Nerve growth factor is a signaling neurotrophin that is an important mediator in the sensation of various types of pain, including pain attributable to osteoarthritis.1 Therefore, neutralization of NGF activity may decrease sensation of pain in animals with osteoarthritis. Neutralization of NGF activity has been investigated in other studies1–8 because of the potential analgesic effects in humans and veterinary patients.
Blockade of NGF activity is effective for reduction of signs of pain in rats with experimentally induced osteoarthritis,5 suggesting NGF signaling is important for sensation of pain attributable to osteoarthritis.5 Administration of antibodies against NGF significantly decreases pain associated with osteoarthritis of knee joints and improves overall joint function in humans.4 Antibodies against NGF have greater analgesic effects than NSAIDs or placebo treatments for humans with lower back pain.2 Concentrations of NGF in synovial fluid samples obtained from dogs with chronic osteoarthritis are significantly higher than those in such samples obtained from control dogs,9 suggesting NGF signaling is involved in the sensation of pain associated with osteoarthritis in dogs.
The use of mAbs for the treatment of humans is becoming more common, and such treatments have been developed for various conditions, including inflammatory diseases and several types of cancer.10 During 2012, 5 of the 15 prescription pharmaceuticals with the highest monetary sales in the world were mAb products.11 The use of mAb products for treatment of veterinary patients has been hindered by difficulties and costs associated with development and manufacturing of species-specific antibodies. However, mAb products developed for use in laboratory animals and humans have recently been adapted for use in companion animals.12 Investigators in one of our laboratories have developed a novel method for the rapid conversion of antibodies for use in companion animals species, without loss of affinity or immunogenicity.12 By use of this method, an mAb against NGF was developed for administration to dogs.12 That proprietary mAb is stable, has affinity at picomolar concentrations for canine NGF, has a long serum half-life (9 days), and is not immunogenic in dogs following repeated administration.12 Administration of a single dose of that mAb (0.2 mg/kg, IV) reduces lameness in dogs with a short duration of experimentally induced foot pad inflammation, versus dogs that receive a placebo treatment12; the magnitude of the effect is comparable to that detected after daily oral administration of the NSAID meloxicam.12
Because responses of animals to acute inflammation may differ from their responses to chronic inflammation (including concentrations of NGF in synovial fluid),9 the study reported here was conducted to determine the effects of an mAb against NGF modified by use of a proprietary process for administration to dogs on the CBPI scores for such animals with chronic osteoarthritis. Our null hypothesis was that CBPI PS and PI scores13–15 determined by owners would not significantly change after administration of that mAb against canine NGF, compared with scores determined before administration.
Materials and Methods
Animals—The University of Adelaide institutional Animal Ethics Committee approved the study protocol, and owners gave written consent for inclusion of their dogs in the study. Adult (≥ 1 years) dogs brought to either the University of Adelaide School of Animal and Veterinary Sciences or SA Veterinary Referrals that had chronic lameness (duration, > 3 months), a history consistent with osteoarthritis, and a previous diagnosis of osteoarthritis in at least 1 limb as determined by a veterinarian were evaluated for inclusion in the study. Dogs were included in the study if results of evaluation by the primary investigator (RPW) indicated detectable lameness at a walk that was attributable to signs of pain in a joint of the appendicular skeleton. Dogs were excluded if they did not have detectable lameness at a walk at the time of evaluation, had lameness that could not be attributed to signs of pain in a joint of the appendicular skeleton, had neurologic abnormalities, or had other concurrent medical conditions for which they were receiving medication. In addition, dogs were excluded if owner-determined CBPI PS or PI scores were ≤ 1 at the time of the first evaluation in the study, if substantial abnormalities were detected on the basis of results of a CBC and serum biochemical electrolyte analyses,a or if analgesic medication had been administered during the 2 weeks prior to the first evaluation. Eleven dogs started the study.
Production of anti-NGF mAb—The anti-NGF mAbb for administration to dogs was prepared in accordance with methods used in another study.12 Briefly, the mAb was produced in Chinese hamster ovary cells, purified by means of multistage chromatography, suspended in PBS solution, and filtered to yield a sterile product.
Study protocol—All dogs started the study on the same date. Each dog was allocated to 1 of 3 treatment groups by use of a random-number functionc prior to collection of data. Dogs were evaluated by the primary investigator (RPW) and received treatments every 2 weeks during a 10-week study period. At each of those times, dogs received the anti-NGF mAbb suspended in sterile PBS solution (0.2 mg/kg, IV) or an equivalent volume of sterile PBS solution IV (placebo treatment). The time of administration of the anti-NGF mAb varied among treatment groups. Dogs in one of the groups (n = 3) received the mAb during the first evaluation (0 weeks), dogs in another group (3) received the mAb during the second evaluation (2 weeks), and dogs in another group (3) received the mAb during the third evaluation (4 weeks). Dogs received the placebo treatment during all other evaluation times. Owners were aware that dogs would receive the anti-NGF mAb once during the study; they were informed that it would be administered at 1 of the 6 evaluation times and were unaware of the time at which the mAb was administered. The date of mAb administration varied among the groups of dogs to reduce bias attributable to expectations of owners regarding treatment responses.
At each evaluation time, owners were asked to complete the CBPI questionnaire; the same owner for each dog completed questionnaires throughout the study period. Dogs received treatments in accordance with the study protocol. Blood samples (5 mL) were collected for performance of a CBC and serum biochemical and electrolyte analysesa at the time of enrollment in the study, at the time of administration of the anti-NGF mAb, and at the end of the study. Results were evaluated to identify substantial changes that may have been attributable to administration of the anti-NGF mAb. Rescue analgesia (tramadol hydrochloride [2 to 4 mg/kg, PO, q 8 to 12 h as needed]) was provided if the owners considered that their dog had unacceptable signs of pain at any time during the study period.
For each evaluation, mean values for owner responses to the first 4 questions of the CBPI regarding PS were calculated to determine a PS score, and mean values for responses to the following 6 questions regarding changes in function attributable to pain were calculated to determine a PI score, as previously described.15 The CBPI scores determined on the date of anti-NGF mAb administration (determined prior to administration) were baseline scores. These scores were compared with scores determined during subsequent evaluations after anti-NGF mAb administration.
Statistical analysis—Data determined during evaluations after administration of the anti-NGF mAb were compared with baseline data with the Wilcoxon signed rank test by use of an online statistical analysis program.d This analysis yielded a Wilcoxon value (ie, W value) and a number-of-signed-ranks value. Because the sample size was < 10 (data were available for only 9 dogs because 2 dogs did not complete the study), we used the W value and number-of-signed-ranks value in conjunction with a table of critical values of W to determine whether values of P were ≤ 0.05. Analyses were performed with a 2-tailed test. Values of P ≤ 0.05 were considered significant.
Results
Eleven dogs were enrolled in the study. One dog was withdrawn by the owner owing to circumstances unrelated to the study. Another dog was withdrawn from the study by the owner because of unacceptable signs of pain despite administration of rescue analgesia during the study period before anti-NGF mAb administration. This dog's pain was successfully managed with additional analgesia outside the study. Therefore, data for 9 dogs (3 dogs in each treatment group) for evaluations 2, 4, and 6 weeks after mAb administration were analyzed. Dogs included 3 Golden Retrievers and 1 Rottweiler, Cavalier King Charles Spaniel, Australian Cattle Dog, Border Collie, Greyhound, and crossbred dog. Four dogs were male and 5 were female. The median weight of the dogs was 32.5 kg (range, 6.8 to 35.2 kg), and the median age of the dogs was 5 years (range, 1 to 10 years).
During the study, values of measured physical examination and laboratory analysis variables for the dogs were unremarkable, including body weight, rectal temperature, heart rate, respiratory rate, and results of CBCs and serum biochemical and electrolyte analyses. In addition, the owners of the dogs reported no adverse events attributable to anti-NGF mAb administration. None of the dogs that completed the study required administration of rescue analgesics during the evaluation period.
Compared with baseline PS scores (median, 4.75; range, 0.75 to 8.5), dogs had significantly lower PS scores 2 weeks (median, 3; range, 1 to 5.5) and 4 weeks (median, 2.25; range, 0.25 to 7.25) after administration of anti-NGF mAb. The PS scores 6 weeks after administration (median, 3.75; range, 0.5 to 8) were lower than baseline scores, but values were not significantly different (Figure 1).
Compared with baseline PI scores (median, 5.33; range, 1.17 to 9.33), dogs had significantly lower PI scores 2 weeks (median, 3; range, 0.67 to 6.83) and 4 weeks (median, 3.33; range, 0.67 to 6.67) after administration of anti-NGF mAb. The PI scores 6 weeks after administration (median, 4.83; range, 0.83 to 8) were lower than baseline scores, although values were not significantly different (Figure 2).
Median CBPI PS scores for 9 dogs immediately before (baseline) and 2, 4, and 6 weeks after administration of an anti-NGF mAb modified by means of a proprietary process for administration to dogs. *Value is significantly (P ≤ 0.05) different from the baseline value.
Citation: American Journal of Veterinary Research 75, 6; 10.2460/ajvr.75.6.532
Median CBPI PS scores for 9 dogs immediately before (baseline) and 2, 4, and 6 weeks after administration of an anti-NGF mAb modified by means of a proprietary process for administration to dogs. *Value is significantly (P ≤ 0.05) different from the baseline value.
Citation: American Journal of Veterinary Research 75, 6; 10.2460/ajvr.75.6.532
Median CBPI PS scores for 9 dogs immediately before (baseline) and 2, 4, and 6 weeks after administration of an anti-NGF mAb modified by means of a proprietary process for administration to dogs. *Value is significantly (P ≤ 0.05) different from the baseline value.
Citation: American Journal of Veterinary Research 75, 6; 10.2460/ajvr.75.6.532
Median CBPI PI scores for 9 dogs immediately before (baseline) and 2, 4, and 6 weeks after administration of an anti-NGF mAb modified by means of a proprietary process for administration to dogs. *Value is significantly (P ≤ 0.05) different from the baseline value.
Citation: American Journal of Veterinary Research 75, 6; 10.2460/ajvr.75.6.532
Median CBPI PI scores for 9 dogs immediately before (baseline) and 2, 4, and 6 weeks after administration of an anti-NGF mAb modified by means of a proprietary process for administration to dogs. *Value is significantly (P ≤ 0.05) different from the baseline value.
Citation: American Journal of Veterinary Research 75, 6; 10.2460/ajvr.75.6.532
Median CBPI PI scores for 9 dogs immediately before (baseline) and 2, 4, and 6 weeks after administration of an anti-NGF mAb modified by means of a proprietary process for administration to dogs. *Value is significantly (P ≤ 0.05) different from the baseline value.
Citation: American Journal of Veterinary Research 75, 6; 10.2460/ajvr.75.6.532
Discussion
Results of the present study indicated the evaluated anti-NGF mAb decreased CBPI PS and PI scores for dogs with chronic osteoarthritis 2 and 4 weeks after administration. Six weeks after administration, the PS and PI scores were lower than baseline scores, but values were not significantly different. Inclusion of a larger number of dogs in the study may have allowed detection of significant differences in values for that time. Results suggested that the duration of analgesic effects after IV administration of 1 dose of the anti-NGF mAb (0.2 mg/kg) was at least 4 weeks. The mAb was administered at various times for dogs in the study, and owners were unaware of the time of administration; this was intended to decrease the chance that observed improvements of dogs were attributable to a placebo effect or confounding factors such as a period of warm weather. Further studies are warranted to confirm the duration of such effects.
Compared with baseline scores, 6 of 9 (67%) dogs had lower PS scores 2 weeks after anti-NGF mAb administration, and 9 (100%) dogs had lower PS scores 4 weeks after administration. Compared with baseline scores, 8 of 9 (89%) dogs had lower PI scores 2 weeks after administration, and 9 (100%) dogs had lower PI scores 4 weeks after administration. These results suggested improvements in scores were attributable to the anti-NGF mAb.
Monoclonal antibody treatments have several purported advantages, compared with conventional chemical drug treatments, including good safety, high specificity of binding with target antigens, and long half-lives. High binding specificity allows precise action of the agent and potentially lowers rates of adverse events, and a long half-life allows a long duration of action.16
Adverse events for humans receiving treatments to neutralize effects of NGF are typically mild and include headaches and transient abnormalities in peripheral sensation.1,2 We did not identify adverse events attributable to administration of the anti-NGF mAb to dogs in the present study. However, adverse events similar to those detected in humans may be difficult to detect in dogs. The lack of detected adverse events after anti-NGF mAb administration may be a potential advantage of such treatments for dogs with osteoarthritis, compared with other analgesics such as NSAIDs. Other potential indications for treatments that neutralize NGF include pain attributable to bone cancer, fractures, and pancreatitis.1,2
The percentage decrease in median PS and PI values between the baseline evaluation and the evaluation 2 weeks after anti-NGF mAb administration for dogs in the present study (range, 37% to 44%) was similar to the percentage decrease in CBPI scores for dogs in another study15 after administration of carprofen (38% to 47%). However, those results are not directly comparable because of differences in study design and statistical analysis methods.
A limitation of the present study was the lack of a control group. Further study is warranted to compare effects of anti-NGF mAbs with those of control treatments, such as NSAIDs or placebo treatments, for dogs with signs of pain attributable to osteoarthritis. Other limitations of the study were the small sample size and use of a single outcome measure determined with a subjective assessment tool. Future studies should include a larger number of animals and objective outcome measures such as kinetic assessment variables determined with a force plate or pressure-sensitive walkway. Results of the present study suggested that administration of an mAb against NGF may be effective for providing analgesia in dogs with osteoarthritis, and further studies of this treatment may be warranted.
ABBREVIATIONS
| CBPI | Canine Brief Pain Inventory |
| mAb | Monoclonal antibody |
| NGF | Nerve growth factor |
| PI | Pain interference |
| PS | Pain severity |
Wellness Profile, Gribbles Veterinary Pathology, Clayton, VIC, Australia.
NV-01 caninized mAb against canine NGF, Nexvet Biopharma Pty Ltd, Melbourne, Australia.
Microsoft Excel, Microsoft Corp, Redmond, Wash.
Wilcoxon signed rank test, VassarStats, Poughkeepsie, NY. Available at: vassarstats.net/wilcoxon.html. Accessed Dec 7, 2013.
References
1. Watson JJ, Allen SJ & Dawbarn D. Targeting nerve growth factor in pain: what is the therapeutic potential? BioDrugs 2008; 6: 349–359.
2. Katz N, Borenstein DG, Birbara C, et al. Efficacy and safety of tanezumab in the treatment of chronic low back pain. Pain 2011; 10: 2248–2258.
3. Kumar V, Mahal BA. NGF—the TrkA to successful pain treatment. J Pain Res 2012; 5: 279–287.
4. Lane NE, Schnitzer TJ, Birbara CA, et al. Tanezumab for the treatment of pain from osteoarthritis of the knee. N Engl J Med 2010; 16: 1521–1531.
5. McNamee KE, Burleigh A, Gompels LL, et al. Treatment of murine osteoarthritis withTrkAd5 reveals a pivotal role for nerve growth factor in non-inflammatory joint pain. Pain 2010; 2: 386–392.
6. Sevcik MA, Ghilardi JR, Peters CM, et al. Anti-NGF therapy profoundly reduces bone cancer pain and the accompanying increase in markers of peripheral and central sensitization. Pain 2005; 1: 128–141.
7. Wild KD, Bian D, Zhu D, et al. Antibodies to nerve growth factor reverse established tactile allodynia in rodent models of neuropathic pain without tolerance. J Pharmacol Exp Ther 2007; 1: 282–287.
8. Woolf CJ, Safieh-Garabedian B, Ma Q-P, et al. Nerve growth factor contributes to the generation of inflammatory sensory hypersensitivity. Neuroscience 1994; 2: 327–331.
9. Isola M, Ferrari V, Miolo A, et al. Nerve growth factor concentrations in the synovial fluid from healthy dogs and dogs with secondary osteoarthritis. Vet Comp Orthop Traumatol 2011; 4: 279–284.
10. Nelson AL, Dhimolea E, Reichert JM. Development trends for human monoclonal antibody therapeutics. Nat Rev Drug Discov 2010; 10: 767–774.
11. Intercontinental Marketing Services Health website. Top 20 global products 2012. Available at: www.imshealth.com/portal/site/ims/menuitem.5ad1c081663fdf9b41d84b903208c22a/?vgnextoid=fbc65890d33ee210VgnVCM10000071812ca2RCRD. Accessed Jul 18, 2013.
12. Gearing DP, Virtue ER, Gearing RP, et al. A fully caninised anti-NGF monoclonal antibody for pain relief in dogs. BMC Vet Res [serial online]. 2013; 9: 226. Available at: www.biomedcentral.com/1746-6148/9/226. Accessed Dec 25, 2013.
13. Brown DC, Boston RC, Farrar JT. Comparison of force plate gait analysis and owner assessment of pain using the canine brief pain inventory in dogs with osteoarthritis. J Vet Intern Med 2013; 1: 22–30.
14. Brown DC, Boston RC, Coyne JC, et al. Development and psychometric testing of an instrument designed to measure chronic pain in dogs with osteoarthritis. Am J Vet Res 2007; 6: 631–637.
15. Brown DC, Boston RC, Coyne JC, et al. Ability of the canine brief pain inventory to detect response to treatment in dogs with osteoarthritis. J Am Vet Med Assoc 2008; 8: 1278–1283.
16. Hansel TT, Kropshofer H, Singer T, et al. The safety and side effects of monoclonal antibodies. Nat Rev Drug Discov 2010; 9: 325–338.
