During the investigation, the CBD product was easily administered and well tolerated by all ponies, with no side effects observed throughout the experiment. CBD was detected in all subjects 48 h after administration. In 6 ponies, CBD concentrations were below LOQ after 72 h (3 days), and in 4 after 168 h (7 days) (Figure 1). The next sampling occurred on day 12, which is a limitation of the present study as the exact time of CBD elimination from all ponies is unknown. However, CBD was not detectable above the quantitation limit (0.100 ng/mL) in any of the horses on day 12. In the course of the research, the pharmacokinetic analysis showed a rapid increase of CBD in plasma and the mean maximum plasma concentration of CBD was 31.472 ± 8.080 ng/mL following the administration of 2 mg/kg CBD oil. As previously mentioned, commonly used oral CBD doses in horses range from 0.1 to 3 mg/kg, with the most frequently reported maximum serum plasma CBD concentrations being less than 20 ng/mL (32, 36–45). The plasma concentration achieved in the present study is, therefore, considered relatively high. Due to the small amount administered orally with a dosing syringe, oromucosal absorption cannot be ruled out. This, by bypassing first-pass metabolism, may have contributed to the relatively high plasma concentration. The influence of high-fat meals on the intestinal absorption of CBD has been well-documented (59), with absorption enhanced due to its lipophilic nature. Accordingly, a coconut oil-based formulation containing medium-chain triglycerides (MCTs) was used. MCTs, a type of medium-chain fatty acid, do not require bile acids for digestion, allowing them to be quickly absorbed and used as an immediate energy source. This may have contributed to the observed peak plasma concentration in the present study.

The pharmacokinetic profile of CBD is influenced by several factors, including the dose, pharmaceutical formulation, route of administration, the type of use (acute vs. chronic), and the individual characteristics of the patient (15). Previous observations have shown that with oral administration, an increase in dose results in a corresponding rise in maximum plasma concentration (34). Thomson et al. (60) reported a plasma concentration of 40.350 (27.700–52.170) ng/mL after administering 8 mg/kg of CBD via nasogastric tube. In comparison, Sánchez de Medina et al. (3) achieved higher plasma concentrations following the administration of 10 mg/kg CBD orally on an empty stomach. They observed plasma concentrations of 55.660 (35.760–94.340) ng/mL with an oil-based preparation and 142.730 (104.600–354.410) ng/mL with a specific micellar formulation. The C_max of approximately 143 ng/mL is notably higher than most reported values, suggesting that using higher doses and specialized formulations, such as micellar preparations, may be effective strategies for enhancing CBD bioavailability and achieving higher plasma concentrations. In another study, a low dose of CBD oil (0.1 mg/kg) was administered transmucosally, resulting in a significant plasma concentration of 27 ng/mL. This effect was likely due to the bypassing of first-pass metabolism in the liver (33). Williams et al. (34) achieved a C_max of 51 (± 14) ng/mL by administering a 2 mg/kg dose of full-spectrum CBD pellets once daily for 7 days. The maximal plasma concentration was measured after the final dose, which may contribute to the higher observed levels, as CBD can accumulate in organs and adipose tissue. Additionally, the pellet formulation could have enhanced absorption.

However, even the highest plasma concentrations achieved in horses remain lower than those observed in dogs receiving comparable doses. Deabold et al. (61) reported a maximal plasma concentration of 301 ng/mL following the administration of 2 mg/kg CBD to dogs. The low bioavailability of orally administered medication in horses can be attributed to several factors, including low gastric pH, slow gastrointestinal transit time, the lower fat content of their forage, drug binding to intestinal contents, and the unique digestive enzymes present in horses (44). Interestingly, lower plasma concentrations of CBD have also been observed in guinea pigs. The significantly lower C_max in horses and guinea pigs, compared to dogs, may suggest a pronounced first-pass effect. Since both guinea pigs and horses are hindgut fermenters, this indicates that these species may require higher doses of CBD to achieve higher plasma concentrations (45). In humans, the oral bioavailability of CBD is reduced to 13–19% due to first-pass metabolism in the liver and poor solubility (15). In horses, only two studies have reported bioavailability, with values of approximately 8 and 14% (3, 44). Based on studies so far, it appears that the pharmacokinetics of CBD in horses are more similar to humans than dogs (27).

Low CBD plasma concentration does not necessarily indicate inadequate efficacy. It may depend heavily on factors such as species (e.g., receptor distribution) and the desired therapeutic effect. Although the exact mechanism of CBD action remains unclear, receptor localization varies between species. For example, CB1 and CB2 receptors are evenly distributed across the canine epidermis, while in humans, CB1 is found in the spinosum and granulosum layers and CB2 in basal keratinocytes (62). Differences in ligand binding between human and rat CB1/CB2 receptors have also been reported, suggesting that efficacy may vary across species (63). Cannabinoids may exert dose-dependent effects by activating, antagonizing, or inhibiting diverse cellular targets (64). As mentioned in a previous study, a 300 mg oral dose of CBD had anxiolytic effects in humans, while 100 mg and 900 mg were ineffective (46), suggesting that different conditions may require different plasma concentrations. For example, lower doses may alleviate anxiety, while higher doses may be needed for seizure control. In studies where there is a solid rationale for CBD use (e.g., Crohn’s disease and chronic pain), neutral results could be due to subtherapeutic dosing. Therefore, dose-escalation trials with integrated pharmacokinetic studies are the logical next step (65). In horses two case reports described the positive effects of CBD in treating mechanical allodynia and chronic crib-biting and windsucking, using 0.5 mg/kg of CBD administered orally twice a day (28, 29). While these reports did not measure CBD plasma concentrations, previously mentioned studies indicate that maximum CBD concentrations are typically less than 20 ng/mL following oral administration of up to 3 mg/kg. However, as discussed above, the low plasma concentrations do not necessarily preclude effectiveness.

According to the present research, the t_max was 2.111 ± 0.928 h, which is consistent with most previous reports. The average time to reach maximum plasma concentration varies from 1–5 h (15). In one aged mare, the CBD concentration peaked 6 h after administration, reaching a notably lower level (8.740 ng/mL) than the average. Significant interindividual variation in both CBD concentrations and concentration-time curves has been reported in previous studies, which could explain the longer t_max and the lower maximal plasma concentration (66). The average half-life (t_1/2) was 12.563 ± 3.054 h, which is significantly longer than in dogs but aligns closely with findings in horses. These findings should be considered when defining dosing regimens and may support a 12-h dosing interval in horses over once-daily administration.

In the present study, the faster absorption and elimination of CBD observed in horses with smaller doses and lower body weight may be related to the accumulation of CBD in adipose tissue. However, body condition scoring, a more accurate indicator of obesity, was not performed. Despite this, the horses’ heights were similar (145–154 cm), suggesting that the variations in body weight were more likely due to differences in body condition rather than height.

The plasma concentration–time curves of THC and its metabolites show a characteristic pattern for oral drug consumption. However, variability was observed between individuals. Similar irregularities were found in the concentration-time curves of CBD and its metabolites (66, 67). In most cases, THC or CBD, after reaching peak plasma concentrations, show a steady decrease. However, in some cases, delayed resorption, intermittent resorption with a shoulder, or a second maximum was observed (66, 67). Similar variability in the resorption kinetics was observed in the present study. A second CBD peak was noticeable in 5 out of 10 animals plasma concentration–time curves. The secondary increase in CBD plasma concentration was between 4 and 6 h after administration. This elevation could be related to the redistribution from fatty tissues.

Cannabinoids are highly lipophilic and lipid-soluble compounds, which leads them to accumulate in fatty tissues and penetrate well-vascularized organs such as adipose tissue, heart, brain, liver, lungs, and spleen (68). A recent research conducted on rats provides clear evidence that CBD accumulates in fat, liver, and muscle tissue. Moreover, at any given dose, the increase in adipose tissue is greater than in muscle or liver (1). This distribution results in a rapid decrease in their plasma concentration (15). After an initial distribution phase, THC redistributes from lipid depots back into blood. CBD metabolism is very similar to THC (68).

To investigate the distinctive pattern observed in the plasma concentration–time curves, a comparison was made between the five ponies exhibiting a double CBD concentration peak and those with a single peak, focusing on body weight and TG levels before and after CBD administration. Ponies with a double peak had slightly lower average body weight compared to those with a single peak (mean±SD, 490.600 ± 43.002 vs. 486.400 ± 20.217 kg; test statistic = −2.889, p = 0.004). However, pre-administration TG levels were significantly higher in the double peak group (0.731 mmol/L) than in the single peak group (0.531 mmol/L; t = −2.518, df = 13.358, p = 0.025). One aged mare showed notably different values—body weight of 448 kg and TG level of 1.140 mmol/L—indicating a potential outlier due to the combination of low body weight and elevated TG. When this individual was excluded, the differences were no longer statistically significant (body weight mean±SD: 490.600 ± 43.002 vs. 496.000 ± 6.946 kg) (test statistic = −0.854, p = 0.394) and TG mean±SD: 0.531 vs. 0.644 mmol/L (t = −1.833, df = 13.363, p-value = 0.089).

Due to the small sample size, this is only an observation, and the long-term relevance remains unclear. In a single-dose pharmacokinetic study conducted on dogs, a prominent increase in CBD and THC plasma concentrations was observed between 12 and 24 h after administration in 5 out of 18 animals. However, this represents a significantly longer period of time compared to the present study. The authors suggested enterohepatic recycling of cannabinoids or coprophagia; however, neither of these theories has been conclusively proven (69).

The main cannabinoid metabolism takes place in the liver. CBD is hydroxylated by CYP450 enzymes to the active metabolite 7-OH-CBD which is further metabolized to the inactive metabolite 7-COOH-CBD (15). The inactive metabolite is substantial in cannabinoid metabolism because it is present in the body for a longer period of time and thus can be a very useful biomarker of cannabis exposure. CBD also undergoes direct glucuronidation via UGTs (9). Cannabinoids mostly excreted in the feces (approx. 65%) and in the urine (approx. 20%) (9, 10). CBD is mainly eliminated unchanged in the feces and as both unchanged and glucuronidated CBD in the urine (9).

The specific metabolite profile can vary significantly between species (42). There is currently no research available that outlines the precise steps of CBD metabolism in horses. In humans, CBD is extensively metabolized, and the rapid increase in metabolite concentrations is due to the extensive first-pass metabolism of the parent compound, which is specific to oral intake (66, 67).

As part of the analysis, the authors expected the inactive metabolite to be detectable in plasma for a longer period. 7-COOH-CBD was above the quantification limit in all horses, even 16 days after a single oral CBD administration. Further investigation is needed to determine the precise elimination time of the 7-COOH-CBD metabolite. The 7-OH-CBD metabolite was only present in the plasma between 1 and 24 h after administration. Eichler et al. (37) explained the low serum plasma value of 7-OH-CBD by the partial metabolism into 7-COOH-CBD. Ryan et al. (42) reported that 7-OH-CBD concentrations remained low in blood throughout the post-administration period, with the carboxylated metabolite being the predominant form. Although urine was not analyzed in the present study, both Ryan et al. (42) and Eichler et al. (37) found that, in equine urine samples, the 7-OH-CBD metabolite was more prevalent than 7-COOH-CBD and CBD itself.

Cannabinoid use is prohibited in human sports competitions, with carboxy-THC serving as the primary marker for cannabis misuse in urine. According to the World Anti-Doping Agency (WADA), concentrations exceeding 180 ng/mL constitute an Adverse Analytical Finding (AAF) (70). An AAF indicates the presence of a prohibited substance or method and may lead to anti-doping rule violations. While no thresholds exist for minor cannabinoids, their detection during in-competition testing may also result in an AAF and subsequent investigation (70). Although CBD was removed from WADA’s Prohibited List in 2018, CBD products derived from cannabis extracts can contain trace amounts of THC or other banned cannabinoids, posing a risk of AAFs if identified in doping controls (70).

Most international equine sport associations list all natural and synthetic cannabinoids as banned substances. However, in 2022, the Fédération Equestre Internationale (FEI) reclassified cannabidiol (CBD) and cannabidiolic acid (CBDA)—the precursor to CBD—as controlled medications and included them among specified substances (37). This change underscores the importance of understanding CBD pharmacokinetics in horses. Detection of a specified substance may still result in an AAF, but such substances are subject to different rules and penalties compared to prohibited substances. This classification accounts for the possibility that the substance may be present due to accidental contamination, incorrect administration, or other factors. Nonetheless, any violation of FEI regulations concerning controlled medications or specified substances can still lead to sanctions, including fines, suspensions, or disqualification, depending on the specific circumstances of the case.

A significant decrease in heart rate was observed 24 h after CBD administration (Figure 1). However, there was no change in the auditory and tactile stimulation response scores, indicating that the response to external stimuli did not reduced. The decrease in heart rate may suggest a calming effect of CBD, but the evidence is low grade, and its clinical relevance remains questionable. Investigating CBD’s calming effect was not a primary objective of the study, but it is an observation worth noting. Further research is warranted, including more detailed evaluations (e.g., reactivity scoring, novel object tests, and heart rate variability).

Hematological and biochemical variations observed in the present study may be attributed to species-specific differences. Lower baseline RBC and HGB levels align with previous findings showing that ponies typically exhibit lower erythrogram values than Thoroughbreds or other horse breeds (71). Elevated baseline triglyceride (TG) levels, seen in 9 subjects, reflect known metabolic differences between ponies and horses, though recent studies challenge the belief that healthy ponies have higher TG levels (71, 72). Elevated LDH levels were noted throughout the study, with concurrent mild CK elevations in some ponies, suggesting potential muscle involvement. Given that ponies tend to have higher lactate levels and LDH is linked to lactate metabolism, proportionally elevated LDH may reflect physiological norms rather than pathology (71, 73, 74).

Table 3 summarizes all hematological and biochemical changes observed between baseline and 24 h, including elevations in urea, chloride, GGT, TBR, and LDH, as well as a slight reduction in WBC count.

Urea is synthesized in the liver and serves as the primary route of nitrogen excretion, making protein catabolism directly related to urea concentrations. Its elimination is mainly renal, as it is freely filtered by the glomeruli and undergoes passive tubular reabsorption. Consequently, reduced tubular flow rates can increase urea reabsorption, thereby elevating blood urea concentrations (75). Dietary protein intake and muscle catabolism have a slight positive effect on urea levels in horses. However, once dietary and metabolic factors are excluded, the primary cause of elevated urea levels is a reduced glomerular filtration rate (75). Urea concentrations may rise earlier than creatinine, particularly in pre-renal azotemia, due to enhanced tubular reabsorption (75).

To the authors knowledge, no definitive evidence links CBD use to adverse renal outcomes, although several studies suggest its potential therapeutic benefits for kidney health. Research on doxorubicin-induced kidney damage in rats showed that CBD reduced oxidative stress, serum creatinine, and inflammatory markers like IL-6 (76). In a mouse model of diabetic nephropathy, CBD hydroxypentylester improved kidney function by reducing creatinine, urea levels, hyperglycemia, apoptosis, and inflammation (77). However, one study indicated that CBD worsened renal damage in streptozotocin-induced diabetic nephropathy, suggesting that CBD may exacerbate nephropathy in type 1 diabetes. While CBD appears safe for kidney function in healthy individuals, caution is advised for those with chronic kidney disease (78).

Elevated urea concentration can be linked to high protein intake or dehydration; however, in the present study, these factors are not relevant. Draeger et al. (32) observed similar changes in blood urea concentrations. Within reference ranges, urea consistently increased with both treatment concentration (they administered 50 mg, 100 mg, or 250 mg CBD to their subjects) and time progression, while creatinine levels did not. They assumed that typically, kidney dysfunction would be indicated by a concurrent rise in both urea and creatinine. They concluded that the administration of each dose in their study caused only minimal changes in blood chemistry concentrations. However, given the slight variations observed in both their study and the present study, it would be prudent to monitor these parameters during prolonged CBD treatment.

Yocom et al. (45) found elevation in liver enzymes (GGT, AST, SDH) while administered sunflower lecithin oil-based CBD at 0.5 mg/kg or 1.5 mg/kg q12 orally for 6 weeks, however the increases were not dose dependent and all values returned to reference intervals or decreased 10 days after the last treatment. In the present study only a slight elevation in GGT, TBR, and LDH values was noted, and none of them were relevant. A study investigated liver safety in healthy adults (n = 16) taking therapeutic (1,500 mg) daily doses of CBD for approximately 3.5 weeks. Seven subjects experienced elevated serum alanine aminotransferase (ALT) levels and five exceeded the threshold for drug-induced liver injury. These ALT elevations occurred within 2–4 weeks of CBD initial exposure, but no correlation was found between the elevations and baseline characteristics, CYP2C19 genotype (the primary enzyme involved in phase I metabolism of CBD), or CBD plasma concentrations (79).

Ewing et al. (80) demonstrated that CBD, when orally administered to mice has the potential to cause liver injury. The administration of CBD led to dose-dependent induction of cytochromes and UDP-glucuronosyltransferases, which have been linked to drug metabolism and toxicity. Their 10-day sub-acute study also revealed that CBD doses above 50 mg/kg CBD, while initially tolerated, caused toxicity when repeatedly administered, manifesting as liver injury with elevated liver enzymes (ALT, AST, TBR) and increased liver-to-body weight ratios. Additional research in horses is warranted to assess whether oral CBD administration at higher doses, or over extended durations, could lead to further changes in liver enzymes that might prevent long-term use. The potential effectiveness of CBD or any other co-administered medication may also be affected by the horse’s liver health.

CBD has become widely popular as an alternative or adjunct treatment in both human and veterinary medicine. The authors would like to highlight the pain management related to laminitis among the previously mentioned therapeutic targets of CBD in equine medicine. Equine metabolic syndrome (EMS) is a common underlying cause of laminitis, which is the primary clinical consequence of EMS (81, 82). Additionally, horses suffering from EMS may be at risk for other complications, including hyperlipemia and critical care-associated metabolic derangements such as hyperglycemia and hypertriglyceridemia (81). Ponies are more prone to develop EMS than standardbreds. It has been demonstrated that baseline insulin levels are correlated with height. A similar correlation has been observed in humans, where shorter individuals are at greater risk of developing metabolic diseases (82). In ponies, the correlation between height and several biochemical parameters showed a negative relationship with insulin, glucose, adiponectin, and ACTH levels, and a positive relationship with triglyceride and leptin concentrations (82). Ponies have higher plasma lipoprotein concentrations than standardbreds and appear to be more prone to developing hyperlipidemia when in a negative energy balance (83). Given these differences in carbohydrate and lipid metabolism between ponies and horses, the authors considered it important to study whether these variations could influence the pharmacokinetics of CBD in ponies. To the autohrs knowledge, this is the first study to examine the pharmacokinetic properties of CBD in ponies. The results of the present investigation could serve as a foundation for future pharmacodynamic studies.

The main limitation of the present study was the small sample size (n = 10). However, it is important to note that previous research on this topic has also used similar sample sizes (3, 27, 32, 34, 36–45, 60). The alignment of the present findings with existing literature suggests that the sample size employed is acceptable within this context. Another limitation was that only blood, not urine samples were tested. Previous research has shown that cannabinoid metabolism and patient responses can vary significantly, even with comparable doses and formulations—a variability potentially explained by genetic polymorphisms in genes involved in cannabinoid metabolism. Personalized medicine aims to improve drug efficacy and safety, and understanding the pharmacogenomics of cannabinoids is crucial for optimizing dosing, minimizing adverse effects, and avoiding treatment failure. Although pharmacogenomic data related to horses exist in the scientific literature, to the authors’ knowledge, no studies have specifically explored its application to cannabinoids; this remains an important and promising area for future investigation, though it was beyond the scope of the present study (84–86).