Clinical Studies

Shelley Turner*, Virginia Day Barker, and Amanda A. Adams

M. H. Gluck Equine Research Center, Department of Veterinary Science, University of
Kentucky, Lexington, KY 40546, USA

Abstract:

Cannabis sativa L. contains cannabidiol (CBD), a compound that has many
anti-inflammatory properties. In this study, 99.9% CBD powder was used to determine its in
vitro efficacy as an anti- inflammatory agent. Heparinized blood was collected via jugular
venipuncture from senior horses. PBMCs were isolated then incubated for 24 hours with
increasing dilutions of CBD dissolved in DMSO. PBMCs were stimulated the last 4 hours of incubation with PMA/IO and Brefeldin A. A Vicell
counter was used to evaluate viability after incubation. PBMCs were stained intracellularly
for IFNγ and TNFα then analyzed via flow cytometry. RT-PCR was used to analyze samples for
gene expression. Five equine-specific intron-spanning primers/probes used are: CB1, CB2,
TNFα, IFNγ, IL-10, and Beta-glucuronidase. Data was analyzed using RM One-way ANOVA
(significance P< 0.05). Viability of PBMCs with CBD was completed to determine cytotoxicity.
The dilution of CBD that did not affect cell viability was 4 μg/mL (P<0.05). CBD at 4 μg/mL
significantly reduced production of IFN-γ and TNF-α (P<0.05). RT-PCR results for TNFα and IFNγ
at 4 μg/mL showed a reduction compared with the positive control and IL-10 showed a
similar reduction at 2 μg/mL and 4 μg/mL. RT-PCR gene expression results showed
significance for 10 μg/mL CBD in CB1 and CB2. CBD at 4μg/mL reduced in vitro production of
inflammatory cytokines from senior horses. This in vitro study supports further investigation
of CBD to determine if it may be effective as an anti-inflammatory treatment for chronic
inflammation in the horse.
Keywords: inflammation, cannabidiol, equine, Cannabis sativa L.

*Corresponding author at: 108 Gluck Equine Research Center, Department of Veterinary
Science, University of Kentucky, Lexington, KY 40546-0099, United States.

Dr. James Baumgartner, a Ph.D.,Biochemistry and Pharmacology from Washington State
University and Lawrence Dukes, MAS, Johns Hopkins University

Abstract— Cannabidiol (CBD) is one of the major non-psychoactive cannabinoids
produced by Cannabis sativa L. Hemp is classified as a Cannabis varietal because it has
less than 0.3% tetrahydrocannabinol (THC) – the psychoactive component found in
Cannabis. Recent studies have shown that CBD has good tolerability and a wide range
of reported health benefits. The purpose of this study was to measure the effects of CBD
increase movement and promote calmness in horses. CBD was studied in horses after
the administration of two oral doses (50 mg each) each day over a two-week
timeframe. Fortyone horses were studied across the United States and participants were
obtained through relationships with horse shelters, rescues, farms with retired show and
race horses, and general pleasure horse farms. In all of the cases, the horses were
evaluated and determined, at the outset, to have impaired movement & presentation
and/or demonstrated common vices of anxiety like cribbing, pacing or reluctance to
load to trailers or gates.

Morris Elizabeth M., Kitts-Morgan Susanna E., Spangler Dawn M., Ogunade Ibukun M., McLeod Kyle
R., Harmon David L.

Frontiers in Veterinary Science Volume 8 Year 2021
https://www.frontiersin.org/article/10.3389/fvets.2021.685606

DOI: 10.3389/fvets.2021.685606 ISSN: 2297-1769

Despite the increased interest and widespread use of cannabidiol (CBD) in humans and
companion animals, much remains to be learned about its effects on health and physiology.
Metabolomics is a useful tool to evaluate changes in the health status of animals and to
analyze metabolic alterations caused by diet, disease, or other factors. Thus, the purpose of this
investigation was to evaluate the impact of CBD supplementation on the canine plasma
metabolome. Sixteen dogs (18.2 ± 3.4 kg BW) were utilized in a completely randomized design
with treatments consisting of control and 4.5 mg CBD/kg BW/d. After 21 d of treatment, blood
was collected ~2 h after treat consumption. Plasma collected from samples was analyzed using
CIL/LC-MS-based untargeted metabolomics to analyze amine/phenol- and
carbonyl-containing metabolites. Metabolites that differed — fold change (FC) ≥ 1.2 or ≤ 0.83 and
false discovery ratio (FDR) ≤ 0.05 — between the two treatments were identified using a volcano
plot. Biomarker analysis based on receiver operating characteristic (ROC) curves was
performed to identify biomarker candidates (area under ROC ≥ 0.90) of the effects of CBD
supplementation. Volcano plot analysis revealed that 32 amine/phenol-containing metabolites
and five carbonyl-containing metabolites were differentially altered (FC ≥ 1.2 or ≤ 0.83, FDR ≤
0.05) by CBD; these metabolites are involved in the metabolism of amino acids, glucose,
vitamins, nucleotides, and hydroxycinnamic acid derivatives. Biomarker analysis identified 24
amine/phenol-containing metabolites and 1 carbonyl-containing metabolite as candidate
biomarkers of the effects of CBD (area under ROC ≥ 0.90; P < 0.01). Results of this study indicate
that 3 weeks of 4.5 mg CBD/kg BW/d supplementation altered the canine metabolome.
Additional work is warranted to investigate the physiological relevance of these changes.

Morris Elizabeth M., Kitts-Morgan Susanna E., Spangler Dawn M., Gebert Jessica, Vanzant Eric S.,
McLeod Kyle R., Harmon David L.

Frontiers in Veterinary Science Volume 8: Year: 2021

URL: https://www.frontiersin.org/article/10.3389/fvets.2021.645667
DOI: 10.3389/fvets.2021.645667 ISSN: 2297-1769

Abstract: Growing public interest in the use of cannabidiol (CBD) for companion animals has
amplified the need to elucidate potential impacts. The purpose of this investigation was to
determine the influence of CBD on the daily activity of adult dogs. Twenty-four dogs (18.0 ± 3.4
kg, 9 months−4 years old) of various mixed breeds were utilized in a randomized complete
block design with treatments targeted at 0 and 2.5 mg (LOW) and at 5.0 mg (HIGH) CBD/kg
body weight (BW) per day split between two treats administered after twice-daily exercise
(0700–0900 and 1,700–1,900 h). Four hours each day [1,000–1,200 h (a.m.) and 1,330–1,530 h
(p.m.)] were designated as times when no people entered the kennels, with 2 h designated as
Quiet time and the other 2 h as Music time, when calming music played over speakers. Quiet
and Music sessions were randomly allotted to daily a.m. or p.m. times. Activity monitors were
fitted to dogs' collars for continuous collection of activity data. Data were collected over a
14-day baseline period to establish the activity patterns and block dogs by activity level (high
or low) before randomly assigning dogs within each block to treatments. After 7 days of
treatment acclimation, activity data were collected for 14 days. Data were examined for
differences using the MIXED procedure in SAS including effects of treatment, day, session
(Quiet or Music), time of day (a.m. or p.m.), and accompanying interactions. CBD (LOW and
HIGH) did not alter the total daily activity points (P = 0.985) or activity duration (P = 0.882). CBD
tended (P = 0.071) to reduce total daily scratching compared with the control. Dogs were more
active in p.m. sessions than in a.m. sessions (P < 0.001). During the p.m. session, dogs receiving
HIGH tended (P = 0.091) to be less active than the control (CON). During the a.m. and p.m.
sessions, CBD reduced scratching compared with CON (P = 0.030). CBD did not affect the
activity duration during exercise periods (P = 0.143). These results indicate that, when
supplemented with up to 4.5 mg CBD/kg BW/day, CBD does not impact the daily activity of
adult dogs but may exert an antipruritic effect.

Morris Elizabeth M., Kitts-Morgan Susanna E., Spangler Dawn M., McLeod Kyle R., Costa Joao H.
C., Harmon David L. Frontiers in Veterinary Science Volume 7, Year 2020
https://www.frontiersin.org/article/10.3389/fvets.2020.569565

DOI: 10.3389/fvets.2020.569565 ISSN: 2297-1769

Abstract: Interest is increasing regarding use of Cannabidiol (CBD) in companion animals
due to anecdotal evidence of beneficial behavioral and health effects. The purpose of this
investigation was to evaluate the influence of CBD on behavioral responses to fear-inducing
stimuli in dogs. Sixteen dogs (18.1 ± 0.2 kg) were utilized in a replicated 4 × 4 Latin square
design experiment with treatments arranged in a 2 × 2 factorial, consisting of control, 25 mg
CBD, trazodone (100 mg for 10–20 kg BW, 200 mg for 20.1–40 kg BW), and the combination of
CBD and trazodone. A fireworks model of noise-induced fear was used to assess CBD
effectiveness after 7 d of supplementation. Each test lasted a total of 6 min and consisted of
a 3 min environmental habituation phase with no noise and a 3 min noise phase with a
fireworks track. Plasma was collected 1 h before, immediately after, and 1 h following testing
for cortisol analysis. Behaviors in each 3 min block were video recorded, and heart rate (HR)
sensors were fitted for collection of HR and HR variability parameters. Research personnel
administering treats and analyzing behavioral data were blinded as to the treatments
administered. Data were tested for normality using the UNIVARIATE procedure in SAS, then
differences examined using the MIXED procedure with fixed effects of treatment, period, time,
and treatment x time interaction. Inactivity duration and HR increased during the first minute
of the fireworks track compared with 1 min prior (P < 0.001 and P = 0.011, respectively),
indicating the fireworks model successfully generated a fear response. Trazodone lowered
plasma cortisol (P < 0.001), which was unaffected by CBD (P = 0.104) or the combination with
CBD (P = 0.238). Neither CBD nor trazodone affected the duration of inactivity (P = 0.918 and
0.329, respectively). Trazodone increased time spent with tail relaxed (P = 0.001). CBD tended
to increase HR (P = 0.093) and decreased the peak of low- and high-frequency bands (LF
and HF, P = 0.011 and 0.022, respectively). These results do not support an anxiolytic effect of
CBD in dogs given 1.4 mg CBD/kg BW/d.

E Gutierre, N Crosignani, C García-Carnelli, A di Mateo, L Recchi
First published: 31 March 2023 https://doi.org/10.1002/vms3.1057

Case Study A 10-year-old mixed breed male neutered cat presented with clinical signs
related to chronic orthopaedic pain. The cat ́s owner reported a decrease in its usual
activity level, difficulty jumping and vocalisation during defecation, amongst others. Upon
physical examination, pain was noted, based on the feline Musculoskeletal Pain Index (FMPI).
Treatment was with CBD at 0.25 mg/kg. Pain score decreased from 13 to 5 points on the FMPI
with 30 day treatment.

NB Full spectrum Cannabis sativa oil containing CBD and THC exhibited an analgesic effect
in a cat with chronic pain. The owner stated that the improvement in the patient's quality of
life outweighed the few adverse effects presented.

Cannabis-based pharmaceutical formulations with analgesic properties have been proved
in to be useful humans with chronic pain, but there is lack of literature in veterinary
medicine. To our knowledge, this is the first reported case about the use of full spectrum
cannabis sativa oil in a cat with osteoarthritic pain.”

EquiCeutica's Feline CBD contains zero THC, however, patients present similar treatment
results without observable side effects

Joana Chambel Coelho, ,Noélia Duarte, Andreia Bento da Silva, Maria do Rosário Bronze and
Lisa Alexandra Mestrinho

Animals 2023, 13(17), 2716; https://doi.org/10.3390/ani13172716

Abstract

A placebo-controlled study evaluated the clinical efficacy and safety of a commercially
available cannabidiol (CBD) oral formulation as an adjunctive treatment for pain
management for feline chronic gingivostomatitis (FCGS). CBD was included in a multimodal
treatment routinely performed on client-owned cats with FCGS that were submitted to
dental extractions. Twenty-two cats were consecutively included in the study. The first group
was treated using a fixed dosage of 4 mg per cat every 12 h for 15 consecutive days, and the
second received a placebo of similar features. Treatments began 2 h before dental
extractions. Pain and disease severity were assessed at days 0 and 15 using the Composite
Oral Pain Scale (COPS-C/F) and the Stomatitis Disease Activity Index score (SDAI). Weight,
vital and biochemistry parameters, and analgesic reinforcement needs were also registered
at the same time points. In the treated cats, blood was collected after 4, 8, and 12 h to
determine CBD serum concentrations using ultra-high-performance liquid
chromatography–mass spectrometry (UHPLC-MS/MS). After data analysis using mixed
models, a significant improvement in the SDAI scores of cats medicated with CBD was
found. The protocol is safe since severe adverse effects and biochemical changes were not
observed during the treatment period. This study suggests that the cats benefited from this
treatment.

Silver RJ. The Endocannabinoid System of Animals. Animals (Basel). 2019;9(9):686. Published
2019 Sep 16. doi:10.3390/ani9090686

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770351/

Williams MR. et al. Pharmacokinetic Evaluation of a Cannabidiol Supplement in Horses. J
Equine Vet Sci. 2022.

Mackie K. Cannabinoid receptors: where they are and what they do. J Neuroendocrinol. 2008.

Pagano, C. et al. Cannabinoids: Therapeutic Use in Clinical Practice. Int J Mol Sci.

2022.Cannabidiol (CBD). Government of Canada. Accessed on 02/28/23.

Lu, H. C., & Mackie, K. An Introduction to the Endogenous Cannabinoid System. Biological
psychiatry. 2016.

Fabio, L. et al. Endocannabinoids and endocannabinoid-related mediators: Targets,
metabolism and role in neurological disorders. Progress in Lipid Research. 2016.

.Howlett, A. C., & Abood, M. E. CB1 and CB2 Receptor Pharmacology. Advances in
pharmacology. 2017.

Ellis, K.L. and Contino, E.K. Treatment using cannabidiol in a horse with mechanical allodynia.
Equine Veterinary Education. 2020.

Meissner, H, & Cascella, M. Cannabidiol (CBD). StatPearls Publishing LLC. 2022.

Williams MR. et al. Pharmacokinetic Evaluation of a Cannabidiol Supplement in Horses. J
Equine Vet Sci. 2022.

St Blanc, MP. Et al. Effects of a Supplement Containing Cannabidiol (CBD) on Sedation and
Ataxia Scores and Health. J Equine Vet Sci. 2022.

Ryan, D. et al. Pharmacokinetics and effects on arachidonic acid metabolism of low doses of
cannabidiol following oral administration to horses. Drug Test Anal. 2021.

Turner, S. et al. Effects of Cannabidiol on the In Vitro Lymphocyte Pro-Inflammatory Cytokine
Production of Senior Horses. J Equine Vet Sci. 2021.

Turner, S. et al. The effects of cannabidiol on immune function and health parameters in
senior horses. Vet Immunol Immunopathol. 2023.

Carrier, E. J. et al. Inhibition of an equilibrative nucleoside transporter by cannabidiol: a
mechanism of cannabinoid immunosuppression. Proceedings of the National Academy of
Sciences of the United States of America. 2006.

Malfait, A. M. et al. The nonpsychoactive cannabis constituent cannabidiol is an oral
anti-arthritic therapeutic in murine collagen-induced arthritis. Proceedings of the
National Academy of Sciences of the United States of America. 2000.

Verrico, CD. Et al. A randomized, double-blind, placebo-controlled study of daily cannabidiol
for the treatment of canine osteoarthritis pain. Pain. 2020.

Yu, CHJ. & Rupasinghe, HPV. Cannabidiol-based natural health products for companion
animals: Recent advances in the management of anxiety, pain, and inflammation. Res Vet
Sci. 2021.

Peyravian N. et al. The Anti-Inflammatory Effects of Cannabidiol (CBD) on Acne. J Inflamm
Res. 2022.

Pagano, E. et al. An Orally Active Cannabis Extract with High Content in Cannabidiol
attenuates Chemically-induced Intestinal Inflammation and Hypermotility in the Mouse.
Front Pharmacol. 2016.

Xu DH. et al. The Effectiveness of Topical Cannabidiol Oil in Symptomatic Relief of Peripheral
Neuropathy of the Lower Extremities. Curr Pharm Biotechnol. 2020.

Hammell, D. C. et al. Transdermal cannabidiol reduces inflammation and pain-related
behaviors in a rat model of arthritis. European journal of pain. 2016.

Maroon J, Bost J. Review of the neurological benefits of phytocannabinoids. Surg Neurol Int.
2018.

Campos AC. et al. Cannabidiol, neuroprotection and neuropsychiatric disorders. Pharmacol
Res. 2016.

Potschka H, Bhatti SFM, Tipold A, McGrath S. Cannabidiol in canine epilepsy. Vet J. 2022.

de Almeida DL. & Devi LA. Diversity of molecular targets and signaling pathways for CBD.
Pharmacol Res Perspect. 2020.

Sholler DJ. et al. Therapeutic Efficacy of Cannabidiol (CBD): A Review of the Evidence from
Clinical Trials and Human Laboratory Studies. Curr Addict Rep. 2020.

Iffland, K., & Grotenhermen, F. An Update on Safety and Side Effects of Cannabidiol: A Review
of Clinical Data and Relevant Animal Studies. Cannabis and cannabinoid research. 2017.

Chesney, E. et al. Adverse effects of cannabidiol: a systematic review and meta-analysis of
randomized clinical trials. Neuropsychopharmacol. 2020.