Nobuhiro Takagi

Mechanisms and pathologic significances in increase in serum interleukin-6 (IL-6) and soluble IL-6 receptor after administration of an anti-IL-6 receptor antibody, tocilizumab, in patients with rheumatoid arthritis and Castleman disease.

Interleukin-6 (IL-6) plays pathologic roles in immune-inflammatory diseases such as rheumatoid arthritis (RA) and Castleman disease. By inhibiting IL-6 receptors (IL-6Rs), tocilizumab (a humanized anti-IL-6R antibody) ameliorates the symptoms of these diseases and normalizes acute-phase proteins, including C-reactive protein (CRP). We found that tocilizumab treatment increased serum levels of IL-6 and soluble IL-6R (sIL-6R). To investigate the pathologic significance of these increases, we analyzed the kinetics of serum IL-6 and sIL-6R and the proportion of sIL-6R saturated with tocilizumab after tocilizumab administration in patients with RA and Castleman disease and then compared the results with the CRP values. Serum IL-6 and sIL-6R markedly increased after tocilizumab administration in both RA and Castleman disease. As long as free tocilizumab was detectable, sIL-6R was saturated with tocilizumab and IL-6 signaling was completely inhibited. We concluded that it is likely that sIL-6R increased because its elimination half-life was prolonged by the formation of tocilizumab/sIL-6R immune complex, and that free serum IL-6 increased because IL-6R-mediated consumption of IL-6 was inhibited by the unavailability of tocilizumab-free IL-6R. We also concluded that the increased level of free IL-6 during tocilizumab treatment closely reflects the actual endogenous IL-6 production and true disease activity.

Blockage of interleukin-6 receptor ameliorates joint disease in murine collagen-induced arthritis

OBJECTIVE To clarify the role of interleukin-6 (IL-6) in the pathogenesis of collagen-induced arthritis (CIA). METHODS CIA was induced by immunizing twice at a 3-week interval with bovine type II collagen (CII) emulsified with complete adjuvant. Rat anti-mouse IL-6 receptor (anti-IL-6R) monoclonal antibody MR16-1 or isotype-matched control antibody KH-5 was then injected once intraperitoneally. Symptoms of arthritis were evaluated with a visual scoring system, and serum anti-CII antibody and IL-6 levels were measured by enzyme-linked immunosorbent assay. In addition, the CII responsiveness of splenic lymphocytes from mice with CIA was examined. RESULTS In mice with CIA, excess production of IL-6 in sera was observed within 24 hours after the first CII immunization, and then rapidly decreased. Serum IL-6 increased again beginning 14 days after immunization, in conjunction with the onset of arthritis. When MR16-1 was injected immediately after immunization with CII, it inhibited the development of arthritis in a dose-dependent manner. Furthermore, MR16-1-treated mice exhibited lower serum levels of IgG anti-CII antibody and reduced responsiveness of lymphocytes to CII. This suppressive effect was observed when MR16-1 was injected on day 0 or 3, but not when injected on day 7 or 14. CONCLUSION IL-6 produced after CII immunization appears to play an essential role in the immunity to CII, and anti-IL-6R antibody reduces the development of CIA by suppressing IL-6 signal transduction.

IL‐6 receptor blockage inhibits the onset of autoimmune kidney disease in NZB/W F1 mice

In the present study, we examined the preventive effect of anti‐mouse IL‐6 receptor (IL‐6R) antibody, MR16‐1, on the development of autoimmune kidney disease in female NZB/W F1 (BWF1) mice. Immunological tolerance to MR16‐1 or isotype‐matched control antibody, KH‐5, was induced by the simultaneous administration of anti‐CD4 MoAb in mice. Thereafter, mice were intraperitoneally given 0.5 mg of MR16‐1, 0.5 mg of KH‐5 or saline once a week from 13 to 64 weeks of age. MR16‐1 treatment dramatically suppressed proteinuria and prolonged the survival time of BWF1 mice. Only one out of 10 mice died with high levels of proteinuria throughout the experiment. MR16‐1 almost completely suppressed the production of IgG forms of anti‐DNA and anti‐TNP antibodies, but not the IgM forms of these antibodies. In particular, all IgG subclasses (IgG1, IgG2a, IgG2b and IgG3) of anti‐DNA antibody production were significantly suppressed. Moreover, serum IgG1, IgG2a and IgG3 levels in MR16‐1‐treated mice were lower than those in saline‐ and KH‐5‐treated mice, whereas serum IgM and IgA levels were not influenced. In conclusion, MR16‐1 potently suppressed the development of autoimmune disease in BWF1 mice, and this was attributed to its effect of specific suppression of IgG class antibody production.

In vitro and in vivo biological activities of a novel nonpolyglutamable anti-folate, MX-68

MX-68 is a newly synthesized anti-folate, chemically designed not to undergo intracellular polyglutamation and to have increased affinity to dihydrofolate reductase (DHFR). In the present study, we examined the in vitro and in vivo biological activities of MX-68 compared with methotrexate (MTX) which forms several polyglutamates intracellularly. MX-68 dose-dependently inhibited the proliferation of PHA-, anti-CD3-, or PMA plus ionomycin-stimulated peripheral blood mononuclear cells (PBMC) and endothelial cells (EC) from normal subjects as well as IL-1 beta- or TNF alpha-stimulated synovial fibroblastic cells (SC) from rheumatoid arthritis (RA) patients. Coaddition of folinic acid completely reversed the anti-proliferative effects of both MX-68 and MTX. Although the anti-proliferative activities of MX-68 were almost comparable to those of MTX, the washout study clearly showed the characteristic nature of MX-68. When drugs were removed during culture, the suppressive effect of MX-68 completely disappeared, whereas suppression by MTX was merely weakened. MX-68 dramatically suppressed the onset of collagen-induced arthritis (CIA) in mice when the drug was orally administered three times a week. starting from the day of first immunization. In this model, 2 mg/kg of MX-68 was sufficient to completely suppress arthritis, whereas suppression by the same dose of MTX was partial. These lines of evidence suggest that polyglutamation is not always a prerequisite in the anti-rheumatic effects of anti-folate. In addition, since intracellular accumulation of polyglutamates is thought to have adverse effects, MX-68 may become a more potent and less toxic anti-rheumatic drug than MTX.

Effectiveness and safety of tocilizumab in achieving clinical and functional remission, and sustaining efficacy in biologics-naive patients with rheumatoid arthritis: The FIRST Bio study

Abstract Objective: To evaluate effectiveness and safety of tocilizumab (TCZ) in biologic-naive Japanese patients with rheumatoid arthritis (RA) in real-world settings, and to analyze the relationship between disease duration and clinical outcomes. Methods: The FIRST Bio study was a postmarketing surveillance study of intravenous TCZ in biologics-naive patients who had a prior inadequate response or were intolerant to ≥1 conventional synthetic disease-modifying antirheumatic drug (csDMARD). Effectiveness, safety, and concomitant csDMARD administration were assessed. Results: Of the 839 patients analyzed, 72.3% completed 52 weeks of treatment. The Clinical Disease Activity Index (CDAI) remission rate at week 52 was 36.8%. Contributing factors for CDAI remission were younger age, early disease stage, and no comorbidities. Health Assessment Questionnaire Disability Index ≤0.5 was achieved in 65.1% of patients, and was significantly associated with disease duration. Discontinuation of concomitant methotrexate (MTX) and glucocorticoids (GCs) was possible in 19.3% and 34.1% of patients, respectively, without decreasing remission rate. The incidence (events/100 patient-years) of serious adverse events was 18.09, the most common being infection. Conclusion: These data validate the importance of TCZ treatment in the early stages of RA in biologic-naive patients to achieve increased effectiveness. The safety profile of TCZ was reconfirmed. Furthermore, TCZ therapy may allow discontinuation of concomitant MTX and GCs without affecting remission.

Polyglutamation of a novel antifolate, MX-68, is not necessary for its anti-arthritic effect

N-[[4-[(2,4-diaminopteridin-6-yl)methyl]-3,4-dihydro-2H-1,4-benzothiazin-7-yl]-carbonyl]-L-homoglutamic acid (MX-68), a derivative of methotrexate, was chemically designed to resist polyglutamation and to have a high affinity for dihydrofolate reductase, in an attempt to reduce the side effects of methotrexate. We confirmed that MX-68 did not undergo polyglutamation and investigated the pharmacological activities of MX-68 compared with methotrexate. (1) In vitro: MX-68 inhibited the activity of dihydrofolate reductase to the same degree as methotrexate-tetraglutamate. MX-68 treatment produced a similar anti-proliferative effect to that of methotrexate. However, the intracellular concentration of MX-68 was much lower than the sum of the levels of methotrexate and its polyglutamate, and the effects of MX-68 disappeared when it was removed from the culture medium. (2) In vivo: Oral administration of MX-68 suppressed the development of collagen-induced arthritis in mice and adjuvant-induced arthritis in rats, in a similar fashion to that of methotrexate. These results indicate that polyglutamation is not essential for the anti-arthritic effect of antifolates.

Preventive effect of a novel antifolate, MX-68, in murine systemic lupus erythematosus (SLE).

We evaluated the preventive effects of a novel nonpolyglutamatable antifolate, MX-68, on two experimental murine models of systemic lupus erythematosus (SLE); NZBxNZW F1 (BWF1) mice and chronic graft-versus-host disease (GVHD) mice, in comparison with classical antifolate methotrexate (MTX). The oral administration of 2 mg/kg MX-68, three times a week from 12 to 40 or 60 weeks of age, significantly delayed the onset of proteinuria and prolonged the life-span of BWF1 mice. The elevation of serum blood urea nitrogen (BUN) and cholesterol levels resulting from the development of lupus nephritis was also inhibited. However, MX-68 did not suppress the increase of serum anti-DNA or anti-TNP antibodies or total IgG isotype (IgG1, IgG2 and IgG3) levels. In chronic GVHD mice, MX-68 given three times a week from the day of first cell injection, for 9 weeks, dose-dependently delayed the appearance of proteinuria. The elevation of BUN and cholesterol levels was also inhibited. Furthermore, in the 4 mg/kg MX-68 group, the production of IgG anti-DNA and anti-TNP antibodies was significantly inhibited, but this was not observed in the 2 mg/kg MX-68 and the 4 mg/kg MTX groups. These beneficial effects of MX-68 were much greater than those of MTX in both models. These results suggest that MX-68 might be a more useful drug for the treatment of SLE.

Prodromal signs and symptoms of serious infections with tocilizumab treatment for rheumatoid arthritis: Text mining of the Japanese postmarketing adverse event-reporting database

Abstract Objective: To search for signs and symptoms before serious infection (SI) occurs in tocilizumab (TCZ)-treated rheumatoid arthritis (RA) patients. Methods: Individual case safety reports, including structured (age, sex, adverse event [AE]) and unstructured (clinical narratives) data, were analyzed by automated text mining from a Japanese post-marketing AE-reporting database (16 April 2008–10 April 2015) assuming the following: treated in Japan; TCZ RA treatment; ≥1 SI; unable to exclude causality between TCZ and SIs. Results: The database included 7653 RA patients; 1221 reports met four criteria, encompassing 1591 SIs. Frequent SIs were pneumonia (15.9%), cellulitis (9.9%), and sepsis (5.0%). Reports for 782 patients included SI onset date; 60.7% of patients had signs/symptoms ≤28 days before SI diagnosis, 32.7% had signs/symptoms with date unidentified, 1.7% were asymptomatic, and 4.9% had unknown signs/symptoms. The most frequent signs/symptoms were for skin (swelling and pain) and respiratory (cough and pyrexia) infections. Among 68 patients who had normal laboratory results for C-reactive protein, body temperature, and white blood cell count, 94.1% had signs or symptoms of infection. Conclusion: This study identified prodromal signs and symptoms of SIs in RA patients receiving TCZ. Data mining clinical narratives from post-marketing AE databases may be beneficial in characterizing SIs.