Therapeutic Effect of Colony Stimulating Factor 1 Receptor Kinase Inhibitor, JTE-952, on Methotrexate-Refractory Pathology in a Rat Model of Rheumatoid Arthritis

Abstract

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation and the destruction of bone and cartilage in affected joints. One of the unmet medical needs in the treatment of RA is to effectively prevent the structural destruction of joints, especially bone, which progresses because of resistance to conventional drugs that mainly have anti-inflammatory effects, and directly leads to a decline in the QOL of patients. We previously developed a novel and orally available type II kinase inhibitor of colony-stimulating factor-1 receptor (CSF1R), JTE-952. CSF1R is specifically expressed by monocytic-lineage cells, including bone-resorbing osteoclasts, and is important for promoting the differentiation and proliferation of osteoclasts. In the present study, we investigated the therapeutic effect of JTE-952 on methotrexate (MTX)-refractory joint destruction in a clinically established adjuvant-induced arthritis rat model. JTE-952 did not suppress paw swelling under inflammatory conditions, but it inhibited the destruction of joint structural components including bone and cartilage in the inflamed joints. In addition, decreased range of joint motion and mechanical hyperalgesia after disease onset were suppressed by JTE-952. These results suggest that JTE-952 is expected to prevent the progression of the structural destruction of joints and its associated effects on joint motion and pain by inhibiting CSF1/CSF1R signaling in RA pathology, which is resistant to conventional disease-modifying anti-rheumatic drugs such as MTX.

INTRODUCTION

Rheumatoid arthritis (RA), an autoimmune-mediated, chronic inflammatory joint disease affecting 0.5–1.0% of the adult population in the U.S., is characterized by joint stiffness, deformity, and pain.^1–3) In general, RA begins with inflammation of the synovium and hyperplasia of the epithelium, progressing to the structural destruction of the joints, including cartilage and bone.^3,4) Notably, the structural destruction of joints in RA leads to functional impairment and loss of flexibility, and the severity of joint destruction is associated with pain.^5–7) Therefore, an improvement in the structural destruction of joints and pain that lead to a limited QOL is the most important issue when treating RA.

Recently, drug therapy for RA has shown markedly improved clinical efficacy related to reducing joint inflammation and disease activity. Traditional disease-modifying anti-rheumatic drugs (DMARDs) such as methotrexate (MTX) and biological DMARDs including tumor necrosis factor (TNF) α blockers, anti-CD20 antibody, anti-interleukin (IL) 6 receptor antibody, and cytotoxic T-lymphocyte-associated antigen 4 fusion protein are commonly used to treat RA.⁵⁾ However, despite treatment with these drugs, some patients do not derive sufficient therapeutic benefit, and cartilage and bone damage gradually worsens, resulting in the irreversible structural destruction of the inflamed joints.^5,8–11) Accordingly, drugs that effectively prevent the structural destruction of joints, which can be resistant to conventional anti-inflammatory drugs, have the potential to address the unmet clinical needs of RA patients.

Colony-stimulating factor-1 receptor (CSF1R), a receptor protein tyrosine kinase specifically expressed by monocytic-lineage cells, is important for the differentiation, proliferation, and survival of cytokine-producing monocytes, macrophages, and bone-resorbing osteoclasts.^12–15) CSF1R is also expressed by fibroblast-like synovial cells, and its ligand, colony-stimulating factor-1 (CSF1; also known as macrophage colony-stimulating factor), and IL34, a cytokine with a similar function to CSF1, share the same receptor.^16,17) Indeed, these ligands are present in higher concentrations in the peripheral blood and synovial fluid of RA patients compared with healthy subjects, and there is a strong correlation between CSF1 concentration in synovial fluid and bone damage.^18–20) Thus, blockade of the CSF1/CSF1R signaling pathway is an attractive therapeutic target to prevent the structural destruction of joints in RA.

Recently, we developed a novel and orally available CSF1R Type II inhibitor, JTE-952, which specifically inhibits human CSF1R kinase activity in vitro, with no apparent inhibitory effect on other kinases, except tropomyosin-related kinase A (TrkA).²¹⁾ In previous studies, JTE-952 exhibited an anti-inflammatory effect in vitro and in vivo.²²⁾ Furthermore, JTE-952 inhibited osteoclastogenesis in vitro and suppressed bone destruction in a mouse model of RA.²³⁾ On the basis of these findings, we investigated the therapeutic effects of JTE-952 on the MTX-refractory structural destruction of joints using a rat model of RA, adjuvant-induced arthritis (AIA). The extent of joint destruction was examined histologically and radiologically, joint function was assessed by the range of joint motion (ROM) of the foot, and pain was evaluated by the mechanical paw withdrawal threshold (PWT).

MATERIALS AND METHODS

Compounds

JTE-952, (2S)-3-{[2-({3-[4-(4-cyclopropylbenzyloxy)-3-methoxyphenyl]azetidine-1-yl}carbonyl)pyridin-4-yl]methoxy}propane-1,2-diol was chemically synthesized at the Central Pharmaceutical Research Institute, Japan Tobacco Inc. (Osaka, Japan). Methotrexate hydrate (MTX) was purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). JTE-952 (purity 97.5%) and MTX were suspended in a 0.5% (w/v) aqueous solution of methylcellulose.

Animals

Female Lewis rats were supplied by Jackson Laboratory Japan, Inc. (Kanagawa, Japan). All the animals were maintained under specific-pathogen-free conditions at a room temperature of 23 ± 3 °C and air humidity of 55 ± 15% under a 12-h light/dark cycle and were given access to a standard laboratory chow diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) and water ad libitum. All experimental procedures related to the use of animals in this study were reviewed and approved according to the Institutional Animal Care and Use Committee guidelines at Japan Tobacco Inc. and were performed in accordance with standards published by the National Research Council (Guide for the Care and Use of Laboratory Animals, NIH OACU) and the National Institutes of Health Policy on Human Care and Use of Laboratory Animals.

Induction of Adjuvant-Induced Arthritis in Rats

AIA was induced in rats aged 7 weeks as previously described.²⁴⁾ Briefly, on day 1, an adjuvant in which heat-killed Mycobacterium tuberculosis H37Ra (Difco Laboratories, Detroit, MI, U.S.A.) was suspended at 5 mg/mL in liquid paraffin was intradermally administered into the base of the rat tail at a volume of 0.1 mL under isoflurane anesthesia. Administration of adjuvant was performed in all rats except for normal untreated rats that were used as negative controls. An increase in the volume of both hind paws (paw swelling), an indication of joint inflammation, was measured in a blinded manner by a water displacement method with a plethysmometer (MK-101P, Muromachi Kikai Co., Ltd., Tokyo, Japan).

Drug Treatment

On day 14, rats were randomly assigned to a group based on the left-right average of paw swelling. From day 15 onward, rats received JTE-952 (1, 3, 10, and 30 mg/kg), MTX (0.3 mg/kg), or vehicle (0.5% methylcellulose) orally once a day for 14 d.

Histological Evaluation

On day 28, rats were sacrificed, and their left hind paws were dissected above the ankle joint and fixed in 10% neutral buffered formalin. After decalcification, the formalin-fixed left ankle specimens were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and evaluated by light microscopy. Histological analysis of tarsal joints was evaluated for bone and cartilage destruction as an indication of joint destruction, and for granulation tissue formation as another indicator of joint inflammation in addition to paw swelling. The severity of each histological change was scored in a blinded manner using a five-point scale ranging from 0 to 4 (0, normal; 1, minimal solitary (and very small) lesions; 2, slight focal (and small) lesions; 3, moderate scattered lesions; 4, severe extensive lesions).

Range of Joint Motion Measurements

The right hindlimb was used to assess the ROM using a method reported by Hashmi et al. with some modifications.²⁵⁾ Briefly, the tibia was aligned with the x-axis of a protractor and the heel of the hind paw was anchored to coincide with the intersection of the x and y axes. The ROM of the ankle joint was measured in a blinded manner in degrees from the beginning position (maximum dorsiflexion) to its position at the end (maximum plantar flexion) of the full range of motion.

X-Ray Analysis

After ROM measurements, the right hindlimb was dissected above the ankle joint and used for micro-computed topographic evaluation. Bone images of tarsal joints were acquired using a microfocus X-ray cone-beam computed tomography (CT) scanner (MCT-CB100MF, Hitachi Medical Corporation, Tokyo, Japan). Based on these images, the severity of bone erosion was scored in a blinded manner using a three-point scale ranging from 0 to 3 (0, normal; 1, mild; 2, moderate; 3, severe) for each tarsal bone and calcaneal bone. For each rat, the scores for the tarsal and calcaneal bones were summed to obtain a bone erosion score (maximum possible score: 6).

Hyperalgesia Measurements

On day 27, the intensity of mechanical hyperalgesia in both hindlimbs was assessed in a blinded manner by measuring the PWT using the method of Randall and Selitto with a pressure analgesy-meter (Model TK-201, UNICOM, Chiba, Japan).²⁶⁾ After the mechanical stimulation of each plantar foot was increased at a constant rate, the mean load value (mmHg) at which escape behavior or a vocal response was initiated was used as the mechanical pain threshold.

Pharmacokinetic Study

JTE-952 was orally administered to normal Lewis rats at dosages of 1, 3, 10, or 30 mg/kg in a volume of 5 mL/kg. Serial blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, and 24 h after dosing. The concentrations of JTE-952 in the plasma were analyzed by LC-tandem mass spectrometry.

Ex Vivo Whole Blood TNFα Production Assay

At 2 h after the administration of JTE-952 on day 28, whole blood collected from the abdominal vena cava with sodium citrate (3.8% (w/v)) was plated into a 96-well microplate (90 µL/well), and then a lipopolysaccharide (LPS) solution as a stimulant was added at 10 µL/well. After incubation for 6 h at 37 °C with 5% CO₂, the concentrations of TNFα in the plasma were measured with a specific enzyme-linked immunosorbent assay (ELISA) kit (Quantikine^®, R&D Systems, Inc., Minneapolis, MN, U.S.A.). The ED₅₀ was calculated from the concentration of JTE-952 and the residual activity (%) was calculated using a logistic function in SAS software (SAS Institute Japan Ltd., Tokyo, Japan).

Statistical Analysis

Data are expressed as means ± standard deviation (S.D.) of the indicated numbers of samples. The statistical significance between two groups was determined with the Student’s t-test (for homoscedastic parameters) or Welch’s t-test (for heteroscedastic parameters) after homoscedasticity analysis by the F-test, and differences between multiple groups were assessed by Dunnett’s test (for homoscedastic parameters) or Steel test (for heteroscedastic parameters) after homoscedasticity analysis by Bartlett’s test. For radiographic or histological scores, statistical significance between two groups was determined with the Wilcoxon rank-sum test, and differences between multiple groups were assessed by the Steel test. Correlation coefficients were calculated from the individual values as a measure of the association between each parameter. All statistical analyses were performed with SAS System version 8.2 and SAS preclinical package version 5.0 (SAS Institute Japan Ltd., Tokyo, Japan). A two-tailed p-value <0.05 was considered statistically significant.

RESULTS

JTE-952 Does Not Suppress Paw Swelling in Clinically Established AIA

The therapeutic efficacy of JTE-952 on the increase in paw swelling, an indicator of joint inflammation, was investigated. Arthritis was induced by the intradermal injection of a heat-killed M. tuberculosis suspension into the base of the rat tail on day 1, and JTE-952, or MTX, as a reference drug, was orally administered once daily for 2 weeks from day 15 when arthritis was established. Paw swelling near to maximum levels remained almost constant until day 28. No inhibitory effect of JTE-952 treatment on paw swelling was observed until the last day of evaluation (day 28) compared with the vehicle-treated AIA rats (Fig. 1). Even a dose of 0.3 mg/kg MTX, which can completely suppress swelling with prophylactic administration (in house data not shown), showed no obvious effect on established paw swelling.

Fig. 1. Effects of JTE-952 on Paw Swelling in Established Adjuvant-Induced Arthritis

Rats were treated as described in the Materials and Methods. JTE-952, methotrexate (MTX), or vehicle (0.5% methylcellulose) were orally administered at the indicated doses once daily for 14 d from day 15 when adjuvant-induced arthritis (AIA) was induced and the mean increase in the volume of both hind paws (paw swelling) was nearly maximal. Normal untreated rats used as negative controls did not receive adjuvant. On day 14 to 28 after AIA induction, the change in paw swelling was measured in a blinded manner using a water displacement plethysmometer. Paw swelling for each group (n = 10) is expressed as the mean paw volume (mL). ‡ p < 0.01 (vs. Normal group, Welch’s t-test).

JTE-952 Reduces Bone and Cartilage Destruction

To examine the effects of JTE-952 on the structural damage of inflamed joints, bone and cartilage destruction of the tarsal joint were evaluated histologically, and its effect on the formation of granulation tissue that reflects joint inflammation was examined. On day 28, each score of bone destruction, cartilage degradation, and granulation tissue for AIA rats treated with vehicle were significantly higher compared with normal untreated rats, in which arthritis was not induced (Figs. 2A–C). Enlarged cavities caused by tissue destruction were filled with granulation tissues (Fig. 2D, vehicle). The bone and cartilage destruction scores were reduced in JTE-952-treated rats compared with vehicle-treated AIA rats, and a significant reduction in these scores was observed at dosages of ≥3 mg/kg. In addition, JTE-952 tended to ameliorate granulation tissue formation, although this did not reach statistical significance compared with vehicle-treated AIA rats. Of note, MTX-treated AIA rats showed severe bone and cartilage destruction and granulation tissue formation, similar to that in vehicle-treated AIA rats.

Fig. 2. Effects of JTE-952 on Histological Changes

The ankle joint in formalin-fixed left hindlimbs taken on day 28 was used for histological analyses. Paraffin-embedded ankle specimens were stained with hematoxylin and eosin and evaluated under a light microscope. Histological severity of tarsal joints was evaluated for (A) bone and (B) cartilage destruction as an indication of joint destruction, and for (C) granulation tissue formation as another indicator of joint inflammation in addition to paw swelling. Each histological change was scored in a blinded manner (0, normal; 1, minimal solitary (and very small) lesions; 2, slight focal (and small) lesions; 3, moderate scattered lesions; 4, severe extensive lesions). Results are expressed as group means and values for individual rats. ‡ p < 0.01 (vs. [N] group, Welch’s t-test). * p < 0.05 (vs. [V] group, Steel test). (D) Representative staining images of rats with bone destruction closest to the mean of the indicated group. Abbreviations in the vehicle image: Bo_Ta (tarsal bone), Bo_Ti (tibia bone), Ca (cartilage), Gr (granulation).

JTE-952 Reduces Bone Erosion

The effect of JTE-952 therapeutic treatment on the erosion of tarsal and calcaneal bones was examined radiographically. On day 28, vehicle-treated AIA rats showed a marked increase in bone erosion scores compared with those of normal untreated rats (Fig. 3A). Bone erosion scores of JTE-952-treated rats (≥3 mg/kg) were significantly reduced compared with the scores of vehicle-treated AIA rats, and ≥10 mg/kg almost completely suppressed the increase in bone erosion scores. However, MTX administration had little effect on bone erosion, with a slight increasing trend in scores. Representative micro-CT (μCT) images are shown in Fig. 3B. In vehicle-treated AIA rats, radiotranslucency was seen in the talus and calcaneus areas, whereas in AIA rats treated with JTE-952, it was almost indistinguishable from normal untreated rats. In MTX-treated AIA rats, the bone erosion was as severe as or worse than that in vehicle-treated AIA rats.

Fig. 3. Effects of JTE-952 on Bone Erosion

(A) Bone erosion scores obtained from bone images taken with a microfocal cone-beam X-ray computed tomography (CT) scanner. The severity of bone erosion of the left ankle joint was scored in a blinded manner for each tarsal bone and calcaneal bone (0, normal; 1, mild; 2, moderate; 3, severe). Results are expressed as group means and values for individual rats. ‡ p < 0.01 (vs. [N] group, Welch’s t-test). ** p < 0.01 (vs. [V] group, Steel test). (B) Representative X-ray CT images of rats with bone erosion closest to the mean of each group (upper panel, upper surface of instep; lower panel, left side of instep).

JTE-952 Increases the Range of Joint Motion

To investigate the effect of JTE-952 on joint flexibility influenced by joint inflammation and the structural destruction of bone and cartilage, we evaluated the ROM of ankle joints. A significant decrease in the ROM of vehicle-treated AIA rats was observed compared with that of normal untreated rats (Fig. 4). In JTE-952-treated rats, the ROM was increased at doses of ≥3 mg/kg with significant changes relative to vehicle-treated AIA rats, but even dose of 30 mg/kg did not lead to a complete recovery. MTX treatment had no effect on the ROM of AIA rats.

Fig. 4. Effects of JTE-952 on the Range of Joint Motion

Measurement of the range of joint motion (ROM) was based on a report by Hashmi et al. with some modifications.²⁵⁾ The ROM of the right ankle joint was measured in a blinded manner in degrees from the beginning position (maximum dorsiflexion) to its position at the end (maximum plantar flexion) of the full range of motion by a protractor. The ROM for each group (n = 10) was expressed as the mean (+ S.D.). ‡ p < 0.01 (vs. [N] group, Welch’s t-test). ** p < 0.01 (vs. [V] group, Steel test).

JTE-952 Reduces Joint Hyperalgesia

To evaluate the effect of JTE-952 on joint hyperalgesia, one of the causes of a poor QOL in RA, we measured hyperalgesia as a reduction in the pain threshold. A significant decrease in the pain threshold in AIA rats treated with vehicle was observed compared with that of the normal untreated rats (Fig. 5). JTE-952 increased the pain threshold in a dose-related manner, with significant changes relative to vehicle-treated AIA rats at dosages of ≥3 mg/kg, but no complete recovery was observed at 30 mg/kg. MTX treatment had no effects on the pain threshold.

Fig. 5. Effects of JTE-952 on Joint Hyperalgesia

On day 27 of AIA, the paw withdrawal threshold (PWT), as a measure of hyperalgesia, was assessed in both hindlimbs in a blinded manner by a digital pressure analgesy-meter. The PWT for each group (n = 10) was expressed as the mean (+ S.D.). ‡ p < 0.01 (vs. [N] group, Welch’s t-test). ** p < 0.01 (vs. [V] group, Steel test).

Correlation between JTE-952 Treatment and Clinical Variables

We then investigated an association between the therapeutic effect of JTE-952 and each clinical variable using Spearman rank correlation coefficients (Table 1). The effects of JTE-952 on the ROM, a measure of joint destruction, correlated highly with effects on bone erosion or hyperalgesia (r = 0.44–0.60), but less so with paw swelling (r = 0.12).

Table 1. Cross-Correlation Matrix between Each Parameter

	PS	BE	ROM	H
PS	1	0.35*	−0.12	−0.37**
BE	—	1	−0.44**	−0.53**
ROM	—	—	1	0.60**
H	—	—	—	1

Spearman rank correlation coefficients, * p < 0.05, ** p < 0.01. PS, paw swelling; BE, bone erosion; ROM, range of joint motion; H, hyperalgesia.

Pharmacokinetic Profile of JTE-952

Plasma concentrations of JTE-952 after a single oral administration to normal rats were elevated dose dependently. The C_2h value was 0.16 ± 0.01, 0.46 ± 0.01, 1.00 ± 0.27, and 1.60 ± 0.59 µmol/L at dosages of 1, 3, 10, and 30 mg/kg, respectively (Fig. 6). The area under the curve (AUC)_0–24h value was 0.64 ± 0.06, 1.87 ± 0.17, 3.67 ± 0.46, and 6.89 ± 0.86 µg·h/mL at dosages of 1, 3, 10, and 30 mg/kg, respectively.

Fig. 6. Pharmacokinetic Properties of JTE-952 in Rats

Change in plasma JTE-952 concentrations after the oral administration of the indicated doses of JTE-952. Results are expressed as the mean JTE-952 concentration (n = 6).

JTE-952 Inhibits TNFα Production from ex Vivo Whole Blood

On day 28, peripheral blood collected at 2 h after the oral administration of JTE-952, which provides the C_max, was used for an ex vivo assay. LPS-induced TNFα production in the whole blood of vehicle-treated AIA rats was higher than that of normal untreated rats, and a decrease in TNFα levels was observed in response to increasing doses of JTE-952, with a marked decrease at 30 mg/kg compared with normal rats (Fig. 7). The ED₅₀ value of JTE-952 for TNFα levels was 1.4 mg/kg. Furthermore, TNFα levels in the MTX-treated AIA rats were lower than those in rats treated with 30 mg/kg JTE-952.

Fig. 7. Ex Vivo Measurement of TNFα Production from Whole Blood

Whole blood was collected on day 28 of AIA, 2 h after JTE-952 was administered orally. Whole blood for each rat including the [N] group was plated into a 96-well plate, and then the lipopolysaccharide (LPS) solution was added. Supernatants were collected after 6 h of culture, and TNFα concentrations were measured by ELISA. Results are expressed as the mean (+ S.D.) TNFα level in the plasma (n = 10). * p < 0.05, ** p < 0.01 (vs. [V] group, Steel test).

DISCUSSION

It is well known that the CSF1/CSF1R pathway is one of the most important signaling pathways for osteoclast differentiation or proliferation.²⁷⁾ Furthermore, CSF1R-mediated signals have a critical role in the production of inflammatory mediators, such as TNFα, IL1β, and IL6, from myelomonocytic-linage cells such as monocytes and macrophages. Therefore, the pharmacological inhibition of the CSF1/CSF1R pathway might be a beneficial therapy for several autoimmune diseases, including RA.²⁸⁾ We previously developed a novel orally available CSF1R Type II inhibitor, JTE-952, which binds to the DFG-out state of CSF1R kinase and shows high kinase selectivity.²¹⁾ We confirmed the effect of JTE-952 on LPS-stimulated TNFα production using human, mouse, and rat bone marrow-derived macrophages and found that it suppressed TNFα and IL6 production without any species differences.²²⁾ Furthermore, we revealed that JTE-952 inhibited the CSF1-induced differentiation of human bone marrow cells into osteoclasts in vitro and inhibited bone destruction in a mouse collagen-induced arthritis model, a representative in vivo model of RA.²³⁾ Because JTE-952 inhibits CSF1R-induced functional activity without species differences, we consider that the evaluation of its in vivo efficacy in rats is feasible. In this study, we investigated the anti-arthritis properties of JTE-952 in rats, with a particular focus on RA.

The greatest unmet need in RA is the control of disease progression by addressing treatment resistance.²⁹⁾ AIA, a widely used animal model of RA, is used as a preclinical model for anti-rheumatic drugs.³⁰⁾ MTX, a traditional DMARD and the most widely used anchor drug for the treatment of RA, administered orally at 0.3 mg/kg once daily before the onset of paw swelling in AIA rats, completely inhibited the progression of symptoms (in house data not shown).³¹⁾ In the present study, as in a previous report,³²⁾ the daily administration of MTX (0.3 mg/kg) from day 15, when paw swelling was established in this model, did not suppress all clinical endpoints including paw swelling. Regarding the dose of MTX, we have previously observed that doses of MTX above 1 mg/kg are toxic doses with obvious weight loss when administered continuously to normal rats. Thus, we consider that 0.3 mg/kg MTX is the maximum tolerated dose (MTD) in rats that can be evaluated pharmacologically without obvious adverse effects on the general condition. Since these facts, 0.3 mg/kg was used as the evaluation dose of MTX in this study.

FK506, an immunosuppressant that specifically inhibits T-cell activation, was reported to suppress swelling when administered after the onset of AIA rats.³³⁾ In this regard, the pathogenesis of the AIA rat model is thought to be T cell-dependent and mediated by inflammatory factors produced by the interaction between activated T cells and synovial fibroblasts, monocytes, or macrophages.^33,34) Compared with FK506, MTX, which inhibits T-cell activation, was ineffective in this study when administered after the establishment of swelling, probably because MTX and FK506 mediate their effects through different mechanisms³⁵⁾; thus, MTX might not inhibit T cells that have already been activated. The post-onset pathology of the AIA rat model partially mimics the mechanism of resistance to treatment in human RA, which is a T cell-dependent disease.^34,36) Therefore, in this study, we used rat AIA as a pathological model to evaluate drug efficacy and investigated the therapeutic effects of JTE-952 in the post-onset phase, when MTX is ineffective. In this study, MTX of 0.3 mg/kg, the MTD, was ineffective on the paw swelling when administered after the onset of AIA. On the other hand, steroids or calcineurin inhibitors, which have shown effective in MTX-ineffective pathology in RA, suppress paw swelling and proinflammatory cytokine levels in paw tissue in this condition (in house data not shown). Thus, we specifically described this condition as ‘MTX-refractory pathology’ because the post-onset phase of rat AIA is not unresponsive to all drugs and some drugs that are effective in RA have also been found to be effective in this condition. JTE-952, similar to MTX, did not appreciably suppress paw swelling when administered orally after the establishment of swelling. This suggested that blockade of the CSF1/CSF1R pathway, which is mainly involved in monocyte-macrophage cells, may have little effect on pathological conditions where T cells are the drivers and subsequent mediator production by the inflammatory response has developed. T cells also produce TNFα and IL6, indicating they might be involved in the inflammatory response during the establishment of swelling. In support of this, it was reported that anti-TNFα antibodies suppressed paw swelling in established AIA rats.³⁷⁾

JTE-952 significantly inhibited the bone erosion of affected joints as assessed by μCT at a dose of 3 mg/kg and almost completely at doses of ≥10 mg/kg. Furthermore, histopathological evaluation showed that cartilage destruction in addition to bone destruction was significantly suppressed at dosages of ≥3 mg/kg JTE-952; however, only a partial inhibitory effect on bone destruction was observed, in contrast to the bone erosion assessed by μCT. These differences might be explained by differences in the evaluation targets for each method evaluated: μCT evaluates entire structural changes in the imaged bones, whereas histology evaluates local changes, including soft tissue around the bones. In the present study, JTE-952 was effective against bone destruction when assessed using both methods, and it also showed inhibitory effects on cartilage. For the cartilage, we further measured a blood biomarker reflecting the level of cartilage destruction, c-terminal telopeptides of type 2 collagen (CTX-II), a degradation product of type II collagen which constitutes the extracellular matrix of cartilage (data not shown). Administration of JTE-952 after the establishment of AIA suppressed the increase in CTX-II suggests that JTE-952 also suppresses cartilage destruction from the viewpoint of blood biomarker. Enzymes such as matrix metalloproteinases and cathepsins are thought to be involved in the progression of cartilage destruction.³⁸⁾ Because anti-CSF1R antibodies were shown to inhibit the production of these inflammatory mediators in RA synovial tissues,³⁹⁾ it was assumed that the suppression of the production of these factors by JTE-952 may contribute to its effectiveness on cartilage destruction and granulation formation. In fact, we confirmed that JTE-952 partially suppressed the increase in IL1β levels in the arthritis tissue (data not shown). In the present study, MTX showed a tendency to worsen bone erosion (Fig. 3). King et al. confirmed that MTX promoted osteoclast differentiation in rats via increased TNFα and IL6 levels and the activation of nuclear factor-kappaB (NF-κB), inducing osteoclastogenesis in bone and the circulation.⁴⁰⁾ Although no clear increase in plasma TNFα levels was observed in vehicle-treated AIA rats in our study (data not shown), the possibility that these mechanisms were involved in the worsening of bone erosion caused by MTX cannot be ruled out. These inflammatory cytokines might be increased locally in the affected bone, rather than in the plasma, and this will require future study. Above results suggest that JTE-952 may have some efficacy against ongoing bone and cartilage destruction in treatment-resistant RA in which MTX is ineffective.

Both the ROM and hyperalgesia were significantly improved by JTE-952 at dosages of ≥3 mg/kg, albeit partially. When we evaluated correlations between the dose of JTE-952 and its efficacy on each parameter (paw swelling, bone erosion, ROM, hyperalgesia), the ROM as a parameter of joint function showed a mutually high correlation (r = 0.44–0.60) with bone erosion and hyperalgesia, but a relatively low correlation with paw swelling (r = 0.12). Regarding hyperalgesia, JTE-952 inhibited TrkA kinase activity approximately 20-fold more weakly than CSF1R kinase.²²⁾ Nerve growth factor, a ligand for TrkA, was reported to be involved in joint pain in AIA rats.⁴¹⁾ Based on the relationship between JTE-952 dose and the kinase inhibitory activity ratio, the contribution of TrkA inhibition to the improvement of hyperalgesia in AIA rats by JTE-952 is small, and we speculate that it is due to the inhibition of bone erosion by inhibiting CSF1R. These results suggest that JTE-952 administered after the establishment of paw swelling improved bone erosion, ROM, and hyperalgesia, along with a series of associations. Suppression of the structural destruction of joints by JTE-952 may lead to clinical improvements in joint function as measured by the ROM and symptomatic pain.

The inhibitory effect of JTE-952 on cytokine production in the ex vivo whole blood of rats treated with JTE-952 was linked to an increase in the dose and plasma concentrations of JTE-952. In AIA rats, JTE-952 significantly inhibited the structural destruction of joints and changes in joint functions at doses of ≥3 mg/kg. The C_max value after a single oral dose of 3 mg/kg JTE-952 was 0.46 µmol/L, which was consistent with the previously reported IC₅₀ value of JTE-952 (0.6 µmol/L) for in vitro human whole blood. Therefore, based on the results of the human whole blood assay and animal studies, the effective plasma concentration of JTE-952 to prevent structural destruction and enhance the function of joints in RA patients was estimated to be 0.46 µmol/L or higher, which was also consistent with the C_max value of the effective blood concentration in the mouse collagen-induced arthritis model reported previously (0.54 µmol/L).

For the treatment of RA, the efficacy of biologics targeting single inflammatory cytokines such as anti-TNFα and anti-IL6 antibodies has been confirmed, and IL6 and TNFα are also known to act directly on osteoclasts and promote their differentiation and function.⁴²⁾ However, biologics targeting these single cytokines are assumed to have limited efficacy against bone and cartilage destruction, either directly via osteoclasts, which are under the influence of a complex interplay of various inflammatory cytokines, or indirectly through the production of metalloproteinases from synovial fibroblasts. Because MTX resistance in rat AIA is unlikely to reflect the entire treatment-resistant pathology of RA, it is difficult to predict the extent to which the effects of JTE-952 obtained in this study will be reflected in the resistant pathology in humans. Future studies should clarify the differences between human and model pathologies, the contribution of CSF1/CSF1R signaling and its change over time, and the identification of patient populations in which JTE-952 is expected to be effective.

In conclusion, we investigated the therapeutic effect of JTE-952 in clinically established AIA rats, which mimic the MTX-resistant RA pathology. The blockade of CSF1/CSF1R signaling by JTE-952 did not suppress paw swelling under inflammatory conditions, but inhibited the destruction of joint structural components including bone and cartilage in the inflamed joints and thus, may improve the subsequent functional impairment of joints related to the ROM and pain. In clinical practice, it may be possible to demonstrate the benefits of JTE-952 in combination with general DMARDs or by selecting patients with RA who are in a more advanced phase of joint destruction.

Acknowledgments

We thank Kayoko Takagi, Hiromi Yoshiuchi, Yuichi Naka, Tomokazu Kanehisa, Chika Oki, and Yusuke Kadota for their excellent technical assistance.

Conflict of Interest

The authors declare no conflict of interest.

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