Effects of β-caryophyllene, A Dietary Cannabinoid, in Animal Models of Drug Addiction

Abstract

Background: β-caryophyllene (BCP) is a natural bicyclic sesquiterpene found in Cannabis and other plants. BCP is currently used as a food additive, although pharmacological studies suggest its potential therapeutic application for the treatment of certain brain disorders. The mechanisms of action of BCP remain uncertain, possibly including full agonism at the cannabinoid CB₂ receptor (CB₂R).

Objective: The study aims to investigate BCP’s potential as a new drug for the treatment of substance use disorders by reviewing preclinical studies with animal models.

Results: BCP has been investigated in behavioral paradigms, including drug self-administration, conditioned place preference, and intracranial self-stimulation; the drugs tested were cocaine, nicotine, alcohol, and methamphetamine. Remarkably, BCP prevented or reversed behavioral changes resulting from drug exposure. As expected, the mechanism of action entails CB₂R activation, although this is unlikely to constitute the only molecular target to explain such effects. Another potential target is the peroxisome proliferator-activated receptor.

Conclusion: Preclinical studies have reported promising results with BCP in animal models of substance use disorders. Further research, including studies in humans, are warranted to establish its therapeutic potential and its mechanisms of action.

1. DRUG ADDICTION AND THE ENDOCANNABINOID SYSTEM

Drug addiction is a major health and social issue in most contemporary societies (WHO, 2020). According to the Diagnostic and Statistical Manual of Mental Disorders (DSM-5), it is characterized by the loss of inhibitory control and compulsive drug intake regardless of the long-term deleterious impact [1]. Long-term drug consumption results in molecular modifications in several brain areas, such as the mesolimbic system, amygdala, hippocampus, and prefrontal cortex [2, 3], which result in behavioral responses of binge and intoxication followed by withdrawal and negative affect, preoccupation and craving. All these neurochemical and behavioral modifications often lead to relapse and the inability to quit drug use [4, 5]. Currently, few medications can efficiently treat compulsive drug intake, withdrawal symptoms, and relapse [6]. Thus, preclinical research has been focusing on new strategies for pharmacologically targeting the brain reward system and related circuits disrupted in addiction [7]. Among these new potential targets is the endocannabinoid system (eCBS). The eCBS may be involved in various aspects of drug addiction, including reward, contextual memories precipitating drug seeking, drug tolerance, and withdrawal effects [8, 9]. The eCBS comprises two main cannabinoid receptor subtypes (CB₁R and CB₂R), their endogenous ligands, termed endocannabinoids, and the proteins and enzymes responsible for endocannabinoid transport, biosynthesis, and degradation [10]. CB₁R are densely expressed throughout brain regions related to reward and drug addiction [10-13], where they modulate the dopaminergic mesolimbic circuit and drug-associated reward [14]. Accordingly, CB₁R antagonism reduces dopamine release in the mesolimbic pathway, possibly by disinhibiting GABAergic afferent neurons projecting onto the ventral tegmental area (VTA) and nucleus accumbens [15]. As such, much effort was put into the development of CB₁R modulators and their investigation of drug-related responses. In fact, CB₁R antagonists and inverse agonists, such as rimonabant, taranabant, surinabant, and AM251, show robust effects in inhibiting drug-induced behaviors [15-17]. Unfortunately, however, the enthusiasm for these drugshas been dumped by the occurrence of clinically significant psychiatric side effects [18]. Contrary to CB₁R, CB₂R were initially considered a peripheral receptor, absent from neurons in the central nervous system [18, 19]. As a result, initial pharmacological studies focused on CB₂R agonists primarily as analgesic drugs devoid of central side effects. Later on, evidence from histological, genetic, electrophysiological, and pharmacological approaches challenged the view of CB₂R as a “peripheral receptor”. CB₂R were identified in neurons in specific brain regions, including those involved in addiction, such as the midbrain VTA neurons [20-23]. In this pathway, CB₂R expression occurs in the cell bodies of dopaminergic neurons [20, 21]. Moreover, their pharmacological activation counteracts the dopamine release in the nucleus accumbens induced by drugs of abuse [20]. CB₂R agonists prevent drug reward [17, 24] and reduce the reinforcing effects of cocaine and ethanol [25-27]. In addition, CB₂R knockout mice were more likely to develop ethanol-induced conditioned place preference (CPP), higher sensitivity to withdrawal after the ingestion of acute doses of ethanol, and higher ethanol intake in comparison to wild type control group [28]. Thus, CB₂R activation could represent a novel mechanism for drug addiction treatment. Moreover, CB₂R agonists do not induce the psychiatric side effects related to the blockade of CB₁R. In fact, they reduce anxiety-like behaviors in rodents [29]. This review aims to discuss the effects of a CB₂R agonist, the compound (1R,9S,E)-4,11,11-Trimethyl-8-methylenebicyclo[7.2.0]undec-4-ene, β-caryophyllene (BCP). BCP is a natural bicyclic sesquiterpene found in Cannabis and other plants [29, 30]. It was approved as a food additive and classified as “generally recognized as safe” by the Food and Drug Administration (FDA). As a terpenoid, the synergistic interaction between BCP and eCBS is presumed to result in important pharmacological properties and therapeutic potential [31]. BCP acts as a CB₂R agonist with an insignificant affinity towards CB₁R [31, 32]. As such, its administration seems to be devoid of abuse potential [31-33] and of psychotropic side effects related to CB₁R binding [34]. In addition, preclinical results have shown that BCP induces anxiolytic, antidepressant [29], and analgesic effects [35]. Moreover, BCP has been investigated regarding its ability to counteract the effects of drugs of abuse [32, 36]. This paper will review preclinical studies investigating the potential use of BCP for the treatment of substance use disorders and discuss the involvement of CB₂R in such responses.

2. EFFECTS OF BCP IN ANIMAL MODELS OF ADDICTION

BCP has been investigated for its effects in animal models of addiction to ethanol, cocaine, nicotine, and methamphetamine. The main characteristics of each study are summarized in Table 1. Regarding BCP effects in animal models of ethanol addiction, Al Mansouri and co-workers [26] found that BCP dose-dependently reduced ethanol consumption and preference in C57BL/6 mice in the two-bottle choice test in addition to decreased the acquisition of ethanol-induced CPP. Remarkably, AM630, a potent and selective CB₂R antagonist, inhibited both BCP effects, suggesting the role of CB₂R activation in BCP action. Overall, the results suggested intraperitoneal BCP injections reduced ethanol addiction-related responses in a CB₂R dependent manner. However, in contrast with the results from two bottle choice and CPP tests, BCP exacerbated sensitivity to very low doses of ethanol in the loss of righting reflex test besides inducing an increase in latency for mice recovering from the impairment in motor coordination induced by ethanol. These results are corroborated by a second study investigating the effects of BCP on the ethanol-induced loss of righting reflex test in Swiss-Webster mice [37]. In this paper, a high dose of BCP (300 mg/kg) increased sensitivity to the ethanol sedative effect and the reflex recovery time. Therefore, the effects of BCP on ethanol addiction seem to be dependent on the behavioral test. While BCP induced positive outcomes in two bottle choice and CPP tests in a CB₂R dependent manner, it was not able to improve ethanol-induced loss of righting reflex. Only one study focused on BCP effects on cocaine addiction [33]. While the intraperitoneal injection of BCP inhibited cocaine self-administration and shifted the cocaine dose-response curve downwards. BCP intragastric administration displayed a less effective response against cocaine self-administration in Long-Evans rats. BCP was also able to impair the reinstatement of cocaine seeking induced by a prime injection of the drug in rats previously extinguished from cocaine self-administration besides reducing the acquisition of cocaine-induced CPP. To evaluate the involvement of CB₂R on BCP effects, knockout mice for CB₂R (CB₂-KO) were used. However, the tests with the gene-knocked mice challenged the conception that BCP effects on cocaine self-administration could be mediated by cannabinoid receptors since CB₂-KO mice did not show attenuation of BCP effects. In contrast, the peroxisome proliferator activated receptors α and γ (PPARα or PPARγ) were suggested to be involved in the BCP mechanisms to reduce cocaine self-administration. Therefore, the effect of BCP on cocaine addiction may not be modulated by only CB₂R. However, more studies are needed to confirm these results. He and co-workers 2020 used pharmacological, transgenic, and optogenetic approaches to evaluate the effects of BCP in animal models of nicotine addiction [38]. Systemic administration of BCP dose-dependently inhibited nicotine self-administration and motivation for nicotine seeking in Long-Evans rats and C57BL/6J mice. This study suggests that the involvement of CB₂R on BCP’s mechanism of action is dependent on BCP dose once the genetic inhibition of CB₂R in CB₂R knockout mice blocked the effects of 25, but not 50 mg/kg of BCP. BCP also attenuated reward in the intracranial self-stimulation test in mice and in an optogenetic VTA dopaminergic neuron stimulation model. This result suggests the involvement of dopamine in BCP reduction of brain reward. Thus, BCP action of reducing nicotine addiction may involve both CB₂R and non-CB₂R dependent mechanisms. Finally, He and co-workers 2021 focused on the effects of BCP on responses related to methamphetamine [39]. The authors used long-Evans rats and C57BL/6J mice in self-administration and electrical brain-stimulation reward tests. BCP attenuated methamphetamine self-administration and methamphetamine-primed reinstatement of drug seeking. The effects of BCP on methamphetamine infusions and in the progressive-ratio break-point level for methamphetamine self-administration were impaired by AM630, suggesting CB₂R involvement. However, the higher dose of BCP also inhibited methamphetamine self-administration in CB₂-KO mice. Lastly, BCP decreased methamphetamine-enhanced brain-stimulation reward. As for cocaine, these results suggest that the dose-dependent effects of BCP on methamphetamine-related behaviors are only partially mediated by CB₂R dependent mechanisms.

Table 1.

Summary of the main results found in the reviewed papers.

Dose BCP	Drug of Abuse	Behavioral Test	Result	CB2R Blockade		Specie	Refs.
				Pharmacological	Genetic
25, 50, and 100 mg/kg	Ethanol	Two-bottle choice	↓ (50, 100)	x (50)	No	Mice	Al Mansouri et al. 2014 [26]
		CPP	↓ (50)	x (50)	No
		Loss of righting reflex	↑ (50)	x (50)	No
30, 100, 178, and 300 mg/kg	Ethanol	Loss of righting reflex	↑ (300)	No	No	Mice	Oppong-Damoah et al. 2019 [37]
3, 10, 25, 50, and 100 mg/kg	Nicotine	Self-administration	↓ (10, 25, 50)	x (25)	x (25) = (50)	Mice and rat	He et al. 2020 [38]
		Seeking during extinction	↓ (25, 50)	No	No	Mice
		Food self-administration	↓ (50)	No	= (50)	Mice
		Electrical brain-stimulation	↓ (50, 100)	No	No	Rat
		Optical brain-stimulation	↓ (50, 100)	No	No	Mice
		Open field	-	No	No	Mice and rat
		Rotarod	↓ (100)	No	No	Mice
25, 50, and100mg/kg	Cocaine	Self-administration	↓ (50, 100)	= (100)	No	Rat	Galaj et al. 2020 [33]
			↓ (100)	No	= (100)	Mice
		Reinstatement	↓ (25, 50)	No	No	Rat
		BCP self-administration	-	No	No	Rat
		CPP	↓ (50, 100)	No	No	Rat
		Food self-administration	-	No	No	Rat
		Electrical brain-stimulation	-	No	No	Rat
		Optical brain-stimulation	↓ (50, 100)	No	No	Mice
		Cocaine-induced hyperlocomotion	-	No	No	Rat
10, 25, 50, and 100 mg/kg	Methamphetamine	Self-administration	↓ (25, 50, 100)	x (50, 100)	No	Rat	He et al. 2021 [39]
			↓ (25, 50, 100)	No	x (25, 50)	Mice
		Reinstatement	↓ (25, 50)	No	No	Rat
		Cue-induced methamphetamine seeking	↓ (50, 100)	No	No	Rat
		Electrical brain-stimulation	↓ (50, 100)	No	No	Rat
		Methamphetamine-enhanced dopamine in NaC	↓ (50)	No	No	Rat
		Open-field	-	No	No	Rat

Note: ↓: BCP reduced the behavioral measure. ↑: BCP increased the behavioral measure. -: BCP did not have an effect. x: CB₂R blockade impared BCP effect. =: same effect in WT and CB₂KO or AM630. no: not measured. Number in parenthesis: dose of BCP with significant effect.

3. DISCUSSION

BCP has attracted attention as a potential drug candidate for treating substance use disorders, even though its mechanism of action has remained uncertain. Compared to synthetic CB₂R agonists (such as JWH133), BCP’s presence in essential oils of plants and herbs makes it an easily accessible substance [30] with demonstrated safety in humans [40]. BCP effects have been investigated in experimental animals related to addiction to nicotine [38], ethanol [26, 37], cocaine [33], and methamphetamine [39]. With doses ranging from 25 to 100 mg/kg, BCP was able to prevent and/or reverse drug effects as evaluated in different animal models, such as self-administration, conditioned place preference, loss of righting reflex, and intracranial self-stimulation. To the best of our knowledge, there is no clinical study investigating BCP effects on drug abuse. However, Rose and co-workers (1994) evaluated the role of black pepper (which contains high levels of BCP) on nicotine smokers. They found that the group with the device containing black pepper essential oil craved significantly less nicotine cigarettes than the control group [41]. Pharmacological and genetic blockade of CB₂R suggests that BCP effects may be dependent on CB₂R [26, 38]. Accordingly, the modulation of CB₂R was already shown to affect rewarding and reinforcing effects of drugs of abuse in different animal models [25, 42-44] besides regulating common neuropsychiatric comorbidities of addiction, such as anxiety [45, 54] and impulsivity [46]. Thus, the results from the literature support the therapeutic use of CB₂R agonists for drug use disorders. In addition, studies with optogenetic stimulation of dopaminergic neurons in the VTA suggested that dopamine may be involved in BCP effects [38]. Indeed, CB₂R is expressed in dopaminergic cell bodies in this brain region [20]. Thus, it is possible that BCP effects in preventing reward and reinforcing effects of drugs of abuse are modulated by CB₂R expressed in dopaminergic neurons located in the mesolimbic pathway. CB₂R mechanisms have been proposed to underlie the effects of BCP also in other mental disorders, such as anxiety and depression [29, 47]. However, the mechanism by which BCP modulates drug reward is not a consensus yet. Despite BCP displaying high selectivity for CB₂R compared to CB₁R, it also targets other brain receptors. As reported in Galaj and co-workers 2021 and He and co-workers 2021, BCP was effective in CB₂-KO mice, especially in high doses, challenging the conception of the CB₂R agonism as the only mechanism of action by which BCP acts in the addiction. The activation of PPARα or PPARγ might also contribute to its overall effects. Accordingly, these receptors counteract the effects of several drugs of abuse, such as nicotine [48], ethanol [49], and heroin [50], possibly by modulating dopamine release on the mesolimbic pathway [51]. It is worth mentioning that the direct antagonism of dopaminergic receptors did not show promising results for addiction treatment [52]. Indirect strategies to regulate dopaminergic activities have been investigated, especially for psychostimulant addiction treatment [53]. The ability of BCP to target different receptors able to modulate dopaminergic tonus on VTA, such as CB₂R and PPARα, provides a mechanistic rationale for the development of addiction pharmacotherapy based on this compound [53].

CONCLUSION

In conclusion, there is potential for BCP to be effective in the treatment of substance use disorders. However, the data is still limited to a few preclinical studies. Further studies in both animals and humans are needed to assess the possible outcomes of addiction, evaluating long-term effects, optimal dosing, alternative routes of administration, and different drugs. Finally, the role of CB₂R should be further investigated. Although BCP acts as a full agonist at this receptor, non-CB₂R mechanisms also seem to play a role in its effects, particularly at high doses.

LIST OF ABBREVIATIONS

CPP

Conditioned Place Preference

eCBS

Endocannabinoid System

FDA

Food and Drug Administration

VTA

Ventral Tegmental Area

CONSENT FOR PUBLICATION

Not applicable.

FUNDING

This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq (grant number 406122/2016-4); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES (grant number 88882.315961/2019-01); and Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP (grant number 2017/24304-0).

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.