The treatment of pain is an ongoing challenge for clinicians. Use of nonsteroidal anti-inflammatory drugs are widely used and effective for treatment of both acute and chronic pain in dogs because of their potent analgesic effects.1 Treatment with NSAIDs inhibits cyclooxygenase enzymes and reduces the amounts of PGs, including PGE2, and thus blunts the inflammatory pathways as well as the pain associated with these pathways. Given that a large variety of biological functions are mediated by PGE2 through signaling at 4 distinct EPs, the development of selective EP antagonists offers an opportunity for therapeutic intervention.2 Anti-inflammatory actions of EP2 have been mainly ascribed to EP43; however, EP2 may play a similar, redundant role.2,4 Several agents have been developed as specific antagonists of EP4.2,5 Although these EP4 antagonists are NSAIDs, they target PGE2 receptor-specific pathways and are effective in ameliorating pain and dysfunction associated with osteoarthritis in dogs.6
To the authors’ knowledge, no studies have been conducted to assess the ability of EP4 inhibitors to treat acute pain in dogs. The purpose of the study reported here was to examine the ability of 2 EP4 antagonists (L-776 and grapiprant) to attenuate lameness attributable to urate-induced synovitis in the stifle joint of dogs and to compare the effects of those 2 antagonists with effects of an NSAID (carprofen) known to successfully attenuate signs of pain in dogs with urate-induced synovitis.7–12 The injection of urate induces a consistent self-limiting lameness and provides a predictable method for detecting changes in lameness associated with transient inflammatory synovitis over a moderate period (48 hours). Our hypothesis was that the 2 EP4 antagonists (L-776 and grapiprant) would be as effective as the NSAID (carprofen) for the attenuation of urate-induced lameness as assessed by both subjective measurements and objective gait analysis.
Materials and Methods
Animals
Five 2-year-old male hound-cross dogs were used in the study. Body weight of the dogs ranged between 25 and 30 kg. An a priori sample size calculation was performed (80% power) to detect a difference among treatments in a crossover study design. Calculations were performed on the basis of data from previous studies7–10 that involved evaluation of ground reaction forces (PVF and VI) in dogs with urate-induced lameness. Objective gait data were deemed the primary outcome measurements. For PVF, a change of 4.0 units (percentage of body weight) was deemed a clinically meaningful change, and the desired sample size was 5 (SD = 1.5; α = 0.05).a For VI, a change of 0.3 units (percentage of body weight × seconds) was deemed a clinically meaningful change, and the desired sample size for this variable was 5 (SD = 0.15; α = 0.05).a
Physical and orthopedic examinations as well as bilateral radiographic evaluation of the hip and stifle joints were performed on all dogs to confirm that there were no abnormalities. All dogs were housed in a climate-controlled animal housing facility at our institution, received routine vaccinations and anthelmintics, were fed a commercially available maintenance diet, and were provided with water ad libitum. The study was approved by the University of Georgia Institutional Animal Care and Use Committee.
Study design
A blinded 3-way crossover study was performed. Dogs were randomly assigned by use of a randomization programb to receive each of 3 treatments. Treatments comprised L-766c (a proprietary EP4 antagonist; 4 mg/kg), grapiprantd (an EP4 antagonist; 2.0 mg/kg), and carprofene (4.4 mg/kg). During all 3 treatment arms, dogs received a single dose of the assigned treatment at 6 pm on day 0 (14 hours before urate injection), 6 am on day 1 (2 hours prior to urate injection), 6 am on day 2 (22 hours after urate injection), and 6 am on day 3 (46 hours after urate injection).
Synovitis was induced at 8 am on day 1. Anesthesia was induced with propofol. An intra-articular injection of sodium urate (2.0 mL of a 17.0-mg/mL solution prepared as described elsewhere7–9) was performed into a stifle joint of each dog. The stifle joint selected for injection was randomly assigned by use of the aforementioned randomization programb for the first treatment and then alternated for the subsequent 2 treatments (eg, the left stifle joint was assigned for the first treatment, then the right stifle joint was used for the second treatment and then the left stifle was used again for the third treatment). Measurements were obtained immediately before (0 hours; baseline) and 6, 12, 24, 36, and 48 hours after sodium urate injection. There was a minimum washout period of 21 days between treatments (21 days between sodium urate injection and subsequent collection of baseline data for the subsequent treatment). Dogs were monitored closely throughout the experimental period. When a dog had a lameness score > 13 (scale, 0 to 15), rescue analgesia was instituted, and the dog was withdrawn from the study and treated with carprofen (4.4 mg/kg).
Data collection
Clinical lameness scores were obtained and recorded for each dog at each time point by use of a subjective lameness scoring system described elsewhere.7–9 Lameness scores for each of the dogs were assigned by 1 investigator, who assigned scores for the same dogs throughout the study. Investigators were not aware of the treatment administered to each dog.
Ground reaction force data were collected by use of 2 force platesf mounted in series and a computer and softwareg; ground reaction force data were used to determine PVF and VI. All trials were performed with dogs trotting at 1.70 to 2.10 m/s and acceleration of ± 0.50 m/s/s. Each dog was led across the force plates by the same handler during all periods of the experiment. Trials were accepted only when there was a single footfall of a hind limb on each force platform while the standard trotting gait was maintained and there were no extraneous movements by the dog or handler. At each time point for each dog, 5 observations were recorded for both hind limbs (untreated and sodium urate-injected stifle joints).
Statistical analysis
A linear mixed model was used to test for differences in PVF, VI, and lameness score among treatments at each time point and between baseline and each time point for each treatment. The full linear mixed model included fixed factors for the treatment, time, and treatment-by-time interaction and a random intercept for each dog. A diagonal residual covariance structure was also used for analysis of lameness scores and force plate data to allow for heterogeneous variability at various time points and for each treatment. A restricted maximum likelihood estimation method and Satterthwaite degrees of freedom method were used. Simple differences between treatments or time points were adjusted for multiple comparisons by use of a Tukey correction. All hypothesis tests were 2-sided, and significance was set at values of P < 0.05. All analyses were performed by use of a commercial statistical program.h
Results
None of the dogs required rescue analgesia. Evaluation of lameness scores revealed that dogs had significantly higher lameness scores 6 hours after induction of synovitis, compared with baseline lameness scores, for all 3 treatments (carprofen, P = 0.01; grapiprant, P = 0.001; and L-766, P = 0.01; Figure 1). Additionally, lameness scores 12 hours after induction of synovitis were significantly higher than baseline lameness scores when dogs received grapiprant (P = 0.04) and L-766 (P = 0.003). Lameness scores 24 hours after induction of synovitis were still significantly (P = 0.04) higher than baseline lameness scores for the grapiprant treatment. Lameness scores for the carprofen treatment were significantly lower than lameness scores for the grapiprant treatment but not significantly (P = 0.08) different from lameness scores for the L-766 treatment 6 hours after induction of synovitis. Lameness scores for the carprofen treatment were significantly lower than lameness scores for the grapiprant treatment 12 and 24 hours after induction of synovitis and significantly (P = 0.003) lower than lameness scores for the L-766 treatment 12 hours after induction of synovitis.
Mean ± SD cumulative lameness scores for 5 dogs receiving each of 3 treatments (L-766, 4.0 mg/kg [triangles]; grapiprant, 2.0 mg/kg [crosses]; and carprofen, 4.4 mg/kg [diamonds]) 14 and 2 hours before and 22 and 46 hours after intra-articular injection of sodium urate into a stifle joint to induce synovitis. Injections were administered at 0 hours (baseline). Lameness was scored on a scale of 0 to 15. There was a 21-day washout period between successive treatments. *Within the carprofen treatment, value differs significantly (P < 0.05) from the value at baseline. †Within the L-766 treatment, value differs significantly (P < 0.05) from the value at baseline. ‡Within the grapiprant treatment, value differs significantly (P < 0.05) from the value at baseline. §Within a time point, value for carprofen differs significantly (P < 0.05) from the value for L-766. ||Within a time point, value for carprofen differs significantly (P < 0.05) from the value for grapiprant.
Citation: American Journal of Veterinary Research 80, 11; 10.2460/ajvr.80.11.1001
Mean ± SD cumulative lameness scores for 5 dogs receiving each of 3 treatments (L-766, 4.0 mg/kg [triangles]; grapiprant, 2.0 mg/kg [crosses]; and carprofen, 4.4 mg/kg [diamonds]) 14 and 2 hours before and 22 and 46 hours after intra-articular injection of sodium urate into a stifle joint to induce synovitis. Injections were administered at 0 hours (baseline). Lameness was scored on a scale of 0 to 15. There was a 21-day washout period between successive treatments. *Within the carprofen treatment, value differs significantly (P < 0.05) from the value at baseline. †Within the L-766 treatment, value differs significantly (P < 0.05) from the value at baseline. ‡Within the grapiprant treatment, value differs significantly (P < 0.05) from the value at baseline. §Within a time point, value for carprofen differs significantly (P < 0.05) from the value for L-766. ||Within a time point, value for carprofen differs significantly (P < 0.05) from the value for grapiprant.
Citation: American Journal of Veterinary Research 80, 11; 10.2460/ajvr.80.11.1001
Mean ± SD cumulative lameness scores for 5 dogs receiving each of 3 treatments (L-766, 4.0 mg/kg [triangles]; grapiprant, 2.0 mg/kg [crosses]; and carprofen, 4.4 mg/kg [diamonds]) 14 and 2 hours before and 22 and 46 hours after intra-articular injection of sodium urate into a stifle joint to induce synovitis. Injections were administered at 0 hours (baseline). Lameness was scored on a scale of 0 to 15. There was a 21-day washout period between successive treatments. *Within the carprofen treatment, value differs significantly (P < 0.05) from the value at baseline. †Within the L-766 treatment, value differs significantly (P < 0.05) from the value at baseline. ‡Within the grapiprant treatment, value differs significantly (P < 0.05) from the value at baseline. §Within a time point, value for carprofen differs significantly (P < 0.05) from the value for L-766. ||Within a time point, value for carprofen differs significantly (P < 0.05) from the value for grapiprant.
Citation: American Journal of Veterinary Research 80, 11; 10.2460/ajvr.80.11.1001
Analysis of PVF data revealed results consistent with the lameness scores (Figure 2). Values 6 hours after induction of synovitis were significantly lower, compared with baseline values, for all 3 treatments (carprofen, P = 0.003; grapiprant, < 0.001; and L-766, P < 0.001). Furthermore, grapiprant treatment resulted in a significant (P = 0.01) decrease in PVF 12 hours after induction of synovitis, compared with the PVF at baseline. At 24 hours after induction of synovitis, PVF was significantly (P < 0.001) lower for the grapiprant and L-766 treatments, compared with PVF for the carprofen treatment.
Mean ± SD values for PVF of 5 dogs receiving each of 3 treatments 14 and 2 hours before and 22 and 46 hours after intra-articular injection of sodium urate to induce synovitis. BW = Body weight. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 80, 11; 10.2460/ajvr.80.11.1001
Mean ± SD values for PVF of 5 dogs receiving each of 3 treatments 14 and 2 hours before and 22 and 46 hours after intra-articular injection of sodium urate to induce synovitis. BW = Body weight. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 80, 11; 10.2460/ajvr.80.11.1001
Mean ± SD values for PVF of 5 dogs receiving each of 3 treatments 14 and 2 hours before and 22 and 46 hours after intra-articular injection of sodium urate to induce synovitis. BW = Body weight. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 80, 11; 10.2460/ajvr.80.11.1001
Analysis of VI data revealed that values 6 hours after induction of synovitis were significantly lower than baseline values for all 3 treatments (carprofen, P = 0.001; grapiprant, P < 0.001; and L-766, P < 0.001; Figure 3). At 6 hours after induction of synovitis, VI was significantly (P < 0.001) lower for the grapiprant and L-766 treatments than for the carprofen treatment. The VI was significantly (P = 0.002) higher for the L-766 treatment than for the carprofen treatment 24 and 48 hours after induction of synovitis and significantly (P = 0.003) higher than for the grapiprant treatment 48 hours after induction of synovitis.
Mean ± SD values for VI of 5 dogs receiving each of 3 treatments 14 and 2 hours before and 22 and 46 hours after intra-articular injection of sodium urate to induce synovitis. ¶Within a time point, value for L-766 differs significantly (P < 0.05) from the value for grapiprant. See Figures 1 and 2 for remainder of key.
Citation: American Journal of Veterinary Research 80, 11; 10.2460/ajvr.80.11.1001
Mean ± SD values for VI of 5 dogs receiving each of 3 treatments 14 and 2 hours before and 22 and 46 hours after intra-articular injection of sodium urate to induce synovitis. ¶Within a time point, value for L-766 differs significantly (P < 0.05) from the value for grapiprant. See Figures 1 and 2 for remainder of key.
Citation: American Journal of Veterinary Research 80, 11; 10.2460/ajvr.80.11.1001
Mean ± SD values for VI of 5 dogs receiving each of 3 treatments 14 and 2 hours before and 22 and 46 hours after intra-articular injection of sodium urate to induce synovitis. ¶Within a time point, value for L-766 differs significantly (P < 0.05) from the value for grapiprant. See Figures 1 and 2 for remainder of key.
Citation: American Journal of Veterinary Research 80, 11; 10.2460/ajvr.80.11.1001
Discussion
Intra-articular injection of sodium urate in the stifle joints of dogs of the study reported here induced reproducible hind limb lameness, which was consistent with results of other studies.7–13 The hypothesis tested in the present study was rejected because L-766 and grapiprant were not as effective as carprofen for attenuation of the sodium urate-induced lameness. The ability of carprofen to attenuate the lameness induced by intra-articular injection of sodium urate was consistent with results of other studies,10,12,14 which can provide investigators with a reliable positive control treatment for the evaluation of sodium urate-induced lameness. Additionally, investigations that involved the use of deracoxib,13 firocoxib,11,12 meloxicam,8 and robenacoxib15 also have detected early and consistent attenuation of lameness attributable to urate-induced synovitis. It was interesting that both of the EP4 antagonists were not able to attenuate the lameness, compared with lameness at baseline, until L-766 caused an attenuation of the lameness 24 hours after induction of synovitis. Grapiprant treatment resulted in dogs that were lame until 36 hours after induction of synovitis, compared with baseline results.
Lack of efficacy of the EP4 antagonist grapiprant was puzzling. Studies16–19 of rodents have revealed anti-inflammatory effects of EP4 antagonists. More specifically, grapiprant (previously known as CJ-023,423) resulted in a reduction of signs of pain and dysfunction in rats.5,20,21 Possible explanations for this treatment failure included species variation in pharmacokinetics or pharmacodynamics as well as a potential difference in the dose response, as has been noted for deracoxib,13 meloxicam,8 and robenacoxib15 for animals with urate-induced lameness. It also may have been attributable to the fact that inflammation induced by PGE2 is mediated by more than only EP4.4,22–24 Several studies2,4,24–26 have found that EP1, EP2, and EP4 are involved in the regulation of inflammation, with EP4 being predominant. It should be mentioned that there were small differences between the 2 EP4 antagonists in the ability to attenuate lameness in the present study. Treatment with L-766 was able to diminish the lameness score to baseline values by 24 hours after induction of synovitis, whereas grapiprant was not.
Findings for objective ground reaction force data were similar to those for the subjective lameness evaluation. However, there were some additional differences within and among treatments. Carprofen was significantly better at attenuating lameness and increasing both PVF and VI. Six hours after induction of synovitis, carprofen resulted in higher PVF and VI than for either EP4 antagonist. Interestingly, L-766 resulted in a significantly higher PVF than carprofen 36 hours after induction of synovitis and significantly higher VI than carprofen 24 hours and both carprofen and grapiprant 48 hours after induction of synovitis. Although PVF and VI did not differ significantly from baseline values for any treatment 24 hours after induction of synovitis, it was surprising to see subsequent increases for L-766. The exact reason for the increases is unknown, but it could have been an accumulation of drug, which then caused increased modulation of EP4. Again, the lack of early attenuation of lameness by both of the EP4 antagonists was confirmed by analysis of the objective gait data.
Interestingly, all agents approved by the US FDA for the treatment of pain and dysfunction attributable to osteoarthritis (including carprofen, deracoxib, etodolac, firocoxib, meloxicam, and robenacoxib) rapidly attenuate lameness attributable to urate-induced synovitis within the first 6 hours after induction (they were administered at the same dose used for treatment of pain and dysfunction attributable to osteoarthritis in clinical patients).6,8–15,27–32 However, in the present study, grapiprant administered at the same dosage listed on the label of the approved product did not provide attenuation as quickly.
Limitations of the study reported here included sample size. In other studies,7–15 investigators have used between 5 and 10 dogs/group. However, our power calculations revealed a minimum of 5 dogs was necessary to detect differences among treatments for the objective data (primary outcome measurements). Inability of the lameness score to enable us to detect some of the smaller changes that were detected in the objective gait analysis may have been attributable to the minimum number of dogs used in the present study combined with the large variance for that subjective measurement. Additionally, clinical translation of lameness scores may not be the same as scores for acute pain associated with surgery or more chronic pain associated with osteoarthritis. However, the ability to attenuate signs of pain and lameness for dogs with sodium urate-induced synovitis has been found to be comparable to positive responses in clinical trials conducted to evaluate the ability of several drugs to relieve pain and dysfunction attributable to osteoarthritis in dogs.8–15,27–32
It was also interesting that the same comparable positive results (urate-induced lameness and chronic osteoarthritis in a clinical trial) were not detected with grapiprant at the same dose.6 The clinical importance of this disparity is unknown. Furthermore, the same positive association between data for pain attributable to urate-induced lameness and clinical trials for acute surgical pain has been noted.8–15,33–40 The authors are not aware of any EP4 antagonists that have been evaluated in a clinical trial of acute surgical pain in dogs that can be used for comparison with the urate-induced lameness of the present study.
Data for the study reported here provided evidence that the EP4 antagonists L-766 and grapiprant did not alleviate signs of pain and lameness induced by intra-articular injection of urate in stifle joints of dogs as well as carprofen did. The method used for induction of lameness provided a robust inflammatory arthropathy consistent with results of other studies.7–15 Additionally, at several time points, grapiprant was the least effective treatment for attenuating lameness induced in the study reported here.
Acknowledgments
The authors declare that there were no conflicts of interest.
ABBREVIATIONS
| EP | E-type prostanoid receptor |
| PG | Prostaglandin |
| PVF | Peak vertical force |
| VI | Vertical impulse |
Footnotes
Statistical considerations for clinical trials and scientific experiments, MGH Biostatistics Center, Boston, Mass. Available at: hedwig.mgh.harvard.edu/sample_size. Accessed Jul 17, 2017.
Random integer generator, Random.org, Randomness and Integrity Services Ltd, Dublin, Ireland.
Merck Animal Health, Madison, NJ.
Galliprant, Elanco Animal Health, Greenfield, Ind.
Rimadyl, Zoetis, Parsippany, NJ.
Model OR-6–6, Advanced Mechanical Technology Inc, Watertown, Mass.
Acquire, version 7.3, Sharon Software Inc, East Lansing, Mich.
SAS, version 9.3, SAS Institute Inc, Cary, NC.
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