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.

Radiation-induced pulmonary gene expression changes are attenuated by the CTGF antibody Pamrevlumab.

BACKGROUND: Fibrosis is a delayed side effect of radiation therapy (RT). Connective tissue growth factor (CTGF) promotes the development of fibrosis in multiple settings, including pulmonary radiation injury. METHODS: To better understand the cellular interactions involved in RT-induced lung injury and the role of CTGF in these responses, microarray expression profiling was performed on lungs of irradiated and non-irradiated mice, including mice treated with the anti-CTGF antibody pamrevlumab (FG-3019). Between group comparisons (Welch's t-tests) and principal components analyses were performed in Genespring. RESULTS: At the mRNA level, the ability of pamrevlumab to prolong survival and ameliorate RT-induced radiologic, histologic and functional lung deficits was correlated with the reversal of a clear enrichment in mast cell, macrophage, dendritic cell and mesenchymal gene signatures. Cytokine, growth factor and matrix remodeling genes that are likely to contribute to RT pneumonitis and fibrosis were elevated by RT and attenuated by pamrevlumab, and likely contribute to the cross-talk between enriched cell-types in injured lung. CONCLUSIONS: CTGF inhibition had a normalizing effect on select cell-types, including immune cells not typically regarded as being regulated by CTGF. These results suggest that interactions between RT-recruited cell-types are critical to maintaining the injured state; that CTGF plays a key role in this process; and that pamrevlumab can ameliorate RT-induced lung injury in mice and may provide therapeutic benefit in other immune and fibrotic disorders.

CTGF antagonism with mAb FG-3019 enhances chemotherapy response without increasing drug delivery in murine ductal pancreas cancer.

Pancreatic ductal adenocarcinoma (PDA) is characterized by abundant desmoplasia and poor tissue perfusion. These features are proposed to limit the access of therapies to neoplastic cells and blunt treatment efficacy. Indeed, several agents that target the PDA tumor microenvironment promote concomitant chemotherapy delivery and increased antineoplastic response in murine models of PDA. Prior studies could not determine whether chemotherapy delivery or microenvironment modulation per se were the dominant features in treatment response, and such information could guide the optimal translation of these preclinical findings to patients. To distinguish between these possibilities, we used a chemical inhibitor of cytidine deaminase to stabilize and thereby artificially elevate gemcitabine levels in murine PDA tumors without disrupting the tumor microenvironment. Additionally, we used the FG-3019 monoclonal antibody (mAb) that is directed against the pleiotropic matricellular signaling protein connective tissue growth factor (CTGF/CCN2). Inhibition of cytidine deaminase raised the levels of activated gemcitabine within PDA tumors without stimulating neoplastic cell killing or decreasing the growth of tumors, whereas FG-3019 increased PDA cell killing and led to a dramatic tumor response without altering gemcitabine delivery. The response to FG-3019 correlated with the decreased expression of a previously described promoter of PDA chemotherapy resistance, the X-linked inhibitor of apoptosis protein. Therefore, alterations in survival cues following targeting of tumor microenvironmental factors may play an important role in treatment responses in animal models, and by extension in PDA patients.

Connective tissue growth factor regulates cardiac function and tissue remodeling in a mouse model of dilated cardiomyopathy.

Cardiac structural changes associated with dilated cardiomyopathy (DCM) include cardiomyocyte hypertrophy and myocardial fibrosis. Connective tissue growth factor (CTGF) has been associated with tissue remodeling and is highly expressed in failing hearts. Our aim was to test if inhibition of CTGF would alter the course of cardiac remodeling and preserve cardiac function in the protein kinase Cε (PKCε) mouse model of DCM. Transgenic mice expressing constitutively active PKCε in cardiomyocytes develop cardiac dysfunction that was evident by 3 months of age, and that progressed to cardiac fibrosis, heart failure, and increased mortality. Beginning at 3 months of age, PKCε mice were treated with a neutralizing monoclonal antibody to CTGF (FG-3149) for an additional 3 months. CTGF inhibition significantly improved left ventricular (LV) systolic and diastolic functions in PKCε mice, and slowed the progression of LV dilatation. Using gene arrays and quantitative PCR, the expression of many genes associated with tissue remodeling was elevated in PKCε mice, but significantly decreased by CTGF inhibition. However total collagen deposition was not attenuated. The observation of significantly improved LV function by CTGF inhibition in PKCε mice suggests that CTGF inhibition may benefit patients with DCM. Additional studies to explore this potential are warranted.

Connective Tissue Growth Factor Inhibition Enhances Cardiac Repair and Limits Fibrosis After Myocardial Infarction.

Myocardial infarction (MI)-induced cardiac fibrosis attenuates cardiac contractile function, and predisposes to arrhythmias and sudden cardiac death. Expression of connective tissue growth factor (CTGF) is elevated in affected organs in virtually every fibrotic disorder and in the diseased human myocardium. Mice were subjected to treatment with a CTGF monoclonal antibody (mAb) during infarct repair, post-MI left ventricular (LV) remodeling, or acute ischemia-reperfusion injury. CTGF mAb therapy during infarct repair improved survival and reduced LV dysfunction, and reduced post-MI LV hypertrophy and fibrosis. Mechanistically, CTGF mAb therapy induced expression of cardiac developmental and/or repair genes and attenuated expression of inflammatory and/or fibrotic genes.

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.

Effects of CTGF Blockade on Attenuation and Reversal of Radiation-Induced Pulmonary Fibrosis.

Background: Radiotherapy is a mainstay for the treatment of lung cancer that can induce pneumonitis or pulmonary fibrosis. The matricellular protein connective tissue growth factor (CTGF) is a central mediator of tissue remodeling. Methods: A radiation-induced mouse model of pulmonary fibrosis was used to determine if transient administration of a human antibody to CTGF (FG-3019) started at different times before or after 20 Gy thoracic irradiation reduced acute and chronic radiation toxicity. Mice (25 mice/group; 10 mice/group in a confirmation study) were examined by computed tomography, histology, gene expression changes, and for survival. In vitro experiments were performed to directly study the interaction of CTGF blockade and radiation. All statistical tests were two-sided. Results: Administration of FG-3019 prevented (∼50%-80%) or reversed (∼50%) lung remodeling, improved lung function, improved mouse health, and rescued mice from lethal irradiation ( P < .01). Importantly, when antibody treatment was initiated at 16 weeks after thoracic irradiation, FG-3019 reversed established lung remodeling and restored lung function. CTGF blockade abrogated M2 polarized macrophage influx, normalized radiation-induced gene expression changes, and reduced myofibroblast abundance and Osteopontin expression. Conclusion: These results indicate that blocking CTGF attenuates radiation-induced pulmonary remodeling and can reverse the process after initiation. CTGF has a central role in radiation-induced fibrogenesis, and FG-3019 may benefit patients with radiation-induced pulmonary fibrosis or patients with other forms or origin of chronic fibrotic diseases.

Effect of desferrioxamine and metals on the hydroxylases in the oxygen sensing pathway.

Hypoxia-inducible transcription factor (HIF) is regulated by two oxygen-dependent events that are catalyzed by the HIF prolyl 4-hydroxylases (HIF-P4Hs) and HIF asparaginyl hydroxylase (FIH). We have purified the three recombinant human HIF-P4Hs to near homogeneity and characterized their catalytic properties and inhibition and those of FIH. The specific activities of the HIF-P4Hs were at least 40-50 mol/mol/min, and they and FIH catalyzed an uncoupled decarboxylation of 2-oxoglutarate in the absence of any peptide substrate. The purified HIF-P4Hs showed considerable activities even without added Fe2+, their apparent Km values for iron being markedly lower than that of FIH. Desferrioxamine and several metals were effective inhibitors of FIH, but surprisingly, ineffective inhibitors of the HIF-P4Hs in vitro, especially of HIF-P4H-2. Desferrioxamine and cobalt were more effective in cultured insect cells synthesizing recombinant HIF-P4H-2, but complete inhibition was not achieved and most of the enzyme was inactivated irreversibly. Cobalt also rapidly inactivated HIF-P4Hs during storage at 4 degrees C. The well-known stabilization of HIF-alpha by cobalt and nickel is thus not due to a simple competitive inhibition of HIF-P4Hs. The effective inhibition of FIH by these metals and zinc probably leads to full transcriptional activity of HIF-alpha even in concentrations that produce no stabilization of HIF-alpha.

Cooperative interaction of CTGF and TGF-ß in animal models of fibrotic disease.

BACKGROUND: Connective tissue growth factor (CTGF) is widely thought to promote the development of fibrosis in collaboration with transforming growth factor (TGF)-ß; however, most of the evidence for its involvement comes from correlative and culture-based studies. In this study, the importance of CTGF in tissue fibrosis was directly examined in three murine models of fibrotic disease: a novel model of multiorgan fibrosis induced by repeated intraperitoneal injections of CTGF and TGF-ß2; the unilateral ureteral obstruction (UUO) renal fibrosis model; and an intratracheal bleomycin instillation model of pulmonary fibrosis. RESULTS: Intraperitoneal coadministration of CTGF and TGF-ß2 elicited a profound fibrotic response that was inhibited by the human anti-CTGF antibody FG-3019, as indicated by the ability of FG-3019 to ameliorate the histologic signs of fibrosis and reduce the otherwise increased hydroxyproline:proline (Hyp:Pro) ratios by 25% in kidney (P < 0.05), 30% in liver (P < 0.01) and 63% in lung (P < 0.05). Moreover, administration of either cytokine alone failed to elicit a fibrotic response, thus demonstrating that CTGF is both necessary and sufficient to initiate fibrosis in the presence of TGF-ß and vice versa. In keeping with this requirement for CTGF function in fibrosis, FG-3019 also reduced the renal Hyp:Pro response up to 20% after UUO (P < 0.05). In bleomycin-injured animals, a similar trend towards a FG-3019 treatment effect was observed (38% reduction in total lung Hyp, P = 0.056). Thus, FG-3019 antibody treatment consistently reduced excessive collagen deposition and the pathologic severity of fibrosis in all models. CONCLUSION: Cooperative interactions between CTGF and TGF-ß signaling are required to elicit overt tissue fibrosis. This interdependence and the observed anti-fibrotic effects of FG-3019 indicate that anti-CTGF therapy may provide therapeutic benefit in different forms of fibroproliferative disease.

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.