JTE-952 Suppresses Bone Destruction in Collagen-Induced

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1884 Biol. Pharm. Bull. 43, 1884–1892 (2020) Vol. 43, No. 12 Regular Article

JTE-952 Suppresses Bone Destruction in Collagen-Induced Arthritis in Mice by Inhibiting Colony Stimulating Factor 1 Receptor Naofumi Uesato,* Koji Inagaki, Naoki Miyagawa, Yoshihiro Kitagawa, Reina Kakefuda, Yushi Matsuo,† Takayuki Yamaguchi, Takahiro Hata, Kazutaka Ikegashira, and Mutsuyoshi Matsushita Central Pharmaceutical Research Institute, Japan Tobacco Inc.; 1–1 Murasaki-cho, Takatsuki, Osaka 569–1125, Japan. Received June 22, 2020; accepted September 6, 2020

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation and structural destruction of the joints. Bone damage occurs in an early stage after onset and osteoclast activation plays a substantial role in its progression. Colony stimulating factor 1 receptor (CSF1R) is a receptor protein tyrosine kinase specifically expressed in monocytic-lineage cells such as macrophages and osteoclasts. Here, we investigated the effect of JTE-952, a novel CSF1R tyrosine kinase inhibitor, on osteoclast formation in vitro and on bone destruction in a mouse model of collagen-induced arthritis. JTE-952 completely inhibited osteoclast differentiation from human monocytes, with an IC50 of 2.8 nmol/L, and reduced osteoclast formation from the synovial cells of RA patients. Detectable levels of colony stimulating factor 1 (CSF1), a ligand of CSF1R, were observed in the synovial tissues of the arthritis model, similar to those observed in the pathology of human RA. JTE-952 significantly suppressed increases in the bone destruction score, the number of tartrate-resistant-acid-phosphatase-positive cells, and the severity of arthritis in the model mice. We also examined the efficacy of JTE-952 combined with methotrexate. This combination therapy more effectively reduced the severity of bone destruction and arthritis than monotherapy with either agent alone. In summary, JTE-952 potently inhibited human osteoclast formation in vitro and suppressed bone destruction in an experimental arthritis model, especially when combined with methotrexate. These results indicate that JTE-952 should strongly inhibit bone destruction and joint inflammation in RA patients and effectively prevent the progression of the structural destruction of joints. Key words JTE-952; colony stimulating factor 1 receptor; osteoclast; bone destruction; rheumatoid arthritis; collagen-induced arthritis

INTRODUCTION effective in all patients with RA, and even if joint inflammation is controlled, bone destruction gradually progresses Rheumatoid arthritis (RA) is a chronic systemic inflam- in the long term, which results in the irreversible structural matory disease characterized by synovial inflammation and destruction of the joints,10–13) and these patients may ulti- progressive structural destruction of the cartilage and bone mately require orthopedic treatments, such as artificial joints. in multiple affected joints.1) It is widely acknowledged that Therefore, drugs that effectively inhibit bone destruction and the progression of joint damage occurs in an early stage, soon prevent the structural destruction of the inflamed joints may after onset.2–4) The pathological processes of joint damage are satisfy unmet needs in terms of improving of QOL in RA influenced by direct or indirect interactions with synovial cells patients. and a wide range of cell types associated with the adaptive Colony stimulating factor 1 receptor (CSF1R) is a receptor and innate immune systems and are mediated by inflamma- protein tyrosine kinase specifically expressed in monocytic- tory factors such as tumor necrosis factor alpha (TNF-α), in- lineage cells, including monocytes, dendritic cells, and mac- terleukin 1 (IL1), IL6, IL17, receptor activator of nuclear fac- rophages. Osteoclasts, which are large multinucleated cells tor-kappaB ligand (RANKL), and matrix metalloproteinase.5,6) responsible for the dissolution and absorption of bone, dif- In clinical practice, traditional disease-modifying anti- ferentiate from monocytic-lineage cells, and the activation rheumatic drugs (DMARDs) such as methotrexate (MTX) are of CSF1R signals increases the number of the osteoclasts frequently used to suppress joint inflammation in the treat- formed.14,15) The levels of colony stimulating factor 1 (CSF1), ment of RA and attenuate disease progression.7) Biological a ligand of CSF1R, are higher in the peripheral blood or sy- DMARDs such as etanercept (a soluble TNF-α receptor) and novial fluid of RA patients than in that of healthy subjects or tocilizumab (an anti-IL6 receptor antibody), which inhibit patients with other joint diseases, such as osteoarthritis.16) Re- disease activity and lead to remarkable clinical improvement, cently, IL34 was identified as a second ligand of CSF1R.17,18) have been approved.8,9) However, these agents are not always IL34 is also expressed in the synovial tissues and sera of RA patients,19,20) although its role in arthritis is not fully under- † Present address: Toxicology Research Laboratories, Central Pharmaceuti- stood. Therefore, osteoclast differentiation and activation, cal Research Institute, Japan Tobacco Inc.; 1–13–2 Fukuura, Kanazawa- which are induced by signals from CSF1R, are thought to ku, Yokohama 236–0004, Japan. occur in the joint lesions of RA patients and may be respon-

* To whom correspondence should be addressed. e-mail: [email protected] © 2020 The Pharmaceutical Society of Japan Vol. 43, No. 12 (2020) Biol. Pharm. Bull. 1885 sible for the progression of bone destruction and consequent formed according to the Declaration of Helsinki. joint damage. Numerous small-molecule CSF1R inhibitors Synovial Tissues from Patients with RA Synovial tis- have been developed that inhibit both joint inflammation and sues from RA patients who had undergone surgery were inflammatory bone erosion in animal models of arthritis, but obtained from the Health Science Research Resources Bank these are limited by poor kinase selectivity.21) (HSRRB; Osaka, Japan). The synovial tissues, which were JTE-952 is a novel, orally available type II kinase in- immersed in a Hanks’ balanced salt solution, were delivered hibitor of CSF1R developed by Japan Tobacco Inc.22) JTE-952 to our laboratory under cool conditions on ice on the day of clearly inhibits human CSF1R kinase activity (at an IC50 synovectomy. The collection and use of these tissues were of 11.1 nmol/L) in vitro, with no obvious inhibitory activity according to the principles of the HSRRB Ethical Committee against other kinases, except tropomyosin-related kinase A.23) for the Use of Human-Derived Samples and the Declaration of JTE-952 (≥3 mg/kg administered orally) has an anti-inflam- Helsinki. matory effect in vivo in mice, evident as the suppression of Osteoclast Formation Assay with Synovial Cells from CSF1-mediated enhancement of lipopolysaccharide (LPS)- RA Patients Synovial cells from the knee-joint syno- induced TNF-α production. Based on these findings, the aim vial tissues of two RA patients were isolated by dispersal of this study was to clarify the effect of JTE-952 on osteoclast in α-minimum essential medium containing 2 mg/mL col- formation in vitro and on bone destruction in an animal model lagenase for 90 min at 37 °C. The synovial cells were then of arthritis, focusing on its effects on the pathology of RA. suspended in DMEM supplemented with 10% FCS, seeded at a density of 5 × 104 cells/well in 96-well flat-bottomed plates, MATERIALS AND METHODS and cultured in the presence of 25 ng/mL recombinant human CSF1 and 30 ng/mL recombinant human RANKL with vari-

Compounds JTE-952, (2S)-3-{[2-({3-[4-(4-cyclopropyl- ous concentrations of JTE-952 for 15 d at 37 °C under 5% CO2. benzyloxy)-3-methoxyphenyl]azetidine-1-yl}carbonyl)- The cells were then stained for TRAP with a TRAP staining pyridin-4-yl] methoxy}propane-1,2-diol, was chemically syn- kit. TRAP-positive and multinucleated cells were counted as thesized at the Central Pharmaceutical Research Institute, osteoclasts under a microscope by a blinded observer, and the Japan Tobacco Inc. (Osaka, Japan). For the cell-based assays, average number for duplicate wells was considered the number JTE-952 (purity 97.5%) was dissolved in dimethyl sulfoxide of osteoclasts from each donor. and diluted in culture medium immediately before use. The Measurement of Cytokine Levels in Synovial Tissues of final concentration of dimethyl sulfoxide was less than 0.1%. Patients with RA The synovial tissues of six RA patients For the animal studies, JTE-952 and MTX (Sigma-Aldrich Co. aged 61–77 years were obtained from HSRRB (five of the LLC, St. Louis, MO, U.S.A.) were suspended in a 0.5% (w/v) six patients were women, knee joints; one of the six patients aqueous solution of methylcellulose (MC). was a man, a wrist joint). These patients had been taking In Vitro Human Osteoclast Formation Assay Human DMARDs such as MTX, selective cyclooxygenase-2 inhibi- peripheral blood was collected from healthy in-house volun- tor, and/or corticosteroid, but none were taking any biological teers in tubes containing citric acid and was used for experi- agent. A part of each synovial tissue was placed in a separate ments on the same day. The monocytes were purified from the tube and 15 µL of ice-cold lysis buffer containing 2 mmol/L peripheral blood by negative selection with immunomagnetic ethylenediaminetetraacetic acid (EDTA) and protease inhibi- beads (human Monocyte Isolation Kit II, Miltenyi Biotec K.K., tors in phosphate-buffered saline was added per 1 mg of tis- Tokyo, Japan) and suspended in Dulbecco’s modified Eagle’s sue. The tissues were homogenized with a mixer mill (Retsch medium (DMEM; Wako Pure Chemical Industries, Ltd., MM400, Verder Scientific Co., Ltd., Tokyo, Japan) for 2 min Osaka, Japan) supplemented with 10% fetal calf serum (FCS). at 30 Hz. After centrifugation (10000 × g, 4 °C), the concen- The monocytes were seeded at a density of 5 × 104 cells/well trations of granulocyte-macrophage colony-stimulating factor in 96-well flat-bottomed plates and cultured in the presence of (GM-CSF) and CSF1 in the supernatant were measured with 25 ng/mL recombinant human CSF1 (PeproTech Inc., Rocky specific enzyme-linked immunosorbent assay (ELISA) kits Hill, NJ, U.S.A.) and 30 ng/mL recombinant human RANKL (Quantikine® ELISA, R&D Systems, Inc., Minneapolis, MN, (PeproTech Inc.) with various concentrations of JTE-952 for U.S.A.). Levels below the quantification limit were coded as

7 d at 37 °C under 5% CO2. Tartrate-resistant acid phosphatase 0 pg/mL. (TRAP) staining was then performed with the TRAP Staining Animals Male DBA/1J mice were supplied by Charles Kit (Primary Cell Co., Ltd., Sapporo, Japan). TRAP-positive River Laboratories Japan, Inc. (Yokohama, Japan) when 6–7 multinucleated cells with at least three nuclei were counted as weeks old. All animals were housed under specific-pathogen- osteoclasts under a microscope by an observer blinded to the free conditions at a room temperature of 23 ± 3 °C and air hu- cell treatment, and the average count for triplicate wells was midity of 55 ± 15% under a 12-h light/dark cycle and given ac- considered the number of osteoclasts in each group. Because cess to standard laboratory chow diet (CRF-1, Oriental Yeast it has been reported that TRAP-5b activity in the culture Co., Ltd., Tokyo, Japan) and water ad libitum. All procedures supernatant of osteoclasts reflects the number of osteoclasts related to the use of animals in this study were reviewed and present,24) the TRAP-5b activity in the culture supernatants approved by the Institutional Animal Care and Use Commit- was also determined with a specific kit for the quantitative tee at Japan Tobacco Inc. and were performed in accordance determination of the active isoform 5b of TRAP (BoneTRAP® with standards published by the National Research Council assay, Immunodiagnostic Systems Ltd., Boldon, U.K.). The (Guide for the Care and Use of Laboratory Animals, NIH experiment was repeated three times. All procedures in this OACU) and the National Institutes of Health Policy on Human study were approved by the Ethics Committee for the Use of Care and Use of Laboratory Animals. Human-Derived Samples at Japan Tobacco Inc. and were per- Induction of Collagen-Induced Arthritis in Mice Col - 1886 Biol. Pharm. Bull. Vol. 43, No. 12 (2020) lagen-induced arthritis (CIA) was induced in DBA/1J mice of knee joints were embedded in paraffin and sectioned. The aged 10 weeks with a previously described method.23) Type II paraffin sections were then stained for TRAP with a TRAP collagen (Collagen Research Center, Kiyose, Japan) derived staining kit (Primary Cell Co., Ltd.). The TRAP-positive cells from bovine articular cartilage was dissolved at a concentra- around the knee joints were counted under a microscope by a tion of 4 mg/mL in a 0.01 mol/L aqueous acetic acid solution blinded observer. and emulsified with an equal volume of Freund’s complete IC50 Determination The IC50 was calculated from the adjuvant containing 2 mg/mL heat-killed Mycobacterium tu- concentration of JTE-952 and the residual (%) osteoclast for- berculosis H37Ra (Difco Laboratories, Detroit, MI, U.S.A.). mation activity or the residual (%) TRAP-5b activity, with Arthritis was induced by the intradermal injection of 100 µL a logistic function in the SAS software (SAS Institute Japan of 2 mg/mL type II collagen emulsion at the base of the tail on Ltd., Tokyo, Japan). days 1 and 22 of the experiment. All mice were immunized, Statistical Analysis All statistical analyses were per- except those in the normal group, in which arthritis was not formed with SAS version 8.2 and SAS preclinical package induced. The severity of arthritis was scored for the digits of version 5.0 (SAS Institute Japan Ltd.). For the bone destruc- each limb using a three-point scale ranging from 0 to 2 (0, tion scores and arthritis scores (nonparametric data), the normal; 1, swelling of one finger; 2, swelling of two or more significance of the differences between two groups was de- fingers) on days 22, 25, 27, 29, 32, and 35. The severity of ar- termined with the Wilcoxon rank-sum test and the differences thritis was scored for the carpus and tarsus of each limb on a among multiple groups were evaluated using the Steel test or three-point scale ranging from 0 to 2 (0, normal; 1, erythema Steel–Dwass test. The significance of the differences between and mild-to-medium swelling; 2, erythema and severe swell- two groups in the numbers of TRAP-positive cells (parametric ing). The total arthritis score for each mouse was expressed data) was determined using Welch’s t test and the differences as the sum of the scores for the four limbs (maximum possible among multiple groups were evaluated using the Steel test. A score: 16). Scoring was blind for each group. two-tailed p value <0.05 was considered statistically signifi- Drug Treatment Arthritic mice were randomized and cant. grouped for drug treatment based on their body weight on day 22 when arthritis had not developed. JTE-952 (1, 3, 10, and RESULTS 30 mg/kg) and vehicle (0.5% MC) were orally administered once a day for 14 d, from day 22 onwards. MTX (0.3 mg/kg), Effects of JTE-952 on Human Osteoclast Formation in a synthetic DMARD, was also orally administered as a mono- Vitro To examine the effects of JTE-952 on human osteo- therapy or together with JTE-952 (3 mg/kg). The experiment clast formation, monocytes were stimulated with CSF1 and was repeated three times. RANKL for 7 d in the presence of JTE-952. Human mono- Measurement of Cytokine Levels in Synovial Tissues of cytes that had differentiated into osteoclasts (red staining), CIA Mice On days 29 and 35 of CIA, the arthritis scores which were identified by TRAP-positive staining and multi- were measured and the synovial tissues from the knee joints nucleation under a microscope (Fig. 1A, Control). However, of each mouse (15 mice per group) were dissected. These tis- negligible osteoclasts were observed in the CSF1-unstimulated sues were pooled in a single tube per group and then 15 µL of group ([−] CSF1; stimulated with RANKL alone). JTE-952 ice-cold lysis buffer containing 2 mmol/L EDTA and protease reduced the number of osteoclasts in a concentration-depen- inhibitors in phosphate-buffered saline was added per 1 mg dent manner and completely inhibited osteoclast formation at of tissue. The tissues were homogenized with a mixer mill concentrations of ≥30 nmol/L (Figs. 1A, B). The IC50 value (Retsch MM400). After centrifugation (10000 × g, 4 °C), the for JTE-952 on osteoclast formation was 2.8 ± 1.0 nmol/L. concentrations of GM-CSF and CSF1 in the supernatant were JTE-952 also reduced the TRAP-5b activity in the culture ® measured with specific ELISA kits (Quantikine ELISA). Lev- supernatant, with an IC50 value of 3.5 ± 1.1 nmol/L (Fig. 1C). els below the limit of quantification were coded as 0 pg/mL. Effects of JTE-952 on Osteoclast Formation among Scoring Bone Destruction The right forelimb of each Synovial Cells of RA Patients When synovial cells iso- CIA mouse was removed and an X-ray transmission image lated from two RA patients were cultured for 15 d, osteoclasts, of the bone was obtained with a microfocal cone-beam X-ray which were characterized by TRAP-positive staining and CT scanner (MCT-CB100MF, Hitachi Medical Corporation, multinucleation, formed among the fibroblast-like cells, as ob- Tokyo, Japan). Based on the bone image, the severity of bone served with microscopy (data not shown). Osteoclasts formed destruction was scored on a three-point scale ranging from even without CSF1 or RANKL ([−] group; 8 and 24 cells/well 0 to 2 (0, normal; 1, mild-to-medium destructive abnormal- in patient 1 and patient 2, respectively) and the addition of ity with bone erosion; 2, severe destructive abnormality with CSF1 and RANKL increased the numbers of osteoclasts (Con- definite bone erosion or complete erosion) for each of five trol group; cultured with CSF1 and RANKL, 36 and 52 cells/ joints (second to fifth metacarpophalangeal joints and carpal well in patient 1 and patient 2, respectively; Fig. 2). JTE-952 joint). The scores for these five joints were summed to obtain concentration-dependently reduced the number of RA-syno- the bone destruction score (maximum possible score: 10) for vial-cell-derived TRAP-positive osteoclasts and completely each mouse. Imaging and scoring were performed in a blinded inhibited their formation at concentrations of ≥100 nmol/L. manner. However, 300 nmol/L JTE-952 did not affect the number of Histological Evaluation The right hindlimb of each CIA fibroblast-like cells. mouse was removed and fixed by immersing it in 4% parafor- GM-CSF and CSF1 Protein Levels in Synovial Tissues maldehyde in phosphate-buffered saline. The specimens were of RA Patients In the synovial tissues from six RA patients then decalcified with 10% EDTA in 0.1 mol/L Tris-buffered who had undergone synovectomy, GM-CSF levels in the sy- saline (pH 7.4) for 4 weeks at 4 °C. All the tissue specimens novia were below the limit of quantification in all patients Vol. 43, No. 12 (2020) Biol. Pharm. Bull. 1887

Fig. 1. Effects of JTE-952 on Osteoclast Formation from Human Monocytes Human monocytes purified from peripheral blood were cultured in the presence of CSF1 and RANKL with the indicated concentrations of JTE-952 for 7 d. (A) Repre- sentative pictures of osteoclasts detected with TRAP staining. The cell shown with an arrow is a representative osteoclast. Cells of the (−) CSF1 group were cultured in the presence of RANKL alone. (B) Effect of JTE-952 on number of TRAP-positive multinucleated cells (with at least three nuclei). (C) Effect of JTE-952 on TRAP-5b activity in the culture supernatant. Results are expressed as mean percentages (±standard error of the mean (S.E.M.)) of the vehicle control value (n = 3).

Fig. 3. GM-CSF and CSF1 Protein Levels in the Synovial Tissues of RA Patients Fig. 2. Effects of JTE-952 on Osteoclast Formation from Synovial Cells Synovial tissues from six RA patients were homogenized with a mixer mill. of RA Patients After centrifugation, the concentrations of GM-CSF and CSF1 in the supernatant Synovial cells from two RA patients, isolated with enzymatic dispersion, were were measured with specific ELISA kits. Levels below the limit of quantification cultured in the presence of CSF1 and RANKL with the indicated concentrations were coded as 0 pg/mL. Results are expressed as the mean (ng/mg tissue) and indi- of JTE-952 for 15 d. Osteoclasts were detected with TRAP staining, and the num- vidual values (○). ber of TRAP-positive multinucleated cells (at least three nuclei) was counted in a blinded manner. The cells of the (−) group were cultured with MEM alone. Results are expressed as the mean number of TRAP-positive cells from the two RA patients and their individual values (patient 1, ○; patient 2, △). of CIA Mice The GM-CSF levels in the synovia were below the limit of quantification (≤0.5 ng/mg tissue) on days 29 and 35, whereas CSF1 was clearly detected at both time points (8.9 (≤1.0 ng/mg tissue), whereas CSF1 was clearly detected in all and 8.0 ng/mg tissue on days 29 and 35, respectively; Fig. 4). donors at an average level of 72.7 ± 17.9 ng/mg tissue (Fig. 3). The average arthritis scores for the mouse synovia were 8.9 GM-CSF and CSF1 Protein Levels in Synovial Tissues on day 29 and 13.2 on day 35 (maximum possible score: 16). 1888 Biol. Pharm. Bull. Vol. 43, No. 12 (2020)

Effects of JTE-952 on Bone Destruction and Joint Inflammation in CIA Mice Prophylactic Effects JTE-952 was orally administered at a dose of 1, 3, 10, or 30 mg/kg once daily for 14 d, from day 22 onwards. The bone destruction score of the vehicle-treated group on day 35 was significantly higher than that of the normal group (Fig. 5A). JTE-952 dose-dependently inhibited the increase in the bone destruction score. Statistically significant differences were observed at JTE-952 doses of ≥3 mg/kg and the percentage inhibition was 35.3, 68.4, 72.9, and 83.5% at doses of 1, 3, 10, and 30 mg/kg, respectively. Osteolytic bone lesions were observed in the metacarpophalangeal joints and carpal joints of the vehicle-treated group, and these radiographic findings were suppressed by JTE-952 (Fig. 5B). JTE-952 also dose-dependently Fig. 4. GM-CSF and CSF1 Protein Levels in the Synovial Tissue of inhibited the increase in the number of TRAP-positive cells in CIA Mice the knee joints (Fig. 5C). Statistically significant differences CIA was induced as described in the Materials and Methods. On days 29 and 35 were observed at JTE-952 doses of ≥3 mg/kg, and the per- of CIA, the synovial tissues were collected from both knee joints of each mouse centage inhibition was 20.3, 37.7, 71.0, and 88.0% at doses of (15 mice per group). The tissues were pooled per group and homogenized with a mixer mill. After centrifugation, the concentrations of GM-CSF and CSF1 in the 1, 3, 10, and 30 mg/kg, respectively. The vehicle-treated CIA supernatant were measured with specific ELISA kits. Levels below the limit of mice showed an arthritis score of 2.4 ± 0.5 on day 27, which quantification were coded as 0 pg/mL. The results are expressed as the cytokine contents (ng/mg tissue) of the pooled synovial tissues. gradually increased to a maximum score of 12.0 ± 0.9 on day 35 (Fig. 5D). JTE-952 inhibited this increase in the arthritis score in an approximately dose-related manner, with signifi-

Fig. 5. Eﬀects of Prophylactic Treatment with JTE-952 JTE-952 was orally administered at the indicated doses once daily for 14 d from day 22 after CIA induction. On day 35, the mice were dissected and the subsequent analysis performed. (A) The bone destruction scores, obtained from the bone images taken with a microfocal cone-beam X-ray CT scanner, are expressed as group means and the values for individual mice (○; normal group, n = 10; vehicle- or JTE-952-administered group, n = 25). (B) Representative X-ray CT images of the mice in each group, showing bone destruction. (C) Numbers of TRAP-positive cells in the knee joints are expressed as means and S.E.M. (D) The arthritis scores are expressed as mean of the mice in each group at diﬀerent time point. ## p < 0.01 (vs. normal group, Wilcoxon rank-sum test). * p < 0.05, ** p < 0.01 (vs. vehicle-treated group, Steel test). ‡p < 0.01 (vs. normal group, Welch’s t test). Vol. 43, No. 12 (2020) Biol. Pharm. Bull. 1889

DISCUSSION

Bone destruction in RA leads to structural destruction of the joint.10–13) Therefore, it is important to control the progression of this bone destruction by suppressing synovial inflammation in the early stage of RA.2–4) Bone destruction is strictly regulated by the process of bone resorption by osteoclasts, and the CSF1/CSF1R axis is specifically involved in the differentiation of monocytic-lineage cells into osteoclasts and their activation. We have previously reported that JTE-952 potently inhibits the tyrosine kinase activity of CSF1R, which results in a potent anti-inflammatory effect in vivo. This effect is highly selective and JTE-952 displays no significant inhibitory activity against other kinases, except tropomyosin-related kinase A.23) Therefore, in the present study, we investigated the effects of JTE-952 on osteoclast formation in vitro and on bone destruction in an experimental model of arthritis, focusing on its effects on RA. JTE-952 inhibited human osteoclast formation from monocytes obtained from the peripheral blood and the TRAP-5b ac-

tivity in the culture supernatants, with IC50 values of 2.8 and 3.5 nmol/L, respectively. In this study, no apparent progression of osteoclast formation was observed in the absence of CSF1, even in the presence of RANKL, which indicates that CSF1 plays an important role in the process of differentiation of monocyte-lineage cells into osteoclasts. In RA, synovial cells, including tissue macrophages and synovial fibroblasts, con- tribute to osteoclast formation at sites of bone erosion,25) and synovial fibroblasts are sites of CSF1 production in the joints Fig. 6. Effects of Combined Treatment with JTE-952 and MTX of patients with inflammatory arthritis.26,27) Therefore, we JTE-952 and/or MTX were orally administered at doses of 3 mg/kg JTE-952 and 0.3 mg/kg MTX, once daily for 14 d from day 22 after CIA induction. The mice next examined the effects of JTE-952 on osteoclast formation were dissected on day 35. (A) The bone destruction scores, obtained from bone in synovial cells obtained from RA patients. Interestingly, as images taken with a microfocal cone-beam X-ray CT scanner, are expressed as the well as an increase in number of fibroblast-like cells, the syno- group means and the values for individual mice (○; normal group, n = 10; vehicle- or drug-administered group, n = 20). (B) The arthritis scores are expressed as mean vial cells derived from the synovial tissues of RA patients dif- of the mice in each group at different time point. ## p < 0.01 (vs. normal group, ferentiated into osteoclasts in the absence of exogenous CSF1 Wilcoxon rank-sum test). $ p < 0.05, $$ p < 0.01 (Steel–Dwass test). and RANKL, and the addition of CSF1 and RANKL further increased the number of osteoclasts. Although JTE-952 had no cant differences at doses of ≥3 mg/kg. apparent inhibitory effect on the proliferation of the fibroblast- Combined Effects with MTX JTE-952 (3 mg/kg), MTX like cells, it inhibited osteoclast formation from synovial cells (0.3 mg/kg), or a JTE-952 (3 mg/kg)/MTX (0.3 mg/kg) mixture in all the donors examined. These results suggest that JTE-952 was orally administered to mice once daily for 14 d, from inhibits osteoclastogenesis in the RA synovium. day 22 onwards. The bone destruction score of the vehicle- Monocytes in the peripheral blood can differentiate into treated group on day 35 was significantly higher than that inflammatory M1 or anti-inflammatory M2 macrophage sub- of the normal group in which arthritis was not induced (Fig. types.28) In a GM-CSF environment, tissue macrophages are 6A). JTE-952 monotherapy inhibited the increase in the bone differentiated and activated toward M1 macrophages, which destruction score, but not significantly, compared with the ve- produce large amounts of proinflammatory cytokines, such as hicle-treated group. Conversely, MTX alone had no apparent TNF-α and IL6. Conversely, polarization to M2 macrophages effect on the bone destruction score at a dose of 0.3 mg/kg, al- that produce low levels of proinflammatory cytokines was ob- though this dose suppressed the increase in the arthritis score served in a CSF1 environment.29) Notably, CSF1 levels in the to the same extent as 3 mg/kg JTE-952 (Fig. 6B). JTE-952 peripheral blood and synovial fluid of RA patients are higher used in combination with MTX significantly inhibited the than in healthy subjects or patients with other joint diseases increase in the bone destruction score compared with that of such as osteoarthritis, and these levels correlate with disease the vehicle-treated group. The vehicle-treated mice showed an severity.16,30) GM-CSF was also detected in the synovial fluid arthritis score of 3.3 on day 27, which gradually increased to and plasma of RA patients,31–33) and a GM-CSF-blocking a maximum score of 11.6 on day 35. When JTE-952 was used antibody was effective against joint inflammation and bone in combination with MTX, the arthritis score on day 35 was destruction in experimental arthritis.34) Regarding osteoclas- 7.2, which was clearly suppressed compared with that in the togenic ability, there is a significantly positive correlation vehicle-treated group. The inhibitory effect of the combined between the ratio of M1/M2 monocytic-linage cells and the drugs on day 35 was statistically significantly different from number of osteoclasts in RA patients.35) These findings imply that of JTE-952 or MTX monotherapy. that the local cytokine microenvironment in the synovial tissues of RA patients, including the quantitative balance of 1890 Biol. Pharm. Bull. Vol. 43, No. 12 (2020)

CSF1 and GM-CSF, is closely associated with osteoclastogenic tion in RA.39) Therefore, JTE-952 strongly suppresses bone activity. Therefore, before considering the effects of JTE-952, destruction in RA patients and may also maximally inhibit we evaluated the protein levels of GM-CSF and CSF1 in sy- the associated joint inflammation, depending on the contribu- novial tissues obtained from RA patients. We found higher tion of CSF1. Further studies are required to evaluate whether levels of CSF1 compared with GM-CSF in all the RA synovia JTE-952 has beneficial effects on global joint functions in evaluated. The differentiation to osteoclasts in the absence of arthritis, including cartilage destruction and joint movement. exogenous factors during osteoclast formation from RA syn- RANKL is another molecule essential for osteoclastogen- oviocytes (Fig. 2) can be explained, in part, by the influence esis. The role of RANKL in the bone destruction associated of endogenous CSF1 produced by the synovial fibroblasts of with RA has been suggested by several observations.40–42) RA patients. These results are also consistent with a previous RANKL is upregulated in RA lesions, acts directly on os- report36) that suggested that osteoclasts in the synovia of RA teoclast precursor cells to stimulate their differentiation into patients are strongly affected by regional levels of CSF1. mature osteoclasts, and regulates the activation of mature To test the possibility that JTE-952 inhibits bone destruc- osteoclasts. In Japan, denosumab, a neutralizing anti-RANKL tion in vivo, we used a mouse model of CIA. Mouse CIA is antibody, has been used clinically to suppress the progres- a disease model of chronic arthritis, with synovial inflamma- sion of the bone erosion associated with RA. However, al- tion and bone erosion, which are similar to the symptoms of though denosumab reduces the progression of bone erosion, human RA.37) First, to estimate the cytokine environment at it reportedly has no protective effect on the progression of the joint locus, we evaluated the protein levels of GM-CSF joint-space narrowing or RA disease activity.43) Furthermore, and CSF1 in the synovial tissues in the early (day 29) and late although there was little evidence of bone destruction in a (day 35) clinical stages after CIA onset. The levels of CSF1 serum-transfer arthritis model with RANKL knockout, joint in the synovial tissues were equally high in both stages, un- inflammation occurred to the same extent as in wild-type like those of GM-CSF. The marked increase in CSF1 from the mice, when measured as the increase in the clinical score.44) onset of CIA demonstrated that CSF1 plays a pivotal role in These lines of evidence indicate that anti-RANKL therapy the pathological process of CIA. These results are similar to may have little effect on joint inflammation or disease activ- those of the pathological analysis of human RA, shown in Fig. ity. Conversely, as demonstrated in our present and previous 3. Therefore, it was reasonable to use the CIA mouse model studies,23) JTE-952 exerts an inhibitory effect on joint inflam- to predict the effect of JTE-952 on bone destruction and joint mation in the CIA mouse model. Indeed, LPS-stimulated inflammation in human RA pathology. TNF-α production by whole blood obtained from CIA mice We then evaluated the inhibitory effect of orally admin- treated with JTE-952 was suppressed by up to 50% compared istered JTE-952 on bone destruction and joint inflammation with that of vehicle-treated CIA mice (data not shown). The in the CIA model. JTE-952 was administered before disease percentage inhibition of TNF-α production was similar to the onset (days 22–35) and significantly and dose-dependently maximum efficacy of inhibition mediated by JTE-952 against suppressed the bone destruction score and TRAP-positive cell joint inflammation, which was measured as an increase in number in the knee joints at doses of ≥3 mg/kg. The disease the arthritis score. These results demonstrate that JTE-952 severity after treatment with 30 mg/kg JTE-952 was almost suppressed proinflammatory cytokine production in the CIA identical to that in the normal group, in which arthritis was mouse model. JTE-952 completely inhibited the production of not induced. These results indicate that JTE-952 reduces the proinflammatory cytokines that were induced by LPS-stim- osteoclast number in the synovium and inhibits bone destruc- ulated monocytes/macrophages in vitro, such as TNF-α and tion by inhibiting the CSF1R signal in vivo. JTE-952 also IL6. TNF-α was reported to cooperate with IL6 to regulate attenuated the increase in the arthritis score, which seems to the differentiation of osteoclasts and osteoclast-mediated bone reflect the severity of joint inflammation. The anti-inflamma- erosion, independent of RANK.45) Therefore, these results tory effect of JTE-952 was statistically significant at doses of suggest that unlike anti-RANKL antibodies, JTE-952 might ≥3 mg/kg, although at maximum efficacy, inhibition was only provide benefit by inhibiting joint inflammation, in addition to partial. The effect of a neutralizing anti-CSF1R antibody on its direct suppressive effect on osteoclastogenesis by inhibiting mouse CIA has been demonstrated in a previous report by CSF1R. To test this hypothesis, further detailed research with Tho et al.38) In that study, although the anti-CSF1R antibody appropriate reference agents is required. fully inhibited bone destruction, the arthritis score was only MTX is frequently used as an anchor drug in the treatment partially suppressed. The partial efficacy of the anti-CSF1R of RA.46,47) However, although monotherapy with MTX has a antibody on the arthritis score was thought to reflect the strong anti-inflammatory effect, its effect on the progression maximum possible contribution of CSF1 to joint inflammation of bone destruction is small.48) In this study, we examined the in mouse CIA. The inhibitory effect of JTE-952 (30 mg/kg) efficacy of 3 mg/kg JTE-952 combined with 0.3 mg/kg MTX on the arthritis score was also partial in our study and was (equivalent to the dose of JTE-952 [3 mg/kg] that suppressed similar to that of the anti-CSF1R antibody. Inflammatory the arthritis score) in the treatment of bone destruction and factors produced by cells that rarely express CSF1R, such as joint inflammation after their repeated oral administration. T cells, may be partly responsible for the proportion of joint Monotherapy with JTE-952 inhibited bone destruction, but not inflammation that failed to respond to JTE-952. Future stud- significantly, in this experiment. When combined with a dose ies should measure CSF1 levels in the synovial tissue after of MTX that had no overt inhibitory effect on bone destruc- JTE-952 treatment and the inflammatory response involved tion when used as a monotherapy, JTE-952 exerted a greater in joint inflammation not controlled by JTE-952. Recent evi- inhibitory effect than JTE-952 alone. The combined effect dence indicates that osteoclasts accumulate and are activated was not statistically significantly different from the effect of in the synovial tissue and play a pivotal role in joint destruc- JTE-952 alone but differed significantly from that in the vehi- Vol. 43, No. 12 (2020) Biol. Pharm. Bull. 1891 cle-treated group. Furthermore, JTE-952 combined with MTX 9) Navarro-Millán I, Singh JA, Curtis JR. Systematic review of tocili- complementarily and synergistically inhibited the increase in zumab for rheumatoid arthritis: a new biologic agent targeting the the arthritis score. Thus, JTE-952 inhibited bone destruction interleukin-6 receptor. Clin. Ther., 34, 788–802.e3 (2012). 10) Granger B, Combe B, Le Loet X, Saraux A, Guillemin F, Fautrel B. and joint inflammation more effectively in the CIA mouse Performance of matrices developed to identify patients with early model when it was used in combination with MTX. MTX sup- rheumatoid arthritis with rapid radiographic progression despite presses the expression of RANKL and increases the secretion methotrexate therapy: an external validation study based on the of osteoprotegerin, a natural inhibitor of RANKL, by synovial ESPOIR cohort data. RMD Open, 2, e000245 (2016). 49) fibroblasts. MTX also inhibits the production of cytokines, 11) Smolen JS, Van Der Heijde DMFM, St Clair EW, Emery P, Bathon such as interferon-γ, by pathogenic T cells and suppresses JM, Keystone E, Maini RN, Kalden JR, Schiff M, Baker D, Han arthritis in the mouse CIA model.50) Therefore, the combined C, Han J, Bala M. Predictors of joint damage in patients with early effects of JTE-952 and MTX are probably attributable to the rheumatoid arthritis treated with high-dose methotrexate with or synergistic effects of the different mechanisms of action of without concomitant infliximab: results from the ASPIRE trial. Ar- these agents. Hence, the combination may be more beneficial thritis Rheum., 54, 702–710 (2006). for the joint destruction and inflammation associated with RA 12) Ramiro S, Sepriano A, Chatzidionysiou K, Nam JL, Smolen JS, van der Heijde D, Dougados M, van Vollenhoven R, Bijlsma JW, than a monotherapy with either agent alone. Burmester GR, Scholte-Voshaar M, Falzon L, Landewe RBM. In conclusion, we have demonstrated that JTE-952, a novel Safety of synthetic and biological DMARDs: a systematic literature CSF1R kinase inhibitor, suppresses osteoclast formation from review informing the 2016 update of the EULAR recommendations monocytes and synovial cells in RA patients in vitro. The oral for management of rheumatoid arthritis. Ann. Rheum. Dis., 76, administration of JTE-952 reduced the number of osteoclasts 1101–1136 (2017). in the CIA mouse model and suppressed bone destruction. 13) Komano Y, Tanaka M, Nanki T, et al. 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