Jim Finnel
Fallen Cannabis Warrior & Ex News Moderator
Robert E. Vann, Charles D. Cook, Billy R. Martin, and Jenny L. Wiley
Abstract
Side effects of marijuana-based drugs and synthetic analogs of Δ9-tetrahydrocannabinol (Δ9-THC), including sedation and dysphoria, have limited their therapeutic application. Ajulemic acid (AJA), a side-chain synthetic analog of Δ8-THC-11-oic acid, has been reported to have anti-inflammatory properties without producing undesired psychoactive effects. Moreover, it has been suggested that AJA does not interact with cannabinoid receptors to produce its pharmacological effects. The aim of the present study was to conduct a thorough evaluation of the pharmacological effects of AJA then to determine whether actions at cannabinoid receptor (CB)1 mediated these effects. This study evaluated the psychoactive and analgesic effects of AJA by examining its cannabimimetic properties in ICR mice (i.e., antinociception, catalepsy, hypothermia, and hypomobility), its discriminative stimulus effects in Long Evans rats trained in a two-lever Δ9-THC (3.0 mg/kg) versus vehicle drug discrimination procedure, and its antihyperalgesia effects in a rat model of inflammatory pain [complete Freund's adjuvant (CFA)-induced mechanical hyperalgesia]. Lastly, antagonism tests with SR 141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride], CB1 receptor antagonist, were conducted. These studies demonstrated that AJA shares a number of CB1-mediated pharmacological properties with Δ9-THC, including cannabimimetic, discriminative stimulus, and antihyperalgesic effects. Furthermore, a separation between doses that produced antinociception and those that produced the other pharmacological effects in mice was not observed. Moreover, AJA showed nearly equipotency for therapeutic efficacy in the CFA model and for substitution in Δ9-THC discrimination. In summary, this study shows that AJA, like Δ9-THC, exhibits psychoactive and therapeutic effects at nearly equal doses in preclinical models, suggesting similar limitations in their putative therapeutic profiles.
Cannabis sativa (marijuana plant) has been used since antiquity for its presumed therapeutic, as well as for its euphoric effects. Although Δ9-tetrahydrocannabinol (Δ9-THC) has been identified as the major psychoactive ingredient in C. sativa (Gaoni and Mechoulam, 1971), difficulty in dissociating unwanted side effects, such as sedation and psychotropic effects, from therapeutic effects has limited clinical application of Δ9-THC-based drugs. For example, dronabinol, an orally administered synthetic version of Δ9-THC, has been developed as an appetite stimulant and antiemetic for use in chronic diseases such as AIDS and cancer (for review, see Ben Amar, 2006). In addition, recent evidence suggests oral Δ9-THC may be effective as an adjunct to opioid analgesics (Roberts et al., 2006). The therapeutic utility of Δ9-THC, however, has been limited due to patient complaints of dysphoria and unpleasant subjective effects (Ben Amar, 2006). Previous research has suggested that Δ9-THC carboxylic acid, one of the acid metabolites of Δ9-THC, lacks psychoactive properties of the parent compound and yet retains antinociceptive and other effects (Burstein et al., 1988; Doyle et al., 1990). Since this metabolite has a relatively low potency, structural changes that increased potency and stability of Δ9-THC analogs in previous structure-activity relationship studies (Loev et al., 1973; Mechoulam et al., 1988; Compton et al., 1993) were applied to the structure Δ9-THC carboxylic acid. The resulting compound, ajulemic acid (AJA), substitutes a 1′,1-dimethylheptyl side chain for the pentyl group of Δ9-THC and changes the Δ9-THC core structure to a more stable confirmation, Δ8-THC (Fig. 1). Fig. 1
Chemical structures of Δ9-THC and AJA.
To date, the efficacy of AJA has been demonstrated in numerous pain and inflammation studies (for review, see Wiley, 2005). These findings are equivocal, however, in that effects have been found with a single dose or a restricted range of doses or have been reported in different strains or species, making comparisons and conclusions problematic. For example, a number of studies have reported that AJA exhibits efficacy similar to that of Δ9-THC in acute rodent models of pain, including hot-plate, formalin, writhing, and tail-clip tests in the mouse (Burstein et al., 1992, 1998; Sumariwalla et al., 2004; Dyson et al., 2005) and hot-plate and tail-clip tests in rats (Dajani et al., 1999). In an experimental model of rheumatoid arthritis, complete Freund's adjuvant (CFA), chronic dosing with AJA reduced the degree of joint deformity, as measured by histological analysis (Zurier et al., 1998). In addition, several acute dosing studies showed that AJA reduced experimentally induced pain or inflammation in rats (Burstein et al., 1998; Dyson et al., 2005; Mitchell et al., 2005). Potencies for AJA reported in two of these studies (Burstein et al., 1998; Dyson et al., 2005), however, were 10-fold greater in magnitude than the single dose reported to be effective in the only other published study that used similar methods (Mitchell et al., 2005).
Unfortunately, previous analgesic studies with AJA have not included a careful evaluation of behavioral effects that might be either present or absent in the therapeutic dose range. Therefore, the present study was designed to elucidate more fully the analgesic and side-effect profile of AJA in the same species and with similar dosing parameters. First, the effects of AJA were compared with Δ9-THC in mice in a well established tetrad of tests, in which cannabinoids produce antinociception ("therapeutic effect") and locomotor suppression, hypothermia, and catalepsy (presumed indices of "side effects") (Martin et al., 1991). Second, doses of AJA that produced antihyperalgesic effects in a rat model of chronic pain were compared with those that produced substitution for Δ9-THC in a rat drug discrimination procedure. Since the results of Δ9-THC drug discrimination in animals has been shown to predict marijuana-like intoxication in humans (Balster and Prescott, 1992), the combined use of these two procedures (rat model of chronic pain and Δ9-THC discrimination) were used to determine the degree of separation between a therapeutic effect (analgesia) of AJA and its unwanted psychotropic effects in rats. Taken together, these studies represent a thorough pharmacological evaluation of AJA, a synthetic analog of a Δ9-THC metabolite, that address the question of whether or not the analgesic effects of this compound can be accessed without inducing unwanted side effects typically associated with Δ9-THC.
Materials and Methods
Subjects
Male ICR mice (25—32 g), purchased from Harlan (Dublin, VA), were housed in groups of five. All mice were kept in a temperature-controlled (20—22°C) environment with a 14-/10-h light/dark cycle and received food and water ad libitum. Separate mice were used for testing each drug dose in the in vivo procedures.
Naive adult male Long Evans rats obtained from Harlan were individually housed in a temperature-controlled (20—22°C) vivarium with a 12-h light/dark cycle. All rats were allowed to acclimate to the vivarium for at least a week before the experiments commenced. During the drug discrimination studies, rats were maintained within a weight range of 400 to 450g by restricted postsession feeding. They had ad libitum water in their home cages. Separate rats were used for the CFA studies. These rats had food and water ad libitum in their home cages, except during test sessions. Guidelines of the International Association for the Study of Pain (IASP, 1983), regarding the ethics of animal experimentation, were followed throughout the study. All animals used in this study were cared for in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Virginia Commonwealth University and the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy Press, 1996).
Apparatus
Measurement of spontaneous activity in mice occurred in standard activity chambers interfaced with a Digiscan Animal Activity Monitor (Omnitech Electronics, Inc., Columbus, OH). A standard tail-flick apparatus and a digital thermometer (Fisher Scientific, Pittsburgh, PA) were used to measure antinociception and rectal temperature, respectively. The ring immobility device consisted of an elevated metal ring (diameter, 5.5 cm; height, 16 cm) attached to a wooden stand.
For the drug discrimination studies, rats were trained and tested in standard operant conditioning chambers (BRS/LVE Inc., Laurel, MD or Lafayette Instruments Co., Lafayette, IN) housed in sound-attenuated cubicles. Pellet dispensers delivered 45-mg BIO SERV (Frenchtown, NJ) food pellets to a food cup on the front wall of the chamber between two response levers. Fan motors provided ventilation and masking noise for each chamber. House lights located above the food cup were illuminated during training and testing sessions. A microcomputer with Logic 1 interface (MED Associates, Georgia, Vermont) and MED-PC software (MED Associates) were used to control schedule contingencies and to record data.
Nociceptive testing in CFA-treated rats occurred in response to mechanical stimulation of the hindpaw. A mechanical stimulus was applied to the right (affected) hindpaw with an analgesy meter (Ugo Basile, Varese, Italy), a device with a dome-shaped plastic tip (diameter, 1 mm) that applies a linearly increasing pressure (acceleration caused by gravity) to the dorsal surface of the hindpaw, with the tip applied to the region of the paw just proximal to the third digit. The amount of force (acceleration caused by gravity) required to elicit a withdrawal attempt was recorded.
Procedure
Mouse Tetrad
Before testing in the pharmacological procedures, mice were acclimated to the experimental setting (ambient temperature, 22—24°C) for at least 1 h. The effects of oral and i.v. administration of Δ9-THC and AJA on spontaneous locomotion, tail-flick, ring immobility, and rectal temperature (the "tetrad") were assessed. Preinjection control values were determined for rectal temperature and tail-flick latency (in seconds). Five minutes following i.v. or 15 min following p.o. administration, individual mice were placed into one of six locomotor activity chambers. Spontaneous activity was measured as total number of interruptions of 16 photocell beams per chamber during the 10-min test and was expressed as percentage inhibition of activity of the vehicle group. Then, tail-flick latencies were redetermined 35 min after drug administration. Antinociception was calculated as percentage of maximal possible effect [%MPE = [(test − control latency)/(10 − control)] × 100]. Control latencies typically ranged from 1.5 to 4.0 s. Rectal temperatures were reassessed 45 min after drug administration. Rectal temperature values were expressed as the difference between control temperature (before injection) and temperatures following drug administration (change in degrees Celsius). Next, at 55 min postdrug administration, mice were placed on the ring immobility apparatus for 5 min. The total amount of time (in seconds) that the mouse remained motionless was measured. This value was divided by 300 s and multiplied by 100 to obtain a percentage immobility rating. The criterion for ring immobility was the absence of all voluntary movement, including snout and whisker movement. Different mice were tested with each dose of each drug. For antagonist tests, SR 141716A (3 and 10 mg/kg), SR144528 (10 mg/kg), or vehicle were administered i.v. 10 min before an i.v. injection of Δ9-THC (10 mg/kg) or AJA (10 mg/kg; a dose that rendered maximal effects when administered alone).
Drug Discrimination
All rats were trained to press one lever following administration of 3 mg/kg Δ9-THC and to press another lever after injection with vehicle (1
18 ratio of Emulphor:ethanol: saline), each according to a fixed-ratio 10 (FR-10) schedule of food reinforcement. Completion of 10 consecutive responses on the injection-appropriate lever resulted in delivery of a food reinforcer. Each response on the incorrect lever reset the response requirement on the correct lever. The position of the drug lever was varied among the group of rats. The daily injections for each rat were administered in a double alternation sequence of training drug and vehicle. Rats were injected and returned to their home cages until the start of the experimental session. Training occurred during 15-min sessions conducted 5 days a week (Monday to Friday) until the rats had met three criteria during 8 of 10 consecutive sessions: first completed FR-10 on the correct lever, percentage of correct lever responding greater than 80% for the entire session, and response rate greater than 0.4 responses/s.
Following successful acquisition of the discrimination, stimulus substitution tests with test compounds were conducted on Tuesdays and Fridays during 15-min test sessions. Training continued on Mondays, Wednesdays, and Thursdays. During test sessions, responses on either lever delivered reinforcement according to an FR-10 schedule. To be tested, rats must have completed the first FR and made at least 80% of all responses on the injection-appropriate lever on the preceding day's training session. In addition, the rat must have met these same criteria during at least one of the training sessions with the alternate training compound (Δ9-THC or vehicle) earlier in the week. Dose-effect determinations with the training drug Δ9-THC and, then, with AJA were conducted in each rat. Doses of each compound were administered in ascending order. Subsequently, antagonist tests were conducted with Δ9-THC and AJA to evaluate the ability of SR 141716A or SR144528 to block the Δ9-THC-like discriminative stimulus effects of AJA. Throughout the study, control tests with vehicle and the training drug were conducted during the week before the start of each dose-effect curve determination.
Nociceptive Testing in Complete Freund's Adjuvant-Treated Rats
Before induction of the arthritic or nonarthritic state on day 0, 14 male rats went through a habituation procedure that consisted of being handled, weighed, and exposed to the paw pressure test on four separate occasions prior to the induction of the arthritic or nonarthritic state on day 0. Subsequently, eight rats were injected s.c. with 0.1 ml of 5.0 mg/ml CFA (heat-killed Mycobacterium; Difco Laboratories, Detroit, MI), and six rats were injected s.c. with 0.1 ml of vehicle (VEH) (mineral oil) in the dorsal surface of the right hindpaw under light isoflurane anesthesia. On day 0, the day of the CFA/VEH injection, the mean weight of the rats was 273 g (± 4.3 S.E.M.).
Nociceptive testing occurred between days 12 and 28, inclusive. Before each test session, paw thickness measurements (millimeters) of the hindpaws were obtained with a digital caliper. Then, each rat was lightly restrained in a towel for testing. Preliminary tests indicated that in VEH- and CFA-treated rats, injection of the vehicle followed by paw pressure testing at 30-min intervals for up to six times resulted in no systematic change in paw pressure thresholds; hence, Δ9-THC and AJA were tested using a cumulative dosing schedule. Δ9-THC was tested on day 12, and AJA was tested on day 14. Immediately after the baseline, paw pressure threshold of the right hindpaw was recorded, rats were injected with the first dose of Δ9-THC or AJA, and thresholds of the right hindpaw were redetermined 30 min later. Immediately after these tests, the next drug dose was administered such that each successive dose increased the total drug concentration by 0.5 log units. This cycle of drug administration followed by threshold determination continued every 30 min until maximal antinociception was achieved or until the largest dose to be tested was administered. Solubility issues with AJA prevented testing doses higher than 30 mg/kg.
Antagonism studies were conducted in CFA-treated rats using the CB1 receptor antagonist SR 141716A (10 mg/kg). On day 26, following the determination of paw thickness measurements, baseline paw pressure thresholds were determined for the right hindpaw. Next, SR 141716A was injected, and thresholds of the right hindpaw were redetermined 30 min later. Subsequently, the highest dose of AJA (30 mg/kg) was injected, and 30 min later, thresholds were redetermined. On day 28, a similar testing protocol was used with the exception that a dose of 10 mg/kg Δ9-THC was administered instead of 30 mg/kg AJA.
Drugs
Δ9-THC (National Institute on Drug Abuse, Rockville, MD) was suspended in a vehicle of absolute ethanol, Emulphor-620 (Rhone-Poulenc, Inc., Princeton, NJ), and saline in a ratio of 118. SR 141716A (National Institute on Drug Abuse) and AJA (Organix, Inc., Woburn, MA) were also mixed in 118 vehicle. For the drug discrimination and the CFA studies, Δ9-THC or AJA were injected i.p. 30 min pretest. For behavioral tests with mice, all drugs were administered i.v. in the tail vein or p.o. by oral gavage, as indicated, at a volume of 0.1 ml/10 g. Presession injection intervals for each drug were chosen based upon previous research with these drugs in our lab or on values obtained in the literature and were as follows. For i.v. and p.o. dose-effect determinations in the mouse tetrad, mice were injected 5 or 15 min, respectively, prior to the start of tests. For antagonist tests in the tetrad, mice were injected i.v. with SR 141716A, SR144528, or vehicle followed 10 min later by injection with Δ9-THC, AJA, or vehicle. For antagonist tests in drug discrimination, rats were injected i.p. with SR 141716A, SR144528, or vehicle followed 10 min later by injection with Δ9-THC or AJA or vehicle.
Data Analysis
Mouse Tetrad
Antinociception was calculated as percentage of maximal possible effect [%MPE = [(test − control latency)/(10 − control)] × 100]. Rectal temperature values were expressed as the difference between control temperature (before injection) and temperatures following drug administration (change in degrees Celsius). Spontaneous activity was expressed as percentage inhibition of activity of the vehicle group. The total amount of time that the mouse remained motionless was divided by 300 s and multiplied by 100 to obtain a percentage immobility rating. ED50 values for Δ9-THC and AJA to produce percentage MPE, change in degrees Celsius, percentage inhibition, and percentage immobility were obtained using least-squares linear regression analysis, followed by calculation of 95% confidence limits by the method of Bliss (1967). Potency ratio values with a 95% confidence interval were calculated by the method of Colquhoun (1971). Based on data obtained from numerous previous studies with Δ9-THC (Compton et al., 1993), maximal effects were estimated as follows: 100% inhibition of spontaneous activity and 100% maximal possible effect in the tail-flick procedure. Maximal change in rectal temperature was estimated at −6°C, and maximal percentage ring immobility was 60%. ANOVAs were conducted separately for p.o. and i.v. tests. Dunnett's test was used for post hoc comparison when appropriate. Antagonism tests also were evaluated with ANOVAs as a function of drug followed by Dunnett's test when appropriate.
Drug Discrimination
For each test session, percentage of responses on the drug lever and response rate (responses per second) was calculated. Full substitution was defined as ≥80% Δ9-THC lever responding. ED50 values were calculated separately for each drug using least-squares linear regression analysis, followed by calculation of 95% confidence limits by the method of Bliss (1967). Potency ratio values with a 95% confidence interval were calculated by the method of Colquhoun (1971). Repeated measures ANOVAs with Dunnett's post hoc tests (α = 0.05) were used to determine differences in drug lever responding during antagonism tests and response rates both compared with vehicle control. Since rats that responded less than 10 times during a test session did not press either lever a sufficient number of times to earn a reinforcer, their drug lever selection data were excluded from analysis, but their response rate data were included in mean response rate.
Nociceptive Testing Complete Freund's Adjuvant-Treated Rats
Right hindpaw thickness measurements and baseline paw pressure thresholds for the right hindpaw of VEH- and CFA-treated rats were averaged across the test days, and differences in the paw thickness and paw pressure between VEH- and CFA-treated rats were determined using a Student's t test. For dose-effect testing in VEH- and CFA-treated rats, right hindpaw pressure thresholds following administration of the Δ9-THC or AJA were converted to a percentage of a maximal possible effect based upon a maximal 500g of pressure using the following equation: % antinociception = [(observed − baseline)/(500g − baseline)] × 100. Antinociception was operationally defined as drug-induced increases in paw pressure thresholds above nondrug baseline thresholds with 100% antinociception equal to 500g. The dose-effect data from CFA-treated rats were also analyzed to determine the antihyperalgesic effects of Δ9-THC and AJA.
Antihyperalgesia was operationally defined as drug-induced increases in paw pressure thresholds in CFA-treated rats above nondrug baseline thresholds with 100% antihyperalgesia equal to the nondrug baseline paw pressure threshold of the VEH-treated group. This was an examination of the ability of the drug to return the nociceptive sensitivity of CFA-treated rats to the baseline nociceptive sensitivity of VEH-treated rats. For mean threshold calculations when the resulting threshold following drug administration was greater than the mean threshold for the nondrug baseline of the respective VEH-treated group (e.g., maximal antihyperalgesia cutoff), this value was replaced with the mean VEH-treated threshold value for calculation of the mean threshold in CFA-treated rats. The percentage antihyperalgesic effect of each drug was determined based upon the following equation: percentage antihyperalgesia = [(observed − CFA baseline)/(VEH baseline − CFA baseline)] × 100.
ED50 values for Δ9-THC to produce antinociception and for Δ9-THC and AJA to produce antihyperalgesia were obtained using least-squares linear regression analysis, followed by calculation of 95% confidence limits by the method of Bliss (1967). Potency ratio values with a 95% confidence interval were calculated by the method of Colquhoun (1971). Differences in the relative potency of Δ9-THC and AJA were considered to be significant if the 95% confidence interval did not overlap. Differences in the maximal percentage antinociceptive effect obtained at the highest dose tested for Δ9-THC (10 mg/kg; AJA, 30 mg/kg) in VEH- and CFA-treated rats were determined using an ANOVA followed by a Dunnett post hoc test. The significance level was set at p ≤ 0.05.
Differences between the baseline right hindpaw pressure thresholds and thresholds following SR 141716A alone and SR 141716A in combination with Δ9-THC or AJA in CFA-treated rats were determined using a repeated measures ANOVA followed by a Bonferroni post hoc test. The significance level was set at p ≤ 0.05.
Results
Mouse Tetrad Dose-Effect Testing of Δ9-THC and AJA following p.o. and i.v. Administration
AJA and Δ9-THC were active in all four tests following p.o. (Fig. 2) and i.v.
(Fig. 3) administration. With each route of administration, both drugs
significantly (p < 0.05) and dose-dependently decreased spontaneous activity and rectal temperature and produced antinociception and catalepsy. ED50 values for Δ9-THC and AJA were consistently higher following p.o. administration (Table 1) than after i.v. injection (Table 2). Fig. 2
Effects of oral gavage administration of Δ9-THC and AJA on spontaneous activity (A), antinociception (B), rectal temperature (C), and catalepsy (D). Mice were pretreated p.o. with Δ9-THC or AJA 15 min prior to the beginning of tests. Values (more ...)
Fig. 3
Effects of i.v. administration of Δ9-THC and AJA on spontaneous activity (A), antinociception (B), rectal temperature (C), and catalepsy (D). Mice were pretreated i.v. with Δ9-THC or AJA 15 min prior to the beginning of tests. Values represent (more ...)
TABLE 1
ED50s and potency ratios for Δ9-THC and AJA in the mouse tetrad after p.o. administration
TABLE 2
ED50s and potency ratios for Δ9-THC and AJA in the mouse tetrad after i.v. administration
To assess the separation between analgesia and other pharmacological effects, potency ratios of antinociception to other effects were calculated. These potency ratios revealed that orally administered AJA was equally potent at producing antinociception and catalepsy but was significantly more potent at producing antinociception than at decreasing spontaneous activity (Table 1). With i.v. administration, AJA was equipotent at decreasing spontaneous activity and producing antinociception but was significantly more potent at producing antinociception than at producing catalepsy (Table 2). In comparison, oral Δ9-THC produced a similar pattern of potency ratios as oral AJA, although the exact values were different. Intravenously administered Δ9-THC produced antinociception and catalepsy at approximately equal potencies but was significantly less potent at producing antinociception than at decreasing spontaneous activity.
To determine whether the cannabimimetic effects induced by Δ9-THC and AJA were mediated via CB1 or CB2 receptors, a dose of 10 mg/kg Δ9-THC or AJA, shown to produce maximal effects in the mouse tetrad, was administered i.v. followed by injections of vehicle, 3 or 10 mg/kg SR 141716A, or 10 mg/kg SR144528. As shown in Table 3, SR 141716A dose-dependently blocked all four of the cannabimimetic effects of both Δ9-THC and AJA. In contrast, the CB2 antagonist SR144528 (10 mg/kg) failed to block any of these cannabimimetic effects. TABLE 3
Effects of pretreatment of vehicle, SR 141716A, or SR144528 on tetrad effects produced by i.v. administration of 10 mg/kg Δ9-THC or 10 mg/kg AJA in mice
Drug Discrimination
Both Δ9-THC and AJA fully and dose-dependently substituted for Δ9-THC, with ED50 = 0.5 (95% CL, 0.3—1) and 3.6 (95% CL, 2.1—6.3) mg/kg, respectively (Fig. 4, top). SR 141716A (10 mg/kg; p < 0.05) blocked the Δ9-THC-like
discriminative stimulus effects exhibited by AJA (10 mg/kg), whereas SR144258 (10 mg/kg) did not (p > 0 0.05). Response rates were not significantly decreased by any dose of Δ9-THC whereas a significant response rate decrease was observed following administration of the 30 mg/kg dose of AJA (p < 0.05). Hence, for both AJA and Δ9-THC, complete substitution was observed at doses that did not produce significant response rate suppression. Fig. 4
Effects of AJA and Δ9-THC on percentage of Δ9-THC lever responding (top) and response rates (bottom) in rats trained to discriminate 3.0 mg/kg Δ9-THC from vehicle. Points above VEH and Δ9-THC represent the results of control (more ...)
Paw Thickness and Baseline Paw Pressure Thresholds in VEH- and CFA-Treated Rats
A significant difference in mean right hindpaw thickness between VEH- and CFA-treated rats was observed across the test days (3.9 ± 0.1 versus 7.5 ± 0.3 mm, respectively). Baseline paw pressure thresholds were significantly different between VEH- and CFA-treated rats across the test days (143 ± 10g versus 73 ± 6g, respectively; p < 0.05).
Dose-Effect Testing of Δ9-THC and AJA in VEH- and CFA-Treated Rats
In CFA-treated rats, both Δ9-THC and AJA produced dose-dependent increases in antihyperalgesia with Δ9-THC producing a 97% antihyperalgesic effect at a dose of 3.0 mg/kg [ED50 value, 0.09 mg/kg (95% CL, 0.05—0.16)], whereas AJA produced a 67% antihyperalgesic effect at the highest dose tested (30 mg/kg) (ED50 value, 5.6 mg/kg; 95% CL, 5.6—24) (Fig. 5). Fig. 5
Antihyperalgesic effects of Δ9-THC and AJA in CFA-treated rats (n = 10). This graph represents the ability of each drug to return baseline thresholds of CFA-treated rats to the baseline thresholds of VEH-treated rats. For tests with Δ (more ...)
Based upon a 500g maximal possible effect, Δ9-THC produced dose-dependent increases in antinociception in CFA-treated rats (ED50 value, 4 mg/kg; 95% CL, 2.7—6.1), whereas in VEH-treated rats, Δ9-THC's maximal effect was less than 45% (data not shown). AJA failed to produce marked antinociception in VEH- or CFA-treated rats (data not shown). Analysis of the maximal antinociceptive effect at the highest dose tested for Δ9-THC and AJA in VEH- and CFA-treated rats indicate that there was a main effect of drug treatment, such that the antinociceptive effect obtained with Δ9-THC in CFA-treated rats (75 ± 13%) was greater than the antinociceptive effect obtained with Δ9-THC in VEH-treated rats (17 ± 5%) and was greater than the antinociceptive effect obtained with AJA in VEH-treated (12 ± 3%) and CFA-treated (30 ± 13%) rats.
Antagonism of Δ9-THC and AJA with SR 141716A
SR 141716A antagonized the Δ9-THC - and AJA-induced increases in paw pressure thresholds in CFA-treated rats (Table 4). Thresholds following SR 141716A administration alone and in combination with Δ9-THC were not significantly different from baseline paw pressure thresholds. Thresholds following SR 141716A administration alone and in combination with AJA were not significantly different from baseline paw pressure thresholds. TABLE 4
Effects of 10 mg/kg SR 141716A on mean paw pressure thresholds (acceleration caused by gravity) (± S.E.M.) alone or in combination with 10 mg/kg Δ9-THC or 30 mg/kg AJA in CFA-treated rats
Discussion
Similar to previous studies (Compton et al., 1992, 1993; Wiley et al., 1998; Martin et al., 1999, 2002), this study demonstrated that Δ9-THC produced characteristic cannabinoid pharmacological effects in mice, including antinociception, catalepsy, hypothermia, and hypomobility (Martin et al., 1991). This cannabimimetic profile occurred with oral and i.v. routes of administration, with Δ9-THC being approximately 8- to 76-fold more potent following i.v. administration. AJA shared Δ9-THC's profile of cannabinoid effects in mice after oral and i.v. administration and was also more potent after i.v. injection. These results are consistent with their moderate binding affinities for CB1 receptor (Ki = 32.3 nM for AJA and 80.3 nM for Δ9-THC) (Rhee et al., 1997).
Previous studies with AJA have shown that it produces antinociceptive effects in acute models of pain, including hot-plate, formalin, and writhing tests and tail clip in the mouse (Burstein et al., 1992, 1998; Sumariwalla et al., 2004; Dyson et al., 2005) and in rats using the hot-plate and tail-clip tests (Dajani et al., 1999). In addition, previous reports have shown decreased activity or other indication of motor impairment; however, in most studies, but not all (Sumariwalla et al., 2004; Mitchell et al., 2005), these deficits in motor behavior occurred at doses higher than the antinociceptive ED50 dose (Dajani et al., 1999; Dyson et al., 2005). Although these results seem to indicate therapeutic selectivity or separation of effects, most previous studies did not include full dose-effect determinations. Our results are consistent with previous reports in that AJA produced antinociceptive and locomotor suppressive effects; however, unlike some of the other studies, our data indicate little separation of antinociceptive and other pharmacological effects including locomotor suppression, hypothermia, and catalepsy. Potency ratios for AJA showed that, across routes of administration, potency for either decreasing activity (i.v.) or inducing catalepsy (p.o.) did not differ significantly from potency to produce antinociception. These results suggest that AJA may be efficacious as an analgesic agent in acute pain (as is Δ9-THC), but that it is likely to produce side effects such as sedation. This pharmacological profile is similar to that observed with dronabinol, synthetic Δ9-THC (for review, see Ben Amar, 2006).
Several mechanisms of actions for the various pharmacological effects of AJA have been suggested, including activation of CB1 or CB2 receptors (Rhee et al., 1997), interaction with peroxisome proliferated-activated receptor γ (Liu et al., 2003), and inhibition of cyclooxygenase-2 or 5-lipoxygenase (Zurier et al., 1998). The present study demonstrates that AJA acts at the CB1 receptor to produce typical cannabimimetic effects and that peripheral CB2 receptors do not play a role in these effects. Specifically, the CB1 receptor antagonist SR 141716A attenuated the ability of AJA to produce antinociception and catalepsy and to decrease spontaneous activity and rectal temperatures, whereas the CB2 antagonist, SR144528, did not. The present study underscores the ability of the mouse tetrad to reveal CB1-mediated cannabimimetic profiles of novel drugs such as AJA. Similarity of tetrad effects, however, may be limited in its application as a tool for determining therapeutic indices of novel compounds. The tetrad has been shown to reveal false positives in classes of drugs pharmacologically distinct from cannabinoids (e.g., central nervous system depressants and antipsychotic drugs), although positive results across tests are less consistent for noncannabinoid drugs, and their effects are not blocked by the CB1 antagonist SR 141716A (Wiley and Martin, 2003).
Although some promise has been demonstrated for AJA as an acute antinociceptive agent, much research has focused on AJA's effects in chronic pain conditions. In rats, Mitchell et al. (2005) tested a single dose of AJA (10 mg/kg) and demonstrated that it reduced allodynia in neuropathic and inflammatory pain models. Interestingly, this dose was smaller than the 30 mg/kg dose of AJA that reversed CFA-induced hyperalgesia in the present study. Another important finding from the current study is that SR 141716A antagonized AJA-induced increases in paw pressure thresholds in CFA-treated rats. This finding is consistent with the report by Dyson et al. (2005) that demonstrated that SR 141716A reversed the effects of AJA on the mechanical hyperalgesia induced by partial sciatic ligation. As discussed above, the mechanism by which AJA produces its therapeutic effects remains unclear. However, the blockade of AJA-induced increases in paw pressure thresholds of pain threshold by SR 141716A in the present study, taken together with the findings of Dyson et al. (2005), strongly suggest that CB1 receptors are intricately involved in the antihyperalgesic effects of AJA.
To date, preclinical research has shown that AJA is a potentially promising therapeutic agent for the treatment of chronic inflammation and pain. The present study supports that notion by demonstrating that AJA produced an antihyperalgesic effect at the highest dose tested, although this was not a complete blockade of pain. Given that AJA and Δ9-THC showed promising antinociceptive/antihyperalgesic effects in CFA and other models of chronic pain in the present study and others (Burstein et al., 1998; Zurier et al., 1998; Dajani et al., 1999; Mitchell et al. 1998, 2005), determination of the degree to which AJA also shares Δ9-THC's intoxicating effects is crucial.
This crucial evaluation was determined using Δ9-THC versus vehicle two-lever drug discrimination, a well accepted animal model of subjective effects of marijuana in humans (Balster and Prescott, 1992). In contrast with the tetrad tests, Δ9-THC is selective for psychoactive cannabinoids, with no false positives reported (Wiley, 1999). Although previous reports have suggested that AJA does not possess psychoactive properties at therapeutic doses (Burstein et al., 2004), the present study demonstrates that AJA shares Δ9-THC's discriminative stimulus properties, suggesting that it may produce intoxicating side effects in humans. Previously, Martin et al. (1995) demonstrated that in a study of 11-nor-Δ9-THC dimethyheptyl carboxylic acid fully substituted for Δ9-THC at a dose of 10 mg/kg. This compound only differs from AJA in that the double bond within the aromatic ring is in the ninth position for 11- nor-Δ9-THC dimethyheptyl carboxylic acid, whereas it is in the eighth position in AJA. Unfortunately, in that study, only one dose was tested; thus, comparisons of ED50 values are impossible. In this study, AJA produced full and dose-dependent substitution, and nearly equal ED50 values were obtained for discriminative stimulus effects (substitution for Δ9-THC) and therapeutic effects (antihyperalgesia in the CFA model). Interestingly, AJA did not produce antinociception in nonarthritic rats, whereas Δ9-THC did. The results of the present study also do not support the hypothesis that the role of CB1 receptors is insignificant at therapeutically relevant doses of AJA (Burstein et al., 2004) since its discriminative stimulus effects and antihyperalgesic effects were blocked by the CB1 receptor antagonist, SR 141716A.
AJA has been tested in phase I and phase II clinical trials. Results from published clinical trials were inconclusive. Although the authors reported analgesia in neuropathic pain patients in the absence of psychotropic effects (Karst et al., 2003), this conclusion is somewhat misleading because it is likely that subthreshold doses were used (for further discussion, see Wiley, 2005). The absence of psychotropic effects in these patients is clear from presentation of the incidence of side-effect profile; however, AJA showed only minimal efficacy, if any, as an analgesic. AJA-induced analgesic effects were statistically significant only for one of the two daily assessments. Only a trend was evident for the other time point. In addition, the magnitude of the significant effect was notably low and approximated the magnitude of changes in analgesia that were observed with different sequences of presentation of drug and vehicle in this crossover design. Based upon these findings, it is likely that a higher dose would be required for adequate pain control. Since the psychoactivity of higher doses was not assessed in this study, it is currently impossible to determine whether or not this type of side effect would co-occur with clinical efficacy of AJA in neuropathic pain patients. Based upon the results of our study, we would predict that marijuana-like intoxication would accompany clinical analgesic efficacy for AJA.
Comprehensive evaluation of the therapeutic versus deleterious effects of AJA has been a main goal of the present study. Although it has been suggested that AJA is therapeutically active at doses lower than those doses that produce undesired effects, the present data provide little evidence of separation of analgesic activity and other effects. Specifically, this study has shown that AJA, like Δ9-THC, induces psychoactive properties including a decrease in spontaneous activity, catalepsy, and Δ9-THC-like discriminative stimulus effects (undesired effects) at doses similar to those that produce antinociception or antihyperalgesia, respectively (desired effects). Moreover, contrary to previous research that has suggested that AJA's therapeutic effects are not mediated via action at CB1 receptors, this study demonstrated that AJA's effects, both therapeutically for pain and psychoactively, are CB1-mediated. These findings also underscore the importance of thoroughly evaluating the pharmacological characteristics of novel Δ9-THC-like compounds, such that therapeutic and deleterious effects are considered simultaneously.
Acknowledgments
This work was supported by National Institute on Drug Abuse Grants DA-05488 and DA-03672 and by Virginia Commonwealth University Institute of Drug and Alcohol Studies.
ABBREVIATIONS
Δ9-THC Δ9-tetrahydrocannabinol
AJA ajulemic acid (1′,1′-dimethylheptyl-Δ8-tetrahydrocannabinol-11-oic acid)
CFA complete Freund's adjuvant
MPE percentage of maximal possible effect in tail-flick test
SR 141716A N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride
SR144528 N-[-(1S)-endo-1,3,3-trimethyl bicyclo [2,2,1] heptan-2-yl]-5-(4-chloro-3-methyl-phenyl)-1-(4methylbenzyl)-pyrazole-3-carboxamide
FR-10 fixed-ratio 10
VEH vehicle
CB cannabinoid receptor
ANOVA analysis of variance
CL confidence limit
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Source with Charts, Graphs and Links: Cannabimimetic Properties of Ajulemic Acid
Abstract
Side effects of marijuana-based drugs and synthetic analogs of Δ9-tetrahydrocannabinol (Δ9-THC), including sedation and dysphoria, have limited their therapeutic application. Ajulemic acid (AJA), a side-chain synthetic analog of Δ8-THC-11-oic acid, has been reported to have anti-inflammatory properties without producing undesired psychoactive effects. Moreover, it has been suggested that AJA does not interact with cannabinoid receptors to produce its pharmacological effects. The aim of the present study was to conduct a thorough evaluation of the pharmacological effects of AJA then to determine whether actions at cannabinoid receptor (CB)1 mediated these effects. This study evaluated the psychoactive and analgesic effects of AJA by examining its cannabimimetic properties in ICR mice (i.e., antinociception, catalepsy, hypothermia, and hypomobility), its discriminative stimulus effects in Long Evans rats trained in a two-lever Δ9-THC (3.0 mg/kg) versus vehicle drug discrimination procedure, and its antihyperalgesia effects in a rat model of inflammatory pain [complete Freund's adjuvant (CFA)-induced mechanical hyperalgesia]. Lastly, antagonism tests with SR 141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride], CB1 receptor antagonist, were conducted. These studies demonstrated that AJA shares a number of CB1-mediated pharmacological properties with Δ9-THC, including cannabimimetic, discriminative stimulus, and antihyperalgesic effects. Furthermore, a separation between doses that produced antinociception and those that produced the other pharmacological effects in mice was not observed. Moreover, AJA showed nearly equipotency for therapeutic efficacy in the CFA model and for substitution in Δ9-THC discrimination. In summary, this study shows that AJA, like Δ9-THC, exhibits psychoactive and therapeutic effects at nearly equal doses in preclinical models, suggesting similar limitations in their putative therapeutic profiles.
Cannabis sativa (marijuana plant) has been used since antiquity for its presumed therapeutic, as well as for its euphoric effects. Although Δ9-tetrahydrocannabinol (Δ9-THC) has been identified as the major psychoactive ingredient in C. sativa (Gaoni and Mechoulam, 1971), difficulty in dissociating unwanted side effects, such as sedation and psychotropic effects, from therapeutic effects has limited clinical application of Δ9-THC-based drugs. For example, dronabinol, an orally administered synthetic version of Δ9-THC, has been developed as an appetite stimulant and antiemetic for use in chronic diseases such as AIDS and cancer (for review, see Ben Amar, 2006). In addition, recent evidence suggests oral Δ9-THC may be effective as an adjunct to opioid analgesics (Roberts et al., 2006). The therapeutic utility of Δ9-THC, however, has been limited due to patient complaints of dysphoria and unpleasant subjective effects (Ben Amar, 2006). Previous research has suggested that Δ9-THC carboxylic acid, one of the acid metabolites of Δ9-THC, lacks psychoactive properties of the parent compound and yet retains antinociceptive and other effects (Burstein et al., 1988; Doyle et al., 1990). Since this metabolite has a relatively low potency, structural changes that increased potency and stability of Δ9-THC analogs in previous structure-activity relationship studies (Loev et al., 1973; Mechoulam et al., 1988; Compton et al., 1993) were applied to the structure Δ9-THC carboxylic acid. The resulting compound, ajulemic acid (AJA), substitutes a 1′,1-dimethylheptyl side chain for the pentyl group of Δ9-THC and changes the Δ9-THC core structure to a more stable confirmation, Δ8-THC (Fig. 1). Fig. 1
Chemical structures of Δ9-THC and AJA.
To date, the efficacy of AJA has been demonstrated in numerous pain and inflammation studies (for review, see Wiley, 2005). These findings are equivocal, however, in that effects have been found with a single dose or a restricted range of doses or have been reported in different strains or species, making comparisons and conclusions problematic. For example, a number of studies have reported that AJA exhibits efficacy similar to that of Δ9-THC in acute rodent models of pain, including hot-plate, formalin, writhing, and tail-clip tests in the mouse (Burstein et al., 1992, 1998; Sumariwalla et al., 2004; Dyson et al., 2005) and hot-plate and tail-clip tests in rats (Dajani et al., 1999). In an experimental model of rheumatoid arthritis, complete Freund's adjuvant (CFA), chronic dosing with AJA reduced the degree of joint deformity, as measured by histological analysis (Zurier et al., 1998). In addition, several acute dosing studies showed that AJA reduced experimentally induced pain or inflammation in rats (Burstein et al., 1998; Dyson et al., 2005; Mitchell et al., 2005). Potencies for AJA reported in two of these studies (Burstein et al., 1998; Dyson et al., 2005), however, were 10-fold greater in magnitude than the single dose reported to be effective in the only other published study that used similar methods (Mitchell et al., 2005).
Unfortunately, previous analgesic studies with AJA have not included a careful evaluation of behavioral effects that might be either present or absent in the therapeutic dose range. Therefore, the present study was designed to elucidate more fully the analgesic and side-effect profile of AJA in the same species and with similar dosing parameters. First, the effects of AJA were compared with Δ9-THC in mice in a well established tetrad of tests, in which cannabinoids produce antinociception ("therapeutic effect") and locomotor suppression, hypothermia, and catalepsy (presumed indices of "side effects") (Martin et al., 1991). Second, doses of AJA that produced antihyperalgesic effects in a rat model of chronic pain were compared with those that produced substitution for Δ9-THC in a rat drug discrimination procedure. Since the results of Δ9-THC drug discrimination in animals has been shown to predict marijuana-like intoxication in humans (Balster and Prescott, 1992), the combined use of these two procedures (rat model of chronic pain and Δ9-THC discrimination) were used to determine the degree of separation between a therapeutic effect (analgesia) of AJA and its unwanted psychotropic effects in rats. Taken together, these studies represent a thorough pharmacological evaluation of AJA, a synthetic analog of a Δ9-THC metabolite, that address the question of whether or not the analgesic effects of this compound can be accessed without inducing unwanted side effects typically associated with Δ9-THC.
Materials and Methods
Subjects
Male ICR mice (25—32 g), purchased from Harlan (Dublin, VA), were housed in groups of five. All mice were kept in a temperature-controlled (20—22°C) environment with a 14-/10-h light/dark cycle and received food and water ad libitum. Separate mice were used for testing each drug dose in the in vivo procedures.
Naive adult male Long Evans rats obtained from Harlan were individually housed in a temperature-controlled (20—22°C) vivarium with a 12-h light/dark cycle. All rats were allowed to acclimate to the vivarium for at least a week before the experiments commenced. During the drug discrimination studies, rats were maintained within a weight range of 400 to 450g by restricted postsession feeding. They had ad libitum water in their home cages. Separate rats were used for the CFA studies. These rats had food and water ad libitum in their home cages, except during test sessions. Guidelines of the International Association for the Study of Pain (IASP, 1983), regarding the ethics of animal experimentation, were followed throughout the study. All animals used in this study were cared for in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Virginia Commonwealth University and the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy Press, 1996).
Apparatus
Measurement of spontaneous activity in mice occurred in standard activity chambers interfaced with a Digiscan Animal Activity Monitor (Omnitech Electronics, Inc., Columbus, OH). A standard tail-flick apparatus and a digital thermometer (Fisher Scientific, Pittsburgh, PA) were used to measure antinociception and rectal temperature, respectively. The ring immobility device consisted of an elevated metal ring (diameter, 5.5 cm; height, 16 cm) attached to a wooden stand.
For the drug discrimination studies, rats were trained and tested in standard operant conditioning chambers (BRS/LVE Inc., Laurel, MD or Lafayette Instruments Co., Lafayette, IN) housed in sound-attenuated cubicles. Pellet dispensers delivered 45-mg BIO SERV (Frenchtown, NJ) food pellets to a food cup on the front wall of the chamber between two response levers. Fan motors provided ventilation and masking noise for each chamber. House lights located above the food cup were illuminated during training and testing sessions. A microcomputer with Logic 1 interface (MED Associates, Georgia, Vermont) and MED-PC software (MED Associates) were used to control schedule contingencies and to record data.
Nociceptive testing in CFA-treated rats occurred in response to mechanical stimulation of the hindpaw. A mechanical stimulus was applied to the right (affected) hindpaw with an analgesy meter (Ugo Basile, Varese, Italy), a device with a dome-shaped plastic tip (diameter, 1 mm) that applies a linearly increasing pressure (acceleration caused by gravity) to the dorsal surface of the hindpaw, with the tip applied to the region of the paw just proximal to the third digit. The amount of force (acceleration caused by gravity) required to elicit a withdrawal attempt was recorded.
Procedure
Mouse Tetrad
Before testing in the pharmacological procedures, mice were acclimated to the experimental setting (ambient temperature, 22—24°C) for at least 1 h. The effects of oral and i.v. administration of Δ9-THC and AJA on spontaneous locomotion, tail-flick, ring immobility, and rectal temperature (the "tetrad") were assessed. Preinjection control values were determined for rectal temperature and tail-flick latency (in seconds). Five minutes following i.v. or 15 min following p.o. administration, individual mice were placed into one of six locomotor activity chambers. Spontaneous activity was measured as total number of interruptions of 16 photocell beams per chamber during the 10-min test and was expressed as percentage inhibition of activity of the vehicle group. Then, tail-flick latencies were redetermined 35 min after drug administration. Antinociception was calculated as percentage of maximal possible effect [%MPE = [(test − control latency)/(10 − control)] × 100]. Control latencies typically ranged from 1.5 to 4.0 s. Rectal temperatures were reassessed 45 min after drug administration. Rectal temperature values were expressed as the difference between control temperature (before injection) and temperatures following drug administration (change in degrees Celsius). Next, at 55 min postdrug administration, mice were placed on the ring immobility apparatus for 5 min. The total amount of time (in seconds) that the mouse remained motionless was measured. This value was divided by 300 s and multiplied by 100 to obtain a percentage immobility rating. The criterion for ring immobility was the absence of all voluntary movement, including snout and whisker movement. Different mice were tested with each dose of each drug. For antagonist tests, SR 141716A (3 and 10 mg/kg), SR144528 (10 mg/kg), or vehicle were administered i.v. 10 min before an i.v. injection of Δ9-THC (10 mg/kg) or AJA (10 mg/kg; a dose that rendered maximal effects when administered alone).
Drug Discrimination
All rats were trained to press one lever following administration of 3 mg/kg Δ9-THC and to press another lever after injection with vehicle (1
18 ratio of Emulphor:ethanol: saline), each according to a fixed-ratio 10 (FR-10) schedule of food reinforcement. Completion of 10 consecutive responses on the injection-appropriate lever resulted in delivery of a food reinforcer. Each response on the incorrect lever reset the response requirement on the correct lever. The position of the drug lever was varied among the group of rats. The daily injections for each rat were administered in a double alternation sequence of training drug and vehicle. Rats were injected and returned to their home cages until the start of the experimental session. Training occurred during 15-min sessions conducted 5 days a week (Monday to Friday) until the rats had met three criteria during 8 of 10 consecutive sessions: first completed FR-10 on the correct lever, percentage of correct lever responding greater than 80% for the entire session, and response rate greater than 0.4 responses/s.Following successful acquisition of the discrimination, stimulus substitution tests with test compounds were conducted on Tuesdays and Fridays during 15-min test sessions. Training continued on Mondays, Wednesdays, and Thursdays. During test sessions, responses on either lever delivered reinforcement according to an FR-10 schedule. To be tested, rats must have completed the first FR and made at least 80% of all responses on the injection-appropriate lever on the preceding day's training session. In addition, the rat must have met these same criteria during at least one of the training sessions with the alternate training compound (Δ9-THC or vehicle) earlier in the week. Dose-effect determinations with the training drug Δ9-THC and, then, with AJA were conducted in each rat. Doses of each compound were administered in ascending order. Subsequently, antagonist tests were conducted with Δ9-THC and AJA to evaluate the ability of SR 141716A or SR144528 to block the Δ9-THC-like discriminative stimulus effects of AJA. Throughout the study, control tests with vehicle and the training drug were conducted during the week before the start of each dose-effect curve determination.
Nociceptive Testing in Complete Freund's Adjuvant-Treated Rats
Before induction of the arthritic or nonarthritic state on day 0, 14 male rats went through a habituation procedure that consisted of being handled, weighed, and exposed to the paw pressure test on four separate occasions prior to the induction of the arthritic or nonarthritic state on day 0. Subsequently, eight rats were injected s.c. with 0.1 ml of 5.0 mg/ml CFA (heat-killed Mycobacterium; Difco Laboratories, Detroit, MI), and six rats were injected s.c. with 0.1 ml of vehicle (VEH) (mineral oil) in the dorsal surface of the right hindpaw under light isoflurane anesthesia. On day 0, the day of the CFA/VEH injection, the mean weight of the rats was 273 g (± 4.3 S.E.M.).
Nociceptive testing occurred between days 12 and 28, inclusive. Before each test session, paw thickness measurements (millimeters) of the hindpaws were obtained with a digital caliper. Then, each rat was lightly restrained in a towel for testing. Preliminary tests indicated that in VEH- and CFA-treated rats, injection of the vehicle followed by paw pressure testing at 30-min intervals for up to six times resulted in no systematic change in paw pressure thresholds; hence, Δ9-THC and AJA were tested using a cumulative dosing schedule. Δ9-THC was tested on day 12, and AJA was tested on day 14. Immediately after the baseline, paw pressure threshold of the right hindpaw was recorded, rats were injected with the first dose of Δ9-THC or AJA, and thresholds of the right hindpaw were redetermined 30 min later. Immediately after these tests, the next drug dose was administered such that each successive dose increased the total drug concentration by 0.5 log units. This cycle of drug administration followed by threshold determination continued every 30 min until maximal antinociception was achieved or until the largest dose to be tested was administered. Solubility issues with AJA prevented testing doses higher than 30 mg/kg.
Antagonism studies were conducted in CFA-treated rats using the CB1 receptor antagonist SR 141716A (10 mg/kg). On day 26, following the determination of paw thickness measurements, baseline paw pressure thresholds were determined for the right hindpaw. Next, SR 141716A was injected, and thresholds of the right hindpaw were redetermined 30 min later. Subsequently, the highest dose of AJA (30 mg/kg) was injected, and 30 min later, thresholds were redetermined. On day 28, a similar testing protocol was used with the exception that a dose of 10 mg/kg Δ9-THC was administered instead of 30 mg/kg AJA.
Drugs
Δ9-THC (National Institute on Drug Abuse, Rockville, MD) was suspended in a vehicle of absolute ethanol, Emulphor-620 (Rhone-Poulenc, Inc., Princeton, NJ), and saline in a ratio of 1
18. SR 141716A (National Institute on Drug Abuse) and AJA (Organix, Inc., Woburn, MA) were also mixed in 118 vehicle. For the drug discrimination and the CFA studies, Δ9-THC or AJA were injected i.p. 30 min pretest. For behavioral tests with mice, all drugs were administered i.v. in the tail vein or p.o. by oral gavage, as indicated, at a volume of 0.1 ml/10 g. Presession injection intervals for each drug were chosen based upon previous research with these drugs in our lab or on values obtained in the literature and were as follows. For i.v. and p.o. dose-effect determinations in the mouse tetrad, mice were injected 5 or 15 min, respectively, prior to the start of tests. For antagonist tests in the tetrad, mice were injected i.v. with SR 141716A, SR144528, or vehicle followed 10 min later by injection with Δ9-THC, AJA, or vehicle. For antagonist tests in drug discrimination, rats were injected i.p. with SR 141716A, SR144528, or vehicle followed 10 min later by injection with Δ9-THC or AJA or vehicle.Data Analysis
Mouse Tetrad
Antinociception was calculated as percentage of maximal possible effect [%MPE = [(test − control latency)/(10 − control)] × 100]. Rectal temperature values were expressed as the difference between control temperature (before injection) and temperatures following drug administration (change in degrees Celsius). Spontaneous activity was expressed as percentage inhibition of activity of the vehicle group. The total amount of time that the mouse remained motionless was divided by 300 s and multiplied by 100 to obtain a percentage immobility rating. ED50 values for Δ9-THC and AJA to produce percentage MPE, change in degrees Celsius, percentage inhibition, and percentage immobility were obtained using least-squares linear regression analysis, followed by calculation of 95% confidence limits by the method of Bliss (1967). Potency ratio values with a 95% confidence interval were calculated by the method of Colquhoun (1971). Based on data obtained from numerous previous studies with Δ9-THC (Compton et al., 1993), maximal effects were estimated as follows: 100% inhibition of spontaneous activity and 100% maximal possible effect in the tail-flick procedure. Maximal change in rectal temperature was estimated at −6°C, and maximal percentage ring immobility was 60%. ANOVAs were conducted separately for p.o. and i.v. tests. Dunnett's test was used for post hoc comparison when appropriate. Antagonism tests also were evaluated with ANOVAs as a function of drug followed by Dunnett's test when appropriate.
Drug Discrimination
For each test session, percentage of responses on the drug lever and response rate (responses per second) was calculated. Full substitution was defined as ≥80% Δ9-THC lever responding. ED50 values were calculated separately for each drug using least-squares linear regression analysis, followed by calculation of 95% confidence limits by the method of Bliss (1967). Potency ratio values with a 95% confidence interval were calculated by the method of Colquhoun (1971). Repeated measures ANOVAs with Dunnett's post hoc tests (α = 0.05) were used to determine differences in drug lever responding during antagonism tests and response rates both compared with vehicle control. Since rats that responded less than 10 times during a test session did not press either lever a sufficient number of times to earn a reinforcer, their drug lever selection data were excluded from analysis, but their response rate data were included in mean response rate.
Nociceptive Testing Complete Freund's Adjuvant-Treated Rats
Right hindpaw thickness measurements and baseline paw pressure thresholds for the right hindpaw of VEH- and CFA-treated rats were averaged across the test days, and differences in the paw thickness and paw pressure between VEH- and CFA-treated rats were determined using a Student's t test. For dose-effect testing in VEH- and CFA-treated rats, right hindpaw pressure thresholds following administration of the Δ9-THC or AJA were converted to a percentage of a maximal possible effect based upon a maximal 500g of pressure using the following equation: % antinociception = [(observed − baseline)/(500g − baseline)] × 100. Antinociception was operationally defined as drug-induced increases in paw pressure thresholds above nondrug baseline thresholds with 100% antinociception equal to 500g. The dose-effect data from CFA-treated rats were also analyzed to determine the antihyperalgesic effects of Δ9-THC and AJA.
Antihyperalgesia was operationally defined as drug-induced increases in paw pressure thresholds in CFA-treated rats above nondrug baseline thresholds with 100% antihyperalgesia equal to the nondrug baseline paw pressure threshold of the VEH-treated group. This was an examination of the ability of the drug to return the nociceptive sensitivity of CFA-treated rats to the baseline nociceptive sensitivity of VEH-treated rats. For mean threshold calculations when the resulting threshold following drug administration was greater than the mean threshold for the nondrug baseline of the respective VEH-treated group (e.g., maximal antihyperalgesia cutoff), this value was replaced with the mean VEH-treated threshold value for calculation of the mean threshold in CFA-treated rats. The percentage antihyperalgesic effect of each drug was determined based upon the following equation: percentage antihyperalgesia = [(observed − CFA baseline)/(VEH baseline − CFA baseline)] × 100.
ED50 values for Δ9-THC to produce antinociception and for Δ9-THC and AJA to produce antihyperalgesia were obtained using least-squares linear regression analysis, followed by calculation of 95% confidence limits by the method of Bliss (1967). Potency ratio values with a 95% confidence interval were calculated by the method of Colquhoun (1971). Differences in the relative potency of Δ9-THC and AJA were considered to be significant if the 95% confidence interval did not overlap. Differences in the maximal percentage antinociceptive effect obtained at the highest dose tested for Δ9-THC (10 mg/kg; AJA, 30 mg/kg) in VEH- and CFA-treated rats were determined using an ANOVA followed by a Dunnett post hoc test. The significance level was set at p ≤ 0.05.
Differences between the baseline right hindpaw pressure thresholds and thresholds following SR 141716A alone and SR 141716A in combination with Δ9-THC or AJA in CFA-treated rats were determined using a repeated measures ANOVA followed by a Bonferroni post hoc test. The significance level was set at p ≤ 0.05.
Results
Mouse Tetrad Dose-Effect Testing of Δ9-THC and AJA following p.o. and i.v. Administration
AJA and Δ9-THC were active in all four tests following p.o. (Fig. 2) and i.v.
(Fig. 3) administration. With each route of administration, both drugs
significantly (p < 0.05) and dose-dependently decreased spontaneous activity and rectal temperature and produced antinociception and catalepsy. ED50 values for Δ9-THC and AJA were consistently higher following p.o. administration (Table 1) than after i.v. injection (Table 2). Fig. 2
Effects of oral gavage administration of Δ9-THC and AJA on spontaneous activity (A), antinociception (B), rectal temperature (C), and catalepsy (D). Mice were pretreated p.o. with Δ9-THC or AJA 15 min prior to the beginning of tests. Values (more ...)
Fig. 3
Effects of i.v. administration of Δ9-THC and AJA on spontaneous activity (A), antinociception (B), rectal temperature (C), and catalepsy (D). Mice were pretreated i.v. with Δ9-THC or AJA 15 min prior to the beginning of tests. Values represent (more ...)
TABLE 1
ED50s and potency ratios for Δ9-THC and AJA in the mouse tetrad after p.o. administration
TABLE 2
ED50s and potency ratios for Δ9-THC and AJA in the mouse tetrad after i.v. administration
To assess the separation between analgesia and other pharmacological effects, potency ratios of antinociception to other effects were calculated. These potency ratios revealed that orally administered AJA was equally potent at producing antinociception and catalepsy but was significantly more potent at producing antinociception than at decreasing spontaneous activity (Table 1). With i.v. administration, AJA was equipotent at decreasing spontaneous activity and producing antinociception but was significantly more potent at producing antinociception than at producing catalepsy (Table 2). In comparison, oral Δ9-THC produced a similar pattern of potency ratios as oral AJA, although the exact values were different. Intravenously administered Δ9-THC produced antinociception and catalepsy at approximately equal potencies but was significantly less potent at producing antinociception than at decreasing spontaneous activity.
To determine whether the cannabimimetic effects induced by Δ9-THC and AJA were mediated via CB1 or CB2 receptors, a dose of 10 mg/kg Δ9-THC or AJA, shown to produce maximal effects in the mouse tetrad, was administered i.v. followed by injections of vehicle, 3 or 10 mg/kg SR 141716A, or 10 mg/kg SR144528. As shown in Table 3, SR 141716A dose-dependently blocked all four of the cannabimimetic effects of both Δ9-THC and AJA. In contrast, the CB2 antagonist SR144528 (10 mg/kg) failed to block any of these cannabimimetic effects. TABLE 3
Effects of pretreatment of vehicle, SR 141716A, or SR144528 on tetrad effects produced by i.v. administration of 10 mg/kg Δ9-THC or 10 mg/kg AJA in mice
Drug Discrimination
Both Δ9-THC and AJA fully and dose-dependently substituted for Δ9-THC, with ED50 = 0.5 (95% CL, 0.3—1) and 3.6 (95% CL, 2.1—6.3) mg/kg, respectively (Fig. 4, top). SR 141716A (10 mg/kg; p < 0.05) blocked the Δ9-THC-like
discriminative stimulus effects exhibited by AJA (10 mg/kg), whereas SR144258 (10 mg/kg) did not (p > 0 0.05). Response rates were not significantly decreased by any dose of Δ9-THC whereas a significant response rate decrease was observed following administration of the 30 mg/kg dose of AJA (p < 0.05). Hence, for both AJA and Δ9-THC, complete substitution was observed at doses that did not produce significant response rate suppression. Fig. 4
Effects of AJA and Δ9-THC on percentage of Δ9-THC lever responding (top) and response rates (bottom) in rats trained to discriminate 3.0 mg/kg Δ9-THC from vehicle. Points above VEH and Δ9-THC represent the results of control (more ...)
Paw Thickness and Baseline Paw Pressure Thresholds in VEH- and CFA-Treated Rats
A significant difference in mean right hindpaw thickness between VEH- and CFA-treated rats was observed across the test days (3.9 ± 0.1 versus 7.5 ± 0.3 mm, respectively). Baseline paw pressure thresholds were significantly different between VEH- and CFA-treated rats across the test days (143 ± 10g versus 73 ± 6g, respectively; p < 0.05).
Dose-Effect Testing of Δ9-THC and AJA in VEH- and CFA-Treated Rats
In CFA-treated rats, both Δ9-THC and AJA produced dose-dependent increases in antihyperalgesia with Δ9-THC producing a 97% antihyperalgesic effect at a dose of 3.0 mg/kg [ED50 value, 0.09 mg/kg (95% CL, 0.05—0.16)], whereas AJA produced a 67% antihyperalgesic effect at the highest dose tested (30 mg/kg) (ED50 value, 5.6 mg/kg; 95% CL, 5.6—24) (Fig. 5). Fig. 5
Antihyperalgesic effects of Δ9-THC and AJA in CFA-treated rats (n = 10). This graph represents the ability of each drug to return baseline thresholds of CFA-treated rats to the baseline thresholds of VEH-treated rats. For tests with Δ (more ...)
Based upon a 500g maximal possible effect, Δ9-THC produced dose-dependent increases in antinociception in CFA-treated rats (ED50 value, 4 mg/kg; 95% CL, 2.7—6.1), whereas in VEH-treated rats, Δ9-THC's maximal effect was less than 45% (data not shown). AJA failed to produce marked antinociception in VEH- or CFA-treated rats (data not shown). Analysis of the maximal antinociceptive effect at the highest dose tested for Δ9-THC and AJA in VEH- and CFA-treated rats indicate that there was a main effect of drug treatment, such that the antinociceptive effect obtained with Δ9-THC in CFA-treated rats (75 ± 13%) was greater than the antinociceptive effect obtained with Δ9-THC in VEH-treated rats (17 ± 5%) and was greater than the antinociceptive effect obtained with AJA in VEH-treated (12 ± 3%) and CFA-treated (30 ± 13%) rats.
Antagonism of Δ9-THC and AJA with SR 141716A
SR 141716A antagonized the Δ9-THC - and AJA-induced increases in paw pressure thresholds in CFA-treated rats (Table 4). Thresholds following SR 141716A administration alone and in combination with Δ9-THC were not significantly different from baseline paw pressure thresholds. Thresholds following SR 141716A administration alone and in combination with AJA were not significantly different from baseline paw pressure thresholds. TABLE 4
Effects of 10 mg/kg SR 141716A on mean paw pressure thresholds (acceleration caused by gravity) (± S.E.M.) alone or in combination with 10 mg/kg Δ9-THC or 30 mg/kg AJA in CFA-treated rats
Discussion
Similar to previous studies (Compton et al., 1992, 1993; Wiley et al., 1998; Martin et al., 1999, 2002), this study demonstrated that Δ9-THC produced characteristic cannabinoid pharmacological effects in mice, including antinociception, catalepsy, hypothermia, and hypomobility (Martin et al., 1991). This cannabimimetic profile occurred with oral and i.v. routes of administration, with Δ9-THC being approximately 8- to 76-fold more potent following i.v. administration. AJA shared Δ9-THC's profile of cannabinoid effects in mice after oral and i.v. administration and was also more potent after i.v. injection. These results are consistent with their moderate binding affinities for CB1 receptor (Ki = 32.3 nM for AJA and 80.3 nM for Δ9-THC) (Rhee et al., 1997).
Previous studies with AJA have shown that it produces antinociceptive effects in acute models of pain, including hot-plate, formalin, and writhing tests and tail clip in the mouse (Burstein et al., 1992, 1998; Sumariwalla et al., 2004; Dyson et al., 2005) and in rats using the hot-plate and tail-clip tests (Dajani et al., 1999). In addition, previous reports have shown decreased activity or other indication of motor impairment; however, in most studies, but not all (Sumariwalla et al., 2004; Mitchell et al., 2005), these deficits in motor behavior occurred at doses higher than the antinociceptive ED50 dose (Dajani et al., 1999; Dyson et al., 2005). Although these results seem to indicate therapeutic selectivity or separation of effects, most previous studies did not include full dose-effect determinations. Our results are consistent with previous reports in that AJA produced antinociceptive and locomotor suppressive effects; however, unlike some of the other studies, our data indicate little separation of antinociceptive and other pharmacological effects including locomotor suppression, hypothermia, and catalepsy. Potency ratios for AJA showed that, across routes of administration, potency for either decreasing activity (i.v.) or inducing catalepsy (p.o.) did not differ significantly from potency to produce antinociception. These results suggest that AJA may be efficacious as an analgesic agent in acute pain (as is Δ9-THC), but that it is likely to produce side effects such as sedation. This pharmacological profile is similar to that observed with dronabinol, synthetic Δ9-THC (for review, see Ben Amar, 2006).
Several mechanisms of actions for the various pharmacological effects of AJA have been suggested, including activation of CB1 or CB2 receptors (Rhee et al., 1997), interaction with peroxisome proliferated-activated receptor γ (Liu et al., 2003), and inhibition of cyclooxygenase-2 or 5-lipoxygenase (Zurier et al., 1998). The present study demonstrates that AJA acts at the CB1 receptor to produce typical cannabimimetic effects and that peripheral CB2 receptors do not play a role in these effects. Specifically, the CB1 receptor antagonist SR 141716A attenuated the ability of AJA to produce antinociception and catalepsy and to decrease spontaneous activity and rectal temperatures, whereas the CB2 antagonist, SR144528, did not. The present study underscores the ability of the mouse tetrad to reveal CB1-mediated cannabimimetic profiles of novel drugs such as AJA. Similarity of tetrad effects, however, may be limited in its application as a tool for determining therapeutic indices of novel compounds. The tetrad has been shown to reveal false positives in classes of drugs pharmacologically distinct from cannabinoids (e.g., central nervous system depressants and antipsychotic drugs), although positive results across tests are less consistent for noncannabinoid drugs, and their effects are not blocked by the CB1 antagonist SR 141716A (Wiley and Martin, 2003).
Although some promise has been demonstrated for AJA as an acute antinociceptive agent, much research has focused on AJA's effects in chronic pain conditions. In rats, Mitchell et al. (2005) tested a single dose of AJA (10 mg/kg) and demonstrated that it reduced allodynia in neuropathic and inflammatory pain models. Interestingly, this dose was smaller than the 30 mg/kg dose of AJA that reversed CFA-induced hyperalgesia in the present study. Another important finding from the current study is that SR 141716A antagonized AJA-induced increases in paw pressure thresholds in CFA-treated rats. This finding is consistent with the report by Dyson et al. (2005) that demonstrated that SR 141716A reversed the effects of AJA on the mechanical hyperalgesia induced by partial sciatic ligation. As discussed above, the mechanism by which AJA produces its therapeutic effects remains unclear. However, the blockade of AJA-induced increases in paw pressure thresholds of pain threshold by SR 141716A in the present study, taken together with the findings of Dyson et al. (2005), strongly suggest that CB1 receptors are intricately involved in the antihyperalgesic effects of AJA.
To date, preclinical research has shown that AJA is a potentially promising therapeutic agent for the treatment of chronic inflammation and pain. The present study supports that notion by demonstrating that AJA produced an antihyperalgesic effect at the highest dose tested, although this was not a complete blockade of pain. Given that AJA and Δ9-THC showed promising antinociceptive/antihyperalgesic effects in CFA and other models of chronic pain in the present study and others (Burstein et al., 1998; Zurier et al., 1998; Dajani et al., 1999; Mitchell et al. 1998, 2005), determination of the degree to which AJA also shares Δ9-THC's intoxicating effects is crucial.
This crucial evaluation was determined using Δ9-THC versus vehicle two-lever drug discrimination, a well accepted animal model of subjective effects of marijuana in humans (Balster and Prescott, 1992). In contrast with the tetrad tests, Δ9-THC is selective for psychoactive cannabinoids, with no false positives reported (Wiley, 1999). Although previous reports have suggested that AJA does not possess psychoactive properties at therapeutic doses (Burstein et al., 2004), the present study demonstrates that AJA shares Δ9-THC's discriminative stimulus properties, suggesting that it may produce intoxicating side effects in humans. Previously, Martin et al. (1995) demonstrated that in a study of 11-nor-Δ9-THC dimethyheptyl carboxylic acid fully substituted for Δ9-THC at a dose of 10 mg/kg. This compound only differs from AJA in that the double bond within the aromatic ring is in the ninth position for 11- nor-Δ9-THC dimethyheptyl carboxylic acid, whereas it is in the eighth position in AJA. Unfortunately, in that study, only one dose was tested; thus, comparisons of ED50 values are impossible. In this study, AJA produced full and dose-dependent substitution, and nearly equal ED50 values were obtained for discriminative stimulus effects (substitution for Δ9-THC) and therapeutic effects (antihyperalgesia in the CFA model). Interestingly, AJA did not produce antinociception in nonarthritic rats, whereas Δ9-THC did. The results of the present study also do not support the hypothesis that the role of CB1 receptors is insignificant at therapeutically relevant doses of AJA (Burstein et al., 2004) since its discriminative stimulus effects and antihyperalgesic effects were blocked by the CB1 receptor antagonist, SR 141716A.
AJA has been tested in phase I and phase II clinical trials. Results from published clinical trials were inconclusive. Although the authors reported analgesia in neuropathic pain patients in the absence of psychotropic effects (Karst et al., 2003), this conclusion is somewhat misleading because it is likely that subthreshold doses were used (for further discussion, see Wiley, 2005). The absence of psychotropic effects in these patients is clear from presentation of the incidence of side-effect profile; however, AJA showed only minimal efficacy, if any, as an analgesic. AJA-induced analgesic effects were statistically significant only for one of the two daily assessments. Only a trend was evident for the other time point. In addition, the magnitude of the significant effect was notably low and approximated the magnitude of changes in analgesia that were observed with different sequences of presentation of drug and vehicle in this crossover design. Based upon these findings, it is likely that a higher dose would be required for adequate pain control. Since the psychoactivity of higher doses was not assessed in this study, it is currently impossible to determine whether or not this type of side effect would co-occur with clinical efficacy of AJA in neuropathic pain patients. Based upon the results of our study, we would predict that marijuana-like intoxication would accompany clinical analgesic efficacy for AJA.
Comprehensive evaluation of the therapeutic versus deleterious effects of AJA has been a main goal of the present study. Although it has been suggested that AJA is therapeutically active at doses lower than those doses that produce undesired effects, the present data provide little evidence of separation of analgesic activity and other effects. Specifically, this study has shown that AJA, like Δ9-THC, induces psychoactive properties including a decrease in spontaneous activity, catalepsy, and Δ9-THC-like discriminative stimulus effects (undesired effects) at doses similar to those that produce antinociception or antihyperalgesia, respectively (desired effects). Moreover, contrary to previous research that has suggested that AJA's therapeutic effects are not mediated via action at CB1 receptors, this study demonstrated that AJA's effects, both therapeutically for pain and psychoactively, are CB1-mediated. These findings also underscore the importance of thoroughly evaluating the pharmacological characteristics of novel Δ9-THC-like compounds, such that therapeutic and deleterious effects are considered simultaneously.
Acknowledgments
This work was supported by National Institute on Drug Abuse Grants DA-05488 and DA-03672 and by Virginia Commonwealth University Institute of Drug and Alcohol Studies.
ABBREVIATIONS
Δ9-THC Δ9-tetrahydrocannabinol
AJA ajulemic acid (1′,1′-dimethylheptyl-Δ8-tetrahydrocannabinol-11-oic acid)
CFA complete Freund's adjuvant
MPE percentage of maximal possible effect in tail-flick test
SR 141716A N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride
SR144528 N-[-(1S)-endo-1,3,3-trimethyl bicyclo [2,2,1] heptan-2-yl]-5-(4-chloro-3-methyl-phenyl)-1-(4methylbenzyl)-pyrazole-3-carboxamide
FR-10 fixed-ratio 10
VEH vehicle
CB cannabinoid receptor
ANOVA analysis of variance
CL confidence limit
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Source with Charts, Graphs and Links: Cannabimimetic Properties of Ajulemic Acid
