Nonclinical Characterization of the Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor Roxadustat, a Novel Treatment of Anemia of Chronic Kidney Disease.

Anemia of chronic kidney disease (CKD) is a multifactorial disorder caused by impaired erythropoietin (EPO) production and altered iron homeostasis associated with inflammation. Hypoxia-inducible factor (HIF) is a transcription factor that stimulates erythropoiesis via a coordinated response involving increased EPO production and enhanced iron availability for Hb synthesis. HIF degradation is regulated by HIF-prolyl hydroxylase (HIF-PH) enzymes. We hypothesized that roxadustat, an orally available small-molecule inhibitor of HIF-PH, would increase EPO production and promote erythropoiesis in animal models of anemia. In cells, roxadustat increased both HIF-1α and HIF-2α proteins, leading to an increase in EPO production, even in the presence of EPO-suppressing inflammatory cytokines. Roxadustat administered intermittently to healthy rats and cynomolgus monkeys increased circulating EPO levels, reticulocytes, blood Hb, and hematocrit in a dose-dependent manner. Roxadustat corrected anemia in a rat model of CKD after five-sixth nephrectomy and in a rat model of anemia of inflammation with impaired iron metabolism induced by peptidoglycan-polysaccharide (PG-PS). In the PG-PS model, roxadustat significantly decreased hepatic expression of hepcidin, a hormone responsible for iron sequestration and functional iron deficiency, and increased expression of two genes involved in duodenal iron absorption: divalent metal transporter 1 and duodenal cytochrome b. In conclusion, by activating the HIF pathway, roxadustat increased EPO production, elevated Hb, corrected anemia, and improved iron homeostasis. The coordinated erythropoietic response stimulated by roxadustat, involving both EPO production and mobilization of iron stores, makes this compound a promising treatment of anemia of CKD and anemia associated with functional iron deficiency. SIGNIFICANCE STATEMENT: Roxadustat is a novel orally available small-molecule inhibitor of HIF prolyl hydroxylase enzymes that reversibly stabilizes HIF-α, thus activating transcription of HIF-dependent genes, including EPO and regulators of iron homeostasis. Activation of the HIF pathway by roxadustat induces erythropoiesis in healthy rats and monkeys and corrects experimentally induced anemia in rats. The coordinated erythropoietic response that increases EPO production and mobilizes iron stores makes roxadustat a promising treatment for anemia of chronic kidney disease and anemia associated with functional iron deficiency.

Evaluation of the Carcinogenic Potential of Roxadustat (FG-4592), a Small Molecule Inhibitor of Hypoxia-Inducible Factor Prolyl Hydroxylase in CD-1 Mice and Sprague Dawley Rats.

The carcinogenic potential of roxadustat (FG-4592), a novel orally active, heterocyclic small molecule inhibitor of hypoxia-inducible factor prolyl hydroxylase (HIF-PH) enzymes in clinical development for treatment of anemia, was evaluated in CD-1 mice and Sprague Dawley rats. Inhibition of HIF-PH by roxadustat leads to a rapid increase in cytoplasmic HIF-α concentrations, followed by translocation of HIF-α to the nucleus and upregulation of HIF-responsive genes, including erythropoietin. Roxadustat was dosed by oral gavage 3 times weekly (TIW) for up to 104 weeks in mice at 0, 15, 30, and 60 mg/kg and in rats at 0, 2.5, 5, and 10 mg/kg. Treatment-associated changes in hematology parameters were consistent with the pharmacologic activity of roxadustat and included elevations in hematocrit in mice at 30 and 60 mg/kg TIW and elevations in erythrocyte count, hemoglobin, hematocrit, and red cell distribution width in rats at 10 mg/kg TIW. No increase in mortality or neoplastic effects compared with vehicle controls was observed after roxadustat treatment in either species. No treatment-related nonneoplastic findings were observed in mice, whereas nonneoplastic microscopic findings in rats were limited to atrial/aortic thromboses at 10 mg/kg TIW males and bone marrow hypercellularity in all treated male and female groups, consistent with the pharmacology of roxadustat. In conclusion, roxadustat administered by oral gavage to mice and rats TIW for up to 104 weeks resulted in dose-dependent exposure and hematologic effects with no effect on survival or development of neoplastic lesions at up to 60 mg/kg in mice and up to 10 mg/kg in rats.

A small-molecule inhibitor of hypoxia-inducible factor prolyl hydroxylase improves obesity, nephropathy and cardiomyopathy in obese ZSF1 rats.

Prolyl hydroxylase (PH) enzymes control the degradation of hypoxia-inducible factor (HIF), a transcription factor known to regulate erythropoiesis, angiogenesis, glucose metabolism, cell proliferation, and apoptosis. HIF-PH inhibitors (HIF-PHIs) correct anemia in patients with renal disease and in animal models of anemia and kidney disease. However, the effects of HIF-PHIs on comorbidities associated with kidney disease remain largely unknown. We evaluated the effects of the HIF-PHI FG-2216 in obese ZSF1 (Ob-ZSF1) rats, an established model of kidney failure with metabolic syndrome. Following unilateral nephrectomy (Nx) at 8 weeks of age, rats were treated with 40 mg/kg FG-2216 or vehicle by oral gavage three times per week for up to 18 weeks. FG-2216 corrected blood hemoglobin levels and improved kidney function and histopathology in Nx-Ob-ZSF1 rats by increasing the glomerular filtration rate, decreasing proteinuria, and reducing peritubular fibrosis, tubular damage, glomerulosclerosis and mesangial expansion. FG-2216 increased renal glucose excretion and decreased body weight, fat pad weight, and serum cholesterol in Nx-Ob-ZSF1 rats. Additionally, FG-2216 corrected hypertension, improved diastolic and systolic heart function, and reduced cardiac hypertrophy and fibrosis. In conclusion, the HIF-PHI FG-2216 improved renal and cardiovascular outcomes, and reduced obesity in a rat model of kidney disease with metabolic syndrome. Thus, in addition to correcting anemia, HIF-PHIs may provide renal and cardiac protection to patients suffering from kidney disease with metabolic syndrome.

Assessment of coronary stenoses of graded severity by myocardial contrast echocardiography.

BACKGROUND: Myocardial contrast echocardiography (MCE) has potential value in the assessment and quantitation of myocardial perfusion defects. However, the severity of stenosis detectable by MCE and its diagnostic accuracy remain undefined. Thus, we produced coronary stenoses of variable severity and quantified their effect on MCE. METHODS AND RESULTS: Three grades of left anterior descending (LAD) obstructions were produced in 7 open-chest swine. The stenoses were nonflow-limiting at rest, but decreased coronary hyperemia by 31.3% +/- 4.7%, 69.9% +/- 5.3% and 98.9% +/- 1.1%, respectively. Regional myocardial blood flow (RBF) was measured with fluorescent microspheres and was expressed as the ratio of LAD and control (LCx) beds. MCE was performed with 0.3 mg/kg intravenous AF0150 during ECG-gated harmonic imaging in short-axis view. Background-subtracted peak intensity (PI) was expressed as the ratio of LAD/LCx beds. Both RBF and PI ratios progressively decreased with increasing grades of stenosis. MCE showed a significant correlation with RBF (r = 0.74; P <.0001). Ratios of both PI and RBF differed significantly from baseline when coronary hyperemia was reduced more than 50%. An LAD/LCx ratio less than 0.6 by MCE yielded 61% and 83% sensitivity and 85% and 76% specificity with stenosis that reduced coronary hyperemia more than 50% and more than 75%, respectively. CONCLUSION: MCE with intravenous AF0150 during vasodilation correctly depicted the progressive reduction of flow ratios produced by graded coronary stenoses. A significant reduction of PI ratio was observed with stenosis causing more than 50% reduction of coronary hyperemia. An MCE ratio in stenosed/control beds could be selected, which exhibited good sensitivity and specificity in the identification of coronary stenosis.