Efficacy of nerve growth factor antibody in a knee osteoarthritis pain model in mice

Knee osteoarthritis (OA) is a common chronic degenerative disease characterized by degeneration of articular cartilage components, synovitis, remodeling of subchondral bone and atrophy of joint muscles. Knee OA patients usually suffer from knee pain and are treated with several treatment modes, including medication, intraarticular injection of hyaluronic acid, and surgery [1]. Understanding of the mechanism of knee OA pain is incomplete, and medication for knee OA pain were sometimes insufficient.

As part of studies into new targets of knee OA pain, we have focused on pain-related molecules, including nerve growth factor (NGF) [2]. NGF not only plays an important role in the maintenance and development of the sensory nervous system [3] but is also a major contributor to inflammation and nociception [4]. Lewin et al. reported that the systemic injection of NGF induced thermal and mechanical hyperalgesia [5]. In addition, systemic injection of anti-NGF antibody reduced allodynia and hyperalgesia in animal models of neuropathic pain, including nerve trunk or spinal nerve ligation [6‐8].

Basic research into knee OA has been aided by the development of several animal models, including the anterior cruciate ligament transection model [9], destabilization of the medial meniscus model [10], the rat medical meniscal tear model [11], GDF5 deficiency mice [12], and monoiodoacetate (MIA) injection model [13‐16]. The MIA injection model has been reported to result in progressive joint damage, with some features that may be considered similar to OA [14‐16] and significant pain-related behavior [13, 16]. The MIA injection model is superior for evaluating knee pain; although it seems animal models including the medical meniscus model better approximate the anatomic pathology found in OA in humans. Some of these previous reports evaluated pain-related behavior using the von Frey test, which is for evaluation of mechanical allodynia. Clinically, however, knee OA patients suffer from knee pain that includes gait impairment but not mechanical allodynia. Several authors have evaluated knee pain behavior using weight bearing assays [17‐20]. For instance, the CatWalk gait analysis system can provide quantitative assessment of gait and motor function in rats and mice. This system has recently been used to assay impaired gait function in knee OA pain models [17‐19]. Regarding the pathological mechanism of knee OA pain, our previous immunohistochemical analysis showed that the expression of pain-related molecules in the sensory nervous system was increased in a knee OA pain model [16]. Thus, this finding demonstrated the up-regulation of pain-related molecules in the sensory nervous system in the pain state.

We therefore hypothesized that anti-NGF therapy was effective for knee pain in a mouse model of knee OA. The aim of the current study is to evaluate the efficacy of NGF antibody in a knee OA pain model using the CatWalk gait analysis system and immunohistochemical analysis of the sensory nervous system in mice.

In this study, intraarticular injection of MIA to the knee joints induced gait disturbances, including a small duty cycle, small swing speed, and small print area, and the up-regulation of neuropeptides in DRG neurons innervating the knee joints. Systemic injection of anti-NGF antibody improved these gait disturbances and the expression of neuropeptides in DRG neurons in this model.

Regarding the Knee OA model, multiple authors have reported various surgery-induced animal models. Myers et al. reported that anterior cruciate ligament transection induced changes in the articular cartilage of the unstable knee that are consistent with OA [23]. In addition, Marijnissen et al. reported the usability of the “Groove” model, in which knee articular cartilage is damaged as a knee OA model [24]. Further, Bendele AM report that medial meniscal tear in rats results in rapidly progressive degenerative changes to the cartilage with similarities to OA [11]. These surgically induced knee OA models are suitable for basic research, to the extent that they simulate clinical situations. Nevertheless, the MIA injection model which results in severe joint damage seems superior for evaluating knee pain. MIA injection to the knee joints has been reported to cause joint pathology via the inhibition of glycolysis, and thereby targets avascular cartilage and causes chondrocyte death [25]. We also previously reported that MIA-treated knees exhibited behavioral disturbances within four days and osteoarthritic histological changes within four weeks [16]. We therefore used the MIA injection model as a knee OA model in this study.

Regarding knee pain behavior, several authors reported that knee pain models exhibited mechanical allodynia using the von Frey test [13, 16]. In addition, Shelton DL et al. report NGF induced mechanical allodynia in auto-immune arthritis [26]. Therefore, evaluating knee pain using the von Frey test seems to be appropriate for basic research using animal models. However, there is doubt about whether these models of knee pain simulate clinical situations, because knee OA patients do not typically complain of mechanical allodynia in their foot. Recently, gait analysis studies have focused on behavioral studies to evaluate pain behavior. The CatWalk system was initially developed to evaluate motor function, such as that modified in a spinal cord injury model, a Parkinson’s disease model, or an olivocerebellar degeneration model [19, 27‐29]. Recently, this system has been applied to various models to evaluate behavior associated with pain, such as mechanical allodynia models [30]. We also previously evaluated low back pain behavior using the CatWalk system, and concluded that rat models of low back pain exhibit changes in rat gait, including a long standing time and short strides [21, 31]. Recently several authors applied the CatWalk system to evaluate behavior associated with knee pain. Ferland et al. reported that the MIA-induced knee pain model shows gait changes, including a short Duty cycle and a small Swing Speed. In our previous preliminary study, the MIA-induced knee pain model exhibited a small duty cycle, small swing speed, and small print area. (un-published date) Therefore, in this study, we compared the ratio of ipsi and contralateral hind paw values for the three variables, namely duty cycle, swing speed, and print area among the three groups to evaluate the effect of anti-NGF therapy.

Regarding the up-regulation of CGRP in the sensory nervous system, we previously evaluated potential pain-like states by evaluating the up-regulation of pain-related neuropeptides such as CGRP and substance P in the sensory nervous system using a combination of immunohistochemistry and retrograde tracing instead of evaluating pain behavior [22]. CGRP-IR DRG neurons have been reported to be NGF-dependent DRG neurons involved in pain perception related to inflammation [32], suggesting that CGRP is a marker of inflammatory pain. We previously reported that MIA injection to the knee joint induced the up-regulation of CGRP in DRGs [16]. These results suggest that the animals in the group receiving MIA injection to their knee joints were in a state of knee pain.

We focused on NGF as target for the treatment for knee OA pain based on our previous report that MIA injection induced the up-regulation not only inflammatory cytokines but also NGF in the knee joints [16]. Regarding the efficacy of anti-NGF therapy for knee OA pain, NGF is generally involved in chronic inflammatory or neuropathic pain states [33]. Several basic and clinical research had been reported the efficacy of anti-NGF therapy for knee OA pain [34, 35]. However, the pathological mechanism of knee OA pain was not fully understood. NGF is physically produced in articular structures and expressed in both normal and OA synovial tissues, but is increased in synovial inflammation, especially upon synovial tissue exposure to inflammatory cytokines including TNF-alpha [36, 37]. Thus, the up-regulation of inflammatory cytokines in OA knee joints promoted NGF production, which induced the up-regulation of CGRP in the sensory nervous system and chronic inflammatory or neuropathic pain states. Ashraf S et al. report that NGF injection into knee OA joints results in gait disturbance as compared to NGF injection into non-OA joints. This group used two models of knee OA, including an MIA injection model and a meniscal transection model [38]. Further, McNamee KE et al. report NGF expression is correlated with knee pain but that TNF alpha is not up-regulated in the late stages of OA [39]. These findings indicate inflammatory cytokines including TNF might only develop during the pain state, but that NGF might play a role in maintaining as well as in development of the pain state in knee OA patients. Therefore, NGF represents a possible important therapeutic target in the treatment of knee OA pain. In preclinical studies, newly developed tanezumab and its murine precursor muMab-911 have effectively targeted the NGF pathway in various chronic and inflammatory pain models [40]. In addition, phase I and II clinical trials for osteoarthritic pain and chronic lower back pain have demonstrated efficacy for the compound, as well as a good safety and tolerability profile [41]. In the present study, anti-NGF therapy suppressed the impairment gait and up-regulation of CGRP in DRG neurons. These findings may explain the pathological mechanism of knee OA pain.

There were some limitations in this study. First, we should have used tanezumab and its murine precursor muMab-911 for this study. We also did not evaluate differences in efficacy between muMab-911 and anti-NGF antibody, which we used in this study. Second, we did not evaluate the efficacy of positive control including non-steroid anti-inflammatory drugs for knee OA pain. Third, we also evaluated the efficacy of only a one-time injection of anti-NGF antibody. In the future, it will be necessary to evaluate the effect of repeated injections for knee pain. Fourth, we did not evaluate the histopathology of the knee joint. In clinical trials of the monoclonal NGF antibody tanezumab for knee and hip OA pain, osteonecrosis was reported as a side effect [42]. Therefore, further study focusing on side effects of anti-NGF therapy is necessary. Fifth, we did not include a power calculation to justify the group sizes. Further, due to ethical concerns related to using the animals, we combined our behavioral and immunohistochemical evaluations. Therefore, with regard to behavioral evaluation, some experiments ended after 4 weeks and others ended after 5 weeks.

MIA injection into the knee joint induced gait impairment and the up-regulation of CGRP in DRG neurons in a knee OA pain model in mice. Further, intraperitoneal injection of anti-NGF antibody suppressed gait impairment and the up-regulation of CGRP in DRG neurons. These finding indicate that anti-NGF therapy might be valuable in the treatment of OA pain in the knee.