This prospective, single-centre RCT used positive (meloxicam) and negative (placebo) control groups to investigate the effectiveness of APPA for the management of naturally-occurring OA in dogs. As the study involved client-owned dogs (primarily pets), written owner consent was obtained following explanation of the study aims and protocols. Owners were free to withdraw their dogs from the study at any time and for any reason, but full participation was encouraged by education about the study design and purpose before enrolment. The study design was approved by the institutional ethics committee, in accordance with Good Scientific Practice guidelines and national legislation.
Eligible dogs were enrolled in the study between September 2013 and September 2014. Dogs were considered eligible if they had a chronic unilateral lameness of at least grade 2 on a scale from 0 (normal) to 5 (nonweight-bearing lameness), clinical and radiographic evidence of OA in the lame limb, and absence of other serious medical conditions in the patient history and on clinical examination that would preclude NSAID use or affect the dog's gait (e.g., neurologic disorders). Dogs with OA in more than one limb were included if the lameness was clearly and consistently manifested in a single limb.
On enrolment, dogs were randomly assigned to one of three treatment groups (meloxicam, APPA, or placebo) using computerised randomisation (Research Randomizer; https://www.randomizer.org). A 1-week "run-in" period preceded the 4-week treatment period. During run-in, the dogs received no NSAIDs or other OA medications that may affect lameness severity or gait. The dogs were evaluated as described below at the end of the run-in period (Day 0) and on Days 14 and 28 of treatment.
During the 4-week treatment period, dogs in the meloxicam group received the recommended label dosage of Metacam® (meloxicam 1.5 mg/ml oral suspension for dogs; Boehringer Ingelheim Vetmedica GmbH, Ingelheim/Rhein, Germany): a single loading dose of 0.2 mg/kg per os (PO), then 0.1 mg/kg PO q24h. As a gastroprotectant, those dogs also received famotidine, 0.5 mg/kg PO q24h. Dogs in the APPA group received APPA (apocynin-paeonol, 20:80 w/w [patent protected]; AKL International, Stevenage, Herts, UK) at a dosage of 40 mg/kg PO q12h, combined in the capsule with animal protein concentrate to mask the odour of the apocynin (an aromatic molecule). Dogs in the placebo group received capsulated animal protein concentrate at a dosage designed to mimic that of APPA. In this way, the investigators and dog owners were blinded to treatment for both the APPA and placebo groups for the duration of the study.
No other OA medications were permitted during the study period, and owners were instructed to continue any joint supplements currently in use (fish oil, glucosamine-chondroitin, etc.) without any additions or increases in dosage, so for ethical reasons the study would conclude for any dog if clinical signs worsened such that acute pain management was needed.
Variables and Outcome Measures:
The following variables were recorded at enrolment for each dog: age (yrs), body weight (kg), gender, breed, and joint(s) primarily affected. Response to treatment was evaluated using the following outcome measures.
Orthopaedic Score (OS)
An orthopaedic score was determined by veterinary examination of the dog's musculoskeletal system and gait, using a standardised orthopaedic examination comprising five components: standing posture (graded from 0 to 3), lameness at the walk (0-3), lameness at the trot (0-3), willingness to lift the contralateral limb (0-4), and pain on palpation/manipulation of the affected joint (0-3), where 0 is normal and the maximum score represents marked abnormality. The complete scoring system is detailed in Table S1. Both the cumulative score (out of 16) and scores for the individual components were analysed. To minimise operator variability, the same experienced veterinarian performed the examination each time. The veterinarian was blinded to treatment group throughout the study.
Canine Brief Pain Inventory (CBPI)
The CBPI is a validated means of classifying and quantitating the owner's assessment of the dog's pain, functional impairment, and overall quality of life in the last 7 days. A Pain Severity (PS) score was generated by averaging the scores for 4 questions about the dog's pain (rate pain at its worst, least, average, and current), each scored on a scale from 0 ("no pain") to 10 ("extreme pain"). A Pain Interference (PI) score was similarly generated by averaging the scores for 6 questions about how the dog's pain has interfered with daily life (general activity, enjoyment of life, and ability to rise to standing from lying down, walk, run, and climb stairs, curbs, etc.), each scored from 0 ("does not interfere") to 10 ("completely interferes"). Overall Quality of Life (QOL) was rated from 1 (excellent) to 5 (poor). The full CBPI questionnaire is shown in Figure S1.
Descriptive statistics were generated using Excel version 14.2 (Microsoft Corp., Redmond, Washington, USA) and statistical analysis was performed using Prism 7 (GraphPad Software, Inc., La Jolla, California, USA). Mean values are presented with their standard deviations (SD) and median values are graphically depicted within the full dataset for each treatment group and assessment day. Data were compared between treatment groups and assessment days using two-way analysis of variance (ANOVA) for repeated measures with post hoc Tukey's multiple comparisons test. Statistical significance was set at p<0.05 and trends toward significance at p≥0.05 but <0.10.
Post hoc power calculations
Using Orthopaedic Score (OS) as the index, post hoc power calculations showed that at least 23 dogs per group would have been needed in order to identify a difference in mean OS of at least 2 points, with significance of <0.05 and 80% power, given a baseline SD of 2.4 points.
Post hoc power calculations using the largest SD identified on each of the assessment days indicated that as few as 14 and as many as 48 dogs per group would need to have completed the 28-day study period, depending on the variable (Table S11).
Thus, while our study was too small to show any significant differences between the positive-control (meloxicam) and negative-control (placebo) groups, it nevertheless identified some statistically significant treatment responses within groups (Figure 1, Figure 3, Figure 5, Figure 7), and statistically significant differences between the APPA and placebo groups for two of the OS components (Figure 2).
As discussed under Limitations, it was difficult to elicit the participation of even 60 owners who were willing to risk having their arthritic dogs receive placebo for 1 month. While not ideal, we proceeded with only 20 dogs per group, and ended up with 16-20 dogs per group for analysis. This decision will undoubtedly have meant that our study missed identifying some statistically significant differences within and between groups, but it nonetheless identified some differences that were not only statistically significant but are clinically relevant.