Nonclinical Characterization of the Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor Roxadustat, a Novel Treatment of Anemia of Chronic Kidney Disease S
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Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2020/06/02/jpet.120.265181.DC1 1521-0103/374/2/342–353$35.00 https://doi.org/10.1124/jpet.120.265181 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 374:342–353, August 2020 Copyright ª 2020 by The Author(s) This is an open access article distributed under the CC BY Attribution 4.0 International license. Nonclinical Characterization of the Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor Roxadustat, a Novel Treatment of Anemia of Chronic Kidney Disease s Ughetta del Balzo,1 Pierre E. Signore,1 Gail Walkinshaw, Todd W. Seeley, Mitchell C. Brenner, Qingjian Wang, Guangjie Guo, Michael P. Arend, Lee A. Flippin, F. Aisha Chow, David C. Gervasi, Christian H. Kjaergaard, Ingrid Langsetmo, Volkmar Guenzler, David Y. Liu, Steve J. Klaus, Al Lin, and Thomas B. Neff FibroGen, Inc., San Francisco, California Downloaded from Received January 16, 2020; accepted May 13, 2020 ABSTRACT Anemia of chronic kidney disease (CKD) is a multifactorial functional iron deficiency, and increased expression of two disorder caused by impaired erythropoietin (EPO) production genes involved in duodenal iron absorption: divalent metal jpet.aspetjournals.org and altered iron homeostasis associated with inflammation. transporter 1 and duodenal cytochrome b. In conclusion, by Hypoxia-inducible factor (HIF) is a transcription factor that activating the HIF pathway, roxadustat increased EPO pro- stimulates erythropoiesis via a coordinated response involving duction, elevated Hb, corrected anemia, and improved iron increased EPO production and enhanced iron availability for Hb homeostasis. The coordinated erythropoietic response stimu- synthesis. HIF degradation is regulated by HIF-prolyl hydroxy- lated by roxadustat, involving both EPO production and lase (HIF-PH) enzymes. We hypothesized that roxadustat, an mobilization of iron stores, makes this compound a promising orally available small-molecule inhibitor of HIF-PH, would in- treatment of anemia of CKD and anemia associated with crease EPO production and promote erythropoiesis in animal functional iron deficiency. at ASPET Journals on September 27, 2021 models of anemia. In cells, roxadustat increased both HIF-1a and HIF-2a proteins, leading to an increase in EPO production, SIGNIFICANCE STATEMENT even in the presence of EPO-suppressing inflammatory cyto- Roxadustat is a novel orally available small-molecule inhibitor of kines. Roxadustat administered intermittently to healthy rats HIF prolyl hydroxylase enzymes that reversibly stabilizes HIF-a, and cynomolgus monkeys increased circulating EPO levels, thus activating transcription of HIF-dependent genes, including reticulocytes, blood Hb, and hematocrit in a dose-dependent EPO and regulators of iron homeostasis. Activation of the HIF manner. Roxadustat corrected anemia in a rat model of CKD pathway by roxadustat induces erythropoiesis in healthy rats after five-sixth nephrectomy and in a rat model of anemia of and monkeys and corrects experimentally induced anemia in inflammation with impaired iron metabolism induced by rats. The coordinated erythropoietic response that increases peptidoglycan-polysaccharide (PG-PS). In the PG-PS model, EPO production and mobilizes iron stores makes roxadustat roxadustat significantly decreased hepatic expression of hep- a promising treatment for anemia of chronic kidney disease and cidin, a hormone responsible for iron sequestration and anemia associated with functional iron deficiency. Introduction Anemia is a frequent and serious complication of chronic kidney disease (CKD) that affects millions of patients world- wide (Thomas et al., 2008; Kassebaum et al., 2014; Stauffer This research was funded by FibroGen, Inc. U.d.B., P.E.S., G.W., T.W.S., M.C.B., Q.W., G.G., M.P.A., L.A.F., F.A.C., D.C.G., C.H.K., I.L., V.G., D.Y.L., and Fan, 2014). Anemia of CKD contributes to decreased S.J.K., A.L., and T.B.N. are employees, consultants, and/or stock owners of quality of life and increased risk of morbidity and mortality FibroGen, Inc. (Finkelstein et al., 2009; Thorp et al., 2009). Key factors 1U.d.B. and P.E.S. contributed equally to this work. FibroGen, Inc., San Francisco, CA. responsible for anemia of CKD are a relative deficiency in https://doi.org/10.1124/jpet.120.265181. erythropoietin (EPO) production and a decrease in iron s This article has supplemental material available at jpet.aspetjournals.org. ABBREVIATIONS: ACD, anemia of chronic disease; BUN, blood urea nitrogen; CKD, chronic kidney disease; DCYTB, duodenal cytochrome b; Dcytb, gene coding for DCYTB protein; DMT1, divalent metal transporter 1; Dmt1, gene coding for DMT1 protein; EGLN egl 9 homolog EPO, erythropoietin; ESA, erythropoiesis-stimulating agent; FIH, factor inhibiting HIF-1a; Hamp, gene coding for hepcidin; Hep3B hepatocellular carcinoma cell lineHIF, hypoxia-inducible factor; HIF-PH, HIF prolyl hydroxylaseHK-2 human proximal tubule kidney cell line; HRP horseradish peroxidaseIL, interleukin; aKG, a-ketoglutarate; MCHC, mean corpuscular Hb concentration; MCV, mean corpuscular volume; MIP, macrophage inflammatory protein; PCR, polymerase chain reaction; PG-PS, peptidoglycan-polysaccharide; PHD, prolyl hydroxylase; Slc11a2, gene coding for DMT1 protein; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor; WBC, white blood cell. 342 Nonclinical Profile of Roxadustat for Anemia 343 availability for Hb synthesis (Yilmaz et al., 2011; Babitt and for iron homeostasis. Elevated hepcidin, which is driven by Lin, 2012; Locatelli et al., 2017). Currently, anemia of CKD is chronic inflammation, leads to sequestering of iron in enter- managed by iron supplementation and, in more severe cases, ocytes, macrophages, and hepatocytes, thereby causing func- by administration of supraphysiologic doses of erythropoiesis- tional iron deficiency. Downregulation of hepcidin by stimulating agents (ESAs), such as human recombinant EPO, activation of the HIF pathway releases iron trapped in cells, in combination with adjuvant iron therapy (Biggar and Kim, thus increasing iron availability for Hb synthesis (Peysson- 2017). However, some patients are hyporesponsive to ESAs naux et al., 2008). and need even larger doses because of functional iron de- Roxadustat was developed as an orally active inhibitor of ficiency associated with inflammation (MacDougall and Coo- the HIF-PH enzymes. In this series of in vitro and in vivo per, 2005; Adamson, 2009; Yilmaz et al., 2011). High doses of studies, we studied the effects of roxadustat on activation of ESAs increase the risk of serious adverse events, including the HIF pathway, EPO secretion, and stimulation of erythro- death, myocardial infarction, congestive heart failure, and poiesis in normal rats and monkeys. We also evaluated the stroke (Szczech et al., 2008; Pfeffer et al., 2009). Thus, to ability of roxadustat to correct anemia in two different rodent effectively treat anemia of CKD, new therapies need to models. The five-sixth nephrectomy model was used to address both impaired EPO production and functional iron demonstrate that roxadustat can correct anemia of CKD. deficiency (Locatelli et al., 2017). The model of anemia of chronic disease (ACD) induced by The observation that low blood oxygen levels lead to administration of proteoglycan-polysaccharide (PG-PS) was increased EPO production was made decades ago (Prentice used to show that roxadustat can reverse anemia associated Downloaded from and Mirand, 1961; Naets, 1963), but the transcription factors with inflammation and functional iron deficiency. responsible for this hypoxic response were not identified until the 1990s (Semenza and Wang, 1992; Wang and Semenza, 1995; Wang et al., 1995; Tian et al., 1997). Since then, these Materials and Methods hypoxia-inducible factors (HIFs) have been found to be part In Vitro Studies of an oxygen-sensing pathway that is evolutionarily conser- jpet.aspetjournals.org ved among metazoans (Kaelin, 2005; Kaelin and Ratcliffe, HIF Hydroxylase Enzyme Assays. Activity of HIF-PH and FIH 14 2008). HIFs function as heterodimers comprising an oxygen- enzymes was determined based on the capture of CO2 released by 14 a a b the decarboxylation of [1- C] KG (Zhang et al., 1999). Reaction regulated -subunit and a constitutively expressed -subunit. 14 a a a a mixtures included Fe(SO4) (Sigma-Aldrich, St. Louis, MO), [1- C] There are two major HIF- isoforms, HIF-1 and HIF-2 , both KG (Perkin Elmer, Waltham, MA), nonlabeled aKG (Sigma-Aldrich), b of which dimerize with the same -subunit to form functional ascorbate (Sigma-Aldrich), peptide substrate (PHD1,2,3: acetyl- HIF-1 and HIF-2 transcription factors, respectively. In the DLDLEMLAPYIPMDDDFQL-amide, FIH: DESGLPQLTSYDCEV- presence of oxygen, HIF-a subunits are hydroxylated on NAPIQGSRNLLQGEELLRAL-biotin) (Mimotopes, Mulgrave, at ASPET Journals on September 27, 2021 specific proline residues by a family of HIF prolyl hydroxy- Australia), and catalase (catalog number C100; Sigma-Aldrich) in lase (HIF-PH) enzymes comprising three isozymes: PHD1 50 mM HEPES buffer (Corning Life Sciences, Tewksbury, MA), pH (Egl nine homolog [EGLN]2), PHD2 (EGLN1), and PHD3 7.4. Enzymatic reactions were initiated by addition of full-length (EGLN3) (Kaelin and Ratcliffe, 2008; Myllyharju, 2013). recombinant human HIF-PH or FIH enzyme to the reaction mixture This hydroxylation enables binding of the von Hippel-Lindau (Hirsilä et al., 2005). The reactions were performed