• Building a Scientific Basis for Oral Fluid Cannabinoid Testing; Cannabinoid Disposition in Oral Fluid Following Smoked Cannabis, Oral Delta-9-tetrahydrocannabinol (THC), and Sativex® and During Extended Cannabis Abstinence

      Lee, Dayong; Huestis, Marilyn (2013)
      Oral fluid (OF) testing offers simple, non-invasive and observable sample collection. Cannabinoids are usually the most prevalent analytes in illicit drug testing. Thus, scientific data are required to document sensitive and specific cannabinoids detection in OF if this technology is to be accepted for drug monitoring. The NIDA Chemistry and Drug Metabolism Section developed multiple clinical protocols to evaluate OF cannabinoid disposition during extended abstinence and following smoked cannabis, oral THC, and Sativex® (oromucosal spray containing approximately 1:1 THC and cannabidiol [CBD]). During extended abstinence, median THC and 11-nor-9-carboxy-THC (THCCOOH) detection windows were 24 h and 13 days, respectively, in chronic cannabis smokers; CBD and cannabinol (CBN) were detected only on admission. After a single smoked 6.8% THC cigarette, THC concentrations were 10-fold higher than CBD and CBN concentrations. Cannabinoids rapidly declined within 3 h; THCCOOH showed a more delayed elimination pattern. After single and multiple oral THC doses, parent cannabinoid concentrations reflected residual excretion from previous self-administered smoked cannabis, whereas THCCOOH concentrations were influenced by oral THC dose regimen. Conversely, parent cannabinoid concentrations were highly increased after Sativex, with CBD concentrations as high as THC's, distinguishing Sativex intake from smoked cannabis. Different cannabinoids had distinct elimination profiles and detection windows, which were influenced by administration route, dose, and prior drug intake history. Quantification of multiple cannabinoids is useful for establishing cutoff criteria for different drug testing programs; CBD and CBN may identify recent intake, while THCCOOH minimizes the potential for false positive results due to passive environmental exposure. Dry mouth, bloody OF samples, and OF cannabinoid stability may influence accurate cannabinoid quantification and should be controlled. OF/plasma cannabinoid ratios exhibited large inter-subject variability after smoking and during oral THC dosing, precluding blood concentration prediction from OF concentrations. There was a possible association among OF cannabinoids, smoking behavior, and cannabis tolerance/withdrawal/residual drug effects, suggesting that OF testing could be valuable for monitoring in cannabis dependence treatment programs. The present research provides an important body of data on cannabinoid OF disposition, improving interpretation of cannabinoid OF test results, formulating evidence-based drug control policy, and managing cannabinoid pharmacotherapy.
    • Free and Glucuronidated Cannabinoids, Subjective, Physiological and Psychomotor Effects and In Vitro Cannabinoid Stability Following Controlled Smoked Cannabis Administration

      Schwope, David M.; Huestis, Marilyn (2011)
      Delta9-tetrahydrocannabinol (THC) is the illicit drug most frequently observed in accident and driving under the influence of drugs (DUID) investigations. Whole blood is often the only available specimen collected during such investigations. However, whole blood cannabinoid concentrations (including phase II cannabinoid glucuronide metabolites) and relationships between subjective and psychomotor effects and cannabinoid blood concentrations following cannabis smoking have not been examined. Furthermore, there was no analytical method to investigate whole blood cannabinoid glucuronides directly. Nine male and one female chronic, heavy cannabis smokers resided on a closed research unit and smoked ad libitum one 6.8% THC cannabis cigarette. Whole blood and plasma specimens were collected up to 22 h after smoking. THC, 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-THC (THCCOOH), cannabidiol (CBD), cannabinol (CBN), THC-glucuronide and THCCOOH-glucuronide were directly quantified by a new, validated liquid chromatography-tandem mass spectrometry method. Specimens were analyzed within 24 h of collection to obviate stability issues. Assessments were performed before and up to 6 h after smoking, including subjective (visual analog scales [VAS] and Likert scales), physiological (heart rate, blood pressure, respirations) and psychomotor (critical tracking and divided attention tasks) measures. Analytical method linearity (R<super>2</super> ≥ 0.997) ranged from 0.5-50 μg/L (THC-glucuronide), 1.0-100 μg/L (THC, 11-OH-THC, THCCOOH, CBD and CBN) and 5.0-250 μg/L (THCCOOH-glucuronide). Median whole blood (plasma) observed C<sub>max</sub> were 50 (76), 6.4 (10), 41 (67), 1.3 (2.0), 2.4 (3.6), 89 (190), and 0.7 (1.4) μg/L 0.25 h after starting smoking for THC, 11-OH-THC, THCCOOH, CBD, CBN, and THCCOOH-glucuronide, respectively, and 0.5 h for THC-glucuronide. CBD and CBN were not detectable after 1 h. THC significantly increased VAS responses and heart rate, with concentration-effect curves demonstrating counter-clockwise hysteresis. No differences were observed for critical tracking or divided attention task performance in this cohort of heavy, chronic cannabis smokers. These are the first authentic human whole blood cannabinoid metabolite data following controlled cannabis smoking. Minor cannabinoids and cannabinoid glucuronide data were evaluated as markers of recent cannabis use. These findings will improve interpretation of whole blood and plasma cannabinoid results, further identification of recent cannabis intake, and inform our understanding of impairment and subjective effects following acute smoked cannabis.
    • Pharmacodynamic and Pharmacokinetic Characterization of Sativex and Oral THC

      Karschner, Erin; Levine, Barry, 1957-; Huestis, Marilyn (2010)
      Sativex is an oromucosal cannabis-plant extract delivering delta-9-tetrahydrocannabinol (THC), the primary psychoactive component of cannabis, and cannabidiol (CBD), a non-psychotropic cannabinoid, in an approximate 1:1 ratio. Previous research indicated that CBD attenuated THC's pharmacodynamic effects, but the mechanism of interaction is unidentified. A placebo-controlled, double blind, double dummy administration study was developed to compare pharmacodynamic and pharmacokinetic profiles of oral THC, an FDA-approved pharmaceutical, and Sativex, an investigational medication for pain refractory to opiates in cancer patients. Experienced cannabis smokers (n=9) were randomly administered placebo, 5 and 15 mg oral synthetic THC and low (5.4 mg THC and 5.0 mg CBD) and high (16.2 mg THC and 15.0 mg CBD) dose Sativex over five sessions. A sensitive two-dimensional gas chromatography mass spectrometry method was validated to simultaneously quantify plasma CBD, THC, 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) at sub-nanogram concentrations expected after cannabinoid pharmacotherapy administration. Low calibration curves were linear from 0.25-25 ng/mL for CBD and THC, 0.125-25 ng/mL for 11-OH-THC and 0.25-50 ng/mL for THCCOOH. Analytical imprecision was <9.5%CV and bias was <9.2% of target. Inter-individual cannabinoid concentrations and pharmacodynamic responses were highly variable. Cmax and AUC0 to 10.5 were higher for all analytes after both high doses of oral THC and Sativex compared to low doses. Also, no significant pharmacokinetic differences were observed between similar oral THC and Sativex doses. THC bioavailability tended to be higher following oromucosal versus oral administration, potentially due to reduced first pass metabolism to 11-OH-THC. Similar, but moderate heart rate, "good drug effect" and anxiety increases were observed after oral THC and Sativex. Contrary to 15 mg oral THC, high dose Sativex did not significantly increase subjective "high" feelings, suggesting that CBD may attenuate euphoria. Weak linear relationships between pharmacodynamic effects and plasma THC concentrations were noted, limiting the ability to infer responses from low plasma cannabinoid concentrations. CBD did not significantly alter THC pharmacokinetics or pharmacodynamics at low, therapeutic cannabinoid doses. No strong cardiovascular or intoxication effects or serious adverse events were observed, indicating that Sativex has a pharmacodynamic safety profile comparable to oral THC at 5 and 15 mg doses.
    • Pharmacodynamics, Pharmacokinetics, and Cannabinoid Stability Following Smoked Cannabis in Occasional and Frequent Cannabis Smokers

      Desrosiers, Nathalie Anne; Huestis, Marilyn (2014)
      Cannabis is the most commonly consumed illicit drug worldwide. As few studies characterized differences between occasional and frequent cannabis smokers, we developed a controlled cannabis administration study to investigate pharmacodynamic and pharmacokinetic differences between these groups. Eleven occasional and 14 frequent smokers smoked one 6.8% delta9-tetrahydrocannabinol (THC) cannabis cigarette ad libitum over 10 min. Psychomotor, working memory, risk-taking, subjective, and physiological effects were monitored, and blood, plasma, oral fluid (OF), urine, and breath collected for up to 30 h after smoking. Optimal storage conditions for urinary cannabinoid stability were defined. Draeger DrugTest 5000 onsite OF testing device performance was determined, and a new OF quantification assay for six cannabinoids was developed. Occasional cannabis smokers had increased psychomotor tracking errors and impaired divided attention and reaction time after controlled cannabis smoking, while frequent smokers demonstrated partial tolerance to cannabis' psychomotor effects. Occasional smokers reported longer and more intense subjective effects compared to frequent smokers. Frequent smokers had higher blood and plasma cannabinoid concentrations, even after accounting for residual baseline concentrations. In OF, frequent smokers had significantly longer windows of cannabinoid detection with various screening and confirmation cutoffs. The DrugTest 5000's diagnostic sensitivity, specificity, and efficiency with a 5-µg/L screening cutoff and THC confirmation at 2 µg/L with Quantisal, Statsure and Oral-Eze OF collection devices were 74.0-90.7, 75.0-94.1, and 80.0-87.9%, respectively. Performance varied based on the different analytes included in the confirmation analysis. Frequent smokers had significantly higher urinary THC-glucuronide, 11-nor-9-carboxy-THC (THCCOOH), THCCOOH-glucuronide, and total THCCOOH concentrations than occasional smokers after smoking cannabis, even after normalizing for urinary creatinine concentrations. Frozen storage provided optimal cannabinoid stability in urine, with THC-glucuronide and THCCOOH-glucuronide being stable for up to 6 months. Our new validated method for the analysis of cannabinoids in OF is the most comprehensive method developed to date. Limits of quantification (LOQ) were 15 ng/L for THCCOOH and 0.2 µg/L for THC, 11-hydroxy-THC, tetrahydrocannabivarin, cannabidiol, and cannabigerol. Our data will aid scientists in cannabinoids identification and interpretation in clinical, driving under the influence of cannabis, criminal justice, anti-doping, and workplace drug testing settings and in the development of evidence-based drug policy.
    • Teenage Brains on Pot: Adolescent Cannabinoid Exposure to Mice and Maturation of Cortical Functions

      Raver, Sylvina Mullins; Keller, Asaf (2014)
      Regular use of marijuana during adolescence -- but not adulthood -- permanently impairs cognitive functions, and significantly elevates the risk for developing severe psychiatric diseases, such as schizophrenia, in some users. This vulnerable adolescent period coincides with the emergence of synchronous, network activity in the neocortex, termed cortical oscillations, as well as the anatomical and physiological maturation of the neural networks, neurotransmitter systems, and the endocannabinoid (eCB) system that shape oscillations. Cortical oscillations are implicated in cognitive and sensory processing, and are abnormal in patients with schizophrenia, in which these functions are impaired. We therefore proposed a link between adolescent use of marijuana, and abnormal cortical network activity in adulthood. Specifically, we hypothesized that repeated cannabinoid administration to adolescent mice would permanently alter cortical oscillations and related cognitive behaviors in adult animals. We tested this hypothesis by administering cannabinoid receptor ligands to adolescent mice, and recording oscillations both in vitro from isolated cortical preparations, and in vivo from intact, behaving mice once they reached adulthood. We find that chronic cannabinoid exposure to adolescent, but not adult animals, persistently suppresses pharmacologically-evoked cortical oscillations in vitro, preferentially in rostral neocortical areas that are less developed at the time of drug exposure. In awake, behaving adult mice, chronic exposure to cannabinoids in adolescence attenuates pharmacologically-evoked cortical oscillations, impairs working memory, and alters oscillations associated with cognitive behaviors. We reveal that attenuation of cortical oscillations in adulthood by a shorter-period of cannabinoid exposure during early adolescence, or by chronic exposure to the primary active ingredient in marijuana, Δ9 THC, is mediated by the cannabinoid-1 receptor (CB1R), and can be reversed with a CB1R antagonist. However, chronic exposure to a more potent cannabinoid receptor agonist cannot be reversed by chronic exposure to cannabinoid receptor antagonists, as antagonists also persistently attenuate oscillations in adulthood when administered alone. These data support the hypothesis that marijuana use in adolescence persistently alters synchronous activity in cortical networks in adulthood, and serve as a novel link between early cannabis use and alterations in cortical network activity implicated in cognitive processing and psychiatric disease.