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  • The small molecule d mannuronic acid


    The small molecule β-d-mannuronic carnosic acid mg (M2000), patented (DE-102016113018.4), has been tested as an anti-inflammatory and a novel immunosuppressive agent in various experimental models such as animal models of immune complex glomerulonephritis, nephrotic syndrome, multiple sclerosis and rheumatoid arthritis. In the immune complex glomerulonephritis and experimental nephrotic syndrome, it has been indicated that the administration of M2000 leads to a significant decrease in blood urea nitrogen (BUN), proteinuria, serum creatinine, and cholesterol, as well as a reduction in the glomerular lesion in M2000-treated rats [[25], [31]]. The immunosuppressive properties of M2000 could significantly lessen clinical scoring and histological lesion in the experimental autoimmune encephalomyelitis (EAE) model [32]. The results of lymph node cell proliferation assays in this model revealed the immunosuppressive efficacy of M2000. The oral and/or intraperitoneal (ip) administration of this drug considerably decreased edema and histopathological parameters in arthritic rats [33]. In another research on Wistar rats and NMRI mice, the outcomes in chronic and subchronic toxicity studies demonstrated no significant clinical or histopathological findings and the assessment of the hematological and biochemical indices illustrated no evidence of adverse effects systemically due to M2000 therapy. In addition, these results suggested that the oral administration of M2000 at level up to 1250 mg/kg BW/does, the highest tested dose, did not cause any adverse effects in animals. Therefore, the use of appropriate levels of this agent can be considered relatively safe for administration in human [29]. The tolerability and biocompatibility of WHI-164, as a sensitive cell line, against increasing quantities of M2000, diclofenac, piroxicam, and dexamethasone have been examined previously. Mirshafiey et al. showed that 50% of cells died when diclofenac, dexamethasone, and piroxicam were added to tissue culture at doses of 25 mg/ml, 80 mg/ml, and 80 mg/ml, respectively, whereas, the M2000 showed no cytotoxic effect with 200 mg/ml compared with steroidal and non-steroidal drugs tested. These pharmaco-toxicological studies showed that M2000 is the safest anti-inflammatory and immunosuppressive drug in comparison with dexamethasone and conventional NSAIDs tested [34]. Additionally, M2000 has no ulcerogenic effect on the rat stomach [34]. Our previous outcomes using the zymo analysis method indicated that M2000 was a potent matrix metalloproteinase (MMP) inhibitor, implicated in some inflammatory renal disorders and arthritis [[25], [31], [33]]. In an investigation, our findings showed that M2000 can be used as NSAID in LPS induced inflammation and decrease inflammatory cytokines production by targeting suppressor of cytokine signaling 1 (SOCS1), phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 1 (SHIP1) and microRNA-155 in auto-immune and inflammatory diseases [35]. Moreover, in another study, our results showed that M2000 has an antagonistic effect on TLR2 and TLR4 downstream signaling transduction pathway in HEK-Blue hTLR2 and hTLR4 cell lines. The M2000 effectively inhibited mRNA expression of MyD88 and p65, major subunit of NF-кB, in HEK-Blue hTLR2 and hTLR4 cells stimulated by lipoteichoic acid (LTA, a TLR2 agonist) and lipopolysaccharide (LPS, a TLR4 agonist) with no evidence of cytotoxicity [36]. At present, this anti-inflammatory drug has been tested in phase 1/2 clinical trial with the registered No IRCT2013062213739N1 in ankylosing spondylitis patients and phase 1/2 clinical trial with the registered No IRCT2014011213739N2 in rheumatoid arthritis patients with no any side effects, even without cardiovascular problems, which their results will soon be published, whereas cardiomyopathy could be on the most important NSAIDs side effects. Moreover, it is now being run two another clinical trials using M2000 on osteoarthritis and multiple sclerosis in Iranian patients with registered No (IRCT2014113013739N3, IRCT2016111313739N6), respectively. Since many studies have shown the important role of COX isoforms in inflammatory and autoimmune diseases, this research intended to study the effects of M2000 on the gene expression and activity of COX-1/COX-2 enzymes in order to introduce a novel NSAID for treating inflammatory diseases. The results showed that the low and high dose of this drug (2.5 and 12.5 mMol/ml) could significantly reduce the gene expression level of the COX-2 compared to the control-treated with LPS (Fig. 2), whereas, surprisingly no significant reduction was observed in the gene expression level of COX-1 compared to the control-treated with LPS (Fig. 3). Furthermore, the results demonstrated that this drug at the three concentrations 5, 50 and 500 mMol/ml was able to strongly decrease the activity of the COX1/COX-2 enzyme compared to the control-treated with LPS and AA (Fig. 4, middle panel) (Fig. 4, right panel). In this figure (Fig. 4, left panel) the cause of increasing level of PGE2 by only M2000 is due to the effect of its solvent (NaOH 27 mM) since this fact could be seen in vehicle graph in this panel.