A Class Of Drugs Carboxylase Amines Triazole Anti-inflammatory Effects In A Variety Of Models And Its Mechanism Research

BackgroundThe incidence of the autoimmune diseases, such as rheumatoid arthritis, non-specific inflammatory bowel disease, is increasing constantly in china cross past decades. However, we know little about its etiology and pathogenesis until now. Glucocorticoids, non-steroidal anti-inflammatory drugs are commonly used agents for the autoimmune diseases, but their long-term application is limited by adverse effects and resistance. There is no doubt that a potent agent with favorable safety profile will fulfill this unmet clinical need. Carboxyamidotriazole (CAI) is a compound that inhibits calcium influx and proliferation of tumor cell. CAI has been proven active against proliferation and metastasis in both in vitro and in vivo models. Its other activities, as reported by literatures, include anti-angiogenesis, inhibition of arachidonic acid release and NF-κB pathway. In our early studies, the potential anti-inflammatory activity of CAI was established in a battery of models that cover acute and chronic inflammation. This study is a step forward on our way to explore CAI's anti-inflammatory effect in inflammatory diseases such as ulcerative colitis and adjuvant arthritis. Animal models and in vitro testing systems were established for mechanism investigation, and CAI was canvassed in multiple levels from in vivo through molecular.MethodsRat adjuvant arthritis model was established and validated. The effect of CAI on primarily and secondary lesion was evaluated using criteria of paw volume, body weight, food consumption and disease activity score. After collecting blood samples, rats were sacrificed and synovial membrane was separated for HE staining. The specimens of serum or paw homogenate were subjected to ELISA for cytokines and mediators including:tumor necrosis factor alpha (TNF-a), interleukin 1β(IL-1β), interleukin 6 (IL-6), prostaglandin E2 (PGE2) and nitric oxide (NO). TNBS-induced colitis model of rat was established and validated. The effect of CAI was evaluated using criteria of mortality, body weight, food consumption, clinical score, macroscopic and microscopic finding. After collecting blood samples, rats were sacrificed and colon section was removed for HE staining and evaluation. The following parameters of serum or colon homogenate were determined:MPO activity, TNF-a level (ELISA), IL-6 level (ELISA), TGFβ1 level (ELISA), NO content, MDA concentration and SOD activity. IHC was employed to evaluate the status of NF-κB and ICAM-1 in colon tissues. Collagen accumulation in rat colon was evaluated by Masson's staining. For mechanism studies in peritoneal macrophages, the cytotoxicity of CAI was evaluated by MTT assay. ELISA was performed to quantify cytokines and mediators. Western Blot was conducted to investigate the expression and activation of inflammation-related proteins in peritoneal macrophages. The status of NF-κB was profiled by Immunocytochemistry. An in vitro neutral red assay was employed to evaluate the influence of CAI on phagocytosis. To address the concern about potential obstruction to normal immune system, CAI was tested in carbon clearance assay, CRBCs-induced hemolysin formation assay and DNFB-induced contact hypersensitivity assay.Results1. CAI (10,20 and 40 mg/kg) improved both primarily and secondary lesion in rat adjuvant arthritis model in a dose-dependent manner. In doses> 10 mg/kg (20 and 40 mg/kg), the effect of CAI on paw edema, body weight, food consumption and joint score was statistically significant when compared with vehicle. CAI (20 mg/kg) was of therapeutic value to those affected but not irreversible joint damage caused by arthritis. The combination of CAI (20 mg/kg) and dexamethasone has a superior efficacy over each alone, and a comparable safety profile with dexamethasone.2.20 mg/kg CAI had little effect on the PGE2 level of rat paw tissue, which was significantly decreased by dexamethasone (p<0.001). Both CAI (20 mg/kg) and dexamethasone suppressed NO level in rat paw tissue (p＜0.01 and p＜0.001, respectively).3. After treated with 20 mg/kg CAI, paw tissue TNF-a and IL-1βlevels decreased by 54.1% and 26.3% (both p<0.05), respectively. A similar repression effect was seen in dexamethasone group. These observations are consistent with what was found in serum samples.4. In the model validation study,75 mg/kg TNBS/EtOH was found to be the optimal dose to induce rat ulcerative colitis. The intestinal pathological changes in model animals were representative of human ulcerative colitis, including intestinal hyperemia, edema, mucosal erosion and ulceration and inflammatory cell infiltration (mainly neutrophils) and the crypt abscess formation. CAI (20,30 and 40 mg/kg) dose-dependently protected model animal from acute symptoms and fibrosis of colon. In CAI 30 mg/kg group and 40 mg/kg group, the improvement achieved was reflected in criteria of mortality, body weight, food consumption, clinical score, macroscopic finding and microscopic finding. The rat treated with CAI (dose≥30 mg/kg) showed alleviated enteremia and edema in colon and diminished mucous membrane lesions in macroscopic examination. Microscopy results revealed less severe telangiectasis, lymph foilicie hyperplasia, and submucosa edema and crypt disorganization compared with control.5. The activity of MPO in colon homogenate prepared from TNBS-treated rat was higher than normal control. The difference in MPO activity between CAI (40 mg/kg) group and control group was statistically significant (p<0.05), with a lower activity in CAI group.6. In 40 mg/kg CAI group, the TNF-a and IL-6 levels in colon homogenate were decreased by 52.68% (p＜0.01) and 57.15% (p<0.01), respectively. The inhibition rate of CAI on TNF-a was greater than that of SASP, but inferior to Infliximab. For IL-6, CAI was shown to be a more potent inhibitor than both SASP and Infliximab. The TNF-a and IL-6 levels in serum samples were also decreased after CAI treatment, with calculated inhibition rate of 75.35% (p＜0.001) and 32.57% (p＜0.01), respectively.7. After treatment with CAI (40 mg/kg), the MDA and NO contents in colon tissue collected from colitis rats were decreased by 25.97% and 41.58% (both p＜0.01, compared with PEG400), respectively. Meanwhile, SOD as measured by its activity, was 49.23% (p＜0.01) higher compared with PEG400.8. The results from IHC indicated that, the altered NF-κB p65 translocation and ICAM-1 expression in model rat colon tissue were significantly reversed in after CAI administration.9. In 40 mg/kg CAI group, the content of TGF-β1 in colon homogenate was significant lower than control group (p<0.01). Results from Masson's stain suggested CAI is active in blocking collagen fiber accumulation in colon.10. Irrespective of the origin of the macrophages (source from normal rats or adjuvant arthritis), CAI did not affect the vitality nor the phagocytosis of the macrophages.11. In in vitro studies focusing on macrophages, the TNF-a concentration in the conditioned medium of normal rat macrophages was significantly decreased after 20 and 40μmol/L CAI treatment (for 18 h; p<0.05 and p＜0.01, respectively). Similar effect was observed for IL-6 and INF-γ. For 10,20 and 40μmol/L CAI, the significance of inhibition to IL-6 was p<0.05, p＜0.01 and p＜0.001, respectively. The significance of inhibition to INF-y was p<0.05, p＜0.01 and p<0.01, respectively. In studies of same design but with macrophages from adjuvant arthritis rats, the effect of CAI (20 mg/kg) on TNF-a, IL-6 and INF-y levels in conditioned medium of macrophages was consistent with what was established with normal rat macrophages. The inhibition rate was 63.56% (p＜0.01),14.52% (p<0.05) and 80.81% (p＜0.01), for TNF-a, IL-6 and INF-y respectively. Although elevated IL-10 level in the CAI group was evident, this result was not significantly different from the PEG400 group. The production of TNF-a and IL-6 in macrophages from TNBS-induced colitis rats was significantly diminished by CAI (40 mg/kg) with inhibition rate of 77.05% and 56.66%, respectively (both p<0.001).12. NO production (elicited by LPS stimulation) of normal rat macrophages was down-regulated after 18-hr incubation with 5,10,20 and 40μmol/L CAI in a dose dependent manner, as compared with control group. A similar inhibition effect of CAI was observed on iNOS expression and NO production with correlation between each other. For peritoneal macrophages collected from adjuvant arthritis rats, the difference between CAI group (20 mg/kg) and PEG400 group on the PGE2 or COX-2 content was negligible. The data on NO production and iNOS expression was consistent with previous findings made on normal rat macrophages.13. In normal rat peritoneal macrophages, NF-κB p65 staining was found to be present in both nuclei and cytoplasm in DMSO-treated (control) group, while barely distinguishable in nuclei and very weak in cytoplasm in 20μmol/L CAI-treated group; indicating a blockade of p65 translocation by CAI.5,10,20 and 40μmol/L CAI was added into rat peritoneal macrophages and incubated for 18 hrs followed by LPS stimulation. The degradation of IκBa and formation of pIκBa induced by LPS in peritoneal macrophages can be significantly inhibited by CAI in a dose-dependent manner. The p65 translocation, IκBa degradation and pIκBa formation were also repressed by CAI (20 mg/kg) in adjuvant arthritis rat's peritoneal macrophages. CAI (20 mg/kg) also inhibited the phosphorylation of JNK and p38 MAPK in adjuvant arthritis rat's peritoneal macrophages, rather than the expression of JNK and p38.14. CAI at a dose of 20 mg/kg was proven to be neutral in following in vitro tests: carbon clearance assay, CRBCs-induced hemolysin formation assay and DNFB-induced contact hypersensitivity assay.Conclusions CAI was proven to be effective to adjuvant arthritis and TNBS-induced ulcerative colitis. As evidenced by data collected in in vivo and in vitro studies, the effect of CAI includes:NF-κB inhibition, active oxygen clearance and inflammatory cytokine (such as TNF-a and IL-6) suppression. For the macrophages, the phagocytosis will not be disturbed when CAI inhibits macrophage's secretion of cytokines, which is the fuel of inflammation. The anti-inflammatory activity of CAI is free from immusupperession and quite different from classic NSAIDs in terms of mechanism. In summary, the data presented herein highlight CAI a promising oral available and economical compound that is likely to become a rising star in therapy of autoimmune diseases.