Yasuhisa Takeda

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.

A Novel Antifolate, MX-68, Inhibits the Development of Autoimmune Disease in MRL/lpr Mice

We compared a novel unpolyglutamable antifolate, MX-68, with polyglutamable antifolate, methotrexate (MTX), for treatment of an autoimmune kidney disease which develops spontaneously in MRL/Mp-lpr/lpr (MRL/lpr) mice. Oral administration of either MX-68 or MTX was commenced in 8-week-old female mice and continued 3 times a week until they reached 30 weeks of age. MX-68 delayed the onset of proteinuria and prolonged life span dose-dependently. Furthermore, it suppressed the elevation of serum blood urea nitrogen and cholesterol levels. MX-68 was as effective as MTX at ameliorating events which accompany the development of lupus nephritis, despite that MX-68 did not undergo polyglutamation. These ameliorative effects of MX-68 and MTX did not occur via inhibition of either autoantibody production or cell proliferation. Neither compound suppressed age-dependent elevation of immune complexes or antibodies for single-stranded DNA and TNP in serum nor did they influence the associated enlargement of lymph nodes and spleen. We conclude that MX-68 is beneficial for the treatment of autoimmune kidney disease in mice and may be useful for other related diseases such as systemic lupus erythematosus.

Polyglutamation of antifolates is not required for induction of extracellular release of adenosine or expression of their anti-inflammatory effects.

Methotrexate (MTX) exerts an anti-inflammatory effect, reportedly by enhancing the release of adenosine, through an accumulation of 5-amino-4-imidazolecarboxamide ribonucleotide (AICAR). To examine the role of polyglutamation in promoting anti-inflammatory effects by antifolates, we tested whether a new antifolate designed to be resistant to intracellular polyglutamation (MX-68) exhibited anti-inflammatory effects and stimulated adenosine release. Both MX-68 and MTX (at concentrations greater than 0.1 microM) increased the release of adenosine from Daudi cells in vitro. Inhibition of AICAR synthesis suppressed adenosine release by MX-68 and MTX. The anti-inflammatory effects of antifolates were estimated using the murine air pouch model, in which a BALB/c mouse was intraperitoneally injected with MX-68 or MTX once a week for 3 weeks. MX-68 (0.5 mg kg(-1) week(-1)), as well as MTX, inhibited infiltration of leukocytes into the air pouch. This inhibitory effect was suppressed in the presence of an adenosine A(2) receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX). These results suggest that MX-68, like MTX, exerts anti-inflammatory effects through the accumulation of AICAR and release of adenosine. These results suggest that polyglutamation of antifolate is not required for expression of anti-inflammatory effects.

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.

Synthesis of tritiated N‐[4‐(2,4‐diaminopteridine‐6‐methyl)‐3,4‐dihydro‐2H‐1,4‐benzothiazine‐7‐carbonyl]‐L‐homo glutamic acid (MX‐68)

Synthesis of tritiated N-[4-(2,4-diaminopteridine-6-methyl)-3,4-dihydro-2H-1,4-benzothiazine-7-carbonyl]-L-homoglutamic acid, [9-3H1]MX-68 (3), is described. [9-3H1]MX-68 (3) was prepared via tritiation at the carbonyl group in 2,4-bis-(butanoylamino)-6-formylpteridine (7) with sodium borotritiide.

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.

Inhibitory effect of CCA on immunoglobulin-production induced by B 151-TRF2.

Lobenzarit disodium (CCA) is an immunomodulating anti-rheumatic drug, and is reported to show inhibitory effects on B-cell activation. To further analyze the direct effects of CCA on B cells, we examined the effects of CCA on Ig-production in vitro by murine B cells stimulated with murine T cell-derived B-cell stimulation factor, B 151-TRF 2 which induces a polyclonal differentiation into Ig-secreting cells including autoantibody production.CCA inhibited Ig-production induced by B 151-TRF 2. This result suggests that the inhibitory effect of CCA on Ig-production is one of the mechanism of CCA action on autoimmune disease, because B 151-TRF 2 is reported to be closely associated with the onset of autoimmune disease. In contrast, CCA did not inhibit Ig-production induced by LPS.These results demonstrate that the induction process of Ig-production by B 151-TRF 2 is differ from that of Ig-production by LPS, and that the effective point of CCA is associated with this difference.

Effects of CCA (Disodium 4-chloro-2, 2'-iminodibenzoate) on the biological activities of both lymphocytes and macrophages were investigated

In the in vitro studies of murine lymphocytes, CCA showed a tendency to increase the viability of spleen cells and an activity to enhance the mitogenic responses of those cells by LPS, B-mitogen. Enhancing effects of CCA on T-mitogen-induced lymphocytes reaction have also been reported in other papers. These results were considered in connection with a phenomenon that CCA increased the RNA polymerase activity of nucleus from rat lymphnode cells. CCA also enhanced thein vivo mitogenic reaction by Con A. CCA was not mytogenic by itself.As for the activities of macrophages, CCA did not almost show any effects inin vitrostudies while it did in the carbon clearance test using both normal and tumor-bearing animals.