Cannabidiol (CBD) and other minor cannabinoids deriving from Cannabis sativa chemovars have sparked considerable interest in the field of pet care and veterinary medicine.1 Two common cannabinoids, cannabidiolic acid (CBDA) and tetrahydrocannabinolic acid (THCA), are thermally unstable and decarboxylated to cannabidiol (CBD) or tetrahydrocannabinol (THC) during extraction and processing.1 While THC has psychoactive toxicity, CBD is a nonintoxicating compound with potential therapeutic applications via interaction with the homeostatic endocannabinoid system regulating, among others, neuronal development, nociception, and inflammation.2
Endocannabinoids and phytocannabinoids bind to specific receptors, like cannabinoid receptor 1 (CB1), mainly located in the brain and spinal cord, and cannabinoid receptor 2 (CB2) in the peripheral nervous system and immune cells.3 Cannabinoids have different affinity at the CB1 or CB2 receptor sites, either as an agonist or antagonist; for example, CBD functions as a CB1 negative allosteric modulator and CB2 partial agonist. Both CBD and CBDA also modulate non-CB1/CB2 receptors, like the peroxisome proliferator-activated receptor γ, 5-HT1A serotonin receptor, and multiple transient receptor potential channels, expressed in primary afferent nociceptors.4–9 In addition, CBDA has been reported as a selective cyclooxygenase-2 inhibitor.10
Due to its distinctive inverse cannabimimetic effect, CBD has gained significant interest in pharmaceutical and nutraceutical fields for both humans and animals. Studies11–13 on dogs demonstrated that CBD or full-spectrum hemp extract CBD/CBDA can reduce seizure frequency in idiopathic epilepsy and mitigate osteoarthritis-related pain. The use of CBD in dogs and cats is supported by absorption and retention studies14–16 that also confirmed its safety, although reversible rises in alkaline phosphatase have been reported. However, there is a noticeable scarcity of research on cannabinoids in horses.
In recent studies,17–19 no adverse effects were found for both adult and senior horses following CBD IV and oral administration. Acute single-dose oral administration of CBD/CBDA did not produce neurologic, behavioral, or gastrointestinal effects in horses.20 In senior horses, the oral bioavailability for CBD is relatively low (7.92%), with both IV and oral administration showing no meaningful effects on hemogram or chemistry analyses.17 Other acute and long-term pharmacokinetic (PK) studies17–21 in horses show low serum concentrations achieved at similar dosing to canine and feline studies, with rapid elimination of CBD, suggesting differences in absorption and retention of CBD in horses. In the study by Thomson et al,20 the absorption and retention of CBDA were superior to CBD, but there is currently no available research on chronic oral dosing of CBDA regarding its bioavailability and safety for adult horses.
The aim of our study was twofold: first, to measure the immediate and long-term PK of full-spectrum CBD oil versus full-spectrum, CBDA-predominant oil to determine if the absorption/retention kinetics of CBDA are superior, and second, to examine the safety of chronic administration of full-spectrum CBD and CBDA oils over a 6-week period of time recording any adverse events as observed in dogs and people.16,22,23
Methods
Study design
All experimental procedures were approved by the Cornell University IACUC (protocol #2020-0075).
Seven horses received 2 full-spectrum hemp oils (CBD and CBDA rich), each oil for a 6-week trial period, using a crossover, randomized design with a 2-week washout period. The safety of chronic administration was assessed via physical examination, periodic bloodwork, and liver biopsy at the beginning and end of the study.
A 12-hour PK analysis was conducted on day 1 of each 6-week trial period, and every 1 to 2 weeks, serum cannabinoid levels were measured prior to and at 1 and 2 hours after the morning dose to measure nadir and peak concentrations.
Animals
Seven adult female horses (3 Warmbloods, 2 Quarter Horses, and 2 mixed breeds; median age, 25 years; range, 12 to 28 years; median body weight [BW], 566 kg; range, 434 to 649 kg) of the Cornell University teaching herd were enrolled in the study. Each horse received, in computer-generated, randomized order (https://www.random.org), CBD- and CBDA-based oils (ElleVet Sciences LLC) for 6 weeks each at a dose of 1 mg/kg BW cannabinoids every 12 hours, administered orally. The oil was administered orally via a catheter tip syringe. No feeding restriction was implemented before or after the oil administration. The cannabinoid oil dose was calculated based on the horse’s BW on day 0 of each trial period. A washout period of 2 weeks separated the 2 trial periods. During the study trials, horses were housed in single-box stalls with daily turnout on a dry lot, had free access to hay and water, and were supplemented with a pelleted complete feed provided at 0.5% of their BW. During the washout period, horses were moved to pasture.
Cannabinoid products
Two cannabis extracts were tested: a CBD-rich oil and a CBDA-rich oil (ElleVet Sciences LLC). Each milliliter of CBD-rich oil contained 59.6 mg cannabinoids with the following profile: 99.2% CBD, 0.15% cannabidivarin, 0.02% Δ9-THC, and 0.01% cannabinol. Each milliliter of CBDA-rich oil contained 66.5 mg cannabinoids with the following profile: 83.5% CBDA, 12.3% CBD, 1.18% cannabigerolic acid, 0.5% cannabigerol, 0.16% cannabidivarin, 0.06% cannabinol, 0.02% D9-THC, and 0.01% THCA. The carrier ingredients consisted of sesame oil and lecithin. The components and concentration of each oil were confirmed by third-party analysis via liquid chromatography by a certified International Organization for Standardization and International Electrotechnical Commission 17025 laboratory (ProVerde Laboratory) at the beginning and end of the study, with less than 5% variability in cannabinoid results, and deemed safe for oral consumption.
Pharmacokinetics sample collection
Horses underwent a 12-hour PK assessment with blood draws at time 0, 30 minutes, and 1, 2, 4, 8, and 12 hours, with twice-a-day dosing starting immediately after the 12-hour PK sampling. Blood was drawn by direct venipuncture of the left jugular vein using an 18-gauge vacutainer needle in a red-top coagulation tube and allowed to clot. Serum was obtained after centrifugation at 2,000 X g for 10 minutes and immediately frozen at −80 °C. Serum was also obtained prior to and 1 and 2 hours after the morning dose at the end of weeks 1, 2, 4, and 6 (days 7, 14, 28, and 42) of cannabinoid oil administration to measure nadir and peak concentrations.
Serum cannabinoid analysis
Serum analysis of cannabinoids, including CBD, CBDA, THC, THCA, and their metabolite 7-COOH-CBD, was performed by LC-MS-MS (Nexera X2 and LCMS 8050; Shimadzu Corp) at the University of Illinois at Chicago Toxicology Research Laboratory as previously described.20,24 Reference standards for CBD and CBDA were obtained from Restek Corporation, whereas other reference and internal standards were obtained from Cerilliant Corporation. Concentrations of cannabinoids were calculated by LabSolutions software (version 5.65, Shimadzu Corp) using a quadratic calibration curve with 1/c2 weighing based on relative response (peak area of cannabinoids/peak area of internal standards). The calibration curve range and corresponding lowest limit of quantification and upper limit of quantification were respectively 1 ng/mL to 1,000 ng/mL for CBD, CBDA, and THC; 0.25 ng/mL and 1,000 ng/mL for THCA; and 1 ng/mL and 5,000 ng/mL for 7-COOH-CBD. Lower limits of detection for CBD, CBDA, THC, THCA, and 7-COOH-CBD were 0.5 ng/mL, 0.5 ng/mL, 0.5 ng/mL, 0.1 ng/mL, and 0.5 ng/mL, respectively.
Pharmacokinetic analysis
Serum concentrations of the various analytes versus time data for each horse were plotted on linear and semilogarithmic graphs for analysis. Data were subjected to a noncompartmental PK analysis using commercially available software (PK Solutions, version 2.0; Summit Research). Data determined for each horse included maximum serum concentration (Cmax), time of Cmax (Tmax), elimination half-life (T1/2), area under the serum concentration curve (AUC0–12h), and mean residence time (MRT). Based on data from a prior study,20 the known peak of absorption was between 1 and 2 hours of administration. Therefore, blood was collected on days 7, 14, 28, and 42 to assess serum concentrations on horses immediately before the morning dose and at 1 and 2 hours after administration to assess nadir and peak concentrations over the 42-day trial.
Safety monitoring
The health of the horses was assessed daily via physical examinations and monitoring for eventual changes in behavior and appetite. A CBC and serum chemistry panel were performed on days 0, 7, 14, 28, and 42 of each treatment trial. The CBCs were obtained from EDTA-anticoagulated blood with an automated hematology analyzer (Element HT5; Heska) and included WBC count with differential count of the individual leucocytes, RBC count and mass, hemoglobin, RBC indices and size variation, and platelet count and size. Serum chemistry measured sodium, potassium, chloride, bicarbonate, urea nitrogen, creatinine, calcium, phosphate, magnesium, total protein, albumin, globulin, glucose, AST, sorbitol dehydrogenase, glutamate dehydrogenase, GGT, total bilirubin, direct bilirubin, indirect bilirubin, creatine kinase, iron, total iron-binding capacity, and iron saturation. Biochemical analysis was performed by the Cornell Animal Health Diagnostic Center Clinical Pathology Laboratory on plasma (from heparin-anticoagulated blood) with an automated chemistry analyzer (Hitachi Modular P; Roche Diagnostics) using the manufacturer’s reagents, with the exception of sorbitol dehydrogenase (Sigma-Aldrich) and glutamate dehydrogenase (Randox Laboratories Ltd).
Liver biopsy
Ultrasonography and biopsy of the liver were performed 1 week prior to starting the oil administration and the day after the last dose (day 99). The horses were sedated with detomidine (0.01 mg/kg) and butorphanol (0.01 mg/kg), IV. A liver ultrasound (2-to-9-MHz curved array transducer; Samsung HS 60; Samsung Healthcare) evaluated the structure and echogenicity on both the left and right side of the abdomen. After the identification of a suitable window to perform the biopsy, the area was clipped and prepared for sterile procedure, followed by a 2% lidocaine injection to anesthetize the skin, SC tissue, and intercostal muscle.
Percutaneous biopsies of the liver were obtained under ultrasound guidance using a standard Tru-Cut biopsy needle (10 cm, 14 gauge; JorVet Jorgensen Laboratories). The specimens were fixed in 10% neutral-buffered formalin for at least 24 hours, then processed routinely and paraffin embedded. Sections, 6 μm in thickness, were stained with H&E, Masson trichrome, and periodic acid–Schiff for routine light microscopy. The sections were examined by a board-certified pathologist and scored for the presence of various pathologic features, including portal inflammation, lobular inflammation, individual necrotic hepatocytes, pigment accumulation, ductular reaction, cell swelling, and fibrosis, as previously described.25 Portal tract inflammation was defined as the presence of more than 5 inflammatory cells within a portal stroma. Lobular inflammation was defined as the presence of aggregates of 2 or more inflammatory cells within hepatic cords. Hepatocytes were considered necrotic if showing hypereosinophilic cytoplasm, nuclear changes (pyknosis, karyolysis, karyorrhexis), and cellular fragmentation, whereas cell swelling was defined as hepatocytes greater than 2 times the size of adjacent representative hepatocytes. Ductular reaction was characterized as proliferation of small, double-wide cords of bipotential progenitor cells breaching the limiting plate and extending into adjacent hepatic parenchyma. Fibrosis was present if immature or mature collagen bands expanded within portal stroma. For each pathologic feature, an individual score of 1 was applied if detected and 0 if absent; a total biopsy score, ranging from 0 (no detectable abnormalities) to 7 (presence of all the pathologic features), was calculated by summing the scores of each pathologic feature. The biopsy grading was performed without knowledge of the timing of collection (pre- and post-CBD/CBDA chronic administration).
Statistical analysis
Pharmacokinetic parameters, including Cmax, Tmax, T1/2, AUC0–12h, and MRT, were assessed for normality of distribution using the Shapiro-Wilk test. Only T1/2 and MRT were normally distributed (P = .241; P = .353); therefore, all PK parameters were log transformed prior to statistical analysis. Differences between treatment groups (CBD and CBDA) were determined by ANOVA using a mixed-effect model, including trial period, treatment, and treatment by trial period interaction as fixed effects and horse as a random effect. Differences between treatments were considered significant at P < .05 and tendency for difference at P < .1. Additional PK parameters for THCA were reported, and partial PK parameters for metabolite 7-COOH-CBD (Cmax, Tmax) were summarized and presented as mean ± SD.
The statistical model to determine the difference between nadir and peak serum concentration of CBD and CBDA at different time points (days 7, 14, 21, 28, and 42) included trial period, treatment, and day as fixed effects and horse nested within treatment as random effect. A covariance structure was added to ensure that the correlation between observations within the same group were constant and equal across all groups. Differences were tested using ANOVA. For 7-COOH-CBD nadir and peak concentrations, the mean ± SE was calculated.
For CBC and serum chemistry data, a Kruskal-Wallis test followed by a post hoc Dunn test with Bonferroni correction was performed to evaluate the changes in parameters over the study time. The Wilcoxon matched pairs test was used to compare the histopathology score of the liver biopsy pre- and postcannabinoid administration. The statistical analysis of PK parameters, nadir and peak serum concentrations of cannabinoids, CBC, and chemistry was performed using RStudio, version 4.3.2 (RStudio PBC). Histopathology scores are reported as mean ± SD, and analysis was conducted using GraphPad Prism, version 9 (GraphPad Software Inc).
Results
Pharmacokinetics
Pharmacokinetics data for CBD and CBDA following oral dosage are presented across the 2 trial periods as there was no treatment by trial period interaction for most parameters (Table 1). Compared to CBD, over the 2 periods, CBDA exhibited a greater Cmax (103.10 ± 87.56 ng/mL vs 12.01 ± 6.17 ng/mL; P < .001) and AUC0–12h (258.50 ± 213.80 ng/h/mL vs 61.67 ± 39.22 ng/h/mL; P < .02) and a lower Tmax (0.79 ± 0.27 hours vs 1.86 ± 0.38 hours; P < .001) and T1/2 (2.82 ± 1.48 hours vs 4.12 ± 1.37 hours; P = .018). The greater AUC0–12h value for CBDA was attributed to trial period 1. In trial period 1, the Cmax was 173.3 ± 84.33 ng/mL and 12.11 ± 8.36 ng/mL, respectively, for CBDA and CBD (P = .025). In trial period 2, the Cmax did not differ between treatments (50.47 ± 44.47 ng/mL for CBDA and 11.88 ± 3.06 ng/mL for CBD; P = .148). Mean residence time did not differ between CBDA and CBD (4.37 ± 2.14 hours vs 4.30 ± 1.19 hours; P = .122).
Twelve-hour pharmacokinetics of cannabidiol (CBD) and cannabidiolic acid (CBDA) in horses administered 1 mg/kg of CBD- or CBDA-rich hemp oils over 2 trial periods.
| Treatment | |||||
|---|---|---|---|---|---|
| Parameter | CBD | CBDA | Treatment P valuea | Period P valuea | Period X treatment P valueb |
| n | 7 | 7 | |||
| Cmax (ng/mL) | 12.01 ± 6.17 | 103.10 ± 87.56 | < .001c | .682 | .148 |
| Tmax (h) | 1.86 ± 0.38 | 0.79 ± 0.27 | < .001c | .288 | .756 |
| T1/2 (h) | 4.12 ± 1.37 | 2.82 ± 1.48 | .018c | .852 | .762 |
| AUC0–12h (ng·h/mL) | 61.67 ± 39.22 | 258.50 ± 213.80 | .020c | .523 | .089 |
| MRT (h) | 5.20 ± 1.57 | 3.93 ± 1.66 | .122 | .153 | .192 |
Values reported are mean ± SD.
AUC0–12h = Area under the serum concentration curve from 0 to 12 hours. Cmax = Maximum serum concentration. MRT = Mean residence time. T1/2 = Elimination half-life. Tmax = Time of Cmax.
Tetrahydrocannabinolic acid was measurable for 12-hour PK assessment following typical PK modeling, whereas 7-COOH-CBD peaked between 4 and 12 hours and plateaued afterwards, not allowing for calculations of all PK parameters (Table 2). The CBD metabolite 7-COOH-CBD showed a higher Cmax (224.6 ± 146.67 ng/mL vs 19.88 ± 9.74 ng/mL) after CBD-rich oil administration compared to CBDA-rich oil (P = .016).
Twelve-hour pharmacokinetics of tetrahydrocannabinolic acid (THCA) and 7-COOH-CBD in horses administered 1 mg/kg of CBDA- and CBD-rich hemp oils.
| CBDA | CBD | ||
|---|---|---|---|
| Parameter | THCAa | 7-COOH-CBDb | 7-COOH-CBDb |
| n | 7 | 7 | 7 |
| Cmax (ng/mL) | 17.16 ± 16.47 | 19.98 ± 9.74 | 224.6 ± 146.67c |
| Tmax (h) | 0.86 ± 0.56 | 10.86 ± 1.96 | 9.71 ± 3.15 |
| T1/2 (h) | 6.47 ± 3.03 | NA | NA |
| AUC0–12h (ng·h/mL) | 67.33 ± 51.51 | NA | NA |
| MRT (h) | 8.73 ± 3.09 | NA | NA |
Chronic dosing peak and nadir
Nadir and peak values for CBD, CBDA, and 7-COOH-CBD over the 42-day trial period were summarized as mean ± SE (Tables 3 and 4). For CBD, day was not significant except for a trend for higher peak serum concentration on day 7 (P = .055); peak concentrations were lower on days 14 (23.60 ± 3.57 ng/mL; P = .045) and 42 (22.30 ± 3.36 ng/mL; P = .021) compared to day 7 (38.10 ± 5.74 ng/mL). Regarding CBDA treatment, a significant effect of day was found across the 2 periods of treatment (P = .034); peak concentration was lower on day 42 (68.70 ± 10.37 ng/mL) compared to days 14 (122.30 ± 18.45 ng/mL; P = .011) and 28 (117.70 ± 17.77 ng/mL; P = .020; Figure 1). For both CBD and CBDA, nadir concentrations did not differ between days 7, 14, 28, and 42 (P = .868). There were no changes in nadir and peak concentrations for 7-COOH-CBD in both CBD and CBDA treatments.
Nadir and peak serum concentrations (nanograms per milliliter) of horses administered 1 mg/kg of CBD- or CBDA-rich hemp oils over both 42-day trial periods.
| CBD | CBDA | |||
|---|---|---|---|---|
| Day | Nadir | Peak | Nadir | Peak |
| 7 | 16.70 ± 3.07a | 38.10 ± 5.74a | 46.00 ± 8.44a | 90.40 ± 13.64a,b |
| 14 | 13.90 ± 2.55a | 23.60 ± 3.57b | 52.30 ± 9.60a | 122.30 ± 18.45a |
| 28 | 16.50 ± 3.03a | 26.30 ± 3.97a,b | 65.50 ± 12.02a | 117.70 ± 17.77a |
| 42 | 12.60 ± 2.31a | 22.30 ± 3.36b | 53.70 ± 9.86a | 68.70 ± 10.37b |
Values reported are mean ± SE.
Within each column, values with different superscripts are significantly different (P < .05).
Trial period was not significant except for peak serum concentration on day 7 for CBD (P = .055) and day 14 for CBDA (P = .034).
Nadir and peak serum concentrations (nanograms per milliliter) of 7-COOH-CBD in horses administered 1 mg/kg of CBD- or CBDA-rich hemp oils over both 42-day trial periods.
| CBD | CBDA | |||
|---|---|---|---|---|
| Day | Nadir | Peak | Nadir | Peak |
| 7 | 1,531.60 ± 263.68 | 1,787.61 ± 304.01 | 376.89 ± 32.57 | 386.34 ± 40.37 |
| 14 | 1,804.05 ± 339.62 | 1,961.27 ± 384.80 | 519.75 ± 36.87 | 524.36 ± 45.32 |
| 28 | 1,736.70 ± 311.70 | 1,840.38 ± 298.27 | 675.00 ± 130.94 | 678.76 ± 137.00 |
| 42 | 1,606.14 ± 228.97 | 1,700.95 ± 223.97 | 545.74 ± 59.15 | 543.63 ± 55.90 |
Values reported are mean ± SE.
Serum concentrations of cannabidiol (CBD) and cannabidiolic acid (CBDA) during 6 weeks of oral supplementation at 1 mg/kg every 12 hours in 7 horses. The graph illustrates the 12-hour pharmacokinetic after the first dose, followed by peak concentration at 7, 14, 28, and 42 days of chronic administration. Data are reported as mean ± SD, with the y-axis logarithmically scaled.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.08.0235
Complete blood count and serum chemistry
None of the horses demonstrated behavioral changes or complications related to the administration of the hemp extracts. One horse developed a traumatic injury on the right hind on day 28 of the first treatment trial while under CBD treatment, and a second horse developed cellulitis of the left hind on day 18 of the second treatment trial (CBDA); both horses recovered uneventfully within a few days.
The CBC at different time points (days 7, 14, 21, 28, and 42) following CBD and CBDA administration showed no clinically relevant changes (Supplementary Tables S1 and S2). One horse had a platelet count below the reference range on the automated hemogram at every time point, but smear evaluation by a board-certified pathologist confirmed platelet clumping and adequate platelet estimation.
The blood chemistry profile showed no significant changes over time for parameters reflecting renal, liver, and muscle function (Supplementary Tables S3 and S4). Electrolytes and mineral homeostasis were maintained over time except for the chloride, which had a small increase on day 28 compared to day 1 (103.30 ± 1.38 mEq/L vs 101.10 ± 1.46 mEq/L; P = .041) during CBD treatment and a mild increase during treatment with CBDA-rich oil on days 14 (103.7 ± 1.38 mEq/L; P = .0072), 28 (103.40 ± 0.53 mEq/L; P .0235), and 42 (103.10 ± 1.22 mEq/L; P = .023).
Liver biopsy
The liver could be visualized on both the right and left side only in 5 of 7 horses (71%), whereas in 2 horses it could be visualized only on the left side. No ultrasonographic abnormalities in parenchymal structure and echogenicity or within the biliary tract were detected at time 0. The biopsy was performed on the right side of 5 horses and on the left side of 2 horses. Ultrasound was repeated after the procedure to evaluate potential bleeding; no significant complications developed after the biopsies besides a small SC hematoma in 2 horses that resolved spontaneously within 48 hours. Ultrasonographic evaluation of the liver at the end of the study showed no appreciable changes in liver structure, echogenicity, and biliary tract compared to time 0.
Histopathologic evaluation of the liver biopsy revealed overall no evidence of significant inflammation or necrosis at the end of the study (Figure 2). No signs of ductular reaction or fibrosis were found in any of the biopsies. Two horses had mild sinusoidal neutrophilia at the beginning of the study and one of them also at the end. The main changes observed, already at time 0 in 6 of 7 horses, were vacuolation of hepatocytes, initially attributed to glycogen but not confirmed on periodic acid–Schiff stain. Some features scored lower in the biopsy at the end of the study. No significant difference was found between pre- and poststudy total histologic score, which was 2.57 ± 1.27 before starting cannabinoid administration and 2.14 ± 1.07 at the end of the study.
Representative photomicrographs of liver biopsy before (T0) and after (study end) chronic administration of CBD- and CBDA-rich oil in horses. Images are from 1 single horse that was representative of the series. All sections were evaluated and assigned a histologic score based on the presence of histopathologic features (0 through 7, where 0 = no detectable abnormalities and 7 = presence of all the pathologic features). At T0 (A, C, and E), pigment accumulation and cell swelling were detected (total score, 2/7); at the end of 12 weeks of hemp oil administration, no significant changes were observed except for signs of mild lobular inflammation (score, 3/7). A and B—H&E stain; scale bar = 100 µm. C and D—Masson trichrome stain; scale bar = 100 µm. E and F—Periodic acid–Schiff stain; scale bar = 100 µm.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.08.0235
Discussion
The objective of this study was to assess the PK of CBD and CBDA and their safety over a 6-week oral administration period. A recent study suggested that CBDA, when present with full hemp extract, provides a natural vehicle to enhance the plasma absorption of cannabinoids, and many species appear to preferentially absorb CBDA when compared to CBD in full-spectrum hemp extracts.11,26,27 We confirmed a higher absorption of CBDA compared to CBD in horses, maintained during chronic administration.
Compared to a previous PK study19 in horses administered the same dosage of CBD at 1 mg/kg BW, CBD had a greater Cmax (12.01 ± 6.17 ng/mL vs 4.3 ± 2.1 ng/mL); lower Tmax (1.86 ± 0.38 hours vs 4.1 ± 4.1 hours), T1/2 (4.12 ± 1.37 hours vs 14.8 ± 4.6 hours), and MRT (5.20 ± 1.57 hours vs 13.5 ± 2.3 hours); and a similar AUC0–12h (61.67 ± 39.22 ng/h/mL vs 51.6 ± 21.1 ng/h/mL). These differences in Cmax, Tmax, and MRT might be caused by the variations in the CBD oil profile and the presence of minor cannabinoid compounds between studies or differences in feeding regimens between the 2 studies. In contrast, CBDA consistently exhibited a higher Cmax (103.10 ± 87.56 ng/mL vs 12.01 ± 6.17 ng/mL) and AUC0–12h (258.50 ± 213.80 ng/h/mL vs 61.67 ± 39.22 ng/h/mL), suggesting increased bioavailability and potentially altered uptake or retention compared to CBD. Comparable findings were reported with CBD/CBDA-rich, full-spectrum hemp oil at a total dose of 2 mg/kg.20 Additionally, a shorter Tmax, T1/2, and MRT were observed for CBDA, indicating its potential as a compound with more rapid onset and clearance compared to CBD.
The predicted average steady-state serum concentration based on 12-hour PK is not presented in this study because actual serum concentrations were measured periodically over the 42-day study. Cannabidiolic acid serum concentrations were greater than CBD over the 42-day study (Figure 1; Table 3), with nadir serum concentrations of both cannabinoids remaining relatively constant throughout the 6-week period. These data suggest that there is not a significant bioaccumulation of the more lipophilic CBD over time, although there is a recent study28 suggesting mild accumulation. Eichler et al28 calculated a terminal half-life of 161.29 ± 43.65 hours, with an accumulation ratio of 2.63, after a CBD oral paste trial at 3 mg/kg twice daily for 15 days. Data regarding CBDA accumulation is not currently available in horses, and in our study CBD and CBDA peak concentrations behaved differently over time. While peak serum concentration for CBD decreased after the first week, CBDA peak concentration initially increased on day 14 and later decreased to reach a minimum on day 42. The cause of the decrease in CBDA peak concentration after a few weeks is unknown but could reflect acquired changes in its absorption, distribution, or metabolism, potentially involving the induction of specific metabolizing enzymes. Cannabinoids can interact with the cytochrome P450 (CYP) enzyme system, which is present in the small intestine epithelium and responsible for drug metabolism. Indeed, CYPs are major metabolic pathways for phytocannabinoids, especially for CYP3A4, which is more abundant in the small intestine and liver.29 Several species of CYPs are inducible by increased synthesis of mRNAs coding for specific enzymes, and the induction of drug-metabolizing enzymes has been described and considered one of the factors involved in development of drug tolerance.30 This suggests that CBDA post dosing serum concentration might not remain steady by week 6, and further study is required to understand long-term dosing of CBDA and its metabolism when considering the potential for therapeutic dosing.
Compared to dogs and humans, horses exhibit lower plasma concentrations of CBD.12,16,19,31,32 It is possible that horses have greater hepatic carboxylation of CBD to 7-COOH-CBD. However, small intestinal absorption of cannabinoids via the transepithelial portal circulation was reported to be limited to 2.1% to 9.3% compared via lymphatic chylomicrons absorption in a rat study; lymphatic CBD concentrations can be up to 1,000 times higher than serum concentrations.33 If there is greater hepatic carboxylation in horses, such a process would be limited to second-pass metabolism rather than first pass. In addition, dietary fat consumption in horses is relatively low, and chylomicron transport may be limited by low dietary fat intake. Given the large abundance of squamous epithelium and a resident microbiota, CBD carboxylation via microbial metabolism in the horse stomach should be considered as a possible mechanism in addition to the intestinal CYP enzyme system. Regarding the matrix and absorption of neutral (CBD) and acidic (CBDA) cannabinoids, little work has been performed to assess protein binding or differences in delivery vehicle between the two. However, it has been shown that protein emulsification in conjunction with various forms of lipid may enhance the absorption and retention of CBD in rats, whereas CBDA appears to be found in biological fluids in humans at higher concentrations when delivered in an aqueous versus a lipid matrix.34,35 These limited studies show that there may be ways to improve the absorption of CBD and CBDA by utilizing ideal vehicles for delivery and that the study of optimal delivery is in its infancy across species.
In horses, only a few longer-term PK studies17,19,28,36 were conducted with doses of 0.5 to 3 mg CBD/kg BW. Turner et al36 measured PK over 264 hours after CBD administration of a single dose at 2 mg/kg BW in senior horses. Compared to our study, CBD appeared to have a similar Cmax (18.54 ± 9.80 ng/mL vs 12.01 ± 6.17 ng/mL) and Tmax (2.46 ± 1.62 hours vs 1.86 ± 0.38 hours), and by 72 hours after oral administration, the plasma CBD concentration was either below the limit of quantitation of the assay or nondetectable. In a consecutive study,36 the chronic use of a once-a-day oral administration of CBD oil (2 mg/kg) for 90 days was found to maintain a much lower mean CBD plasma concentration, around 3 ng/mL. In Yocom et al,19 during chronic administration of 1.5 mg/kg twice daily of oil-based CBD, only nadir steady-state plasma CBD concentration at weeks 2 and 4 were presented and are comparable to our current study. In Eichler et al,28 after CBD paste (3 mg/kg BW) oral administration twice daily for 15 days, CBD and 7-COOH-CBD serum concentrations on days 7 and 14 were comparable to our study.28
As shown in previous studies,17,20,28 7-COOH CBD is the predominant CBD metabolite in horses, with accumulation within the first week of oil administration. This finding is not surprising considering the long terminal half-life (43 ± 16 hours) reported after a single dose of CBD oil.17 From a metabolic standpoint, an interesting finding in this study was a relatively high 7-COOH-CBD serum concentration in the CBDA group during the 7-, 14-, 28-, and 42-day time points that were not evident during the acute 12-hour PK analysis. The reason for this is unclear as one would have expected some 7-COOH-CBD within the first 12 hours also in the CBDA group since the CBDA oil had approximately 12% CBD as a constituent, meaning that horses would have received approximately a 0.1-mg/kg dose of CBD from that oil. It is possible that the horses cleared the 7-COOH-CBD quickly after the acute 12-hour PK, making it undetectable while accumulation could be happening over time, or there is a decarboxylation and rapid carboxylation event occurring in the intestinal epithelium or liver that turns CBDA into 7-COOH-CBD as a metabolite. The mechanism is unclear, and further studies utilizing pure CBDA are needed.
We confirmed the safety of chronic CBD and CBDA administration in horses, ruling out adverse effects via clinical assessment and bloodwork. The CBC and chemistry profile remained stable over the 6-week study without significant effects on albumin or calcium as described in other studies.19,36,37 We detected relative increases in sodium and chloride over time without clinically relevant consequence. Considering that this electrolyte variation was not accompanied by changes in renal values or bicarbonate, the relative increase in chloride was attributed to the change in horse management. During each trial period, the horses were moved from 24 hours’ continuous access to grass at pasture to stall housing with turnout in a dry lot; removing grass from the diet potentially reduced the overall body free water with relative increases in sodium and chloride.
More importantly, in our study we detected no increase in the serum liver enzymes that have been reported as potential issues in 3 studies of horses.19,31,37 Our dosing was relatively low at 1 mg/kg for both cannabinoids, providing longer-term data that these modest doses appear safe. In view of hepatic adverse events noted in the human literature and recent work in dogs and horses reporting rises in liver enzymes, it was important to examine hepatic biopsies.19,38,39 Considering the horses’ welfare, we only performed hepatic biopsies immediately prior to and after the entire 14-week study period, including both CBD at 1 mg/kg and CBDA at 1 mg/kg twice daily. Cannabidiol use in humans at relatively high doses (above 5 mg/kg) has been associated with an increase in liver enzymes, in some cases consistent with drug-induced liver injury, and CBD-induced cellular damage of human hepatocytes has been reported in vitro.38,40,41 A dose-dependent CBD toxicity has been described in rodents with cytoplasmic swelling and centrilobular hypertrophy.37 The most common drug-induced liver injury histopathologic lesions also include cholestatic hepatitis, fibrosis, ductular reaction, portal inflammation, and necrosis.42 The liver histopathology in our study detected some cell swelling in samples collected prior to the treatment period and confirmed no significant hepatocellular changes associated with the CBD and CBDA use. Additionally, special stains for fibrosis and glycogen also confirmed that there were no negative effects on the liver with CBD and CBDA at our dosing regimen.
Products that contain CBD or hemp extract (other cannabinoids) are commonly seen in the equine market for potential alternative treatments for arthritis, pain relief, or anxiety,37 and both CBDA and CBD have been reported to be useful in the treatment of epilepsy and other neurological disorders in humans and dogs.13,23 In cows, CBDA has been shown to have positive effects on behavior and biomarkers of inflammation, piquing interest in CBDA therapeutic use in ruminants.43 In addition, CBDA at high doses has the potential to work through the cyclooxygenase-2 pathway, potentially regulating pain and modulating the inflammatory process.4,44 The pharmaceutical use of hemp extract or cannabinoids remains unexplored in horses as the efficacy and therapeutic concentrations of these compounds have not been established for any common condition in this species except for 1 case report on horse cribbing and a recent study by Interlandi et al46 examining the treatment of horses in conjunction with phenylbutazone.45,46 From very few efficacy studies it could be extrapolated, for CBD only, that a serum concentration of 102 ng/mL would be an appropriate therapeutic level for arthritis pain treatment in dogs and 54.8 to 78.9 ng/mL to control seizures in humans.12,47 The 12-hour PK and long-term administration of this study suggest that CBDA bioavailability is superior to CBD and should be further evaluated as a more physiologically pertinent cannabinoid supplement for horses. Alternatively, potentially far higher doses of CBD should be used to obtain similar responses that have been observed in other species.
Despite the significant findings, our dataset is characterized by high variance in the observed cannabinoid serum concentrations. This wide variability in the oil absorption between horses could be attributed to different factors, starting from the administration protocol. The oil was administered orally via a catheter tip syringe, without any feeding restriction, about 1 to 2 hours after pelleted feed, and while the horses had free access to hay. We cannot rule out potential partial loss of oil during administration or interference in the oil absorption due to the stomach content. The amount of forage in the gastrointestinal tract, pH variations in the gastrointestinal tract, protein binding, and enzymes specific to the equine digestive tract have already been considered factors affecting cannabinoid oral bioavailability in the horses.17 Moreover, even if the horses were healthy on physical examination and bloodwork, the group included several senior horses, whose intestinal absorption and metabolism could be affected by age-related changes in gastric secretion and small intestinal absorption as described in humans.48 In fact, a similar high variance in cannabinoid serum concentration with low bioavailability after oral administration has been already observed in senior horses.17
This study represents an initial assessment of long-term oral administration of CBD and CBDA in adult horses. The results indicate that there were no clinically significant adverse effects observed with multidose administration. Horses appeared to tolerate full-spectrum CBD or CBDA hemp oil extract well when dosed at 1 mg/kg BW. Notably, the nadir and peak serum concentrations over 6 weeks for CBD and CBDA comparison suggest a better absorption for CBDA, highlighting the need for further evaluations before providing a comprehensive guide for clinical use. Additional studies are necessary to elucidate the therapeutic benefits and determine the optimal dosage for adult horses as well as to evaluate the PK and potential adverse effects associated with longer-term dosing.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
The authors would like to thank Stephen Parry from the Cornell Statistical Consulting Center for guidance in selecting statistical models.
Disclosures
Dr. Wakshlag and Dr. Schwark are paid consultants of ElleVet Sciences. Dr. Chevalier was an Associate Editor for AJVR. The current AJVR Editor-in-Chief, Dr Lisa Fortier, was a coprincipal investigator of this project. The other authors have nothing to disclose.
Funding
This study was funded by ElleVet Sciences.
ORCID
J. M. Chevalier https://orcid.org/0000-0002-7278-5463
M. Cercone https://orcid.org/0000-0002-8922-7927
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