Serum-soluble folate receptor ß as a biomarker for the activity of rheumatoid arthritis synovitis and the response to anti-TNF agents.

This study aims to develop a sandwich ELISA system for the measurement of soluble folate receptor ß (sFRß) and evaluate whether base line levels of serum sFRß are a biomarker for the activity of RA synovitis and the response to anti-TNF agents. Serum sFRß from normal controls (41 samples), patients with OA (29 samples), and patients with RA (27 samples) and synovial fluid sFRß from patients with RA (17 samples) were measured by sandwich ELISA, using anti-FRαß and anti-FRß antibodies as capture and detection antibodies, respectively. Baseline levels of serum sFRß before therapy were evaluated in relation with DAS28-CRP or CRP and response to anti-TNF agents at 3-month follow-up. sFRß levels in RA synovial fluids were higher than those in RA sera, and sFRß levels in RA sera were higher than those in osteoarthritis and normal control sera. A significant relationship was observed between serum sFRß levels and the DAS28-CRP scores or CRP values. The area under curve (AUC) values for receiver-operating characteristic curves defined using the serum sFRß levels of RA patients before therapy had a higher predictive capacity than DAS28-CRP and CRP for the effective response of anti-TNF agents. The high serum sFRß levels with a cutoff value of 8 ng/mL were 100% specificity for the effective response of anti-TNF agents. The findings support that the serum sFRß levels in patients with RA act as a disease activation biomarker and that high serum sFRß levels act as a predictive biomarker for the response to anti-TNF agents.

Risk of low bone mineral density in patients with rheumatoid arthritis treated with biologics.

BACKGROUND: Osteoporosis is a complication of rheumatoid arthritis (RA). We identified risk factors for osteoporosis during treatment with biologics. METHODS: Femoral neck bone mineral density (BMD) was measured in 186 patients with biologics-treated RA. We compared the characteristics of those with BMD ≥70% of young adult mean (YAM) and those with BMD <70% of YAM, and undertook multivariable logistic regression analysis to identify risk factors for bone loss. RESULTS: Mean age and disease duration, the proportion of females, scores in the Modified Health Assessment Questionnaire and history of vertebral fracture were significantly greater in the BMD <70% of YAM group, but body mass index (BMI) was significantly lower in the BMD <70% of YAM group. There was no significant difference between the groups in terms of other biomarkers of RA activity, the proportion treated with methylprednisolone, or the duration or choice of biologics. The proportions of patients treated with anti-osteoporosis drugs and parathyroid hormone were significantly higher in the BMD <70% of YAM group. In the multivariable analysis, advanced age, female, longer disease duration, history of past thoracic or lumbar vertebral fracture, higher Steinbrocker classification and lower BMI were significant factors for BMD <70% of YAM. DISCUSSION: We identified risk factors for bone loss in patients with RA treated with biologics. Before suppression of disease activity by biologics, bone loss might already be advanced. CONCLUSIONS: We recommend that patients with RA who possess these risk factors be considered for earlier and more intense treatment to prevent bone loss, as well as addressing RA disease progression.

Assessment of peripheral blood CD4+ adenosine triphosphate activity in patients with rheumatoid arthritis.

OBJECTIVE: The ability of the ImmuKnow (Cylex) assay to predict the risk of infection in rheumatoid arthritis (RA) patients receiving synthetic or biological disease-modifying antirheumatic drugs (DMARDs) was examined. METHODS: The amount of adenosine triphosphate (ATP) produced by CD4+ cells in response to phytohemagglutinin was measured in whole blood from 117 RA patients without infection versus 17 RA patients with infection, and compared with results in 75 healthy controls. RESULTS: The mean ATP level was significantly lower in patients with infection compared to both healthy controls (P < 0.0005) and patients without infection (P = 0.040). Also, the mean ATP level in patients without infection was significantly lower than that in healthy controls (P = 0.012). There was no correlation between the ATP level and the Disease Activity Score in 28 joints. CONCLUSION: ImmuKnow assay results may be effective in identifying RA patients at increased risk of infection, but the results showed no correlation with RA activity. Larger studies are required to establish the clinical advantages of this assay in RA treatment.

An investigation of the correlation between blood concentration of mizoribine and its efficacy in treatment of rheumatoid arthritis based on indices of drug survival and improvement in DAS28-CRP.

OBJECTIVES: The efficacy of mizoribine (MZR) in treatment of rheumatoid arthritis (RA) was retrospectively investigated in terms of drug survival, improvement in Disease Activity Score-28 (DAS28)-C-reactive protein (CRP), and blood MZR concentration obtained 3 h after dosing (MZR-C3). METHODS: To compare the efficacy of MZR administered via different regimens, the subjects were divided into 2 groups: those receiving a single dose of MZR at 100-150 mg every other day (group A) and those receiving 2 or 3 divided doses of the drug on consecutive days, which is the usual dosing method of the drug (group B). RESULTS: Group A had significantly higher MZR-C3 levels compared with group B, as well as significantly greater improvement in DAS28-CRP. Moreover, drug survival was significantly longer in group A. The primary regression equation suggested that the effective blood MZR concentration in RA treatment is MZR-C3 of 1.47 µg/mL or more. CONCLUSIONS: The results of the present study indicate that it is possible to increase the efficacy of MZR in a blood concentration-dependent manner, and also to control RA over a prolonged period, using single administration of MZR on alternate days at an increased dose.

Factors associated with fatal outcome of leflunomide-induced lung injury in Japanese patients with rheumatoid arthritis.

OBJECTIVE: To elucidate the factors associated with poor prognosis of LEF-induced lung injury in patients with RA. METHODS: The background and clinical and laboratory features of LEF-induced lung injury were examined and compared between patients who died of and who recovered from it. RESULTS: Among 22 patients who developed LEF-induced lung injury, 9 died of and 13 recovered from it. The patients who died tended to have pre-existing interstitial pneumonia (8/9 vs 6/13, P = 0.07). The loading and maintenance doses, serum concentration of the LEF metabolite A771726 and administration period did not differ between the groups. Patients who died had more frequently hypoxaemia of <60 Torr and mechanical ventilation, and had a high serum CRP level (19.3 +/- 9.4 vs 10.1 +/- 8.1 mg/dl, P = 0.03) and a low albumin level (2.7 +/- 0.6 vs 3.3 +/- 0.5 g/dl, P = 0.03) at the lung injury onset. The peripheral blood lymphocyte count decreased in both groups at the lung injury onset, and it remained low until fatal outcome, in contrast to a re-increase upon recovery (406 +/- 394 vs 1203 +/- 399/microl, P = 0.006). The main histopathological finding in two autopsied patients was diffuse alveolar damage, in contrast to the alveolitis observed in a biopsied patient who recovered. CONCLUSIONS: Pre-existing interstitial pneumonia, extremely high serum CRP and low albumin levels, severe hypoxaemia and mechanical ventilation indicated poor prognosis. Peripheral blood lymphocytopenia developed in association with lung injury, and a sustained low lymphocyte count indicated a fatal outcome.

Leflunomide-induced interstitial lung disease: prevalence and risk factors in Japanese patients with rheumatoid arthritis.

OBJECTIVES: The possible link between LEF and interstitial lung disease (ILD) has evoked increasing concern. The aim of the present study was to elucidate the prevalence and risk factors for newly developed and/or exacerbated ILD, based on post-marketing surveillance data, in which all RA patients receiving LEF were pre-registered and monitored for 24 weeks in Japan. METHODS: We analysed data from a cohort of 5054 RA patients who were prescribed LEF since its launch in September 2003 in Japan. Multivariable logistic analysis was performed to identify the risk factors for newly developed and/or exacerbation of ILD. RESULTS: Sixty-one (1.2%) of 5054 RA patients who received LEF were reported to have development and/or exacerbation of ILD as an adverse drug reaction to LEF, judged by the attending physicians. Multivariable logistic regression analysis identified pre-existing ILD [odds ratio (OR) 8.17; 95% CI 4.63, 14.4], cigarette smoking (3.12; 95% CI 1.73, 5.60), a low body weight (<40 kg vs >50 kg) (2.91; 95% CI 1.15, 7.37) and the use of a loading dose (3.97; 95% CI 1.22, 12.9) as independent risk factors for LEF-induced ILD. CONCLUSIONS: Pre-existing ILD was the most important risk factor for LEF-induced ILD. We suggest that LEF should not be prescribed for RA patients complicated with ILD.

Proposals for leflunomide use to avoid lung injury in patients with rheumatoid arthritis.

Among the 5,043 consecutive patients registered in the postmarketing surveillance for leflunomide, 61 were reported to have lung injury and 24 died from it. The adjusted multivariate logistic regression analysis of the risk factors showed that preexisting interstitial lung disease posed the greatest risk, as well as loading dose, smoking history, and low body weight of 40 kg or less with odds ratios of 8.17, 3.97, 3.12, and 2.91, respectively. In 12 patients, lung injury developed even 2 months after leflunomide withdrawal. When patients with (n=9) and without (n=13) fatal outcome were compared, eight out of the former, and six out of the latter had preexisting interstitial lung disease; the former showed severe hypoxemia, high serum C-reactive protein level, hypoalbuminemia, and continuous lymphocytopenia, and required mechanical ventilation. On the basis of these results and literature review, the committee proposes that leflunomide should only be recommended as a second-line drug, should not be administered to patients with preexisting interstitial lung disease, should also not be administered to patients with smoking history or those with low body weight, and should be administered without loading dose. Careful monitoring is necessary, and when lung injury develops, leflunomide elimination using colestyramine is mandatory.

[Effective combination therapy of TNF antagonists with DMARDs].

Efficacy and safety of anti-tumor necrosis factor (TNF) antagonists (infliximab, etanercept, adalimumab) in combination with methotrexate for rheumatoid arthritis is well determined. But methotrexate is not tolerable nor effective in some cases. Leflunomide in combination with anti-TNF antagonists is the alternative combination therapy in the world. But unfortunately, leflunomide itself is not tolerable for some Japanese cases because of fatal interstitial pneumonitis. A prospective open-label study of sulfasalazine, hydroxychloroquine, intramusculer gold in combination with etanercept indicated the efficacy and tolerability. Azathiopurine and bucillamine may be used when methotrexate is not effective or intolerable, although they need more examination.

In vitro and in vivo efficacy of a recombinant immunotoxin against folate receptor beta on the activation and proliferation of rheumatoid arthritis synovial cells.

OBJECTIVE: To investigate the effects of the recombinant immunotoxin dsFv anti-FRbeta-PE38, which consists of the disulfide-stabilized Fv fragment (dsFv) of the anti-folate receptor beta (anti-FRbeta) antibody and the 38-kd portion of Pseudomonas exotoxin A (PE38), on the activation and proliferation of cells that function in inflammatory and degradative processes in rheumatoid arthritis (RA) synovial tissue. METHODS: The Ig VH-PE38 fusion protein and the Ig VL protein were produced in Escherichia coli, and then joined with a disulfide bond by engineering cysteine residues in the framework regions of these proteins. The effects of dsFv anti-FRbeta-PE38 on the activation and proliferation of cells in RA synovial tissue were investigated by immunohistochemistry; the numbers of cells expressing CD68, vascular cell adhesion molecule 1, angiopoietin 1, CD34, proliferating cell nuclear antigen, and interleukin-6 and the numbers of apoptotic cells were counted in RA synovial tissue engrafted into SCID mice treated or not treated with dsFv anti-FRbeta-PE38. The effects of dsFv anti-FRbeta-PE38 on the generation of osteoclasts from RA adherent synovial mononuclear cells in vitro was investigated by counting the number of resorption pits on dentin slices treated or not treated with dsFv anti-FRbeta-PE38. RESULTS: Administration of dsFv anti-FRbeta-PE38 reduced the numbers of macrophages, activated fibroblast-like cells, endothelial cells, and proliferating cells and increased the numbers of apoptotic cells in RA synovial tissue engrafted into SCID mice. In vitro, the generation of osteoclasts from RA adherent synovial mononuclear cells was largely suppressed by treatment with dsFv anti-FRbeta-PE38. CONCLUSION: Our findings show that dsFv anti-FRbeta-PE38 immunotoxin would be a promising tool for the treatment of RA synovitis, especially when administered intraarticularly.

Anti-interleukin-6 receptor antibody therapy favors adrenal androgen secretion in patients with rheumatoid arthritis: a randomized, double-blind, placebo-controlled study.

OBJECTIVE: Proinflammatory cytokines such as tumor necrosis factor (TNF) were demonstrated to inhibit adrenal steroidogenesis in patients with rheumatoid arthritis (RA), and this was particularly evident in the increase in adrenal androgen levels during anti-TNF therapy. This study investigated the influence on steroidogenesis of an interleukin-6 (IL-6)-neutralizing strategy using IL-6 receptor monoclonal antibodies (referred to as MRA). METHODS: In a placebo-controlled, double-blind, randomized study over 12 weeks in 29 patients with RA being treated with prednisolone, 13 of whom received placebo and 16 of whom received 8 mg MRA/kg body weight, the effects of MRA on serum levels of adrenocorticotropic hormone (ACTH), cortisol, 17-hydroxyprogesterone (17OHP), dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), androstenedione (ASD), estrone, and 17beta-estradiol, as well as their respective molar ratios, were determined. RESULTS: MRA therapy markedly improved clinical signs of inflammation (the erythrocyte sedimentation rate, swollen joint score, and Disease Activity Score in 28 joints). Serum levels of ACTH and cortisol and the molar ratio of cortisol to ACTH did not change. Although serum levels of DHEA and DHEAS remained stable during therapy, the DHEAS:DHEA molar ratio significantly decreased in treated patients (P = 0.048). Serum levels of ASD as well as the ASD:cortisol and ASD:17OHP molar ratios increased in MRA-treated patients (minimum P < 0.004). Serum levels of estrone and 17beta-estradiol did not change. but the estrone:ASD molar ratio (an indicator of aromatization) decreased during 12 weeks of MRA treatment (P = 0.001). CONCLUSION: Neutralization of IL-6 increases secretion of biologically active adrenal androgens in relation to that of precursor hormones and estrogens. This is another important indication that proinflammatory cytokines interfere with adrenal androgen steroidogenesis in patients with RA.

Effectiveness of anti-folate receptor beta antibody conjugated with truncated Pseudomonas exotoxin in the targeting of rheumatoid arthritis synovial macrophages.

OBJECTIVE: To define the distribution of folate receptor beta (FRbeta)-expressing cells in various tissues, including rheumatoid arthritis (RA) synovial tissues, and to verify the effects of an immunotoxin composed of an anti-FRbeta monoclonal antibody (mAb) and truncated Pseudomonas exotoxin A (PEA) on apoptosis and tumor necrosis factor alpha (TNFalpha) production by adherent synovial mononuclear cells from RA patients. METHODS: Anti-FRbeta mAb were produced by immunizing mice with FRbeta-transfected murine pre-B cells. The distribution of the FRbeta antigen was examined by immunohistochemical analysis using anti-FRbeta mAb and macrophage-specific anti-CD163 mAb. Anti-FRbeta mAb was chemically crosslinked with truncated PEA. FRbeta-expressing macrophages were produced by the transfection of adenovirus vector containing the FRbeta gene. Apoptotic cells were detected by staining with propidium iodide. TNFalpha was measured by enzyme-linked immunosorbent assay. RESULTS: FRbeta-expressing cells were not present in peripheral blood leukocytes and their activated cells. In all of the tissues examined, most FRbeta-expressing cells were CD163+. The immunotoxin significantly induced the apoptosis of FRbeta-transfected macrophages and adherent RA synovial mononuclear cells and inhibited TNFalpha production by adherent RA synovial mononuclear cells. CONCLUSION: We demonstrated the limited distribution of FRbeta-expressing cells in various tissues. The immunotoxin targeting FRbeta-expressing cells will provide a therapeutic tool for rheumatoid synovitis.