| Budget Amount *help |
¥48,360,000 (Direct Cost: ¥37,200,000、Indirect Cost: ¥11,160,000)
Fiscal Year 2006: ¥17,290,000 (Direct Cost: ¥13,300,000、Indirect Cost: ¥3,990,000)
Fiscal Year 2005: ¥31,070,000 (Direct Cost: ¥23,900,000、Indirect Cost: ¥7,170,000)
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| Research Abstract |
Oxygen supply declines in many human diseases including ischemic heart disease, kidney diseases such as diabetic nephropathy, and stroke. The resulting hypoxia causes functional impairment of cells as well as structural tissue damage, and triggers a broad spectrum of cellular defenses such as angiogenesis, erythropoiesis, glycolysis, and anti-oxidative enzymes. Hypoxia inducible factor (HIF) plays a pivotal role in the adaptation to hypoxia and in hypoxic tissue injury. Its activity is modulated by an oxygen-dependent hydroxylation of proline residues by prolyl hydroxylases (PHD).
We evaluate the effects of long-term stimulation of HIF in diabetic nephropathy (DN). For this, we utilized an hypertensive, type 2 diabetic rat model with nephropathy (SHR/NDmcr-cp rat) and cobalt chloride (Co), a compound known to inhibit PHD and enhance the HIF activity. Co, given for 26 weeks in rats, did not correct obesity, hyperglycemia, hyperlipidemia, or hypertension but reduced proteinuria as well as
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histological kidney injury. Immunohistological analysis demonstrated that cobalt up-regulated renal HIF-1 expression and Increased the expression of HIF-regulated genes, including erythropoietin, vascular endothelial growth factor, and heme oxygenase (HO)-1. Co thus protects the kidney in a type 2 diabetic rat model, independently of metabolic status and blood pressure.
Because Co is very toxic and can not be used in clinical medicine. More safe, specific inhibitors of PHD might thus prove therapeutic. We have therefore searched for non-toxic low-molecular-weight PHD Inhibitors by a novel approach of combination of virtual screening based on the three-dimensional structure of PHD by structure based drug design (SBDD), biological in vitro screening and combinatorial chemistry. Three unique compounds (TM6008, TM6010 and TM6089) were discovered in the course of this study. SBDD and docking simulations demonstrated that these compounds could preferentially bind to the active site of PHD. Among the three compounds, TM6089 should be a unique PHD inhibitor because It inhibits the PHD activity without chelating irons (a potential side effect). In vitro TM6089 stabilized HIF activity by preventing the interaction between PHD and HIF. In vivo TM6089 enhanced angiogenesis in the subcutaneously implanted sponge after the local administration, and stimulated HIF activities in various organs of a hypoxia-sensing transgenic rat expressing a hypoxia-responsive reporter vector after oral administration. Furthermore, given orally, TM6089 rescued neurons from post-ischemic cell death in gerbil without any adverse effect.
In conclusion, we demonstrated pathological involvements of chronic hypoxia in several animal experiments, and identified, by a unique approach, a novel PHD inhibitor which ameliorates chronic hypoxia by enhancing HIF activity In vitro and in vivo. Less
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