Long-term effects of a diet supplement containing Cannabis sativa oil and Boswellia serrata in dogs with osteoarthritis following physiotherapy treatments: a randomised, placebo-controlled and double-blind clinical trial

Abstract Dogs are commonly affected by Osteoarthritis (OA). Different approaches can be used to alleviate animals’ symptoms. In this randomised, placebo-controlled and double-blind clinical trial, we performed a three months follow-up study assessing the efficacy of a food supplement containing natural ingredients (Cannabis sativa oil, Boswellia serrata Roxb. Phytosome® and Zingiber officinale extract) in dogs with OA after the interruption of physiotherapy that was performed during the previous three months. Inflammation and oxidative stress were reduced in the treated group (higher glutathione (GSH) and lower C-reactive protein [CRP] levels in blood) as well as chronic pain. Graphical Abstract


Introduction
Osteoarthritis (OA) is a chronic, degenerative and inflammatory disease. It causes chronic pain and leads to a decrease in mobility (Rychel 2010). Chronic inflammation has a key role in the pathogenesis of OA (Zhang 2018), together with oxidative stress. In the literature, it has been confirmed that the production of excessive reactive oxygen species (ROS) associated to pro-inflammatory cytokines and prostaglandins could be deleterious in the case of articular diseases (Hui et al. 2015). To-date, there are different markers of oxidative stress, and the level of antioxidant enzymes could change in the case of human and animal patients affected by OA. A simple blood test can be performed to monitor two elective serum markers prone to decreasing or increasing in the case of OA: the glutathione (GSH) (Goranov 2007;Sarban et al. 2005) and the serum C-reactive protein (CRP) (Zhang 2018). New and non-conventional therapeutic approaches to counter-act disease progression and to improve animal well-being are in continuous development. Human and animal trials confirmed the positive effect of natural products for supporting different clinical conditions. (Ernst 2009;Kline 2002;Lana et al. 2006;Mosavat et al. 2018;Kline 2002. Several natural ingredients extracted from plants have been repeatedly reported in literature as having relevant anti-microbial, antioxidant, immunomodulatory and anti-inflammatory proprieties (Tsai et al. 2011;Valdivieso-Ugarte and Gomez-Llorente 2019), also reducing chronic pain associated with OA (Caterino et al. 2021;Martello et al. 2022Martello et al. , 2018Martello et al. , 2019. Additionally, other approaches have been used in veterinary medicine including physiotherapy, acupuncture, and hydrotherapy (Cimino Brown 2017; Silva et al. 2017). In this randomised, placebo-controlled, double-blind clinical study, we conducted a three-month follow-up study assessing the efficacy of a dietary supplement (Evexia Plus, Candioli s.r.l., Beinasco, Italy) containing Cannabis sativa oil, Boswellia serrata Roxb. Phytosome V R and Zingiber officinale extract (Supplementary material -Table S1) in maintaining or even improving the clinical condition of dogs with OA after the interruption of physiotherapy sessions. Physiotherapy had been combined with the administration of the same supplement tested also in this study (Evexia Plus, Candioli s.r.l., Beinasco, Italy) in the previous three months, as already reported (Martello et al. 2021).

Results and discussion
Details on animal inclusion and recruitment process for this study are summarised in the Figure S1 (Supplementary material). Dogs aged 8-15 years (TRT: mean 10 ± 2.4 SD; CTR mean 9.5 ± 1.7 SD). There were 14 females (6 TRT and 8 CTR groups) and 13 males (8 TRT and 5 CTR group). Dogs weight ranged between 5 and 54 kg (TRT: mean 33.9 kg ± 16.3 SD; CTR 29.5 kg ± 14.5 SD). This difference in weights was considered in the statistical model for the correct interpretation of the results on the HCPI and blood parameters. No dogs had to be excluded during the 170 d of trial and owners did not report any adverse effect of the supplement (no vomiting or diarrhoea). The complete blood analysis did not reveal alteration of the values for all dogs, clarifying that there were no ongoing pathologies at T0. In addition, values were within the normal ranges at T0, T4, T5, T6 and T7. This data gives us a further confirmation of the safeness of the supplement. The two serum inflammatory and oxidative stress biomarkers (CRP and GSH), together with the HCPI, were specifically used to evaluate the long-term effects of the supplement after the interruption of physiotherapy treatment commenced at T4.
Based on the HCPI results, the level of pain significantly (p < 0.05) and progressively decreased during the study, starting from T3 and even more when the physiotherapy ended during the follow-up period (T5-T7, Figure 1; Supplementary material - Table S2). Thus, the sole supplement can maintain, and even improve, the already-demonstrated positive and synergic effect of physiotherapy with the supplement highlighted also in the first study (Figure 1; Supplementary material -Table S3 (Martello et al. 2021)). The use of herbal treatments in humans (Cameron and Chrubasik 2014) and dogs (Beths et al. 2020) with OA have been increasing to reduce the administration of drugs with known sideeffects, like non-steroidal anti-inflammatory drugs (NSAIDs) (Lascelles et al. 2005). Our data suggest that the use of natural products together with physiotherapy is probably needed when a dog is first diagnosed with OA, and when the pain is high, based on the HCPI scores. On the other hand, once an animal's condition starts to improve, the administration of the supplement is enough to ensure excellent maintenance, or even improvement, of the animal's wellbeing in the following three months. To support the results on chronic pain, objective data on two blood parameters, CRP and GSH, respectively showed a significant (p < 0.05) decrease and increase from T4 compared to T0 at each time point during the entire study. Boswellia serrata gum resin extract containing boswellic acids, has been already demonstrated to be effective in the management of pain and inflammation of the musculoskeletal system (Riva et al. 2017). This effect is linked to the ability to inhibit the synthesis of leukotrienes, the chemical mediators of the inflammatory process in various inflammatory diseases, including arthritis (Musco et al. 2019). Recently, medical interest in alternative way to reduce pain has led many pet owners to seek hemp related products rich in cannabinoids. Cannabis sativa oil that is present in the tested supplement, may have shown its antioxidant activity increasing the GSH values and reducing chronic pain in the TRT group (Nafis et al. 2019-10-01).
These results confirm once again, that the supplement has an excellent effect on reducing the inflammation and oxidative stress in dogs affected by OA, and that the use of nutraceuticals is promising in veterinary medicine (Cardeccia et al. 2022;Johnson et al. 2020).
Specifically, the positive synergic effect of natural products included in the supplement with physiotherapy in dogs with OA, and the excellent capacity of the sole supplement to guarantee long-term maintenance of this improved condition without physiotherapy. The small number of included animals did not allow to perform any subgroup analysis (by age, sex, affected join, etc.). Further studies would be useful to see the effect of the sole supplement on a larger population and over a longer time.

Experiment
See Supplementary material.