A.J. de Brum-Fernandes

A comparative study of the antipyretic effects of indomethacin and dipyrone in rats

Abstract. Objective: Compare the antipyretic effects of dipyrone and indomethacin.¶Materials and methods: Fever was induced in rats by i.v. LPS or i.c.v. interleukins (IL), prostaglandins (PG), arachidonic acid (AA), pre-formed pyrogenic factor (PFPF), tumour necrosis factor-α (TNF-α) or corticotrophin releasing hormone (CRH). Dipyrone and indomethacin were administered i.p., arginine vasopressin V1-receptor antagonist, d(CH2)5 Tyr(Me)AVP, into the ventral septal area. Cyclooxygenase (COX-1/-2) blocking activity was assessed in transfected COS-7 cells. CRH release from isolated hypothalami was determined by ELISA.¶Results: Indomethacin or dipyrone reduced LPS, IL-1β, IL-6 or TNF-α induced fever and CRH release from rat hypothalamus. Only dipyrone inhibited IL-8, PFPF or PGF2α fever. Only indomethacin inhibited fever induced by AA or IL-1β plus AA. Neither antipyretic affected fever caused by PGE2 or CRH. d(CH2)5Tyr(Me)AVP only blocked antipyresis induced by indomethacin. Dipyrone at a very high concentration (10 mM) inhibited only COX-1, while indomethacin (0.1 μM) blocked COX-1 and COX-2 in COS-7 cells.¶Conclusion: The antipyretic effect of dipyrone differs from that of indomethacin in that it does not depend on AVP release or inhibition of PG synthesis.

Systematic pharmacological approach to the characterization of NSAIDs.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of inflammatory diseases. NSAIDs inhibit cyclooxygenase (COX), the rate limiting enzyme responsible for the conversion of arachidonic acid into prostaglandins. Recent studies have shown the existence of two isoforms of cyclooxygenase: COX-1, now often referred to as the constitutive form, and COX-2, an inducible form which is the major isoenzyme involved in prostaglandin synthesis in inflammation and other pathological situations. Since inhibition of prostaglandin production in tissues where they play a physiological role leads to important side effects, a COX-2 preferential inhibitor would present therapeutical advantages. In the present study, we evaluated the inhibitory properties of cyclooxygenase inhibitors on human COX-1 and COX-2 using a heterologous expression system. We investigated instantaneous inhibition and pre-incubation inhibition as well as time recovery of cyclooxygenase activity assays with the aid of four NSAIDs: mefenamic acid, indomethacin, aspirin and NS-398. Our results demonstrate that instantaneous inhibition assays have little correlation with clinical results. Inhibition assays using pre-incubation with the drugs tested, however, more closely resemble the data from in vivo studies. Cyclooxygenase recovery assays enabled better characterization of simple competitive inhibitors, competitive reversible time-dependent inhibitors and irreversible time-dependent inhibitors. The data illustrate the usefulness of our system in allowing a better determination of the pharmacological characteristics of NSAIDs as well as permitting a comparison among different drugs.

Contribution of B2 receptors for bradykinin in Arthus reaction-induced plasma extravasation in wild-type or B2 transgenic knockout mice

The aim of the present study was to investigate the contribution of bradykinin (BK) B1 and B2 receptors in a model of type III hypersensitivity, the reverse passive Arthus reaction (RPA), in wild‐type mice and transgenic B2 knockout littermates. BK (10 μg mouse−1) or bovine serum albumin (0.5 mg mouse−1) induced a sustained Evans blue extravasation for more than 80 min in naive or rabbit anti‐bovine serum albumin‐treated mice (RPA model), respectively. The response to the two stimuli was prevented by the B2 receptor antagonist, HOE‐140, but not by [Leu8]desArg9‐BK (B1 receptor antagonist). In contrast to the wild‐type littermates, RPA and bradykinin were unable to trigger an increase in plasma extravasation in B2 knockout mice. Furthermore, endothelin‐1 (5 μg mouse−1) and a selective NK‐1 receptor agonist [Sar9,Met (O2)11]‐SP (20 μg mouse−1), triggered a significant increase in peritoneal plasma extravasation in both wild‐type and B2 knockout animals. A pretreatment with indomethacin (200 μg mouse−1) significantly reduced the RPA‐induced but not the BK‐induced increase in Evans blue extravasation. Furthermore, RPA, but not BK, triggered a significant indomethacin‐sensitive increase in peritoneal prostaglandin E2 content. Our results suggest a pivotal role for B2 receptors in the mechanism of plasma extravasation which occurs during the reverse passive Arthus reaction in the mouse. Moreover, our results suggest an important contribution of prostanoids in the plasma leakage mechanisms triggered by RPA but not by bradykinin.

Expression of recombinant human cyclooxygenase isoenzymes in transfected COS-7 cells in vitro and inhibition by tenoxicam, indomethacin and aspirin

The recent discovery of cyclooxygenase-2 (COX-2), an isoenzyme associated mainly with inflammation created the need to reevaluate cyclooxygenase inhibitors with reliable screening methods. In the present study we standardized a technique to determine the IC50S of cyclooxygenase inhibitors on recombinant human COX-1 and COX-2 expressed in mammalian cells and used it to study the compounds tenoxicam, aspirin and indomethacin. The IC50S of aspirin, indomethacin and tenoxicam for human COX-1 were 0.41 +/- 0.07 microgram/ml, 0.008 +/- 0.003 microgram/ml, and 7.94 +/- 3.28 micrograms/ml, respectively, and for human COX-20.64 +/- 0.16 microgram/ml, 0.09 +/- 0.05 microgram/ml, and 10.61 +/- 1.50 micrograms/ml, for aspirin, indomethacin, and tenoxicam. Tenoxicam had the lowest IC50hCOX-2/IC50hCOX-1 ratio (1.34), followed by aspirin (1.53) and indomethacin (10.82). The system described in the present study provides a simple and efficient way to determine the specificity of NSAID inhibition for each of the human cyclooxygenase isoenzymes separately.

Leukotriene B4 binding sites in guinea-pig alveolar macrophages

Leukotriene B4 binding sites were investigated in alveolar macrophages obtained from guinea-pigs by brochoalveolar lavage. Analysis of the binding data was compatible with a two-receptors model. Best-fit computer-assisted evaluation of the results yielded a KD = 0.33 +/- 0.18 nM with 618 +/- 138 binding sites/cell for the high-affinity receptor, and KD = 52.9 +/- 12.3 nM with 95,400 +/- 37,900 sites/cell for the low-affinity binding site. Study of the dissociation rate of labelled ligand induced by dilution only and by dilution plus excess unlabelled ligand showed no differences in the two situations. These data suggest that the finding of two receptors is not due to negative cooperativity. Since most studies failed to demonstrate two distinct LTB4-binding proteins, the present results reinforces the hypothesis of LTB4 receptors in guinea-pig alveolar macrophages being a single protein with interchangeable affinity states.

OP0243 Single Nucleotide Polymorphisms (SNP) in The IL-4 Receptor, IL-4, and CD40 Loci and Outcome Prediction in Patients with Early Immune-Mediated Inflammatory Polyarthritis

Background Clinicians need reliable biomarkers of disease progression contributing additional information sufficient to personalize treatments in patients with early immune-mediated inflammatory polyarthritis (EPA) and rheumatoid arthritis. Some single nucleotide polymorphisms (SNP) in immune-related genes impact the expression of encoded proteins. Objectives To determine whether SNP in the Interleukin (IL)-4 Receptor (IL-4R), IL-4, and CD40 genes are associated with EPA disease outcomes. Methods We studied patients from the Early Undifferentiated PolyArthritis (EUPA) cohort. These patients were evaluated early (median 3.8 months), treated to remission and followed up at pre-specified intervals. Radiographs were scored according to Sharp/van der Heijde (SvH); significant damage was ≥5. SNPs were genotyped using an allele-specific primer extension (ASPE) assay and Luminex 100 analyzer. HLA-DR alleles were determined using the LABType rSSO assay (One Lambda). SNP and HLA-DR alleles were correlated to total (SvH ≥5) or erosive (Erosion ≥5) joint damage, functional incapacity (Modified Health Assessment Questionnaire (M-HAQ) ≥1.0) and remission according to Simplified disease activity index (SDAI ≤3.3) using generalized estimating equation (GEE) with repeated measures over 5 years. Results were presented with relative risk (RR) and 95% confidence intervals (CI). General linear model (GLM) with repeated measures was used for continuous outcomes. Results The allelic distribution for the SNPs is presented in Table. IL-4 SNPs rs2243250 and rs2070874 were strongly correlated (r2=0.99); only rs2243250 is presented. The (G) allele at rs1801275 (IL-4R) was significantly associated with decreased radiographic damage (GLM: Estimate (standard error): 0.035 (0.017), p=0.043). A (T) allele at rs2243250 (IL-4) was positively associated with erosive damage (GEE: RR 1.26 (1.02–1.55), p=0.034) and functional incapacity (M-HAQ ≥1.0; GEE: RR 1.25 (1.00–1.55), p=0.048). A (T) allele at rs4810485 (CD40) was associated with a higher likelihood of reaching SDAI remission (GEE: RR 1.36 (1.13–1.64), p=0.001). As previously published1, the presence of at least one copy of the DERAA protective alleles was significantly associated with decreased erosive damage (GEE: RR 0.75 (0.58–0.96), p=0.02), a trend to decreased functional incapacity (GLM: M-HAQ ≥1.0; RR: 0.79 (0.61–1.02), p=0.07) and increased chance of SDAI remission (GEE: RR 1.19 (1.01–1.4), p=0.04). Combined presence of at least one copy of the DERAA alleles and two copies of rs2243250 (C) allele of IL-4 further reduced erosive damage (GEE: RR 0.67 (0.51–0.90), p=0.007), functional incapacity (GLM: RR 0.75 (0.56–1.00), p=0.051) and slightly increased the chance for SDAI remission (GEE: RR 1.21 (1.02–1.44), p=0.03). Conclusions Genotyping the SNP rs1801275 (IL-4R), rs2243250 (IL-4), rs1883832/rs4810485 (CD40) and determining the HLA-DR allele of EUPA patients provided a set of stable genetic predictors of disease progression, both clinically and on radiographs. Their association together and with other stable predictors may contribute to develop reliable biomarker signatures suggesting significant associations with poor disease outcomes. References Carrier N et al. Arthritis Rheum 2009;60:698–707 Disclosure of Interest None declared

SAT0041 14-3-3Eta Predicts Radiographic Progression in Recent-Onset Polyarthritis Patients

Background Early RA diagnosis and timely treatment can improve patient outcomes. Markers are needed to identify patients with early disease who are at high-risk of joint damage progression. Current clinical and serological variables only explain approximately 32% of the risk1, underscoring the need for new disease specific markers that add to the rheumatologists prognostic armamentarium. 14-3-3η is a mechanistic, joint-derived, serological marker that potently induces cytokines and joint damage factors, directly implicating it in RA disease pathophysiology. Objectives The aim of this study was to determine whether 14-3-3η serum levels are predictive of joint damage progression in this recent-onset polyarthritis cohort. Methods Serum 14-3-3η levels were measured at baseline in 33 patients with recent-onset polyarthritis (EPA) from the Sherbrooke EUPA cohort. Patients were rapidly treated with conventional DMARDs to achieve clinical remission; 3 also received biologic agents. Radiographic progression was defined as a change in Sharp/van der Heijde score (ΔSHS) ≥1 at 30 months. Differences in median 14-3-3η between progressors and non-progressors were analyzed by 2-tailed Mann-Whitney U-test. The relationship between serum 14-3-3η and radiographic progression was investigated by univariate analysis using 14-3-3η cut-offs selected as follows: the manufacturers (Augurex 14-3-3η ELISA) reported diagnostic cut-off ≥0.19 ng/ml and 2X that,0.40 ng/ml. Stepwise multivariate analyses were performed to determine 14-3-3ηs contribution to predicting joint damage progression amongst other clinical and serological variables, including titres of 14-3-3η, RF, CCP, CRP, ESR, together with age, gender and disease duration. Results Median age was 51 years and 70% were female. Twenty of the 33 (61%) patients progressed after 30 months while 13 did not. Progressors had a median (IQR) ΔSHS of 7.0 (3.3-12.8). 14-3-3η and RF median (IQR) levels at baseline were significantly higher in progressors than non-progressors [2.7ng/ml (0.1-15.9) vs. 0.1ng/ml (0.1–0.2), p=0.006] and [160 IU/ml (40-320) vs. 0 IU/ml (0–160), p=0.006]. Univariate analyses revealed that RF positivity was associated with radiographic progression; relative risk (RR) of 2.8 (95%CI: 1.1-7.6), p=0.035. 14-3-3η, at both cut-offs, was also associated with radiographic progression; ≥0.19 ng/ml, RR=2.0 (1.1-3.7), p=0.02; ≥0.40 ng/ml, and RR=2.2 (1.2-4.1), p=0.006, respectively. 14-3-3η titres were associated with joint damage progression, LR of 5.2, p=0.02. Stepwise multivariate analysis returned 14-3-3η titres (LR=5.6, p=0.02), ESR (LR=6.4, p=0.01), CRP (LR=4.6, p=0.03) and gender (LR=4.4, p=0.04) as independent predictors of radiographic progression, together informing 29.4% of the total variance (R2) in radiographic progression. When 14-3-3η titres were excluded, the R2 for ESR, CRP and gender was 16.8%, indicating that of the 4 significant variables, 14-3-3η accounted for 43% (12.6%/29.4%) of the models predictive power. Conclusions Serum 14-3-3η in recent-onset polyarthritis was associated with an increased risk of joint damage progression at 30 months. When added to current prognostic markers, is 14-3-3η an independent predictor of radiographic progression adding 43% to their prognostic predictive power. References de Rooy DPC et al. Rheum. 2011. 50:93. Disclosure of Interest G. Boire: None declared, N. Carrier: None declared, A. de Brum-Fernandes: None declared, P. Liang: None declared, A. Masetto: None declared, Y. Gui Employee of: Augurex Life Sciences Corp, M. Murphy Employee of: Augurex Life Sciences Corp, W. Maksymowych Consultant for: Augurex Life Sciences Corp, A. Marotta Employee of: Augurex Life Sciences Corp DOI 10.1136/annrheumdis-2014-eular.3299