Sex, species and age: Effects of rodent demographics on the pharmacology of ∆9-tetrahydrocanabinol

https://doi.org/10.1016/j.pnpbp.2020.110064Get rights and content

Highlights

  • Choice of animal model is important in study of THC-containing edibles.

  • We compared the effects of THC and 11-OH-THC in mice and rats of both sexes.

  • 11-OH-THC had marginally higher affinity than THC for cerebellar CB1 receptors.

  • Sex differences in THC-like in vivo effects occurred in rats, but not in mice.

  • Results support consideration of sex and species in choice of animal model.

Abstract

Cannabis edibles are becoming more common in an increasingly diverse population of users, and the impact of first pass metabolism on cannabis's pharmacological profile across age and sex is not well understood. The present study examined the impact of age, sex and rodent species on the effects of intraperitoneal (i.p.) delta-9-tetrahydrocannabinol (THC) and its primary psychoactive metabolite, 11-OH-THC, in rodent models of psychoactivity and molecular assays of cannabinoid receptor type-1 (CB1) pharmacology. Like oral THC, i.p. THC also undergoes first pass metabolism. In both species and sexes, 11-OH-THC exhibited marginally higher affinity (~1.5 fold) than THC and both served as partial agonists in [35S]GTPγS binding with equivalent potency; 11-OH-THC exhibited slightly greater efficacy in rat brain tissue. In ICR mice, 11-OH-THC exhibited greater potency than THC in assays of catalepsy (7- to 15-fold) and hypothermia (7- to 31-fold). Further, 11-OH-THC was more potent in THC drug discrimination (7- to 9-fold) in C57Bl/6 J mice, with THC-like discriminative stimulus effects being CB1-, but not CB2-, mediated. THC's discriminative stimulus also was stable across age in mice, as its potency did not change over the course of the experiment (~17 months). While sex differences in THC's effects were not revealed in mice, THC was significantly more potent in females Sprague-Dawley rats than in males trained to discriminate THC from vehicle. This study demonstrates a cross-species in the psychoactive effects of i.p. THC across sex that may be related to differential metabolism of THC into its psychoactive metabolite 11-OH-THC, suggesting that species is a crucial design consideration in the preclinical study of phytocannabinoids.

Introduction

Recent policy shifts in the U.S. and internationally have loosened legal restrictions on access to cannabis and cannabis-based products, resulting in corresponding increases in use in some demographic groups (Smart and Pacula, 2019). While recreational use in young men (aged ≤ 24) still outpaces use in women and older adults (Carliner et al., 2017), the population of cannabis users is diverse and use is increasing in middle-aged adults (aged 50–64) and in seniors (≥65) of both sexes (Han and Palamar, 2018; Han and Palamar, 2020). Further, methods of cannabis delivery have diversified. Although the most common route of cannabis administration for non-medical use continues to be inhalation through smoking or vaping, ingestion of cannabis edibles and multi-modal use has become more prevalent (Borodovsky et al., 2017; Schauer et al., 2020), with an estimated 25% of adults in states where cannabis is legal for recreational use reporting consumption of edibles (Schauer et al., 2020).

Unlike inhalation, oral consumption is accompanied by first pass metabolism, which raises the issue of the potential role of metabolites in determination of the effects of edibles containing ∆9-tetrahydrocanabinol (THC), the major psychoactive constituent of Cannabis sativa. Metabolism of orally administered THC occurs primarily in the liver and is extensive, with almost 100 metabolites having been identified (Grotenhermen, 2003). The two most referenced metabolites are 11-OH-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (11-COOH-Δ9-THC or THC-COOH). 11-OH-THC, formed by the cytochrome-P450-(CYP)-mediated hydroxylation of the C-11 site, is the initial step of THC biotransformation in most species, including humans (Harvey and Brown, 1991; Yamamoto et al., 1984). A secondary metabolite, THC-COOH, is formed through subsequent oxidation of 11-OH-THC (Watanabe et al., 1980). Whereas THC-COOH lacks cannabimimetic effects (Huestis, 2007; Mazur et al., 2009), 11-OH-THC readily crosses the blood-brain barrier, perhaps with even greater brain penetrance than THC (Huestis, 2007; Schou et al., 1977).

Given the increasing use of THC-infused edibles by both women and men in the U.S. (Schauer et al., 2020), identifying biological or pharmacological mechanisms through which sex influences THC's behavioral effects is crucial for development of appropriate cannabis therapeutics or treatments for cannabis dependence. Design of such mechanistic studies in humans is complicated by self-selection of users and lack of control over their prior history; hence, animal models are often employed. To date, the most commonly used research model in this effort has been rats (Cooper and Craft, 2018; Craft et al., 2013b). For example, prior research has revealed that female rats are more sensitive to analgesic and discriminative stimulus effects of THC (Craft et al., 2013a; Wiley et al., 2017). In contrast, mice have been used in relatively few studies investigating sex differences in cannabinoid effects, albeit their use is increasing concurrent with the U.S. federal mandate to consider sex as a biological variable in NIH-funded research (Clayton and Collins, 2014).

The goal of the present study was two-fold: (1) to evaluate the effects of sex, age, and species in an animal model of the psychoactive effects of THC and (2) to provide data on the in vitro effects of THC and 11-OH-THC, the primary psychoactive metabolite of THC, in male and female rodents. Because one of our interests was in the behavioral effects of a THC metabolite, we chose a route of administration (intraperitoneal, i.p.) wherein THC undergoes first-pass metabolism (Lukas et al., 1971). Although oral gavage may have been more translationally relevant, it has been shown to be stressful for rodents (Walker et al., 2012) and results in more variable absorption than i.p. (Daublain et al., 2017). Subjective effects were assessed using drug discrimination, in which mice and rats were trained to discriminate between the effects of THC and vehicle. Subsequently, we evaluated sex differences in THC's discriminative stimulus effects in both species. Because cannabinoid research on sex differences in mice is sparse, we also examined additional pharmacological effects of cannabinoids in mice, including antagonist tests and tests with 11-OH-THC in drug discrimination and evaluation of mice in a classical cannabinoid tetrad of in vivo assays for which centrally acting cannabinoids typically produce a characteristic profile of effects: reduction in body temperature, reduction in locomotor activity, antinociception, and catalepsy (Martin et al., 1991). We also assessed the binding affinity of 11-OH-THC and THC for the CB1 cannabinoid receptor and their activation of this G-protein coupled receptor (GPCR) in both sexes in mice and rats.

Section snippets

Subjects

Adult drug naïve male and female ICR mice (31-34 g; Envigo, Frederick, MD) and C57/Bl6J mice (20-25 g; Jackson Laboratories, Bar Harbor, ME) were used in the tetrad battery and mouse drug discrimination experiments, respectively. Adult drug naïve male and female Sprague-Dawley rats (200–274 g; Envigo, Frederick, MD) were used in the rat drug discrimination study. While mice in the tetrad battery experiment received free access to food in their home cage, mice and rats in the drug discrimination

CB1 receptor binding and activation

As shown in Table 1, both THC and its psychoactive metabolite 11-OH-THC bind to CB1 receptors in the cerebella of mice of both sexes with moderate affinity (Ki = 27.3–41.3 nM). Both compounds exhibited similar affinities across sex, with 11-OH-THC showing slightly (1.3–1.5-fold) greater affinity than THC in both sexes. Functionally, THC and 11-OH-THC were partial agonists, as measured in the [35S]GTPγS assay, and activated the CB1 receptor at similar potencies across sex (Table 1).

Similarly,

Discussion

Given the increasingly widespread use of cannabis edibles therapeutically and their continued abuse by both females and males in the U.S., identifying how sex influences cannabinoid pharmacology is imperative. One of the first steps in achieving this goal is to choose the most appropriate animal model. To date, most studies that have examined sex differences in the behavioral effects of cannabinoids have been conducted in rats (Cooper and Craft, 2018; Craft et al., 2013b), with findings

Acknowledgements

Research was supported by U.S. National Institutes of Health/National Institute on Drug Abuse [grants DA-045003, DA-003672]. NIDA had no further role in the writing of the manuscript or in the decision to submit the paper for publication.

Author statement

All authors have read and approved the final version of the manuscript.

Participated in research design: Wiley, Lefever, Gamage.

Performed data analysis: Wiley, Gamage.

Wrote or contributed to the writing of the manuscript: Wiley, Gamage, Barrus.

Contributed to data collection: Barrus, Farquhar, Lefever.

Ethical statement

This manuscript and the data it contains are original, are not under current consideration for publication in any other journal, and have not previously been published. The studies reported in this manuscript were carried out in accordance with National Institutes of Health guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to

Declaration of Competing Interest

None of the authors have any conflicts of interest with respect to the research described in this manuscript. The funding sources provided funding for the research, but did not otherwise influence the conduct of the experiments, writing on the manuscript, or decision to submit.

References (50)

  • G.L. Schauer et al.

    Modes of marijuana use - smoking, vaping, eating, and dabbing: Results from the 2016 BRFSS in 12 states

    Drug Alcohol Depend.

    (2020)
  • L. Silva et al.

    Sex-specific alterations in hippocampal cannabinoid 1 receptor expression following adolescent delta-9-tetrahydrocannabinol treatment in the rat

    Neurosci. Lett.

    (2015)
  • A.H. Tseng et al.

    Sex differences in antinociceptive and motoric effects of cannabinoids

    Eur. J. Pharmacol.

    (2001)
  • A.H. Tseng et al.

    Pharmacokinetic factors in sex differences in Delta 9-tetrahydrocannabinol-induced behavioral effects in rats

    Behav. Brain Res.

    (2004)
  • R.E. Vann et al.

    Discriminative stimulus properties of Delta 9-tetrahydrocannabinol (THC) in C57Bl/6J mice

    Eur. J. Pharmacol.

    (2009)
  • M.K. Walker et al.

    A less stressful alternative to oral gavage for pharmacological and toxicological studies in mice

    Toxicol. Appl. Pharmacol.

    (2012)
  • K. Watanabe et al.

    Comparison in mice of pharmacological effects of delta 8-tetrahydrocannabinol and its metabolites oxidized at 11-position

    Eur. J. Pharmacol.

    (1980)
  • J.L. Wiley et al.

    Sex differences in Delta(9)-tetrahydrocannabinol metabolism and in vivo pharmacology following acute and repeated dosing in adolescent rats

    Neurosci. Lett.

    (2014)
  • J.L. Wiley et al.

    Comparison of the discriminative stimulus and response rate effects of Delta9-tetrahydrocannabinol and synthetic cannabinoids in female and male rats

    Drug Alcohol Depend.

    (2017)
  • J.L. Wiley et al.

    Do you feel it now? Route of administration and Delta(9)-tetrahydrocannabinol-like discriminative stimulus effects of synthetic cannabinoids in mice

    Neurotoxicology

    (2019)
  • P.J. Winsauer et al.

    Ovarian hormones and chronic administration during adolescence modify the discriminative stimulus effects of delta-9-tetrahydrocannabinol (Delta(9)-THC) in adult female rats

    Pharmacol. Biochem. Behav.

    (2012)
  • R.G. Browne et al.

    Discriminative stimulus properties of ∆9-THC: Mechanistic studies

    J. Clin. Pharmacol.

    (1981)
  • J.J. Burston et al.

    Regional enhancement of CB1 receptor desensitization in female adolescent rats following repeated Δ9-tetrahydrocannabinol exposure

    Br. J. Pharmacol.

    (2010)
  • M.P. Castelli et al.

    Male and female rats differ in brain cannabinoid CB1 receptor density and function and in behavioural traits predisposing to drug addiction: Effect of ovarian hormones

    Curr. Pharm. Des.

    (2014)
  • J.A. Clayton et al.

    Policy: NIH to balance sex in cell and animal studies

    Nature

    (2014)
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