Sex Differences in Plasma, Adipose Tissue, and Central Accumulation of Cannabinoids, and Behavioral Effects of Oral Cannabis Consumption in Male and Female C57BL/6 Mice
Department of Physiology and Pharmacology, The University of Calgary, Calgary, Canada
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Cairo University, Cairo, Egypt
Department of Cell Biology and Anatomy, The University of Calgary, Calgary, Canada
Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka, Japan
Department of Cell Biology and Anatomy, The University of Calgary, Calgary, Canada (Dr Hill and Ms Baglot)
Correspondence: Stephanie L. Borgland, PhD., Hotchkiss Brain Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, AB, Canada T2N 4N1 (s.borgland@ucalgary.ca)Abstract
Background
Cannabis edibles are an increasingly popular form of cannabis consumption. Oral consumption of cannabis has distinct physiological and behavioral effects compared with injection or inhalation. An animal model is needed to understand the pharmacokinetics and physiological effects of oral cannabis consumption in rodents as a model for human cannabis edible use.
Methods
Adult male and female C57BL/6 mice received a single dose of commercially available cannabis oil (5 mg/kg Δ⁹-tetrahydrocannabinol [THC]) by oral gavage. At 0.5, 1, 2, 3, and 6 hours post exposure, plasma, hippocampus, and adipose tissue were collected for THC, 11-OH-THC, and THC-COOH measures.
Results
We report delayed time to peak THC and 11-OH-THC concentrations in plasma, brain, and adipose tissue, which is consistent with human pharmacokinetics studies. We also found sex differences in the cannabis tetrad: (1) female mice had a delayed hypothermic effect 6 hours post consumption, which was not present in males; (2) females had stronger catalepsy than males; (3) males were less mobile following cannabis exposure, whereas female mice showed no difference in locomotion but an anxiogenic effect at 3 hours post exposure; and (4) male mice displayed a longer-lasting antinociceptive effect of oral cannabis.
Conclusions
Oral cannabis consumption is a translationally relevant form of administration that produces similar physiological effects as injection or vaping administration and thus should be considered as a viable approach for examining the physiological effects of cannabis moving forward. Furthermore, given the strong sex differences in metabolism of oral cannabis, these factors should be carefully considered when designing animal studies on the effects of cannabis.
Boxed Text
Oral delivery of cannabis oil in mice is a translational model that increases plasma, hippocampal, and adipose cannabinoids. Furthermore, oral cannabis produces lasting psychoactive effects, including sex-dependent effects on hypothermia, cataplexy, locomotor activity, and nociception.
INTRODUCTION
Cannabis edibles are a popular mode of consumption among cannabis users. In 2021, oral cannabis consumption was second only to smoking as the most popular method among those who consumed cannabis (Statistics Canada, 2021). With legalization of cannabis in Canada, self-reported edible cannabis use rose from 32% in 2017 to 53% in 2021 (Statistics Canada, 2021). Nearly one-third of Canadian and US 16- to 19-year-olds report eating or drinking cannabis in the past 30 days in 2017, 2018, and 2019 (Wadsworth et al., 2022). Furthermore, oral administration is the most common method of use for medicinal users (Sznitman, 2017; Boehnke et al., 2019). Therefore, fully characterized mouse models of oral use are important to understand the acute and long-term physiological consequences of oral cannabis use.
Human pharmacokinetics studies of oral cannabis use have found significant differences in the peak blood and oral fluid concentrations compared with inhalation routes of administration (Swortwood et al., 2017; Spindle et al., 2021; Bidwell et al., 2022). Specifically, the active metabolites Δ⁹-tetrahydrocannabinol (THC), 11-OH-THC, and THC-COOH peak blood levels are considerably delayed compared with inhalation (Ohlsson et al., 1980; Spindle et al., 2021). This is consistent with reports of subjective “drug effect” peaking 1 to 2 hours after oral consumption (Vandrey et al., 2017; Schlienz et al., 2020) compared with less than an hour following cannabis inhalation (Ohlsson et al., 1980). Orally administered THC undergoes first-pass metabolism to 11-OH-THC, a metabolite with similar bioavailability at cannabinoid receptor 1 to THC (Nadulski et al., 2005b). Thus, the behavioral and physiological consequences may be distinct from cannabis inhalation or injection. This may also account for differences in some subjective effects of cannabis taken orally versus by inhalation (Hart et al., 2002; Spindle et al., 2021).
Preclinical rodent models are useful for studying the effects of cannabis as they allow for fine control of the environment, cannabinoid concentrations, and timing. Moreover, animal research allows us to investigate the biological underpinnings of the effects of cannabis and takes advantage of novel genetic tools and transgenic lines to target neuronal populations, receptors, or enzymes. Many preclinical studies have examined injections of cannabis extract, THC, or cannabinoid receptor 1 receptor agonists. However, humans rarely administer cannabis through injection, and the pharmacokinetic properties differ between oral and injected cannabis products. Furthermore, while inhalation models have been recently validated (Manwell et al., 2014; Nguyen et al., 2016) and the pharmacokinetics assessed in rats (Baglot et al., 2021), oral cannabis has different pharmacokinetic properties that may have unique behavioral and biological implications. Vaporized cannabis administration models are also complex, challenging to implement, and not accessible to all laboratories. Therefore, to explore additional ways to model human use, we characterized oral administration in mice as this approach is easily tractable across laboratories and is still relevant for modeling the effects of cannabis in humans.
Several rodent cannabis studies have used oral consumption, though doses vary considerably between studies (Fairbairn and Pickens, 1979; Abel et al., 1980; Steffens et al., 2005; Mitchell et al., 2021), and it is unclear how these doses compare with human use, given potential differences in rodent metabolism. A 2017 study in Wistar rats examined pharmacokinetic differences in 10 mg/kg THC by route, including oral consumption (Hložek et al., 2017); however, these experiments were only performed in male rats, which did not allow for identification of potential sex differences in the metabolism, tissue disposition, and behavioral response to cannabis. Another study examined the effects of voluntary THC administration in gelatin (1–2 mg/15 mL gelatin) 3 days per week over 33 days and found that the average daily consumption of THC by male and female adolescent rats ranged from 1 to 5 mg/kg and rats consumed greater quantities of the control gelatin than the THC gelatin (Kruse et al., 2019). THC oral administration triggers hypothermia and analgesia and increases in locomotor activity measured after 12 days of chronic use, 1 hour after daily access to THC gelatin (Kruse et al., 2019). Peak plasma concentration following oral administration of 20 mg THC to male subjects, equivalent to 0.33 mg/kg (using 60 kg bodyweight), was 15 ± 10 ng/mL (Grotenhermen, 2003). We converted this to a mouse dose of 4.1 mg/kg using the allometric scaling approach by (Nair and Jacob, 2016) to advise us on the approximate dose to test. Furthermore, an oral THC dose of 5 mg/kg in rats produced locomotor effects and significant antinociception on a tail flick assay (Moore and Weerts, 2022), produced hypothermia and hypolocomotor activity in male and female C57BL/6 mice (Smoker et al., 2019), and was calculated as the ED50 for THC’s antinociceptive effect in male mice (Chesher et al., 1973). However, it is unknown what the tissue distribution or physiological effects of oral cannabis are after a single administration in male and female mice. Therefore, for this study, we administered an oral dose of 5 mg/kg whole cannabis THC.
Given that mode of cannabis consumption can produce vastly different pharmacokinetic profiles (Swortwood et al., 2017; Spindle et al., 2021; Bidwell et al., 2022) and that oral consumption of cannabis is an increasingly popular method of cannabis consumption among recreational and medical cannabis users, it is critically important to understand the pharmacokinetics and behavior effect of edible cannabis in mouse models. Here, we analyzed the time- and sex-dependent changes in relevant behaviors measuring cannabis intoxication and levels of THC, 11-OH-THC, and THC-COOH in plasma, adipose, and brain tissue in C57BL/6 mice, a common inbred mouse strain, following oral consumption of commercially available cannabis oil containing 95% THC.
METHODS
Animals
All protocols were in accordance with the ethical guidelines established by the Canadian Council for Animal Care and were approved by the University of Calgary Animal Care Committee. Cannabis use for research was approved under license LIC-IYGANQJY09-2022. Adult (PD90+) male and female C57Bl/6 mice were obtained from Charles River Laboratories (St. Constant, QC, Canada). Mice were group housed (3–4 mice per cage) in a reverse light-dark cycle room (12-hour-light/-dark cycle; lights on: 10 pm MST; lights off: 10 am MST). All experiments were performed during the dark cycle. Following 2 weeks of acclimation, mice were split into 2 groups (cannabis and vehicle). For all experiments, commercially available cannabis oil purchased from Tweed Inc. (Smith Falls, Ontario, Canada) containing 25 mg/mL THC in medium chain triglyceride (MCT) oil and less than 1 mg/mL cannabidiol(CBD) was used. A single dose of 5 mg/kg THC or MCT oil (from coconut oil; Kirkland) was administered by oral gavage at 2.5 hours after lights-off (12:30 pm MST) following 2 hours of fasting to ensure gastric emptying. Mice were habituated to oral gavage, transfer between procedure rooms, and use of the rectal thermometer (for body temperature experiments only) for 3 days before the test day. Behavioral experiments were performed either immediately after cannabis administration or at the indicated timepoints. Mice were divided into 3 cohorts. One cohort was used to measure rectal temperature then underwent catalepsy testing and was immediately killed for tissue collection thereafter. Two measurements per mouse were taken (at baseline and another time point). The second cohort was used for the open field test. The third cohort was used for the hot plate test. Different mice were used at different timepoints allowing for collection of plasma and hippocampal samples at each timepoint or to avoid timepoints that would be influenced by prior activity at earlier timepoints (e.g., hot plate test). Mice were killed by CO2 directly after behavioral testing. The timepoints were selected based on a previous THC pharmacokinetic study in mice (Torrens et al., 2020; Dumbraveanu et al., 2023).
Open field
Mice were habituated to the behavioral room for 15 minutes before testing. The open field test was used to assess cannabis-induced changes in locomotion and thigmotaxis. Mice were placed in the center of an arena measuring 30 cm × 30 cm × 30 cm (L × H × W) and allowed to move freely for 10 minutes at 0.5, 1, 2, 3, and 6 hours post cannabis exposure. Movement was recorded with a Basler GenICam mounted above the arena and was analyzed using Noldus Ethovision XT 11.5 software (Noldus Information Technology, Leesburg, VA, USA). Locomotor activity was measured as total distance travelled and mean velocity, and the time spent in the center of the chamber. Open field arenas were cleaned with the disinfectant Virkon between time points and animals to reduce scents.
Hot plate
Preexposure and 3 or 6 hours post exposure, mice were placed in a 10-cm-wide glass cylinder on a hot plate set to 52°C. The latency to reaction (paw licking or jumping), to a maximum of 30 seconds, was recorded by an observer blinded to the experimental treatment. The apparatus was cleaned with ethanol between animals to remove animal scents.
Body temperature
Body temperature was taken via a rectal thermometer at baseline and before killing for plasma and hippocampus collection at the 0.5-, 1-, 2-, 3-, and 6-hour time points.
Catalepsy
Catalepsy was assessed by placing the forepaws of the mouse on a bar in the center of a cage and its hind paws on the floor. The time to move forepaws on or off the bar was recorded as latency to move. The maximum cutoff time was 5 minutes (300 seconds) (Metna-Laurent et al., 2017). The catalepsy apparatus consisted of a modified home cage measuring 28 cm × 12.5 cm × 17.8 cm (L × H × W) with a bar fixed 3.5 cm off the floor in the center of the cage. Catalepsy measurements were taken in the same mice as body temperature measurements and immediately before killing for sample collection at the 0.5-, 1-, 2-, 3-, and 6-hour time points. The catalepsy chamber was cleaned with 20% ethanol between mice.
Sample preparation
On the day of experiments, mice were fasted for 2 hours to ensure gastric emptying, and their baseline temperature and catalepsy were assessed. Male (60) and female (60) mice were dosed with commercially available cannabis oil or MCT alone by oral gavage. At 0.5, 1, 2, 3, or 6 hours (5–6 mice per time point, per treatment, per sex), rectal body temperature and catalepsy (latency to move) were recorded, the mouse was killed, whole blood was collected in heparin-lined tubes, the brain rapidly was removed, and the hippocampus was dissected out. Visceral gonadal adipose tissue was also obtained. The hippocampus or adipose tissue was immediately placed in a tube on dry ice until it was moved to a −80°C freezer to be stored until analysis. Whole blood was stored on ice for no longer than 2 hours, then centrifuged at 4°C for 20 minutes at 2500 rpm and the plasma removed and stored at −80°C until analysis. Samples were prepared as previously described (Baglot et al., 2021). Briefly, samples were thawed at room temperature (plasma) or weighed frozen (hippocampus or adipose tissue) and placed into glass tubes with 2 mL acetonitrile and 100 μL deuterated internal standard (Cerilliant, Round Rock, TX, USA). Hippocampal or adipose samples were first homogenized with a glass rod. Plasma, adipose, and brain samples were sonicated in an ice bath for 30 minutes then stored at −20°C overnight. Tubes were centrifuged at 1800 rpm at 4°C for 3 to 4 minutes, and the supernatant was transferred to a new tube. The supernatant was then evaporated under nitrogen gas, washed with acetonitrile, evaporated, and washed again to collect lipids on the tube walls. Samples were then suspended in 1:1 methanol and deionized water, centrifuged twice at 15 000 rpm at 4°C for 20 minutes, and the supernatant stored at −80ºC until analysis.
LC-MS/MS analysis
Samples were prepared for liquid-chromatography-mass spectrometry (LC-MS/MS; multiple reaction monitoring) by the Southern Alberta Mass Spectrometry (SAMs) facility at the University of Calgary for measurement of THC, 11-OH-THC, and THC-COOH levels. LC-MS/MS analysis was performed using an Eksigent Micro LC200 coupled to an AB Sciex QTRAP 5500 mass spectrometry (AB Sciex, Ontario, Canada). Analyte concentrations (in pmol/µL) were normalized to sample volume/weight and converted to ng/mL.
Statistics
All data were expressed as mean ± SEM and were analyzed and graphed using GraphPad Prism 9.3.1. Normality was determined using a Shapiro-Wilk test. Time-dependent changes in THC, 11-OH-THC, and THC-COOH in males and females were examined using mixed-effects analysis. Three-way ANOVA was used to compare male and female responses of cannabis and time on temperature, catalepsy, and locomotor activity. Time course measurements were not repeated in mice due to sample collection (THC and metabolite levels), repeated stress (administering rectal probe for temperature), or the effect of repeated measures on behavioral performance (catalepsy, locomotor activity). Hot plate data were compared between baseline and 3 hours or baseline and 6 hours using a repeated-measures 2-way ANOVA. Sidak’s posthoc tests were used to assess for multiple comparisons. In the figures, asterisks refer to significant P values as follows: *P < .05, ** P < .01, *** P < .001, **** P < .0001.
RESULTS
THC, 11-OH-THC, and THC-COOH Plasma, Brain, and Adipose Tissue Levels
Plasma analyte levels were compared between sexes and examined over time within sexes. A main effect of sex was present in plasma levels of THC (F(1, 49) = 6.134 at P = .0168; Figure 1A), with females showing increased levels at 1 hour (P = .01) after exposure and 11-OH-THC (F(1, 49) = 60.81, P < .0001; Figure 1B) with significant differences at 1 (P = .0002), 2 (P < .0001), and 3 hours (P = .003) after exposure, but not THC-COOH (F(1, 51) = 0.15, P = .699; Figure 1C). There was no significant main effect of time on THC levels (F(4, 49) = 2.09, P = .09). However, there was a significant effect of time on 11-OH-THC levels (F(4, 49) = 3.46, P = .01) and THC-COOH levels (F(4, 51) = 4.1, P = .006). In females, the time taken to reach maximum concentation in plasma (Tmax) of THC occurred at 1 hour (Maximum plasma concentration (Cmax): 27.0 ± 9.6 ng/mL, n = 7), whereas Tmax of 11-OH-THC (Cmax: 24.3 ± 5.1 ng/mL, n = 6) and peak THC-COOH (Cmax: 25.8 ± 4.3 ng/mL, n = 6) occurred at 2 hours. In males, plasma THC reached Tmax at 3 hours (Cmax: 12.6 ± 3.1 ng/mL, n = 6), but Tmax of 11-OH-THC was earlier at 1 hour (Cmax: 1.9 ± 0.9 ng/mL, n = 6) and THC-COOH at 2 hours (Cmax: 22.4 ± 2.2 ng/mL, n = 6). Taken together, females have higher plasma THC and 11-OH THC than males after oral administration.
We next analyzed cannabinoid levels in the brain. We chose to analyze the hippocampus because it is the site for many of the neurobehavioral effects of cannabinoids and has a high expression of cannabinoid receptors (Herkenham et al., 1991). Additionally, hippocampus dissection is straightforward and consistent. In the hippocampus, there was a main effect of sex on THC levels (F(1,50) = 26.86, P < .0001; Figure 1D), with males showing increased levels at 2 (P = .003) and 3 hours (P = .008) after exposure and 11-OH-THC (F(1,51) = 15.64 at P = .0002; Figure 1E), with significant differences at 1 (P = .003) and 2 hours (P = .04) after exposure but not THC-COOH (F(1, 50) = 0.025 at P = .88; Figure 1F). Hippocampal levels of THC reached Tmax at 2 hours in males (Cmax: 39.4 ± 6.8 ng/g, n = 7) and females (Cmax: 18.2 ± 2.6 ng/g, n = 6). However, hippocampal levels of 11-OH-THC reached Tmax at 2 hours in females (Cmax: 12.4 ± 1.9 ng/g, n = 6) and 1 hour in males (49.4 ± 21.7 ng/g, n = 6). Hippocampal THC-COOH levels reached Tmax at 2 hours in males (Cmax: 15.9 ± 1.2 ng/g, n = 7) and females (Cmax: 14.8 ± 3.7 ng/g, n = 6). Taken together, hippocampal levels of THC and 11-OH-THC are higher in males than females, suggesting that uptake to the brain from plasma is much greater in males than in females.
In adipose tissue, there was a significant sex × time interaction on THC levels (F(4,48)=3.69, P = .011; Figure 1G) and main effects of sex (F(1,48)=36.39, P < .0001) and time (F(4,48)=3.35, P = .013), with males showing increased adipose THC accumulation at 2 (P = .0005), 3 (P = .021), and 6 hours (P < .0001). Adipose levels of THC reached Tmax at 1 hour in females (Cmax: 275.5 ± 70 ng/g, n = 6) and 6 hours in males (Cmax: 927.3 ± 113 ng/g, n = 7). Metabolite levels were considerably lower, with 11-OH-THC reaching Tmax at 2 hours in females (Cmax: 14.3 ± 1.3 ng/g, n = 6) and males (Cmax: 23.1 ± 4.2 ng/g, n = 6). Similarly, THC-COOH reached Tmax at 2 hours in females (Cmax: 8.6 ± 0.9 ng/g, n = 6) and males (Cmax: 11.9 ± 0.8 ng/g, n = 6).
Cannabis Tetrad Test Battery
We next assessed for sex differences in the physiological and behavioral effects of oral cannabis consumption between male and female mice on 4 measures: body temperature, cataplexy, locomotor activity, and nociception. THC administered by injection or inhalation typically induces hypothermia in rats (Nguyen et al., 2020; Ruiz et al., 2021). A 3-way ANOVA indicated there was a significant time × sex interaction (F(5, 97) = 2.72, P = .02), a time × cannabis interaction (F(5, 104) = 3.93, P = .003), but no sex × cannabis interaction (F(1, 97) = 2.67, P = .11). There were main effects of time (F(5, 104) = 2.51, P = .02), sex (F(1, 97) = 5.97, P = .02), and cannabis (F(1, 104) = 5.16, P = .03). While there were no significant changes in body temperature after cannabis exposure in males, female mice had lower temperature after cannabis only at the 6-hour timepoint (P = .003) (Figure 2).
To determine if oral cannabis exposure influenced catalepsy in male or female mice, we placed mice on a horizontal bar and measured the time until forepaws were off the bar. A 3-way ANOVA indicated that there were significant time × cannabis (F(5, 117) = 13.10, P < .0001), sex × cannabis (F(1, 90) = 39.34, P < .0001), and time × sex × cannabis interactions (F(5, 90) = 10.56, P < .0001). There were also main effects of time (F(5, 117) = 11.38, P < .0001), sex (F(1, 90) = 28.98, P < .0001), and cannabis (F(1, 117) = 22.29, P < .0001). Oral consumption of cannabis oil resulted in significant catalepsy in male (Figure 3A) and female mice (Figure 3B). However, the cataleptic effect of cannabis was greater, had an earlier onset, and was longer lasting in females, with significant differences from vehicle at 2 (P < .0001) and 3 hours (P < .0001) after oral administration.
To determine if oral cannabis exposure altered locomotor behavior, we measured distance travelled within 10 minutes on the open field test. A 3-way ANOVA indicated that there were significant main effects of time (F(4,179) = 23.47, P < .0001), cannabis (F(1,170) = 10.71, P = .0013), and sex (F(1,170) = 19.74, P < .0001) on distance travelled but no significant interactions of time × cannabis (F(4,170) = 1.08, P = .37), time × sex (F(4,170) = 0.24, P = .92), or cannabis × sex (F(1,170) = 0.77, P = .38). Whole cannabis oil significantly decreased locomotion in the open field task at 3 hours in male (Figure 4A; P = .041) but not female mice (Figure 4B; P = .19).
We then examined the effects of oral cannabis consumption on velocity in male (Figure 4C) and female mice (Figure 4D). A 3-way ANOVA indicated that there were significant main effects of time (F(4,170) = 23.18, P = .38), cannabis (F(1,170) = 12.03, P = .0007), and sex (F(1,170) = 19.18, P < .0001). However, there were no interactions of time × cannabis (F(4,170) = 1.24, P = .29), time × sex (F(4,170) = 0.26, P = .91), or cannabis × sex (F(1,170) = 1.21, P = .27). Oral cannabis significantly decreased velocity at 3 hours in male (Figure 4C; P = .03) but not female mice (Figure 4D; P = .36).
We next measured distance travelled in the center zone, which is suggestive of anxiolytic behavior as mice become more thigmotaxic, remaining near the walls of the open field apparatus with higher anxiety. A 3-way ANOVA indicated that there were significant main effects of time (F(4,126) = 6.35, P = .0001), cannabis (F(1,126) = 6.84, P = .01), and sex (F(1,126) = 46.44, P < .0001) and a significant cannabis × sex interaction (F(1,126) = 4.92, P = .028). There were no interactions of time × cannabis (F(4,126) = 0.73, P = .57) or time × sex (F(4,126) = 0.20, P = .93). Oral cannabis significantly decreased time in center at 3 hours in female (Figure 4F; P = .046) but not male mice (Figure 4E; P = .91). This suggests that oral cannabis may be anxiogenic in female mice.
Anti-nociception (Hot Plate Test)
We tested for cannabis-induced anti-nociception using a hot plate test. Oral cannabis consumption produced a sex-specific anti-nociceptive effect. At 3 hours post exposure in male mice (Figure 5A), there was a cannabis × time interaction (F(1, 17) = 5.47, P = .032) but no main effects of cannabis (F(1, 17) = 1.6, P = .22) or time (F(1, 17) = 1.22, P = .16). A Sidak’s posthoc test revealed a significant increase in latency to evoke nociceptive behaviors (paw licking or jumping) of cannabis at 3 hours (P = .036). In female mice (Figure 5B), there was a cannabis × time interaction (F(1, 16) = 4.5, P = .049) and a main effect of time (F(1, 17) = 6.89, P = .018) but no main effect of cannabis (F(1, 16) = 1.35, P = .26). A Sidak’s posthoc test revealed a significant increase in latency to evoke nociceptive behaviors of cannabis at 3 hours (P = .008). At 6 hours post cannabis exposure, male mice displayed significantly increased latency to evoke nociceptive behaviors (cannabis × time interaction [F(1, 13) = 11.63, P = .005]; cannabis effect: F(1, 13) = 7.27, P = .02, time effect: F(1, 13) = 10.02, P = .007); Sidak’s posthoc: P = .002), whereas female mice showed no difference (cannabis × time interaction [F(1, 18) = 0.001, P = .97]; cannabis effect: F(1, 18) = 0.29, P = .59, time effect: F(1, 18) = 0.31, P = .58), suggesting that the antinociceptive effects of oral cannabis last longer in males than females.
DISCUSSION
In the present study, we demonstrated that oral cannabis produces significant sex differences in plasma and brain concentrations of THC, 11-OH-THC, and THC-COOH. Furthermore, these differences in pharmacokinetic effect of oral cannabis were observed in behavioral responses such that females had a stronger hypothermic, cataleptic, and anxiogenic response than males but a shorter-lasting antinociceptive effect. Thus, oral cannabis shows significant sex differences in the levels and tissue dispersal of THC and its metabolite 11-OH-THC, as well as behavioral effects in the cannabis tetrad.
Sex Differences in Cannabis Tetrad
We also saw significant differences in the behavioral and physiological effects of oral cannabis consumption in the cannabis tetrad test. Specifically, we saw that female mice had a delayed hypothermic effect of cannabis 6 hours post consumption, which was not present in males; that although both male and female mice displayed a cataleptic effect of cannabis, it was more pronounced in females; and males had slightly decreased activity following cannabis exposure, whereas female mice showed no difference in locomotion, although a decrease in distance traveled in the center zone was evident. Finally, although both male and female mice displayed an antinociceptive effect of oral cannabis, it was longer lasting in males.
Despite lower brain THC and 11-OH-THC levels, female mice seem to be more sensitive to the cataleptic and hypothermic effects of THC and 11-OH-THC. The lack of hypothermic effect of cannabis in males is not unprecedented. A recent publication that examined the cannabis tetrad response after oral cannabis in rats found that at a 5.6-mg/kg THC dose, male rats showed no hypothermic effect of cannabis at any point. However, there was a significant hypothermic effect in females after 5 hours (Moore and Weerts, 2022), similar to what we have reported here. Although this study also noted that CBD increased body temperature in male rats, the doses used in this study (3, 10, and 30 mg/kg CBD) are considerably higher than the CBD present in the whole cannabis oil we have used, which contained <1 mg/mL CBD. Thus, CBD does not likely contribute to the sex differences in the hypothermic effect of cannabis we report here. Cataleptic effects of cannabinoids have been reported from i.p. injections (Tseng and Craft, 2001; Tseng et al., 2004) or oral administration of THC (Cohn et al., 1972), i.p. injection of 11-OH -THC or the CBR1/2 agonist CP55940 (Tseng and Craft, 2001) in rats. Consistent with our results, there was a greater cataleptic effect of i.p. THC in female rats compared with male rats (Tseng et al., 2004). However, when the cytochrome P450 inhibitor blocked conversion of THC to 11-OH-THC, this sex difference was abolished, suggesting that this sex difference in catalepsy is likely due to the psychoactive metabolite. Notably, we observed significantly lower levels of brain 11-OH-THC in females than in males; therefore, factors other than metabolite concentration likely also play a role in cataleptic response. One factor for this difference may include the contribution of the estrous cycle, which was previously shown to impact cannabinoid receptor expression in some regions (de Fonseca et al., 1994; Castelli et al., 2014) but which we did not control for.
Some behavioral sex differences are consistent with our observation of increased male brain levels of THC and its primary active metabolite, 11-OH-THC. Namely, male mice reduced their locomotor activity and had longer-lasting antinociception following oral cannabis consumption. Decreased locomotor activity was also observed in male Wistar rats 2 hours after oral THC (Tseng et al., 2004). Furthermore, consistent with our results, a recent study of oral cannabis tetrad effects in rats also found that male rats had a stronger anti-nociceptive effect of 5.6 mg/kg THC oral cannabis in response to thermal stimuli 5 hours post consumption (Moore and Weerts, 2022); however, both sexes showed a similar anti-nociceptive response at 2 hours post consumption and in response to a physical stimulus.
Limitations and Conclusions
Previous pharmacokinetic studies have largely used rats, making it difficult to directly compare pharmacokinetic responses of cannabis because species differences between rats and mice likely contribute to differences in metabolism and distribution of cannabis. Furthermore, our mice were fasted before oral gavage to ensure gastric emptying to control for food consumption before cannabis dosing. In humans, oral consumption of cannabis while fasted results in earlier peak THC and 11-OH-THC times (Lunn et al., 2019); thus, the time to peak THC and metabolites we report may be shorter than expected compared with fed mice. Furthermore, although we administered cannabis oil via oral gavage to tightly control the time that mice received cannabis, we recognize this is a stressful procedure (Brown et al., 2000). Therefore, our behavioral results may differ from those produced by a voluntary oral cannabis consumption model. This study used only a single dose of THC. There is evidence of biphasic effects of THC on behavior with injection models (Patel and Hillard, 2006; Rubino et al., 2007); however, this has not been well investigated in oral models. Future studies should address the possibility of biphasic effects with different THC concentrations in the oral administration model. Finally, because this study design collected both blood and brain samples at each time point rather than taking repeated measures from the same animal over time, we were unable to effectively measure repeated pharmacokinetic parameters such as the half-life of elimination, volume of distribution, clearance, or elimination rate constant. Future studies should address these parameters with oral administration in mice compared with other routes of administration as well as other developmental periods.
In conclusion, using a commercially available cannabis oil at a common dose used in rodent studies (5 mg/kg THC), we have characterized the pharmacokinetics and behavioral effects of oral cannabis consumption in male and female C57BL/6 mice. We have also demonstrated significant sex differences in the pharmacokinetics of THC and report different behavioral responses to oral cannabis consumption in male versus female mice in agreement with previous research (Smoker et al., 2019; Wiley et al., 2021). It is unclear to what extent sex differences in response to cannabis are due to differences in THC metabolism versus the impact of THC and its metabolites on sex differences in neural circuits, and/or expression of cannabinoid receptors within these circuits, underlying these behaviors. These findings should be considered by researchers interested in using preclinical models of cannabis consumption because this oral administration model is low-cost, easy to use, and translationally relevant. We encourage researchers of future animal and human oral cannabis studies to carefully consider potential sex differences in their experimental design and analyses given the significant sex-dependent effects observed here.
Acknowledgments
The authors acknowledge the Southern Alberta Mass Spectrometry facility. This research was performed at the University of Calgary, which is located on the unceded traditional territories of the people of the Treaty 7 region in Southern Alberta, which includes the Blackfoot Confederacy (including the Siksika, Piikuni, Kainai First Nations), the Tsuut’ina, and the Stoney Nakoda (including the Chiniki, Bearspaw, and Goodstoney First Nations). The City of Calgary is also home to Metis Nation of Alberta, Region III.
This work is supported by a University of Calgary VPR Catalyst Grant and a Matheson Centre Research Grant on Cannabis (S.L.B), Alberta Innovates Research Grant -mCannabis and CIHR (PJT-162271) to T.T., CIHR Catalyst grant-Cannabis to M.H., and Tier 1 Canada Research Chair (S.L.B. 950-232211). C.P. was supported by an Alberta Innovates Graduate Studentship in Health Innovation.
Conflict of Interest Statement
The authors declare no competing financial or other conflicts of interest.
Data Availability
Data is available upon request.