The efficacy of a nutritional supplement containing green‐lipped mussel, curcumin and blackcurrant leaf extract in dogs and cats with osteoarthritis

Abstract Background Osteoarthritis is a common disease in dogs and cats, and the search for novel treatment options is needed. The combination of green‐lipped mussel, curcumin and blackcurrant leaf extract has to date not been studied in dogs and cats. Objectives The aim of this study was to test the effect of a supplement containing green‐lipped mussel (Perna canaliculus), curcumin (Curcuma longa) and blackcurrant (Ribes nigrum) leaf extract on locomotion and behaviour in client‐owned dogs and cats suffering from mild to moderate osteoarthritis. Methods To this end, 32 dogs and 16 cats were enrolled in a double‐blinded, randomised, crossover, placebo‐controlled trial for 10 weeks in cats and 16 weeks in dogs. Outcome parameters were the Helsinki Chronic Pain Index (HCPI) by pet owners in dogs and cats, Canine OsteoArthritis Staging Tool (COAST) by a veterinarian in dogs and Force Plate Analysis (FPA) in 18 dogs. Results In dogs, the COAST improved significantly in the supplement group compared to baseline but was not different than the placebo group. In cats, the ability to groom, activity level, playfulness and walking up the stairs improved in the supplement group. No differences were found on HCPI scores and FPA in dogs. Several non‐responders were noted in both species, which were irrespective of the stage of osteoarthritis. Conclusions Overall, the supplement had only partial positive effects in client‐owned dogs and cats with mild to moderate osteoarthritis. Further research with a larger sample size and longer duration is needed to expand these findings.

Osteoarthritis is a chronic progressive disease, which is treated by adaptation of the environment, weight reduction, exercise instructions, physical therapy and the use of non-steroidal anti-inflammatory drugs.
As these drugs have side effects, especially with long-term use, there is a need for alternative treatment options. Especially treatment options that focus on reducing the pathophysiological mechanisms of cartilage destruction, which also provide anabolic factors for cartilage repair, become more of interest. These treatment options may also be useful for prevention of osteoarthritis (Wang et al., 2018).
Glucosamine and chondroitin are the most commonly used supplements, with a recent systematic review demonstrating meaningful pain relief compared with control in humans (Ton et al., 2020). Suggested mechanisms of action of glucosamine and chondroitin are being building blocks of the cartilage glycosaminoglycan matrix, and reducing the activity of the cartilage degrading matrix metalloproteinases (MMPs).
In green-lipped mussel, glucosamine and chondroitin are combined with eicosatetraenoic acid (ETA) which is a long-chain polyunsaturated omega-3 fatty acid, and a precursor of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). These fatty acids are known for their anti-inflammatory properties. Other components of green-lipped mussel are furan fatty acids (F-acids), sphingolipids, phytosterols, diacylglycerols, diterpenes, sesquiterpenes and saponins, alongside antioxidants such as carotenoids, xanthophylls and anthocyanins (Eason et al., 2018). Green-lipped mussel was proven to be effective in dogs with naturally occurring osteoarthritis (Railland et al., 2013).
Curcumin has anti-inflammatory properties and was proven to be effective in dogs with naturally occurring osteoarthritis (Innes et al., 2003). More recently, a study on 18 dogs with osteoarthritis has been performed. A phytosome complex form of curcumin was used, where a phospholipid was added to curcumin to enhance bioavailability. The outcome of this study showed a promising in vivo effect of curcumin. TNF-α, NF-kB1, IL-8 and prostaglandin-endoperoxide synthase 2 (PTGS2) all decreased in the curcumin-treated group (Sgorlon et al., 2016).
Blackcurrant leaf extract contains several phytochemicals such as ProAnthoCyanidins (PACs) which dampen the inflammatory response and also have a protective effect on cartilage of people with osteoarthritis (Garbacki et al., 2002). These PACs also reduced adhesion molecules (ICAM-1 and VCAM-1), thus preventing immune cells to bind to the blood vessel wall (Garbacki et al., 2005).
A synergistic effect of phytochemicals and omega-3 fatty acids has been suggested, as they all inhibit the inflammatory response and production of MMPs. The positive effects of these combinations on locomotion and pain in dogs and cats with osteoarthritis have been studied, both in therapeutic diets, as well as in supplements in both dogs and cats (Johnson et al., 2020). The combination of green-lipped mussel, curcumin and blackcurrant leaf extract has to date not been studied in dogs and cats.
The aim of this study was to test the effect of a supplement containing green-lipped mussel (Perna canaliculus), curcumin (Curcuma longa) and blackcurrant (Ribes nigrum) leaf extract on locomotion and behaviour in client-owned dogs and cats suffering from mild to moderate osteoarthritis.

MATERIALS AND METHODS
Based on previous studies, we aimed at a minimum of 16 animals per group to finish the trials (Corbee et al. 2013;Musco et al. 2019 The effect of the supplement was tested in a double-blinded, randomised, crossover, placebo-controlled trial for 10 weeks per period in cats and 16 weeks per period in dogs. The animals first received either placebo or product, followed by a 2-week wash-out period, and then the other (similar to the protocol of Innes et al., 2003). Randomisation occurred according to the scheduled appointments. Both supplement and placebo had to be administered orally and in the same volume.
The product and placebo were packed in identical packaging and were labelled with different lot numbers that were only known by the manufacturer and revealed after statistical analysis of the data. The liquid could either be mixed with the food or directly sprayed into the mouth of the dog using a syringe, as recommended by the manufacturer. The dosing schedule was based on weight and can be found in Table 1.
Owners were required to keep a logbook to record each day whether the treatment was administered, if rescue analgesia were used and if there were any particularities.
All assessments and measurements were performed at the Univer-

Helsinki Chronic Pain Index
At each visit, owners were asked to fill in the HCPI score. This is a questionnaire consisting of 11 questions that relate to the mood, vocalisation of pain and several activities that are more difficult for dogs with chronic pain caused by osteoarthritis. An adapted HCPI score was used for cats (Corbee et al., 2013), where items were scored on a 5-point scale (i.e. 1 = much worse, 2 = worse, 3 = same, 4 = better, 5 = much better) compared to baseline. To determine the stage of osteoarthritis at baseline, the cat owners were asked to give these scores compared to the period prior to osteoarthritis diagnosis. Both HCPI questionnaires are demonstrated in Appendix A. The dog HCPI questionnaire was adapted to giving a 1-10 score, as this is a common grading in the Netherlands.
A score of 1 represents the worst condition, whereas a score of 10 represents the best condition. The final HCPI score of the items was divided by the number of items to get an overall average score between 1 and 10.

Canine OsteoArthritis Staging Tool
Clinical examination of dogs was used for staging the level of osteoarthritis by using COAST at each visit (Appendix B). Because there is quite a lot of variance of clinical symptoms within each stage (normal, mild, moderate and severe), half grades were added to allow for better distinction. A grade of 2.5, for example, would represent a dog with mild to moderate osteoarthritis. Because the supplement was not intended to affect the bony deterioration in osteoarthritis, radiography was not used in COAST staging of osteoarthritis during this study.

Force Plate Analysis
FPA was performed according to Corbee et al. (2014

Statistical analysis
Statistical analysis was performed using SPSS statistics 25. All data were tested for normal distribution by a Kolmogorov-Smirnov test.
Furthermore, data were looked at graphically using a distribution graph and compared to the standard normal distribution in a quantile-

RESULTS
Of the 19 recruited dogs for the full study, 18 completed the study ( Note: Individual dog data. Group is Placebo-Supplement (PS) or Supplement-Placebo (SP), respectively. B are baseline data, S is the difference between Baseline and Supplement and P is the difference between Baseline and Placebo. HCPI is the Helsinki Chronic Pain Index score, modified to get a score between 1 (severe pain) and 10 (no pain). COAST is the Canine OsteoArthritis Staging Tool with a score between 0 (no osteoarthritis) and 4 (severe osteoarthritis). FPA Fzmax is the maximum peak vertical force (average of the front limbs) on Force Plate Analysis expressed in N/kg. Differences between a and b were considered significant (p = 0.011), whereas groups with similar letters did not differ.  Figure 1). Nine dogs did not show improvement in overall HCPI score (non-responders), which was irrespective of their stage of osteoarthritis. None of the dogs received rescue NSAIDs during the trial.
Of the 26 recruited cats, 16 completed the study (

DISCUSSION
The aim of this study was to test the effect of a supplement containing green-lipped mussel (Perna canaliculus), curcumin (Curcuma longa) and blackcurrant (Ribes nigrum) leaf extract on locomotion and behaviour in client-owned dogs and cats suffering from mild to moderate osteoarthritis. Based on the trend for improvement of the overall HCPI score in dogs, and improvement on several HCPI items in cats, the owners noted clinical improvement. COAST scores improved when using the supplement, but there was also improvement in the placebo group resulting in no significant difference between supplement and placebo. Furthermore, FPA did not show significant changes.
Non-responders were present in both dogs and cats irrespective of the stage of osteoarthritis. The results of non-responders should not be ignored or censored but should be taken into account when interpreting the overall effect, as non-responders have a negative impact on the overall observed effect of the supplement. In some individuals, TA B L E 4 Results of the dogs from the HCPI only study, and combined with the HCPI results from Note: Individual dog data. Group is Placebo-Supplement (PS) or Supplement-Placebo (SP), respectively. B are baseline data, S is the difference between Baseline and Supplement and P is the difference between Baseline and Placebo. HCPI is the Helsinki Chronic Pain Index score, modified to get a score between 1 (severe pain) and 10 (no pain). Differences between a and b were considered a trend (p = 0.100). Differences between x and y were considered a trend (p = 0.098).
improvement was also demonstrated with placebo. This can be explained by the caregiver placebo effect (Conzemius & Evans 2012, Gruen et al., 2017. This effect narrows the difference between the supplement group and the placebo group, which also stresses the importance of comparison with baseline. Several nutraceuticals have been proven to be effective in dogs, of which omega-3 fatty acids are considered among the most promising (Johnson et al., 2020).
Little research has been done on the effects of nutraceuticals in cats with osteoarthritis. The results of this study were similar to the findings in cats that were given omega-3 fatty acids, as with both supplements cats revealed a higher activity level, and more walking up the stairs. Effects that were demonstrated with the supplement in this study and not with omega-3 supplementation were increased ability to groom, playfulness and overall level of satisfaction, whereas with omega-3 supplementation less stiffness during gait, more interaction with the owner and higher jumps were noted, which were not seen with the supplement in this study (Corbee et al., 2013). Comparison with baseline was not possible due to the setup of the questionnaire, which was a limitation of the study.
Because of the high variation between individual responses, a significant difference could not be found in HCPI and FPA. For HCPI, a significant difference might be expected to be found with a larger sample size, as the HCPI results of the full study already demon-strated a trend. It could be noted that COAST is designed as a staging tool, and maybe more useful for selecting the patients for the study rather than evaluating the effect of nutraceuticals or other therapeutic agents. Whether the observed average 0.44-point improvement of the COAST score is clinically relevant therefore remains speculative.
FPA results did not show significant differences between groups, nor between supplement, placebo and baseline. FPA is complex and its results are dependent on the stage of osteoarthritis, the location of the osteoarthritis and the compensation by other limbs. The peak vertical force is mostly used as outcome parameter; however, this parameter is also influenced by several factors, such as symmetry, division of the weight bearing over the four limbs, the impulse and walking speed.
Only a single dosage of the supplement was tested. It is possible that a higher dosage would have given a better result. Also, the duration of the study was quite short, as usually supplements for osteoarthritis in dogs and cats are evaluated after a 3-to 6-month period.
Further research with a larger sample size, different dosage and longer duration is therefore needed to expand the findings from this study.
In conclusion, the supplement had some positive effects but minimal significant findings in reducing pain and lameness according to the owners' observations in client-owned dogs and cats with mild to moderate osteoarthritis.

ACKNOWLEDGEMENTS
The authors would like to acknowledge all the pet owners that had their pets enrolled in this study. Remco Consemulder and Miriam Dieperink are acknowledged for their master thesis work, which were part of the work presented in this article.

ETHICAL STATEMENT
The study was approved according to Dutch legislation and registered under number AVD1080020184847.

AUTHOR CONTRIBUTION
Dr. R.J. Corbee was responsible for conceptualization, formal analysis, funding acquisition, investigation, methodology, project administration and writing.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

PEER REVIEW
The peer review history for this article is available at https://publons.