Weekly split-dose regimen for oral methotrexate reduced polyglutamation in red blood cells in patients with rheumatoid arthritis compared with single-dose regimen: Results from a multicentered randomized control trial.

AIMS: We compared the incidence of adverse events between single and divided-dose regimens of methotrexate (MTX) by using a multicenter randomized controlled trial. METHODS: Eighty-nine patients with insufficient control on MTX 8 mg/wk were randomly assigned into single-dose (39 patients) or triple dose (39 patients) groups. The MTX dose for all patients was gradually increased to 16 mg/wk. The primary endpoint was the occurrence of liver dysfunction during the observation period (20 weeks). RESULTS: There were no differences in baseline data and MTX dose at Week 20 between groups. There was no significant difference in the incidence of liver dysfunction between groups (single dose, 3 [7.7%] patients vs. triple dose, 5 [13.2%] patients; P = .455). The incidence of adverse event increased in triple dose (single dose, 12 [30.8%] patients vs. triple dose, 20 [51.3%]), but the difference was not significant (P = .066). There was no significant difference in disease activity between groups, although MTX-triglutamate (PG3), MTX-PG4, and MTX-PG5 were significantly higher in the single dose group. CONCLUSIONS: Weekly split dosing reduced the polyglutamation of MTX. There was no significant difference in efficacy and safety between the 2 groups.

Concomitant Treatment with Etanercept and Tacrolimus Synergistically Attenuates Arthritis Progression via Inhibition of Matrix Metalloproteinase-3 Production and Osteoclastogenesis in Human TNF-α Transgenic Mice.

In the present study, we investigated the effects and mechanisms of action of a combined treatment with etanercept, a soluble tumor necrosis factor receptor (p75) Fc fusion protein, and tacrolimus, a calcineurin inhibitor on the progression of arthritis in human tumor necrosis factor-α (TNF-α) transgenic (hTNF-Tg) mice. Single-drug treatments with etanercept and tacrolimus attenuated the clinical signs but not the radiographic changes associated with the development of arthritis in mice. On the contrary, combined treatment significantly suppressed the radiographic progression and also improved the clinical signs. The combined treatment exhibited synergistic effects of the two drugs in reducing the serum matrix metalloproteinase-3 level and the number of peripheral CD11bhigh osteoclast precursor cells. Moreover, tacrolimus inhibited the cytokine-induced osteoclast differentiation in synergy with etanercept in an in vitro assay. Interestingly, tacrolimus did not inhibit the production of antidrug antibodies (ADAs) against etanercept in the hTNF-Tg mice. This result implies that the synergistic effects of etanercept and tacrolimus are not due to secondary effects derived from the suppression of ADA production by tacrolimus but are due to their primary effects. These findings suggest that concomitant treatment with etanercept and tacrolimus may be one of preferable treatment options to control disease activities for patients with rheumatoid arthritis, especially for those with bone resorption.

Bucillamine inhibits CD40-mediated Akt activation and antibody production in mouse B-cell lymphoma.

The improvement of rheumatoid factor titers in patients with rheumatoid arthritis is one of the significant clinical effects of bucillamine (Buc). In this study, we investigated the effects of SA981, an active metabolite of Buc, and methotrexate (MTX) on CD40-mediated antibody production using mouse B-cell lymphoma, BCL1. SA981 significantly attenuated CD40-mediated antibody production in a concentration-dependent manner, but weakly affected cell proliferation. In contrast, MTX did not attenuate CD40-mediated antibody production until it had strongly inhibited cell proliferation at a concentration of 100 nM. CD40 signaling induced protein phosphorylation, including Akt phosphorylation, p38 mitogen-activated protein kinase (p38MAPK), and IκBα. SA981 at a concentration of 30 µM attenuated CD40-mediated Akt phosphorylation, but not p38MAPK or IκBα phosphorylation. MTX at a concentration of 100 nM did not affect CD40-mediated Akt, p38MAPK, or IκBα phosphorylation. Commercially available Akt inhibitor VIII significantly attenuated CD40-mediated IgM production at a concentration of 100 nM without significant inhibition of cell proliferation. These results suggest that SA981 inhibits CD40-mediated antibody production in mouse B-cell lymphoma, at least in part, by attenuation of Akt phosphorylation.

The kappa opioid receptor agonist SA14867 has antinociceptive and weak sedative effects in models of acute and chronic pain.

We examined the analgesic effect of the selective kappa opioid receptor agonist SA14867 and the balance of its antinociceptive and sedative effects. The ED(50) values of SA14867 after oral administration for acetic acid-induced writhing, first and second phases of the formalin test, and rotarod test in mice were 6.1, 9.3, 2.7, and 19.5mg/kg, respectively. These values were smaller than those of the conventional kappa receptor agonists asimadoline and U-50488H. However, the balance of the antinociceptive and sedative effects of SA14867 was better than those of the other two drugs. Orally administered SA14867 (0.1-1mg/kg) significantly improved the decreased pain threshold in a specific alternation of rhythm in an environmental temperature (SART)-stressed model by prophylactic and therapeutic treatment. Improvement in the decreased pain threshold of SA14867-treated animals was attenuated by the opioid receptor antagonist naloxone. Furthermore, orally administered asimadoline (10-100mg/kg) improved the decreased pain threshold in a SART-stressed model, but the doses were close to those known to induce sedative effects. In addition, SA14867 (0.1-1mg/kg) significantly inhibited the arthritis-induced decrease in the pain threshold. Subcutaneously administered morphine (0.1-1mg/kg) improved the decreased pain threshold in a SART-stressed model; on the contrary, morphine did not inhibit the arthritis-induced decrease in the pain threshold. Moreover, orally administered SA14867 (0.1-1mg/kg) strongly attenuated mechanical allodynia and thermal hyperalgesia in a sciatic nerve ligation model. These results suggest that SA14867 has analgesic effects on chronic pain and may serve as a new therapeutic agent for pain treatment.

ARG098, a novel anti-human Fas antibody, suppresses synovial hyperplasia and prevents cartilage destruction in a severe combined immunodeficient-HuRAg mouse model.

BACKGROUND: The anti-human Fas/APO-1/CD95 (Fas) mouse/human chimeric monoclonal IgM antibody ARG098 (ARG098) targets the human Fas molecule. The cytotoxic effects of ARG098 on cells isolated from RA patients, on normal cells in vitro, and on RA synovial tissue and cartilage in vivo using implanted rheumatoid tissues in an SCID mouse model (SCID-HuRAg) were investigated to examine the potential of ARG098 as a therapy for RA. METHODS: ARG098 binding to each cell was analyzed by cytometry. The effects of ARG098 on several cells were assessed by a cell viability assay in vitro. Effects on the RA synovium, lymphocytes, and cartilage were assessed in vivo using the SCID-HuRAg mouse model. RESULTS: ARG098 bound to cell surface Fas molecules, and induced apoptosis in Fas-expressing RA synoviocytes and infiltrating lymphocytes in the RA synovium in a dose-dependent manner. However, ARG098 did not affect the cell viability of peripheral blood mononuclear cells of RA patients or normal chondrocytes. ARG098 also induced apoptosis in RA synoviocytes and infiltrating lymphocytes in the RA synovium in vivo. The destruction of cartilage due to synovial invasion was inhibited by ARG098 injection in the modified SCID-HuRAg mouse model. CONCLUSIONS: ARG098 treatment suppressed RA synovial hyperplasia through the induction of apoptosis and prevented cartilage destruction in vivo. These results suggest that ARG098 might become a new therapy for RA.

SA14867, a newly synthesized kappa-opioid receptor agonist with antinociceptive and antipruritic effects.

SA14867 ((+)-3-Acetyl-6-chloro-2-[2-(3-(N-(2-ethoxyethyl)-N-isopropylamino)propoxy)-5-methoxyphenyl]benzothiazoline O,O'-diacetyl-L-tartrate), a selective kappa-opioid receptor agonist, was synthesized and its antinociceptive and antipruritic effects were investigated. In a functional binding assay, SA14867 showed approximately more than 31,000 and 2200 fold higher affinity for the kappa-opioid receptor than for the mu- and delta-opioid receptors, respectively. SA14867 inhibited acetic acid-induced writhing and formalin test results after oral administration. The ED(50) values of SA14867 for acetic acid-induced writhing and for formalin test first phase and second phase were 1.9, 9.4, and 6.4 mg/kg, respectively. These values were smaller than those of asimadoline, U-50488H, and tramadol. SA14867 also showed antinociceptive effects in silver nitrate-induced arthritis that were as strong as U-50488H, tramadol, and morphine, and were stronger than asimadoline. The ED(50) value of SA14867 for hyperalgesia of arthritis was approximately 10 mg/kg. In addition, SA14867 showed antipruritic effects on 5-hydroperoxyeicosatetraenoic acid (HPETE) and substance P-induced pruritic models at 1 to 3 mg/kg. SA14867 also attenuated scratching reactions in a morphine-induced pruritic model in monkeys. Some of the inhibitory effects of SA14867 on nociceptive and pruritic models were attenuated by a kappa-opioid receptor antagonist, nor-BNI. These results suggest that SA14867 is a potential antinociceptive and antipruritic drug.

Effects of SA13353, a transient receptor potential vanilloid 1 agonist, on leukocyte infiltration in lipopolysaccharide-induced acute lung injury and ovalbumin-induced allergic airway inflammation.

We recently demonstrated that SA13353, a transient receptor potential vanilloid 1 (TRPV1) agonist, reduced the severity of the symptoms of kidney injury, arthritis, and encephalomyelitis in disease models. Here, we investigated the effects of orally administered SA13353 on leukocyte infiltration in lipopolysaccharide (LPS)-induced acute lung injury and ovalbumin-induced allergic airway inflammation. In LPS-induced lung injury, SA13353 attenuated neutrophil infiltration and the increase of TNF-alpha and CINC-1 levels. In allergic airway inflammation, SA13353 tended to inhibit leukocyte infiltration and attenuated the increase of IL-4 and IL-12p40. These results suggest that somatosensory TRPV1 may play an anti-inflammatory role in lung inflammation.

Transient receptor potential vanilloid 1 agonists as candidates for anti-inflammatory and immunomodulatory agents.

We recently demonstrated that SA13353 [1-[2-(1-adamantyl)ethyl]-1-pentyl-3-[3-(4-pyridyl)propyl]urea], a novel transient receptor potential vanilloid 1 (TRPV1) agonist, inhibits TNF-alpha production through the activation of capsaicin-sensitive afferent neurons. In the present study, we investigated the effects of SA13353 on lipopolysaccharide (LPS)-induced cytokine production and a murine model of experimental autoimmune encephalomyelitis (EAE). SA13353 inhibited LPS-induced TNF-alpha and interleukin (IL)-1beta production while augmenting IL-10 production in mice. It also inhibited TNF-alpha and IL-1beta mRNA expression, and increased IL-10 mRNA expression in LPS-treated murine liver. These effects were not observed in TRPV1 KO and sensory denervated mice. Capsaicin and SA13353 increased serum neuropeptide levels, and calcitonin gene-related peptide fragment 8-37 (CGRP(8)(-)(37)), a CGRP antagonist, partially blocked the inhibitory effects of capsaicin and SA13353 on LPS-induced TNF-alpha production. These results suggest that the TPPV1 agonistic effects inhibit TNF-alpha production, at least partially, via neuropeptide release. SA13353 did not directly affect LPS-induced cytokine production in vitro using RAW264.7 macrophages, which do not express TRPV1. Therefore, we consider SA13353 to be a good tool for the investigation of the value of TRPV1 agonists for the treatment of chronic inflammation. In a murine EAE model, SA13353 attenuated clinical signs and histopathological changes. SA13353 attenuated cytokine levels, including TNF-alpha, IL-1beta, IL-12p40, IL-17, and interferon (IFN)-gamma, after proteolipid protein (PLP) immunization. In addition, SA13353 attenuated the increase of IL-17-producing cells. These results suggest that TRPV1 agonists may act as anti-inflammatory and immunomodulatory agents in vivo in certain inflammatory diseases.

The investigation of synovial genomic targets of bucillamine with microarray technique.

OBJECTIVE: To identify the molecular mechanisms of bucillamine activity, global gene expression analysis and pathway analysis were conducted using IL-1 beta-stimulated human fibroblast-like synovial cells (FLS). METHODS: Normal human FLS were treated with IL-1 beta in the presence or absence of 10 and 100 microM bucillamine for 6 h. Total RNA was extracted and global gene expression levels were detected using a 44 k human whole genome array. Data were analyzed using Ingenuity pathway analysis. RESULTS: Numerous pathways were activated by IL-1 beta stimulation. At both concentrations, bucillamine suppressed nine signal pathways stimulated by IL-1 beta. CONCLUSIONS: Bucillamine effectively inhibited fibroblast growth factor (FGF) signaling and tight junction signaling activated by IL-1 beta in FLS. Suppression of these signal pathways may correlate with the pharmacologic mechanisms of bucillamine. In particular, the suppression of FGF signaling by bucillamine is remarkable because the activation of FGF signaling may be involved in rheumatoid arthritis pathology.

SA13353 (1-[2-(1-Adamantyl)ethyl]-1-pentyl-3-[3-(4-pyridyl)propyl]urea) inhibits TNF-alpha production through the activation of capsaicin-sensitive afferent neurons mediated via transient receptor potential vanilloid 1 in vivo.

Tumor necrosis factor-alpha (TNF-alpha) is known to play a crucial role in the pathogenesis of rheumatoid arthritis. In the present study, we demonstrate the effects of SA13353 (1-[2-(1-Adamantyl)ethyl]-1-pentyl-3-[3-(4-pyridyl)propyl]urea), a novel orally active inhibitor of TNF-alpha production, in animal models, and its mechanism of action on TNF-alpha production. SA13353 significantly inhibited lipopolysaccharide (LPS)-induced TNF-alpha production in a dose-dependent manner in rats. Moreover, SA13353 exhibited a binding affinity for the rat vanilloid receptor and increased neuropeptide release from the rat dorsal root ganglion neurons. However, its effects were blocked by pretreatment with the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine. The ability of SA13353 and capsaicin to inhibit LPS-induced TNF-alpha production was eliminated by sensory denervation or capsazepine pretreatment in vivo. Although they inhibited LPS-induced TNF-alpha production in mice, these effects were not observed in TRPV1 knockout mice. SA13353 provoked the release of neuropeptides without nerve inactivation, even when chronically administered to rats. These results suggest that SA13353 inhibits TNF-alpha production through activation of capsaicin-sensitive afferent neurons mediated via TRPV1 in vivo. Post-onset treatment of SA13353 strongly reduced the hindpaw swelling and joint destruction associated with collagen-induced arthritis in rats. Thus, SA13353 is expected to be a novel anti-arthritic agent with a unique mechanism of action.

Bucillamine mechanism inhibiting IL-1beta-induced VEGF production from fibroblast-like synoviocytes.

We investigated the bucillamine (Buc) mechanism inhibiting interleukin (IL)-1beta-induced vascular endothelial growth factor (VEGF) production from human fibroblast-like synoviocytes (HFLS) which derived from the inflamed synovium of an RA patient using SA981, its active metabolite. HFLS did not produce IL-1beta, spontaneously. While SA981 partially inhibited IL-1beta-induced VEGF production at concentrations of 10 to 100 microM (10.1% and 14.2% inhibition of total VEGF production under IL-1beta coexistence condition, respectively), it failed to inhibit IL-1beta-induced IL-6 production at the same concentrations. IL-1beta induced phosphorylation of the mitogen-activated protein (MAP) kinases, IkappaBalpha, c-Jun and Akt. SA981 at a concentration of 100 microM partially inhibited IL-1beta-induced phosphorylation of p38MAPK and Akt (12.0% and 36.1% inhibition of each total amount of phosphoprotein under IL-1beta coexistence condition, respectively). The VEGF promoter includes four transcription factors: AP1, hypoxia-inducible factor (HIF), Sp1 and AP2 binding elements. HIF-1beta, Sp1 and AP1 increased under IL-1beta coexistence conditions. At a concentration of 100 microM, SA981 attenuated increases in HIF-1beta and Sp1 (10.1% and 19.8% inhibition of each total amount of transcription factor under IL-1beta coexistence condition, respectively), but not AP1. These results suggest that SA981 partially inhibits VEGF production via modifications on IL-1beta signaling. Attenuation of the expression of HIF-1beta and Sp1 (but not AP1) may be a key with respect to SA981's selective inhibition of VEGF production.

Differential effects between marimastat, a TNF-alpha converting enzyme inhibitor, and anti-TNF-alpha antibody on murine models for sepsis and arthritis.

We investigated the effects of marimastat, an inhibitor of TNF-alpha converting enzyme and matrix metalloproteinases, and anti-TNF-alpha antibodies on a murine model for sepsis, and on arthritis in human TNF-alpha transgenic mice. Marimastat (25-200 mg/kg) inhibited lipopolysaccharide (LPS)-induced soluble TNF-alpha production in mice in a dose-dependent manner. At an oral dose of 200 mg/kg, marimastat almost completely inhibited LPS-induced soluble TNF-alpha production, but only slightly delayed LPS lethality. On the other hand, anti-TNF-alpha antibodies completely abolished LPS-induced morbidity. In addition, anti-TNF-alpha antibodies, but not marimastat (200 mg/kg/day), inhibited the development of arthritis in human TNF-alpha transgenic mice. These results suggest that cell surface TNF-alpha may be important in the pathogenesis of murine models for sepsis and arthritis.