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BEHAVIORAL PHARMACOLOGY

⁹-Tetrahydrocannbinol Accounts for the Antinociceptive, Hypothermic, and Cataleptic Effects of Marijuana in Mice

Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia (S.A.V., D.T.B., Q.T., B.R.M., A.H.L.); and Research Triangle Institute, Research Triangle Park, North Carolina (B.F.T.)

Received November 15, 2004; accepted April 5, 2005.

	Abstract

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Although it is widely accepted that ⁹-tetrahydrocannabinol (⁹-THC) is the primary psychoactive constituent of marijuana, questions persist as to whether other components contribute to marijuana's pharmacological activity. The present experiments assessed the cannabinoid activity of marijuana smoke exposure in mice and tested the hypothesis that ⁹-THC mediates these effects through a CB₁ receptor mechanism of action. First, the effects of ⁹-THC on analgesia, hypothermia, and catalepsy were compared with those of a marijuana extract with equated ⁹-THC content after either i.v. administration or inhalation exposure. Second, mice were exposed to smoke of an ethanol-extracted placebo plant material or low-grade marijuana (with minimal ⁹-THC but similar levels of other cannabinoids) that were impregnated with varying quantities of ⁹-THC. To assess doses, ⁹-THC levels in the blood and brains of drug-exposed mice were determined following both i.v. and inhalation routes of administration. Both marijuana and ⁹-THC produced comparable levels of antinociception, hypothermia, and catalepsy regardless of the route of administration, and these effects were blocked by pretreatment with the CB₁ antagonist SR141716 [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl]. Importantly, the blood and brain levels of ⁹-THC were similar in mice exhibiting similar pharmacological effects, regardless of the presence of non-⁹-THC marijuana constituents. The present experiments provide evidence that the acute cannabinoid effects of marijuana smoke exposure on analgesia, hypothermia, and catalepsy in mice result from ⁹-THC content acting at CB₁ receptors and that the non-⁹-THC constituents of marijuana (at concentrations relevant to those typically consumed) influence these effects only minimally, if at all.

Several hundred compounds have been isolated in various plant preparations, including 66 distinct phytocannabinoids (ElSohly, 2002). Of these, ⁹-THC, cannabidiol (CBD), cannnabichromene (CBC), cannabigerol, and cannabinol (CBN) are perhaps the most quantitatively important. It has been suggested that these other cannabinoids may act to modulate the effects of ⁹-THC, providing benefits that cannot be obtained with ⁹-THC alone. In fact, evidence in humans and animal models have shown that other constituents of marijuana can modify the effects of ⁹-THC. Particular interest has been given to CBD, which has been shown to antagonize some of the effects of ⁹-THC in mice such as catalepsy (Karniol and Carlini, 1973) and antinociception as assessed by suppression of an abdominal constriction response (Welburn et al., 1976), while potentiating other effects such as hot plate antinociception (Karniol and Carlini, 1973). It was recently reported that a CBD-rich marijuana extract produced no working memory deficits in rats trained to perform a delayed matching-to-place water maze task, even at doses that contained levels of ⁹-THC that by itself did produce deficits, suggesting an attenuation of ⁹-THC's memory-impairing effects (Fadda et al., 2004). In humans, CBD has been shown to attenuate most of the subjective effects of ⁹-THC, whereas it did not affect other effects such as tachycardia (Dalton et al., 1976; Zuardi et al., 1982). Also, CBD is known to alter the metabolism of ⁹-THC (Bornheim et al., 1995) and has been shown to have some antagonist activity at cannabinoid CB₁ receptors at high doses (Petitet et al., 1998). However, these interactions are usually only seen at doses of CBD that are as high or higher than the dose of ⁹-THC, a combination that is not often the case with recreationally available marijuana (e.g., ElSohly et al., 2000). Also, several studies have reported opposite or no such interactions between ⁹-THC and CBD (e.g., Welburn et al., 1976; Jarbe et al., 1977; Zuardi et al., 1984; Fadda et al., 2004; Finn et al., 2004). Other constituents of marijuana have also been evaluated for their own activity and their ability to modulate the effects of ⁹-THC. For example, CBN has been shown to bind weakly to CB₁ receptors and can produce effects similar to and additive with those of ⁹-THC, although again only at doses equal to or greater than the dose of ⁹-THC (Takahashi and Karniol, 1975; Petitet et al., 1998). The question of whether the relatively low concentrations of non-⁹-THC constituents commonly found in marijuana may act together to modify ⁹-THC's effects remains unanswered.

The development of a variety of pharmacological tools, particularly selective CB₁ antagonists such as SR141716 (Rinaldi-Carmona et al., 1994) and well-validated models of in vivo CB₁ receptor activation allow a direct evaluation of the hypothesis that the effects of inhaled marijuana smoke are mediated via its ⁹-THC content acting at CB₁ receptors. The present experiments were designed to address this question, as well as to examine whether or not other marijuana constituents may interact with ⁹-THC to modulate any of its effects, and whether any such interaction may differ following i.v. or inhalation exposures. The important issue of establishing the relationship between inhalation and i.v. administration on dose was addressed by measuring blood and brain levels of ⁹-THC. By quantifying blood and brain levels of drug at doses that elicited comparable pharmacological effects, we sought to determine the degree to which ⁹-THC contributes to the potency of several of marijuana's acute pharmacological effects.

	Materials and Methods

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Subjects. Male ICR mice weighing 22 to 30 g (Harlan, Indianapolis, IN) were housed in stainless steel cages in groups of five in a temperature-controlled vivarium on a 12-h light/dark cycle. Food and water were available ad libitum. All animal protocols were approved by the Virginia Commonwealth University Institutional Animal Care and Use Committee.

Drugs. Marijuana (Cannabis sativa, containing 5.19% ⁹-THC, 0.37% CBD, 0.20% cannabigerol, and 0.23% CBC), low-grade marijuana (containing 0.45% ⁹-THC, 0.58% CBD, 0.71% CBN, and 0.09% CBC), ethanol-extracted marijuana (placebo material), marijuana extract (containing 33.5% ⁹-THC, 1.01% CBN, and less than 1% of other cannabinoids), ⁹-THC, and SR141716 were obtained from the National Institute on Drug Abuse (Bethesda, MD). For i.v. injections, ⁹-THC and SR141716 were dissolved in a 1:1 mixture of absolute ethanol and alkamuls-620 (Aventis, Strasbourg, France) and diluted with saline to a final ratio of 1:1:18 (ethanol/alkamuls/saline). Injections were given at a volume of 10 ml/kg.

Apparatus. The smoke exposure system was developed in this laboratory and is based on an apparatus previously described (Meng et al., 1997), with a few modifications. The smoke was drawn through a 27.5-cm length of Tygon tubing to the manifold at a flow rate of 1.0 liter/min using a vacuum pump and flow regulator. The flow rate was modified from previous experiments (i.e., where a 0.4 liter/min flow rate was used) to reduce the amount of time required to burn a 200-mg marijuana sample from 2 to 4 to 1 to 2 min and consequently minimize hypoxia and associated behavioral toxicity. As before, a solenoid puffing device was used to alternate the flow of smoke and fresh air to the animals every 8 s. The subjects were placed into holding tubes that fit snugly into a manifold, consisting of six ports for a nose-only exposure. Tygon tubing, containing 0.5 g of glass wool fiber to sequester the smoke, was connected to the exhaust of the manifold. If the pipe or cigarette ceased to burn at any time, it was lit again until completely consumed.

Procedures. The purpose of experiment 1 was to evaluate the effects of marijuana smoke exposure and to determine whether these effects were mediated via CB₁ receptors. For the first experiment, three control groups were used: a naive group that received no treatment, an air group that was restrained in the exposure tubes and exposed to air only for 2 min, and a placebo group that was exposed to the smoke from 200 mg of the placebo plant material. In an effort to conserve mice, the naive group was not repeated in subsequent inhalation experiments. For the dose-effect determination of marijuana, the amount of total plant material was held constant at 200 mg by burning different ratios of placebo to marijuana (e.g., the 100-mg marijuana condition contained 100 mg of marijuana + 100 mg of placebo). Mice were exposed to 10, 50, 100, or 200 mg of marijuana, and assessments of drug effects were conducted as described below. In separate animals, mice were pretreated i.p. with either 10 mg/kg SR141716 or vehicle 10 min prior to exposure to smoke from 200 mg of marijuana, which had produced the largest behavioral and physiological effects in the previous experiment. This dose of SR141716 has been shown previously to block the antinociceptive effects of this amount of marijuana under similar conditions, whereas it had no effects on its own or in combination with exposure to placebo smoke (Lichtman et al., 2001).

The effects of ⁹-THC and marijuana extract when administered i.v. were compared in experiment 2. Vehicle or 0.03, 0.1, 0.3, 1, 3, 10, or 20 mg/kg ⁹-THC or the corresponding doses of marijuana extract (equated based on ⁹-THC content) were injected into the tail vein, and behavioral and physiological evaluations were conducted as described below. For the antagonism test, separate mice were pretreated i.p. with 10 mg/kg SR141716 or vehicle 10 min prior to i.v. administration of 10 mg/kg ⁹-THC or the equivalent dose (based on ⁹-THC content) of marijuana extract, the lowest dose to produce maximal effects.

In experiment 3, the acute pharmacological effects of ⁹-THC and marijuana extract upon inhalation exposure were compared by exposing mice to smoke from placebo material rolled into cigarettes with papers (Zig-Zag brand; North Atlantic Operating Company, Louisville, KY) that had been laced with varying amounts of either pure ⁹-THC (in ethanol) or an equivalent amount of ⁹-THC in marijuana extract (0.625, 1.25, 2.5, 5, 10, or 20 mg). Next, separate mice were pretreated i.p. with 10 mg/kg SR141716 or vehicle 10 min prior to inhalation exposure of placebo cigarettes laced with either 20 mg of ⁹-THC or the equivalent of marijuana extract. This amount of ⁹-THC was used for the antagonism tests in experiments 3 and 4 because it was the only amount to produce full effects in experiment 4.

Experiment 4 addressed a similar question by applying varying amounts of ⁹-THC (0.9, 1.25, 2.5, 5, 10, or 20 mg) directly onto placebo plant material or low-grade marijuana. Since the low-grade marijuana naturally contained a small amount of ⁹-THC already (determined to be 0.9 mg in a 200-mg sample), the low-grade marijuana samples were impregnated with 0.9 mg less ⁹-THC than its corresponding placebo group (i.e., the 10-mg ⁹-THC condition consisted of 10 mg of ⁹-THC added to placebo material, but only 9.1 mg of ⁹-THC was added to the low-grade marijuana). As before, antagonism tests were conducted by pretreating mice i.p. with either 10 mg/kg SR141716 or vehicle before inhalation exposure to placebo or low-grade marijuana impregnated with 20 mg of ⁹-THC. The various amounts of ⁹-THC or marijuana extract added in experiments 3 and 4 were prepared in a 200-µl volume of ethanol and were evenly pipetted directly onto 200 mg of plant material or each cigarette paper. This procedure was performed the afternoon before each test session, and the impregnated plant material/paper was stored overnight at room temperature in a dark cabinet so that the ethanol had completely evaporated prior to the exposure.

Behavioral and Physiological Evaluation. Control tail-flick latencies in the radiant heat nociceptive test (D'Amour and Smith, 1941), and core body temperatures were assessed in each subject prior to smoke exposure or i.v. administration. The intensity of the stimulus from the heat lamp in the tail-flick test was fixed to yield control latencies of 2 to 4 s, and an automatic 10-s cut-off was used. Core temperatures were measured to the nearest 0.1°C by inserting a rectal probe, connected to a telethermometer (YSI Inc., Yellow Springs, OH) to a depth of 1.8 cm. Five minutes following inhalation exposure, individual mice were placed into one of six locomotor activity chambers (MED Associates, St. Albans, VT) for a total duration of 10 min, containing photocell beams monitored with a Digiscan Animal Activity Monitor (MED Associates). Activity in the chamber was expressed as the total number of beam interruptions. As discussed below, locomotor activity was not assessed in experiments 2 to 4 because of hypolocomotion observed in control conditions during experiment 1. At 20 min postexposure, tail-flick latencies were reassessed. At 40 min, a ring test procedure was used to evaluate catalepsy (Pertwee, 1972) in which each subject was placed on a ring (5.5-cm diameter) that was elevated 16 cm from a table top for a 5-min observation period. The amount of time each subject remained motionless, except for respiratory movements, was recorded to the nearest second. Finally, core temperatures were reassessed at 60 min. All measures were taken in each animal.

Determination of ⁹-THC Blood and Brain Levels. Separate groups of mice were used to determine blood and brain levels of ⁹-THC following exposure to the various drug conditions. Mice were sacrificed 20 min following drug administration to mimic conditions at the time point when analgesia testing was carried out in the previous experiments.

Methods for extraction and LC-MS quantification of ⁹-THC from whole blood and brain tissue were modified from a previously described procedure (Lichtman et al., 2001). Calibration standards were prepared from mouse whole blood and homogenized brain (2:1, water/brain, v/w) obtained from untreated animals. Fifty nanograms of deuterated ⁹-THC (Radian Corporation, Austin, TX) was added as an internal standard to the blood samples and brain homogenates that contained varying quantities of ⁹-THC. The same quantity of deuterated ⁹-THC was added to each sample prepared from treated animals. Following an equilibration period, 2.5 ml of cold acetonitrile (high-performance liquid chromatography grade; Fisher Scientific Co., Pittsburgh, PA) was added drop-wise while vortexing. The samples were then centrifuged (Precision Vari-Hi-Speed Centricone; Precision Scientific, Winchester, VA) at 2500 rpm for 15 min to precipitate solids and then stored in a freezer (-20°C) overnight that allowed the acetonitrile layer to separate from aqueous layers. The next day the acetonitrile layer was removed and evaporated under nitrogen. Finally, the ⁹-THC/deuterated ⁹-THC was resolublized in 0.1 ml of methanol (high-performance liquid chromatography grade; Fisher Scientific Co.).

LC-MS identification was used for quantification of ⁹-THC and deuterated ⁹-THC in blood and brain matrices using an 85:15 methanol/1% glacial acetic acid (0.1% formic acid) mobile phase. A guard column was used inline with the standard reverse-phase C18 column. The mass spectrometer was run in APCI+ mode. Ions analyzed in single-ion monitoring mode were 315 for ⁹-THC and 318 for deuterated ⁹-THC. A calibration curve was constructed for each assay based on linear regression using the peak area ratios of ⁹-THC to deuterated ⁹-THC of the extracted calibration samples. No peaks were detected above background in the blank control samples, blank blood samples, or blank brain samples. The extracted standard curves ranged from 25 to 5000 ng ⁹-THC/g sample and were included in each run for the determination of ⁹-THC concentrations in blood and brain. Samples containing concentrations falling outside of this range were excluded from the analysis. Standard curves were almost always linear (r 0.99), but in the rare cases that they were not linear, the experimental samples were excluded from analysis.

Statistical Analysis. Separate ANOVAs were conducted for each condition to determine the effects of ⁹-THC dose. For experiment 1, all three control conditions (e.g., naive, air only, and placebo smoke) were included in the analyses, which were followed by Tukey's post hoc tests. For the subsequent experiments, significant ANOVAs were followed by Dunnett's post hoc tests comparing each dose against vehicle (experiment 2) or placebo smoke (experiments 3 and 4). ED₅₀ values were determined by least-squares linear regression followed by calculation of 95% confidence limits (Bliss, 1967), which were used for potency ratio analyses to compare the effects of ⁹-THC alone versus marijuana extract or of marijuana versus low-grade marijuana (Colquhoun, 1971). Student's t tests were used to analyze results from the antagonism tests. Rectal temperatures were expressed as the difference between pre- and postinjection values obtained from each mouse. Antinociception was calculated by transforming the tail-flick data to the percentage of maximum possible effect (%MPE), where %MPE = 100 x ([postinjection latency - preinjection latency]/[cut-off time - preinjection latency]). Blood and brain ⁹-THC content was analyzed with two-way ANOVAs comparing the effects of dose and type of treatment.

	Results

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Experiment 1: Effects of Marijuana Smoke. The effects of exposure to marijuana smoke on antinociception, hypothermia, catalepsy, and hypomotility are shown in Fig. 1, and the ED₅₀ values for these effects are listed in Table 1. Marijuana produced a significant and dose-dependent antinociception [F(6,47) = 5.61, p < 0.001]. Post hoc analysis showed that the antinociception produced after exposure to the 200 mg of marijuana was significantly higher than after exposure to placebo smoke or in naive mice. Marijuana smoke also produced significant decreases in rectal temperatures [F(6,47) = 6.25, p < 0.001], with the smoke from 100 and 200 mg of marijuana lowering temperatures significantly below those of the placebo and naive groups. Catalepsy was also observed following exposure to marijuana smoke [F(6,47) = 18.42, p < 0.001], which was significantly higher in 200 mg of marijuana compared with placebo, whereas almost every dose tested (except for 50 mg) produced greater catalepsy than the naive group. Locomotor activity was depressed by all doses of marijuana as well as by exposure to placebo smoke [F(6,47) = 5.36, p < 0.001]. Subjects exposed to air only also showed a tendency toward decreased activity, although this group did not significantly differ from naive mice.

View larger version (33K): [in this window] [in a new window]   Fig. 1. The effects of inhalation of marijuana smoke on antinociception (top left, %MPE), hypothermia (top right), catalepsy (bottom left), and hypoactivity (bottom right). Control conditions included a naive group (N), an air exposure group (A), and an ethanol-extracted marijuana placebo exposure group (P). Asterisks represent significant differences compared with the placebo smoke condition (p < 0.05), and plus signs represent significant differences compared with the treatment naive group (p < 0.05). The results are presented as means ± S.E. with six to nine mice per group.

View this table: [in this window] [in a new window]   TABLE 1 ED50 values (with 95% confidence limits) for antinociception, hypothermia, and catalepsy following i.v. administration of 9-THC or marijuana extract, inhaled marijuana smoke, inhaled 9-THC, or marijuana extract on placebo paper (method 1) or inhaled 9-THC on placebo or LGM material (method 2) Potency ratios (with 95% confidence limits) are listed for each comparison. No significant potency differences between treatments (at each administration route) were observed. Intravenous ED50 values are expressed as milligrams per kilogram. Inhalation ED50 values are expressed as milligrams of 9-THC in the matrix prior to burning (shared between six mice).

As shown in Fig. 2, pretreatment with 10 mg/kg SR141716 significantly blocked the antinociceptive [t(10) = 3.7, p < 0.01], hypothermic [t(10) = 2.4, p < 0.05], and cataleptic [t(10) = 6.6, p < 0.001] but not locomotor depressive effects of 200 mg of marijuana smoke exposure. Because exposure to smoke produced hypomotility irrespective of marijuana content, locomotor activity was not assessed in any subsequent experiments.

View larger version (30K): [in this window] [in a new window]   Fig. 2. The effects of pretreatment (10 min before exposure) of 10 mg/kg SR 141716 (SR) on the effects of inhalation exposure to the smoke generated by 200 mg of marijuana on antinociception (top left), hypothermia (top right), catalepsy (bottom left), and locomotor activity (bottom right). *, p < 0.05; **, p < 0.01; ***, p < 0.001 for vehicle versus SR pretreatment. The results are presented as means ± S.E. with six to nine mice per group.

Experiment 2: Acute Pharmacological Effects following i.v. Administration of ⁹-THC or Marijuana Extract.

View larger version (25K): [in this window] [in a new window]   Fig. 3. Acute pharmacological effects of pure 9-THC or marijuana extract containing an equivalent amount of 9-THC following i.v. administration. Mice were given an i.v. injection of vehicle or drug and evaluated for antinociception in the tail-flick test (left), rectal temperature (middle), and catalepsy (right). Asterisks, significant differences in the 9-THC group compared with vehicle; open stars, significant differences in the extract group compared with vehicle (p < 0.05). The results are presented as means ± S.E. with six to nine mice per group.

Figure 4 depicts the impact of SR141716 (10 mg/kg) pretreatment on the pharmacological effects following an i.v injection of either ⁹-THC (10 mg/kg) or marijuana extract that contained 10 mg/kg ⁹-THC. Planned comparisons revealed that SR141716 blocked the antinociceptive [t(10) = 13.9, p < 0.001], hypothermic [t(10) = 9.4, p < 0.001], and cataleptic [t(10) = 3.1, p < 0.05] effects of ⁹-THC. SR141716 also blocked the antinociceptive [t(10) 6.4, p < 0.001], hypothermic [t(10) = 7.3, p < 0.001], and cataleptic [t(10) 2.6, p < 0.05] effects of marijuana extract.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Effects of pretreatment (10 min before exposure) of 10 mg/kg SR on the effects of 10 mg/kg 9-THC or the equivalent amount of marijuana extract after i.v. administration on antinociception (left), hypothermia (middle), and catalepsy (right). *, p < 0.05; ***, p < 0.001 for vehicle versus SR pretreatment. The results are presented as means ± S.E. with six mice per group.

Experiment 3: Acute Pharmacological Effects following an Inhalation Exposure to Smoke from Cigarettes Containing Either ⁹-THC or Marijuana Extract. The goal of this experiment was to compare the acute pharmacological effects between ⁹-THC and marijuana extract upon inhalation exposure. Mice were exposed to air alone, placebo smoke, or smoke containing various amounts of cannabinoids. As can be seen in Fig. 5, both ⁹-THC and marijuana extract produced dose-related increases in antinociception, hypothermia, and catalepsy. Significant effects of ⁹-THC were found for antinociception [F(7,87) = 18, p < 0.001; all doses were higher than placebo], hypothermia [F(7,87) = 13, p < 0.001; 10 and 20 mg/kg greater than placebo], and catalepsy [F(7,87) = 13, p < 0.001; 2.5, 10, and 20 mg/kg greater than placebo]. Similarly, significant effects of marijuana extract were found for antinociception [F(7,94) = 23, p < 0.001; all doses but 0.5 mg/kg greater than placebo], hypothermia [F(7,94) = 11, p < 001; 2.5, 5, and 20 mg/kg greater than placebo], and catalepsy [F(7,94) = 30, p < 0.001; 5, 10, and 20 mg/kg greater than placebo]. Potency ratios calculated from the ED₅₀ values (see Table 1) revealed no significant differences between ⁹-THC and marijuana extract on any measure.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Effects of inhalation of placebo cigarettes laced with either pure 9-THC or marijuana extract containing an equivalent amount of 9-THC. Mice were exposed to air alone (A), placebo smoke (P), or smoke containing either 9-THC or 9-THC in marijuana extract and assessed for antinociception (left), hypothermia (middle), and catalepsy (right). Asterisks denote significant differences in the 9-THC group compared with placebo (p < 0.05). Open stars, significant differences in the extract group compared with placebo (p < 0.05). The results are presented as means ± S.E. with six mice per group.

Figure 6 depicts the impact of SR141716 (10 mg/kg) pretreatment on the pharmacological effects of smoke from cigarettes containing either ⁹-THC (20 mg) or marijuana extract that contained 20 mg of ⁹-THC. SR141716 blocked the antinociceptive [t(10) = 12.0, p < 0.001], hypothermic [t(10) = 4.4, p < 0.001], and cataleptic [t(10) = 4.7, p < 0.001] effects of ⁹-THC and also blocked the antinociceptive [t(22) = 5.2, p < 0.001] and cataleptic [t(22) = 3.6, p < 0.01] effects of marijuana extract. However, in this experiment, the magnitude of the hypothermia produced by the marijuana extract was relatively small; consequently, the effects of SR141716 were not significant on this measure.

View larger version (18K): [in this window] [in a new window]   Fig. 6. Effects of pretreatment (10 min before exposure) of 10 mg/kg SR on the effects of 9-THC and marijuana extract (both samples containing 20 mg of 9-THC) after inhalation exposure on antinociception (left), hypothermia (middle), and catalepsy (right). **, p < 0.01; ***, p < 0.001 for vehicle versus SR pretreatment. The results are presented as means ± S.E. with 6 to 12 mice per group.

Experiment 4: Effects following Inhalation Exposure to Placebo or Low-Grade Marijuana Impregnated with ⁹-THC.

View larger version (21K): [in this window] [in a new window]   Fig. 7. Effects of inhalation exposure to equal amounts of 9-THC applied to either an ethanol-extracted marijuana placebo or low-grade marijuana (LGM). Mice were exposed to air alone (A), placebo smoke (P), or smoke containing various amounts of 9-THC. The lowest dose of 9-THC (0.9 mg) is the amount naturally occurring in 200 mg of low-grade marijuana and represents a low-grade marijuana-only exposure. Asterisks, significant differences in the placebo + 9-THC group compared with placebo. Open stars, significant differences in the LGM + 9-THC group compared with placebo. *, p < 0.05. The results are presented as means ± S.E. with 6 to 12 mice per group.

View larger version (17K): [in this window] [in a new window]   Fig. 8. Effects of pretreatment (10 min before exposure) of 10 mg/kg SR on the effects of inhaled 9-THC (20 mg/sample) that had been applied to either an ethanol-extracted marijuana placebo or LGM on antinociception (left), hypothermia (middle), and catalepsy (right). *, p < 0.05; ***, p < 0.001 for vehicle versus SR pretreatment. The results are presented as means ± S.E. with 15 mice per group.

Analysis of ⁹-THC Content in Blood and Brain. Levels of ⁹-THC in the blood and brains of mice receiving treatments similar to those in the experiments above are presented in Table 2. Analysis of blood and brain ⁹-THC levels following i.v. administration of ⁹-THC or marijuana extract revealed a significant main effect of dose [brain, F(2,50) = 42, p < 0.001; blood, F(2,49) = 52, p < 0.001], but no significant effect of treatment. Similarly, when administered via the inhalation route (solutions were applied to placebo cigarettes), main effects of dose were found for brain [F(1,82) = 131, p < 0.001] and blood [F(1,75) = 62, p < 0.001] levels, but there were no differences found between ⁹-THC and marijuana extract for either blood or brain.

View this table: [in this window] [in a new window]   TABLE 2 Levels of 9-THC measured in blood and brain of mice under different exposure conditions No significant differences were found between 9-THC and marijuana extract for either route of administration. The levels of 9-THC in blood and brain are depicted as mean ± S.E. Sample sizes for each condition are in parentheses. In the inhalation conditions, listed doses of 9-THC reflect the total amount of drug in the samples prior to burning. This total was then exposed to groups of six mice.

	Discussion

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Results from these experiments strongly suggest that the acute pharmacological effects of marijuana on tail-flick latencies, catalepsy, and hypothermia in mice can be accounted for solely by its ⁹-THC content acting via CB₁ receptors. Furthermore, we have found no evidence to support the hypothesis that other constituents of marijuana modulate those effects to any relevant degree, at least at relative concentrations of those constituents that are most commonly found in marijuana in circulation.

The effects of inhaled marijuana smoke were shown to be dose-dependent and were completely reversed by pretreatment with SR141716. These results confirm and extend those published earlier from this lab (Lichtman et al., 2001). One difference between these results and those previously reported is that in the present study, no significant hypothermia was observed following exposure to placebo smoke. This difference is most likely due to slight modifications of our exposure system, most notably an increase in the air flow rate that reduced the amount of time necessary to burn a 200-mg sample and allowed us to minimize the toxicity associated with the placebo smoke condition. This is an important consideration because many early studies examining the effects of smoke inhalation were plagued by toxicity in control conditions (e.g., Weinberg et al., 1977; Rosenkrantz and Hayden, 1979). As reported previously (Lichtman et al., 2001), decreases in locomotor activity were observed after placebo smoke exposure and even to a lesser degree following air only exposures, indicating that some degree of behavioral impairment was still present, possibly resulting from the stress associated with the inhalation apparatus. For this reason, subsequent assessments of cannabimimetic activity were restricted to tests of antinociception, hypothermia, and catalepsy.

The present data allow several comparisons to be made that support the contention that administration via the inhalation or i.v. routes does not alter the relative potency or specificity of the activity of either marijuana or ⁹-THC. Marijuana, whether inhaled or administered i.v. in extract form, produced significant degrees of antinociception, hypothermia, and catalepsy, all of which were reversed by SR141716. Similarly, i.v. administration of ⁹-THC produced the same effects as inhalation exposures to smoke from ⁹-THC-impregnated placebo material; again, all effects were blocked by SR141716. Importantly, levels of ⁹-THC in the blood and brain were similar after doses that produced comparable behavioral and physiological effects, regardless of the route of administration and regardless of the presence of non-⁹-THC marijuana constituents. This relationship between tissue levels of ⁹-THC and behavioral and physiological effects is consistent with other work from our laboratory in which subchronic dosing of either exposure to marijuana smoke or i.v. ⁹-THC was used to show that brain levels of ⁹-THC decreased at a comparable rate for both routes of administration (D. M. Wilson, D. T. Bridgen, S. A. Varvel, and A. H. Lichtman, manuscript submitted for publication). Much of the preclinical work with ⁹-THC has employed an i.v. route of administration, making this a logical first choice to compare with inhalation. However, the more clinically relevant comparison will be to compare inhalation with the oral route, which is the only formulation in which ⁹-THC has currently been approved for use. Research in this area is ongoing.

The present data also demonstrate that the effects of marijuana on antinociception, hypothermia, and catalepsy can be accounted for by its ⁹-THC content and that marijuana's other constituents do not seem to modify these effects, at least at the time points at which they were assessed in the present study. Specifically, the pharmacological profiles of i.v. and inhaled ⁹-THC were not altered significantly by the presence of other marijuana constituents, whether marijuana's alkaloid constituents were added (in the form of the marijuana extract) to the placebo plant material or when the non-⁹-THC constituents were embedded within the matrix of the plant material itself, as in the low-grade marijuana + ⁹-THC condition. Again, comparable levels of ⁹-THC in blood and brain produced comparable behavioral and physiological effects, regardless of the presence or absence of non-⁹-THC marijuana constituents. The only differences observed in these procedures were in the antagonism studies where the extract + placebo cigarettes produced a smaller than expected hypothermic effect and where the cataleptic effects of ⁹-THC + low-grade marijuana were not significantly reduced by SR 141716. However, the trends exhibited in those two instances were still consistent with the other data, and despite their failure to reach statistical significance, it does not seem that these results constitute evidence of any ⁹-THC-constituent interaction. It was anticipated that by using both approaches to these inhalation route comparisons, limitations inherent to each approach could be overcome. For example, it was not clear whether the constituents soaked on the cigarette paper or the plant material would be pyrolyzed and released as evenly as those embedded within the matrix of the plant material or whether impregnation procedure used with the low-grade marijuana experiments would yield reliable results. However, both procedures yielded comparable outcomes.

Although it may be predicted from these and other experiments that ⁹-THC and marijuana should possess similar clinical profiles (both with respect to therapeutic potential as well as side effect liability), several factors limit the generality of the present findings and emphasize the need for further research. For example, each of the behavioral and physiological indices used to assess the effects of marijuana in the present study were selected because of their sensitivity to cannabinoid agonists. This particular constellation of effects of CB₁ agonists: antinociception, hypothermia, and catalepsy (along with hypoactivity) has been well validated as a marker of CB₁ activity and shown to be highly stereospecific (e.g., Wiley et al., 1998). Although the present study provides no evidence of interactions between ⁹-THC and other marijuana constituents, it does not preclude the possibility that such interactions may influence other mechanisms that are not reflected in these particular assays, such as possible activity at non-CB₁ sites or at CB₁ receptors that are part of neural circuits distinct from those involved with analgesia, hypothermia, or catalepsy. Just as importantly, no attempts were made in the present experiments to determine the rate of onset or the duration of the effects that were assessed, factors that could be just as clinically relevant as a change in maximal efficacy. For example, the antispasticity effects of a marijuana extract in a mouse model of multiple sclerosis were reportedly accounted for by its ⁹-THC content, but the presence of low levels of other marijuana constituents seemed to enhance the rate of onset of this effect (Wilkinson et al., 2003). Furthermore, the onset and potency of anticonvulsant effects of ⁹-THC in an in vitro rat brain slice model of epilepsy were enhanced by the presence of these other marijuana constituents (Wilkinson et al., 2003).

Another factor worthy of note is that all of the preparations used in the present study to represent the natural combination of marijuana constituents (including the marijuana itself, the marijuana extract, and the ⁹-THC-impregnated low-grade marijuana) were composed of levels of non-⁹-THC constituents that were much lower than levels of ⁹-THC. It is perhaps not surprising in these instances that ⁹-THC-induced effects were not significantly affected by these other components, and it might be argued that varieties of marijuana with higher relative levels of other constituents may show interactions that were not observed in the present study. In fact, some strains of marijuana do indeed have higher relative quantities of other constituents. For example, some Turkish varieties of marijuana have been reported to contain quantities of CBD much higher than ⁹-THC (Rosenkrantz and Hayden, 1979). Other examples of perhaps greater relevance include some strains of marijuana that have been specifically bred for particular ratios of ⁹-THC to CBD (e.g., GW Pharmaceuticals, 2003). It is possible that the present study may have identified interactions if such strains had been analyzed. However, as a practical matter, the marijuana used in the present study is representative of the vast majority of marijuana in circulation. A survey of 1500 marijuana seizures from 1970 to 1997 showed that although the ⁹-THC content has progressively increased, levels of other constituents have not and constitute a shrinking percentage of the total (ElSohly et al., 2000). Whether or not chronic use of these marijuana strains may lead to accumulation of non-⁹-THC constituents in the brain that do rise to levels that may modulate ⁹-THC's effects remains an unanswered question.

In conclusion, marijuana smoke produces cannabinergic effects in mice that seem to be at least primarily mediated by ⁹-THC acting via CB₁ receptors. Despite anecdotal reports of increased efficacy and decreased side effects of smoked marijuana compared with orally administered ⁹-THC, we found no evidence of any such interactions between ⁹-THC and other marijuana constituents when evaluated acutely in the present study.

	Footnotes

 
This work was supported by National Institute on Drug Abuse Grant DA02396.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.104.080739.

ABBREVIATIONS: ⁹-THC, ⁹-tetrahydrocannabinol; CBD, cannabidiol; CBC, cannnabichromene; CBN, cannabinol; SR141716, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl; ANOVA, analysis of variance; %MPE, percentage of maximum possible effect; LGM, low-grade marijuana.

Address correspondence to: Dr. Aron H. Lichtman, Department of Pharmacology and Toxicology, Virginia Commonwealth University, Box 980613, Richmond, VA 23298-0613. E-mail: alichtma{at}hsc.vcu.edu

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