Masaki Nakamura

The new sepsis marker, sCD14-ST, induction mechanism in the rabbit sepsis models

Soluble CD14 subtype (sCD14-ST) is a fragment of CD14 and is markedly increased in sepsis patients. We developed a new immunoassay to detect sCD14-ST and evaluated the efficacy of this marker for diagnosis of sepsis. For developing the strategies of sCD14-ST as a sepsis diagnostic marker, the induction mechanism must be known.

The novel Nrf2 inducer TFM-735 ameliorates experimental autoimmune encephalomyelitis in mice

ABSTRACT The transcription factor NF‐E2‐related factor 2 (Nrf2) is a key regulator of cellular defense mechanisms against oxidative stress. Multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system, is characterized by progressive demyelination and neurodegeneration induced by inflammation and oxidative stress. The induction of Nrf2 signaling has been shown to inhibit disease development and progression in the experimental autoimmune encephalomyelitis (EAE) model of MS in mice. In the present study, we performed a high‐throughput screening assay using a chimeric construct of the N‐terminal portion of Nrf2 fused to LacZ. Using this approach, we identified the novel Nrf2 inducer TFM‐735. Using human primary cell profiling systems, we found that TFM‐735 inhibited T cell proliferation and exerted immuno‐modulatory effects by inhibiting the production of IL‐6 and IL‐17. TFM‐735 also inhibited IL‐17 secretion from human peripheral blood mononuclear cells stimulated with anti‐CD3 and anti‐CD28. In EAE mice treated with TFM‐735, the expression of the Nrf2 target gene Nqo1 increased in the brain and spleen, disease severity was ameliorated, and plasma IL‐17 levels decreased. Furthermore, TFM‐735 inhibited luciferase activity in Wim‐6 transgenic EAE mice expressing the human interleukin 6‐luciferase (hIL6‐BAC‐Luc) reporter. Therefore, these findings indicate that TFM‐735 is a potent Nrf2 inducer that inhibits inflammatory cytokine production and disease progression in mice with EAE and that TFM‐735 is a promising therapeutic agent for MS.

A novel free fatty acid receptor 1 (GPR40/FFAR1) agonist, MR1704, enhances glucose‐dependent insulin secretion and improves glucose homeostasis in rats

Activation of G protein‐coupled receptor 40/Free fatty acid receptor 1 (GPR40/FFAR1), which is highly expressed in pancreatic β cells, is considered an important pharmacologic target for the treatment of type 2 diabetes mellitus. The aim of this study was to determine the effect of MR1704, a novel GPR40/FFAR1 agonist, on glucose homeostasis in rats. MR1704 is a highly potent and selective, orally bioavailable agonist with similar in vitro potencies among humans, mice, and rats. Treatment of rat islets with MR1704 increased glucose‐dependent insulin secretion. Augmentation of glucose‐dependent insulin secretion was abolished by adding a GPR40/FFAR1 antagonist. In mouse, insulinoma MIN6 cells, palmitic acid induced the activity of caspase 3/7 after a 72‐h exposure, while pharmacologically active concentrations of MR1704 did not. In an oral glucose tolerance test in normal Sprague‐Dawley rats, orally administered MR1704 (1–10 mg·kg−1) reduced plasma glucose excursion and enhanced insulin secretion, but MR1704 did not induce hypoglycemia, even at 300 mg·kg−1, in fasted Sprague‐Dawley rats. In addition, orally administered MR1704 reduced plasma glucose excursion and enhanced insulin secretion in diabetic Goto‐Kakizaki rats. Oral administration of MR1704 once daily to Goto‐Kakizaki rats reduced their blood glucose levels during a 5‐week treatment period without reducing pancreatic insulin content; as a result, hemoglobin A1C levels significantly decreased. These results suggest that MR1704 improves glucose homeostasis through glucose‐dependent insulin secretion with a low risk of hypoglycemia and pancreatic toxicity. MR1704 shows promise as a new, glucose‐lowering drug to treat type 2 diabetes mellitus.

Anti-human CD14 monoclonal antibody improves survival following sepsis induced by endotoxin, but not following polymicrobial infection

Abstract Cluster of differentiation 14 (CD14), a pattern recognition receptor expressed on myeloid cells and a critical component of the innate immune system, mediates local and systemic host responses to gram‐negative bacterial products, including lipopolysaccharide (LPS). Therefore, CD14 is an attractive target for development of sepsis therapies, and several monoclonal anti‐CD14 antibodies have been reported. In this study, we prepared an anti‐human CD14 monoclonal antibody, F1024‐1‐3, which suppressed LPS‐induced upregulation of pro‐inflammatory cytokines and an adhesion molecule in human peripheral mononuclear cells and human vascular endothelial cells. Half‐maximal inhibitory concentrations in these assays ranged from 0.1 to 1 &mgr;g/ml. In rabbits, intravenous administration (3 mg/kg) as well as in vitro exposure of F1024‐1‐3 suppressed LPS‐induced cytokine production in whole blood. In endotoxemia models generated by three sequential injections of LPS, intravenous administration of F1024‐1‐3 at 0.3–3 mg/kg sharply reduced pro‐inflammatory responses in a dose‐dependent manner and moderately attenuated pro‐coagulant responses; at 1 mg/kg, the protein protected rabbits from lethality even when administered 2 h after the initial LPS injection. However, F1024‐1‐3 (10 mg/kg) given 2 h post‐surgery did not prevent death of rabbits in a cecal ligation and puncture model. Thus, suppression of CD14‐mediated activation of leukocytes and endothelial cells alone may not be clinically efficacious for the treatment of severe sepsis and septic shock.

Simultaneous targeting of CD14 and factor XIa by a fusion protein consisting of an anti-CD14 antibody and the modified second domain of bikunin improves survival in rabbit sepsis models

ABSTRACT Severe sepsis is a complex, multifactorial, and rapidly progressing disease characterized by excessive inflammation and coagulation following bacterial infection. To simultaneously suppress pro‐inflammatory and pro‐coagulant responses, we genetically engineered a novel fusion protein (MR1007) consisting of an anti‐CD14 antibody and the modified second domain of bikunin, and evaluated the potential of MR1007 as an anti‐sepsis agent. Suppressive effects of MR1007 on lipopolysaccharide (LPS)‐induced inflammatory responses were assessed using peripheral blood mononuclear cells or endothelial cells. Its inhibitory activity against the coagulation factor XIa was assessed using a purified enzyme and a chromogenic substrate. Anticoagulant activity was assessed using human or rabbit plasma. Anti‐inflammatory and anti‐coagulant effects and/or survival benefits were evaluated in an endotoxemia model and a cecal ligation and puncture model. MR1007 inhibited LPS‐induced cytokine production in peripheral blood mononuclear cells and endothelial cells, inhibited factor XIa, and exhibited anticoagulant activity. In an endotoxemia model, 0.3–3 mg/kg MR1007 suppressed pro‐inflammatory and pro‐coagulant responses in a dose‐dependent manner; at a dose of 3 mg/kg, the protein improved survival even when administered 8 h after the LPS injection. In addition, 10 mg/kg MR1007 administered 2 h post cecal ligation and puncture improved survival. However, MR1007 administered at doses up to 30 mg/kg did not increase ear bleeding time or bacterial counts in the cecal ligation and puncture model. Thus, simultaneous targeting of CD14 and factor XIa improves survival in the rabbit endotoxemia and sepsis models and represents a promising approach for the treatment of severe sepsis.

Free fatty acid receptor 1 agonist, MR1704, lowers blood glucose levels in rats unresponsive to the sulfonylurea, glibenclamide

Preclinical Research & Development