SEX DIFFERENCES AND THE ENDOCANNABINOID SYSTEM IN PAIN

Abstract

Cannabis use has been increasing in recent years, particularly among women, and one of the most common uses of cannabis for medical purposes is pain relief. Pain conditions and response to analgesics have been demonstrated to be influenced by sex, and evidence is emerging that this is also true with cannabinoid-mediated analgesia. In this review we evaluate the preclinical evidence supporting sex differences in cannabinoid pharmacology, as well as emerging evidence from human studies, both clinical and observational. Numerous animal studies have reported sex differences in the antinociceptive response to natural and synthetic cannabinoids that may correlate to sex differences in expression, and function, of endocannabinoid system components. Female rodents have generally been found to be more sensitive to the effects of Δ⁹-THC. This finding is likely a function of both pharmacokinetic and pharmacodynamics factors including differences in metabolism, differences in cannabinoid receptor expression, and influence of ovarian hormones including estradiol and progesterone. Preclinical evidence supporting direct interactions between sex hormones and the endocannabinoid system may translate to sex differences in response to cannabis and cannabinoid use in men and women. Further research into the role of sex in endocannabinoid system function is critical as we gain a deeper understanding of the impact of the endocannabinoid system in various disease states, including chronic pain.

1. Introduction

Self-reported cannabis use in adults is rising in the United States and the rate of increase is faster among women compared to men (SAMHSA, 2019). The ever increasing number of states with legalized medical (35) and adult-recreational (15) cannabis use have likely contributed to these increases in reported use. In recent years cannabis and associated products have increased in availability and decreased in cost, and a softening of prior social-stigma around the usage of cannabis has taken place. Among states with medical cannabis programs, chronic pain is the most common qualifying medical condition reported among patients by a wide margin (Boehnke et al., 2019). Understanding male/female differences in endocannabinoid system physiology and response to cannabinoid analgesia is a timely topic as numerous studies have reported sex differences in pain perception and prevalence (Sorge and Totsch, 2017) as well as sex differences in response to other analgesics (Soldin et al., 2011). Thus, the focus of this review is about the influence of sex as a biological variable in pain processes, and addressing emerging evidence supporting sex and sex hormones as contributing factors in cannabinoid pharmacology.

In this review we begin by providing an introduction to sex as a biological variable in pain and analgesic response. Next, we introduce the endocannabinoid system and provide pre-clinical and clinical evidence for sex differences in cannabinoid-mediated analgesia in acute, inflammatory, and chronic pain models. Finally, we evaluate pre-clinical evidence supporting sex differences in endocannabinoid system expression, and interactions between the endocannabinoid and hormone systems. Additionally, within this review we will identify emerging areas where more research is needed to elucidate how sex and sex hormones influence the endocannabinoid system as well as pain processing.

2. Sex Differences in Pain and Impact of Hormones

Pain as a concept is complex and ever-evolving; in 2020 an updated definition was proposed by the International Association for the Study of Pain (IASP) as “an unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage” (Raja et al., 2020). Pain encompasses sensory, affective, and cognitive aspects, which cannot always sufficiently be evaluated in preclinical animal models. Animal models typically focus on nociception, the sensory transmission and processing of pain information, by measuring the nocifensive behavioral responses resulting from nociceptive stimuli, or by measuring activity in neurons in the pain pathway such as the dorsal root ganglia, or pain processing areas of the brain. Compounds which inhibit these nocifensive responses, or decrease activity in the neurons in the nociceptive pathways, are referred to as antinociceptive. Analgesia typically is used when referring to pain relief in humans, i.e. a compound that is analgesic may relieve the sensory (nociceptive) aspects of pain, but may also impact affective or cognitive aspects of pain. In human studies cannabis use has been reported to alter the affective aspect of pain, rendering experimental pain less unpleasant (De Vita et al., 2018). This type of subjective analgesic response can’t be measured in animals, thus the term antinociceptive more accurately reflects the responses measured in animal pain models.

2.1. Sex as a Biological Variable in Pain

The contribution of sex in relation to pain and analgesia is a rapidly expanding topic of pre-clinical and clinical research. While pre-clinical studies largely focus on the sensory (nociceptive) aspect of pain, it is important to acknowledge that the concept of pain also encompasses cognitive and emotional dimensions, which can influence the severity, duration, and recovery from pain. Clinically, it has been reported that women are more sensitive, and less tolerant to experimental pain than men, and women make up a larger percentage of patients with chronic pain than men (Mogil, 2012). The exclusion of females from both pre-clinical and clinical research prior to the latter part of the last century was predicated on the idea that the fluctuation of hormones in females would create an additional burden of variability in data (Mogil and Chanda, 2005). Recent developments have brought about a heightened focus into our disparate understanding of the role of sex as a biological variable. In particular, a scientific spotlight has become focused on the influence of sex as it relates to pain differences and outcomes. As sex hormones are hypothesized to contribute to sex differences in experimental outcomes, investigations into the effects of hormone manipulation in various pain models have been a key focus of preclinical studies into pain and analgesia, including studies with cannabinoids (Figure 1).

Figure 1: Mechanisms underlying sex differences in cannabinoid antinociception in rodents.

Sex differences in cannabinoid antinociception in rodents have been found to vary based on a variety of factors including pain model, cannabinoid used, and effect of sex hormones, among others. The most widely studied cannabinoid, THC, has been found to be generally more potent in female rats, possibly as a function of differences in metabolism and effects of sex hormones. Abbreviations: Estradiol (E2); Progesterone (P4); Ovariectomy (OVX); WIN 55, 212-2 (WIN).

2.2. The Effects of Sex Hormones in Pain

Female rodents undergo hormone fluctuations across the 4-6 days of their 4 stage reproductive estrous cycle, with a peak in estradiol in the proestrus and estrus phases (corresponding to the human follicular/proliferative phase) followed by a sharp drop, and a peak in progesterone in metestrus and diestrus phases (corresponding to the human secretory/luteal phase) (Wood et al., 2007; Ajayi and Akhigbe, 2020). Conversely, testosterone levels in males have been reported to be much more stable; levels peaking following interaction with a sexually-receptive (estrous phase) female (Shulman and Spritzer, 2014).

These differences in hormone fluctuations may account for some of the differences between male and female measures of nociception. Estrous phase has been reported to affect baseline nociception to acute thermal (Frye et al., 1993; Martinez-Gomez et al., 1994), mechanical (Kayser et al., 1996), visceral (Sapsed-Byrne et al., 1996; Bradshaw et al., 1999), and electrical pain (Vincler et al., 2001). However, a seminal 2005 meta-analysis of pain studies across various rodent strains, experimenters, and time argued against sex as the most impactful factor on variability between studies. Rather, while males displayed slightly longer tail flick response latencies, and females spent slightly less time displaying nocifensive behaviors in the acute pain phase of the formalin test, the factors of genotype and experimenter contributed much more significantly to variation in experiments than did sex (Mogil and Chanda, 2005). Nevertheless, much of the research on sex differences in pain sensitivity has focused on investigating sex hormones as a variable in pain and analgesic response.

A common approach to address this question is the use of surgical gonadectomy in conjunction with exogenous hormone replacement (testosterone, estrogen, and progesterone, primarily), to study the contribution of hormones to baseline nociceptive thresholds and response to analgesics. Much of the work using this approach has been in the study of opioid-mediated antinociception. Opioid-mediated antinociception has long been established to have a sex-dependent component, with female rats typically requiring higher doses of morphine compared to their male counterparts (Kasson and George, 1984). Gonadectomy has been reported to differentially affect morphine responsiveness between sexes; gonadectomized female rats require smaller doses of morphine compared to their sham counterparts, while males may either require larger doses (Islam et al., 1993), or doses may be unaffected by treatment (Kasson and George, 1984).

2.3. Sex Differences in Neuroimmune-mediated Pain Processes

Sex and gonadal hormones have also been found to impact neuroimmune-mediated processes involved in pain and response to analgesics. Neuroimmune-mediated neuroinflammation has been demonstrated to both directly induce pain and to contribute to sensitization in chronic pain, with noted sex differences in the functioning of this system (Ji et al., 2018; Rosen et al., 2017; Spychala et al., 2017). The effects of gonadal sex hormones on neuroimmune-mediated pain processes have been found to vary based on concentration and site of action. Estrogen has been found to be both pro-nociceptive and antinociceptive due to its regulation of macrophages, natural killer cells, dendritic cells, and T lymphocytes (Amandusson and Blomqvist, 2013; Kovats, 2015). It has been found that females have higher levels of inflammation due to pro-inflammatory effects of estrogen; this higher level of inflammation has been suggested to contribute to the higher rates of reported chronic pain among females (Fillingim et al., 2009; Mogil, 2012).

Sex differences have been shown in the immune cells responsible for the development of neuropathic pain, with sensitization mediated through activation of TLR4 (toll-like receptor 4)-positive spinal microglia in male mice (Sorge et al., 2011) and via infiltrating T lymphocytes in female mice (Sorge et al., 2015). Differences in TLR4 signaling have also been hypothesized to underlie sex differences in opioid antinociception in preclinical studies; females generally requiring higher morphine doses compared to males. At baseline, female rats were found to have more activated TLR4-positive microglia in the periaqueductal gray (PAG) versus males, and antagonism of TLR4 by (+)-naloxone potentiated morphine antinociception in females but not males (Doyle et al., 2017). The endocannabinoid system, particularly via CB₂ (cannabinoid receptor 2) receptor signaling, is highly involved in modulation of neuroimmune processes, including inflammatory responses (Rom and Persidsky, 2013; Zoppi et al., 2014). Thus, sex differences in cannabinoid antinociception may also involve differences in neuroimmune mechanisms as seen with morphine (Doyle et al., 2017; Rosen et al., 2017).

3. Sex Differences in Cannabinoid Antinociception: Pre-clinical Studies

3.1. Understanding Cannabinoid Analgesia: A Primer on the Endocannabinoid System

Our understanding of the breadth and depth of endocannabinoid system is in continual expansion, however, at its core it may be defined by three main components; the ligands (endocannabinoids), the receptors for those ligands (cannabinoid receptors) and the enzymes responsible for the synthesis and degradation of those ligands (Cristino et al., 2020). The two most characterized endocannabinoids are the lipid signaling molecules 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamide (AEA), also referred to as anandamide (Devane et al., 1992; Mechoulam et al., 1995; Sugiura et al., 1995). These lipid signaling molecules are produced on demand through the enzymes diacylglycerol lipase alpha (DAGLα) for 2-AG, and via N-acylphosphatidylethanolamine (NAPE)-specific phospholipase D-like hydrolase (NAPE-PLD) for anandamide (Bisogno et al., 2003; Okamoto et al., 2004). Endocannabinoid degradation occurs through the enzymes monoacylglycerol lipase (MAGL) for 2-AG, and fatty acid amide hydrolase (FAAH) for anandamide (Cravatt et al., 1996; Dinh et al., 2002). Endocannabinoids act in a retrograde signaling matter; synthesis occurs post-synaptically and the endocannabinoids act at cannabinoid receptors located pre-synaptically (Wilson et al., 2001). The two primary receptors for endocannabinoid actions are the G-protein coupled CB₁ and CB₂ cannabinoid receptors, which primarily couple to inhibitory Gα_i, G proteins (Felder et al., 1995; Glass and Northup, 1999). The CB₁ receptor is highly expressed in the neurons of the central nervous system, mediating numerous effects, including the psychotropic effects produced by cannabis and Δ⁹-THC (Devane et al., 1988). Conversely, CB₂ receptor expression in CNS neurons is lower compared to CB₁; and is expressed to a much greater extent in glia of the CNS and PNS, and throughout the immune system (Matsuda et al., 1990; Munro et al., 1993). Agonist binding to cannabinoid receptors produces a net inhibitory effect on cell activity through various mechanisms including inhibition of adenylyl cyclase-mediated cAMP formation, and modulation of potassium and calcium channels (Felder et al., 1995).

The endocannabinoid system is expressed throughout the ascending and descending pain pathways (Guindon and Hohmann, 2009) in addition to the hypothalamus-pituitary-gonadal (HPG) sex hormone axis (du Plessis et al., 2015; Walker et al., 2019). Moreover, numerous studies have reported sex differences in endocannabinoid system expression and activity in the brain; some as a direct function of hormone status (Figure 2). Despite this, studies directly comparing male and female responses to cannabinoid analgesia are relatively limited, and have largely been conducted by a few select research groups (see Table 1). Antinociception is one component of the tetrad measurement of centrally- (CB₁ receptor) mediated cannabinoid effects along with hypothermia, hypolocomotion, and catalepsy (Little et al., 1988), although cannabinoids such as CBD (cannabidiol), peripherally-restricted CB₁ (cannabinoid receptor 1) agonists, and CB₂- (cannabinoid receptor 2) selective agonists may produce antinociception without affecting body temperature or motor activity.

Figure 2: Sex differences in brain endocannabinoid system expression and response.

Sex differences in the expression and function of the endocannabinoid system in the brains of rodents have been found in numerous studies. Sex hormones, particularly in females, have been found to affect cannabinoid receptor expression and the affinity and efficacy of cannabinoid ligands. There have been conflicting results concerning cannabinoid receptor expression in brain regions including amygdala, cortex, and hippocampus, supporting the need for further studies. Abbreviations: Anterior (Ant.); Gonadectomy (GDX); Ovariectomy (OVX), Anandamide (AEA); 2-Arachidonylglycerol (2-AG).

Table 1:

Comparison of cannabinoid-mediated antinociception in male and female rodents This table summarizes the studies evaluating sex differences in the antinociceptive effects of natural and synthetic cannabinoids in acute and chronic pain models in rats and mice. Abbreviations: Central Freund’s Adjuvant (CFA); Intraperitoneal (I.P.); Intraplantar (FPL.); Intramuscular (I.M.); Mechanical (Mech.); Intracerebroventricular (I.C.V.); Subcutaneous (S.C.).

Reference	Pain Model	Acute or Chronic	Species	Compound (S)	Dose & ROA	Acute and/or Repeated	Efficacy: Male v Female	Mediated By	Serum Analysis	Estrous Effect	Hormone
Blednov et al., 2003	Hotplate (Paw)	Acute	Mouse	WIN 55, 212-2	6 mg/kg; I.P.	Acute	M = F	GIRK 2	NT	NT	NT
Britch et al., 2017	Pressure (Paw), Heat (Tail Immersion)	Acute	Rat	CBD, THC	CBD (30 mg/kg) + THC (1.8 mg/kg); I.P.	Acute	M = F	NT	CBD, THC, 11-OH-THC, THC-COOH, CBN	No Effect	NT
Britch et al., 2020	Heat (Paw), Mech. Allodynia (Paw), Weight Bearing (paw)	Chronic - CFA (paw)	Rat	CBD, THC	CBD (1.25-10 mg/kg), THC ( 1-4 mg/kg); I.P.	Acute & Repeated	F>M; acute THC heat hyperalgesia	NT	TNF-α, IL-1β, IL=6, IL-10, INFγ	NT	NT
Craft and Leitl, 2008	Pressure (Paw), Heat (Tail Immersion)	Acute	Rat	THC	5, 10 mg/kg; I.P.	Acute	F>M, E2 potentiate in Female	NT	NT	Yes; THC strongest in estrus	M & F Gonadectomy, Testosterone & Estradiol Replacement
Craft et al., 2012	Heat (Tail Immersion), Pressure (Paw)	Acute	Rat	CP 55,940, THC	CP 55,940 (0.05 - 1.6 mg/kg), THC (1.25 - 20 mg/kg); I.P.	Acute	CP 55,940 F>M; THC: F>M	CP55: CB1 - M & F; THC: CB1- Male, CB1 & CB2 - Female	Rimonabant	Yes; THC strongest in estrus	NT
Craft et al., 2013	Mech. Allodynia (Paw), Heat (Paw)	Chronic - CFA (paw)	Rat	THC	0.32 - 3.2 mg/kg I.P.; 0-500 μg I.PL.	Acute & Repeated	THC: F>M	IP: CB1 - M & F; I.PL.: CB1 & CB2 Female, CB1 only Male	NT	No Effect	NT
Craft et al., 2017	Pressure (Paw), Heat (Tail Immersion)	Acute	Rat	THC	THC: 3 mg/kg (Female), 5 mg/kg (Male), Proadifen: 25mg/kg, I.P.	Acute	F>M, E2 potentiate THC in male, T inhibit THC in female	Testosterone, Estradiol, and CYP450 (CYP2C family)	THC, 11-OH-THC, THC-COOH	NT	M & F Gonadectomy, Testosterone & Estradiol Replacement
Craft et al., 2018	Acute: Heat (Tail Immersion), Pressure (Paw), Inflammatory Mech. Allodynia (Paw), Heat (Paw), Weight Bearing (paw)	Acute Chronic - CFA (paw)	Rat	JWH-015	5-40 mg/kg, I.P.	Acute	M=F, except mech. allodynia F>M (10 mg/kg)	CB1 & CB2	NT	NT	NT
Greene et al., 2018	Pressure (Paw), Heat (Tail Immersion)	Acute	Rat	CBD, THC	CBD: 10 mg/kg, THC: 3.6 mg/kg (Female), 9.3 mg/kg (Male); I.P.	Acute & Repeated	THC: F>M (acute), CBD: M>F (repeated - tail flick)	NT	CBD, CBN, THC, 11-OH-THC, THC-COOH	NT	NT
Javadi-Paydar et al., 2018	Heat (Tail Immersion)	Acute	Rat	CBD, THC	CBD: 12.5, 50 mg, THC: 1.5-25mg, Vaporized	Repeated (>lweek washout between)	F>M	NT	THC	No Effect; THC equivalent between Diestrus & Estrus	NT
Kalbasi Anaraki et al., 2008	Heat (Tail Flick)	Acute	Mouse	WIN 55, 212-2	2, 4 mg/kg, I.P.	Acute	F only; OVX enhance analgesia, E2 inhibit, P4 no effect	CB1	NT	NT	F Only OVX, E2 & P4 Replacement
LaFleur et al., 2018	Inflammatory (Formalin-Paw)	Acute	Mouse	CP 55,940,THC	CP 55,940 (0.06 - 0.2 mg/kg), THC (1 - 6 mg/kg); I.P.	Acute & Repeated	CP 55,940: M>F; THC: M>F antinociception & rate of tolerance	NT	NT	NT	NT
Linher-Melville et al., 2020	Mech. Allodynia (Paw)	Chronic - Sciatic Nerve Cuff	Rat	CBD, THC	CBD: 0.41 mg/kg THC: 0.08 mg/kg, Combination: 0.2 mg/kg + 0.2 mg/kg; Oral	Repeated	M>F	NT	NT	NT	NT
Marusich et al., 2015	Heat (Tail Flick)	Acute	Rat	THC	30 mg/kg, I.P.	Acute & Repeated	F>M (Acute); M=F (Chronic)	CB1	NT	Inconclusive	M & F Gonadectomy, Testosterone, Estradiol, & Progesterone Replacement
Mulpuri et al., 2018	Mech. Allodynia (Paw), Cold Allodynia (Paw)	Chronic - Cisplatin (systemic)	Rat	PrNMI	0-2 mg/kg I.P.; 0.25 mg/kg I.PL.; 3 mg/kg Oral	Acute & Repeated	M=F	CB1	NT	NT	NT
Niu et al., 2012	Mech. Allodynia (Masseter)	Chronic - CFA (masseter)	Rat	ACPA	10-300 μg I.M.	Acute & Repeated	M>F	CB1	NT	NT	M & F Gonadectomy, Testosterone, Estradiol Replacement
Parks et al., 2020	Heat (Tail Flick)	Acute	Mouse	THC	10 mg/kg, I.P.	Acute & Repeated	M=F	NT	NT	NT	NT
Romero et al., 2002	Heat (Tail Immersion)	Acute	Rat	CP 55,940	0.1-0.6 mg/kg, S.C.	Acute	M=F	CB1	NT	NT	NT
Smoker et al., 2019	Hotplate (Paw)	Acute	Mouse	THC	1-10 mg/kg; Oral	Acute & Repeated	M=F	CB1	NT	NT	NT
Tseng and Craft, 2001	Pressure (Paw), Heat (Tail Immersion)	Acute	Rat	CP 55,940, THC, 11-OH-THC	CP 55, 940 (0.1-0.56 mg/kg), THC (1-10 mg/kg), 11-OH-THC (0.3-10 mg/kg); I.P.	Acute	F>M	NT	NT	No Effect	NT
Tseng et al., 2004	Heat (Tail Immersion)	Acute	Rat	THC	10 mg/kg, I.P.	Acute	F>M	CYP 450 (Female)	THC, 11-OH-THC	NT	NT
Wakley et al., 2011	Pressure (Paw), Heat (Tail Immersion)	Acute	Rat	THC	100 μg, I.C.V.	Acute	F>M	NT	NT	Yes; F>M when in Late Proestrus	NT
Wakley et al., 2014	Pressure (Paw), Heat (Tail Immersion)	Acute	Rat	THC	5.4 mg/kg (Female), 7.6 mg/kg (Male), I.P.	Acute & Repeated	F>M	NT	NT	No Effect	NT
Wakley et al., 2015	Pressure (Paw), Heat (Tail Immersion)	Acute	Rat	THC	5.7 mg/kg (Female), 9.9 mg/kg (Male), I.P.	Acute & Repeated	F>M antinociception & rate of tolerance; P4 inhibit THC	NT	NT	Inconclusive	M & F Gonadectomy, Testosterone, Estradiol, & Progesterone Replacement
Wiley et al., 2007	Heat (Tail Flick)	Acute	Rat	THC	1-300 mg/kg, I.P.	Acute & Repeated	M=F	CB1	NT	NT	NT
Yuill et al., 2017	Inflammatory (Formalin-Paw)	Acute	Mouse	JWH-133	0.01 - 10 mg/kg (acute), 1 mg/kg repeated; I.P.	Acute & Repeated	M=F	CB2	NT	NT	NT
Zhu et al., 2020	Mech. Allodynia (Paw), Heat (Paw)	Chronic - Sciatic Nerve Cuff	Rat	CBDA-ME	0.01-4 μg/kg, I.P.	Repeated	M>F	NT	NT	NT	NT

Cannabinoids may produce antinociceptive effects through peripheral, spinal, and supraspinal mechanisms. Centrally acting CB₁ receptor agonists (Δ⁹-THC, CP55,940, WIN 55,212-2) may produce antinociception through activation of descending (inhibitory) pain circuits in regions of the brain including the amygdala (Manning et al., 2003), periaqueductal gray (Lichtman et al., 1996), and rostral ventromedial medulla (Martin et al., 1998). Cannabinoids may also block ascending pain signals which are transmitted via activation of nociceptive peripheral nerves which synapse at the dorsal horn of the spinal cord, prior to decussating and ascending to the brain. Local administration of cannabinoids, such as by intraplantar injection, has been demonstrated to be antinociceptive via both CB₁ and CB₂ (Calignano et al., 1998; Guindon et al., 2007). Cannabinoids may also produce antinociception through anti-inflammatory effects via CB₁, CB₂, as well as other receptors. Selective CB₂ receptor agonists show efficacy in inflammatory and neuropathic pain models through inhibition of pro-inflammatory signaling (Malan et al., 2003). Moreover, cannabidiol (CBD), which does not behave as an agonist at CB₁ or CB₂ receptors, produces beneficial effects via numerous targets including vanilloid (TRPV1) and serotonin (5-HT_1A) receptors (De Gregorio et al., 2019). Additionally, orphan GPCRs have also been implicated as targets for cannabinoid-mediated antinociception by endogenous ligands including N-arachidonoylglycine (GPR18) and synthetic cannabinoids such as CP 55,940 (GPR55) (Guerrero-Alba et al., 2019).

In summary, various mechanisms mediate endocannabinoid system antinociception, with targets in the central and peripheral nervous systems, as well as the immune system. Moreover, these mechanisms may also be impacted by sex and sex hormones. The following subsections summarize the sex differences reported in pre-clinical studies of cannabinoid-mediated antinociception in male and female rats and mice. Detailed summaries of each pre-clinical study referenced below are available in Table 1.

3.2. Thermal Antinociception (Acute)

The most commonly employed test for antinociception is through measurement of the latency (time in seconds) until withdrawal from a heat source applied to either the tail or hind paw (Table 1). Cannabinoid-mediated antinociception in this test has been determined to involve both spinal and supraspinal mechanisms (Lichtman and Martin, 1991). In opposition to the sex differences seen with the quintessential opioid, morphine, the quintessential phytocannabinoid, Δ⁹-THC, has been documented to be more potent in female rats versus male rats in this test (Craft et al., 2012, 2013, 2017; Tseng et al., 2001, 2004; Wakley et al., 2011, 2014) (Table 1). Notably, while far fewer studies exist with mice, sex differences in antinociceptive response to Δ⁹-THC were not demonstrated in mice in the tail flick (Parks et al., 2020) or hot plate tests (Smoker et al., 2019), raising the possibility that sex differences seen in rats may not be replicated in mice (Wiley et al., 2020) (Table 1). Cannabidiol (CBD) has been found to be equally efficacious in male and female rats at doses of 10 and 30 mg/kg (Britch et al., 2017; Greene et al., 2018). However, repeated administration of CBD (10 mg/kg) significantly increased baseline tail withdrawal responses latencies in male but not female rats; an effect not seen in the Δ⁹-THC or CBD + Δ⁹-THC combination conditions (Greene et al., 2018) (Table 1).

CP 55,940 and WIN 55,212-2, two of the most commonly used synthetic, dual (CB₁/CB₂) cannabinoid receptor agonists, have shown varied results in terms of sex differences (Table 1). In rats, CP 55,940 has been demonstrated to be more potent in females versus males in some studies (Tseng and Craft, 2001; Craft et al., 2012), while another study reported equal potency between sexes in response to CP 55,940 (Romero et al., 2002) (Table 1). Differences in rat strain (Sprague-Dawley vs Wistar) and route of administration (intraperitoneal vs subcutaneous) could underlie these differences in response to CP 55,940. In mice, sex differences were not seen in response to WIN 55,212-2 in the hotplate test (Blednov et al., 2003), although in a study using ovariectomized females the antinociceptive effect of WIN in the tail flick assay was enhanced by ovariectomy and antagonized by estradiol supplementation, suggesting hormone status could modulate the response to this compound (Kalbasi-Anaraki et al., 2008)(Table 1). Notably, when comparing the antinociceptive effects of Δ⁹-THC and CP 55,950 in rats, the antinociceptive effect of CP 55,950 was mediated by CB₁ only in both sexes, while the antinociceptive effects of Δ⁹-THC were mediated by CB₁ alone in males, but both CB₁ and CB₂ in females (Craft et al., 2012). While the antinociceptive effects Δ⁹-THC might be mediated by CB₂ to a greater extent in females versus males, the CB₂-preferring agonist JWH-015 was equally efficacious in male and female rats, underlying the probability that sex differences in cannabinoid-mediated antinociception are likely compound dependent (Craft et al., 2018)(Table1).

3.3. Inflammatory Pain (Acute and Chronic)

Inflammatory pain conditions commonly affect the skin (ex. dermatitis), joints (arthritis), and gut (ex. inflammatory bowel disease) and are an important area of preclinical pain research (Muley et al., 2016). Sex differences in cannabinoid-mediated antinociception have been demonstrated in models of acute inflammatory pain (ex. Formalin test), as well as chronic inflammatory pain models (ex. Complete Freund’s Adjuvant [CFA]) (Table 1). In the formalin pain model, a small amount (~10 uL) of dilute (2.5 - 5%) formalin solution is injected intraplantar into the hind paw of a rodent. The injection of formalin produces a biphasic pain response consisting of an acute phase (minute 0 to 15) and a longer inflammatory phase (minute 15 to 60) which are observed and recorded over the course of an hour (Dubuisson and Dennis, 1977). While cannabinoids have long been established to be antinociceptive in the formalin model (Moss and Johnson, 1980), very few studies have evaluated sex differences with cannabinoids in this model, unfortunately. In contrast to the results seen in rats in the acute thermal pain models, in mice the mixed CB₁/CB₂ agonists Δ⁹-THC and CP55,940 were more potent in inhibiting formalin-induced pain in males versus females (LaFleur et al., 2018) (Table 1). In agreement with the results seen with the CB₂-preferring agonist JWH-015 in rats in acute thermal pain models, the CB₂-selective agonist JWH-133 produced equal antinociception in male and female mice in the formalin model, suggesting that in acute pain models CB₂-mediated antinociception may not show sex differences (Yuill et al., 2017) (Table 1).

The CFA inflammatory pain model is used to model chronic inflammatory pain conditions such as arthritis. In this model, a small amount of CFA is injected into a site of interest (paw, tail, etc.), producing an inflammatory reaction which leads to the development of hypersensitivity to noxious heat (heat hyperalgesia), and mechanical stimulation (mechanical allodynia), in addition to edema, decreases in weight bearing at the injected site, and ongoing spontaneous pain behaviors (Muley et al., 2016). In rats, CBD was found to be ineffective in relieving CFA-induced heat hyperalgesia but partially reversed mechanical allodynia in both males and females to an equal extent (Britch et al., 2020) (Table 1). Δ⁹-THC was found to be equally efficacious in relieving mechanical allodynia in male and female rats when delivered intraperitoneal (i.p.) and more efficacious in relieving heat hyperalgesia in females versus males (Craft et al., 2013; Britch et al., 2020). Additionally, Δ⁹-THC when delivered via intraplantar (i.pl.) injection was found to be more efficacious in female rats at relieving both heat hyperalgesia and mechanical allodynia (Craft et al., 2013). When CFA was injected into the masseter muscle, the CBM₁-selective agonist ACPA was approximately 30 times more potent in male versus female rats in relieving mechanical allodynia (Niu et al., 2012) (Table 1). Conversely, the CB₂-preferring agonist JWH-015 was equally efficacious in male and female rats in relieving thermal hyperalgesia, but more potent in females in relieving mechanical allodynia (Craft et al., 2018). Finally, sex differences were also shown in the development of tolerance to the analgesic effects of Δ⁹-THC in the CFA model. It was found that female rats developed tolerance in the heat hyperalgesia assay with repeated Δ⁹-THC administration after 4 days of repeated administration, while Δ⁹-THC remained equally efficacious in males from day 1 to day 4 (Britch et al., 2020) (Table 1).

3.4. Neuropathic Pain

Treatment options for neuropathic pain are relatively limited, and often sub-effective, making use of cannabinoids a promising treatment avenue (Blanton et al., 2019). Studies investigating sex differences in cannabinoid antinociception in neuropathic pain models are limited but have been increasing in recent years (Table 1). Using the sciatic nerve cuff model, where the sciatic nerve is surgically restricted to create a neuropathic pain state, sex differences have been demonstrated in response to phytocannabinoid-mediated antinociception. In rats, repeated oral administration of low doses of Δ⁹-THC, CBD, and Δ⁹-THC+CBD, scaled down to represent typical human doses, produced greater relief of mechanical allodynia, and hypersensitivity to mechanical force applied to the paws, in males versus females (Linher-Melville et al., 2020) (Table 1). This sex difference in efficacy in the sciatic nerve cuff model was also produced using a synthetic CBDA derivative, cannabidiolic acid-methyl ester (CBDA-ME), in both mechanical allodynia and heat hyperalgesia; male rats again showing greater relief of symptoms (Zhu et al., 2020)(Table 1). Conversely, in a model of chemotherapy-induced neuropathic pain using cisplatin, the peripherally restricted CB₁ selective agonist PrNMI, was found to be equally efficacious in both male and female rats in relieving mechanical and cold (acetone) allodynia (Mulpuri et al., 2018)(Table 1).

4. Sex Differences in Cannabinoid Analgesia: Human Laboratory Studies and Survey Data

While pre-clinical rodent studies have demonstrated clear antinociceptive effects of cannabinoids, results from randomized controlled trials in humans have shown varied results in terms of efficacy (Fisher et al., 2019). Moreover, evidence supporting sex differences in the analgesic efficacy of cannabinoids in humans is still very limited. Men have been found to have a greater analgesic response to smoked cannabis in the cold-pressor test (Cooper and Haney, 2016). Conversely, orally administered nabilone, a synthetic cannabinoid resembling Δ⁹-THC in structure, was found to produce anti-hyperalgesia in an experimental heat pain model for women only (Redmond et al., 2008). Chronic pain has been reported as the most common qualifying condition for medical cannabis use (Boehnke et al., 2019), and among medical cannabis users, men and women report equally on the perceived efficacy of cannabis for pain (Cuttler et al., 2016). With regard to chronic pain, it has been noted that women are more likely to use non-smoked compounds with higher levels of cannabidiol (CBD), while men were more likely to use smoked compounds with higher levels of Δ⁹-THC (Boehnke et al., 2019). The finding that women are more likely to choose cannabis products with a more balanced Δ⁹-THC:CBD ratio may reflect sex differences in sensitivity as has been seen with animal studies. Indeed, a recent study using smoked cannabis found that women reported equivalent effects to men on measures of changes in cognition and mood, despite consuming less cannabis and displaying lower blood levels of Δ⁹-THC and metabolites (Matheson et al., 2020). Similarly, another study found women were more sensitive to the subjective psychological and physiological effects of low dose (5 mg), orally-administered Δ⁹-THC, versus men (Fogel et al., 2017). Additionally, while controlled studies have only reported modest effects on the nociceptive (sensory) component of pain in humans, cannabis may produce an alteration of the affective component of pain, rendering experimental pain less unpleasant (De Vita et al., 2018). The contribution of sex and gender on the affective and cognitive dimensions of pain has been a topic of debate (Templeton, 2020), and the effect of analgesics—cannabis, for example—on these dimensions of pain can only be adequately studied in humans. Thus, future studies in humans should address sex differences in sensitivity to both physiological and psychological responses caused by cannabis and cannabinoids, and address sex differences in cannabinoid modulation of pain with respect to the sensory, affective, and cognitive dimensions.

5. Impact of Hormones on Cannabinoid Antinociception

5.1. Impact of Estrous Cycle Phase

Determining if estrous cycle stage, which correlates to hormone levels, affects cannabinoid analgesia has only been evaluated by a small number of studies to date. Female rats in the late proestrus/estrus phase (high estradiol) showed an enhanced response to Δ⁹-THC in the tail immersion assay relative to both females in other stages of the estrous cycle as well as males (Craft et al., 2012; Wakley and Craft, 2011). Notably, this effect seen with Δ⁹-THC was not replicated with CP 55,940 in rats, which was more potent in females than males regardless of estrous phase (Craft et al., 2012). Still, other studies have failed to show an effect of estrous stage on cannabinoid analgesia in rats using Δ⁹-THC (Britch et al., 2017; Craft et al., 2013; Javadi-Paydar et al., 2018; Tseng and Craft, 2001; Wakley et al., 2014). In addition to studying the effect of estrous stage on cannabinoid-mediated analgesia, other studies have revealed hormone- and sex-dependent effects of cannabinoids using surgical gonadectomy and/or exogenous hormone supplementation approaches. Using gonadectomized (GDX) male and female rats, it was demonstrated that estradiol supplementation in GDX males increased tail flick latency following Δ⁹-THC administration, while in GDX females testosterone supplementation decreased Δ⁹-THC’s antinociceptive effect (Craft et al., 2017). Furthermore, in GDX females, estradiol supplementation blocked the antinociceptive effect of WIN 55,212-2 in the tail flick assay relative to vehicle and progesterone treated GDX females (Kalbasi Anaraki, et al., 2008). Considering the above, interactions between the endocannabinoid and hormone systems may underlie some of the sex differences in cannabinoid antinociception between males and females.

5.2. Sex Differences in Cannabinoid Metabolism

The cytochrome P450 (CYP) family of enzymes is primarily found in the liver and is responsible for the metabolism of many drugs, CYP 2C9, 2C19, and 3A4 representing the largest contributors to phytocannabinoid (THC, CBD, etc.) metabolism in humans (Stout and Cimino, 2014). There are well documented differences in the expression of CYP isozymes between sexes; CYP 3A4, for example, has been found to be more highly expressed and show greater activity in women versus men (Waxman and Holloway, 2009). When acted on by this family of enzymes Δ⁹-THC may be metabolized into inactive (ex. Δ⁹-THC-COOH) and active (ex. 11-OH-Δ⁹-THC) forms (Zendulka et al., 2016). Notably, females have been shown to produce greater amounts of the psychoactive metabolite 11-OH-Δ⁹-THC versus males in studies of rats (Narimatsu et al., 1991), mice (Watanabe et al., 1992), and humans (Nadulski et al., 2005).

Hormones have been found to directly influence CYP activity; estradiol, for example, has been demonstrated to be an inducer of CYP 2A6 (Higashi et al., 2007). Thus, sex hormone effects on CYP expression and activity may contribute to sex differences in cannabinoid actions. In a study with rats, gonadectomized males that received estradiol produced significantly higher levels of 11-OH-Δ⁹-THC from Δ⁹-THC metabolism compared to vehicle treated counterparts, while estradiol replacement in gonadectomized females did not increase 11-OH-Δ⁹-THC, and suppressed increases in 11-OH-Δ⁹-THC caused by the cytochrome P450 inhibitor proadifen (Craft et al., 2017). In humans, formation of 11-OH-Δ⁹-THC has been found to be largely produced by CYP 2C9 (Patilea-Vrana et al., 2019). However, estrogen and oral contraceptives have been demonstrated to reduce expression and activity of CYP 2C9 both in vitro (Mwinyi et al., 2011) and in women (Sandberg et al., 2004). If increased levels of 11-OH-Δ⁹-THC are responsible for increased potency of cannabis effects, and if estrogen and related compounds suppress CYP 2C9 expression and activity, then it could be hypothesized that the effects of cannabis in women would be more potent in periods of lower circulating estrogen levels. Unfortunately, data in humans to support or refute this line of thinking has yet to be conducted. Additionally, pre-clinical investigation into sex specific differences in cannabinoid metabolism have largely been restricted to the common phytocannabinoids. The metabolism of the commonly researched synthetic cannabinoids (CP55,940, WIN 55,212-2, ACEA, and compounds in the JWH series) has been less thoroughly investigated, and without regard to sex-specific differences in metabolism (Zendulka et al., 2016). Thus, sex differences in metabolism of the synthetic compounds could be contribute to the sex differences in antinociception described throughout this review.

6. Sex Differences in Endocannabinoid System Expression

6.1. Sex differences in Cannabinoid Receptor Expression in the Rodent Brain

The extent to which the endocannabinoid and hormone systems interact and/or regulate one another is an important research focus as cannabinoid receptors are expressed throughout the central and peripheral nervous systems, including in areas responsible for the synthesis and regulation of hormones. Sex differences in the CB₁ receptor have been found between male and female rodents throughout the brain (Figure 2), including in regions involved in hormone regulation; the hypothalamus and anterior pituitary (Rodriguez de Fonseca et al., 1994; Gonzalez et al., 2000) and regions involved in pain processing and modulation; the prefrontal cortex and amygdala (Castelli et al., 2014). The expression of CB₁ receptor protein or mRNA was found to be greater in male versus female rats in the anterior pituitary (Gonzalez et al., 2000), mesencephalon (Rodriguez de Fonseca et al., 1994), and prefrontal cortex and amygdala (Castelli et al., 2014). Conversely, CB₁ receptor protein density did not significantly differ between males and females in the hypothalamus, striatum, and limbic forebrain (Rodriguez de Fonseca et al., 1994).

Studies examining sex differences regarding CB₂ receptors are much less prevalent than those examining CB₁ receptors, although there is evidence for sex differences in the role of the CB₂ receptor in the periphery. In 2013 Craft et al. examined the antinociceptive effects of Δ⁹-THC and SR144528, a selective CB₂ receptor antagonist, administered systemically (intraperitoneal) and locally (intraplantar). Interestingly, locally administered SR144528 partially blocked the local antinociceptive effects of Δ⁹-THC in females, but not males. Further studies by Craft et al. in 2012 and Wiley et al. in 2017 tentatively suggest that CB₂ receptors in females may contribute to the pharmacological effects of Δ⁹-THC to a greater extent than in males, although further studies are required to elucidate if this is a function of sex differences in CB₂ receptor expression.

6.2. Effect of Hormones on Cannabinoid Receptor Expression in the Rodent Brain

Females show variation in CB₁ receptor expression which has been hypothesized to be connected to the hormone fluctuations characteristic of the estrous cycle. The direction of these fluctuations has varied between studies and brain regions. In rats, ovariectomy increased CB₁ receptor density in the prefrontal cortex and amygdala (Castelli et al., 2014) and anterior pituitary (Gonzalez et al., 2000), and decreased CB₁ receptor density in the striatum and limbic forebrain (Rodriguez de Fonseca et al., 1994). In the rat hypothalamus, ovariectomy was found to either have no effect on CB₁ receptor density (Rodriguez de Fonseca et al., 1994), or to increase CB₁ receptor density (Gonzalez et al., 2000).

Administration of estrogen in female rats reversed the increases in CB₁ receptor density produced by ovariectomy in the anterior pituitary and hypothalamus (Gonzalez et al., 2000), and prefrontal cortex and amygdala (Castelli et al., 2014), and reversed the decreases in CB₁ receptor density in the limbic forebrain produced by ovariectomy (Rodriguez de Fonseca et al., 1994). Progesterone produced mixed effects on CB₁ receptor density in female rats; a decrease was observed in the limbic forebrain and an increase was observed in the mesencephalon (Rodriguez de Fonseca et al., 1994). Additionally, progesterone produced mixed effects when co-administered with estradiol depending on whether administered acutely or chronically (Rodriguez de Fonseca et al., 1994).

In contrast to females, orchidectomy in males produced no changes in CB₁ receptor density in rat hypothalamus, striatum, limbic forebrain, or mesencephalon, nor did testosterone replacement affect expression with the exception of the limbic forebrain (Rodriguez de Fonseca et al., 1994). However, in the anterior pituitary of rats, orchidectomy reduced CB₁ receptor density, which was further reduced through testosterone replacement (Gonzalez et al., 2000). Finally, gonadectomy increased the affinity of CP 55,940 for hippocampal CB₁ receptors in both males and females, although the efficacy of CP 55,940 was greater in females than males, further emphasizing sex differences may be region- and/or compound-specific (Farquhar et al., 2019).

6.3. Effect of Hormones on Endocannabinoid Levels in the Rodent Brain

Sex differences have also been characterized for endocannabinoid levels in rodents. When female rats across all stages of the estrous cycle were pooled, females showed higher levels of 2-AG (2-arachidonoylglycerol) in both the hypothalamus and pituitary relative to males (Bradshaw et al., 2006), and higher levels in AEA (anandamide) in the pituitary relative to males (Gonzalez et al., 2000). Endocannabinoid content in female rat brains were found to vary based on estrous stage for 2-AG in the hypothalamus (metestrus and proestrus < diestrus and estrus), and pituitary (estrus < proestrus), and AEA levels varied by estrous stage in the pituitary (metestrus < proestrus) and hypothalamus (proestrus, estrus, metestrus < diestrus) (Bradshaw et al., 2006).

6.4. Sex Differences in Cannabinoid Receptor Expression in Humans

Cannabinoid receptor distribution in humans has also been examined, however, differences in methodologies between the limited studies prevent any definitive conclusions from being drawn. Most of these studies rely on PET (positron emission tomography) radiotracers to examine cannabinoid receptor densities. Recently, these PET studies have been conducted with the radiotracer [¹¹C]OMAR (Normandin et al., 2015; Neumeister et al., 2013), while the radiotracer [¹⁸F]MK-9470 has been used previously (Van Laere et al., 2008). Utilizing [¹¹C]OMAR, CB₁ receptor density was greater in healthy, non-cannabis using women in the follicular phase of the menstrual cycle compared to men (Normandin et al., 2015). Greater CB₁ receptor density was also seen in women with and without PTSD versus men, although menstrual phase was not reported in this study (Neumeister et al., 2013). In opposition to these results, and earlier study using the radiotracer [¹⁸F]MK-9470 reported greater CB₁ receptor density in men versus women among healthy non-cannabis using subjects (Van Laere et al., 2008). Unfortunately, no studies have been conducted to examine sex differences in CB₂ receptor distribution to date. Ultimately, whether sex differences in sensitivity to cannabinoids in humans correlates with differences in cannabinoid receptor expression remains to be determined, as does the impact of menstrual phase on cannabinoid receptor expression in women.

7. Interactions Between Hormone and Endocannabinoid Systems

7.1. Hormone Modulation of Cannabinoid Signaling

In addition to altering cannabinoid receptor expression, sex hormones may interact with cannabinoid receptors more directly, modulating binding and/or signaling. Indeed, there is considerable overlap in pathways that are influenced by cannabinoid and hormone receptors including adenylate cyclase/cAMP, PI3K/Akt, and ERK/MAPK pathways, among others (Dobovisek et al., 2016). Sex differences have been demonstrated in binding affinity and efficacy of cannabinoids in rodents, and these differences may be mediated by sex hormones (Figure 2). In rats, the binding affinity of tritiated CP 55,940 to the CB₁ receptor was greater in males versus females in the striatum, limbic forebrain, and mesencephalon (Rodriguez de Fonseca et al., 1994). Ovariectomy in females increased binding affinity in the striatum which was reversed by an acute dose (150 ng/kg, subcutaneous injection), but not chronic administration (slow release capsule), of estradiol (Rodriguez de Fonseca et al., 1994). Gonadectomy increased affinity and efficacy of CP 55,940 in the hippocampus and prefrontal cortex, respectively, in both male and female rats (Farquhar et al., 2019). Moreover, in gonadectomized rats, administration of estrogen potentiated Δ⁹-THC-mediated antinociception in males, while administration of testosterone inhibited Δ⁹-THC-mediated antinociception in females (Craft et al., 2017).

7.2. Cross Modulation of Endocannabinoid and Hormone Synthesis and Degradation

Steroid hormones and endocannabinoids have also been demonstrated to modulate the activity of the enzymes responsible for the synthesis and degradation of each other. A recently published study showed in vitro inhibition of aromatase-mediated estradiol production by anandamide (AEA), and in silico docking studies supported binding of anandamide to the active site of aromatase (Almada et al., 2019). Steroids have also been demonstrated to affect FAAH (fatty-acid amide hydrolase), the principal degradative enzyme for AEA. Estradiol and progesterone were demonstrated to decrease FAAH activity in mouse uteri through inhibition of FAAH expression at the translation level when given 24 hours prior to sacrifice (Maccarrone et al., 2000). Conversely, an increase in membrane binding of recombinant rat FAAH was demonstrated recently in vitro when physiological levels of pregnenolone, hydrocortisone, and estradiol were applied (Sabatucci et al., 2019).

7.3. Cross Reactivity of Cannabinoids and Hormones

Cannabinoids and steroid hormones have demonstrated cross reactivity; steroids have been demonstrated to bind and modulate cannabinoid receptors directly. Pregnenolone, a precursor to other steroid hormones including estradiol, progesterone, and testosterone, in addition to acting a neurosteroid itself, has been demonstrated to modulate CB₁ receptor activity. Evidence for pregnenolone as a negative allosteric modulator of the CB₁ receptor was demonstrated in vivo in mice and rats through inhibition of the behavioral and physiological effects of Δ⁹-THC as measured by the cannabinoid tetrad test, changes in food intake, and memory impairment (Vallee et al., 2014). These results were further validated through electrophysiology measurements that showed an inhibition of Δ⁹-THC’s inhibitory effect on glutamatergic neuron firing, without affecting binding, suggesting pregnenolone behaved as a CB₁ receptor negative allosteric modulator, not an antagonist (Vallee et al., 2014). However, another study did not find allosteric modulation of the CB₁ receptor by pregnenolone, and instead demonstrated displacement of the radiolabeled CB₁ receptor antagonist, Rimonabant, from the CB₁ receptor (Khajehali et al., 2015). Thus, the exact mechanism of pregnenolone at the CB₁ receptor remains to be determined. In addition to potential modulation by the endogenous steroid pregnenolone, one research group has recently published multiple articles demonstrating CB₁ and/or CB₂ antagonist/inverse agonist activity by a number of synthetic selective estrogen receptor modulators (SERMs) including tamoxifen (Ford et al., 2016; Franks et al., 2016; Franks et al., 2018). As the structures of these SERMs are based on the steroid scaffold the question arises whether endogenous steroids may also be capable of binding to cannabinoid receptors, or vice versa.

8. Discussion

Sex differences research in cannabinoid analgesia is a growing field of interest as cannabinoid-based therapies both pharmaceutical and botanical are being evaluated for a variety of pain conditions. At this time, however, no definitive conclusions can be made about sex differences in efficacy, or the mechanisms mediating these possible differences. While female rats have been shown to display greater sensitivity to Δ⁹-THC compared to male counterparts in many studies (Table 1), this trend appears to be a function of many factors (metabolism, influence of hormones, differences in receptor expression, etc.) rather than any individual mechanism. Moreover, sex differences in sensitivity to Δ⁹-THC may be species-specific; sex differences were not observed in mice in a recent study that evaluated the discriminative effects of Δ⁹-THC in both rats and mice (Wiley et al., 2020). Mice and rats are perhaps the most utilized model organisms in pre-clinical pain research, each with their own advantages and disadvantages, but translation of findings between the two species is not always possible, let alone extrapolation of those findings to humans (Ellenbroek and Youn, 2016). Future studies of cannabinoid pharmacology that evaluate both rats and mice like those of Vallee et al., 2014 and Wiley et al., 2020 would be welcome additions to the field. Additionally, there is a need for more studies investigating sex differences in cannabinoid-mediated antinociception and development of tolerance to cannabinoids in chronic pain models such as chemotherapy-induced neuropathic pain; a condition which is insufficiently addressed by currently available medications (Blanton et al., 2019).

Interactions between endocannabinoid and hormone systems are another important topic for future research. Estrous cycle was only evaluated in one-third of the studies of cannabinoid-mediated antinociception compiled in Table 1. As evidenced by numerous preclinical studies to date, estrous stage, ovariectomy, and hormone replacement can influence both the expression of cannabinoid receptors and ligands, as well as the affinity and efficacy of those ligands at cannabinoid receptors in various brain regions (Figure 2). Unfortunately, human studies have not yet evaluated menstrual stage or hormone status as a factor in cannabinoid-mediated analgesia, but if the data from rats translates to humans, hormone status could be an important contributing factor in cannabinoid response.

As cannabis and cannabinoids continue to become more widely accessible and utilized, understanding sex differences in the behavioral, physiological, and psychological responses to cannabis and cannabinoids, both acute and chronic, is a time sensitive research focus (Cooper and Craft, 2018). Ultimately, the inclusion of both sexes, and performing independent statistical comparisons between sexes in studies using cannabinoids is desperately needed. Indeed, many studies to date have pooled males and females together, or failed to perform the appropriate statistical analyses between the two groups, potentially leaving important sex differences undiscovered. While sex and sex hormones seem to contribute to differences between male and female responsiveness to cannabinoids in rats, at this time a clear consensus on the nature the contribution is unclear, as is the translatability of these findings to humans. It is likely that these pioneering studies have only scratched the surface of revealing the potential mechanisms behind sex differences in the efficacy of cannabinoids in pain modulation, let alone the full scope of the endocannabinoid system in the modulation of sex hormones and hormonal influence on other behavioral and physiological measures. We propose that the findings from the limited studies to date summarized in this review warrant an increased research focus on sex differences in pain and analgesia, and investigation into the interactions between endocannabinoid and hormone systems, in disease states such as chronic pain, as well as other aspects of physiology.

Highlights.

• Female mice mostly found to be more sensitive to Δ⁹-THC

• Preclinical evidence showingo sex differences in cannabinoid pharmacology

• Direct interactions between sex hormones and endocannabinoid system demonstrated from preclinical studies

• Sex differences in the antinociceptive responses leading to changes in expression and function of endocannabinoid system

• Translation of sex differences in the endocannabinoid system to cannabis/cannabinoid use in men and women

Abbreviations

AEA

anandamide; 2-AG, 2-arachidonolyglycerol

CB₁

cannabinoid receptor 1

CB₂

cannabinoid receptor 2

FAAH

fatty-acid amide hydrolase

TLR4

Toll-like receptor 4

PAG

periaqueductal gray

Δ⁹-THC

Δ⁹-tetrahydrocannabinol

CBD

cannabidiol

CFA

Central Freund’s Adjuvant

GDX

gonadectomized

OVX

ovariectomized

CYP/P450

cytochrome P450

PET

positron emission tomography

cAMP

cyclic adenosine monophosphate

PI3K

phosphoinositide 3-kinase

Akt

protein kinase B

ERK

extracellular signal-regulated kinase

MAPK

mitogen-activated protein kinase