The cannabis plant contains more than 400 compounds, including over 120 phytocannabinoids, more than 140 terpenoids, as well as various flavonoids and alkaloids. Cannabigerolic acid (CBGA) is the precursor to other major cannabinoids including Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabichromene (17). While many of these compounds have yet to be fully characterized, isolated forms such as THC and CBD have been well-studied in medicinal research. Although several studies have investigated the safety and tolerability of the decarboxylated forms of cannabinoids, particularly CBD in dogs, data on the safety and tolerability of full-spectrum and acidic forms remain limited (5, 6, 13, 17, 18).

In the cannabis plant, cannabinoids and other compounds function synergistically as secondary metabolites serving as antioxidants and contributing to the plant’s defense against predators (18). Following purification, cannabinoids can be present in various formats, including as isolates of single cannabinoids either in their decarboxylated or acidic forms, as full spectrum extracts preserving the plant’s native cannabinoid and terpene profile, or as mixtures containing two or more cannabinoids. The term “entourage effect” refers to the enhance overall impact when cannabinoids are used in combination with other minor compounds, e.g., terpenoids and flavonoids (9).

For the acidic form, pharmacokinetic studies have demonstrated its safety when administered to rabbits, horses, cats, dogs, humans and parrots (13–15, 19–21), with all of those studies suggesting superior absorption of CBDA (6, 13, 14, 17–19). Prior data from human pharmacokinetics have shown that CBD absorption appears to be enhanced when a portion of CBDA is delivered together (10). Further examination of rodents (mice) has suggested the CBDA absorption is relatively poor as an isolate, and the absorption was nearly 14 fold better while delivering in a full spectrum mixture. It was proposed that the other minor cannabinoids allowed for inhibition of enterocyte efflux enzymes allowing for better absorption as the primary mechanism (9). Therefore it is expected that there may be differing effects of full spectrum products compared to isolates which has been reported in the human literature. Full-spectrum cannabinoids are suggested to enhance symptom relief when treating mood or anxiety disorders, as well as seizures (22). Although we found little evidence to support that the major cannabinoids CBD and CBDA were enhanced by administrated as full spectrum products, there appears to be a numerical trend over 7 days on full spectrum CBDA absorption, therefore the “entourage effect” is not likely to be related to the major cannabinoid absorption, but rather to the minor components found in full spectrum products. However, our data is limited, and additional studies are needed to determine these concentrations at steady state.

Pharmacokinetic data that compares full spectrum cannabinoids to isolate cannabinoid forms was unknown in dogs prior to this study. The objectives of this study were to (1) determine the pharmacokinetics of full-spectrum hemp extract and cannabinoid isolates including quantification of CBD metabolites (6-OH-CBD, 7-COOH-CBD) in healthy adult dogs, and to (2) assess the tolerability and safety of orally dosed cannabinoid isolates (CBD, CBDA) and a hemp extract in adult beagle dogs. It was hypothesized that, compared to the isolate cannabinoid (CBD), acidic cannabinoids (CBDA) and full-spectrum cannabinoids would exhibit greater absorption in adult dogs. No significant changes in complete blood count (CBC) and serum chemistry profiles were anticipated during the one-week period of cannabinoid administration.

Earlier studies have reported that the oral bioavailability of cannabinoids in dogs is low due to extensive first-pass metabolism (23, 24). However, transdermal administration of cannabinoids has not resulted in serum concentrations comparable to those achieved with oral dosing (25). Due to the lipophilic nature of cannabinoids, and the stratum corneum, their absorption through the skin epithelium is limited (16).

In our study, the absorption of orally dosed CBD isolate at 1 mg/kg BW was low (69.8 ± 35.44 ng/mL) and comparable to that of other studies. Gamble et al. reported Cmax value of 102 ng/mL for CBD dosed at 1 mg/kg BW in a long chain triglyceride oil vehicle format to fasted dogs. Wakshlag et al. reported Cmax values of 145 ± 69 and 124 ± 62 ng/mL, respectively, for CBD administered at the same dose (1 mg/kg BW) formulated with either a mixture of medium chain triglycerides (MCT) and a sesame oil or a mixture of sunflower lecithin and sesame oil. Della Rocca et al. reported a Cmax of 206.77 ± 167.07 ng/mL for CBD administered at 1 mg/kg BW in MCT oil. In contrast, Vaughn et al. reported a significantly lower Cmax 30 ± 7 ng/mL for the same CBD dose and carrier oil. In contrast, in the studies by Deabold et al. and Wakshlag et al., when CBD was dosed at 1 mg/kg using an enriched soft chew, Cmax was greater, with values of 301 ± 63 ng/mL and 226 ± 89 ng/mL, respectively. Therefore, the type of carrier vehicle to dose CBD appears to be a driving factor in CBD absorption. In addition to carrier types, various absorption of cannabinoids were observed in other species with fasted animals or fed before dosing (13, 20). In our study, dogs were fed dry kibble before dosing, and wet food immediately after. At the same dosage (1 mg/kg), the CBD AUC observed in this study (420.61 ± 45.52 ng-h/mL) was comparable to that reported by Della Roca et al. (536.05 ng-h/mL) using MCT oil as carrier vehicle. In Doran et al., feeding dogs before dosing showed a twofold increase in AUC at 5, 10, 20 mg/kg dosage. Although additional feeding did not lead to a higher AUC in our study, feeding state and encapsulation materials used could be the other driving factors when considering cannabinoids absorption (3, 26).

In our study, Cmax, AUC, and Css for CBDA isolates were greater than those observed for the CBD isolate. These findings support previous research in dogs, cats, horses and steers, suggesting that the acidic form of cannabinoids exhibit superior absorption compared to their decarboxylated counterparts in mammalian species (7, 14, 15, 19). Similar to results reported by Gamble et al., co-administration of CBDA and CBD (each at 1 mg/kg) did not enhance the Cmax of CBD (99 ± 29.13 ng/mL). Likewise, in our study, the presence of CBDA in the CBD/CBDA FS mixture did not influence the Cmax of CBD when compared to the CBD isolate (64.66 ± 37.05 ng/mL vs. 69.8 ± 35.44 ng/mL, respectively). In contrast, Tittle et al. reported higher Cmax values for CBD in dogs dosed with a CBD-rich hemp extract formulated as either a soft gel (267.6 ± 98.9 ng/mL) or in sesame oil (184.5 ± 55.8 ng/mL), with both formulations containing CBD and CBDA at 1 mg/kg. This discrepancy may indicate that the absorption of acidic cannabinoids is less affected by feeding status compared to their decarboxylated counterparts.

The Cmax and AUC values for CBDA in CBD/CBDA FS were greater compared to those for CBD in CBD/CBDA FS. This is the first study that compares CBD and CBDA pharmacokinetics in a full spectrum mixture. This finding aligns closely with the CBDA isolate treatment, which had greater absorption compared to CBD isolate treatment. CBDA has a low affinity for both CB1 or CB2, is one of the minor cannabinoids that functions as allosteric regulators for 5-HT1α receptors, and reported to be 1,000 time more potent than CBD in binding to these receptor sites (27). CBDA also showed better anti-hyperalgesia, anti-nausea and anti-seizure effects in rodent models (18). Those findings in the rodent studies align with the previous studies in dogs, cats and horses (7, 15, 21, 28), suggesting a better potency in the cannabinoid acidic form.

The Cmax for CBDA in the CBD/CBDA FS mixture remained unaffected with the addition of CBD, other minor cannabinoids, terpenes and flavonoids in the mixture, compared to the CBDA isolate (229.30 ± 145.51 vs. 235.51 ± 65.59) and similarly for Tmax, AUC and Css. When comparing the CBDA FS to CBDA isolate, no differences between these treatments were observed, similar to CBD results, suggesting that FS formulations play little role in the absorption and or/retention of CBD or CBDA.

The time-course serum concentrations of two primary CBD metabolites 6-OH-CBD and 7-COOH-CBD (Supplementary Figures 2–4) indicate potential treatment-dependent differences in metabolite levels. Interpretation of these data should be made with caution, as several time points were below the detection limit, and proper statistical analyses could not be conducted. In Supplementary Figure 2, dogs receiving the CBD isolate for 1 week appeared to show slightly higher 6-OH-CBD levels than those receiving the CBD/CBDA FS treatment, with peak concentration of 6-OH-CBD for both groups occurring between 2 and 4 h. Furthermore, 6-OH-CBD appeared higher than the traditional metabolite 7-COOH-CBD, as supported by previous studies in dogs (3), suggesting that dogs undergo differential metabolism of CBD when compared to humans and horses. These results are consistent with the known first-pass hepatic metabolism, where 6-OH-CBD serves as a primary oxidative metabolite that rapidly forms and either clears through renal excretion or undergoes further unidentified metabolism for biliary excretion (29). Moreover, prior pharmacokinetic studies has shown that dogs exhibit only 1–2% of the 7-COOH-CBD levels observed in humans receiving similar dosages (3, 7).

Further research is warranted to evaluate the biological activity and potency of 6-OH-CBD as a minor metabolite in dogs. In addition, CBDA metabolism has not been thoroughly investigated with only one study in extrahepatic microsomes suggesting that there is direct glucuronidation of CBDA at the acidic carboxylic acid (30). These findings reinforce that cannabinoid metabolism is sensitive to formulation, feeding status, and co-administered compounds, and that species-specific differences in CBD metabolism are likely substantial.

While metabolic differences were observed between treatments, it is important to also consider how the presence of additional cannabinoids and terpenes may influence the pharmacological effects of CBD through the proposed entourage effect. The entourage effect was first suggested in a preclinical study in 1998 when describing metabolites interactions (31). This term was later widely used in the field of medicinal cannabinoids, as the example of polypharmacy (use of multiple drugs to treat a single condition), e.g., full spectrum cannabinoids. The potential entourage effect can be variable (32). One of the common misleading concepts is that the entourage effects are often described as beneficial synergistic effects, while adverse effects, either caused by antagonism or additive effects are often ignored, leading to a public awareness of the beneficial sides only of the entourage effect (33). While many studies have emphasized the entourage effect in both humans and animals, limited research has been done to provide robust evidence. The pharmacokinetic results of this study do not support a better absorption of either CBD or CBDA when provided in a full spectrum. This may be due to the industrial purification methods, or that the specific cannabis chemovars used in this study did not contain the effective terpenoids or flavonoids or proportions of minor cannabinoids to promote enhanced absorption.

In our study, no adverse events were associated with a 7-day administration of CBD or CBDA isolates, or of these cannabinoids in various combinations and full spectrum. There was no elevated serum ALT, which has been observed in previous studies in cats and dogs (5, 25) nor the more innocuous ALP enzyme in this study. However, to thoroughly understand the mechanisms and hepatic safety behind the entourage effect, additional research is needed.

The results of this study are the first of a comprehensive comparison between full-spectrum, isolate, and acidic forms of cannabinoids. The study further supports that CBDA, the acidic form of CBD, is absorbed better in dogs. Previous research in mouse suggests that CBDA interacts with transporter breast cancer resistance protein (BCRP), and that CBG and THC inhibit BCRP-mediated transport of CBDA, thus leading to an elevated plasma concentration for CBDA (9). In our study, we found no evidence that full-spectrum cannabinoids have enhanced absorption in dogs. Therefore, there is a need for further validation with isolate CBDA and addition of CBG to test out the entourage effect in the canine model, as it relates to receptor biology rather than cannabinoids promoting synergistic absorption. Based on the physical assessment, the lack of adverse events, and CBC and chemical profiles falling within reference ranges, CBD, CBDA, and these isolates combined or presented in full spectrum are safe for dogs when provided at 1 mg/kg every 12 h during a one-week treatment protocol.

Due to the complex variations in cannabis chemovars, the findings are only applicable to the specific formulations tested in this study. Additionally, due to the limitations of the small sample size and the short duration of the trial, the generalizability remains limited. Future studies with larger sample sizes, different ages and breeds, and longer trial durations are necessary before providing comprehensive guidelines for cannabinoid usage in veterinary medicine.