Rationale The legalization of medicinal use of Cannabis sativa in most US states and the removal of hemp from the Drug Enforcement Agency (DEA) controlled substances act has resulted in a proliferation of products containing Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) for oral consumption (e.g., edibles, oils, and tinctures) that are being used for recreational and medicinal purposes. Objective This study examined the effects of cannabinoids THC and CBD when administered orally on measures of pain sensitivity, body temperature, locomotor activity, and catalepsy (i.e., cannabinoid tetrad) in male and female Sprague Dawley rats. Methods Rats (N = 24, 6 per sex/drug group) were administered THC (1-20 mg/kg), CBD (3-30 mg/kg), or sesame oil via oral gavage. Thermal and mechanical pain sensitivity (tail flick assay, von Frey test), rectal measurements for body temperature, locomotor activity, and the bar-test of catalepsy were completed. A separate group of rats (N = 8/4 per sex) was administered morphine (5-20 mg/kg; intraperitoneal, IP) and evaluated for pain sensitivity as a positive control. Results We observed classic tetrad effects of antinociception, hypothermia, hyper- and hypolocomotion, and catalepsy after oral administration of THC that were long lasting (> 7 h). CBD modestly increased mechanical pain sensitivity and produced sex-dependent effects on body temperature and locomotor activity. Conclusions Oral THC and CBD produced long lasting effects that differed in magnitude and time course when compared with other routes of administration. Examination of cannabinoid effects administered via different routes of administration, species, and in both males and females is critical to enhance translation.

Cannabinoid tetrad effects of oral Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) in male and female rats: sex, dose-effects and time course evaluations

Tolerance to compounds that target G protein–coupled receptors (GPCRs), such as the cannabinoid type-1 receptor (CB1R), is in part facilitated by receptor desensitization. Processes that mediate CB1R desensitization include phosphorylation of CB1R residues S426 and S430 by a GPCR kinase and subsequent recruitment of the β-arrestin2 scaffolding protein. Tolerance to cannabinoid drugs is reduced in S426A/S430A mutant mice and β-arrestin2 knockout (KO) mice according to previous work in vivo. However, the presence of additional phosphorylatable residues on the CB1R C-terminus made it unclear as to whether recruitment to S426 and S430 accounted for all desensitization and tolerance by β-arrestin2. Therefore, we assessed acute response and tolerance to the cannabinoids delta-9-tetrahydrocannabinol (Δ9-THC) and CP55,940 in S426A/S430A x β-arrestin2 KO double-mutant mice. We observed both delayed tolerance and increased sensitivity to the antinociceptive and hypothermic effects of CP55,940 in male S426A/S430A single- and double-mutant mice compared with wild-type littermates, but not with Δ9-THC. Female S426A/S430A single- and double-mutant mice were more sensitive to acute antinociception (CP55,940 and Δ9-THC) and hypothermia (CP55,940 only) exclusively after chronic dosing and did not differ in the development of tolerance. These results indicate that phosphorylation of S426 and S430 are likely responsible for β-arrestin2–mediated desensitization as double-mutant mice did not differ from the S426A/S430A single-mutant model in respect to cannabinoid tolerance and sensitivity. We also found antinociceptive and hypothermic effects from cannabinoid treatment demonstrated by sex-, agonist-, and duration-dependent features. SIGNIFICANCE STATEMENT A better understanding of the molecular mechanisms involved in tolerance will improve the therapeutic potential of cannabinoid drugs. This study determined that further deletion of β-arrestin2 does not enhance the delay in cannabinoid tolerance observed in CB1R S426A/S430A mutant mice.

Cannabinoid Tolerance in S426A/S430A x β-Arrestin 2 Knockout Double-Mutant Mice

Mechanisms of cannabinoid tolerance.

Abstract Cannabis use is highly prevalent especially among people living with HIV (PLWH). Activation of the anti-inflammatory and neuroprotective endocannabinoid system by phytocannabinoids, i.e. Δ 9 -tetrahydrocannabinol (THC), has been proposed to reduce HIV symptoms. However, THC’s effects on HIV-associated cognitive impairments are unclear. Using HIV-1 Tat transgenic mice, the current study investigates acute THC effects on various behavioral outcomes and the endocannabinoid system. Minor or no effects of THC doses (1, 3, 10 mg/kg) were noted for body mass, body temperature, locomotor activity, and coordination, but spontaneous nociception was significantly decreased, with Tat induction increasing antinociceptive THC effects. Anxiogenic effects of THC (10 mg/kg) were demonstrated in Tat(−) females and males compared to vehicle-treated mice, with overall increased anxiety-like behavior in females compared to males. Object recognition memory was diminished by acute THC (10 mg/kg) injections in Tat(−) but not Tat(+) females, without affecting males. For the endocannabinoid system and related lipids, no effects were noted for acute THC, but female sex and Tat induction was associated with elevated 2-AG, AEA, AA, CB 1 R, CB 2 R, FAAH and/or MAGL expression in various CNS regions. Further, females demonstrated higher AEA levels compared to males in most CNS structures, and AEA levels in the prefrontal cortex of Tat(+) females were negatively associated with recognition memory. Overall, findings indicate that acute THC exposure exerts differential effects on behavior in the context of neuroHIV dependent on sex, potentially due to an altered endocannabinoid system, which may be of relevance in view of potential cannabis-based treatment options for PLWH. 

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Effects of acute Δ9 -tetrahydrocannabinol on behavior and the endocannabinoid system in HIV-1 Tat transgenic female and male mice

Tolerance to cannabinoids could limit their therapeutic potential. Male mice expressing a desensitization-resistant form (S426A/S430A) of the type-1 cannabinoid receptor (CB1R) show delayed tolerance to delta-9-tetrahydrocannabinol (∆9-THC) but not CP55,940. With more women than men using medical cannabis for pain relief, it is essential to understand sex differences in cannabinoid antinociception, hypothermia, and resultant tolerance. Our objective was to determine whether female mice rely on the same molecular mechanisms for tolerance to the antinociceptive and/or hypothermic effects of cannabinoids that we have previously reported in males. We determined whether the S426A/S430A mutation differentially disrupts antinociceptive and/or hypothermic tolerance to CP55,940 and/or Δ9-THC in male and female S426A/S430A mutant and wild-type littermates. The S426A/S430A mutation conferred an enhanced antinociceptive response for ∆9-THC and CP55,940 in both male and female mice. While the S426A/S430A mutation conferred partial resistance to ∆9-THC tolerance in male mice, disruption of CB1R desensitization had no effect on tolerance to ∆9-THC in female mice. The mutation did not alter tolerance to the hypothermic effects of ∆9-THC or CP55,940 in either sex. Interestingly, female mice were markedly less sensitive to the antinociceptive effects of 30 mg/kg ∆9-THC and 0.3 mg/kg CP55,940 compared with male mice. Our results suggest that disruption of the GRK/βarrestin2 pathway of desensitization alters tolerance to Δ9-THC but not CP55,940 in male but not female mice. As tolerance to Δ9-THC appears to develop differently in males and females, sex should be considered when assessing the therapeutic potential and dependence liability of cannabinoids.

Sex-specific mechanisms of tolerance for the cannabinoid agonists CP55,940 and delta-9-tetrahydrocannabinol (Δ9-THC)

Tolerance to the pain-relieving effects of cannabinoids limits the therapeutic potential of these drugs in patients with chronic pain. Recent preclinical research with rodents and clinical studies in humans has suggested important differences between males and females in the development of tolerance to cannabinoids. Our previous work found that male mice expressing a desensitization resistant form (S426A/S430A) of the type 1 cannabinoid receptor (CB1R) show delayed tolerance and increased sensitivity to the antinociceptive effects of delta-9-tetrahydrocannabinol (∆9-THC). Sex differences in tolerance have been reported in rodent models with females acquiring tolerance to ∆9-THC faster than males. However, it remains unknown whether the S426A/S430A mutation alters analgesic tolerance to ∆9-THC in mice with chemotherapy-evoked chronic neuropathic pain, and also whether this tolerance might be different between males and females. Male and female S426A/S430A mutant and wild-type littermates were made neuropathic using four once-weekly injections of 5 mg/kg cisplatin and subsequently assessed for tolerance to the anti-allodynic effects of 6 and/or 10 mg/kg ∆9-THC. Females acquired tolerance to the anti-allodynic effects of both 6 and 10 mg/kg ∆9-THC faster than males. In contrast, the S426A/S430A mutation did not alter tolerance to ∆9-THC in either male or female mice. The anti-allodynic effects of ∆9-THC were blocked following pretreatment with the CB1R antagonist, rimonabant, and partially blocked following pretreatment with the CB2R inverse agonist, SR144528. Our results show that disruption of the GRK/β-arrestin-2 pathway of desensitization did not affect sensitivity and/or tolerance to ∆9-THC in a chronic pain model of neuropathy.

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Angela N Henderson-Redmond

Papers

Sex-specific mechanisms of tolerance for the cannabinoid agonists CP55,940 and delta-9-tetrahydrocannabinol (Δ9-THC)

Sex Differences in Tolerance to Delta-9-Tetrahydrocannabinol in Mice With Cisplatin-Evoked Chronic Neuropathic Pain