Pharmacological inhibition of prolyl hydroxylase protects against inflammation-induced anemia via efficient erythropoiesis and hepcidin downregulation

Author’s Accepted Manuscript Pharmacological inhibition of prolyl hydroxylase protects against inflammation-induced anemia via efficient erythropoiesis and hepcidin downregulation Mukul Jain, Amit Joharapurkar, Vishal Patel, Samadhan Kshirsagar, Brijesh Sutariya, Maulik Patel, Hiren Patel, Pankaj R. Patel

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S0014-2999(18)30678-2 https://doi.org/10.1016/j.ejphar.2018.11.023 EJP72085

To appear in: European Journal of Pharmacology Received date: 20 September 2018 Revised date: 12 November 2018 Accepted date: 16 November 2018 Cite this article as: Mukul Jain, Amit Joharapurkar, Vishal Patel, Samadhan Kshirsagar, Brijesh Sutariya, Maulik Patel, Hiren Patel and Pankaj R. Patel, Pharmacological inhibition of prolyl hydroxylase protects against inflammationinduced anemia via efficient erythropoiesis and hepcidin downregulation, European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2018.11.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Pharmacological inhibition of prolyl hydroxylase protects against inflammationinduced anemia via efficient erythropoiesis and hepcidin downregulation

Mukul Jain*, Amit Joharapurkar, Vishal Patel, Samadhan Kshirsagar, Brijesh Sutariya, Maulik Patel, Hiren Patel, Pankaj R. Patel

Zydus Research Centre, Cadila Healthcare Limited, Sarkhej Bavla NH 8A, Moraiya, Ahmedabad, India 382210

*

Corresponding author. Mukul R. Jain Department of Pharmacology & Toxicology, Zydus

Research Centre, Cadila Healthcare Limited, Sarkhej-Bavla N.H.No.8A, Moraiya, Ahmedabad Tel.: +91 2717665555; fax: +91 2717665155. [email protected]

Abstract Chronic inflammatory diseases are often associated with anemia. In such conditions, anemia is generally treated with erythropoiesis stimulating agents (ESAs) which are associated with potentially hazardous side effects and poor outcomes. Suboptimal erythropoiesis in chronic inflammation is believed to be caused by elevated hepcidin levels, which causes blockade of iron in tissue stores. In the current work using rodent models of inflammation, an orally available small molecule prolyl hydroxylase inhibitor desidustat was assessed as an effective treatment of anemia of inflammation. In BALB/c mice, a single dose treatment of desidustat attenuated the 1

effect of lipopolysaccharide (LPS) - or turpentine oil-induced inflammation and increased serum erythropoietin (EPO), iron, and reticulocyte count, and decreased serum hepcidin levels. In turpentine oil-induced anemia in BALB/c mice, repeated dose desidustat treatment increased hemoglobin, RBC and hematocrit in a dose related manner. In female Lewis rats, treatment with desidustat markedly reduced PGPS-induced anemia and increased hemoglobin, red blood cell (RBC) and white blood cell (WBC) count, hematocrit, serum iron and spleen iron. These effects of desidustat were associated with reduction in hepcidin (HAMP) expression as well as reduction in serum hepcidin, and increased EPO expression in liver and kidneys. Desidustat treatment caused a significant increase in expression of Duodenal cytochrome B (DcytB), ferroportin (FPN1) and divalent metal transporter 1 (DMT1) in duodenum, and FPN1 and monocyte chemoattractant protein-1 (MCP-1) in liver suggesting an overall influence on iron metabolism. Thus, pharmacological inhibition of prolyl hydroxylase enzymes can be useful in treatment of anemia of inflammation.

Keywords: Desidustat; anemia of inflammation; hepcidin; iron

1. Introduction Inflammation associated with anemia is widely observed in patients with infections, malignancies, or autoimmune disorders (Nemeth and Ganz, 2014). In these conditions, enhanced production of cytokines such as interferon-γ, tumor necrosis factor alpha (TNFα), and Interleukin-1 (IL-1) is observed that hampers erythropoiesis (Milner et al., 2010; Nemeth and Ganz, 2014).

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Apart from the inflammatory cytokines, a major cause of iron-restricted anemia is elevated levels of hepcidin (D’Angelo, 2013). Hepcidin is a liver-derived peptide that causes downregulation and degradation of cellular iron exporter ferroportin. Degradation of ferroportin causes reduction in the release of iron from the cellular iron stores, and decreases serum iron. Low serum iron, despite iron supplementation in such conditions is thus called “functional iron deficiency” (Wang and Babitt, 2016). It has been observed that hepcidin overexpression results in severe anemia in mice, which is positively correlated with iron-restriction in tissue stores (Nicolas et al., 2002). Clinically, inflammatory conditions are correlated with hepcidin overproduction and low serum iron due to iron retention in tissue stores like liver, spleen, and macrophages (Kemna et al., 2008). Erythropoiesis-stimulating agents (epoetin or darbepoetin alfa) are used in clinical practice to correct anemia of inflammation (Campbell, 2001; Melnikova, 2006), but they are associated with cardiovascular side effects and risk of thrombosis (Santhanam et al., 2010). In addition, a large patient population suffering from anemia of chronic disease may develop hematological unresponsiveness or resistance to the erythropoietin therapy (Melosky, 2008; Palmer et al., 2014). Also, erythropoiesis-stimulating agents (ESA) require iron supplementation to fulfill erythropoiesis demands (Melnikova, 2006), and increasingly higher doses are required to correct the functional iron deficiency despite iron supplementation. These are associated with adverse effects caused by increased erythropoietin as well as iron levels in serum. Hence, novel therapies that can cure anemia of inflammation without adverse effects are strongly needed. Reports suggest that hypoxia can inhibit hepcidin, which can improve iron utilization by increasing its intestinal uptake as well as release from tissue stores (Nicolas et al., 2002). Hypoxia-inducible factor (HIF) is the regulator of cellular adaptation to hypoxia. HIF can induce

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erythropoietin (EPO) synthesis in kidneys and liver, and also suppress the hepcidin gene (Hamp). Since inhibition of prolyl hydroxylases (PHD) enzymes can stabilize HIF, PHD inhibitors have emerged as a novel and interesting approach for the treatment of anemia (Kim and Yang, 2015; Muchnik and Kaplan, 2011). Many PHD inhibitors are currently undergoing clinical trials supporting the possibility of an orally bioavailable drug for treatment of anemia (Beuck et al., 2012; Jain et al., 2016). Desidustat (also known as ZYAN1) is a novel PHD inhibitor under clinical development (Jain et al., 2016; Kansagra et al., 2018). Desidustat is a pan-PHD inhibitor, which has good oral bioavailability, without any drug related adverse effects in the alternate day dosing regimen (Kansagra et al., 2018). In the current study, we have used a PHD inhibitor, desidustat, as a novel approach to reduce the anemia of inflammation using three different models of inflammation-induced anemia in mice and rats. 2. Material and Methods 2.1. Animals All the animal studies were performed in Zydus Research Centre (Ahmedabad, India) a facility accredited by Association for Assessment and Accreditation of Laboratory Animal Care International. The study protocol was reviewed and approved by the Institutional Animal Ethics Committee. BALB/c mice were housed 6 animals per cage and female Lewis rats were housed 3 animals per cage with sterile corncob bedding in polypropylene cages with freely available food and water with clean disinfected area. At the end of the experiment, animals were euthanized individually by adjusting the isoflourane flow rate to 5%, with continued isoflourane exposure until one minute after cessation of breathing. 2.2. Chemicals and analysis 4

Desidustat was synthesized at Zydus Research Centre (Ahmedabad, India). All other chemicals were purchased from Sigma Aldrich. During in vivo studies, desidustat was administered by oral gavage using 0.25% aqueous carboxymethylcellulose (CMC), while vehicle control was administered 0.25 % CMC for all acute as well as chronic studies. Erythropoietin was measured using a commercial ELISA kit (R&D System, Minneapolis, MN 55413, USA). Hemoglobin was estimated by tail bleeding using QuantiChrom Hemoglobin Assay Kit (Bio Assay systems, Hayward, CA, USA). Reticulocytes, and red blood cell (RBC) count in rodent blood samples were determined using Cell-Dyne 3700 hematology analyzer (Abbott Laboratories. Abbott Park, Illinois, USA). Serum iron was measured using total iron estimation kit (Pointe scientific Inc., Canton MI, USA). Serum hepcidin was measured using rat hepcidin ELISA kit (Cusabio, Wuhan, Hubei Province 430206, China). Tissue iron was measured using a reported method (Rebouche et al., 2004). HIF-1α protein level in liver was measured using HIF-α transcription factor assay kit (Cayman Chemical, Ann Arbor, USA). 2.3. Acute effect of desidustat on EPO release in inflammation Male BALB/c mice were administered lipopolysaccharide (LPS, 1 mg/kg, i.p.). In another experiment, male BALB/c mice were administered turpentine oil (5 ml/kg, s.c.) in intrascapular region. Desidustat (15 mg/kg, p.o.) and vehicle (p.o.) were administered along with LPS or turpentine oil treatment. Animals were bled by retroorbital puncture 6 and 72 h after desidustat treatment and serum was separated. Serum samples were separated for assessment of EPO release and % reticulocyte count. 2.4. Acute effect of desidustat on iron regulation in inflammation Male BALB/c mice were administered turpentine oil (5 ml/kg, s.c.). In another set of experiment, animals were administered LPS (1 mg/kg, i.p.). Animals were treated with vehicle (0.25 % 5

CMC) or desidustat (15 mg/kg, p.o.) along with either turpentine oil or LPS treatment. Animals were killed 16 h after turpentine oil and 6 h after LPS treatment. At the end of treatment, animals were bled by retroorbital puncture. Spleen and liver tissue were collected for estimation of total iron. Serum samples were analyzed for total iron content as mentioned in analysis section. 2.5. Chronic effect of desidustat in turpentine oil-induced anemia Male BALB/c mice were administered turpentine oil (5 ml/kg, s.c.), twice a week for six weeks. After three weeks of turpentine oil administration, animals were randomized based on their hemoglobin levels and were treated with desidustat (5, 10, 15, 20, 30 and 50 mg/kg, p.o., alternate day) for next three weeks. At the end of three weeks of desidustat treatment, all animals were bled by retroorbital puncture for collection of whole blood and analyzed for RBC, hemoglobin, white blood cell (WBC) and mean corpuscular volume (MCV). 2.6. Chronic effect of desidustat in PGPS-induced anemia Six to eight-week-old female Lewis rats were used in the study. They were treated with Peptidoglycan-polysaccharide (PGPS, 15 mg/kg of rhamnose content, i.p.) or vehicle saline. Anemia accompanying chronic, relapsing systemic inflammation induced by PGPS is an established animal model of the anemia of chronic inflammatory disease (Sartor et al., 1989). One week after PGPS injection, animals were randomized based on their hemoglobin content and desidustat (15 and 30 mg/kg, p.o.) was administered to them every alternate day for 4 weeks. Normal control animals were administered vehicle saline. All the animals were bled by retroorbital puncture every week till 4 weeks for estimation of hemoglobin. At the end of study, animals were bled by retroorbital puncture for collection of whole blood and serum. Whole blood was analyzed for complete blood counts, while serum samples were analyzed for hepcidin

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and serum iron. Animals were euthanized and liver, spleen, and duodenum samples were collected in liquid nitrogen and stored at -70ºC. 2.7. Gene expression using quantitative reverse transcription polymerase chain reaction Total RNA was extracted from the duodenum and kidney samples by TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) as per supplier’s instructions. One microgram of total RNA from each sample was taken for first-strand cDNA synthesis using High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA; Part No. 4322171). An equal amount of cDNA from each sample was taken for quantitative real-time PCR using ABIprism-7300 (Applied Biosystems, Foster City, CA, USA). Gene expression of rat hepcidin (HAMP, F:5 GAAGGCAAGATGGCACTAAGCA- 3 and R:5 -TCTCGTCTGTTGCCGGAGATAG-30), EPO (F:5 -ATGGGGGTGCCCGAACG-3 and R:5 -TACCTCTCCAGAACGCGACT-3), Duodenal cytochrome B (DcytB, F:5-GTCATGCCCATACATGTGTATTC -3 and R:5 AGAATCAGAAGGCCCAGGGT-3), ferroportin (FPN1, F:5AGATCGCAGAACCCTTCCGCA-3 and R:5-TGTGGTGATACAGTCGAAGCCCA-3), divalent metal transporter 1 (DMT1, F:5 -CTAAGTAAACACTGGGTCAGCCT -3 and R:5AGGAGGATTGCTGTAAGTTTGAAGG-3) and monocyte chemoattractant protein-1 (MCP-1, F:5 -TGATCCCAATGAGTCGGCTG -3 and R:5-GGTGCTGAAGTCCTTAGGGT-3) was determined using SYBR Green quantitative real-time PCR and QIAGEN QuantiFast SYBR Green kit (Cat. No. 204052, Qiagen, Germantown, MD, USA). Rat β-actin (F:5TGTCACCAACTGGGACGATA-3 and R:5-AACACAGCCTGGATGGCTAC-3) was used as an internal control for normalization of data. Quantitation of the mRNAs was performed using the 2−ΔΔCt method using β-actin as a housekeeping gene. 7

2.8. Data Analysis Quantitative results were expressed as the mean ± S.E.M. Statistical significance was determined by one-way ANOVA followed by Dunnett’s post hoc analysis using GraphPad Prism version 7.03 (GraphPad Software, San Diego, CA, USA). P<0.05 was considered as significant. 3. Results 3.1. Single dose of desidustat improved erythropoietin and reticulocyte count in acute inflammatory condition In BALB/c mice, EPO levels reduced from 35.6 ± 2.9 to 20.8 ± 3.3 and 20.7 ± 1.4 pg/ml in LPS-treated and turpentine oil-treated group, respectively (Fig. 1A). Treatment with desidustat increased EPO (97.5 ± 5.5 pg/ml in LPS-treated group and 95.8 ± 8.2 pg/ml in turpentine oiltreated group, Fig. 1A). Reticulocyte count (%) was 1.3 ± 0.2 in control group, and it was 1.3 ± 0.2 and 1.5 ± 0.2, respectively, in LPS and turpentine oil-group (Fig. 1B). Treatment with desidustat increased reticulocyte count (3.9 ± 0.3 in LPS-treated group and 4.0 ± 0.3 in turpentine oil-treated group, respectively, Fig. 1B). 3.2. Single dose of desidustat improved iron homeostasis in acute inflammatory condition LPS treatment reduced serum iron by 57.2 ± 2.5 %, and increased serum hepcidin by 9.1 ± 0.5 fold, against normal control (Fig. 2A-B). LPS treatment increased spleen iron and liver by 31.6 ± 6.6 % and 17.9 ± 6.8 %, against normal control (Fig. 2 C-D). Turpentine oil administration increased hepcidin by 9.7 ± 1.7-fold, and reduced serum iron by 57.7 ± 2.3%, against normal control (Fig. 2A-B). Turpentine oil treatment increased spleen iron and liver by 42.5 ± 15.3 and 44.9 ± 8.2, against normal control. Desidustat treatment increased serum iron by 68.2 ± 12.7 % in LPS-treated group, and by 106.3 ± 21.9 % in turpentine oil-treated group (Fig. 2A). Desidustat 8

decreased serum hepcidin by 45.7 ± 5.9 % in LPS-treated mice, and by 44.7 ± 5.0 % in turpentine oil-treated mice (Fig. 2B). Desidustat treatment in LPS or Turpentine oil treated animal caused a non-significant reduction in spleen and liver iron (Fig. 2C-D). 3.3. Repeated dose treatment of desidustat improves erythropoiesis in turpentine oil-induced anemia In BALB/c mice, turpentine oil treatment reduced hemoglobin by 23.9 ± 2.0 %, RBC count by 12.5 ± 2.8 %, hematocrit by 16.1 ± 1.6%, MCV by 10.1 ± 0.8 % and increased WBC count by 91.1 ± 14.3%, when compared to normal control (Fig. 3A-E). Desidustat (5 to 50 mg/kg, alternate day for three weeks) treatment increased hemoglobin, RBC count and hematocrit in a dose-related manner (Fig. 3A-C). Desidustat treatment from 10 to 50 mg/kg, increased MCV levels in turpentine oil-treated animals (Fig. 3D). Desidustat did not alter WBC count at all the tested doses, when compared to vehicle control (Fig. 3E). 3.4. Repeated dose treatment of desidustat improves erythropoiesis and iron homeostasis in PGPS-induced anemia After a week of PGPS treatment, the hemoglobin levels reduced from 143.9 ± 2.5 g/l to 129.8 ± 4.0 g/l (Fig. 4A). These animals were then randomized into treatment groups (Desidustat, 15 and 30 mg/kg, alternate day for four weeks). At the end of treatment, PGPS-treated control rats demonstrated decreased hematocrit, RBC, MCV, and increased WBC count when compared with normal control (Fig. 4A-D). Desidustat (15 and 30 mg/kg) treatment caused increase in hemoglobin to 35.6 ± 2.8 and 52.4 ± 2.9 %, hematocrit to 34.5 ± 2.8 and 51.9 ±3.2 %, and RBC count to 14.0 ± 2.4 and 24.9 ± 3.7%, respectively when compared to PGPS control (Fig. 4A-C). Desidustat (15 and 30 mg/kg) treatment increased MCV by 17.9 ± 0.4 and 21.8 ± 1.7 %,

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respectively, when compared to PGPS-treated control group (Fig. 4D). Desidustat (15 and 30 mg/kg) treatment reduced WBC by 42.0 ± 9.1 and 59.4 ± 9.9 %, at 15 and 30 mg/kg dose, respectively, when compared to PGPS control (Fig. 4E). Desidustat (30 mg/kg) completely normalized the increase in WBC-induced by PGPS challenge (Fig. 4E). Desidustat (15 and 30 mg/kg) treatment increased HIF-1α levels by 57.2 ± 7.5 and 65.7 ± 22.3 %, respectively, when compared to PGPS-treated control group (Fig. 4F). PGPS treatment reduced serum iron from 55.3 ± 2.3 to 11.3 ± 1.4 µM, increased spleen iron from 74.3 ± 5.2 to 139.5 ± 5.4 µM/g, and increased serum hepcidin from 5.5 ± 0.6 to 75.2 ± 8.2 ng/ml, when compared to normal control (Fig. 5A-C). Desidustat treatment at 15 and 30 mg/kg caused increase in serum iron by 2.9 ± 0.4 and 3.0 ± 0.4 fold, respectively, when compared with PGPS control (Fig. 5A). Spleen iron was reduced by 38.4 ± 9.0 and 52.1 ± 7.0 % by desidustat treatment at 15 and 30 mg/kg dose, respectively (Fig. 5B). Desidustat treatment suppressed serum hepcidin by 32.2 ± 9.9 and 68.1 ± 7.6 % in 15 and 30 mg/kg respectively in PGPS control (Fig. 5C). 3.5. Repeated dose treatment of desidustat improves expression of genes involved in erythropoiesis and iron homeostasis in PGPS-induced anemia PGPS treatment has reduced DMT1 gene expression by 0.6 ± 0.1 fold in duodenum, while expression of FPN1 and DcytB genes remained unchanged when compared with normal control (Fig. 6A). PGPS treatment increased expression of HAMP by 5.2 ± 0.6 fold and MCP-1 by 4.4 ± 0.6 fold, while decreased expression of EPO by 0.6 ± 0.1 fold and FPN1 by 0.3 ± 0.1 fold, when compared to normal control (Fig. 6B). Desidustat (15 and 30 mg/kg) treatment increased duodenal gene expression of FPN1 by 1.6 ± 0.2 and 2.7 ± 0.3 fold, DcytB by 5.4±1.1 and 13.0 ± 1.1 fold, and DMT1 by 2.2 ± 0.4 and 2.3 ± 0.2 fold, respectively, when compared to PGPS 10

control (Fig. 6A). Desidustat (15 and 30 mg/kg) treatment increased hepatic EPO expression by 2.7 ± 0.1 fold and 4.1 ± 0.9 fold, when compared to vehicle control (Figure 6B). Desidustat (15 and 30 mg/kg) treatment increased hepatic expression of FPN1 by 2.3 ± 0.2 and 2.8 ± 0.5 fold (Fig. 6B). Desidustat (15 and 30 mg/kg) treatment reduced HAMP expression by 0.7 ± 0.1 and 0.3 ± 0.1 fold, and MCP-1 expression to 0.2 ± 0.1 and 0.2 ± 0.1 fold, respectively, when compared with PGPS control (Fig. 6B). 4. Discussion A major reason for anemia in chronic inflammation is blockade of iron in the tissue stores due to increase in hepcidin levels. Due to this iron restriction, efficient erythropoiesis is not possible even after ESA treatment in patients of anemia of chronic diseases. It is reported that systemic inflammation in chronic diseases is associated with hyporesponsiveness of patients to the EPO therapy (Alves et al., 2015). The iron supplementation is not useful in this case, since it further increases hepcidin levels, and causes more restriction of iron in tissue stores. Hence, decrease in hepcidin along with increase in erythropoietin levels is needed for the optimum therapy of anemia of chronic diseases. It has recently been reported that prolyl hydroxylase inhibitors can correct anemia associated with chronic kidney disease by virtue of their erythropoietin releasing effect as well as by decrease in hepcidin levels (Joharapurkar et al., 2018). This study evaluated the effect of desidustat, a novel PHD inhibitor, on erythropoietin, reticulocytes, iron and hepcidin in mice which were treated with two different inflammatory stimuli, namely LPS and turpentine oil. LPS is an endotoxin which functions through the inflammatory cytokines and elicits acute inflammatory response. The cytokine induction patterns in mice and humans are similar which enhance hepcidin production and induces hypoferremia (Kemna et al., 2008; Nicolas et al., 2002). It is also known that LPS-induced inflammation in 11

kidney decreases EPO (Jelkmann, 1998). On the other hand, turpentine oil administration induces sterile abscess and causes local inflammation that gives rise to cytokine induction, which has a different profile than LPS-induced endotoxemia, though interleukin 6 (IL-6) seems to be the common cytokine that is induced by both these toxins (Langdon et al., 2014). Hence, LPS and turpentine oil were used to induce acute inflammation. Repeated treatment of turpentine oil produces anemia by increasing hepcidin (Langdon et al., 2014). We have used turpentine oilinduced anemia model for finding appropriate dose of desidustat for PGPS-induced anemia. Desidustat (15 mg/kg) oral dose improved the serum iron levels that were reduced by acute treatment of either LPS or turpentine oil in BALB/c mice. Desidustat showed non-significant reduction in liver and spleen iron level in LPS and turpentine oil induced acute inflammation. The iron restoring effect of desidustat in mice treated either with LPS or turpentine oil were consistent with the reduction in hepcidin production, and enhanced release of erythropoietin, which was translated in increased reticulocyte production. It appears that desidustat can rescue hepcidin-dependent iron restriction in both these conditions after acute administration. The increase in serum iron seems to be originated from the combination of tissues, including reticuloendothelial system and liver. Anemia of chronic disease is associated with high hepcidin levels in clinic. Hepcidin levels are elevated in patients with chronic inflammation, leading to functional iron deficiency (D’Angelo, 2013). In the rat model of chronic inflammation, inflammatory normocytic, normochromic anemia and low serum iron was manifested in one week after a single i.p. injection of the PGPS (Coccia et al., 2001). The treatment with turpentine oil and PGPS reduced hemoglobin, RBC and MCV indicating development of microcytic hypochromic anemia. The inflammatory anemia induced by PGPS is responsive to the erythropoietin therapy, and is associated with enhanced

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hepcidin levels (Sartor et al., 1989; Coccia et al., 2001). Hepcidin induced regulation of iron metabolism appears mainly in liver and duodenum. Desidustat increases serum iron and suppresses liver HAMP in PGPS-treated female Lewis rats. It has been reported that extrarenal sites can contribute to a marked rise in circulating EPO with pharmacologic activation of HIF. Besides the kidneys, the liver is a site of relevant EPO production, producing most EPO during fetal life and contributing up to one third to total EPO production in animals who are exposed to severe hypoxia. It is therefore considered appropriate that EPO production in anephric patients is likely to be of hepatic origin (Bernhardt et al., 2010). Patients with chronic kidney diseases maintain persistent EPO, most probably due to increased liver EPO synthesis (de Seigneux et al., 2016). The PHD inhibitor desidustat can improve serum EPO in LPS and Turpentine oil-treated inflammatory conditions in mice, indicating enhanced EPO releasing capacity of the organs, i.e., primarily kidneys and liver. Increase in the hepatic expression of EPO, indicates that in addition to kidneys, desidustat treatment can improve erythropoietin release from hepatic stores, an observation which will be useful for positioning PHD inhibitors in anephric or kidney- compromised patients. The elevation in circulating erythropoietin concentrations after desidustat administration was in physiological limits, and translated to increased reticulocyte count. Desidustat treatment for four weeks corrected hemoglobin, RBC and MCV levels towards normal value at a dose of 15 mg/kg and 30 mg/kg in anemia caused by PGPS and at a dose of 10 mg/kg to 50 mg/kg in anemia caused by turpentine oil. In turpentine oil-induced anemia, oral administration of desidustat caused a dose-dependent increase in hemoglobin and RBCs. Inflammation increases nuclear factor-kB (NF-kB) and other cytokines such as TNF-α, IL-6 which suppress EPO release, leading to anemia (Nairz et al., 2001; Rivkin et al, 2016). PHDs are

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positively associated with expression of pro-inflammatory cytokines, by interacting with p65 and thus increase NF-kB mediated expression of TNF-α, Interleukin 1 beta and MCP-1. It is proposed that these effects of PHDs are independent of their HIF-degrading activity (Li et al., 2015). Chronic systemic inflammation causes increased expression of HAMP, a gene for hepcidin (Júnior et al., 2015). Up-regulation of HAMP blocks FPN1, which causes entrapment of iron in tissue stores like intestine, liver and spleen, visa-versa down-regulation of HAMP increases FPN1 mRNA and releases iron from tissue to blood circulation (Wessling-Resnick, 2010; Zughaier et al., 2014). Hepcidin also inhibit the expression of DMT1 and DcytB involved in iron absorption, leading to reduced availability of iron for efficient erythropoiesis (WesslingResnick, 2010; Wollmann et al., 2014). DcytB knockout mice do not demonstrate iron deficiency, but they have impaired maturation of reticulocytes (Gunshin et al., 2005; Choi et al., 2012). Overexpression of DcytB in Caco2 cells increases iron uptake, and coexpression of DcytB and DMT1 show additive increase in iron uptake (Latunde-Dada et al., 2008). It is possible that other reductases are redundant for this process. Expression of DcytB and DMT1 in duodenum regulates iron absorption and mobilization which is needed for maturation of reticulocytes (Mastrogiannaki et al., 2009; Shah et al., 2009). An increase in duodenal DcytB and DMT1 was observed after chronic treatment of desidustat, which indicated efficient iron absorption. Hepatic expression of MCP-1 was reduced after desidustat treatment, which could be mediated by suppression of NF-kB. This is also reflected in normalization of WBC count, indicative of reduced inflammatory load. Increase in duodenal and hepatic FPN1 after desidustat treatment could facilitate release of iron from the tissue stores, which can be incorporated in hemoglobin. FPN1 is regulated both by direct expression and by post-translational modification by hepcidin (Nemeth, 2008; Zhang et

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al., 2011). It has been reported that hepcidin induces the degradation of FPN1 protein as well as reduces FPN1 expression in a dose-dependent manner (Zhang et al., 2011), and a negative correlation exists between hepcidin and ferroportin expression in the inflammatory condition (Zughaier et al., 2014). Though we have not measured ferroportin levels, it can be predicted that desidustat could degrade FPN1 protein levels. Taken together, it appears that by virtue of prolyl hydroxylase inhibition, desidustat regulates hepcidin-ferroportin axis to improve iron homeostasis in chronic inflammatory condition. All the prolyl hydroxylase inhibitors function by stabilizing HIF. Desidustat is able to inhibit liver hepcidin and optimize iron utilization for erythropoiesis by stabilization of HIF-1α in liver and kidney in normal and nephrectomized rats (Jain et al., 2016). In the current study, desidustat treatment increases HIF-1α levels in liver of PGPS-treated rats. It has been reported that direct HIF binding to the hepcidin promoter led to downregulation of hepcidin (Peyssonnaux et al., 2007). It is also possible that a prolyl hydroxylase inhibitor can decrease HAMP protein expression by an increase in erythroid regulator erythroferrone-ERFE (Gammella et al., 2015), and hypoxia-inducible factor regulates hepcidin via erythropoietin-induced erythropoiesis (Liu et al., 2012). Studies have also shown that hepcidin repression can also occur without erythropoietic stimulus, by HIF stabilization (Anderson et al., 2012). It is reported that VhlR200W homozygote humans with enhanced activated HIF signaling demonstrate reduced hepcidin levels that did not correlate with an increase in serum EPO (Gordeuk et al., 2011). This suggests that HIFs could suppress hepcidin independently of erythropoiesis. Taken together, it appears that prolyl hydroxylase inhibition can function as a regulator of interdependent pathways of hepcidin-ferroportin and erythropoietin production through the stabilization of HIF. In chronic inflammatory condition, desidustat increases EPO, and suppresses HAMP and thus enhances

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efficient iron utilization. Since all the PHD inhibitors wok by stabilization of HIF, it can be contemplated that these beneficial effects on iron metabolism could be similarly produced by other PHD inhibitor in clinical trials. In conclusion, oral administration of desidustat, a novel PHD inhibitor, reverses anemia-induced by inflammatory stimuli like PGPS or turpentine oil. Thus, PHD inhibitors can be useful in the treatment of anemia of inflammation by virtue of their actions on efficient erythropoiesis. Thus, desidustat can be an effective treatment for anemia of inflammation.

Acknowledgement No funding received for this manuscript. This is a Zydus Research Centre (ZRC) communication.

Conflict of interest All the authors are employees of Zydus Research Centre, a unit of Cadila Healthcare Ltd. No author of this manuscript has any potential competing interest. Authors are thankful to Dr. Vrajesh Pandya, Mr. Kalpesh Shah, and Dr. Ranjit Desai for their valuable inputs in this work.

Author contributions Mukul R. Jain, Amit Joharapurkar, Pankaj Patel, and Vishal Patel contributed to the conception of the manuscript, design of experiments, and analysis and interpretation of the data, and writing of the manuscript. Amit Joharapurkar, Vishal Patel, Samadhan Kshirsagar, Brijesh Sutariya, Maulik Patel, and Hiren Patel performed the experiments, analyzed the data, and wrote the manuscript. All authors have 16

commented on the initial and final drafts of the manuscript and are responsible for approval of the final version of the manuscript in all aspects.

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