Inhibition of SYK and cSrc kinases can protect bone and cartilage in preclinical models of osteoarthritis and rheumatoid arthritis

The pathophysiology of osteoarthritis (OA) includes the destruction of subchondral bone tissue and inflammation of the synovium. Thus, an effective disease-modifying treatment should act on both of these pathogenetic components. It is known that cSrc kinase is involved in bone and cartilage remodeling, and SYK kinase is associated with the inflammatory component. Thus the aim of this study was to characterize the mechanism of action and efficacy of a small molecule multikinase inhibitor MT-SYK-03 targeting SYK and cSrc kinases among others in different in vitro and in vivo arthritis models. The selectivity of MT-SYK-03 kinase inhibition was assayed on a panel of 341 kinases. The compound was evaluated in a set of in vitro models of OA and in vivo OA and RA models: surgically-induced arthritis (SIA), monosodium iodoacetate-induced arthritis (MIA), collagen-induced arthritis (CIA), adjuvant-induced arthritis (AIA). MT-SYK-03 inhibited cSrc and SYK with IC50 of 14.2 and 23 nM respectively. Only five kinases were inhibited > 90% at 500 nM of MT-SYK-03. In in vitro OA models MT-SYK-03 reduced hypertrophic changes of chondrocytes, bone resorption, and inhibited SYK-mediated inflammatory signaling. MT-SYK-03 showed preferential distribution to joint and bone tissue (in rats) and revealed disease-modifying activity in vivo by halving the depth of cartilage erosion in rat SIA model, and increasing the pain threshold in rat MIA model. Chondroprotective and antiresorptive effects were shown in a monotherapy regime and in combination with methotrexate (MTX) in murine and rat CIA models; an immune-mediated inflammation in rat AIA model was decreased. The obtained preclinical data support inhibition of cSrc and SYK as a viable strategy for disease-modifying treatment of OA. A Phase 2 clinical study of MT-SYK-03 is to be started.


In vitro experiments. SYK inhibition luminescent assay. Luminescent assay was performed at A. N. Bakh
Institute of Biochemistry (Moscow, Russia). Recombinant 6xHis-SYK kinase (expressed in Sf9 cells) was obtained as described previously 28 . The reaction mixture containing the studied compound MT-SYK-03 or known SYK kinase inhibitor R406 19 as a reference (1 nM-10 μM) and 6xHis-SYK (5 μL, 0.7 pM) was incubated for 30 min, then polyE4Y and ATP (up to 10 μM) were added. After incubation (20 min) Kinase-Glo reagent (Promega) was added. The luminescence was detected using the Fusion Universal Microplate Analyzer (PerkinElmer USA). The obtained IC 50 values are given in Table S1, first column. Dose response curve of inhibitory activity of MT-SYK-03 against SYK kinase is presented in Fig. S1a.
SYK inhibition radiometric assay. Radiometric assay was performed at EMD Millipore Corporation (UK) by measuring the extent of substrate phosphorylation by 6xHis-SYK (expressed in Sf21 cells) using the standard protocol 29 . The reaction mixture containing polyE4Y (0.2 mg/mL), GST-labeled kinase (up to 2 nM), the studied compound (MT-SYK-03 or R406) and 32 P ATP was incubated and spotted onto ion exchange filter; unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. The obtained IC 50 values are given in Table S1.
cSrc inhibition and kinase selectivity profile assay. The activity of MT-SYK-03 against cSrc kinase and selectivity profile towards other 341 kinases was determined at Reaction Biology Corporation (Malvern, PA, USA) using the standard kinase assay protocol. The reaction mixture containing 0.1 mg/mL of substrate polyE4Y (Sigma, USA), 6xHis-SYK (0.1-0.7 pM), the studied compound MT-SYK-03 or reference R406 (500 nM) and 33 P ATP (up to 10 μM, final specific activity 0.225-0.360 μCi) was incubated (120 min) and spotted onto ion exchange filter; unbound phosphate was removed by extensive washing of filters in phosphoric acid. Kinase activity data was expressed as the percent remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions based on the radioactivity of reaction products (see Table S2). Experiment was replicated twice. IC 50 values for kinase listed in Table S3 (having less than 50% residual activity) were measured in 10-dose mode with threefold serial dilution starting at 10 μM and curve fits were obtained using Prism4 Software (GraphPad). Dose response curve of inhibitory activity of MT-SYK-03 against cSrc kinase is presented in Fig. S1b.

In-vitro experiments.
Osteoclast-mediated resorption of the mineralized matrix. The experiment was performed at Atlantic Bone Screen (France) and approved by Atlantic Bone Screen ethics committee. Human CD14 + monocytes were isolated from peripheral blood of healthy volunteers (informed consent was obtained from all subjects) using density gradient centrifugation (Ficoll-Hypaque) and magnetic cell sorting (MACS, MiltenyiBiotec). CD14 + monocytes (1.5 × 10 6 cells/well) were cultured for three days in proliferation medium (αMEM/FCS-10% supplemented with 25 ng/mL M-CSF), inducing proliferation and expression of RANK. Secondly, the cells were differentiated into osteoclasts in differentiation medium supplemented with 100 ng/mL RANKL for 4 days. Osteoclasts were detached from the wells using accutase, the cell suspension was homogenized and cells (75 μL/well) were reseeded into: (a) a 96-well plate coated with a synthetic mineralized matrix for an evaluation of osteoclast resorption, or (b) a classic 96-well plastic culture plate, for an assessment of osteoclast number. 75 μL of differentiation medium containing M-CSF and RANKL (at a 2 × concentration) was added to www.nature.com/scientificreports/ each well. The cells were incubated for an hour and were cultured for 48 h in the fresh differentiation medium supplemented with MT-SYK-03 or vehicle (DMSO, 0.3%). All treatments were carried out in quadruple. Then the cell medium was removed, cells were lysed and the surface of the resorbed areas of the mineralized matrix was quantified in each well. TRAP staining was performed for cells in a classic plastic plate and the number of mature osteoclasts (TRAP-positive cells with three or more nuclei) was determined in each well. Reconstruction of images and osteoclast count was performed using Nikon NIS-D software. The obtained data are presented in Table S4.
Hypertrophic-like changes in IL-1β treated primary chondrocytes. The experiment was performed at Atlantic Bone Screen, France. To isolate primary chondrocytes, Sprague Dawley 3 weeks old rats were euthanized and their hind limbs were collected. Knee cartilage shavings were digested with collagenase in serum-free Dulbecco's Modified Eagle's Medium (DMEM). Once digested, the cell suspension was centrifuged and resuspended in DMEM containing 10% fetal bovine serum (FBS). Then the cells were plated on tissue culture plastic at a density of 10 000 cells•cm −2 and were amplified in monolayer in culture medium (DMEM/FCS-10% supplemented with HEPES (25 mM)) until passage 1 and frozen at − 80 °C. Before study rat's chondrocytes were seeded and cultured in monolayers in 12-well plates for 24 h. After that the cells were treated for three days with IL-1β (10 ng/mL) and MT-SYK-03; positive (no treatment at all), negative (only IL-1β) and vehicle (DMSO, 0.3% in cell medium) controls were also carried out in triplicate. At the end of the treatment, chondrocytes were lysed, and total RNA was purified using the NucleoSpin RNA II kit (Macherey Nagel). Total RNAs (2 μg) were retro-transcribed using M-MLV RT (Lifetechnologies) and obtained cDNAs (10 ng) were used for qPCR to determine steady-state levels of type II collagen and aggrecan mRNAs (levels of RPL19 and β-actin mRNAs were used as controls). Each reaction was set up as follows: 5 μL of iQ SYBR Green Supermix (Biorad, ref 1,708,882), 0.6 μL of forward primer (5 μM), and 0.6 μL of reverse primer (5 μM), 1.8 μL H 2 O, and 2 μL of cDNA. All qPCRs (72 qPCRs per marker) were run on a DNA Engine Thermal Cycler Chromo 4 in triplicates and analyzed using REST software 30 . The obtained data are presented in Table S5.   www.nature.com/scientificreports/ concentration of TNF-α in supernatant was measured fluorometrically using BMS223FF immunoanalytical kit by Bender MedSystems and IC 50 value was computed. Each concentration was tested in triplicate; the experiment was replicated twice. For dose-response curve see Fig. S3.

In vivo experiments. Pharmacokinetics of MT-SYK-03 after single oral administration to rats and rab-
bits. Experiments were performed at ChemPartner, Shanghai, China and approved by Institutional Ethics Committee (IEC) of ChemPartner. Male Wistar 10-12 weeks old rats with body weights of 185-220 g and male New Zealand rabbits with body weights of 2.0-2.3 kg were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. MT-SYK-03 was administered to the three groups of rats (six rats per group) via oral gavage at 10 mL/ kg concentration, and in rabbits 5 mL/kg concentration. Blood samples were kept on ice and centrifuged (2000 g, 4 ℃, 5 min) within 15 min post sampling.
Surgically induced meniscal tear OA in rats. Medial meniscal tear (MMT) model was performed at Pharmenterprises, Russia according to the published procedure 31 and approved by Ethics Committee of Pharmenterprises. Male 4-month old Sprague-Dowley rats were anesthetized with Zoletil (Vibrac, France) and Xylazine (Interchemie werken "De Adelaar" BV, Netherlands) mixture. The medial collateral ligament was transected, and the medial meniscus was grasped with a hemostat and reflected proximally toward the femur. The meniscus was transected and then skin was closed with sutures. 60-90 min after surgery rats were subcutaneously injected with flexoprofen (VIC GROUP, Russia). Treatment groups (20 rats each) were intragastrically treated with MT-SYK-03 (100 and 500 mg/kg) or vehicle control (0.5% methylcellulose solution) 24 h before OA induction and then once daily for 21 day. Active control group was subcutaneously injected with zoledronic acid (0.1 mg/kg) 24 h before OA induction and then once every three days for 21 day. After 21 day animals were sacrificed; surgically operated right knee was removed and used for image analysis.

MT-SYK-03 is a potent and selective cSrc/SYK kinases inhibitor.
MT-SYK-03 inhibited activities of cSrc and SYK kinases potently with IC 50 values of 23-40 nM and 14 nM, correspondingly (Fig. 1). Of the other 341 protein kinases tested, at 500 nM 34 kinases were inhibited by more than 50%, 13 kinases-by more than 80% and only five kinases by more than 90% (DDR1, YES1, MAP3K10, LYN, BLK). Some of these kinases were found to be associated with osteoarthritis. DDR1 is expressed in chondrocytes, and is known to bind to type I collagen 33 ; activates p38, ERK1/2, JNK MAP и PI3-AKT signal pathways 34,35 , as well as the Wnt/β-catenin one 36 . Osteoarthritis of temporomandibular joint was shown to develop spontaneously in Ddr1 −/− mice 37 . DDR1 was also recently shown to be involved in periostin-associated elevated MMP-13 expression via an AKT-Wnt/βcatenin pathway 38 . However, it is questionable that inhibition of Ddr1 kinase alone can have clinical effect on OA, because Ddr1 kinase is a common off-target of many approved kinase inhibitors, particularly approved Bcr-Abl inhibitors (imatinib, dasatinib, nilotinib, ponatinib), for which no data on efficacy in OA was reported. MAP3K10 is known to regulate cytokine expression, support homeostasis and regulatory T-cell functions in rheumatoid arthritis pathogenesis 39 ; involved in Jnk and Jun mediated signaling pathways and has potential implications in osteoporosis pathogenesis 40 . Polymorphisms in BLK are potentially associated with increased risk of rheumatoid arthritis (RA) 41 .
A known SYK kinase inhibitor R406 was found to be less selective in the same assay: it inhibited 104 kinases by > 50%, 50 kinases by > 80% and 26 kinases by > 90%.

MT-SYK-03 effectively suppresses osteoclast-mediated bone resorption and inhibits degradation of aggrecan and type II collagen in chondrocytes.
In the human osteoclast resorption assay MT-SYK-03 dose-dependently inhibited osteoclast resorption, reducing the relative resorbed surface more than threefold at 10 μM (Fig. 2). Anti-hypertrophic effect of MT-SYK-03 was assessed in chondrocytes treated with IL-1β where MT-SYK-03 increased the gene expression levels of aggrecan and type II collagen (Table S5). The increase in aggrecan expression was dose-dependent (Fig. S12). www.nature.com/scientificreports/   (Fig. 3). SYK-kinase mediated inflammation induction can be achieved through B-cell receptor signaling and inflammatory cytokines production via FcγR signaling 42 . To confirm inhibition of SYK-dependent B-cells activation in human whole blood it was stimulated with anti-IgD (anti-BCR) or PMA to induce SYK-dependent or SYKindependent activation of B cells. Thus, MT-SYK-03 inhibited BCR-dependent (but not PMA-dependent) activation of B cells with IC 50 value of 0.550 μM (Fig. S2).
As SYK kinase is also a key participant in FcγR-dependent signaling in monocytes, MT-SYK-03 effect was evaluated in the model of inhibition of cytokines production by differentiated monocytes. A dose-dependent inhibition of TNF-α release with IC 50 value of 0.48 μM (Fig. S3) was shown. This effect of MT-SYK-03 may

MT-SYK-03 pharmacokinetics profile and distribution in rats and rabbits. The pharmacokinetic
parameters after single oral administration of MT-SYK-03 are presented in Table 1 and Figs. 4 and 5. AUC and C max were dose-proportional, T max and t 1/2 were dose-independent. The exposure to MT-SYK-03 was threefold higher in target tissues (bone and cartilage) than in plasma (Fig. 5). In rabbits the compound reaches higher plasma concentrations compared to rats.   www.nature.com/scientificreports/

MT-SYK-03 shows anti-resorptive effects on cartilage in surgically induced meniscal tear OA in rats.
The most commonly used rat medial meniscal tear (MMT) model demonstrates cartilage destruction and dynamic subchondral bone changes. Therefore, the cartilage-and bone-preserving activities of therapeutic agents can be evaluated in this model 45 . The model was carried out for 21 day, before gross joint damage becomes evident 46 : moderate to severe knee OA is already a late stage of the disease with advanced tissue changes that may not be amenable to any drug aimed at modifying the disease course 4 . It was shown that using disease-modifying OA therapy as a benchmark, that a positive result in rat MMT model would be predictive of efficacy in human OA 47 . We compared MT-SYK-03 with zoledronic acid. Joint damage was assessed histologically 3 weeks after surgery. Animals treated with vehicle were characterized by thickening of the joint capsule (fibrosis and hyperplasia of the synovial membrane) and erosion of the articular surfaces, down to the lower part of the cartilage (Fig. 6a). MT-SYK-03 therapy (500 mg/kg) halved lesion depth (from 68.5 ± 6.7% in vehicle group to 35.0 ± 3.0% in treatment group) (Fig. 6b). Treatment with zoledronic acid reduced lesion depth to a lesser extent. Lesion area was also smaller for both MT-SYK-03 and zoledronic acid treatment compared to negative control ( Table 2).

MT-SYK-03 is effective in mouse collagen-induced arthritis model. Collagen-induced arthritis
(CIA) model can be used to study all three components (cartilage and bone destruction as well as inflammation) of arthritis pathogenesis 48 . During the CIA development, paw inflammation was evident within 26 to 27 days after antigen boosting, at which time oral administration of MT-SYK-03 was initiated. MT-SYK-03 exhibited chondroprotective and antiresorptive effectiveness even at low dose (103 mg/kg): microphotographs of knees (Fig. 7) indicate decreasing of cartilage damage, bone resorption and inflammation in knees. These results are consistent with decreased histopathology scores for knee and ankle joints for animal treated with MT-SYK-03. In case of a combination therapy with methotrexate (frequently used as reference 49,50 ) these results were statistically significant (Fig. S16). MT-SYK-03 slightly decreased periosteal bone width of paw and knee joints. This effect was statistically significant when MT-SYK-03 was used in combination with MTX (Fig. S17, Table S18).
As for anti-inflammatory activity, MT-SYK-03 at 103 mg/kg reduced the severity of inflammation (Fig. 8a). The effect was larger when combined with MTX (1.5 mg/kg). Reduction of inflammation was more pronounced if measured by only non-involved paws (Fig. 8b), suggesting the autoimmune contribution.  www.nature.com/scientificreports/

MT-SYK-03 is effective in rat collagen-induced arthritis model.
In the rat CIA model, treatment was initiated on study day 6, 5-6 days before the ankle inflammation began (Fig. S9). As pharmacokinetics studies in rats showed fast metabolism of MT-SYK-03 with τ 1/2 value of 5.08 ± 2.39 h at 200 mg/kg dose (Table 1), higher doses of MT-SYK-03 (compared to murine CIA model) were used in this experiment. Blinded histopathology scores for cartilage damage and bone resorption (Fig. 9, see SI for scoring details) indicated that MT-SYK-03 demonstrated chondroprotective and antiresorptive effects, although statistical significance was reached only for the knee bone resorption. MTX alone did not show statistical significance for any parameter, however its combination with 103 mg/kg of MT-SYK-03 was found to have statistically significant effects on all disease progression parameters, both at knees and ankles (Fig. 9). Measurements of periosteal bone width (Fig. 9c) showed that high-dose MT-SYK-03 (207 mg/kg) and MTX similarly decrease periosteal bone formation by ~ 43%, while combination of the latter with low dose of MT-SYK-03 resulted in stronger decrease of ~ 68%.
Histopathology scores of inflammation and pannus formation were also smaller for MT-SYK-03 (207 mg/kg). For a combination of 103 mg/kg MT-SYK-03 with MTX the changes were statistically significant. As opposed to treatment with MT-SYK-03 or MTX alone, the combination yielded the largest effect (10% diameter reduction) which stayed statistically significant from day 11 up to the end of the study, and resulted in statistically significant 56% paw weight reduction at the study end (Figs. S9, S10). Relative weights of thymus, liver and spleen were statistically identical across all groups (Fig. S11), indicating absence of toxic effects of MT-SYK-03 treatment.
MT-SYK-03 shows anti-inflammatory effect in adjuvant-induced OA in rats. Primary and secondary immunological reactions to the adjuvant injection were assessed by measuring volumes of the involved (CFA-injected) and non-involved paws, respectively, on days 0, 14, 18, 21, 25, and 28. MT-SYK-03 in doses 103 and 207 mg/kg and high-dose MTX decreased the volume of non-involved paws on the day 28, while diclofenac and low-dose MTX showed no statistically significant effect compared to control animals (Table S26). Combinations of MTX with MT-SYK-03 have shown statistically significant reduction of volume of paws on the days 21, 25 and 28.
Considering the treated paws (Table S22), only MTX and its combinations with MT-SYK-03 reached statistically significant reductions of paw volume, but only by day 28. As for toxicological profile, MT-SYK-03 demonstrated the best survival rate (0% rats died) compared to sodium diclofenac (20% rats died) and MTX (20% and 70% rats died after 0.2 and 0.5 mg/kg administration, respectively, Table S23). All rats died from bleeding ulcerative lesions of the stomach and intestines, confirming known side effects of diclofenac and MTX and showing lack of this fall-out for MT-SYK-03.
MT-SYK-03 alleviates pain in MIA-induced OA in rats. The development of pain in OA is correlated with joint destruction but it can also be induced by more subtle changes or biochemical events without structural correlates 46 . Injection of sodium mono-iodoacetate (MIA) into rat joint is referred as a chronic pain model caused by cartilage degeneration 46,51 . The initial period of knee swelling in the MIA model associated with a transient synovial inflammation may correlate with early synovial inflammation in clinical setting that predicts development of OA in the knee 46 . Pain syndrome was assessed using three criteria: hind paws grip strength, mechanical allodynia and mechanical hyperalgesia. MT-SYK-03 in all doses showed trend toward restoration of hind paws grip strength; nalgesin and zoledronic acid, in turn, showed stable statistically significant improvements, but only on day 28 (Table S24). According to mechanical hyperalgesia measurements, prophylactic administration of MT-SYK-03 led to statistically significant increase of pain thresholds for both doses from www.nature.com/scientificreports/ day 14. In the therapeutic treatment regimen, a statistically significant improvement appeared only on day 28 for higher dose of MT-SYK-03. Both zoledronic acid and nalgesin demonstrated positive effects, but they were statistically significant only on days 21 and 28. Thus, prophylactic treatment with MT-SYK-03 has shown the best timing of onset (Table S25). According to mechanical allodynia measurements (Table S27), best efficacy of MT-SYK-03 was reached with prophylactic treatment regimen: on day 14 pain threshold was elevated in all measurements, on days 21 and 28-one and three hours after treatment. In the treatment regimen, positive effects of MT-SYK-03 administration became statistically significant on day 28 and only in the higher dose -500 mg/kg. Zoledronic acid and nalgesin increased the pain threshold, but their therapeutic effects were outplayed by those of MT-SYK-03 in the prophylactic treatment regime.

Discussion
The disease-modifying osteoarthritis drugs (DMOADs) being developed currently target only one of three main components of OA: articular cartilage damage, subchondral bone resorption, or synovium inflammation 4,52,53 .
Despite the evidence of multiple phenotypes of OA, the data on how distinct phenotypes respond to current treatment is lacking 4 . In our study we tested the hypothesis whether selective inhibition of cSrc and SYK kinases by MT-SYK-03 can potentially affect all three pathogenetic components of OA.  www.nature.com/scientificreports/  www.nature.com/scientificreports/ MT-SYK-03 selectivity profile was compared with that of a known SYK kinase inhibitor R406 (tamatinib). The narrow therapeutic window for R406 was responsible for its discontinuation in rheumatoid arthritis 54 . A binding assay to profile MT-SYK-03 against 341 other protein kinases demonstrated that the molecule is highly selective. Complete selectivity toward cSrc is hard to reach due to its similarity to the related kinases 55 , but MT-SYK-03 is far more selective compared to R406 toward some of them (Lyn, Fyn, Hck, PDGFR, Lck, Fgr, Blk, Flt4, Ret, EphA1) and moderately inhibits (> 54% residual activity at 500 nM) the rest (BCR-Abl, c-Kit, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB2, EphB3, EphB4, EGFR, FGFR1-4, TIE2, Brk, Frk). Importantly, MT-SYK-03 did not inhibit FLT1 and KDR kinases inhibition of which was associated with hypertension 56 , diarrhea, nausea and increased transaminase 57 . Also, despite the high PO doses used in in vivo experiments, the average plasma concentration after administration in rats is relatively low and lies in 200-700 nM range thereby indicating the achievement of kinase selectivity in vivo. Interestingly, while plasma PK parameters confirmed achievability of effective concentrations in vivo, the drug's exposure to bone and cartilage tissues was found to be 3 times higher. Taken together with promising preclinical toxicology and a relatively selective kinase inhibition profile, there is a possibility of favorable safety of drug candidate in a clinical setting, which was recently confirmed by Phase 1 clinical study of MT-SYK-03 in healthy volunteers [to be published].
In vitro and in vivo studies were conducted to support cSrc and SYK-mediated biological action of MT-SYK-03 and to check its effectivity in alleviation of OA symptoms: cartilage degradation, bone remodeling, inflammation, and pain. Antiresorptive and chondroprotective effects of MT-SYK-03 were demonstrated in osteoclast-mediated bone resorption and chondrocyte hypertrophic-like changes models in vitro and in surgically induced meniscal tear and collagen-induced arthritis models in vivo. Although it was hypothesized earlier that such effects may be associated with inhibiting other kinases (for example, osteoclastogenesis suppression was explained by MEK1, MAPKAPK2, PI3K or PKA inhibition 12 ), MT-SYK-03 did not inhibit any from this list.
It should also be noted that some of in vivo models used to characterize MT-SYK-03, namely AIA and CIA, are well recognized as rheumatoid arthritis models and were used in this study to study features common for both RA and OA: inflamation 58 , immunity 59,60 , subchondral bone resorption [22][23][24] , and pannus 25,26 .
The role of SYK inhibition in the anti-inflammatory properties of MT-SYK-03 was tested in in vitro and in vivo models. In AIA model MT-SYK-03 was compared with methotrexate and diclofenac. Despite not being approved as an OA therapy, methotrexate efficacy was recently investigated in this indication 61 ; some sources report its case studies in knee OA and erosive OA [62][63][64] . Administration of methotrexate and diclofenac was associated with severe adverse effects including lethality. On the contrary, MT-SYK-03 demonstrated anti-inflammatory activity without such adverse effects. MT-SYK-03 combined with MTX demonstrated efficacy in a setting where MTX alone was not effective 65,66 .
Current results demonstrate that MT-SYK-03 is a kinase inhibitor with a relatively selective profile and cSrc and SYK kinases among the main targets associated with OA. To our knowledge, this is the first compound which acts simultaneously on bone and cartilage metabolism and inflammation in the synovium and demonstrates disease-modifying ability (zoledronic acid that is approved only as an antiresorptive therapy showed low disease-modifying ability 4 ; combined with severe adverse effects when administered intravenously 67 ). The phase 2 clinical study of MT-SYK-03 in patients with painful OA is being planned.