Randomized placebo-controlled dose-ranging and pharmacodynamics study of roxadustat (FG-4592) to treat anemia in nondialysis-dependent chronic kidney disease (NDD-CKD) patients.

BACKGROUND: Roxadustat (FG-4592) is an oral hypoxia-inducible factor prolyl hydroxylase inhibitor that stimulates erythropoiesis. This Phase 2a study tested efficacy (Hb response) and safety of roxadustat in anemic nondialysis-dependent chronic kidney disease (NDD-CKD) subjects. METHODS: NDD-CKD subjects with hemoglobin (Hb) ≤11.0 g/dL were sequentially enrolled into four dose cohorts and randomized to roxadustat or placebo two times weekly (BIW) or three times weekly (TIW) for 4 weeks, in an approximate roxadustat:placebo ratio of 3:1. Efficacy was assessed by (i) mean Hb change (ΔHb) from baseline (BL) and (ii) proportion of Hb responders (ΔHb ≥ 1.0 g/dL). Pharmacodynamic evaluation was performed in a subset of subjects. Safety was evaluated by adverse event frequency/severity. RESULTS: Of 116 subjects receiving treatment, 104 completed 4 weeks of dosing and 96 were evaluable for efficacy. BL characteristics for roxadustat and placebo groups were comparable. In roxadustat-treated subjects, Hb levels increased from BL in a dose-related manner in the 0.7, 1.0, 1.5 and 2.0 mg/kg groups. Maximum ΔHb within the first 6 weeks was significantly higher in the 1.5 and 2.0 mg/kg groups than in the placebo subjects. Hb responder rates were dose dependent and ranged from 30% in the 0.7 mg/kg BIW group to 100% in the 2.0 mg/kg BIW and TIW groups versus 13% in placebo. CONCLUSIONS: Roxadustat transiently and moderately increased endogenous erythropoietin and reduced hepcidin. Adverse events were similar in the roxadustat and placebo groups. Roxadustat produced dose-dependent increases in blood Hb among anemic NDD-CKD patients in a placebo-controlled trial. CLINICAL TRIALS REGISTRATION: Clintrials.gov #NCT00761657.

Hypoxia-inducible factor-2alpha-expressing interstitial fibroblasts are the only renal cells that express erythropoietin under hypoxia-inducible factor stabilization.

The adaptation of erythropoietin production to oxygen supply is determined by the abundance of hypoxia-inducible factor (HIF), a regulation that is induced by a prolyl hydroxylase. To identify cells that express HIF subunits (HIF-1alpha and HIF-2alpha) and erythropoietin, we treated Sprague-Dawley rats with the prolyl hydroxylase inhibitor FG-4497 for 6 h to induce HIF-dependent erythropoietin transcription. The kidneys were analyzed for colocalization of erythropoietin mRNA with HIF-1alpha and/or HIF-2alpha protein along with cell-specific identification markers. FG-4497 treatment strongly induced erythropoietin mRNA exclusively in cortical interstitial fibroblasts. Accumulation of HIF-2alpha was observed in these fibroblasts and in endothelial and glomerular cells, whereas HIF-1alpha was induced only in tubular epithelia. A large proportion (over 90% in the juxtamedullary cortex) of erythropoietin-expressing cells coexpressed HIF-2alpha. No colocalization of erythropoietin and HIF-1alpha was found. Hence, we conclude that in the adult kidney, HIF-2alpha and erythropoietin mRNA colocalize only in cortical interstitial fibroblasts, which makes them the key cell type for renal erythropoietin synthesis as regulated by HIF-2alpha.

Activation of hypoxia-inducible factors ameliorates hypoxic distal tubular injury in the isolated perfused rat kidney.

BACKGROUND: Preconditional activation of HIF with specific prolyl-hydroxylase inhibitors (PHD-I) attenuates proximal tubular injury, induced by warm ischaemia/ reperfusion (Bernhardt, JASN, 2006). Distal tubular damage occurs in humans with acute kidney injury (AKI), in experimental contrast media-induced nephropathy (CIN), as well as in cell-free isolated perfused kidneys (IPKs). Since in the IPK distal tubular damage inversely correlates with HIF activation (Rosenberger, KI, 2005), we explored the potential of PHD-I to improve morpho-functional outcome in this model. METHODS: Male SD rats were randomly given the synthetic PHD-inhibitor FG-4497 (FibroGen, 50 mg/kg IV) or its vehicle (CTR, n = 10 per group). Six hours later, the right kidney was perfused for 90 min with cell-free oxygenated medium and subsequently perfusion-fixed for morphologic assessment. The left kidney was used for HIF immunostaining. RESULTS: As compared with CTR kidneys, at 6 h after FG-4497 HIF-alpha isoforms were markedly up-regulated in all renal zones: HIF-1alpha in tubules and in papillary interstitial cells (IC), HIF-2alpha in IC and vascular endothelial cells. FG-4497 treatment resulted in a higher perfusate flow rate (P < 0.04, ANOVA). Tubular injury to medullary thick ascending limbs (mTALs) was significantly attenuated in the treatment versus control group (38.9 +/- 7.4% versus 62.7 +/- 4.9% of mTALs in the mid-inner stripe (P < 0.02); 23.8 +/- 6.8% versus 45.6 +/- 7.4% in the innermost zone of the inner stripe (P < 0.05). CONCLUSIONS: These findings illustrate that PHD-I preconditioning attenuates hypoxic distal tubular injury produced in the IPK in the same fashion in which it protects proximal tubules. mTAL conservation may be related to the stabilization of cellular HIF, as well as to preserved endothelial function and microcirculation.

Connective tissue growth factor-specific antibody attenuates tumor growth, metastasis, and angiogenesis in an orthotopic mouse model of pancreatic cancer.

Connective tissue growth factor (CTGF) plays an important role in fibrosis by modulating cell migration and cell growth but may also modify tumor growth and metastasis. Because CTGF is overexpressed in pancreatic ductal adenocarcinoma, we investigated the in vitro effects of CTGF on the proliferation and invasiveness of PANC-1 pancreatic cancer cells and examined the consequences of its in vivo inhibition on the growth and metastasis of these cells using a fully human CTGF-specific monoclonal antibody (FG-3019) in an orthotopic nude mouse model. Although PANC-1 cells expressed relatively high levels of endogenous CTGF mRNA, the addition of CTGF to conditioned medium increased the proliferation and invasiveness of PANC-1 cells. Moreover, transforming growth factor-beta1 caused a further increase in CTGF expression in these cells. In vivo, the twice weekly i.p. administration of FG-3019 decreased tumor growth and metastasis and attenuated tumor angiogenesis and cancer cell proliferation. FG-3019 did not enhance apoptosis and did not attenuate the inhibitory effects of gemcitabine on tumor growth and metastasis. These findings suggest that CTGF may contribute to aberrant autocrine and paracrine pathways that promote pancreatic cancer cell growth, invasion, metastasis, and angiogenesis. Therefore, blocking CTGF actions with FG-3019 may represent a novel therapeutic approach in pancreatic ductal adenocarcinoma.

Induction of erythropoiesis by hypoxia-inducible factor prolyl hydroxylase inhibitors without promotion of tumor initiation, progression, or metastasis in a VEGF-sensitive model of spontaneous breast cancer.

The effects of pharmacological hypoxia-inducible factor (HIF) stabilization were investigated in the MMTV-Neundl-YD5 (NeuYD) mouse model of breast cancer. This study first confirmed the sensitivity of this model to increased vascular endothelial growth factor (VEGF), using bigenic NeuYD;MMTV-VEGF-25 mice. Tumor initiation was dramatically accelerated in bigenic animals. Bigenic tumors were also more aggressive, with shortened doubling times and increased lung metastasis as compared to NeuYD controls. In separate studies, NeuYD mice were treated three times weekly from 7 weeks of age until study end with two different HIF prolyl hydroxylase inhibitors (HIF-PHIs), FG-4497 or roxadustat (FG-4592). In NeuYD mice, HIF-PHI treatments elevated erythropoiesis markers, but no differences were detected in tumor onset or the phenotypes of established tumors.

An endoplasmic reticulum transmembrane prolyl 4-hydroxylase is induced by hypoxia and acts on hypoxia-inducible factor alpha.

Prolyl 4-hydroxylases (P4Hs) act on collagens (C-P4Hs) and the oxygen-dependent degradation domains (ODDDs) of hypoxia-inducible factor alpha subunits (HIF-P4Hs) leading to degradation of the latter. We report data on a human P4H possessing a transmembrane domain (P4H-TM). Its gene is also found in zebrafish but not in flies and nematodes. Its sequence more closely resembles those of the C-P4Hs than the HIF-P4Hs, but it lacks the peptide substrate-binding domain of the C-P4Hs. P4H-TM levels in cultured cells are increased by hypoxia, and P4H-TM is N-glycosylated and is located in endoplasmic reticulum membranes with its catalytic site inside the lumen, a location differing from those of the HIF-P4Hs. Despite this, P4H-TM overexpression in cultured neuroblastoma cells reduced HIF-alpha ODDD reporter construct levels, and its small interfering RNA increased HIF-1alpha protein level, in the same way as those of HIF-P4Hs. Furthermore, recombinant P4H-TM hydroxylated the two critical prolines in HIF-1alpha ODDD in vitro, with a preference for the C-terminal proline, whereas it did not hydroxylate any prolines in recombinant type I procollagen chains.

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.