Utilizing cannabis-related products to achieve favorable health impacts is rapidly increasing following its legalization in some parts of the world. In June 2022, Thailand removed marijuana from its narcotics list and became the first Asian nation to approve cannabis for medicinal and industrial use (26). Cannabis use is of interest not only in human medicine, but also in veterinary medicine, where extensive research is warranted to better understand the behavior and impact of the drug after administration in animals, since interspecies differences are a main factor influencing PK variation (27). Dogs have been the main companion animal species studied; however, the published PK studies have mainly been on oil-based CBD (11, 15, 28).

The current study was designed to explore the PK patterns of CBD in the plasma from different preparation forms—liquid (the OM, NM, and WM groups) and semi-solid (the CM group)—following a single oral administration in overnight fasted crossbreed dogs. It is known that CBD is a lipophilic compound with limited absorption into circulating blood (12). Several reports on the PK of CBD in dogs have studied CBD in oil-based formulations, microencapsulated-beads, chewable soft capsules, and soft gel capsules (11, 15–18). However, comparison data of oral CBD profiles in companion animals with different formulations are scarce. To the best knowledge of the authors, this is the first report on CBD in nanoemulsion and water-soluble forms in dogs. In addition, according to the limited available information on CBD in semi-solid form, this study has presented plasma CBD behavior following snack-as-treat ingestion in dogs.

Variation in the PK pattern of plasma CBD arises from an extensively first-pass metabolism and its low aqueous solubility that leads to poor bioavailability and a poor biological effect (12, 14). Commonly, inconsistent and variable systemic drug exposure are affected by multiple factors, including route of administration, dosage form, dose range, and health and feeding status. It has been noted that differences in the study design, including animal signalment (breed, sex, age) and status, sampling time point, and determination method, may affect PK outcomes; therefore, it is inappropriate to directly compare those estimated PK parameters between various experiments (29).

The oral bioavailability of CBD in dogs has been estimated to be in range 13–19% (13). Improving CBD delivery into the blood stream and its efficacy via an oral route is challenging to achieve a therapeutic response. Specifically, numerous studies have been conducted with various developed CBD preparations to increase the oral bioavailability and PK evaluation in dogs (11, 15–18).

The current results indicated there were no significant differences between all PK profiles across the CBD delivered in liquid form. However, comparison of the CBD in liquid forms against the semi-solid form identified differences in the Vz/F, CL/F, AUC_0-t, and AUC_0-inf parameters. The current findings showed that the CBD behavior profiles in the WM group were comparable to those in the OM group. Although the highest C_max was in the WM group, it was a noticeably high variation, including for the MRT_inf, T_1/2, and AUC parameters. This might indicate that the estimated rate of absorption in water-soluble dosage form had larger bioavailability variation than for the oil-based form. Coincidentally, another study reported that a water-based formulation of CBD, which had a similar composition to the WM prepared in the current study also reported statistically comparable PK parameters in human plasma compared to that of the CBD in human plasma after oral administration of the oil-based formulation of the CBD (5).

There has been a wide range reported of the CBD maximum concentration following oral administration of oil-based CBD formulations (11). Compared to another experiment, in which there was oil-based administration at the same dose in fasted dogs, the C_max value in the OM group in the current report was about twice that of the earlier report (30). In contrast, another experiment that involved drug administration to fed dogs with an equal adjusted dose of CBD-infused oil had a C_max that was around twice that of the current study (18).

Several factors influence the bioavailability and disposition of CBD, resulting in relatively high intra- and inter-individual variability in the PK profiles. Co-consumption of CBD with food, particularly in a fat meal, may alter the rate and extent of absorption. It has been reported that in humans, CBD plasma levels increased when concomitantly administered with food or in a fed state (31, 32). Likewise, positive food effects have been associated with increased maximum systemic exposure without affecting the AUC_0-t in dogs; however, higher C_max and AUC levels were observed in one out of three fasted dogs (30). Contrary to these results, Vaughn et al. (20) argued that overnight fasting with dogs might enhance the systemic absorption of CBD. In rabbits, it has been reported that feeding decreased systemic CBD absorption (33). In fact, fasted-fed variability is affected by various factors, such as the physiological condition, demographic and genetic factors, chemical, and formulation-related factors (34).

A single dose administration in the current study presented fluctuations of CBD plasma concentrations both within and between groups. Recently, it has been suggested that giving CBD twice daily may reduce the variation in plasma concentration (28). In addition, the maximum CBD plasma concentration has been reported to increase in a dose-dependent manner but some studies seemed not to be linear (16, 28–30).

Notably, the current findings corroborated the phenomenon of the so called ‘secondary peak’ as it was found in one dog in every group except the NM group. At first, this was considered as a possible error in the sample preparation or related to the laboratory process; however, it was confirmed following double checking and determining with different instruments of detection. The explanation for this phenomenon is not yet clearly understood. The double peak of CBD found in the plasma has been suggested to have been caused by a combination mechanism, such as enterohepatic recycling and intestinal lymphatic absorption (35). In addition, the CBD secondary peak has been reported in dogs given a medium (5 mg/kg) or high (10 mg/kg) dose rather than a low (2 mg/kg) dose, with coprophagia being one possible explanation (29).

However, the absence of a secondary peak of CBD in the dog plasma after orally taking NM form may have been due to the small size of the oil droplets in the nanoemulsion. Consequently, they had a larger surface area and so were efficiently exposed to the intestinal lipase at its binding sites (36). Therefore, the CBD in the oil droplets of the nanoemulsion was absorbed after the oil was digested and rapidly transformed into the primary derivatives (CBD-7-COOH and OH-7-CBD) through a first-pass metabolism process that resulted in a rapid decrease in the CBD concentration in the plasma (37). Finally, the content of the plasma CBD in the NM form could not be detected as a second peak, as occurred for CBD in the other dosage forms.

In all four dosage forms, the CBD was rapidly absorbed with mean maximum plasma concentrations occurring in the range 2–3.2 h post-dose. This was in agreement with other reports, where the times to reach the maximum plasma concentration were within 1–4 h, with no effect of dose amount or duration of exposure (6, 15, 16, 29).

The current results showed that the extent and rate of CBD systemic exposure in the OM group was highly absorbed. The lowest extent of absorption for the CM formulation compared to the other formulations was confirmed by the relative bioavailability value. The low plasma levels of the CM group could have been due to the low oral bioavailability of the semi-solid formulation, considering that the AUC was significantly lower than for the oil-based formulation (OM group), with the value for Vz/F and CL/F being significant higher than for the OM group. CBD degradation from the treats snack in the CM group following the heat process in preparation was ruled out because the CBD concentration was re-checked prior to being given to the dogs. Unlike the semi-solid form preparation in the current experiment, another study found that CBD in a soft chew format had high absorption with a delayed time to reach its peak, confirming that differently formulated preparations affect the PK outcome (15). As such, liquid and solid forms may alter the rate of absorption and total bioavailability.

Notably, CBD in the NM group followed by the WM group peaked sooner than the CBD in the OM group, which may support the rapid onset of an effect. The effect of nanoemulsion based on rapid oral absorption of the CBD found in the current study was consistent with the results reported by Yen et al. (38), who found that an andrographolide-loaded nanoemulsion was rapidly absorbed via the gastrointestinal tract because the surfactant molecules in the formulation (Tween 80 and Span 80) could suppress the function of P-gp, which inhibited drug secretion by the P-gp-mediated efflux process in the intestinal tissue. Therefore, the shorter time to reach the CBD peak from oral administration of the NM group may have been due to the effect of these particular surfactants.

In fact, oral drug absorption depends on the conditions in the gastro-intestinal tract. Consequently, it was possible that fasting the animals overnight in the current study might have shortened the time to peak concentration for the NM and WM forms. The interaction of PK properties and physiological features in the empty gastro-intestinal tract, including enteric epithelium and influx-efflux transporters, may have hindered this phenomenon; however, the mechanism has not yet been well elucidated.

Currently, there is a lack of research into using a nanoparticle-based approach with different techniques and routes of application to enhance CBD uptake (12). Development and commercial scale production of cannabinoid-loaded nanoemulsions have been highlighted to improve the absorption rate and efficacy for therapeutic purposes (39). The current findings showed that CBD in a nanoemulsion-based formulation tended to achieve rapid absorption, avoiding any fluctuations in kinetic behavior. A similar finding was recently reported, whereby a nanoemulsifying-CBD formulation had a shorter time to reach C_max compared to CBD in an oil-based form (35). In addition, it has been mentioned that the nanoemulsion formulated may have improved the rate and variability of absorption (40). However, notably, different CBD nanocarriers delivered different C_max and T_max, outcomes at a particular site of action (21).

Little information is available on CBD volume distribution in dogs. The larger values of the apparent volume of distribution in the liquid forms in the current study seem to suggest the CBD was more likely retained in the body than circulated in the blood. The current results had a Vz/F value nearly triple that of an oil-based treatment with the same dose in another study (28). In humans, it is evident that CBD is rapidly distributed throughout the tissues resulting in very high volume of distribution (41). Notably, the nanoemulsion-based treatment had a significantly higher volume of distribution compared to the semi-solid form. The modifying mode of delivery in the nanocarrier formulation may have enhanced the dispersion of CBD throughout the body. Clinically, CBD could be administered in multiple doses over several days up to a month for a therapeutic effect (18). Thus, the tissue distribution ratio should be considered of the CBD dispersed among physiological tissue and accumulated in parts of the body (20). There should be further study of the biodistribution of different CBD preparations within therapeutic sites of interest in the target animal.

Despite scarce scientific information, the use of CBD in dogs has been of broad interest to owners, based on anecdotal evidence of its therapeutic benefits. Notably the current study was conducted with a non-Beagle breed which may not be directly comparable to reported studies involving a Beagle breed. However, based on visual assessment, the crossbreed dog PK parameters of CBD did not show any significant differences from those in the study conducted with Beagle dogs (11, 28). The crossbreed dog population is estimated at 31–53% in the United States, Germany, and the UK (42). In Thailand, based on domestic survey data of pet owners, crossbreed dogs constitute approximately 29%. A limitation of the current study was the small number of crossbreed dogs with only 8 per group. An another limitation is that less frequently in blood sample collection after 10 h post-dose which may cause insufficient data points in the elimination phase. Since there is a wide range of crossbreed dogs, with undoubtedly differences in response to drug behavior, it is unclear whether the plasma concentrations in the current study can be considered representative of the general population of crossbreed or different-sized dogs. The differences in the CBD absorption rate and its metabolic action across dogs has resulted in high variability in plasma concentrations (20). The interpretation and practical use of the available pharmacokinetic data from the current single-dose study should be further investigated. Nonetheless, the novelty of this study is the generation of data relevant to different CBD forms and its behavior in dogs.