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.

Gastrointestinal Inhibition of Sodium-Hydrogen Exchanger 3 Reduces Phosphorus Absorption and Protects against Vascular Calcification in CKD.

In CKD, phosphate retention arising from diminished GFR is a key early step in a pathologic cascade leading to hyperthyroidism, metabolic bone disease, vascular calcification, and cardiovascular mortality. Tenapanor, a minimally systemically available inhibitor of the intestinal sodium-hydrogen exchanger 3, is being evaluated in clinical trials for its potential to (1) lower gastrointestinal sodium absorption, (2) improve fluid overload-related symptoms, such as hypertension and proteinuria, in patients with CKD, and (3) reduce interdialytic weight gain and intradialytic hypotension in ESRD. Here, we report the effects of tenapanor on dietary phosphorous absorption. Oral administration of tenapanor or other intestinal sodium-hydrogen exchanger 3 inhibitors increased fecal phosphorus, decreased urine phosphorus excretion, and reduced [(33)P]orthophosphate uptake in rats. In a rat model of CKD and vascular calcification, tenapanor reduced sodium and phosphorus absorption and significantly decreased ectopic calcification, serum creatinine and serum phosphorus levels, circulating phosphaturic hormone fibroblast growth factor-23 levels, and heart mass. These results indicate that tenapanor is an effective inhibitor of dietary phosphorus absorption and suggest a new approach to phosphate management in renal disease and associated mineral disorders.

HIF prolyl hydroxylase inhibition results in endogenous erythropoietin induction, erythrocytosis, and modest fetal hemoglobin expression in rhesus macaques.

The hypoxia-inducible factor (HIF) pathway is crucial in mitigating the deleterious effects of oxygen deprivation. HIF-alpha is an essential component of the oxygen-sensing mechanisms and under normoxic conditions is targeted for degradation via hydroxylation by HIF-prolyl hydroxylases. Several HIF-prolyl hydroxylase inhibitors (PHIs) induced erythropoietin (epo) expression in vitro and in mice, with peak epo expression ranging from 5.6- to 207-fold above control animals. Furthermore, several PHIs induced fetal hemoglobin (HbF) expression in primary human erythroid cells in vitro, as determined by flow cytometry. One PHI, FG-2216, was further tested in a nonhuman primate model without and with chronic phlebotomy. FG-2216 was orally bioavailable and induced significant and reversible Epo induction in vivo (82- to 309-fold at 60 mg/kg). Chronic oral dosing in male rhesus macaques was well tolerated, significantly increased erythropoiesis, and prevented anemia induced by weekly phlebotomy. Furthermore, modest increases in HbF-containing red cells and reticulocytes were demonstrated by flow cytometry, though significant increases in HbF were not demonstrated by high-pressure liquid chromatography (HPLC). HIF PHIs represent a novel class of molecules with broad potential clinical application for congenital and acquired anemias.

Hypoxia-inducible factor prolyl 4-hydroxylase inhibition. A target for neuroprotection in the central nervous system.

Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases are a family of iron- and 2-oxoglutarate-dependent dioxygenases that negatively regulate the stability of several proteins that have established roles in adaptation to hypoxic or oxidative stress. These proteins include the transcriptional activators HIF-1alpha and HIF-2alpha. The ability of the inhibitors of HIF prolyl 4-hydroxylases to stabilize proteins involved in adaptation in neurons and to prevent neuronal injury remains unclear. We reported that structurally diverse low molecular weight or peptide inhibitors of the HIF prolyl 4-hydroxylases stabilize HIF-1alpha and up-regulate HIF-dependent target genes (e.g. enolase, p21(waf1/cip1), vascular endothelial growth factor, or erythropoietin) in embryonic cortical neurons in vitro or in adult rat brains in vivo. We also showed that structurally diverse HIF prolyl 4-hydroxylase inhibitors prevent oxidative death in vitro and ischemic injury in vivo. Taken together these findings identified low molecular weight and peptide HIF prolyl 4-hydroxylase inhibitors as novel neurological therapeutics for stroke as well as other diseases associated with oxidative stress.