This study investigated CBD and THC tissue distribution at 1, 2, and 3H and the efficacy of EPA/DHA and DHA oil compared to SES carrier oils on the acute incorporation of orally consumed cannabinoids in the tissues of mice. SES oil significantly increased concentrations (p < 0.05) of CBD and THC in all six tissues across most time points compared to either n-3 PUFA carrier oils (Tables 1 and 2). When compared, EPA/DHA oil performed marginally better than DHA oil at increasing CBD and THC tissue concentrations; however, DHA oil notably resulted in higher brain cannabinoid concentrations. THC tissue concentration was highly variable and sometimes below the standard curve range of the analysis method; thus, CBD is primarily discussed. Overall, SES oil is far more effective at increasing tissue concentrations of cannabinoids following oral consumption. However, differential trends of incorporation between the two different ratios of EPA and DHA found in the n-3 PUFA oils demonstrate potential for future modifications to improve or tailor delivery to tissues.

Based on current knowledge, this study is the first to assess n-3 PUFA carrier oil to increase the bioavailability of orally consumed CBD and THC. Several other vegetable oils consisting of long and medium-chain triglycerides have been assessed in rodent models with varying success at increasing the bioavailability of cannabinoids, the most effective being SES and olive oil (Zgair et al. 2016; Izgelov et al. 2020; Feng et al. 2022). SES oil was chosen as the control to compare against the two n-3 PUFA oils as SES oil is established in the literature as an effective carrier oil and is used in the only drug containing CBD (Epidiolex®) approved by the US Food and Drug Administration and European Medicines Agency (Zgair et al. 2016; Lattanzi et al. 2021; Silmore et al. 2021). The results presented here conform with previous research, which shows SES oil is a superior carrier oil and promotes postprandial incorporation of for CBD and THC in the liver, serum, and brain. Additionally, this effect is observed in other tissues, including heart, muscle, and adipose, and the degree and speed of uptake differ between tissues, making it relevant when studying the impact of CBD and THC on a tissue-specific basis, related to mechanisms of action and managing potential risk with oral ingestion of cannabinoids (Froude et al. 2024).

As n-3 PUFA oil contains the long-chain PUFA, EPA and DHA, we hypothesized that the chylomicrons formed during digestion would allow the incorporated cannabinoids to enter directly into the lymphatic system, reducing the first-pass metabolism in the liver, resulting in tissue concentrations that were in a similar range to that of SES oil (Feng et al. 2021). Vegetable oils vary in fatty acid composition and distribution, leading to distinct physical properties (i.e. density of the chylomicrons formed) that alter intestinal lymphatic transport of co-administered cannabinoids post-oral consumption (Feng et al. 2022). SES oil is typically composed of > 80% unsaturated fatty acids, ~ 47% of n-6 PUFA are linoleic acid (LA; 18:2n6) and monounsaturated fatty acid (MUFA) are ~ 37% oleic acid (OA; 18:1n9) (Wei P et al. 2022; Oboulbiga et al. 2023). The EPA/DHA and DHA oil are > 60% n-3 PUFA and < 10% MUFA, a notable difference from SES oil. Previous research in rats found corn oil, consisting of OA and LA, and cod liver oil, of EPA and DHA, have similar quantities of triglycerides recovered from intestinal lymph 6H post-ingestion, indicating similar absorption profiles despite differences in fatty acid profiles. (Degrace et al. 1996). Additionally the largest chylomicrons formed are with corn oil, while cod liver oil results in smaller, more numerous chylomicrons (Degrace et al. 1996). Also, rats fed a diet rich in olive oil, with high levels of OA, had twice the lymphatic transport rate of FA at 8 H compared to those fed a high n-3 PUFA diet (Porsgaard and Hoy 2000). Research conducted in-vitro with Caco-2 cells found that EPA and DHA limit chylomicron assembly and secretion, whereas OA promotes chylomicron formation (Wang et al. 2014; Williams et al. 2004). Similar results in humans found orally consumed EPA and DHA decreased chylomicron size while accelerating clearance and reducing intestinal lymphatic transport of n-3 PUFA (Park and Harris 2003). Therefore, oils high in n-3 PUFAs and low in MUFAs limited lymphatic transport of co-administered cannabinoids, accounting for the lower tissue concentrations observed for EPA/DHA and DHA in the current study compared to that of SES oil.

The lower experimental doses are chosen to reflect quantities toleratable to acute human intake, which still elicit physiological effects reported in rodents (Dumbraveanu et al. 2023; Gorelick et al. 2013; Rock et al. 2020; Moore and Weerts 2022). A greater dose of CBD over THC was chosen because CBD is non-psychoactive, and previous research shows it is tolerated at higher doses in humans, with no significant side effects in adults orally consuming acute doses of 600–1500 mg of CBD (Bergamaschi et al. 2011). If an average adult male weighing 85 kg were to consume the dose used in our study, 5 mg/kg of CBD, that would equate to an acute dose of 425 mg, well within tolerable quantities. Previous studies using rodents provide an oral dose between 20–100 mg/kg, an unrealistic to achieve in humans (Yau et al. 2023). When considering chronic supplementation of CBD, research recommends more conservative doses of 30–160 mg/day CBD to be considered safe in healthy adults (Henderson et al. 2023). Additionally, in countries where recreational cannabis products are sold, the 425 mg quantity used in this study far exceeds the typical quantity of CBD sold in consumable products requiring consumers to purchase numerous items to achieve the same dosage (Tassone et al. 2023). Future studies must consider the upper dose limit of cannabinoids, route of administration, and study duration (acute vs. chronic) while maintaining the safety and applicability of results to healthy adults.

For the first time, the tissue distribution of major cannabinoids in six different tissues following oral consumption of CBD and THC with a lipid carrier shows a wide distribution of CBD and THC after 1H post-oral gavage (Tables 1 and 2). With CB₁ and CB₂ receptors being widely distributed throughout many tissues, it is important to consider the whole-body perspective, as many targets exist for cannabinoids to interact with and influence biological function and outcomes (Kilaru and Chapman 2020). Irrespective of carrier oil, T_max is most common at 2H post-oral gavage, which aligns with previous pharmacokinetic studies reporting T_max between 90–120 min (Millar et al. 2018; Lucas et al. 2018). Comparison between studies is challenging because of time intervals, lipid carrier, digestive conditions of each species, and cannabinoid dose each influence the outcome (Millar et al. 2018). A wide cannabinoid dose range from 1 mg/kg to > 100 mg/kg, is used across experiments, which dramatically impacts C_max in tissues as oral administration of cannabinoids is dose-dependent and variable (Millar et al. 2018; Huestis 2007; Yau et al. 2023). Studies of a similar CBD oral dosage, 15 mg/kg of CBD mixed with SES oil, present a T_max at 4H and a plasma/serum concentrations, C_max of 135.94 ± 38.15 ng/ml in rats (Fu et al. 2022). In rats, a 12 mg/kg CBD dose in SES oil reports a T_max at 3H post-oral gavage and a C_max of 164 ± 142 ng/ml (Brookes et al. 2023). The pharmacokinetic profile of an orally-gavaged 10 mg/kg CBD dose administered to rats in two separate studies both found T_max at 2H post-oral gavage and a C_max of 96.5 ± 17.3 and ~ 275 ng/ml, respectively (Hlozek et al. 2017; Schwotzer et al. 2023). Orally-consumed CBD with SES carrier oil in our study resulted in a T_max at 2H and C_max of 91.6 ± 42.4 ng/ml in serum, while the serum C_max of CBD with n-3 PUFA carrier oils was 9.1 ± 6.5 ng/ml for DHA and 19.3 ± 19.1 ng/ml for EPA/DHA, well below the reported values mentioned above.

The central nervous system contains the highest concentration of cannabinoid receptors and has other receptors that cannabinoids, notably CBD, interact with (e.g., peroxisome proliferator-activated receptor gamma (PPARγ), transient receptor potential vanilloid 1 (TRPV1), and the serotonin 1 A receptor (5-HT1A), making increased delivery to the brain a research priority (Yau et al. 2023; Tambe et al. 2023). Along with serum, the distribution of orally consumed cannabinoids is commonly reported in brain tissue exhibiting dose-dependent responses in humans and animals (Millar et al. 2018). The C_max of CBD in brain tissue is reported as 126 ± 9.5 and ~ 225 ng/g and a T_max at 2H (Hlozek et al. 2017; Schwotzer et al. 2023). Cannabinoids used to treat pain are studied in mice at concentrations ranging from 1 to 20 mg/kg CBD in SES oil; the 20 mg/kg dose C_max is ~ 200 ng/g, while a 1 mg/kg dose C_max is ~ 3 ng/g in brain tissue, and a T_max of 4H and 2H, respectively (Dumbraveanu et al. 2023). Similar to our study, a 5 mg/kg oral CBD dose in SES oil has a C_max of 30.3 ± 11.3 ng/g in brain tissue with a T_max at 2H. SES oil has a significantly greater C_max in brain tissue at all times compared to the n-3 PUFA oils (Table 1). CBD C_max is lowest in the brain across time points compared to the other tissues, which may be due to the selectivity of the BBB compared to other tissues (Calapai et al. 2020). A greater C_max in heart and adipose tissues (Tables 1 and 2) implies cannabinoids are distributed to other tissues before the brain following digestion, reducing the concentration of accessible cannabinoids to the brain.

The non-specific tissue distribution following ingestion limits oral cannabinoid delivery; thus, strategies to increase bioavailability and enhance targeted delivery, including lipid-based nanoparticles and self-emulsifying drug delivery systems, are needed (Reddy et al. 2023). We hypothesized that due to the high concentration of DHA in brain phospholipids (Lacombe et al. 2018), oral CBD combined with a DHA oil compared to an equal ratio of EPA/DHA oil results in a greater C_max in brain tissue, which is confirmed at 2H and 3H (Table 1). The carrier oil used has found to alter cannabinoid delivery to specific brain regions where SES oil more effectively increases CBD in the hippocampus, while lipid-free formulations result in more CBD in the cerebellum (Brookes et al. 2023). Improving the bioavailability and selectivity of oral formulations will yield more targeted tissue distribution of cannabinoids and tailored health outcomes.

The tissue distribution of orally consumed cannabinoids is limited despite the increase in orally ingestible cannabis products on the market (Hammond et al. 2022). Heart tissue has the greatest CBD concentration for all carrier oils at 1H post-oral gavage (Table 1) (SES: 103.4 ± 25.6 ng/g; EPA/DHA: 24.2 ± 7.8 ng/g; DHA: 15.8 ± 5.6 ng/g). Cannabinoids are distributed into organs and tissues that are well vascularized, and since all blood must flow through the heart, the elevated concentration is not unexpected (Chayasirisobhon 2020; Kreuz and Axelrod 1973; Ruiz et al. 2021). CBD elicits many beneficial effects on heart health, such as lowering CVD risk via stimulating anti-inflammatory pathways of CB₂ and TRPV1 receptors (Kicman A 2020; Fulmer and Thewke 2018). Dosages used in previous works report CBD concentration of adipose, liver, and muscle tissue upwards of 40 × greater than this study, making direct comparisons difficult due to the dose-dependent nature of orally consumed cannabinoids. At 2H post-oral gavage, heart tissue has the highest CBD C_max (SES: 231.8 ± 82.4 ng/g; EPA/DHA: 45.5 ± 34.0 ng/g; DHA: 25.1 ± 19.4 ng/g). At 3H, a notable shift shows an increased concentration of CBD in adipose tissue overtaking heart tissue concentration (SES: 205.8 ± 120.7 ng/g; EPA/DHA: 50.4 ± 34.0 ng/g; DHA: 29.0 ± 18.7 ng/g). Adipose tissue effectively stores cannabinoids long-term, with previous studies reporting cannabinoid concentrations in adipose days and even weeks post-ingestion (Kreuz and Axelrod 1973; Gunasekaran et al. 2009). Child and Tallon (Child RB 2022) found that at 28 days post-oral gavage of CBD given to rats significantly increased concentrations in adipose tissue compared to skeletal muscle (gastrocnemius) and the liver, supporting findings herein where adipose tissues reached their peak concentration at 3H. Although liver tissue has the third highest concentration of CBD after heart and adipose tissue, the C_max of THC is consistently the highest in the liver (Table 2). While CBD and THC share similar structural characteristics and metabolism (Huestis 2007), a greater metabolic rate in the liver for THC than CBD, coupled with more CBD excreted in feces, may account for the differences observed herein (Rao Q et al. 2022). Future studies examining the distribution and concentration of orally consumed cannabinoids in the liver and other relevant tissues are necessary to develop specific pharmacokinetic profiles that improve oral dosing.