A randomized, double-blind, placebo-controlled study of daily cannabidiol for the treatment of canine osteoarthritis pain

Abstract

Over the last 2 decades, affirmative diagnoses of osteoarthritis (OA) in the United States have tripled due to increasing rates of obesity and an aging population. Hemp-derived cannabidiol (CBD) is the major nontetrahydrocannabinol component of cannabis and has been promoted as a potential treatment for a wide variety of disparate inflammatory conditions. Here, we evaluated CBD for its ability to modulate the production of proinflammatory cytokines in vitro and in murine models of induced inflammation and further validated the ability of a liposomal formulation to increase bioavailability in mice and in humans. Subsequently, the therapeutic potential of both naked and liposomally encapsulated CBD was explored in a 4-week, randomized placebo-controlled, double-blinded study in a spontaneous canine model of OA. In vitro and in mouse models, CBD significantly attenuated the production of proinflammatory cytokines IL-6 and TNF-α while elevating levels of anti-inflammatory IL-10. In the veterinary study, CBD significantly decreased pain and increased mobility in a dose-dependent fashion among animals with an affirmative diagnosis of OA. Liposomal CBD (20 mg/day) was as effective as the highest dose of nonliposomal CBD (50 mg/day) in improving clinical outcomes. Hematocrit, comprehensive metabolic profile, and clinical chemistry indicated no significant detrimental impact of CBD administration over the 4-week analysis period. This study supports the safety and therapeutic potential of hemp-derived CBD for relieving arthritic pain and suggests follow-up investigations in humans are warranted.

Figure 1.. CBD reduces hallmarks of arthritis-related inflammation in vitro.

5 × 10⁶ cells of the specified type were plated in triplicate in 6-well plate in 5 ml RPMI + 10% FBS followed by addition of either 1 ng/ml LPS for 4 hrs or 100 ng/ml SEB for 6 hrs with or without the addition of 100 ng/ml CBD after 2 hrs. After the incubation period, the media was analyzed using the BD TNF-⍺ Flex set. A. TNF-⍺ levels in murine RAW267.4 macrophage cell line. B. TNF-⍺ levels in primary mouse splenocytes. C. TNF-⍺ levels in human THP-1 monocyte cell line. D. TNF-⍺ levels in primary human PBMC. Representative experiment of three shown. Error bars +/− SD. *p<0.05, **<0.01 by Student’s two-tailed t-test for all A-D. Cohorts of female mice were also treated on one ear with 100 μl 2% croton oil-acetone, and ear edema was allowed to occur for 1 – 4 hrs. At 2 hrs, mice were treated on the swollen ear with either 100 μl of vehicle or 100 μl of 10 mg/ml CBD oil. Additionally, a group of untreated mice also received 100 μl CBD-oil E. At each time point indicated, 4 mm biopsies from the most central portion of swelling was obtained, homogenized, and measured for myeloperoxidase (MPO) activity via ELISA. F. After 4 hrs, each cohort was retro-orbitally bled for analysis of circulating TNF-⍺ concentrations via the BD TNF⍺ Flex set. Each cohort consisted of n=5 mice. Representative experiment of three shown. Error bars +/− SD. *p<0.05 by Student’s two-tailed t-test.

Figure 2.. Intraperitoneal CBD administration reduces inflammatory cytokines and circulating neutrophils in an in vivo LPS inflammatory model.

Cohorts of female mice were treated intraperitoneally with 200 ng LPS for 2 hrs and subsequently administered intraperitoneal CBD, intraperitoneal PBS control, topical CBD, or topical Ben-Gay control as indicated. After an additional 2 hours, mice were retro-orbitally bled and circulating cytokines were analyzed with the appropriate BD Flex set. A. TNF-α. B. IL-6. C. IL-10. D. CXCL1. E. CXCL2. F. Flow cytometry analysis of the cellular portion was performed each hour to determine relative number of neutrophils (CD115^neg CD11b⁺ Ly6G⁺) in circulation. Representative experiment shown. Error bars +/− SD. *p<0.05, **<0.01 by one-way ANOVA.

Figure 3.. Liposomal encapsulation of small molecules enhances bioavailability.

Sunflower lecithin (phosphatidyl choline) was used as a base to make liposomes approximately 100 nm in size that could encapsulate small molecules at a concentration of 10–20 mg/ml and retain polydispersity and size for at least 3 months at 4°C. A. Stable size and polydispersity observed by Transmission Electron Microscopy (TEM). B. Cohorts of mice were implanted subcutaneously injected with 500,000 luc2⁺ cells near the hindquarters. B/C. Twenty-four hours later, D-luciferin (100 μl, 10 mg/ml) or D-luciferin liposomes (100 μl, 10 mg/ml) was applied subcutaneously near the forequarters, and animals were continually imaged by IVIS for 2 hrs with subsequent photon measurement at the target serving as a proxy for absorption and bioavailability. D. The ability of liposomal CBD to reduce TNF-⍺ production relative to controls and naked CBD was determined. For B and C, n=5 mice per cohort. Representative experiment of three shown. For D, n=8 mice per cohort. Representative experiment of two shown. Error bars +/− SD. *p<0.05, **p<0.01 by Student’s two-tailed t-test.

Figure 4.. Daily administration of CBD for 30 days improves owner-perspective QOL scores among large dogs with affirmative diagnosis of osteoarthritis.

Twenty large domestic canines with affirmative diagnosis of osteoarthritis were enrolled in a double-blind, placebo-controlled randomized study. animals were administered coconut oil placebo, 20 mg/day naked CBD, 50 mg/day naked CBD, or 20 mg/day liposomal CBD. Owners assessed their animals by means of the Helsinki Chronic Pain Index (HPCI) on days 0, 30, and 45. A. Individual HPCI values were plotted for each study cohort on days 0 and 30. B. Cohort HPCI values were plotted on days 0, 30, and 45. Error bars +/− SD. *p<0.05, **p<0.01 by Student’s two-tailed t-test.

Figure 5.. Daily administration of CBD for 30 days improves veterinarian-perspective subset QOL scores among large dogs with affirmative diagnosis of osteoarthritis.

Study enrolled canine subjects were scored by the (blinded) study veterinarian on days 0 and 30 using a scale of 1 (best) to 5 (worst) for four different movements consisting of sitting to standing, lying to standing, walking, and running. Subset scale data comparing day 0 and day 30 scores for each task are shown by cohort. Error bars +/− SEM. *p<0.05, **p<0.01 by Student’s two-tailed t-test.

Figure 6.. Daily administration of CBD for 30 days improves veterinarian-perspective overall QOL scores among large dogs with affirmative diagnosis of osteoarthritis.

Study enrolled canine subjects were scored by the (blinded) study veterinarian on days 0 and 30 using a scale of 1 (best) to 5 (worst) for four different movements consisting of sitting to standing, lying to standing, walking, and running. Data are represented as pie charts indicating percent of each cohort that showed improvement, worsening, or no change in condition for the animals enrolled in each study group. **p<0.01, ****p<0.001 by Pearson’s chi-squared.

Figure 7.. Daily administration of CBD for 30 days does not alter alanine aminotransferase (ALT) or alkaline phosphatase (ALKP) levels.

Blood was drawn from animals we enrolled in the clinical study on days 0 and 30, and Chem10 analysis was performed. A. Relative changes in circulating ALT and ALKP values over the 30-day period. B. Specific changes in circulating ALT and ALKP values over the 30-day period. Dark horizontal lines outline normal range. Error bars +/− SD. No statistically significant changes were observed.