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Objectives To evaluate the long-term safety and efficacy of E6011, a humanized anti-fractalkine monoclonal antibody, in patients with rheumatoid arthritis (RA) with an inadequate response to methotrexate (MTX). Methods Active RA patients with an inadequate response to MTX were randomly assigned to the E6011 or placebo group and received the study drug subcutaneously every 2 weeks during a 24-week double-blind study period. Subjects who completed evaluations at Week 24 were rolled over into the extension phase and received open-label E6011 (200 or 400 mg) every 2 weeks until Week 102. The safety analysis was conducted up to Week 104, and the efficacy analysis was conducted up to Week 84. Results A total of 169 subjects completed the double-blind treatment phase and were rolled over into the extension phase. In total, 167 (98.8%) subjects experienced any adverse events, and the incidence of treatment-related adverse events was 56.2%. The American College of Rheumatology 20 response rates were observed between 40 and 70% during the extension phase. Conclusions E6011 was safe and well tolerated with no notable safety concerns up to 102 weeks in RA patients with an inadequate response to MTX.

Objectives The objective of the study is to evaluate the long-term safety and efficacy of E6011, a humanized anti-fractalkine monoclonal antibody, in patients with rheumatoid arthritis with an inadequate response to biological disease-modifying antirheumatic drugs. Methods In the double-blind treatment phase (24 weeks), placebo or E6011 400 mg was administered until Week 10. Thereafter, E6011 200 mg or 400 mg was administered to Week 22. Subjects who completed the evaluation at Week 24 of the treatment phase were rolled over into the extension phase. The extension phase lasted until Week 104, and all subjects received E6011 400 mg or 200 mg every 2 weeks in an open-label manner until Week 102. Results A total of 47 subjects completed the double-blind treatment phase and were rolled over into the extension phase. In total, 46 (97.9%) subjects experienced any adverse events, and the incidence of treatment-related adverse events was 57.4%. No clear efficacy trend in the American College of Rheumatology 20% response rates was observed. Conclusions E6011 was well tolerated in active rheumatoid arthritis patients who had shown an inadequate response to biologic disease-modifying antirheumatic drugs, but no clear benefit in the American College of Rheumatology 20% response rates was observed. Further studies are needed to clarify the clinical benefit of E6011.

The chemokine fractalkine (FKN) is produced by various cell types, including osteoblasts and endothelial cells in bone tissue, and signals through a sole receptor, CX3CR1, which is expressed on monocytes/macrophages, including osteoclast precursors (OCPs). However, the direct effects of FKN signaling on osteoclast lineage cells under homeostatic noninflammatory conditions remain unclear. Here, we report that FKN regulates mouse OCP survival and primes OCPs for subsequent osteoclast differentiation. Wild-type but not CX3CR1-deficient OCPs grown on immobilized FKN showed enhanced osteoclast formation following receptor activator of NF-κB ligand (RANKL) stimulation, with increased expression of osteoclast differentiation markers. Interestingly, the growth of OCPs on immobilized FKN increased the expression of

To evaluate the safety and efficacy of E6011, a novel humanized anti-fractalkine monoclonal antibody, in patients with active rheumatoid arthritis (RA) with an inadequate response to biological disease-modifying antirheumatic drugs (DMARDs).Active RA patients inadequately responding to biological DMARDs were randomly assigned to placebo or E6011 400-mg group at a 1:1 ratio, and administered E6011 at weeks 0, 1, 2, and subsequently every 2 weeks. Primary endpoint was American College of Rheumatology (ACR)20 response at week 12.Of 64, 33 received placebo, 31 received E6011 400-mg. The ACR20 response rate at week 12 (non-responder imputation) was 27.3% and 22.6% in the placebo and E6011 groups, respectively. ACR50, ACR70 response rates at week 12 were 3.0%, 0% in the placebo and 9.7%, 3.2% in the E6011 group. Exploratory PK exposure analysis revealed that the effect of E6011 tended to be clearer in patients with higher serum trough E6011 concentration. E6011 was well tolerated with no notable safety concerns.E6011 400-mg was well tolerated but had no clear efficacy at week 12 in RA patients with inadequate response to biologics. Further investigations are warranted to determine the optimal clinical dose and evaluation period for E6011.

Rheumatoid arthritis (RA) is an autoimmune disorder that affects joints and is characterized by synovial hyperplasia and bone erosion associated with neovascularization and infiltration of proinflammatory cells. The introduction of biological disease-modifying anti-rheumatic drugs has dramatically changed the treatment of RA over the last 20 years. However, fewer than 50% of RA patients enter remission, and 10-15% are treatment refractory. There is currently no cure for RA. Fractalkine (FKN, also known as CX3CL1) is a cell membrane-bound chemokine that can be induced on activated vascular endothelial cells. FKN has dual functions as a cell adhesion molecule and a chemoattractant. FKN binds specifically to the chemokine receptor CX3CR1, which is selectively expressed on subsets of immune cells such as patrolling monocytes and killer lymphocytes. The FKN-CX3CR1 axis is thought to play important roles in the initiation of the inflammatory cascade and can contribute to exacerbation of tissue injury in inflammatory diseases. Accordingly, studies in animal models have shown that inhibition of the FKN-CX3CR1 axis not only improves rheumatic diseases but also reduces associated complications, such as pulmonary fibrosis and cardiovascular disease. Recently, a humanized anti-FKN monoclonal antibody, E6011, showed promising efficacy with a dose-dependent clinical response and favorable safety profile in a Phase 2 clinical trial in patients with RA (NCT02960438). Taken together, the preclinical and clinical results suggest that E6011 may represent a new therapeutic approach for rheumatic diseases by suppressing a major contributor to inflammation and mitigating concomitant cardiovascular and fibrotic diseases. In this review, we describe the role of the FKN-CX3CR1 axis in rheumatic diseases and the therapeutic potential of anti-FKN monoclonal antibodies to fulfill unmet clinical needs.

Objective To elucidate the role of the fractalkine (FKN)/CX3 CR1 pathway in joint destruction in rheumatoid arthritis. Methods We examined the effect of treatment with anti-mouse FKN (anti-mFKN) monoclonal antibody (mAb) on joint destruction and the migration of osteoclast precursors (OCPs) into the joint, using the collagen-induced arthritis (CIA) model. DBA/1 mice were immunized with bovine type II collagen to induce arthritis, and then treated with anti-mFKN mAb. Disease severity was monitored by arthritis score, and joint destruction was evaluated by soft x-ray and histologic analyses. Plasma levels of joint destruction markers were assessed by enzyme-linked immunosorbent assay. FKN expression on endothelial cells was detected by immunohistochemistry. Bone marrow-derived OCPs were labeled with fluorescein and transferred to mice with CIA, and the migration of the OCPs to the joints was then analyzed. Results Both prophylactic and therapeutic treatment with anti-mFKN mAb significantly decreased the arthritis and soft x-ray scores. Plasma levels of cartilage oligomeric matrix protein and matrix metalloproteinase 3 decreased after treatment with anti-mFKN mAb. Histologic analysis revealed that anti-mFKN mAb inhibited synovitis, pannus formation, and cartilage destruction, as well as suppressed bone damage, with a marked reduction in the number of tartrate-resistant acid phosphatase-positive osteoclasts. Anti-mFKN mAb strongly inhibited the migration of bone marrow-derived OCPs into the affected synovium. Conclusion Anti-mFKN mAb notably ameliorates arthritis and joint destruction in the CIA model, as well as inhibits migration of OCPs into the synovium. These results suggest that inhibition of the FKN/CX3 CR1 pathway could be a novel strategy for treatment of both synovitis and joint destruction in rheumatoid arthritis.

Chemokine systems modulate inflammatory and immune responses in inflammatory bowel disease (IBD). The colons of IBD patients show increased levels of fractalkine (FKN) and high numbers of FKN receptor-positive (CX3CR1+) cells; however, the FKN–CX3CR1 axis’s role in intestinal inflammation, especially in intravascular leukocyte behaviors, still remains unclear. Here, we show that interruption of the FKN–CX3CR1 axis by anti-FKN monoclonal antibody (mAb) ameliorates murine colitis through regulation of intravascular monocyte behaviors in murine colitis models. FKN expression was detectable in vascular endothelium and CX3CR1+ macrophages accumulated in the mucosal lamina propria and submucosa of the inflamed colons. CD115+ monocytes tethered to the venous endothelium and expressed pro-inflammatory mediators. The anti-FKN mAb improved colitis symptoms, markedly reduced pro-inflammatory factors in the colon, maintained blood vessel integrity and reduced tethered monocytes in the inflamed veins. Intravital imaging revealed that CD115+Gr-1low/− monocytes crawled on the apical surfaces of venous endothelium, and anti-FKN mAb rapidly dislodged the crawling monocytes and inhibited their patrolling behavior. These findings suggest that the FKN–CX3CR1 axis triggers the patrolling behavior of crawling monocytes on the venous endothelium of inflamed colons, and accelerates the subsequent leukocyte activation and infiltration by locally producing inflammatory cytokines and chemokines. The mAb also ameliorated symptoms in another IBD model, T-cell-transferred colitis. Blocking the FKN–CX3CR1 axis with an anti-FKN mAb considerably inhibits the colitis-triggered inflammatory cascades, which may be an alternative strategy to treat IBD.

Rheumatoid arthritis (RA) is an autoimmune disorder that affects joints and is characterized by synovial hyperplasia and bone erosion associated with neovascularization and infiltration of proinflammatory cells. The introduction of biological disease-modifying anti-rheumatic drugs has dramatically changed the treatment of RA over the last 20 years. However, fewer than 50% of RA patients enter remission, and 10–15% are treatment refractory. There is currently no cure for RA. Fractalkine (FKN, also known as CX3CL1) is a cell membrane-bound chemokine that can be induced on activated vascular endothelial cells. FKN has dual functions as a cell adhesion molecule and a chemoattractant. FKN binds specifically to the chemokine receptor CX3CR1, which is selectively expressed on subsets of immune cells such as patrolling monocytes and killer lymphocytes. The FKN–CX3CR1 axis is thought to play important roles in the initiation of the inflammatory cascade and can contribute to exacerbation of tissue injury in inflammatory diseases. Accordingly, studies in animal models have shown that inhibition of the FKN–CX3CR1 axis not only improves rheumatic diseases but also reduces associated complications, such as pulmonary fibrosis and cardiovascular disease. Recently, a humanized anti-FKN monoclonal antibody, E6011, showed promising efficacy with a dose-dependent clinical response and favorable safety profile in a Phase 2 clinical trial in patients with RA (NCT02960438). Taken together, the preclinical and clinical results suggest that E6011 may represent a new therapeutic approach for rheumatic diseases by suppressing a major contributor to inflammation and mitigating concomitant cardiovascular and fibrotic diseases. In this review, we describe the role of the FKN–CX3CR1 axis in rheumatic diseases and the therapeutic potential of anti-FKN monoclonal antibodies to fulfill unmet clinical needs.

Objective To evaluate the efficacy and safety of E6011, a humanized IgG2 monoclonal antibody against human fractalkine (FKN), in a phase II, double-blind, placebo-controlled study in rheumatoid arthritis (RA) patients. Methods Patients with moderate-to-severe RA who had an inadequate response to methotrexate were randomly assigned to a placebo group or to E6011 100-mg, 200-mg, or 400/200-mg groups at a 2:1:2:2 ratio. During the 24-week period, patients received the study drug subcutaneously at weeks 0, 1, and 2 and then once every 2 weeks. The primary end point was the American College of Rheumatology 20% improvement criteria (ACR20) response rate at week 12. Results Study drugs were administered to 190 patients (placebo, n = 54; E6011 100 mg, n = 28; E6011 200 mg, n = 54; E6011 400/200 mg, n = 54), and 169 patients completed treatment. A significant difference from placebo was not found in ACR20 response rates at week 12 (37.0% [placebo], 39.3% [100 mg], 48.1% [200 mg], and 46.3% [400/200 mg], using nonresponder imputation). As a secondary end point, ACR20 response rate in the 200-mg and 400/200-mg groups attained statistical significance at week 24 (35.2% [placebo], 39.3% [100 mg], 53.7% [200 mg], and 57.4% [400/200 mg]). Subsequent exploratory subgroup analysis revealed greater efficacy of E6011, particularly in patients with a higher baseline proportion of CD16+ monocytes; ACR20 response rates in this patient subgroup at week 24 were 30.0% (placebo), 46.7% (100 mg), 57.7% (200 mg), and 69.6% (400/200 mg). E6011 administered for 24 weeks was well tolerated. Conclusion This is the first evidence that E6011, a novel cell trafficking inhibitor targeting the FKN-CX3 CR1 interaction, is modestly effective with 24 weeks of treatment in RA patients, although the primary end point was not met.

Fractalkine (CX3CL1/FKN) is a chemokine that regulates chemotaxis and adhesion of CX3C chemokine receptor 1 (CX3CR1)-expressing inflammatory cells. We conducted the first phase 1/2, open-label, multiple ascending dose study of E6011, a humanized anti-FKN monoclonal antibody, in Japanese rheumatoid arthritis (RA) patients (clinicaltrial.gov identifier: NCT02196558).Active RA patients with an inadequate response or intolerance to methotrexate or tumor necrosis factor (TNF) inhibitor received E6011 at week 0, 1, 2, and thereafter every 2 weeks for 12 weeks.Twelve, 15, and 10 subjects were enrolled in the 100, 200, and 400 mg cohorts, respectively. No severe adverse events (AEs) or deaths occurred, and no major differences were observed in the incidence or severity of AEs across the cohorts. Serum E6011 concentrations increased dose dependently. American College of Rheumatology (ACR) 20, 50, and 70 responses at week 12 were 75.0%, 33.3%, and 8.3% in the 100 mg cohort; 66.7%, 20.0%, and 13.3% in the 200 mg cohort; and 60.0%, 30.0%, and 20.0% in the 400 mg cohort, respectively.E6011 appeared to be safe and well tolerated in RA patients during this 12-week treatment period, suggesting that E6011 has an effective clinical response in active RA patients.

Background In the Phase 1/2 clinical study, E6011, a novel humanized anti-fractalkine (FKN) mAb demonstrated a promising efficacy in active RA patients who were inadequately controlled by MTX and/or TNF-α inhibitors (NCT 02196558)1.In RA joint tissue, increased expression of fractalkine (FKN) and abundant infiltration of CX3CR1-positive cells were observed2. FKN is expressed on endothelial cells and fibroblast-like synoviocytes in synovium and also expressed on osteoblasts. CX3CR1 is expressed on monocytes/macrophages and osteoclast precursors (OCPs). Therefore, FKN-CX3CR1 interaction could play pivotal roles in migration, differentiation and activation of those cells. However, the precise mechanism(s) of FKN-CX3CR1 axis in RA, especially on joint destruction remains to be elucidated. Objectives To elucidate the roles of FKN-CX3CR1 axis in cartilage destruction, bone damage and proliferated synovial cells by using anti-mouse FKN mAb (anti-mFKN mAb). Methods For the induction of CIA, mice were immunized with bovine type II collagen. Anti-mFKN mAb was injected twice a week. The clinical arthritis score was monitored, and joint destruction was evaluated by soft x-ray and histology. The mRNA expression levels were assessed by quantitative RT-PCR. Blood parameters were measured using ELISA. In in vitro, effect of immobilized FKN on RANK ligand (RANKL)-induced osteoclast differentiation was examined. In in vivo, bone marrow-derived OCPs were fluorescein-labeled and transferred to CIA mice to evaluate the migration of OCPs into synovium. Inhibitory effect of anti-mFKN mAb, etanercept or tofacitinib against OCP migration was assessed. To examine the effect of anti-mFKN mAb against proliferated synovial cells, propidium iodide (PI) was injected to anti-mFKN mAb-treated CIA mice to detect the synovial cell death. Results Anti-mFKN mAb significantly reduced the arthritis and soft x-ray scores in both prophylactic and therapeutic treatment. Anti-mFKN mAb histologically improved synovitis, cartilage destruction and bone damage, with marked reduction of osteoclast numbers. Plasma levels of COMP and MMP-3 were also decreased. Interestingly, anti-mFKN mAb significantly suppressed Tnf and Il6 mRNA expression in the affected joints. In in vitro, RANKL-induced osteoclast differentiation was enhanced by immobilized FKN, and anti-mFKN mAb suppressed FKN-dependent osteoclast formation. In in vivo, anti-mFKN mAb strongly inhibited the migration of OCPs into the proliferated synovial cells of mice with CIA, whereas etanercept or tofacitinib had no significantly effect. Importantly, synovial cell death was abundantly found in proliferated synovial cells of mice with CIA after the anti-mFKN mAb treatment3. Conclusion Anti-mFKN mAb remarkably ameliorated the joint destruction with the marked reduction of osteoclasts by the inhibition of both OCP survival and OCP migration in inflamed joint tissues. In addition, anti-mFKN mAb immediately induced synovial cell death in the proliferated synovial cells, suggesting the direct inhibitory effect in the synovitis. These results strongly indicate that inhibition of FKN-CX3CR1 axis by a humanized anti-FKN mAb, E6011, is an attractive and affected joints-selective therapeutic strategy for the treatment of both inflammatory synovitis, cartilage destruction and bone damage in RA patients. References [1] Tanaka Y, et al., Mod Rheumatol (2018) 28, 58-65 [2] Nanki T, et al., Arthritis Rheum (2002) 46, 2878-83 [3] Hoshino K, et al., Arthritis Rheumatol (2018) Aug 6 [Epub ahead of print] Disclosure of Interests NAOTO ISHII Shareholder of: Eisai Co., Ltd., Employee of: KAN Research Institute, Inc. (subsidiary of Eisai Co., Ltd.), Kana Hoshino-Negishi Shareholder of: Eisai Co., Ltd., Employee of: KAN Research Institute, Inc. (subsidiary of Eisai Co., Ltd.), Masayoshi Ohkuro Employee of: EA Pharma Co., Ltd., Tomoya Nakatani Employee of: KAN Research Institute, Inc. (subsidiary of Eisai Co., Ltd.), Wataru Ikeda Shareholder of: Eisai Co., Ltd., Employee of: KAN Research Institute, Inc. (subsidiary of Eisai Co., Ltd.), Yoshikazu Kuboi Shareholder of: Eisai Co., Ltd., Employee of: KAN Research Institute, Inc. (subsidiary of Eisai Co., Ltd.), Nobuyuki Yasuda Shareholder of: Eisai Co., Ltd., Employee of: KAN Research Institute, Inc. (subsidiary of Eisai Co., Ltd.), Toshio Imai Shareholder of: Eisai Co., Ltd., Employee of: KAN Research Institute, Inc. (subsidiary of Eisai Co., Ltd.)

Background Fractalkine (CX3CL1, designated as FKN hereafter) is the sole member of the CX3C-chemokine which leads to dual actions, chemotaxis and cell adhesion for leukocytes expressing the cognate receptor, CX3CR1, during their migration. We have conducted clinical trials of E6011, a novel humanized anti-FKN monoclonal antibody, for patients with rheumatoid arthritis (RA) in Japan1. This is the first report of efficacy and safety results of E6011 from a Phase 2, multicenter, randomized, double-blind, placebo-controlled, parallel-group comparison study in RA patients inadequately responding to methotrexate (NCT02960438). Objectives To evaluate efficacy and safety of three dose of E6011 compared with placebo. Methods During the 24-week double-blind period, patients with moderately to severely active RA of inadequate response to MTX were randomly assigned to E6011 100 mg, 200 mg, 400/200 mg, or placebo groups at a 1:2:2:2 ratio. In the E6011 100 mg, 200 mg, and placebo groups, subjects received 100 mg, 200 mg, or placebo at Weeks 0, 1, 2, and every 2 weeks subsequently. In the E6011 400/200 mg group, subjects received 400 mg at Weeks 0, 1, 2, 4, 6, 8, 10 and then 200 mg every 2 weeks subsequently. Results A total of 190 subjects (54 in the placebo group, 28 in the 100 mg group, 54 in the 200 mg group, 54 in the 400/200 mg group) received study drug. Of the 190 subjects, 169 completed and 21 discontinued study treatment prematurely during the 24-week double-blind period. The ACR20 response rate at Week 12 (non-responder imputation), the primary endpoint, was 37.0% in the placebo group, 39.3% in the 100 mg group, 48.1% in the 200 mg group, and 46.3% in the 400/200 mg group (not statistically significant), and statistically significant difference from placebo in ACR20 response rate was found in the 200 mg and 400/200 mg groups at Week 24 (35.2% for placebo, 39.3% for 100 mg, 53.7% for 200 mg, 57.4% for 400/200 mg). In addition, we focused on CD16+ monocytes which highly expressing FKN receptor/CX3CR1 as a blood biomarker that linked to the clinical response to E6011. The whole patient population was divided into 2 groups according to the median value of baseline CD16+ monocyte percentage (Median: 10.35%). Much clearer ACR20 response was observed in a dose dependent manner in the subjects who showed higher baseline CD16+ monocytes over the median at Week 24 (NRI) (30.0% for placebo, 46.7% for 100 mg, 57.7% for 200 mg, and 69.6% for 400/200 mg) although such fashion was obscure in the subjects below the median value. Adverse events that occurred in ≥5% of subjects in any E6011 group were nasopharyngitis, upper respiratory tract infection, stomatitis, bronchitis, back pain, pharyngitis, and dental caries. As a results, E6011 was well tolerated with no notable safety concerns at doses of 100, 200, and 400/200 mg when administered subcutaneously for 24 weeks. Conclusion E6011 provided clinical improvements with a good safety profile in RA patients with inadequately responding to MTX. Especially, a higher efficacy of E6011 was suggested in patients with higher baseline CD16+ monocytes (%). This is the world-first evidence suggesting that a novel approach to target FKN/CX3CR1 interaction could be beneficial for RA. References [1] Tanaka Y, et al., Mod Rheumatol (2018) 28, 58-65 Acknowledgement The authors wish to thank the study investigators. Disclosure of Interests Yoshiya Tanaka Grant/research support from: Abbvie, Astellas, Bristol-Myers Squibb, Chugai, Daiichi-Sankyo, Eisai, Mitsubishi-Tanabe, MSD, Ono, Taisho-Toyama, Takeda, Speakers bureau: Abbvie, Asahi-kasei, Astellas, Bristol-Myers Squibb, Chugai, Daiichi-Sankyo, Eli Lilly, Eisai, Glaxo-Smithkline, Janssen, Mitsubishi-Tanabe, Novartis, Pfizer Japan Inc, Sanofi, Takeda, UCB, YL Biologics, Tsutomu Takeuchi Grant/research support from: Astellas Pharma Inc, Chugai Pharmaceutical Co, Ltd., Daiichi Sankyo Co., Ltd., Takeda Pharmaceutical Co., Ltd., AbbVie GK, Asahikasei Pharma Corp., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Eisai Co., Ltd., AYUMI Pharmaceutical Corporation, Nipponkayaku Co. Ltd., Novartis Pharma K.K., Grant/research support from: AbbVie, Asahi Kasei, Astellas, AstraZeneca, AYUMI, Bristol-Myers Squibb, Chugai, Daiichi Sankyo, Eisai, Eli Lilly Japan, Janssen, Mitsubishi Tanabe, Nippon Kayaku, Novartis, Pfizer Japan Inc, Taiho, Taisho Toyama, Takeda, Teijin, Grant/research support from: Astellas Pharma Inc., Bristol Myers Squibb, Chugai Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Santen Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Teijin Pharma Ltd., AbbVie GK, Asahi Kasei Pharma Corp., Taisho Toyama Pharmaceutical Co., Ltd., SymBio Pharmaceuticals Ltd., Janssen Pharmaceutical K.K., Celltrion Inc., Nipponkayaku Co. Ltd., and UCB Japan, Consultant for: Astra Zeneca K.K., Eli Lilly Japan K.K., Novartis Pharma K.K., Mitsubishi Tanabe Pharma Co., Abbivie GK, Nipponkayaku Co.Ltd, Janssen Pharmaceutical K.K., Astellas Pharma Inc., Taiho Pharmaceutical Co. Ltd., Chugai Pharmaceutical Co. Ltd., Taisho Toyama Pharmaceutical Co. Ltd., GlaxoSmithKline K.K., UCB Japan Co. Ltd., Consultant for: AbbVie, Asahi Kasei, Astellas, AstraZeneca, AYUMI, Bristol-Myers Squibb, Chugai, Daiichi Sankyo, Eisai, Eli Lilly Japan, Janssen, Mitsubishi Tanabe, Nippon Kayaku, Novartis, Pfizer Japan Inc, Taiho, Taisho Toyama, Takeda, Teijin, Consultant for: Astra Zeneca K.K., Eli Lilly Japan K.K., Novartis Pharma K.K., Mitsubishi Tanabe Pharma Co., Asahi Kasei Medical K.K., AbbVie GK, Daiichi Sankyo Co., Ltd., Bristol Myers Squibb, and Nipponkayaku Co. Ltd., Speakers bureau: Astellas Pharma Inc., Bristol Myers Squibb, Chugai Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Santen Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Teijin Pharma Ltd., AbbVie GK, Asahi Kasei Pharma Corp., Taisho Toyama Pharmaceutical Co., Ltd., SymBio Pharmaceuticals Ltd., Janssen Pharmaceutical K.K., Celltrion Inc., Nipponkayaku Co. Ltd., and UCB Japan, Speakers bureau: AbbVie, Asahi Kasei, Astellas, AstraZeneca, AYUMI, Bristol-Myers Squibb, Chugai, Daiichi Sankyo, Eisai, Eli Lilly Japan, Janssen, Mitsubishi Tanabe, Nippon Kayaku, Novartis, Pfizer Japan Inc, Taiho, Taisho Toyama, Takeda, Teijin, Speakers bureau: AbbVie GK., Bristol–Myers K.K., Chugai Pharmaceutical Co. Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Astellas Pharma Inc, Diaichi Sankyo Co. Ltd., Eisai Co. Ltd., Sanofi K.K., Teijin Pharma Ltd., Takeda Pharmaceutical Co. Ltd., Novartis Pharma K.K., Hisashi Yamanaka Grant/research support from: AbbVie, Eisai, Bristol-Meyers, Novartis, Behringer, Astellas, Kaken, Nippon-Shinyaku, Pfizer, UCB, Ayumi, Ono, Daiichi-Sankyo, Taisyo-Toyama, Takeda, Tanabe-Mitsubishi, Chugai, Teijin Pharma, Torii, YLbio, Speakers bureau: Bristol-Meyers, Astellas, Pfizer, Daiichi-Sankyo, Takeda, Tanabe-Mitsubishi, Chugai, Teijin Pharma, YLbio, Toshihiro Nanki Grant/research support from: Chugai, Eisai, Takeda, Teijin, Eli Lilly, Bristol-Myers, AbbVie, Ono, Novartis, Asahikasei, Mitsubishi-Tanabe, Astellas, Ayumi, Pfizer, Daiichi Sankyo, Shionogi, Sanofi, Nippon Kayaku, Yutoku, Actelion, UCB, Bayer, Nihon Pharmaceutical., Consultant for: UCB, Eisai, Chugai, Ono, Gilead., Speakers bureau: Mitsubishi-Tanabe, Chugai, Eisai, Takeda, Astellas, Janssen, Eli Lilly, Ayumi, Pfizer, Asahikasei, Sanofi, Daiichi Sankyo, Otsuka, AbbVie, Ono, Teijin, Nippon Kayaku, UCB., Hisanori Umehara: None declared, Nobuyuki Yasuda Shareholder of: Eisai Co., Ltd., Employee of: KAN Research Institute, Inc. (subsidiary of Eisai Co., Ltd.), Fumitoshi Tago Employee of: Eisai Co., Ltd., Yasumi Kitahara Shareholder of: Eisai Co., Ltd., Employee of: Eisai Co., Ltd., Makoto Kawakubo Shareholder of: Eisai Co., Ltd., Employee of: Eisai Co., Ltd., Kentaro Torii Shareholder of: Eisai Co., Ltd., Employee of: Eisai Co., Ltd., Seiichiro Hojo Employee of: Eisai Co., Ltd., Tetsu Kawano Employee of: KAN Research Institute, Inc. (subsidiary of Eisai Co., Ltd.), Toshio Imai Shareholder of: Eisai Co., Ltd., Employee of: KAN Research Institute, Inc. (subsidiary of Eisai Co., Ltd.)

Background Fractalkine (CX3CL1, designated as FKN hereafter) is the sole member of the CX3C-chemokine which leads to dual actions, chemotaxis and cell adhesion for leukocytes expressing the cognate receptor, CX3CR1, during their migration. We have conducted clinical trials of E6011, a novel humanized anti-FKN monoclonal antibody, for patients with rheumatoid arthritis (RA) in Japan1. This is the first report of a Phase 2, multicenter, randomized, double-blind, placebo-controlled, parallel-group comparison study of E6011 in RA patients inadequately responding to biologics (NCT02960490). Objectives To evaluate efficacy and safety of E6011 compared with placebo. Methods During the 24-week double-blind period, patients with moderately to severely active RA of inadequate response to biologics were randomly assigned to E6011 400 mg or placebo groups at a 1:1 ratio. Patients who continued the study beyond Week 12 were further allocated to E6011 200 mg or 400 mg at a 1:1 ratio within the initially assigned group. Patients received either E6011 400 mg or placebo at Weeks 0, 1, 2, and every 2 weeks subsequently until Week 10 and then E6011 200 mg or 400 mg every 2 weeks between Weeks 12 and 22 in a double-blind manner. This abstract reports the results from the first 12-week placebo controlled period. Results A total of 64 subjects (33 in the placebo group and 31 in the E6011 400 mg group) received study drug. Of the 64 subjects, 55 completed and 9 discontinued study treatment prematurely during the 12-week placebo controlled double-blind period. The ACR20 response rate at Week 12 (non-responder imputation), the primary endpoint, was 27.3% (9/33 subjects) in the placebo group and 22.6% (7/31 subjects) in the E6011 group. ACR50 and ACR70 response rate at Week 12 were 3.0%, 0% in the placebo group and 9.7%, 3.2% in the E6011 group, respectively. Numerically greater improvement from baseline was found for some parameters of the ACR components in the E6011 group, however, no statistically significant differences were found in any of the ACR components between the placebo and E6011 groups. From the exploratory PK exposure analysis, there was a tendency that the effect of E6011 became clearer in the subjects who achieved higher serum trough E6011 concentration. Adverse events that occurred in at least 2 subjects in the E6011 group were stomatitis, injection site erythema, nasopharyngitis, and blood creatine phosphokinase increased. As a results, E6011 was well tolerated with no notable safety concerns at doses of 400 mg when administered subcutaneously for 12 weeks. Conclusion E6011 400 mg was well tolerated but did not show clear efficacy at Week 12 compared with placebo in RA patients with inadequately responding to biologics. Further investigation to seek an optimal clinical dose and evaluation period of E6011 are warranted. Reference [1] Tanaka Y, et al., Mod Rheumatol (2018) 28, 58-65 Acknowledgement The authors wish to thank the study investigators. Disclosure of Interests Yoshiya Tanaka Grant/research support from: Abbvie, Astellas, Bristol-Myers Squibb, Chugai, Daiichi-Sankyo, Eisai, Mitsubishi-Tanabe, MSD, Ono, Taisho-Toyama, Takeda, Speakers bureau: Abbvie, Asahi-kasei, Astellas, Bristol-Myers Squibb, Chugai, Daiichi-Sankyo, Eli Lilly, Eisai, Glaxo-Smithkline, Janssen, Mitsubishi-Tanabe, Novartis, Pfizer Japan Inc, Sanofi, Takeda, UCB, YL Biologics, Tsutomu Takeuchi Grant/research support from: Astellas Pharma Inc, Chugai Pharmaceutical Co, Ltd., Daiichi Sankyo Co., Ltd., Takeda Pharmaceutical Co., Ltd., AbbVie GK, Asahikasei Pharma Corp., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Eisai Co., Ltd., AYUMI Pharmaceutical Corporation, Nipponkayaku Co. Ltd., Novartis Pharma K.K., Grant/research support from: AbbVie, Asahi Kasei, Astellas, AstraZeneca, AYUMI, Bristol-Myers Squibb, Chugai, Daiichi Sankyo, Eisai, Eli Lilly Japan, Janssen, Mitsubishi Tanabe, Nippon Kayaku, Novartis, Pfizer Japan Inc, Taiho, Taisho Toyama, Takeda, Teijin, Grant/research support from: Astellas Pharma Inc., Bristol Myers Squibb, Chugai Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Santen Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Teijin Pharma Ltd., AbbVie GK, Asahi Kasei Pharma Corp., Taisho Toyama Pharmaceutical Co., Ltd., SymBio Pharmaceuticals Ltd., Janssen Pharmaceutical K.K., Celltrion Inc., Nipponkayaku Co. Ltd., and UCB Japan, Consultant for: Astra Zeneca K.K., Eli Lilly Japan K.K., Novartis Pharma K.K., Mitsubishi Tanabe Pharma Co., Abbivie GK, Nipponkayaku Co.Ltd, Janssen Pharmaceutical K.K., Astellas Pharma Inc., Taiho Pharmaceutical Co. Ltd., Chugai Pharmaceutical Co. Ltd., Taisho Toyama Pharmaceutical Co. Ltd., GlaxoSmithKline K.K., UCB Japan Co. Ltd., Consultant for: AbbVie, Asahi Kasei, Astellas, AstraZeneca, AYUMI, Bristol-Myers Squibb, Chugai, Daiichi Sankyo, Eisai, Eli Lilly Japan, Janssen, Mitsubishi Tanabe, Nippon Kayaku, Novartis, Pfizer Japan Inc, Taiho, Taisho Toyama, Takeda, Teijin, Consultant for: Astra Zeneca K.K., Eli Lilly Japan K.K., Novartis Pharma K.K., Mitsubishi Tanabe Pharma Co., Asahi Kasei Medical K.K., AbbVie GK, Daiichi Sankyo Co., Ltd., Bristol Myers Squibb, and Nipponkayaku Co. Ltd., Speakers bureau: Astellas Pharma Inc., Bristol Myers Squibb, Chugai Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Santen Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Teijin Pharma Ltd., AbbVie GK, Asahi Kasei Pharma Corp., Taisho Toyama Pharmaceutical Co., Ltd., SymBio Pharmaceuticals Ltd., Janssen Pharmaceutical K.K., Celltrion Inc., Nipponkayaku Co. Ltd., and UCB Japan, Speakers bureau: AbbVie, Asahi Kasei, Astellas, AstraZeneca, AYUMI, Bristol-Myers Squibb, Chugai, Daiichi Sankyo, Eisai, Eli Lilly Japan, Janssen, Mitsubishi Tanabe, Nippon Kayaku, Novartis, Pfizer Japan Inc, Taiho, Taisho Toyama, Takeda, Teijin, Speakers bureau: AbbVie GK., Bristol–Myers K.K., Chugai Pharmaceutical Co. Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Astellas Pharma Inc, Diaichi Sankyo Co. Ltd., Eisai Co. Ltd., Sanofi K.K., Teijin Pharma Ltd., Takeda Pharmaceutical Co. Ltd., Novartis Pharma K.K., Hisashi Yamanaka Grant/research support from: AbbVie, Eisai, Bristol-Meyers, Novartis, Behringer, Astellas, Kaken, Nippon-Shinyaku, Pfizer, UCB, Ayumi, Ono, Daiichi-Sankyo, Taisyo-Toyama, Takeda, Tanabe-Mitsubishi, Chugai, Teijin Pharma, Torii, YLbio, Speakers bureau: Bristol-Meyers, Astellas, Pfizer, Daiichi-Sankyo, Takeda, Tanabe-Mitsubishi, Chugai, Teijin Pharma, YLbio, Toshihiro Nanki Grant/research support from: Chugai, Eisai, Takeda, Teijin, Eli Lilly, Bristol-Myers, AbbVie, Ono, Novartis, Asahikasei, Mitsubishi-Tanabe, Astellas, Ayumi, Pfizer, Daiichi Sankyo, Shionogi, Sanofi, Nippon Kayaku, Yutoku, Actelion, UCB, Bayer, Nihon Pharmaceutical., Consultant for: UCB, Eisai, Chugai, Ono, Gilead., Speakers bureau: Mitsubishi-Tanabe, Chugai, Eisai, Takeda, Astellas, Janssen, Eli Lilly, Ayumi, Pfizer, Asahikasei, Sanofi, Daiichi Sankyo, Otsuka, AbbVie, Ono, Teijin, Nippon Kayaku, UCB., Hisanori Umehara: None declared, Nobuyuki Yasuda Shareholder of: Eisai Co., Ltd., Employee of: KAN Research Institute, Inc. (subsidiary of Eisai Co., Ltd.), Fumitoshi Tago Employee of: Eisai Co., Ltd., Yasumi Kitahara Shareholder of: Eisai Co., Ltd., Employee of: Eisai Co., Ltd., Makoto Kawakubo Shareholder of: Eisai Co., Ltd., Employee of: Eisai Co., Ltd., Hayato Hisaki Shareholder of: Eisai Co., Ltd., Employee of: Eisai Co., Ltd., Seiichiro Hojo Employee of: Eisai Co., Ltd., Tetsu Kawano Employee of: KAN Research Institute, Inc. (subsidiary of Eisai Co., Ltd.), Toshio Imai Shareholder of: Eisai Co., Ltd., Employee of: KAN Research Institute, Inc. (subsidiary of Eisai Co., Ltd.)

Considering current paradigm shifts of comparative jurisprudence from narrow “legal” view to wider “social/political” concerns, this paper proposes two methodological frameworks for comparative legal research from Asian perspectives. First is the idea of “three types of laws” (indigenous, modern, and developmental), “three (legal) principles” (community, market, and command), and “three societal dimensions” (social, economic, and political), which aims at rather static understanding of “law.” Further, in order to understand dynamic socio-legal developments within region, it conceptualizes “two dynamic forces” (market and community), to show the historical development of law and societies, and examines major legal problems under the ongoing current globalization. Second, the paper creates the “three-layered understanding of law”; “law as rule,” “law as institution,” and “law as culture,” which construct the cycling structure of the national legal system. Legal systems function successfully only when “law” cycles these three phases smoothly and inclusively. Finally, discussing how the globalization impacts on the national legal systems, it suggests that “law” would change its nature from “legal or formal” to more “social and political or substantial/reflexive,” illustrating two examples of “transnational law,” on the regulations of transnational corporations and the autonomous rights of indigenous/local people.

Background:Fractalkine (FKN) and its solo receptor CX3CR1 are deeply involved in the pathogenesis of rheumatoid arthritis (RA). FKN is expressed on vascular endothelium, while CX3CR1 is expressed on peripheral blood leukocytes such as monocytes, macrophages, NK cells, effector CD8+T cells and a minor fraction of CD4+T cells. E6011, a novel humanized anti-FKN monoclonal antibody (mAb), is under clinical development in RA.Objectives:In order to continuously assess the E6011 pharmacodynamics by monitoring the alteration of peripheral blood immune cells, including CX3CR1-expressed cell populations, a series of multi-color flow cytometry (FCM) was conducted before and during the course of the E6011 treatment of active RA patients in phase 2 clinical trial.Methods:Immuno-phenotypic changes were explored by FCM during the E6011 administration in 190 Japanese RA patients with inadequate response to MTX (NCT02960438). Patient’s peripheral blood were drawn into fixative tube (Cyto-Chex® BCT, Streck) at each clinics and thereafter transported to the FCM facility at KAN Research Institute, Inc. within 30 hours after the blood collection to operate the FCM analysis by standardized method. Immuno-phenotyping was carried out by multi colors flow cytometry (BD FACSCantoIITM, BD LSRFortessaTM, BD Biosciences).Results:Based on these determined conditions, CX3CR1 expression on monocytes, NK cells and a part of CD8+and CD4+T cells were confirmed in this method. Interestingly, during the E6011 treatment, the proportion of CD16+monocytes, which highly express CX3CR1 within whole monocytes, were significantly decreased at 2 week after initial treatment from the baseline (E6011:p< 0.001, placebo:p> 0.48) and sustained up to 24 week, while that of CD16-monocytes were increased. The reduction of the frequency of CX3CR1+cells in NK cells, CD4+and CD8+T cells were not observed, but in some certain populations like CX3CR1-expressed CD56+CD16+NK cells and terminal differentiated effector CD8+T cells, the frequency of CX3CR1+cells in these populations tended to increase from the baseline at 2 week and kept increasing up to 24 week by the E6011 treatment.Conclusion:E6011 significantly decreased the proportion of CD16+monocytes in whole monocytes. Our results indicated that the reduction of CD16+monocytes after initial treatment might be a sensitive marker of E6011 in peripheral blood, possibly reflecting mechanism of action of E6011, since the CD16+monocytes highly express CX3CR1.References:[1]Tanaka Y, et al., Mod Rheumatol (2018) 28, 58-65Disclosure of Interests:Tomohiro Yamada Employee of: KAN Research Institute, Inc,, Jungo Kakuta Employee of: KAN Research Institute, Inc., Eri Fusaoka-Nishioka Employee of: KAN Research Institute, Inc., Jun-ichi Ito Employee of: EISAI, Nobuyuki Yasuda Employee of: KAN Research Institute, Inc., Tetsu Kawano: None declared, Toshio Imai: None declared

E6011 is a novel humanized antifractalkine (FKN) monoclonal antibody being developed as a therapeutic target for Crohn's disease, rheumatoid arthritis, and primary biliary cholangitis. This study was a randomized, double-blind, placebo-controlled single-ascending-dose study of intravenous administration of E6011 (0.0006-10 mg/kg) in healthy Japanese adult men (n = 64). The starting dose was the minimum anticipated biological effect level (MABEL). MABEL was estimated by extrapolating results of a pharmacokinetic/pharmacodynamic (PK/PD) model relating E6011 exposure and suppression of free soluble FKN using data obtained from cynomolgus monkeys. Safety assessments consisted of monitoring and recording adverse events, laboratory tests, vital signs, intensive electrocardiograms, and chest x-rays. Blood samples to determine PK, PD (serum total FKN concentration), and serum anti-E6011 antibody were collected. Noncompartmental analysis was used to derive PK parameters. Single intravenous infusions of E6011 were safe and well tolerated in healthy subjects. Serum E6011 concentrations showed triphasic elimination. An increase in serum total FKN concentration was observed, confirming target engagement. The dose strategy for patient studies is to select regimens that will attain a minimum serum E6011 exposure of 10 μg/mL, identified as the minimum concentration needed to saturate the target-mediated elimination pathway. Model-based drug development from preclinical stage was successful in identifying dose regimens for clinical testing.

Background In the Phase 1/2 clinical study, E6011, a novel humanised anti-fractalkine (FKN) mAb demonstrated a promising efficacy in active RA patients who were inadequately controlled by MTX and/or TNF-α inhibitors (NCT 02196558). FKN is expressed on endothelial cells and fibroblast-like synoviocytes in synovium and also expressed on osteoblasts. CX3CR1 is expressed on monocytes/macrophages and osteoclast precursor cells (OPCs). Therefore, FKN-CX3CR1 interaction could play pivotal roles in migration, differentiation and activation of those cells. However, the precise mechanism(s) of FKN-CX3CR1 axis in RA, especially on joint destruction remains to be elucidated. Objectives We examined the roles of FKN-CX3CR1 axis in joint destruction, particularly focused on osteoclast precursor cells in in vitro and in vivo by using anti-mouse FKN mAb (anti-mFKN mAb). Methods DBA/1J mice were immunised with intradermal injections of bovine type II collagen to induce arthritis(CIA). Anti-mFKN mAb or control IgG were intraperitoneally injected twice a week. The clinical arthritis score was monitored, and joint destruction was evaluated by soft X-ray and histopathology. Plasma levels of joint destruction markers were assessed by ELISA. FKN expression in joint tissues were assessed by immunohistochemistry. Cell survival of bone marrow-derived OPCs without or with immobilised FKN was also assessed by FACS. In in vivo, OPCs were labelled by fluorescein and transferred to CIA mice to evaluate migration of OPCs into inflamed synovium. Anti-mFKN mAb or control IgG were injected before the cell transfer. The number of fluorescein-labelled OPCs that migrated into the CIA joint tissue were analysed. Results In both prophylactic and therapeutic treatments, anti-mFKN mAb clearly reduced the clinical arthritis score, soft X-ray score. Plasma levels of cartilage oligomeric matrix protein (COMP) and matrix metalloproteinase-3 (MMP-3) were also decreased after treatment with anti-mFKN mAb. Histological analysis revealed that anti-mFKN mAb inhibited synovitis, pannus formation, cartilage destruction, and that it suppressed bone damage with marked reductions in the number of tartrate-resistant acid phosphatase (TRAP)-positive cells. In in vitro, RANKL-induced osteoclast differentiation was enhanced by immobilised FKN, and anti-mFKN mAb suppressed FKN-dependent enhancement of osteoclast formation. FKN also enhanced cell survival of OPCs and eventually increased the number of OPCs. In in vivo, increasing expression of FKN was detected on endothelium in synovial tissue of CIA mice, and fluorescein-labelled OPCs migrated into the affected synovium. Anti-mFKN mAb clearly abrogated their migration into synovium. Conclusions Anti-mFKN mAb remarkably ameliorated the joint destruction with the marked reduction of osteoclasts by the inhibition of both OPC survival and OPC migration in inflamed joint tissues These results strongly indicate that inhibition of FKN-CX3CR1 axis by a humanised anti-FKN mAb, E6011, is an attractive and affected joints-selective therapeutic strategy for the treatment of both inflammatory synovitis and joint destruction in RA patients. Disclosure of Interest None declared

Background In the Phase 1/2 clinical study, E6011, a novel humanized anti-fractalkine (FKN) monoclonal antibody (mAb) demonstrated a promising efficacy in active rheumatoid arthritis (RA) patients who were inadequately controlled by MTX and/or TNF-a inhibitors. However, the effect of anti-FKN mAb on joint destruction remains to be elucidated. In RA, synovium-infiltrated monocytes/macrophages cause synovitis and cartilage damage. Osteoclasts are generated from osteoclast precursor cells (OCPs) and cause bone erosion. FKN is expressed on endothelial cells and fibroblast-like synoviocytes in synovium in both experimental arthritis model and RA patients. FKN is also expressed on osteoblasts in neonatal mouse calvariae. CX3CR1, the receptor for FKN, is expressed on monocytes/macrophages and OCPs. Therefore, the interaction of FKN and CX3CR1 might play important roles in migration, differentiation and activation of these cells, leading to cartilage damage and bone erosion. Objectives We examined the efficacy of an anti-FKN mAb on collagen-induced arthritis (CIA) in mice, especially on joint destruction. Methods For the induction of CIA, DBA/1J mice were immunized with bovine type II collagen. Anti-FKN mAb or control IgG was injected twice a week from the day of the 1st immunization (for the prophylactic treatment) or after the onset of CIA (for the therapeutic treatment). The clinical arthritis score was defined as the sum of the scores of four paws. Plasma concentrations of IL-6, TNF-a, serum amyloid A (SAA), Tartrate-Resistant Acid Phosphatase type 5b (TRAP-5b), Cartilage Oligomeric Matrix Protein (COMP) and Matrix Metalloproteinase 3 (MMP-3) were measured using ELISA. Radiological score was measured by the soft x-ray images of limb bones. Alcian blue/Alizarin red and Tartrate-Resistant Acid Phosphatase (TRAP) staining were performed for histopathological analysis of ankle joints. Results In the prophylactic treatment, anti-FKN mAb markedly reduced the clinical arthritis score, soft x-ray score, and plasma levels of TRAP-5b, COMP and MMP-3, whereas plasma levels of IL-6, TNF-a and SAA were not significantly reduced compared with control IgG-treated mice. Histopathological analysis demonstrated almost complete suppression of joint destruction and dramatic reduction in the number of TRAP-positive cells by the treatment of anti-FKN mAb. Importantly, therapeutic treatment of anti-FKN mAb also significantly ameliorated clinical arthritis score and soft x-ray score. Synovitis, pannus formation, cartilage degradation and bone erosion were also strongly suppressed and the number of TRAP-positive cells in joint was also decreased by the therapeutic treated of anti-FKN mAb. Conclusions Anti-FKN mAb demonstrated a remarkable efficacy in the arthritis score without affecting systemic inflammatory parameters and inhibited the joint destruction with the marked reduction of osteoclasts in CIA model. These results suggest that inhibition of FKN/CX3CR1 axis by a humanized anti-FKN mAb, E6011, is an attractive therapeutic strategy for the treatment of both inflammatory synovitis and joint destruction of RA. Disclosure of Interest None declared

E3024 (3-but-2-ynyl-5-methyl-2-piperazin-1-yl-3,5-dihydro-4H-imidazo[4,5-d]pyridazin-4-one tosylate) is a dipeptidyl peptidase-IV (DPP-IV) inhibitor that was expected to be an antidiabetic agent. Its safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) were investigated in a randomized, double-blind, placebo-controlled, ascending single-dose study in 48 healthy Japanese male subjects. Fasted subjects were orally administered E3024 (5, 10, 20, 40, or 80 mg) or placebo. E3024 was rapidly absorbed, with tmax values ranging between 0.33 and 3 h after dosing. The mean t1/2 ranged from 5.34 to 11.68 h. AUC0-inf and Cmax increased dose-proportionately. PK-PD relationship of E3024 was evaluated by using an Imax model, indicating that plasma E3024 concentrations and inhibitory effects of plasma DPP-IV activity were well correlated. The IC50 value was calculated as 33.7 ng/mL, which was consistent with in vitro data. Thus, E3024 showed a good PK profile and inhibited DPP-IV dose-dependently. Of 30 subjects administered E3024, 12 (40%) experienced adverse events (AEs). Dose escalation to 160 mg was abandoned owing to undesired subjective/objective findings in 4 of 6 subjects receiving 40 mg and 5 of 6 subjects receiving 80 mg. The most prominent AE was rash, but there were no serious AEs or deaths. The maximum tolerated dose was considered to be 20 mg. We hypothesized that histamine was a cause of the rash induction, and examined blood histamine levels of normal Fischer rats treated with E3024. Blood histamine levels were increased significantly by E3024 at 500 mg/kg (p Ws/Ws rats treated with E3024 at 500 mg/kg. In in vitro assays, E3024 induced histamine release from normal rat peritoneal mast cells in a concentration-dependent manner, but not from basophils. The structure-activity relationship study suggested that a piperazine group N-linked to the 2-position of the 5,6-membered fused heterocyclic rings was a key structural element for triggering histamine release.

Inflammatory and immune responses are generated locally by the selective invasion and accumulation of the immune cells into the lesion site. The infiltration process of the immune cells into the tissue from the blood through the vascular endothelial cells is closely regulated by a number of chemotactic factors and cell adhesion molecules. Fractalkine (FKN)/CX3CL1 is a membrane-bound chemokine possessing a chemokine/mucin hybrid structure and a transmembrane domain and has a dual function as an adhesion molecule and a chemoattractant. FKN is mainly expressed on activated endothelial cells, activated fibroblasts, and osteoblasts. Its receptor, CX3CR1, is expressed on cytotoxic effector lymphocytes, monocytes/macrophages, and osteoclasts. To date, a lot of key functional aspects of the FKN-CX3CR1 axis has been identified: (1) the rapid capture and firm adhesion of immune cells to vascular endothelial cells, (2) chemotaxis, (3) the enhancement of the transmigration to other chemokines, (4) the crawling behavior of the monocytes that patrol on vascular endothelial cells, (5) the retention of monocytes as the accessory cells of the inflamed endothelium to recruit inflammatory cells, and (6) the survival of the macrophage. In this review, we will focus on the pathological role of FKN in rheumatoid arthritis (RA) and the physiological role of FKN on osteoclast differentiation. Furthermore, we will discuss the therapeutic potential of anti-FKN mAb for RA patients and its distinct mode of action from other cytokine inhibitors.

Objectives We examined oral N-acetylcysteine effects on contrast-induced nephropathy (CIN) and clinical events in patients undergoing primary angioplasty for acute myocardial infarction. Background Recent studies have reported that N-acetylcysteine reduces CIN and improves the clinical outcome in patients undergoing primary angioplasty. However, additional investigations are warranted to further support these findings. Methods We randomly assigned 76 patients undergoing primary angioplasty into two groups: 38 patients were assigned to N-acetylcysteine (NAC, 705 mg orally administration before and 12, 24, 36 hours after primary angioplasty), and 38 patients to placebo. CIN was defined as an increase in the serum creatinine concentration of 25 percent or more from baseline value within the 72-hour period after primary angioplasty. Results CIN occurred in 7 patients (9.2%). In the NAC group, the incidence of CIN tended to be lower than in the placebo group (NAC; 2/38; 5.3% vs. Placebo; 5/38; 13.2%, p=0.21). The composite endpoints such as death, acute renal failure requiring temporary renal replacement therapy, or need for mechanical ventilation did not occur in either group. Conclusion While N-acetylcysteine might have the possibility to reduce the incidence of contrast-induced nephropathy in patients undergoing primary angioplasty for acute myocardial infarction, the in-hospital mortality and morbidity were not significantly different between the two groups.

Aims Several treadmill exercise testing prognostic parameters have been identified in various populations. However, despite the widespread use of treadmill exercise testing, the prognostic value in very elderly patients has not been well characterized. The aim of this study was to assess the results of treadmill exercise testing in octogenarians, and to examine various parameters in order to identify a prognostic marker of mortality. Methods and results This study included 97 consecutive octogenarians (age, 81.1 ± 1.8 years; 66% male) who were referred for treadmill exercise testing. During the follow-up period (2.6 ± 1.6 years), all-cause death occurred in 20 patients (21%). Univariate Cox proportional hazard regression analysis showed that abnormal heart rate recovery (HRR) (defined as a decreased heart rate of ≤18 beats per minute after peak exercise) [hazard ratio (HR), 2.82; 95% confidence interval (CI), 1.06–7.47; P = 0.037] and ischaemic ST-segment change (HR, 2.56; 95% CI, 1.01–6.46; P = 0.047) were significantly associated with all-cause mortality. After adjusting for age and sex, multivariate Cox proportional hazard analysis showed that abnormal HRR was the only independent predictor of all-cause death (HR, 2.86; 95% CI, 1.01–8.11; P = 0.048). Conclusion Attenuated HRR is a significant prognostic marker for all-cause death among octogenarians. The results may provide helpful support for risk stratification in clinical practice.

Several combination therapies have been tried for treating of type 2 diabetes to control more effectively fasting hyperglycemia and postprandial hyperglycemia. In this study, we have examined the effects of combining a novel, selective, and competitive dipeptidyl peptidase IV (DPP-IV) inhibitor, 3-but-2-ynyl-5-methyl-2-piperazin-1-yl-3,5-dihydro-4H-imidazo[4,5-d]pyridazin-4-one tosylate (E3024), with a representative of one of two types of insulin secretagogues, i.e., either glybenclamide (a sulfonylurea) or nateglinide (a rapid-onset/short-duration insulin secretagogue), on glucose and insulin levels in an oral glucose tolerance test (OGTT) using mice fed a high-fat diet. In addition, we have investigated the effects of these combinations on blood glucose levels in fasting rats. Two-way analysis of variance showed that the combination of E3024 and glybenclamide improved glucose tolerance additively and also caused a synergistic increase in insulin levels in the OGTT in mice fed a high-fat diet. In a similar way, the combination of E3024 and nateglinide ameliorated glucose tolerance additively and raised insulin levels additively. In fasting rats, coadministration of E3024 with glybenclamide or nateglinide treatment did not affect the glucose-lowering effects of the insulin secretagogues. Therefore, a DPP-IV inhibitor in combination with glybenclamide or nateglinide may be a promising option for the treatment of type 2 diabetes, and particularly, for controlling postprandial hyperglycemia in the clinic.

Dipeptidyl peptidase IV (DPP-IV) inhibitors are expected to become a useful new class of anti-diabetic agent. The aim of the present study is to characterize the in vitro and in vivo profile of E3024, 3-but-2-ynyl-5-methyl-2-piperazin-1-yl-3,5-dihydro-4H-imidazo[4,5-d]pyridazin-4-one tosylate, which is a novel imidazopyridazinone-derived DPP-IV inhibitor. E3024 inhibited recombinant human and mouse DPP-IV with IC50 values of approximately 100 nM. E3024 inhibited DPP-IV in human, mouse, rat and canine plasma with IC50 values of 140 to 400 nM. In contrast, E3024 did not inhibit DPP-8 or DPP-9 activity. Kinetic analysis indicated that E3024 is a competitive DPP-IV inhibitor. In Zucker fa/fa rats, E3024 (1 mg/kg) reduced glucose excursion after glucose load, with increases in plasma insulin and active glucagon-like peptide-1 levels. In fasted rats, this compound did not cause hypoglycemia. In a rat 4-week toxicological study, no notable changes were found at doses up to 750 mg/kg. The present preclinical studies indicate that E3024 is a novel selective DPP-IV inhibitor with anti-diabetic effects and a good safety profile.

In type 2 diabetic patients, the administration of glucagon-like peptide-1 (GLP-1), known as an incretin, exerts antidiabetic effects. However, GLP-1 is rapidly degraded by dipeptidyl peptidase IV (DPPIV) after its release. DPPIV inhibition is thought to be a rational strategy to treat type 2 diabetes. In this study, using C57BLKS/J-db/db (db/db) mice as a model of type 2 diabetes, we examined the effect of acute DPPIV inhibition on glucose tolerance at the early and later stages of diabetes, determining plasma active GLP-1 and insulin levels. In addition, we investigated changes of plasma DPPIV activity. Compared with normal C57BL6/J (B6) and db/+ mice, significantly increased plasma DPPIV activities were observed in db/db mice. Expression of the proglucagon gene encoding GLP-1 was significantly upregulated in the colon of db/db mice. The administration of valine-pyrrolidide, a DPPIV inhibitor, resulted in potentiated insulin secretion mediated by increased endogenous GLP-1 action, leading to improved glucose tolerance in db/db mice at 6 weeks of age. However, although acute DPPIV inhibition with valine-pyrrolidide resulted in higher plasma active GLP-1 and insulin levels in db/db mice at 23 weeks of age, it did not improve glucose tolerance. The function of the enteroinsular axis is preserved in both stage of diabetes and the DPPIV inhibitor potentiated it, but the progression of insulin resistance appeared to block the improvement of glucose tolerance through DPPIV inhibition. Our results suggest that DPPIV inhibition is a suitable approach for treatment of impaired glucose tolerance (IGT), and type 2 diabetes in the early stage.

The adenosine receptor subtype mediating glucose production by glycogenolysis and gluconeogenesis was studied in primary cultured rat hepatocytes. Adenosine and adenosine agonists caused cyclic AMP accumulation in rat hepatocytes. The order of potency was 5′- N -ethylcarboxamidoadenosine (NECA)> R (−)- N 6 -(2-phenylisopropyl)adenosine (RPIA)>adenosine>2-[ p -(carboxyethyl)phenylethylamino]-5′- N -ethylcarboxamidoadenosine (CGS21680). Furthermore, adenosine agonists stimulated glycogenolysis and gluconeogenesis. The order of potency was NECA>RPIA>CGS21680. The rank order of potency is typical for adenosine A 2B receptors. Glycogenolysis stimulated by NECA was fully inhibited by nonselective adenosine antagonists, 9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5- c ]quinazolin-5-amine (CGS15943). However, the adenosine A 2A receptor-selective antagonist, 8-(3-chlorostyryl)caffeine (CSC), and the adenosine A 1 receptor-selective antagonist, (+)-( R )-[( E )-3-(2-phenylpyrazolo[1,5-alpha]pyridin-3-yl)acryloyl]-2-piperidine ethanol (FK453), had a low inhibitory potency. A strong correlation was found between the inhibitory effect of adenosine antagonists on NECA-induced glucose production and that on intracellular cyclic AMP generation in rat hepatocytes. Our results suggest that adenosine stimulates cyclic AMP formation and regulates glycogenolysis and gluconeogenesis, most likely through the adenosine A 2B receptor subtype in rat hepatocytes.

A series of 2-alkynyl-8-aryladenine derivatives bearing an amide moiety at the 9-position of adenine was synthesized. These analogues were evaluated for inhibitory activity on N-ethylcarboxamidoadenosine (NECA)-induced glucose production in primary cultured rat hepatocytes. The m-primary benzamide derivative 15f was the most potent compound (IC(50)=0.017 microM), being 15-fold more active than the corresponding 9-methyl derivative (1). Compound 15f showed 72- and 5.2-fold selectivity for human A(2B) receptor versus human A(1) and A(2A) receptors, respectively. Structure-activity relationship (SAR) studies of the synthesized compounds indicated that a three-carbon linker, fixed in the form of a benzene ring, between the adenine core and the amide moiety is important for both A(2B) antagonistic activity and selectivity. The IC(50) values in rat hepatocyte glucose assay correlated well with the IC(50) values in cAMP assay using Chinese hamster ovary cells stably transfected with human A(2B) receptors (r(2)=0.94). The A(1) and A(2A) affinities showed no correlation with the potency to inhibit NECA-induced glucose production. These results strongly support our previous conclusion that adenosine agonist-induced hepatic glucose production in rat hepatocytes is mediated through the A(2B) receptor.

Glucagon-like peptide-1 (GLP-1) is an incretin, which induces glucose-dependent insulin secretion. GLP-1 is rapidly degraded by dipeptidyl peptidase IV (DPPIV) after its release. We investigated whether DPPIV-deficient F344/DuCrj rats show improved glucose tolerance when compared with DPPIV-positive F344/Jcl rats. Oral glucose tolerance test indicated improved glucose tolerance in F344/DuCrj rats, but blood glucose levels of the two strains were almost the same 120 min after the glucose bolus. Valine-pyrrolidide, a DPPIV inhibitor, had no effect on the glucose tolerance of F344/DuCrj rats, but improved that of F344/Jcl rats. Enhanced insulin secretion and high plasma active GLP-1 levels were detected in an intraduodenal glucose tolerance test. Glucose tolerance is improved in DPPIV-deficient F344/DuCrj rats via enhanced insulin release mediated by high active GLP-1 levels. Our results suggest that DPPIV inhibition is a rational strategy to treat diabetic patients by improving glucose tolerance with low risk of hypoglycemia.

In the current aging society, acute heart failure (AHF) is one of the leading causes of hospitalization due to the increased number of older individuals, and the prevalence of HF is increasing, raising a major public health concern in industrialized countries [1,2]. In addition, HF is one of themost important causes of death in elderly patients. The 5year-survival rate after the first admission for HF was reportedly worse than for that for cancer [3]. On the other hand, recent advances in technology and development of medical therapies have improved mortality in heart disease, and their effect include expanding an opportunity for therapy even among very elderly patients. However, the situation in very elderly population, over 90 years of age with heart disease is unclear, and few data are available on outcome and mortality in nonagenarians with HF. The aim of the present of study was to investigate the clinical features of AHF in nonagenarians, and identify prognostic factors by using routine non-invasivemethods and examinations. A retrospective observational review of 146 consecutive patients aged 90 years and older who were admitted to our institution during the period from January 2006 to December 2008 with a diagnosis of AHF was conducted. AHF was defined as new onset decompensated HF or worsening of chronic HF requiring emergency hospitalization. Patients whose course was complicated by acute coronary syndrome and severe renal failure were also included. Clinical records including medical history, co-morbidities, demographic data, and the results of examinations at the time of hospital admission, were retrospectively reviewed. Total mortality was evaluated during the follow-up period, and prognostic factors identified. The study protocol was approved by our institutional review board. Continuous variables were expressed as means±standard deviation, and categorical variables as numbers and proportions. The patients were divided into a survival group and a non-survival group, and characteristics and examination results are compared by Cox proportional hazards regression analysis. The factors with p values b0.10 in univariate analysis were entered in the multivariate Cox proportional hazards regression model to identify independent predictive factors for mortality. Based on the obtained independent predictor, a receiver operating characteristics (ROC) curve was plotted, and cut-off point for ROC curve factor was determined. The

Background Fractalkine (CX3CL1, designated as FKN hereafter) is the sole member of the CX3C-chemokine which leads to dual actions, chemotaxis and cell adhesion for leukocytes expressing the cognate receptor, CX3CR1, during their migration. Accumulating evidence is telling that FKN-CX3CR1 axis plays a pivotal role in leukocyte/lymphocyte accumulation in inflamed tissues in RA1. We developed E6011, a novel humanized anti-FKN monoclonal antibody. Objectives To evaluate safety, pharmacokinetics and efficacy of E6011 in a Phase 1/2, open-label, multiple ascending dose study in RA patients for the first time (NCT02196558). Methods Active RA patients with inadequate response to MTX or TNF inhibitors were received 7 consecutive doses (subcutaneous: SC) of E6011 at week 0, 1, 2 and thereafter every 2 weeks up to week 10. A hundred and 200 mg of E6011 were chosen for the study as putatively appropriate doses for patients by the PK/PD modeling previously obtained in the Ph1 healthy volunteer study of E6011. Results Twelve subjects were enrolled in the cohort of 100 mg and subsequently 15 subjects were enrolled in the 200 mg cohort, in total, 27 subjects received repeated subcutaneous administrations of E6011. As a result, repeated dose of E6011 was found safe and well tolerated. The incidence of AE, treatment-related AE and SAE was 59.3%, 29.6% and 7.4%, respectively. There were no severe AE or deaths, and no significant differences were observed in the incidence or severity of AE between the 100 mg and 200 mg cohorts. After starting multiple SC injection of E6011, serum E6011 concentration reached steady-state at week 2, and its level was maintained up to week 12. Mean trough E6011 concentrations from week 2 through week 12 were 11.6 – 15.7 ug/mL and 29.2 – 38.8 ug/mL after dosing of 100 and 200 mg, respectively. Clinical outcome was also available in the study in which response rates of ACR20, 50 and 70 at week 12 were 75.0%, 33.3%, 8.3% and 80.0%, 26.7%, 20.0% in the cohort of 100 and 200 mg, respectively. At week 12, 33.3% of subjects achieved DAS28-CRP remission in both cohorts, 16.7% and 20.0% for SDAI remission and 8.3% and 26.7% for Boolean remission were observed in the 100 and 200 mg cohorts, respectively. Conclusions E6011 was safe and well tolerated, and demonstrated a promising efficacy in active RA patients with MTX or TNFi-IR. While further clinical studies are required, the results obtained indicate that a novel biological DMARD targeting FKN/CX3CR1 interaction will be clinically beneficial for active RA patients. References Nanki T. Arthritis Rheum. 2002; 46(11):2878–83. Acknowledgement The authors wish to thank the study investigators. Disclosure of Interest Y. Tanaka Grant/research support from: Mitsubishi-Tanabe, Takeda, Chugai, Astellas, Eisai, Taisho-Toyama, Kyowa-Kirin, AbbVie, Bristol-Myers, Speakers bureau: AbbVie, Daiichi-Sankyo, Chugai, Takeda, Mitsubishi-Tanabe, Bristol-Myers, Astellas, Eisai, Janssen, Pfizer, Asahi-kasei, Eli Lilly, GlaxoSmithKline, UCB, Teijin, MSD, Santen, T. Takeuchi Grant/research support from: Astellas, Bristol-Myers, Chugai, Daiichi-Sankyo, Eisai, AYUMI, Takeda, Teijin, AbbVie, Asahikasei, Taisho-Toyama, Consultant for: Astra Zeneca, Eli Lilly, Novartis, Mitsubishi-Tanabe, AsahiKasei, AbbVie, Daiichi-Sankyo, Bristol-Myers, Nipponkayaku, Janssen, Merck Serono, Takeda, Astellas, Speakers bureau: AbbVie, Bristol-Myers, Chugai, Eisai, Janssen, Mitsubishi-Tanabe, Pfizer, Takeda, Astellas, Daiichi-Sankyo, Celtrion, Nipponkayaku, H. Umehara: None declared, T. Nanki Grant/research support from: Chugai, Eisai, Takeda, Teijin, Eli Lilly, Bristol-Myers, AbbVie, Mitsubishi-Tanabe, Ono, Daiichi-Sankyo, Shionogi, Asahikasei, Nippon Boehringer Ingelheim, Ajinomoto, Consultant for: UCB, Eisai, Chugai, Speakers bureau: Mitsubishi-Tanabe, Chugai, Eisai, Takeda, Astellas, Janssen, Eli Lilly, Ono, AbbVie, Bristol-Myers, Daiichi-Sankyo, Santen, H. Akama Shareholder of: EISAI, Employee of: EISAI, N. Yasuda Shareholder of: EISAI, Employee of: EISAI, F. Tago Employee of: EISAI, M. Kawakubo Shareholder of: EISAI, Employee of: EISAI, S. Hojo Employee of: EISAI, T. Kawano Employee of: KAN Research Institute, T. Imai Employee of: KAN Research Institute

1. Electrical field stimulation (EFS; 10 V, 10 Hz, 2 ms) of porcine coronary artery strips precontracted with 10 nM endothelin-1 (ET-1) for 5 min caused a biphasic response, consisting of a slight contraction during EFS and a marked and irreversible relaxation just after EFS. This irreversible relaxation after EFS has never been investigated. In the present study, we have investigated the mechanism of the relaxation after EFS. 2. The EFS-induced response was not affected by the presence or absence of endothelium and was insensitive to 10 microM tetrodotoxin (TTX). 3. In the presence of free radical scavengers (40 u ml-1 superoxide dismutase (SOD), 1200 u ml-1 catalase or 80 mM D-mannitol), the relaxation after EFS was significantly inhibited. Moreover, relaxation after EFS was not observed in porcine coronary artery strips precontracted with 20 mM KCl. 4. In a cascade experiment, EFS of Krebs-Ringer solution containing 10 nM ET-1 induced marked suppression of the contractile activity of ET-1 in porcine coronary artery strips, which was in accord with the observed decrease in release of immunoreactive ET-1 (ir-ET-1). This effect of EFS was significantly inhibited by each of the free radical scavengers, 3 mM vitamin C, 40 u ml-1 SOD, 1200 u ml-1 catalase and 80 mM D-mannitol. 5. The exchange of 95% O2/5% CO2 gas for 95% N2/5% CO2 gas significantly inhibited the EFS-induced decrease in release of ir-ET-1. 6. Neither superoxide anions generated by xanthine (10 JM) plus xanthine oxidase (0.1 micro ml-1) nor hydrogen peroxide (10 microM) exogenously added to Krebs-Ringer solution containing 10 nM ET-1 affected the level of ir-ET-1.7. Generation of hydroxyl radicals was detected in the EFS-applied Krebs-Ringer solution. The EFS-induced generation of hydroxyl radicals was dependent on the period of stimulation and 02-bubbling, and significant generation of hydroxyl radicals was detectable with stimulation of over 5 min.Moreover, hydroxyl radicals generated in 50 mM NaCl solution containing 10 nM ET-1 by H202 plus Fe2 , i.e. the Fenton reaction, significantly decreased the level of ir-ET-l.8. These findings suggest that oxygen-derived hydroxyl radicals generated by EFS of porcine coronary artery strips inactivate ET-1, probably by structural modification. Thus, porcine coronary artery strips precontracted with ET-1 are potently relaxed by EFS.

Although recent progress in emergency surgery has resulted in an increase in the indication for older patients with acute type A aortic dissection (AAD), some patients remain who cannot undergo surgical treatment and little is known about the prognosis of patients with AAD who receive medical treatment, especially in elderly patients. Of the 82 patients with AAD who were admitted to our institution, 48 received medical therapy only. We retrospectively reviewed their clinical data and analyzed the prognostic value of the clinical characteristics in both younger and older patients. The in-hospital and 1-year mortality were significantly lower in the patients who underwent surgical treatment than in those who received medical treatment (6% vs 65%, p0.001; 8% vs 73%, p0.001, respectively). Of the patients with medical treatment, the in-hospital and 1-year mortality rate in the younger (80 years old, n = 27) and older (≥80 years old, n = 21) groups was 70% and 80% and 57% and 65%, respectively. For the younger group, the presence of an open false lumen was significantly associated with in-hospital mortality (89% vs 50%, p = 0.044). In contrast, in the older group, a lower serum albumin level (3.4 ± 0.3 vs 4.0 ± 0.5 g/dl, p = 0.010) and the incidence of an open false lumen (83% vs 33%, p = 0.032) were significantly associated with in-hospital mortality. In conclusion, in addition to an open false lumen as a risk factor, a lower serum albumin level is an important prognostic factor in older patients with AAD.

Law plays an important role in the development process in the Association of Southeast Asian Nations (ASEAN). This article identifies three types of law in terms of their origin: "indigenous law", originating in the "proto states"; "imported law" introduced by colonial rulers; and "development law" arising since the independence of these states. From these types of laws functional principles are deduced to aid in understanding contemporary Third World legal systems more clearly in development perspectives. The historical development of ASEAN law is reviewed in these three time periods and the role which law has played in political, economic and social development in the region is examined. The conclusion suggests reasons why it may be desirable in the long term to integrate or at least co-ordinate the law and policy within the region but on a basis which reflects regional rather than Western tradition.

4-Bromomethyl-4,5-dihydro-1,3-thiazoles and 5-bromo-4H-5,6-dithydro-1,3-thiazines were synthesized by intramolecular cyclization of allylthioimidates by the reaction with N-bromosuccinimide (NBS) or bromine in dichloromethane at room temperature.

7-But-2-ynyl-9-(6-methoxy-pyridin-3-yl)-6-piperazin-1-yl-7,9-dihydro-purin-8-one (ER-319711) is a novel dipeptidyl peptidase (DPP)-IV inhibitor discovered in our laboratories. In this study, we have characterized this DPP-IV inhibitor in vitro and in vivo as an antidiabetic agent. The trifluoroacetate salt form of ER-319711, ER-319711-15, inhibited human DPP-IV with an IC(50) value of 0.089 microM, whereas its IC(50) values toward human DPP8 and DPP9 were >100 microM. Inhibition kinetic pattern analysis indicated that ER-319711-15 inhibited DPP-IV in a competitive manner. ER-319711-15 (1 mg/kg) reduced glucose excursion in an oral glucose tolerance test (OGTT) using Zucker fa/fa rats, with significant increases in plasma insulin and active glucagon-like peptide-1 levels. In an OGTT using mice fed a high-fat diet in which ER-319711-15 (0.1-10 mg/kg) was orally administered at 0 h, and glucose was loaded at 0 and 5 h, this compound improved glucose tolerance dose dependently at both 0- and 5-h glucose loading. Next, we compared efficacy of ER-319711-15, E3024, a competitive DPP-IV inhibitor having an imidazopyridazinone structure, or vildagliptin, a slow-binding and long-acting DPP-IV inhibitor, at the same dose, 10 mg/kg, in the same procedures. At the first glucose challenge, all compounds lowered area under the curve (AUC) values of delta blood glucose between 0 and 2 h significantly to the same degree. At the second glucose load, the AUC values between 5 and 7 h were significantly decreased by ER-319711-15 and vildagliptin, but not by E3024. Therefore, ER-319711 might be a potent, competitive, and selective DPP-IV inhibitor with an antihyperglycemic activity.