Journal of Pharmacological and Toxicological Methods 70 (2014) 112119
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Journal of Pharmacological and Toxicological Methods
j ourna l homepage: www.e lsev ie r .com/ locate / jpharmtoxOriginal articleA vapourized 9-tetrahydrocannabinol (9-THC) delivery system part II:Comparison of behavioural effects of pulmonary versus parenteralcannabinoid exposure in rodentsLaurie A. Manwell a,b,c,, Brittany Ford a, Brittany A. Matthews a, Heather Heipel a, Paul E. Mallet a
a Department of Psychology, Wilfrid Laurier University, Waterloo, ON N2L3C5, Canadab Department of Psychology, University of Guelph, Guelph, ON N1G2W1, Canadac Centre for Addiction and Mental Health, Social Aetiology of Mental Illness Program, University of Toronto, ON M5T1R8, CanadaAbbreviations:9-THC,9-tetrahydrocannabinol; 11-Orahydrocannabinol; IP, intraperitoneal; IV, intravenous;subcutaneous. Unit 1111, 33 Russell Street, Toronto, ON, M5S 2
8501x77632.E-mail addresses: email@example.com, laurieman
http://dx.doi.org/10.1016/j.vascn.2014.06.0041056-8719/ 2014 Elsevier Inc. All rights reserved.a b s t r a c ta r t i c l e i n f oArticle history:
Received 25 February 2014Accepted 4 June 2014Available online 21 June 2014
Keywords:9-Tetrahydrocannabinol (9-THC)Pulmonary administrationLocomotionCross-sensitizationFood consumptionRat
Introduction: Studies of the rewarding and addictive properties of cannabinoids using rodents as animal modelsof human behaviour often fail to replicate findings from human studies. Animal studies typically employ paren-teral routes of administration, whereas humans typically smoke cannabis, thus discrepancies may be related todifferent pharmacokinetics of parenteral and pulmonary routes of administration. Accordingly, a novel deliverysystem of vapourized 9-tetrahydrocannabinol (9-THC) was developed and assessed for its pharmacokinetic,pharmacodynamic, and behavioural effects in rodents. A commercially available vapourizer was used to assessthe effects of pulmonary (vapourized) administration of 9-THC and directly compared to parenteral (intraper-itoneal, IP) administration of 9-THC.Methods: SpragueDawley rats were exposed to pure 9-THC vapour (1, 2, 5, 10, and 20 mg/pad), using a Volca-no vapourizing device (Storz and Bickel, Germany) or IP-administered 9-THC (0.1, 0.3, 0.5, 1.0 mg/kg), anddrug effects on locomotor activity, food and water consumption, and cross-sensitization to morphine (5 mg/kg)
were measured.Results: Vapourized 9-THC significantly increased feeding during the first hour following exposure, whereas IP-administered 9-THC failed to produce a reliable increase in feeding at all doses tested. Acute administration of10 mg of vapourized 9-THC induced a short-lasting stimulation in locomotor activity compared to control inthe first of four hours of testing over 7 days of repeated exposure; this chronic exposure to 10 mg of vapourized9-THC did not induce behavioural sensitization to morphine.Discussion: These results suggest vapourized9-THC administration produces behavioural effects qualitatively dif-ferent from those induced by IP administration in rodents. Furthermore, vapourized 9-THC delivery in rodentsmay produce behavioural effects more comparable to those observed in humans. We conclude that some of theconflicting findings in animal and human cannabinoid studies may be related to pharmacokinetic differences as-sociated with route of administration. 2014 Elsevier Inc. All rights reserved.1. Introduction
Studies in both humans and rodents report significant variability inthe pharmacokinetic, pharmacodynamic and behavioural effects of can-nabinoids, particularly for 9-tetrahydrocannabinol (9-THC), acrossdifferent routes of administration, and only a very few number of stud-ies have directly compared the effects of pulmonary and parenteralH-9-THC, 11-hydroxy-9-tet-MDI, metered dose inhaler; SC,
S1, Canada. Tel.: +1 416 535
firstname.lastname@example.org (L.A. Manwell).administration of 9-THC in humans (e.g., Naef, Russman, Petersen-Felix, & Brenneisen, 2004) or rodents (e.g., Fried, 1976; Fried &Neiman, 1973; Niyuhire et al., 2007; Wilson, Varvel, Harloe, Martin, &Lichtman, 2006; Wilson et al., 2002). In human trials, Naef et al.(2004) reported that pulmonary administration of a 9-THC liquidaerosol (0.053 mg/kg) produced peak plasma levels of 9-THC inthe range of 18.7 7.4 ng/ml for approximately 10 to 20 min afterinhalation, and then rapidly decreased; however, plasma levelsafter intravenous (IV) administration of 9-THC (0.053 mg/kg)were more variable, ranging from 81.6 to 640.6 ng/ml (mean of271.5 61.1 ng/ml) within 5 min after injection (Naef et al.,2004). More importantly, Naef et al. (2004) reported that the psy-chological and somatic side effects of 9-THC differed according tothe route of administration: although parenteral administration of
113L.A. Manwell et al. / Journal of Pharmacological and Toxicological Methods 70 (2014) 1121199-THC produced greater euphoria, it also produced greater anxiety,irritation, confusion and disorientation, hallucination, nausea, andheadache compared to pulmonary administration, which only re-sulted in transient respiratory irritation.
Early studies on rodents, by Fried and Neiman (1973), reported thatrats exposed to inhaled cannabis smoke showed electrical brain activity(via electroencephalographic recordings) in both cortical and hippo-campal regions which was similar, but less pronounced, than that ob-served in rats administered intraperitoneal (IP) injections of 9-THC.Fried and Neiman (1973) also compared the effects of oral and IP ad-ministrated 9-THC, versus exposure to cannabis smoke, on rats in theopen-field test; all conditions significantly reduced exploratory behav-iour, with injected 9-THC having the greatest impact. However, ratsadministered IP or oral 9-THC, but not cannabis smoke, were reportedto be hypersensitive, exhibiting greater vocalizations when handledafter testing. In studies of chronic exposure to inhaled cannabis, Fried(1976) showed that male, but not female, rats developed cross-tolerance to acute exposure to IP administered9-THC.Male and femalerats exposed to cannabis smoke every other day for 32 days showed re-duced locomotor activity on the initial trials (1 to 10) which thenreturned to baseline levels near the last trials (13 to 16); however, re-peated exposure to cannabis smoke significantly increased locomotoractivity after IP injection of 9-THC, indicating tolerance in male butnot female rats (Fried, 1976).
More recent studies in rodents, by Wilson et al. (2002), used ametered dose inhaler (MDI) that aerosolized 9-THC to provide asystematic route of exposure in mice. They found that in malemice, intravenous (IV) administered and inhaled9-THC produced sim-ilar levels of 9-THC in blood and brain tissue at the antinociceptiveED50 dose (median effective dose of 30mg) and that the behavioural ef-fects of inhaled 9-THC antinociception, hypomotility, catalepsy, andhypothermiawere all significantly antagonized by co-administrationof the CB1 antagonist/inverse agonist SR141716. Subsequently, Wilsonet al. (2006) reported that although SR141716 induces withdrawalsigns (e.g., paw tremors) in mice chronically exposed to either inhaledmarijuana smoke or IV administered 9-THC, only co-administrationof IV administered 9-THC reversed these effects; inhaled marijuanasmoke did not prevent precipitated withdrawal induced by SR141716.To account for this unexpected finding, Wilson et al. (2006) suggestedthat levels of 9-THC from the marijuana smoke in the brain afterinhalation were insufficient to reverse the effects of SR141716 andthus themechanism of action was still likely CB1-mediated. This con-clusion was supported by additional findings of a dissociation be-tween 9-THC levels found in blood and brain that were dependentupon the route of administration; although blood and brain levelswere roughly equivalent following inhalation, brain levels were200300% greater in brain than in blood after IV administration(Wilson et al., 2006). In mice, Niyuhire et al. (2007) showed thatthe effects of 9-THC on learning and memory also differed accordingto the route of administration: IP administration of 9-THC (1, 3,10 mg/kg) dose-dependently disrupted both acquisition and recall ofplatform location in the Morris water maze task, whereas inhaledsmoke from marijuana (50, 100, and 200 mg) only impaired perfor-mance at the highest dose (estimated to have 4.2 mg 9-THC beforeburning). Co-administrationwith SR141716 reversed these effects, sug-gesting that the mechanism of action was also CB1 mediated (Niyuhireet al., 2007).
All of these studies, which directly compared the effects of paren-teral versus pulmonary 9-THC demonstrate that the route of ad-ministration produces qualitatively different results, which mayaccount for some of the many conflicting findings in cannabinoid re-search in animal models of human behaviour. Since the combustionof cannabis releases many compounds in addition to 9-THC, includingother cannabinoids (e.g., cannabidiol) andmany toxins and carcinogens(e.g., anthrocyclines, nitrosamines, polycyclic aromatic hydrocarbons,terpenes, and vinyl chloride) (Turner, Bouwsma, Billets, & Elsohly,1980; Turner, Elsohly, & Boeren, 1980; Sarafian et al., 1999; Zhanget al., 1999; Roth et al., 2001; Hashibe et al., 2005; Voirin et al., 2006;Aldington et al., 2008; Berthiller et al., 2008; also reviewed in Reece,2009) which may exert their own effects, vapourization of pure 9-THC provides a more accurate assessment of the direct effects of 9-THC and its primary psychoactive metabolite, 11-hydroxy-9-THC(11-OH-9-THC). In the current study, a novel delivery system ofvapourized 9-THCwas developed and assessed for its pharmacokinet-ic, pharmacodynamic, and behavioural effects in rodents. In addition,studies in animals need to account for actual bioavailable levels of 9-THC in blood after injection and inhalation of pure 9-THC, not justmarijuana smoke containing unknown quantities of 9-THC and nu-merous other toxicants. Previously, we have directly compared canna-binoid recovery levels of 9-THC and 11-OH-9-THC in whole bloodafter IP and vapourization exposure to 9-THC in rodents (Manwellet al., 2014-in this issue); for the applications to animal behaviour stud-ies, we were only interested in the psychoactive cannabinoids 9-THCand itsmetabolite 11-OH-9-THC. A commercially available vapourizer,commonly used by cannabis smokers, was used to assess the effects ofpulmonary administration of 9-THC and directly compared to paren-teral administration of 9-THC; drug effects on locomotor activity,food and water consumption, and cross-sensitization to morphinewere measured.
2. Material and methods
2.1. Materials, standards, and chemicals
9-THC (Dronabinol, N98% purity) was obtained from THC PharmGmbH (Frankfurt, Germany). For experiments involving IP drug ad-ministration, 9-THC was first dissolved in a small volume of ethanoland then mixed with TWEEN-80 (polyoxyethylene sorbitanmonooleate; ICN Biomedicals). The ethanol was evaporated undera stream of nitrogen gas, and the dispersion was then mixed withphysiological saline. The final vehicle contained 15 l TWEEN-80per 2 ml saline. 9-THC was prepared in concentrations of 0.1, 0.30.5, 1.0, 1.5, and 2.0 mg/ml and injected in a volume of 1 ml/kg. Forexperiments involving pulmonary (inhaled) drug administration,9-THC was prepared in ethanol at concentrations of 4, 8, 20, 40and 80 mg/ml, and 250 l of each was applied to small steel woolpads (liquid pads, Storz & Bickel, Tuttlingen, Germany), yieldingfinal amounts of 1, 2, 5,10, and 20 mg/pad. The ethanol was thenallowed to evaporate completely before vapourization. Morphinehydrochloride (CDMV, St. Hyacinth, Quebec) was dissolved in 0.9%saline and administered subcutaneously (SC) at a dose of 5 mg/kgin a volume of 1 ml/kg body weight. Equivalent injections of vehiclewere given for the saline probe.
Male SpragueDawley rats (Charles River, Canada) (n = 120),weighing approximately 200300 g, were used in experiments. Ratswere pair-housed in standard plastic shoebox cages (45 25 20 cm3) maintained at 2122 C in a colony room on a 12-h reversedlightdark cycle (lights off at 7 AM) and fed standard rat chow (Harlan8640) and water ad libitum. Testing was conducted during the darkcycle. Experimental procedures followed Canadian Council on AnimalCare guidelines and were approved by the Wilfrid Laurier UniversityAnimal Care Committee. Rats were acclimatized to the colony and han-dling procedures prior to experimentation.
2.3.1. Vapourization apparatusA Volcano Vapourization device (Storz and Bickel, GmbH and
Co., Tuttlingen, Germany) was used as described by Hazekamp,
114 L.A. Manwell et al. / Journal of Pharmacological anRuhaak, Zuurman, van Gerven, and Verpoorte (2006). Briefly, 9-THC(1, 2, 5, 10 or 20mg) was vapourized (at heat setting 9, approximate-ly 226 C) and collected into detachable plastic balloons (approxi-mately 8 l), which were then fitted with a release valve and thevapour immediately expelled (over 10 s) into enclosed plasticboxes (32 16 12 cm3) containing two rats that inhaled the va-pour for 5 min. After 5 min, the lids were opened for ventilation.This device has been previously reported by Hazekamp et al.(2006) to deliver N50% of the loaded 9-THC into the balloon in a re-produciblemannerwith a pulmonary uptake comparable to smokingcannabis.
2.3.2. Behavioural testing apparatusSix enclosures (292 610 292mm high) equipped with a video
tracking system were used to detect locomotor activity and con-sumption testing. The chambers had ultra-high molecular weight(UHMA) polyethylene sides and acrylonitrile butadiene styrene(ABS) plastic floors, while the tops were made of clear acrylic. Activ-ity under dim red light (0.04 lx) was recorded by three video cam-eras mounted 110.5 cm above the chambers and images weretransmitted to a computer in the neighbouring room running ANY-maze Video Tracking System software (Stoelting Co., Wood Dale, IL,USA). During testing, food was presented in cylindrical glass dishes(120 mm diameter 20 mm high) placed in one corner of the cham-ber, and plastic drinking bottles containing tap water were attachedto one side of the chamber. Dishes were washed daily at the comple-tion of the session with a detergent solution. Each rat received thesame dish for the duration of the experiment. Measurements offood and water were obtained using a digital scale. For all experi-ments, locomotor activity was recorded by ANY-maze using threeoverhead video cameras (110.5 cm above the chambers) and definedby total distance travelled (m), total ti...