psychoactive plants, resulting in the United Nations Office on Drugs and Crime creating a list of "plants of concern." One such material is Sceletium tortuosum and products derived from it. Regulation of these materials is challenging because of their innocuous appearance, the cumbersome sample preparation steps required to render the material into a form amenable to analysis by conventional techniques, the requirement for nuanced sample analysis protocols, and lengthy analysis times. It is demonstrated here that direct analysis in real time-high-resolution mass spectrometry (DART-HRMS) can be used to not only identify S.tortuosum material based on the detection of characteristic biomarkers including hordenine and several mesembrine alkaloids, but also quantify the amount of hordenine present. Using hordenine-d6 as an internal standard, a protocol, validated according to US Food and Drug Administration (FDA) Guidelines for the Development and Validation of Bioanalytical Methods, was devised for the quantification of the psychoactive component hordenine. The method was then applied to the quantification of hordenine in six commercially available products derived from the foliage and stems of S.tortuosum. By this method, the lower limit of quantification (LLOQ) was found to be 1μg/ml. Observed hordenine concentrations ranged from 0.02738 to 1.071 mg of hordenine per gram of plant material. The developed technique provides an effective and quick means for the detection and quantification of hordenine in S.tortuosum, which can be extended to analysis of other hordenine-containing products.

Astroglial CB1 Cannabinoid Receptors Mediate CP 55,940-Induced Conditioned Place Aversion Through Cyclooxygenase-2 Signaling in Mice.

Cannabinoids (CBs), such as phytocannabinoids, synthetic CBs, and endogenous CBs, can be neuroprotective, rewarding, or aversive. The aversive effects of CBs may hinder their medical and recreational applications. It is unknown which type of CB receptors mediates the direct aversive effects of synthetic CB CP 55,940 which is an analog of Δ9-tetrahydrocannabinol, the major psychoactive component of marijuana. In this study, we address this question by taking the advantage of systematic type 1 CB receptor (CB1R) knockout mice and conditional reinstatement of this receptor only in astrocytes. We show that CP 55,940 at a concentration of 1 mg/kg induces conditioned place aversion (CPA) and the CPA effect of CP 55,940 is mediated by the astroglial CB1Rs. Inhibiting cyclooxygenase-2 (COX-2) eliminates CP 55,940-induced CPA in mice that only express CB1Rs in astrocytes. These findings conclude that CPA effect of CP 55,940 is mediated by the astroglial CB1Rs through COX-2 signaling, suggesting that selective COX-2 inhibition or precise isolation of astroglial CB1R activity may be the strategy for treating aversive response of medical and recreational administrations of marijuana.

Cannabinoid distribution in fatally-injured pilots’ postmortem fluids and tissues

The primary psychoactive component of cannabis, Δ9-tetrahydrocannabinol (THC) impairs cognitive function and psychomotor performance, particularly for complex tasks like piloting an aircraft. The Federal Aviation Administration’s (FAA) Forensic Sciences Section at the Civil Aerospace Medical Institute (Oklahoma City, OK) performs toxicological analyses on pilots fatally injured in general aviation incidents, permitting cannabinoids measurement in a broad array of postmortem biological specimens. Cannabinoid concentrations in postmortem fluids and tissues from 10 pilots involved in airplane crashes are presented. Median (range) THC blood concentration was 1.6 (1.0–13.7) ng/mL. Phase I metabolites, 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) and phase II glucuronide metabolite, THCCOOH-glucuronide, had median (range) blood concentrations of 1.4 (0.5–1.8), 9.9 (2.2–72.6) and 36.6 (7.1–160) ng/mL, respectively. Urine analyses revealed positive results for THCCOOH, THC-glucuronide, THCCOOH-glucuronide and 11-nor-9-carboxy-Δ9-tetrahydrocannabivarin (THCVCOOH). THC was readily distributed to lung, brain, kidney, spleen and heart. The psychoactive metabolite, 11-OH-THC, was identified in liver and brain with median (range) concentrations 7.1 (3.5–10.5) and 2.4 (2.0–6.0) ng/g, respectively. Substantial THCCOOH and THCCOOH-glucuronide concentrations were observed in liver, lung, brain, kidney, spleen and heart. These cannabinoid concentrations from multiple types of postmortem specimens add to the limited postmortem cannabinoid research data and suggest useful biological matrices for investigating cannabinoid-related deaths.

32 Accidental ingestions of cannabis in children pre- and post-legalization: Perspectives from a Canadian paediatric hospital

Abstract Primary Subject area Public Health and Preventive Medicine Background Cannabis is one of the most prevalent recreational substances worldwide. In 2018, Canada legalized the use of recreational cannabis. Cannabis is available in numerous forms, including a wide range of ingestible forms (baked goods, candies, gummies, etc.). This increasing availability and palatability of cannabis – coupled with a rise in the concentration of tetrahydrocannabinol (THC; the psychoactive component of cannabis) in many products – has made children particularly vulnerable. Research has demonstrated rising cases of pediatric accidental cannabis exposures, primarily in U.S. states, but there is a paucity of Canadian data. Objectives We sought to: i) Characterize the frequency and clinical features of children presenting with accidental cannabis ingestions to a Canadian tertiary pediatric hospital; and ii) Compare the rates and presentations in the pre- and post-legalization periods. Design/Methods A retrospective chart review of Emergency Department visits at the Children’s Hospital of Eastern Ontario (Ottawa, ON) between March 2013 and September 2020 was undertaken, with institutional ethics approval sought. Inclusion criteria were: (i) Age < 13 years; and (ii) Accidental cannabis ingestion, as identified by specific ICD-10 diagnostic codes (T40.7, X42). Data collected from each chart included basic demographics; type and quantity of cannabis ingested; timing and location of ingestion; vital signs and Glasgow Coma Scale (GCS) rating at triage; presenting symptoms; investigations and procedures performed; as well as length of stay in hospital. Data were analyzed using descriptive statistical methods. Results A total of 39 patients met inclusion criteria (23 male; 59%). The mean age of presentation was 5.2 years (± 3.3; range 1 month–12 years). The vast majority of visits (32/39; 82%) occurred in the last two years of data collection (i.e., after legalization of recreational cannabis in Canada in October 2018). A total of 15 patients (38%) required admission to hospital, with a mean length of stay of 1.3 (± 1.0) days. Details about each patient’s presenting symptoms, investigations, and disposition were analyzed. Conclusion Our data from a Canadian tertiary pediatric centre confirms an increased rate of unintentional cannabis ingestions among young children, particularly post-legalization. This is of significant public health concern. An understanding of the clinical presentation of cannabis intoxication, as well as advocacy for safe storage strategies is prudent for parents, pediatric providers, and public health policymakers.

PSXIV-12 Sources of variation in the nutritive value of hemp seed meal

Abstract The 2018 Farm Bill, with its groundbreaking hemp legislation, offered an alternative protein source to livestock producers. Hemp is the agricultural product of Cannabis sativa L. which must contain less than 0.3% tetrahydrocannabinol, the psychoactive component common to marijuana. Hemp oil, cold-pressed from C. sativa seed, has been gaining popularity over recent years for its use in cosmetics, pharmaceuticals, and human food products. Like most oilseeds, this leaves an oilseed meal with a potential for inclusion in livestock feeds. Thus, our objective was to quantify the sources of variation in the nutritive value of hempseed meal. Hempseed meal samples were obtained from commercial oil processing facilities (n = 4). Across these facilities, samples represented 15 independent hempseed meal batches. Our experimental design was a completely randomized design including the random effects of source, batch within source, and replicate within batch by source (laboratory replication). Samples were assayed for NDF, ADF, ADL, CP, and IVTD. Source had no contribution to variance for NDF, ADF, ADL, or CP (P ≥ 0.20). However, batch within source had a significant contribution to variation for NDF (μ = 50.1%; P = 0.01), ADF (μ = 36.8%; P = 0.01), ADL (μ = 12.9%; P < 0.01), and CP (μ = 30.9%; P < 0.01). Irrespective of the differences in nutritive value, there was no contribution to variation (P ≥ 0.23) of any measured effect on IVTD (μ = 53.0%). Results are interpreted to mean that, while variation exists among hempseed meal samples with respect to nutritive value, the viability of hempseed meal as a livestock feed source is strong.

Paternal Cannabis Exposure Prior to Mating, but Not Δ9-Tetrahydrocannabinol, Elicits Deficits in Dopaminergic Synaptic Activity in the Offspring.

The legalization and increasing availability of cannabis products raises concerns about the impact on offspring of users, and little has appeared on the potential contribution of paternal use. We administered cannabis extract to male rats prior to mating, with two different 28-day exposures, one where there was a 56-day interval between the end of exposure and mating ("Early Cannabis"), and one just prior to mating ("Late Cannabis"); the extract delivered 4 mg/kg/day of the main psychoactive component, Δ9-tetrahydrocannabinol. We then assessed the impact on dopamine (DA) systems in the offspring from the onset of adolescence (postnatal day 30) through middle age (postnatal day 150), measuring the levels of DA and its primary metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC) in various brain regions. Paternal cannabis with either regimen elicited a profound and persistent deficit in DA utilization (DOPAC/DA ratio) in the offspring, indicative of subnormal presynaptic activity. However, the two regimens differed in the underlying mechanism, with Early Cannabis reducing DOPAC whereas Late Cannabis increased DA and elicited a smaller reduction in DOPAC. Effects were restricted to male offspring. The effects of cannabis were not reproduced by equivalent exposure to its Δ9-tetrahydrocannabinol, nor did we see the effects with perinatal exposure to tobacco smoke or some of its fetotoxic contributors (benzo[a]pyrene without or with nicotine). Our studies provide some of the first evidence for adverse effects of paternal cannabis administration on neurodevelopment in the offspring, and reinforce the important consequences of paternal drug use in the preconception period.

Prenatal Δ9-Tetrahydrocannabinol Exposure in Males Leads to Motivational Disturbances Related to Striatal Epigenetic Dysregulation

BackgroundCannabis remains one of the most widely abused drugs during pregnancy. In utero exposure to its principal psychoactive component, Δ9-tetrahydrocannabinol (THC), can result in long-term neuropsychiatric risk for the progeny. This study investigated epigenetic signatures underlying these enduring consequences. MethodsRat dams were exposed daily to THC (0.15 mg/kg) during pregnancy, and adult male offspring were examined for reward and depressive-like behavioral endophenotypes. Using unbiased sequencing approaches, we explored transcriptional and epigenetic profiles in the nucleus accumbens (NAc), a brain area central to reward and emotional processing. An in vitro CRISPR (clustered regularly interspaced short palindromic repeats) activation model coupled with RNA sequencing was also applied to study specific consequences of epigenetic dysregulation, and altered molecular signatures were compared with human major depressive disorder transcriptome datasets. ResultsPrenatal THC exposure induced increased motivation for food, heightened learned helplessness and anhedonia, and altered stress sensitivity. We identified a robust increase specific to males in the expression of Kmt2a (histone-lysine N-methyltransferase 2A) that targets H3K4 (lysine 4 on histone H3) in cellular chromatin. Normalizing Kmt2a in the NAc rescued the motivational phenotype of prenatally THC-exposed animals. Comparison of RNA- and H3K4me3-sequencing datasets from the NAc of rat offspring with the in vitro model of Kmt2a upregulation revealed overlapping, significant disturbances in pathways that mediate synaptic plasticity. Similar transcriptional alterations were detected in human major depressive disorder. ConclusionsThese studies provide direct evidence for the persistent effects of prenatal cannabis exposure on transcriptional and epigenetic deviations in the NAc via Kmt2a dysregulation and associated psychiatric vulnerability.

Epigenetic Mediation of AKT1 rs1130233's Effect on Delta-9-Tetrahydrocannabinol-Induced Medial Temporal Function during Fear Processing.

High doses of delta-9-tetrahydrocannabinol (THC), the main psychoactive component of cannabis, have been shown to have anxiogenic effects. Additionally, THC effects have been shown to be modulated by genotype, including the single nucleotide polymorphism (SNP) rs1130233 at the protein kinase AKT1 gene, a key component of the dopamine signalling cascade. As such, it is likely that epigenetic methylation around this SNP may affect AKT gene expression, which may in turn impact on the acute effects of THC on brain function. We investigated the genetic (AKT1 rs1130233) and epigenetic modulation of brain function during fear processing in a 2-session, double-blind, cross-over, randomized placebo-controlled THC administration, in 36 healthy males. Fear processing was assessed using an emotion (fear processing) paradigm, under functional magnetic resonance imaging (fMRI). Complete genetic and fMRI data were available for 34 participants. THC caused an increase in anxiety and transient psychotomimetic symptoms and para-hippocampal gyrus/amygdala activation. Number of A alleles at the AKT1 rs1130233 SNP, and percentage methylation at the CpG11–12 site, were independently associated with a greater effect of THC on activation in a network of brain regions including left and right parahippocampal gyri, respectively. AKT1 rs1130233 moderation of the THC effect on left parahippocampal activation persisted after covarying for methylation percentage, and was partially mediated in sections of the left parahippocampal gyrus/hippocampus by methylation percentage. These results may offer an example of how genetic and epigenetic variations influence the psychotomimetic and neurofunctional effects of THC.

A biomolecule-free electrochemical sensing approach based on a novel electrode modification technique: Detection of ultra-low concentration of Δ⁹-tetrahydrocannabinol in saliva by turning a sample analyte into a sensor analyte

Cannabis is currently one of the most consumed drugs in many countries. Δ⁹-tetrahydrocannabinol (THC) is the principal psychoactive component of this drug and is present in saliva after consumption. This paper reports a novel biomolecule-free electrochemical approach to detect an ultra-low level of THC in saliva using modified electrodes with molecules of the same analyte (THC) that are detected later via square wave voltammetry. The results from this research revealed that the electrodeposition of THC on the working electrode (sensor analyte) could highly enhance the limit of detection by improving the affinity of the THC molecules present in the sample (sample analyte) to the sensing electrode surface. Detailed descriptions about the optimization of the sensor and its performance in simple media, such as PBS, and complex media, such as simulated and real saliva, are provided. This novel and yet simple electrochemical-based sensing strategy allowed for a low limit of detection of 1.6 ng/mL THC in simulated and real saliva, distinguishing concentrations ranging from 2 to 25 ng/mL, making this technology viable for a real-world application such as roadside testing.

Metabolic Consequences of Gestational Cannabinoid Exposure.

Up to 20% of pregnant women ages 18–24 consume cannabis during pregnancy. Moreover, clinical studies indicate that cannabis consumption during pregnancy leads to fetal growth restriction (FGR), which is associated with an increased risk of obesity, type II diabetes (T2D), and cardiovascular disease in the offspring. This is of great concern considering that the concentration of Δ9- tetrahydrocannabinol (Δ9-THC), a major psychoactive component of cannabis, has doubled over the last decade and can readily cross the placenta and enter fetal circulation, with the potential to negatively impact fetal development via the endocannabinoid (eCB) system. Cannabis exposure in utero could also lead to FGR via placental insufficiency. In this review, we aim to examine current pre-clinical and clinical findings on the direct effects of exposure to cannabis and its constituents on fetal development as well as indirect effects, namely placental insufficiency, on postnatal metabolic diseases.

Genetic Modulation of Initial Sensitivity to Δ9-Tetrahydrocannabinol (THC) Among the BXD Family of Mice.

Cannabinoid receptor 1 activation by the major psychoactive component in cannabis, Δ9-tetrahydrocannabinol (THC), produces motor impairments, hypothermia, and analgesia upon acute exposure. In previous work, we demonstrated significant sex and strain differences in acute responses to THC following administration of a single dose (10 mg/kg, i.p.) in C57BL/6J (B6) and DBA/2J (D2) inbred mice. To determine the extent to which these differences are heritable, we quantified acute responses to a single dose of THC (10 mg/kg, i.p.) in males and females from 20 members of the BXD family of inbred strains derived by crossing and inbreeding B6 and D2 mice. Acute THC responses (initial sensitivity) were quantified as changes from baseline for: 1. spontaneous activity in the open field (mobility), 2. body temperature (hypothermia), and 3. tail withdrawal latency to a thermal stimulus (antinociception). Initial sensitivity to the immobilizing, hypothermic, and antinociceptive effects of THC varied substantially across the BXD family. Heritability was highest for mobility and hypothermia traits, indicating that segregating genetic variants modulate initial sensitivity to THC. We identified genomic loci and candidate genes, including Ndufs2, Scp2, Rps6kb1 or P70S6K, Pde4d, and Pten, that may control variation in THC initial sensitivity. We also detected strong correlations between initial responses to THC and legacy phenotypes related to intake or response to other drugs of abuse (cocaine, ethanol, and morphine). Our study demonstrates the feasibility of mapping genes and variants modulating THC responses in the BXDs to systematically define biological processes and liabilities associated with drug use and abuse.

In Utero Exposure to Δ9-Tetrahydrocannabinol Leads to Postnatal Catch-Up Growth and Dysmetabolism in the Adult Rat Liver.

The rates of gestational cannabis use have increased despite limited evidence for its safety in fetal life. Recent animal studies demonstrate that prenatal exposure to Δ9-tetrahydrocannabinol (Δ9-THC, the psychoactive component of cannabis) promotes intrauterine growth restriction (IUGR), culminating in postnatal metabolic deficits. Given IUGR is associated with impaired hepatic function, we hypothesized that Δ9-THC offspring would exhibit hepatic dyslipidemia. Pregnant Wistar rat dams received daily injections of vehicular control or 3 mg/kg Δ9-THC i.p. from embryonic day (E) 6.5 through E22. Exposure to Δ9-THC decreased the liver to body weight ratio at birth, followed by catch-up growth by three weeks of age. At six months, Δ9-THC-exposed male offspring exhibited increased visceral adiposity and higher hepatic triglycerides. This was instigated by augmented expression of enzymes involved in triglyceride synthesis (ACCα, SCD, FABP1, and DGAT2) at three weeks. Furthermore, the expression of hepatic DGAT1/DGAT2 was sustained at six months, concomitant with mitochondrial dysfunction (i.e., elevated p66shc) and oxidative stress. Interestingly, decreases in miR-203a-3p and miR-29a/b/c, both implicated in dyslipidemia, were also observed in these Δ9-THC-exposed offspring. Collectively, these findings indicate that prenatal Δ9-THC exposure results in long-term dyslipidemia associated with enhanced hepatic lipogenesis. This is attributed by mitochondrial dysfunction and epigenetic mechanisms.

Glucocorticoids, Stress and Delta-9 Tetrahydrocannabinol (THC) during Early Embryonic Development.

Elevated molecular stress in women is known to have negative impacts on the reproductive development of oocytes and the embryos prior to implantation. In recent years, the prevalence of cannabis use among women of reproductive age has risen due to its ability to relieve psychological stress and nausea, which are mediated by its psychoactive component, ∆-9-tetrahydrocannabinol (THC). Although cannabis is the most popular recreational drug of the 21st century, much is unknown about its influence on molecular stress in reproductive tissues. The current literature has demonstrated that THC causes dose- and time-dependent alterations in glucocorticoid signaling, which have the potential to compromise morphology, development, and quality of oocytes and embryos. However, there are inconsistencies across studies regarding the mechanisms for THC-dependent changes in stress hormones and how either compounds may drive or arrest development. Factors such as variability between animal models, physiologically relevant doses, and undiscovered downstream gene targets of both glucocorticoids and THC could account for such inconsistencies. This review evaluates the results of studies which have investigated the effects of glucocorticoids on reproductive development and how THC may alter stress signaling in relevant tissues.

Cannabinoid Modulation of Dopamine Release During Motivation, Periodic Reinforcement, Exploratory Behavior, Habit Formation, and Attention.

Motivational and attentional processes energize action sequences to facilitate evolutionary competition and promote behavioral fitness. Decades of neuropharmacology, electrophysiology and electrochemistry research indicate that the mesocorticolimbic DA pathway modulates both motivation and attention. More recently, it was realized that mesocorticolimbic DA function is tightly regulated by the brain’s endocannabinoid system and greatly influenced by exogenous cannabinoids—which have been harnessed by humanity for medicinal, ritualistic, and recreational uses for 12,000 years. Exogenous cannabinoids, like the primary psychoactive component of cannabis, delta-9-tetrahydrocannabinol, produce their effects by acting at binding sites for naturally occurring endocannabinoids. The brain’s endocannabinoid system consists of two G-protein coupled receptors, endogenous lipid ligands for these receptor targets, and several synthetic and metabolic enzymes involved in their production and degradation. Emerging evidence indicates that the endocannabinoid 2-arachidonoylglycerol is necessary to observe concurrent increases in DA release and motivated behavior. And the historical pharmacology literature indicates a role for cannabinoid signaling in both motivational and attentional processes. While both types of behaviors have been scrutinized under manipulation by either DA or cannabinoid agents, there is considerably less insight into prospective interactions between these two important signaling systems. This review attempts to summate the relevance of cannabinoid modulation of DA release during operant tasks designed to investigate either motivational or attentional control of behavior. We first describe how cannabinoids influence DA release and goal-directed action under a variety of reinforcement contingencies. Then we consider the role that endocannabinoids might play in switching an animal’s motivation from a goal-directed action to the search for an alternative outcome, in addition to the formation of long-term habits. Finally, dissociable features of attentional behavior using both the 5-choice serial reaction time task and the attentional set-shifting task are discussed along with their distinct influences by DA and cannabinoids. We end with discussing potential targets for further research regarding DA-cannabinoid interactions within key substrates involved in motivation and attention.

The Effects of Acute Δ9-Tetrahydrocannabinol on Striatal Glutamatergic Function: A Proton Magnetic Resonance Spectroscopy Study

BackgroundCannabis and its main psychoactive component, Δ9-tetrahydrocannabinol (THC), can elicit transient psychotic symptoms. A key candidate biological mechanism of how THC induces psychotic symptoms is the modulation of glutamate in the brain. We sought to investigate the effects of acute THC administration on striatal glutamate levels and its relationship to the induction of psychotic symptoms. MethodsWe used proton magnetic resonance spectroscopy to measure glutamate levels in the striatum in 20 healthy participants after THC (15 mg, oral) and matched placebo administration in a randomized, double-blind, placebo-controlled design. Psychotic symptoms were measured using the Psychotomimetic States Inventory. ResultsWe found that THC administration did not significantly change glutamate (glutamate plus glutamine relative to creatine) concentration in the striatum (p = .58; scaled Jeffreys-Zellner-Siow Bayes factor = 4.29). THC increased psychotic symptoms, but the severity of these symptoms was not correlated with striatal glutamate levels. ConclusionsThese findings suggest that oral administration of 15 mg of THC does not result in altered striatal glutamate levels. Further work is needed to clarify the effects of THC on striatal glutamate.

Analysis of cannabinoids in conventional and alternative biological matrices by liquid chromatography: Applications and challenges

Cannabis is by far the most widely abused illicit drug globe wide. The analysis of its main psychoactive components in conventional and non-conventional biological matrices has recently gained a great attention in forensic toxicology. Literature states that its abuse causes neurocognitive impairment in the domains of attention and memory, possible macrostructural brain alterations and abnormalities of neural functioning. This suggests the necessity for the development of a sensitive and a reliable analytical method for the detection and quantification of cannabinoids in human biological specimens. In this review, we focus on a number of analytical methods that have, so far, been developed and validated, with particular attention to the new “golden standard” method of forensic analysis, liquid chromatography mass spectrometry or tandem mass spectrometry. In addition, this review provides an overview of the effective and selective methods used for the extraction and isolation of cannabinoids from (i) conventional matrices, such as blood, urine and oral fluid and (ii) alternative biological matrices, such as hair, cerumen and meconium.

Cannabis use, abuse, and withdrawal: Cannabinergic mechanisms, clinical, and preclinical findings.

Cannabis sativa is the most widely used illicit drug in the world. Its main psychoactive component is delta‐9‐tetrahydrocannabinol (THC), one of over 100 phytocannabinoid compounds produced by the cannabis plant. THC is the primary compound that drives cannabis abuse potential and is also used and prescribed medically for therapeutic qualities. Despite its therapeutic potential, a significant subpopulation of frequent cannabis or THC users will develop a drug use syndrome termed cannabis use disorder. Individuals suffering from cannabis use disorder exhibit many of the hallmarks of classical addictions including cravings, tolerance, and withdrawal symptoms. Currently, there are no efficacious treatments for cannabis use disorder or withdrawal symptoms. This makes both clinical and preclinical research on the neurobiological mechanisms of these syndromes ever more pertinent. Indeed, basic research using animal models has provided valuable evidence of the neural molecular and cellular actions of cannabis that mediate its behavioral effects. One of the main components being central action on the cannabinoid type‐one receptor and downstream intracellular signaling related to the endogenous cannabinoid system. Back‐translational studies have provided insight linking preclinical basic and behavioral biology research to better understand symptoms observed at the clinical level. This narrative review aims to summarize major research elucidating the molecular, cellular, and behavioral manifestations of cannabis/THC use that play a role in cannabis use disorder and withdrawal.

Major Metabolites of the Synthetic Cannabinoid 5F‐ADB Retain High Affinity and Full Efficacy at CB1 Receptors; Potential Mechanism Contributing to Enhanced Toxicity?

Synthetic cannabinoids (SCs) are a large group of abused psychoactive compounds that elicit numerous toxic effects not observed with marijuana, including death. SCs are abused worldwide and metabolites of these drugs are often detected as a means of drug identification. SC metabolites resulting from Phase I metabolism have been shown to retain affinity and activity at cannabinoid receptors. However, whether these active compounds contribute to toxicity of SCs is unknown. 5-Fluoro-ADB (5F-ADB), also known as 5-Fluoro-MDMB-PINACA, is a commonly abused third-generation synthetic cannabinoid agonist based on an indazole-structure. Significantly, over 40 recent fatalities have been attributed to 5F-ADB overdose. A potential mechanism underlying 5F-ADB-induced mortality is drug-related dysrhythmia resulting in sudden death. Analysis of postmortem central and peripheral blood samples have revealed abnormally high concentrations of an ester-hydrolysis Phase I metabolite of 5F-ADB, identified as metabolite 7 (5F-ADB-M7). We propose that high concentrations of potent and efficacious active metabolites, such as 5F-ADB-M7 and others, accumulate with the parent drug 5F-ADB and produce additive and/or synergistic effects that possibly contribute to fatal intoxication following overdose. As a first step to test this hypothesis, in vitro studies were conducted to compare the affinity and activity of tetrahydrocannabinol (THC; the primary psychoactive component in marijuana), 5F-ADB, 5F-ADB-M7 and a second major metabolite, 5F-ADB-M2 at cannabinoid type-1 (CB1) receptors. Competition receptor binding studies, using mouse brain membranes, showed that all four cannabinoids bound with high affinity to CB1 receptors with a rank order of affinity (Ki values) of 5F-ADB > 5F-ADB-M2 > THC > 5F-ADB-M7. Furthermore, functional experiments examining modulation of G-protein activity demonstrated that 5F-ADB and both Phase I metabolites acted as full agonists at CB1 receptors, relative to partial agonist effects produced by THC. Subsequently, in vivo studies were conducted utilizing the cannabinoid tetrad in mice to compare the effects of THC with those of 5F-ADB-M2. Both 5F-ADB-M2 and THC produced CB1-mediated dose-dependent hypothermia, analgesia, and locomotor suppression, within 30 minutes of injection; however, catalepsy was not elicited by the metabolite. Consistent with higher CB1 affinity determined from in vitro studies, 5F-ADB-M2 produced tetrad effects with higher potency compared to THC. Future studies are planned to examine in vivo effects of 5F-ADB-M7 compared to THC. If 5F-ADB metabolites exhibit in vivo activity similar to that predicted from the in vitro studies presented here, this suggests that the enhanced, often fatal, effects of 5F-ADB may be due to an accumulation of the parent and active, high affinity metabolites acting at CB1 receptors.

Therapeutic Potential of Cannabidiol and WIN 55, 212‐2‐mesylate for Treatment of Anxiety

As the medicalization of marijuana occurs across the United States, the psychoactive component of the cannabis plant, Delta9-Tetrahydrocannabinol (THC), has gained recognition for its therapeutic potential. Meanwhile, its non-psychoactive counterpart, cannabidiol (CBD), has been marketed to treat Parkinson's disease, Chron's disease, dystonia, attention-deficit hyperactivity disorder (ADHD), inflammation, depression, fibromyalgia, epilepsy, and most commonly, anxiety. Given the over the counter accessibility of CBD and the fact that anxiety disorders are the most common type of mental illness in the United States, our objective was to access the therapeutic potential of cannabinoid receptor agonists and antagonists for treatment of anxiety. We hypothesized that CBD might be anxiolytic when chronically administered to a newly found mouse model of anxiety and attention deficit (Kv1.3-/- mice). Similarly to THC, WIN 55, 212-2-mesylate (WIN) is an agonist for the cannabinoid receptors CB1 and CB2, but it is not known to have any psychoactive properties. CBD is an antagonist for CB1 and CB2 receptors, and we used a CBD isolate to ensure purity. Our methods employed two-month-old Kv1.3-/- or wildtype mice that were phenotyped for anxiety behaviors using three assays, namely the elevated plus maze (EPM), the light-dark box (LDB), and the marble burying test. Mice were dosed daily, 30 minutes prior to performing a behavioral test. Mice of both sexes were separated into cohorts in which they were designated to receive an intraperitoneal injection of either drug (WIN55 = 1 mg/kg; CBD = 5 mg/kg) or vehicle solution at equivalent volume. Acute cohorts were defined as daily dosage for 5 days and chronic cohorts were treated with 20 doses over a 30 day interval. Without drug treatment, both genotypes exhibited anxiety-like behaviors in the LDB and EPM tasks, preferring dark side or closed arms of the apparatuses, respectively. For acute WIN treatment, Kv1.3-/- mice showed lessened anxiety in the LDB (increased time in light, decreased time in dark), however, following chronic WIN treatment, they showed no change in LDB behavior. In contrast, in wildtype mice, neither acute nor chronic WIN treatment changed LDB behaviors. Both acute and chronic WIN treatment in Kv1.3-/- mice mitigated anxiety in the EPM test (mice decreased time in the closed arms), whereas the same in wildtype mice showed no effect of anxiety if acutely administered and heightened anxiety following chronic treatment (mice increased time in closed arms). For cohorts treated acutely with CBD, Kv1.3-/- mice showed enhanced anxiety behaviors in the LDB (mice decreased time in light and increased time in dark). Mice of both genotypes showed enhanced anxiety behaviors in the EPM (mice increased time in the closed arms) following acute CBD treatment.In conclusion, our data did not support our hypothesis and showed that CBD may act as an anxiogenic drug in both wildtype and Kv1.3-/- mice.WIN acted as an anxiolytic drug for mice with trait anxiety (Kv1.3-/-), which was ineffective in these mice long-term, and ineffective acutely in wildtype mice exposed to state anxiety.