Perspectives for the therapy of anemia of chronic diseases

Abstract

The incidence of anemia of chronic disease (ACD) is underestimated, increases with age, and affects about 30% of the elderly. ACD treatment is currently based on the pharmacotherapy of diseases that caused anemia, erythropoiesis-stimulating agents, and parenteral administration of iron supplementation in case of iron deficiency. Increasing knowledge on the pathophysiology of ACD has resulted in the burst of research on the development of new drugs that are focused on three main areas. The first group of drugs includes substances that inhibit hepcidin transcription, namely direct and indirect bone morphogenetic protein 6 (BMP6) inhibitors and/or SMAD signaling pathway inhibitors, and drugs that regulate hepcidin transcription through STAT3 signaling pathway. The second group of drugs includes direct hepcidin inhibitors (e.g., aptamers, anticalin proteins, monoclonal antibodies) or substances that inhibit the binding of hepcidin to ferroportin. The third group of drugs improves erythropoiesis mainly by upregulation of erythropoietin and/or inhibition of proinflammatory cytokines. In the latter group, hypoxia-inducing factor stabilizers and IL-6 or TNFα antagonists are particularly important. This article discusses new drug groups and substances that are in different phases of development, including both preclinical and clinical studies, and focuses on the prospects of their use in ACD. © 2020 Polish Society of Hematology and Transfusion Medicine, Insitute of Hematology and Transfusion Medicine.

Full text

125
Perspectives for the therapy of anemia
of chronic diseases
Department of Pharmacology and Toxicology,
Collegium Medicum, University of Zielona Góra,
Zielona Góra, Poland
Sylwia Sulimiera Michalak
REVIEW ARTICLE
Article history:
Received: 31.01.2020
Accepted: 02.03.2020
journal homepage: https://content.sciendo.com/ahp
Abstract
The incidence of anemia of chronic disease (ACD) is underestimated, increases with age, and aects about 30% of the elderly. ACD
treatment is currently based on the pharmacotherapy of diseases that caused anemia, erythropoiesis-stimulating agents, and parenteral
administration of iron supplementation in case of iron deciency. Increasing knowledge on the pathophysiology of ACD has resulted
in the burst of research on the development of new drugs that are focused on three main areas. The rst group of drugs includes
substances that inhibit hepcidin transcription, namely direct and indirect bone morphogenetic protein
6 (BMP6) inhibitors and/or SMAD signaling pathway inhibitors, and drugs that regulate hepcidin transcription through STAT3 signaling
pathway. The second group of drugs includes direct hepcidin inhibitors (e.g., aptamers, anticalin proteins, monoclonal antibodies) or
substances that inhibit the binding of hepcidin to ferroportin. The third group of drugs improves erythropoiesis mainly by upregulation
of erythropoietin and/or inhibition of proinammatory cytokines. In the latter group, hypoxia-inducing factor stabilizers and IL-6 or
TNFα antagonists are particularly important. This article discusses new drug groups and substances that are in dierent phases of
development, including both preclinical and clinical studies, and focuses on the prospects of their use in ACD.
© 2020 Polish Society of Hematology and Transfusion Medicine, Insitute of Hematology and Transfusion Medicine. Published by Sciendo.
All rights reserved.
Keywords:
anemia of chronic disease, hepcidin, cytokine, hypoxia-inducible factor, treatment
Acta Haematologica Polonica 51(3) • September 2020 • 125–132 • DOI: 10.2478/ahp-2020-0024
Introduction
The global prevalence of anemia of chronic disease (ACD), also known
as anemia of inammation, is not fully estimated [1]. The incidence of
ACD increases with age, and in the elderly population it accounts for
approximately one-third of all anemia cases [2, 3]. ACD can develop
in the course of infections, malignancies, and autoimmune diseases.
The etiology of this type of anemia is complex and it is associated with
a decrease in red blood cells (RBCs) production and decreased survival.
The activity of proinammatory cytokines, including tumor necrosis
factor α (TNFα) and interferons, results in decreased availability of
iron for erythropoiesis, reduced response to erythropoietin (EPO),
and decreased EPO synthesis, which in turn inhibit proliferation and
dierentiation of erythroid progenitor cells and increase erythrocyte
turnover and their degradation by macrophages. Hepcidin plays
a central role in reducing the bioavailability of iron, and its expression
is regulated by proinammatory cytokines, in particular, interleukin 6
(IL-6) [4, 5]. However, other molecules, such as IL-10, interferon γ (IFN-γ),
IL-1β, and lipopolysaccharide, also serve as hepcidin inducers [6]. Many
proteins have been described to be involved in the regulation of hepcidin
synthesis, and thus they may become a target for ACD therapy. These
include bone morphogenetic protein (BMP), hemojuvelin (HJV), activin
receptor-like kinases 2 and 3 (ALK-2 and ALK-3), and erythroferrone
(ERFE) [5, 7]. Also EPO can directly reduce hepcidin production by
inhibiting the BMP-small mothers against decapentaplegic proteins
(BMP-SMAD) pathway [8] and also via hypoxia-inducible factor (HIF)
signaling [9].
Currently, ACD therapy is limited to the treatment of the underlying
disease that is responsible for inammatory response. Additionally,
erythropoiesis-stimulating agents (ESAs), in particular human
recombinant erythropoietin (EPO), are also administered. Sometimes
iron supplementation is necessary. Better understanding of the
regulation of iron metabolism in the presence of inammation, with
particular focus on the role of hepcidin, proinammatory cytokines, and
HIF in erythropoiesis, has caused a burst of research on the development
of new drugs for the treatment of ACD. These studies, focusing on
three major research areas, are in various stage of development from
experimental stage through preclinical research to clinical studies. The
rst group of studies evaluates the inuence of various substances
on hepcidin production. The second group examines the substances
that inhibit the eects of hepcidin release. This group contains direct
hepcidin inhibitors (hepcidin neutralizers and anti-hepcidin monoclonal
antibodies) or substances that are able to inhibit binding of hepcidin to
ferroportin. Finally, the third group of studies focuses on erythropoiesis
inducers that upregulate EPO, stabilize HIF, and block proinammatory
cytokines.
Hepcidin production inhibitors
Hepcidin production inhibitors downregulate hepcidin gene (hepcidin
antimicrobial peptide gene [HAMP]) in hepatocytes (Fig. 1). Hepcidin
transcription can be inhibited as a result of the inhibition of SMAD and/
or signal transducer and activator of transcription (STAT) signaling
pathways. It can be achieved in a variety of mechanisms, the most
important of which seems to be BMP or BMP receptor interaction and
blocking of IL-6 or IL-6 receptor. Downstream signaling from BMP
receptor is mediated through SMAD1/5/8 pathway while signaling
from IL-6 receptor is mediated through Janus-activated kinases (JAK)/
Corresponding author: Sylwia Sulimiera Michalak, Department of Pharmacology and Toxicology, Collegium Medicum, University of Zielona Góra, Zyty 28, 65-046 Zielona Góra, Poland,
phone: +48 68 328 73 77, e-mail: s.michalak@cm.uz.zgora.pl

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STAT3 signaling pathway. The BMP-SMAD pathway is activated
by BMP-2 and BMP-6 through BMP receptors forming complexes
with ALK-2 and ALK-3, and HJV as a BMP receptor on liver cells
enhances the transmission of this signal [7, 10].
Drugs/substances aecting BMP6 pathway
Currently, a number of substances aecting BMP6 pathways
are studied. In two models, murine and rat, the use of anti-BMP6
antibodies led to an increase in hemoglobin (Hb), a decrease in iron
deposition in tissues, and a reduction in ESA doses needed to treat
ACD [11]. Recently, LY3113593, a human anti-BMP6 monoclonal
antibody, is gaining more interest. LY3113593 blocks binding of BMP6
to its receptor, which leads to an increase in iron concentration, an
increase in transferrin saturation, and a decrease in hepcidin level. In
a study performed in a group of patients with chronic kidney disease
(CKD), LY3113593 increased Hb and caused a decrease in ferritin
level in comparison with placebo [12].
Other drugs aecting BMP-6 receptor currently tested in clinical trials
include: LDN-193189, modied heparin (e.g., roneparstat), TP-0184,
and a group of drugs related to HJV. LDN-193189 is a selective
inhibitor of the BMP kinase type 1 receptor [13]. TP-0184 is an ALK-2
inhibitor that reduces hepcidin mRNA and improves Hb concentration
in preclinical studies in mice [14]. Modied heparin with reduced
anticoagulation ability binds BMP-6 and blocks hepcidin expression,
as demonstrated in in vitro and in vivo animal models [15, 16].
The ability to inhibit hepatic BMP-SMAD signaling, reduce hepcidin
levels, increase ferroportin expression, and increase serum iron
levels has also been demonstrated for protein-soluble HJVFc (HJV.
Fc). HJV.Fc consists of extracellular domain of HJV conjugated with
human IgG Fc fragment [10, 17]. Currently, other potential drugs, e.g.,
h5F9.23 and h5F9-AM8, which interact with HJV, are experimentally
investigated [18]. Administration of a single dose of ABT-207 or
H5F9-AM8 in rats and monkeys resulted in prolonged suppression of
hepcidin production and an increase in serum iron concentration [19].
H5F9-AM8 was tested in animal models of ACD and iron-refractory
iron-deciency anemia (IRIDA), resulting in reduction of hepcidin and
improvement of anemia in all cases [20].
Druggable targets associated with proinammatory
cytokines
Clinically available drugs include a humanized anti-IL-6 receptor
antibody, tocilizumab, and a chimeric (human–murine) antibody
against IL-6, siltuximab. Tocilizumab is used in rheumatoid arthritis.
As an improvement of RBC parameters have been observed during
the treatment, tocilizumab has become a new potential drug for
ACD [21, 22]. Tocilizumab was successfully used in the treatment
of anemia in patients with Castleman’s disease [22] and in patients
with malignant tumors [23]. Reduced serum hepcidin level and
improvement of anemia in patients with Castleman’s disease [24, 25],
myeloma, and other lymphoproliferative cancers [26] have been
reported for siltuximab. An improvement in RBC parameters has also
been demonstrated in the treatment of solid tumors [27]. Another IL-6
receptor antibody, MR16-1, was tested in murine models. MR16-1 has
been shown to improve Hb parameters in cancer-associated anemia
[23]. Other substances aecting hepcidin production by modulation of
signaling pathways in hepatocytes have been tested experimentally.
In murine models, natural and synthetic STAT3 inhibitors have been
shown to inhibit hepcidin expression in macrophages [28]. MaR1
Fig. 1. Hepcidin transcription inhibitors
ACVR2A – activin receptor type 2A; ALK3/2 – activin receptor-like kinase 3/2; BMP-6 – bone morphogenetic protein 6; BMPR2 – bone
morphogenetic protein receptor 2; ERFE – erythroferron; EGFR – epidermal growth factor receptor; HAMP – hepcidin antimicrobial peptide;
HJV – hemojuvelin; HFE – human hemochromatosis protein; IL-6 – interleukin6; IL-6R – IL-6 receptor; JAK – Janus-activated kinases; SMAD
– small mothers against decapentaplegic proteins; STAT3 – signal transducer and activator of transcription 3; TFR2 – transferrin receptor 2;
TMPRSS6 – transmembrane protease serine 6

Acta Haematologica Polonica
127
inhibits inammatory response, including hepcidin expression, by
aecting the IL-6/STAT3 signaling pathway and then reducing the
severity of anemia symptoms [29].
Potential role of ERFE
ERFE is a physiological regulator of hepcidin, which is synthesized
and secreted by erythroblasts in the bone marrow and other tissues in
response to EPO. ERFE inhibits hepcidin transcription by an unknown
mechanism that involves BMP6 pathway and probably some known
membrane receptors [30, 31]. ERFE antagonists and agonists, and
also other substances involved in the BMP6 signaling pathway,
may prove useful in the prevention and treatment of iron disorders.
However, a recently published study suggested a correlation
between increased ERFE levels and death/cardiovascular events in
hemodialysis patients with CKD, which is further enhanced by the
administration of ESA [30]. Therefore, the potential use of ERFE in
anemia requires further research and careful determination of patient
groups who may benet from such a therapy.
Other possibilities of hepcidin production inhibition
It has been shown that hepcidin production can be inhibited
by thiazolidinediones, testosterone, estrogens, vitamin D,
and substances of plant origin (e.g., Angelica sinensis). New
thiazolidinediones are studied in the treatment of anemia. These
drugs may aect hepcidin expression through various pathways,
including SMAD1/5/8 signaling pathway, extracellular signal-
regulated kinases 1/2 (ERK1/2) and TMPRSS6, and also probably
by indirectly aecting ERFE-mediated hepcidin production [32].
In vitro and in vivo studies have shown that vitamin D inhibits the
expression of proinammatory cytokines and directly inhibits
hepcidin transcription by interaction of vitamin D receptor with the
HAMP promoter [33, 34]. In a population of children with chronic
inammatory bowel disease, high levels of cholecalciferol (≥30 ng/mL)
were associated with higher Hb levels, while low levels correlated
with elevated hepcidin and lower Hb concentration [35]. However, not
all studies conrmed the eect of vitamin D supplementation on the
levels of proinammatory cytokines, ferritin, or hepcidin [36]. A meta-
analysis and systematic review of literature did not conrm the eect
of cholecalciferol on IL-6 or C-reactive protein (CRP) levels [37].
On the other hand, Smith and colleagues showed that high doses
of vitamin D signicantly reduced hepcidin levels in healthy adults
1 week after the administration of a single dose, without aecting the
levels of proinammatory cytokines or ferritin. The authors concluded
that this may suggest that the eect of vitamin D on hepcidin is
independent from proinammatory cytokines or ferritin [38]. In many
clinical trials, the inverse correlation between vitamin D levels and
anemia has been reported. It is now believed that vitamin D is an
important factor involved in the pathogenesis of anemia [39, 40].
Testosterone inhibits hepcidin by upregulation of the epidermal
growth factor receptor (EGFR) signaling in the liver [41] and
downregulates hepcidin transcription by its inuence on BMP-SMAD
signaling pathway [42]. The administration of testosterone increases
the concentration of Hb in men with reduced testosterone levels, in
case of anemia of both known and unknown cause as demonstrated
in a study of nearly 800 men, including 126 with anemia [43].
Similarly, 17-β-estradiol could be considered a drug for anemia in the
elderly women because it has also been shown to inhibit hepcidin
transcription. Inhibition of hepcidin transcription results from the
activity of estrogens on HAMP-associated gene promoters [44].
Angelica sinensis polysaccharide (ASP), a polysaccharide
obtained from the root of the Angelica sinensis plant, inhibits
hepcidin expression and may potentially improve RBC parameters
in patients with anemia. The ecacy of ASP in the treatment of
anemia associated with CKD has been demonstrated in rats. ASP
inhibited hepcidin production, increased the amount of iron available
for erythropoiesis by its mobilization from the liver and spleen, and
increased EPO levels [45]. Previous data have shown that regular
supplementation of Angelica sinensis in hemodialyzed patients
improved anemia in patients resistant to recombinant human EPO
therapy [46]. It is believed that ASP inhibits hepcidin expression
by inhibiting the expression and/or phosphorylation of JAK1/2,
SMAD1/5/8, and ERK1/2 while upregulating SMAD7 [47].
Direct hepcidin inhibitors and agents
preventing hepcidin from binding to
ferroportin
Another group of drugs that aect hepcidin are direct hepcidin
inhibitors and drugs that prevent hepcidin from binding to ferroportin
(Fig. 2). Direct neutralization of hepcidin is possible with monoclonal
antibodies (AB 12B9M) as well as anticalin proteins (e.g., PRS-080),
aptamers (e.g., NOX-H94), or guanosine 5'-diphosphate (GDP).
Anticalins are a class of ligand-binding proteins designed based on a
lipocalin scaold. Aptamers are oligonucleotides (short DNA or RNA
fragments) or peptides that bind specically to a specic molecule.
PRS-080 is a hepcidin-binding anticalin protein [48]. In cynomolgus
monkeys, PRS-080 has been shown to reduce hepcidin and increase
iron levels [49]. However, there are currently ongoing studies
assessing the eects of PRS-080 anticalin proteins in patients with
anemia in the course of CKD (NCT03325621) [50]. On the other
hand, the aptamer NOX-H94 (lexaptepid pegol [LP]) is a pegylated
oligoribonucleotide that binds to hepcidin with high anity and blocks
its biological function. The eects of this aptamer were conrmed in
vitro and in vivo [51]. The pharmacokinetics, pharmacodynamics,
and safety study of NOX-H94 carried out in 64 healthy volunteers has
shown decrease in hepcidin level and increase in iron and transferrin
saturation in comparison with placebo [52]. LP has been used to
treat ACD in patients with multiple myeloma and lymphoma. LP
caused a signicant increase in Hb (≥1 g/dL) in 5 out of 12 patients.
Responders experienced increase in Hb concentration in RBCs and
reticulocytes, and a decrease in soluble transferrin receptor (sTFR)
level. These results conrm the hypothesis on the ecacy of hepcidin
inhibition in the treatment of cancer-related anemia, especially in
functional iron deciency. However, the patients with symptomatic
iron deciency, hypochromic anemia, without excessive levels of
ferritin, and high sTFR levels were more likely to respond to LP [53].
The group of monoclonal antibodies that serve as direct inhibitors
of hepcidin includes Ab12B9m and LY2787106. Ab12B9m is fully
human monoclonal IgG2 antibody that binds both human and
monkey hepcidin, which is currently under clinical development

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[54]. LY2787106 is also a fully humanized monoclonal antibody. In
phase 1 clinical trials, the use of LY2787106 in tumor-associated
anemia resulted in a dose-dependent increase in iron concentration
and transferrin saturation, with good tolerance prole [55]. GDP also
seems to be a promising agent. It is a direct hepcidin inhibitor with
ecacy conrmed in in vitro studies. In animal experiments in mice
with ACD, administration of GDP in combination with iron sulfate
resulted in an increase in Hb concentration, iron, and ferroportin
(FPN) expression and decrease in ferritin level [56].
Potential drugs that prevent hepcidin from binding to ferroportin
include: LY2928057, fursultiamine, thioxolone, and chloroquine.
These compounds prevent internalization and lysosomal degradation
of ferroportin. As a result, hepcidin cannot inhibit iron transport in
intestinal epithelial cells or macrophages [57, 58]. LY2928057 is
a fully humanized IgG4 monoclonal antibody that binds with human
ferroportin. In patients with anemia in the course of CKD, LY2928057
improved iron parameters but did not increase Hb level. Therefore,
the need for concomitant ESAs treatment was suggested [59].
Agents aecting EPO and proinammatory
cytokines
Another option for treating ACDs is to enhance erythropoiesis by
aecting EPO pathway or interfere with inammatory processes
(Fig. 3). This mechanism is utilized by HIF prolyl hydroxylase
inhibitors (HIF-PHI), i.e., molecules that stabilize HIF subunits. The
use of HIF-PHI, similar to hypoxia, activates genes that regulate EPO
concentration and contribute to the regulation of iron metabolism.
As a result, there is an increase in EPO level and iron availability
for erythropoiesis [9, 60]. EPO stimulates erythropoiesis, and via
activation of EPO receptor it induces ERFE and blocks hepcidin
activity [61].
HIF hydroxylase inhibitors appear to be benecial, especially
for patients with CKD-associated anemia. By increasing EPO
concentration, the need for ESAs that are associated with increased
risk of cardiovascular events is reduced. In addition, HIF-PHI can be
administered orally which is the preferred route for patient treatment
[60]. The following HIF-PHI are currently tested: molidustat [62],
GSK1278863 [63], or FG-4592 [64]. The research is focused mainly
on patients with CKD. Phase 3 trials of the safety, tolerability,
and ecacy of molidustat in patients with CKD (both on and o
dialysis) are currently ongoing [62, 65]. Phase 3 on the ecacy of
GSK1278863 in patients with CKD showed a signicant increase
in the reticulocyte count and other RBC parameters, indicating that
further testing is warranted [63]. Also, phase 2 trials of roxadustat
(FG-4592) has shown its potential for oral therapy in dialyzed anemic
patients with CKD [66].
The trials with HIF hydroxylase inhibitors focus not only in the
treatment of ACDs but also in other studies. HIF stabilization with
HIF-PHI is important in regulating hematopoietic stem cell (HSC)
proliferation and HSC regeneration processes. These properties
can be used to protect the bone marrow during radiation therapy, or
to increase mobilization in transplant procedures using granulocyte
colony-stimulating factor (GCS-F) and plerixafor. Therefore, HIF
Fig. 2. Direct and indirect hepcidin inhibitors

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hydroxylase inhibitors may have a potential to improve the eects
of treatment in transplant recipients or oncology patients [67]. HIF
also participates in the regulation of multiple processes including:
apoptosis, angiogenesis, cell proliferation, pH regulation, cellular
energy metabolism, and glucose transport [9]. Therefore, further
research on the long-term safety of HIF hydroxylase inhibitors is
needed, with particular attention to the assessment of the risk of
tumorigenesis. It should be emphasized that in rat studies, roxadustat
did not show carcinogenic properties [68]. Another potential threat is
that HIF stabilization may be associated with an increase in circulating
broblast growth factor 23 (FGF23) level, and high concentrations
of FGF23 have been associated with cardiovascular complications
and bone mineralization disorders. However, the increase in FGF23
caused by HIF-PHI is lower than its increase after administration of
ESAs [69, 70].
The increase in EPO synthesis can also be caused by monoclonal
antibodies as discussed earlier, i.e., anti-IL-6 siltuximab [24] and
anti-IL-6 receptor tocilizumab [21, 24]. TNFα is a potential target
for anemia of inammatory diseases, especially in the course of
arthritis or inammatory bowel disease (IBD) [22]. Anti-TNFα drugs
include etanercept (ETN), adalimumab (ADA), iniximab (IFX), and
golimumab (GO). ETN is a recombinant receptor protein conjugated
to the Fc fragment of a human immunoglobulin G1. ETN prevents
TNFα from binding to the receptor and blocks its eects. Other
human monoclonal antibodies, ADA and GO, as well as chimeric
monoclonal antibody IFX and humanized pegylated Fab fragment of
monoclonal antibody, certolizumab, exert similar eects. In patients
with IBD, reduced hepcidin level and improved anemia after anti-
TNFα treatment that modulated hepcidin concentration via IL-6
pathway were observed [71]. It has also been reported that the use of
anti-TNFα drugs in IBD patients resolves anemia without the need for
iron supplementation [72]. ETN, ADA, and IFX improved Hb level in
patients with rheumatoid and psoriatic arthritis [73]. Increased risk of
carcinogenesis after anti-TNFα therapy has not been conrmed so far;
however, careful surveillance is needed in the treated patients [74]. It
should be highlighted that the use of biologicals in the course of IBD
is associated with the potential risk of hematological complications
such as neutropenia or the development of lymphomas, especially
when biologicals are used in combination with other drugs. Further
studies are needed to estimate the exact risk of these complications
[75, 76].
Summary
A number of studies on new drugs for the treatment of ACD are
underway, focusing on various factors involved in iron metabolism
and erythropoiesis regulation. Drugs and tested substances are
intended to directly inhibit hepcidin or its transcription, or counteract
hepcidin activity. The eect of various substances on erythropoiesis
is also assessed, mainly by stabilizing the HIF and inhibiting
proinammatory cytokines. Some of these drugs are already used
in the treatment of inammatory bowel disease or in rheumatology,
while others are in the experimental phase and will take many years
before their introduction to the routine practice. Intensive research
Fig. 3. Drugs aecting erythropoietin and pro-inammatory cytokines
EPO – erythropoietin; IL-6 – interleukin 6; IL-6R – IL-6 receptor; TNFα – tumor necrosis factor α; HIF – hypoxia-inducible factor

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and a vast array of tested compounds give hope for the development
of more eective ACD treatments in the coming future.
Authors’ contributions
SSM – the only author.
Conict of interest
None.
Financial support
None.
Ethics
The work described in this article has been carried out in accordance
with The Code of Ethics of the World Medical Association (Declaration
of Helsinki) for experiments involving humans; EU Directive 2010/63/
EU for animal experiments; Uniform requirements for manuscripts
submitted to biomedical journals.
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