Design
This was a randomized, stratified, double masked, placebo controlled, proof of principle clinical pilot study with parallel treatment groups. The Institutional Animal Care and Use Committee (IACUC) approved this study (IACUC #12-149-O), and in all cases owners signed a written consent form following a detailed verbal explanation of the study protocol.
Study population
Dogs ≥1-year old and ≥15 kg with DJD-associated pain and mobility impairment were recruited. Recruitment was performed using advertisements on the NCSU-CVM internal TV monitors, through NCSU websites, e-mail advertisement to NC State university colleges (Education, Engineering), via direct advertising to local veterinarians and newspaper advertisements run in local weekly newspapers. Promotion of the study was also done using the NCSU-CVM social media sites (NCSU-CVM blog, Twitter feed and Facebook page). Recruitment began mid-April 2013 and continued through mid-October 2013.
Inclusion criteria
To be eligible for the study, dogs were required to have impaired mobility of a certain severity, as judged by owners, no detectable systemic disease, and at least one appendicular joint or axial skeleton area that was considered painful and where radiographs showed the presence of DJD (see ‘Study Protocol’). Radiological features used to establish the presence of DJD in appendicular joints were: joint effusion, osteophytes, sclerosis, subluxation, subchondral bone erosions and cysts and presence of intra-articular mineralization. Radiological features used to establish the presence of DJD in the axial skeleton were: osteophytes, spondylosis, disc-associated degeneration (end plate sclerosis, erosion, disc mineralization, narrowing) and subluxation. Additionally, dogs were required to not be currently receiving any anti-inflammatory medications (NSAIDs) or other analgesics (e.g. amantadine, gabapentin, tramadol). Other analgesics (e.g. amantadine, gabapentin, tramadol) were permitted if it was deemed there was currently significant pain associated with the DJD, and the dog had been on the medication(s) for at least 3 weeks. Dogs were required to be either not receiving nutritional supplements, or have been on them for 6 weeks or more before the start of the study. A two week withdrawal period was required prior to study entry for dogs discontinuing nutritional supplements, NSAIDs or other analgesics. If dogs were considered to be mobility impaired but no DJD was detected radiographically, or if they had DJD but the impairment in mobility was not sufficient, they were not enrolled. Other exclusion criteria included known or suspected presence of any of the following conditions: clinically significant cardiovascular disease; severe dental disease; neurological disease, renal disease; liver disease (ALT levels of up to twice the upper normal value and AlkPhos levels of up to four times the upper normal value were considered acceptable in the absence of other signs of liver disease); chronic pulmonary disease; infectious disease; immune-mediated disease; neoplasia; urinary tract infection; hypothyroidism (unless well controlled); diabetes mellitus; skin disease of the foot; obesity (8 or 9 out of the 1-9 Body Condition Score Scale). These were exclusion criteria because they may be associated with decreased activity that would not respond to analgesic treatment. Particular attention was given to ruling out neurological disease through a comprehensive neurological evaluation. Additionally, owners had to agree to not change the management of dogs for the period of the study, and owners were required to have a stable lifestyle for the duration of the study (no planned house moves, vacations, relationship changes or new pets).
Study protocol
The study was conducted over a 28-day period with outcome measures gathered at screening (D -7) and on D0, D14 and D28.
Approximately 7 days prior to starting on the study, potential candidate dogs were screened using a general physical, neurological and orthopedic examination, complete blood count, serum biochemistry and urinalysis. Physical and orthopedic examinations were performed by the same examiner (MF) throughout the study. Pain on manipulation of each appendicular joint and manipulation of each region of the axial skeleton was assessed and recorded using a 5-point scale (Additional file 1). Scores were summed across all appendicular joints (manus, carpus, elbow, shoulder, pes, hock, stifle, hip) and each region of the axial skeleton (cervical, thoracic, thoraco-lumbar, lumbar, lumbo-sacral) to create a total pain score (maximum score 84). In addition, an index joint or region of the axial skeleton was defined, and the index joint/region pain score recorded. The index joint/region was defined based on clinical judgment of what was the most severely affected area, with the predominant criterion being the pain score for that area. Based on the orthopedic examination (pain and function), dogs were designated as being either predominantly ‘Fore’ (forelimb, neck or thoracic spine) or ‘Hind’ (hind limb, thoraco-lumbar, lumbar or lumbo-sacral spine) impaired. Digital radiographs of all clinically abnormal (painful) appendicular joints or areas of the axial skeleton were taken under sedation. Owners completed three clinical metrology instruments (CMIs) - the Client Specific Outcome Measures (CSOM) [1,30] the Canine Brief Pain Inventory (CBPI) [27,28] and the Liverpool Osteoarthritis in Dogs Index (LOAD) [26,29]. Owners also completed a Quality of Life Index (Additional file 2), constructed in a similar manner to previous reports [38,39]. Dogs were required to have a qualifying degree of mobility impairment as reported and scored by the owner on the CSOM (CSOM score of ≥5, based on three activities each scored on a 0-4 scale where 0 = normal, 4 = impossible to perform the activity). Dogs were fitted with a collar-mounted accelerometer (see below). Owners were asked to keep a diary of any unusual events that might affect a dog’s activity.
On D0, owners returned to the clinic with their dogs, and completed the CMIs again. The physical, orthopedic and neurological examinations were repeated to ensure there were no changes and no additional findings. On D0, dogs were administered the anti-nerve growth factor antibody (NV-01) or placebo, at a dose of 200 mcg/kg of a 2 mg/ml solution administered IV over a 1-minute period through an intra-venous 20G catheter. Normal saline was used as the placebo, and administered IV at a volume equivalent to the dose of NV-01. Following test article administration, dogs were observed for a period of 4 hours for any signs of an allergic reaction.
On D14, owners returned to the clinic and completed the CMIs and the physical, orthopedic and neurological examinations were repeated to ensure there were no changes or additional findings.
On D28, owners returned to the clinic and completed the CMIs, the physical, orthopedic and neurological examinations were repeated and joint pain scored. The data from the accelerometer was downloaded, and samples collected for CBC, clinical chemistry and urinalysis.
Randomization to treatment groups
Dogs were randomized to receive the drug or placebo on D0 based on a stratified, blocked design. Stratification was based on predominant site of problem (Fore or Hind), and total CBPI score (<50, ≥ 50). Hence, the stratification groups were: CBPI < 50/Fore; CBPI < 50/Hind; CBPI ≥ 50/Fore; CBPI ≥ 50/Hind.
Within each stratification, randomization of treatment occurred in groups of 2 (e.g. placebo, then NV-01; or, NV-01 then placebo). The randomization schedule and the key were held by the NCSU pharmacy and not disclosed to investigators. The dispensing of drug/placebo was performed by NCSU pharmacy, with all other personnel involved in the evaluation of dogs and collection of data masked to the administration until completion of the statistical analysis. Pharmacy staff prepared unmarked syringes for each patient with the barrel covered in opaque tape. Testing prior to starting the study indicated there was no appreciable difference between the feel of injecting saline versus NV-01 through a 20G catheter, allowing masking to be complete.
Outcome measures
The primary outcome measures were the CMIs and actimetry data. Secondary outcome measures were index joint pain, total joint pain score, owner-assessed side effects, hematology, clinical chemistry and urinalysis.
Clinical metrology instruments
CMIs were completed by the same owner at all visits, and owners were directed to base their answers on their observations of the preceding seven days. The owners completed the CMIs while sitting or standing (as they preferred), in a standard consulting room. At each time point, they were requested to complete the CMIs and the instructions were explained to them in a neutral tone of voice. Owners completed the CMIs while their dog was being examined in a separate room.
The LOAD [26,29] is a 13-item instrument with all items reported on a five-point Likert-type scale. Each item is scored 0 to 4, and the item scores are summed to give an overall instrument score. The CBPI [27,28] is a two-part instrument. The pain severity score (CBPI PSS) is the arithmetic mean of four items scored on an 11-point (0 to 10) numerical scale, and the pain interference score (CBPI PIS) is the mean of six items similarly scored. The CSOM [30] is a CMI that follows 3 activities unique to the individual dog that are determined to be impaired. It is modeled after the Cincinnati Orthopedic Disability Index, CODI [40]. The CSOM was constructed by a single study individual (BC) for each case as previously described [1] but only defining 3 activities as more recently described [30]. At each time point, the difficulty performing each of the 3 activities was scored on a 0-4 scale: 0 = No Problem, 1 = Mildly Problematic, 2 = Moderately Problematic, 3 = Severely Problematic, and 4 = Impossible. The CSOM score resulted from the addition of the score for each activity. Only complete CMIs were considered valid.
Activity monitoring (AM)
As previously reported [23,26,33], the spontaneous activity of each individual dog was measured using an accelerometera, a method termed actimetry. Actimetry commenced following screening and continued for the duration of the study. At D28 the monitor was removed from the collar and placed on a telemetric reader to download the data to a personal computer.
Statistical analysis
This was a pilot study, and the potential degree of improvement associated with anti-NGF antibody was not known, and so the number of dogs was based on the following. From a previous study carried out at our site [23], change in mean pain interference CBPI score following 2-weeks of an NSAID was 1.4 (baseline = 2.9; after NSAID for 14d = 1.5) and the mean standard deviation was 1.2. Using this standard deviation, and data from a study [28] where the improvement in CBPI interference score was 0.2 for placebo, and 1.1 for NSAID, we determined that a total of 24 dogs (12 each group), would give a power of 0.49 which was considered acceptable for a pilot study.
All data were entered into hard-copy notebooks, and then transcribed to digital files. All data underwent 100% quality control by two individuals not associated with the study to verify the digital entries. Descriptive statistics were used to describe the demographic characteristics of the two groups, and appropriate statistical tests used to compare these characteristics.
For all subjective parameters (CMIs, joint pain, QoL), the change within groups over time, and the difference in change between groups were assessed statistically. Data were compared non-parametrically. Data were converted to success/failure where published guidelines were available, and analyzed using a Fisher’s Exact test. For the CBPI data, success/failure at D14 and D28 was defined based on an improvement of at least 1 for CBPI PSS, and at least 2 for CBPI PIS, with inclusion criteria set as a CBPI PSS and CBPI PIS of at least 2 at baseline (D0). For the CSOM, success/failure at D14 and D28 was defined as an improvement of 2 or more on the total CSOM, with no deterioration in any single CSOM activity.
Actimetry data were extracted for days -7 to -1 (baseline), and 20 to 27 (final week of study). For each dog, the average activity count per minute over each 7-day period was calculated and used for analysis. Activity data were normally distributed, and within-group changes evaluated using a paired t-test. Between group comparisons were not performed due to the large inter-dog variations in baseline activity, and recent data from our group indicating significant differences in output between accelerometers (unpublished data). In addition, the within-group change in mean activity per minute for each hour of the day was compared for several segments of the day.
Laboratory data were compared between groups at the start and end of the study (Wilcoxon Rank Sums). Where significant differences between groups were detected, they were further examined for within group changes (Wilcoxon Signed Rank).
In all analyses, the critical P-value was set as 0.05. Due to the pilot nature of the study, the P-value was not adjusted for multiple comparisons within any outcome measure, and additionally, in some results, both the one-sided and two-sided statistical test results are described.
ELISA assay for neutralizing antibodies to NV-01
Plasma samples were assayed in a competition ELISA for inhibition of binding of purified NV-01 to mouse NGF. Purified NV-01 was mixed with dog plasma from placebo- or NV-01-treated dogs and added to plates previously coated with mouse NGF. Following incubation, blocking and washing, binding was detected using secondary anti-canine IgG polyclonal antibody-HRP conjugate [16]. Purified NV-01 without dog plasma, in the absence or presence of a neutralizing anti-NV-01 mouse monoclonal antibody (1RC1) was used as negative and positive controls.