Main Research Findings

1) Administration of URB-597 has the potential to improve the behavioral effects of anandamide, a key endogenous cannabinoid neurotransmitter.

2) Treatment with URB-597 has been shown to enhance endocannabinoid signalling related to improved auditory discrimination sensitivity.

Selected Data
1) The research team of Stewart et al investigated the effects of delta-9-THC, rimonabant, and URB-597 in rhesus monkeys using a drug discrimination procedure. The subjects included both male and female rhesus monkeys, housed individually under controlled light conditions and maintained on a regulated diet. The monkeys had prior exposure to cannabinoids and non-cannabinoids in earlier studies. Monkeys underwent surgical implantation of a chronic indwelling catheter under anesthesia with ketamine and isoflurane. The catheter was inserted into a subclavian or femoral vein and was anchored with suture silk. The catheter was tunneled under the skin and connected to a vascular access port, allowing for drug administration during experiments [1].

For behavioral testing, monkeys were seated in primate chairs within ventilated, sound-attenuating chambers equipped with two response levers and a light above each lever. Brass electrodes in the footrest delivered brief electric shocks as a form of aversive stimulus. A computer system controlled and recorded experimental events using Med-PC software. Two separate groups of monkeys were trained to discriminate either 0.1 mg/kg of delta-9-THC delivered intravenously, or 1 mg/kg of rimonabant delivered intravenously from a vehicle solution. Monkeys responded under a fixed-ratio 5 schedule of stimulus-shock termination, meaning that they had to make five consecutive responses on the correct lever to avoid receiving an electric shock.

For delta-9-THC discrimination, experimental cycles lasted 10 minutes including a 5-minute timeout followed by a 5-minute response period. For rimonabant discrimination, cycles lasted 20 minutes and included a 15-minute timeout followed by a 5-minute response period. The schedule was signaled by the illumination of red lights above the levers. If the monkey responded correctly, the red lights turned off, the electric stimulus was prevented, and a 30-second timeout followed. Incorrect responses reset the response requirement. Each monkey had a fixed correct lever for the duration of the study. Monkeys underwent training to recognize delta-9-THC or rimonabant by receiving drug or vehicle injections at the beginning of experimental cycles. The training criteria required at least 80% correct lever selection over multiple consecutive training sessions. Testing sessions were conducted after the monkeys met these criteria [1].

During test sessions, five consecutive responses on either lever postponed scheduled shock presentations. In delta-9-THC-trained monkeys, dose-response functions for delta-9-THC and anandamide were determined by administering increasing doses in a stepwise manner. The effects of combined delta-9-THC and anandamide were evaluated using a fixed-ratio design, where doses were administered in proportion to their respective ED50 values.To assess the effects of URB-597, monkeys received a fixed dose ranging from 0.32–3.2 mg/kg, followed by increasing doses of delta-9-THC or anandamide. The duration of anandamide effects was examined by administering the drug alone at doses of 5.6 mg/kg and 10 mg/kg or in combination with 3.2 mg/kg of URB-597 [1].

In rimonabant-trained monkeys, dose-response functions for rimonabant were determined similarly, with increasing doses administered sequentially. The effects of delta-9-THC delivered in doses ranging from 0.32 – 3.2 mg/kg and 3.2 mg/kg of URB-597 on rimonabant discrimination were also tested. Because anandamide has a short duration of action of less than 15 minutes, its combined effects with rimonabant were examined in single-cycle tests. The same method was used to evaluate URB-597’s effect on rimonabant discrimination.
The study utilized a within-subject design, meaning that each monkey served as its own control. Discrimination data were expressed as the percentage of responses on the drug lever out of total responses. Response rates were calculated based on the number of responses per second, excluding timeout periods. The effects of drugs on response rates were analyzed using repeated-measures ANOVA, with Dunnett’s test used for post-hoc comparisons [1].

Individual dose-response data were analyzed using linear regression, focusing on doses that spanned the linear portion of the response curve. If dose-response slopes did not differ significantly, a common slope was used for further analyses. ED50 values, potency ratios, and their confidence limits were calculated using parallel line analysis. The study also examined whether the combined effects of delta-9-THC and anandamide followed an additive model. The combined effects of delta-9-THC, rimonabant, and URB-597 were also analyzed to determine shifts in dose-response relationships.
This study provided insight into drug discrimination behavior in rhesus monkeys trained to recognize delta-9-THC and rimonabant. The results demonstrated how cannabinoids and related compounds interact at a behavioral level, particularly in response to changes in anandamide signaling. The role of URB-597 as a modulator of cannabinoid effects was also examined, shedding light on its potential pharmacological significance. These findings contribute to the broader understanding of cannabinoid receptor activity and its implications for neuropharmacology [1].

2) The research team of Sokolic et al investigated the effects of delta-9-THC and fatty acid amide hydrolysis inhibitor URB-597 on the performance of auditory and olfactory discrimination tasks in rats. For the purpose of this study, a total of 32 male Australian Hooded Wistar rats, bred in-house at the University of Sydney, were used in this study. The rats, weighing between 116 and 229 grams and aged 40–50 days at the start, were housed in groups of eight in a temperature-controlled colony room set to 21°C. The room followed a reverse light–dark cycle and the rats were placed on a water deprivation schedule, receiving 10 ml of water per day but having free access to water on most weekends. Experimental training and testing were conducted between 10:00 and 16:00 h [2].

Five different experiments took place in 12-channel air-dilution olfactometers, two of which were modified for auditory discrimination tasks. The test chamber measured 17 cm × 24 cm × 25 cm and contained a stainless steel grid floor, a ventilation fan, a sniff port for odor detection, and a lick tube for water rewards. The olfactometers generated olfactory stimuli by diverting an air stream through saturator bottles containing odorants, mixing the odorized air with a main airflow before delivering it through the sniff port. Rats initiated trials by nose poking into the port, which triggered a 2-second stimulus presentation period.
For odor discrimination tasks, rats sampled one of 16 different odorants, including common food-related scents (e.g., lemon, vanilla, banana) and aromatic oils (e.g., jasmine, honeysuckle). Aldehydes were diluted, while other odorants remained undiluted. Auditory discrimination trials followed the same response protocols but presented sound stimuli instead of odors, using either a beeper or white noise. A custom-written software program controlled the olfactometers and data recording [2].

Initially, rats learned to associate licking the lick tube with a water reward, followed by nose-poking training to ensure adequate sampling of stimuli. The final task was a go/no-go successive discrimination paradigm. The olfactory group was trained to distinguish between lemon (S+) and strawberry (S−), while the auditory group learned to differentiate between a beeper (S+) and white noise (S−). A correct response on an S+ trial was defined as licking the tube and resulted in a water reward, while incorrect responses on S− trials, defined as licking when they shouldn’t, led to no reward and a longer intertrial interval. A performance criterion of 17 correct responses in 20 consecutive trials was set for all tasks.
Once rats reached criterion performance, the first experiment began with dividing the animals into treatment subgroups that received either delta-9-THC or rimonabant at increasing doses of 0.3, 1, and 3 mg/kg. Drug administration occurred 15 minutes before testing, with washout days between drug trials to ensure baseline recovery. Higher doses of delta-9-THC impaired auditory discrimination performance significantly, prompting the use of additional washout days. To assess whether rimonabant could counteract delta-9-THC effects, a final test combined both drugs at 3 mg/kg. The second experiment examined the potential long-term effects of delta-9-THC on auditory discrimination with rats previously tested in Experiment 1 receiving a single high dose of 10 mg/kg of delta-9-THC. Testing occurred 24 hours later and performance was compared between the delta-9-THC-treated group and a vehicle-treated control group [2].
Three weeks after Experiment 2, the third experiment was initiated using the same rats that were tested with URB-597 and increased endocannabinoid levels. On the first test day, all rats received a vehicle injection, while on the second day, they were split into two groups receiving either 0.1 or 0.3 mg/kg of URB-597. Two washout days followed before reversing the dosage groups and repeating the process. A final test examined whether 3 mg/kg of rimonabant could block the effects of URB-597 when coadministered.
Experiment 4 evaluated whether delta-9-THC affects the ability to acquire and reverse novel olfactory discriminations. The olfactory group from Experiment 1 was used, while the auditory group was excluded due to severe impairment under delta-9-THC. Rats were assigned to either drug or control groups for five different odor discriminations. Doses of delta-9-THC at 0.3, 1, and 3 mg/kg were tested, followed by a combined dose of 3 mg/kg of delta-9-THC and 3 mg/kg of rimonabant trial, as well as a rimonabant-alone trial [2].

Acquisition was measured by the number of errors made before reaching the 17-out-of-20 correct response criterion. If rats did not reach criterion within 400 trials on the first day, they continued on subsequent days. Once acquired, the discrimination was tested over four additional days to establish asymptotic performance. The reversal phase reallocated rats to new treatment groups to assess whether drug treatment affected their ability to reverse the learned discrimination.
Finally, the fifth experiment included rats from previous experiments that were divided into an experimental group treated with URB-597 at 0.3 mg/kg and a control group to test their ability to acquire a coffee (S+) vs. jasmine (S−) discrimination. Performance was measured similarly to Experiment 4. After reaching criterion, the discrimination was reversed and a final test assessed whether rimonabant blocked URB-597 effects when coadministered before a frangipani (S+) vs. honeysuckle (S−) discrimination task [2].

Discussion

1) The study conducted by the research team of Stewart et al investigated the discriminative stimulus effects of delta-9-THC, anandamide, and their interactions with other compounds in non-human primates. The results of the experiment revealed that delta-9-THC dose-dependently increased responding on the delta-9-THC lever, reaching a maximum of 96% at a dose of 0.1 mg/kg, while vehicle administration resulted in 0% responses. Anandamide similarly increased responses, with a maximum effect of 90% at 17.8 mg/kg. Despite these similarities, the dose–response slopes for delta-9-THC and anandamide were not significantly different, with ED50 values of 0.039 mg/kg and 5.8 mg/kg, respectively [1]

When delta-9-THC and anandamide were combined at a fixed ratio based on their ED50 values, the results deviated from the expected additive effects. Although a combination of half the ED50 values produced only 5% responding on the delta-9-THC lever, the full ED50 doses resulted in 91% responding. However, isobolographic analysis revealed that the combination had infra-additive effects, particularly at lower doses, as the experimentally derived dose–response curve had a significantly greater slope than the predicted additive curve.
The effects of URB-597, an inhibitor of anandamide metabolism, were examined alone and in combination with delta-9-THC and anandamide. URB-597 alone did not significantly increase responses on the delta-9-THC lever, with a maximum of only 1%. When combined with delta-9-THC, URB-597 did not significantly modify the discriminative stimulus effects of delta-9-THC, as indicated by unchanged ED50 values. However, URB-597 dose-dependently enhanced the potency of anandamide, reducing its ED50 value by 3.1-, 13-, and 32-fold at doses of 0.32, 1, and 3.2 mg/kg, respectively. Importantly, the rate of responding remained unaffected across all conditions [1].

Time-course studies further demonstrated that anandamide delivered in doses of 5.6 and 10 mg/kg produced transient effects, with responding on the delta-9-THC lever peaking within 5–10 minutes and returning to 0% by 15–20 minutes. However, when anandamide in doses of 5.6 mg/kg was combined with URB-597 in doses of 3.2 mg/kg, both the potency and duration of action increased significantly, with responses reaching 100% at 5–10 minutes and remaining above 50% for up to 50 minutes. This combination also significantly decreased response rate to 66% of control at 5–10 minutes, highlighting a behavioral effect not observed with either compound alone [1].

The study also explored the discriminative stimulus effects of rimonabant, a CB1 receptor antagonist. Rimonabant dose-dependently increased responses on the rimonabant lever, reaching 91% at a dose of 1 mg/kg, with an ED50 value of 0.35 mg/kg. Single-dose administration resulted in slightly higher potency compared to cumulative dosing. Neither delta-9-THC nor anandamide fully substituted for rimonabant, with maximum responding of only 1% and 12%, respectively. However, both compounds dose-dependently attenuated the discriminative stimulus effects of rimonabant, increasing its ED50 value by 3.7- to 9.7-fold for delta-9-THC and 6.9-fold for anandamide.
URB-597 alone did not significantly affect rimonabant’s discriminative stimulus effects. When combined with anandamide at a dose of 10 mg/kg, URB-597 increased the ED50 value of rimonabant by 3.9-fold, though this was not significantly different from anandamide alone. Notably, anandamide at a dose of 32 mg/kg significantly decreased response rate to 26% of control, an effect that was antagonized by rimonabant at a dose of 3.2 mg/kg. In contrast, neither URB-597 nor its combination with rimonabant significantly affected response rate [1].

Finally, the magnitude of the shift in the rimonabant dose–response curve was analyzed as a function of delta-9-THC and anandamide doses. Both compounds produced similar shifts, with estimated doses required for a twofold shift being 0.31 mg/kg for delta-9-THC and 8.7 mg/kg for anandamide. These potency values were significantly lower than those required for substitution in delta-9-THC discrimination tests, suggesting differential CB1 receptor-mediated effects. When combined with URB 597 at a dose of 3.2 mg/kg, anandamide’s potency in delta-9-THC-treated monkeys increased 1.6-fold, though this effect was not statistically significant [1].
In conclusion, delta-9-THC and anandamide exhibited similar discriminative stimulus effects, but their combination produced infra-additive interactions. URB-597 significantly enhanced anandamide’s effects while having minimal impact on delta-9-THC. The discriminative effects of rimonabant were attenuated by both delta- 9-THC and anandamide, with URB-597 further modifying anandamide’s interaction with rimonabant [1].

2) Experiment 1 out of 5 experiments conducted by Sokolic et al examined how delta-9-THC affects performance on an auditory discrimination task. The results showed that delta-9-THC significantly impaired task performance at doses of 0.3, 1, and 3 mg/kg. The impairment was also observed the day after administration of the highest dose of 3 mg/kg. However, when rimonabant at 3 mg/kg was coadministered with delta-9-THC at 3 mg/kg, the detrimental effects were significantly reduced, suggesting a role of CB1 receptor blockade in mitigating these cognitive deficits. Notably, rimonabant alone had no significant effect on auditory discrimination performance. Additionally, neither delta-9-THC nor rimonabant affected performance on an olfactory discrimination task, indicating that the observed impairment was task-specific rather than a general cognitive deficit [2].

Experiment 2 assessed the longer-term impact of delta-9-THC, rats were tested on a well-learned go/no-go auditory discrimination task 24 and 48 hours after administration of a high dose of 10 mg/kg. Performance was severely impaired 24 hours post-administration but recovered by 48 hours. This suggests that delta-9-THC can cause a temporary but substantial disruption in auditory discrimination abilities. That being said, the third experiment revealed that URB-597 administered at doses of 0.1 and 0.3 mg/kg significantly impaired auditory discrimination performance, and this impairment persisted for 24 hours after administration. However, when URB-597 was coadministered with rimonabant, the cognitive impairment was prevented, suggesting that endocannabinoid-mediated CB1 receptor activation plays a crucial role in auditory discrimination deficits [2].

The fourth experiment evaluated the effects of delta-9-THC on the acquisition of novel olfactory discrimination tasks. Unlike its effect on auditory tasks, delta-9-THC administered at doses of 0.3, 1, and 3 mg/kg did not significantly impair the acquisition of olfactory discrimination learning. The same was true when delta-9-THC was administered with rimonabant. These findings indicate that these treatments did not impact learning in this domain. However, when rimonabant was administered alone there was a slight tendency to enhance learning [2].

However, in an olfactory discrimination reversal task assessing cognitive flexibility, delta-9-THC at 1 and 3 mg/kg significantly impaired performance, increasing errors during reversals. The lowest dose of 0.3 mg/kg did not significantly affect performance. Notably, the impairment in reversal learning was prevented when delta-9-THC was coadministered with rimonabant, reinforcing the role of CB1 receptor activity in this cognitive function. Again, rimonabant alone had a mild facilitatory effect, potentially improving cognitive flexibility.
The final experiment examined the effects of URB-597 on olfactory discrimination learning. Similar to delta-9-THC, URB-597 administered at a dose of 0.3 mg/kg did not impair the acquisition of novel olfactory discrimination tasks. Additionally, coadministration of URB-597 with rimonabant did not affect acquisition. However, URB-597 did significantly impair performance in the reversal task, in a manner similar to delta-9-THC. This impairment was eliminated when URB-597 was administered with rimonabant, further supporting the hypothesis that CB1 receptor activation underlies the observed cognitive inflexibility [2].

Disclaimer
**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

Citations

[1] Stewart JL, McMahon LR. The fatty acid amide hydrolase inhibitor URB 597: interactions with anandamide in rhesus monkeys. Br J Pharmacol. 2011 Sep;164(2b):655-66. doi: 10.1111/j.1476-5381.2011.01388.x. PMID: 21449917; PMCID: PMC3188916.

[2] Sokolic L, Long LE, Hunt GE, Arnold JC, McGregor IS. Disruptive effects of the prototypical cannabinoid Δ⁹-tetrahydrocannabinol and the fatty acid amide inhibitor URB-597 on go/no-go auditory discrimination performance and olfactory reversal learning in rats. Behav Pharmacol. 2011 Jun;22(3):191-202. doi: 10.1097/FBP.0b013e328345c82b. PMID: 21512341.

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