The findings of the study on canine plasma following a single administration of 5 mg/kg CBD-infused oil (OG) and 50 mg of CBD-mixed as a treat (TG) over 30 days provided valuable insights into the pharmacokinetics of CBD in dogs. The pharmacokinetic profile of CBD in canines has been extensively investigated, with numerous studies elucidating its behavior in this species [10]. Typically, it is characterized by low oral bioavailability [15,17] and moderate distribution [6,25].

The Cmax and AUC values observed in this study are consistent with previous research on CBD pharmacokinetics in dogs, contingent upon the formulation and dosage [5,11,14,17]. Furthermore, the Cmax in the TG group was comparable to the absorption levels reported in earlier studies involving soft chews [11]. The higher Cmax and AUC in the OG group suggest that this formulation may enhance CBD absorption compared to the TG group. This observation aligns with the findings that dogs administered CBD in an oil-based formulation exhibited a higher Cmax and AUC compared to those given a capsule formulation [17]. These results corroborate the existing literature, indicating that the formulation type significantly influences CBD absorption in canines. For instance, a study comparing CBD-infused oil to microencapsulated CBD oil beads found that the microencapsulated beads resulted in lower Cmax and AUC values, suggesting reduced bioavailability compared to the oil formulation [10]. Another investigation evaluated a soft chew containing a 50:50 mixture of CBD and cannabidiolic acid (CBDA) administered at 2 mg/kg, achieving a Cmax of approximately 300 ng/mL for CBD, which was higher than the ~100 ng/mL observed with an oil-based formulation at the same dosage [11]. This suggests that certain edible formulations may enhance CBD absorption. Additionally, a study assessing various CBD preparations found that the liquid formulations (oil-based, nanoemulsion-based, and water-soluble) provided higher bioavailability and faster absorption compared to the semi-solid form [14]. The relative bioavailability of CBD in the TG compared to the OG group indicated a reduced overall exposure and peak concentration in the treat formulation. The current findings confirmed a trend for oil formulations to result in a faster Tmax than treat or soft-gel formulations [11,14]. Furthermore, solid dosage forms tend to have reduced bioavailability, highlighting the challenge of formulating CBD effectively in these forms. The treat formulation likely presented a different matrix that may have hindered the dissolution and absorption of CBD, leading to reduced bioavailability. The treat’s ingredients and excipient interaction with CBD may reduce releasing and/or absorption in the gastrointestinal tract. Collectively, the treat formulation in this study required nearly a two times higher dose than the oil formulation to achieve comparable AUC values, suggesting reduced bioavailability due to food matrix interactions. Despite differing doses, a dose-normalized Cmax and AUC were analyzed to assess relative absorption. The longer Tmax for the snack form indicates a delayed release, which may influence dosing regimens in clinical use.

In contrast, soft-gel capsules showed higher maximum serum concentrations for CBD and its metabolites compared to the oil formulations [13]. These studies underscore the impact of formulation on CBD pharmacokinetics in dogs, with oil-based and certain edible forms demonstrating varying degrees of bioavailability, highlighting the necessity of considering the formulation type when evaluating CBD absorption and therapeutic efficacy in canines.

It is widely recognized that plasma concentrations of CBD in canines are influenced by the route of administration, dosage, formulation characteristics, and individual factors. It is noteworthy that the oral transmucosal administration of 1 mg/kg CBD resulted in a mean Cmax comparable to that observed with oral administration [26]. This finding implies that the administration route may not significantly affect plasma concentrations at relatively low doses.

The present data corroborate the accumulation of CBD in plasma over time, consistent with previous studies that indicate that repeated administration results in elevated plasma levels, thereby supporting the plasma accumulation hypothesis [9,12]. However, upon cessation, CBD levels in the current study declined rapidly, suggesting the presence of active clearance mechanisms. Vaughn and colleagues demonstrated that CBD exhibits linear pharmacokinetics with repeated administration, where plasma concentrations increase proportionally with the dose [12]. As the dose increases, the volume of distribution for CBD may also increase due to its lipophilicity and tendency to accumulate in fatty tissues. This can lead to greater systemic exposure over time with repeated dosing [5].

The observed plasma accumulation and extended elimination half-life suggest that CBD may reach steady-state levels with chronic administration, reinforcing its predictable pharmacokinetic profile in canines. This was further substantiated by a study indicating that chronic administration of CBD to dogs resulted in dose-proportional accumulation over 36 weeks, with an increased half-life, total exposure, mean residence time, and plasma peak level [9].

CBD generally has a short half-life, lasting a few hours after a single administration. The current findings showed that the half-life was similar between the OG and TG groups, with no significant difference, suggesting comparable elimination rates of CBD in both groups. This study confirmed previous findings that the half-life of CBD in dogs ranges from approximately 6 to 10 h [12,14,15,17]. This may support the hypothesis that CBD pharmacokinetics are largely independent of minor formulation changes, at least in terms of elimination.

The present study utilized refined rice bran oil enriched with γ-oryzanol as a carrier for CBD, distinguishing it from prior canine pharmacokinetic studies that predominantly employed different oily vehicles, including sunflower lecithin, medium-chain triglyceride (MCT) oil, and sesame oil [5,11,13,17]. MCT oils are widely used in canine formulations; this may be due to their rapid absorption via portal circulation [27], though their lack of endogenous antioxidants may limit long-term stability compared to plant-derived oils like rice bran oil.

To our knowledge, this represents the first systematic evaluation of rice bran oil for CBD delivery in veterinary applications. Its balanced fatty acid profile (approximately 20% saturated, 40% monounsaturated, and 35% polyunsaturated) coupled with native antioxidant compounds (notably γ-oryzanol at 1.5–2.5% w/w) may offer distinct advantages for chronic administration. γ-Oryzanol not only stabilizes the formulation against oxidative degradation [28] but may also exert synergistic anti-inflammatory effects with CBD, as demonstrated in murine models [29]. These pharmacodynamic interactions warrant further investigation in canine systems.

Additionally, dosing frequency alteration may be necessary to maintain therapeutic effects, particularly for conditions requiring long-term management. However, inter-study variability highlights the impact of multiple factors on pharmacokinetic outcomes. Taken together, CBD pharmacokinetics are known to be highly variable in dogs, both within and between studies. Factors such as breed, individual metabolic differences, dosage, formulation, feeding status, and duration of administration can all influence plasma concentrations [5,14,25,30].

While CBD is generally well-tolerated in canines, its effects on blood parameter profiles have yielded mixed results, particularly concerning the liver enzymes and other biochemical markers [5,15,19,31]. The present study identified alterations in specific hematological parameters. Notably, within the erythrocyte profile, the OG group exhibited a significant increase in red blood cell (RBC) count and hemoglobin levels, along with a near-significant rise in hematocrit, suggesting mild erythropoietic stimulation. CBD interacts with the endocannabinoid system, which plays a role in hematopoiesis. Some studies suggested that endocannabinoids can stimulate hematopoiesis [32]. Controversially, an in vitro study suggested that CBD may affect the integrity of erythrocytes, potentially leading to the formation of hemolytic vesicles [33].

Regarding the leukocyte profile, a significant decrease in eosinophil levels was observed in both groups, indicating potential anti-inflammatory or anti-allergic effects. In a murine asthma model, administration of CBD-X resulted in a significant reduction in eosinophil infiltration within lung tissues, suggesting its potential to mitigate eosinophil-driven inflammation [34]. This finding implies that CBD may exert anti-inflammatory effects by modulating immune cell activity, particularly by reducing eosinophil counts. However, the OG group demonstrated reduced lymphocytes, possibly reflecting immunomodulatory activity. For platelet and coagulation markers, the mean platelet volume (MPV) and platelet large cell ratio (P-LCR) exhibited significant reductions in both groups, suggesting CBD may influence platelet activation or turnover.

In relation to the biochemical safety profiles observed in this study, minor fluctuations were noted in the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Specifically, the AST levels exhibited a slight increase in the OG group, while the ALT levels decreased in the TG group. Alkaline phosphatase (ALP) demonstrated a significant reduction in the TG group, possibly due to formulation-specific effects. However, the levels of ALT, AST, and ALP remained within normal parameters, thereby confirming the absence of hepatotoxicity concerning liver function. Certain studies reported that most hematological and serum chemistry variables remained within normal ranges for both the CBD-treated and control groups; some dogs showed elevated alkaline phosphatase levels in response to CBD treatment [19,20]. This indicates that individual dogs may respond differently to CBD supplementation, possibly due to variations in metabolism or sensitivity. Additionally, blood urea nitrogen (BUN) and creatinine (CRE) exhibited no significant variations, indicating no renal impairment. However, with respect to protein metabolism, there was a significant increase in total protein and albumin levels in both the OG and TG groups, suggesting enhanced protein synthesis or retention. A recent investigation into the safety and pharmacokinetics of a CBD-rich hemp extract in rats over a 90-day period found that female rats developed significant hyperalbuminemia following CBD administration, whereas male rats did not exhibit this increase [35]. This outcome contrasts with findings from another study, which identified significant differences in albumin levels between treatment groups at weeks 18 and 26, with mean concentrations approximately 2 g/L lower in CBD-treated dogs. Nonetheless, these values remained within the normal reference range [19]. A non-significant positive association was observed in several parameters, with no obvious clinical incidence of adverse events in this current study.

Collectively, the OG group demonstrated erythropoietic and anti-inflammatory effects without adverse hepatic or renal impact. Additionally, the TG group exhibited mild immunomodulatory effects (eosinophil reduction) but no significant hematological alterations. Both formulations were well-tolerated over 30 days, supporting their safety in canine use. However, while CBD is generally well-tolerated in dogs, elevated liver enzymes (particularly ALP) are the most notable adverse effect, warranting periodic monitoring.

Certain limitations of this study merit consideration, particularly its small sample size consisting of crossbreed dogs. This may have resulted in the limited statistical power, thereby increasing the risk of not identifying clinically significant differences in the pharmacokinetic and blood parameters between groups. Moreover, this study could not thoroughly assess inter-individual variability in CBD metabolism, which may be influenced by factors such as CYP450 polymorphisms or body composition, thus constraining the generalizability of the results. It should be noted that inter-individual variation in drug responsiveness is not limited to CBD but is a common phenomenon in laboratory animals. Löscher (2024) emphasizes that despite standardized breeding and husbandry, phenotypic diversity exists in laboratory mice and rats, affecting various characteristics of animal disease models, including drug responsiveness. This variation can lead to misinterpretations when data are averaged within experimental groups, as divergent responses may be concealed [36]. The use of different doses between formulations in this study may introduce confounding factors in direct pharmacokinetic comparisons, despite dose-normalization adjustments. Furthermore, the food matrix of the snack formulation (e.g., fat content) may have independently influenced drug absorption, thereby introducing variabilities that are not fully accounted for in the analysis. Additionally, the assumption of dose-linear pharmacokinetics may not be valid if saturation effects occur at higher doses. These factors should be considered when evaluating the therapeutic implications under real-world dosing conditions. Future research involving larger and more diverse cohorts is necessary to validate these preliminary results and to refine the dosing recommendations.