Activin receptor ligand traps in chronic kidney disease.

PURPOSE OF REVIEW: Sotatercept and luspatercept are recombinant soluble activin type-II receptor-IgG-Fc fusion proteins that are tested in clinical trials for the treatment of various types of anemias, including renal anemia. The mechanism of the action of the novel drugs is incompletely understood, but it seems to be based on the inactivation of soluble proteins of the transforming growth factor-ß (TGFß) family. This review considers pros and cons of the clinical use of the drugs in reference to the current therapy with recombinant erythropoiesis-stimulating agents (ESAs). RECENT FINDINGS: One or more activin type-II receptor (ActRII) ligands appear to inhibit erythroid precursors, for example growth and differentiation factor 11. Trapping of these ligands by the recombinant ActRII fusion proteins, sotatercept and luspatercept increases red blood cell numbers and hemoglobin levels in humans. Reportedly, the novel compounds were well tolerated in trials on healthy volunteers and patients suffering from anemia due to chronic kidney disease or malignancies. On approval, the drugs may prove particularly useful in patients suffering from ineffective erythropoiesis, such as in myelodysplastic syndrome, multiple myeloma or ß-thalassemia, where ESAs are of little use. Independent of their effect on erythropoiesis, ActRII ligand traps were found to exert beneficial effects on renal tissue in experimental animals. SUMMARY: ESAs are likely to remain standard of care in renal anemia. There is a need for a better understanding of the effects of ActRII ligand traps on TGFß-like proteins. The novel drugs have not been approved for sale as therapeutics so far. Their long-term efficacy and safety still needs to be proven, particularly with respect to immunogenicity. Antifibrotic effects may be worthy to be investigated in humans.

Watch out for a revival of peginesatide in sports.

The erythropoietin-mimetic peptide (EMP) peginesatide belongs to the group of erythropoiesis-stimulating agents (ESAs) that are prohibited when misused in sports. Peginesatide is a synthetic pegylated homodimer of two cyclic 21-amino acid chains. It was approved for the treatment of anaemic patients with chronic kidney disease in the USA in 2012, but recalled in 2013 due to prevalent cases of acute severe anaphylactoid reactions and associated fatalities (0.02%). The drug was considered obsolete for athletes and part of the anti-doping scene lost sight of it. However, recent research indicates that the adverse events were not caused by the drug substance, but by the drug product formulated in multi-use vials. These vials contained comparably high levels of subvisible particles. Phenol was identified as a critical component of the drug formulation, which caused the release of histamine from mast cells. Tricky athletes might consider peginesatide a pharmacologically safe ESA in an appropriate formulation. In addition, other EMPs may get a second wind for therapy including misuse in sports. Therefore, it is very important to proceed in developing electrophoretic, immunological, and mass spectroscopic methods for detecting peginesatide and other EMPs in human urine and blood samples. Copyright © 2016 John Wiley & Sons, Ltd.

Impact of hypoxia inducible factors on estrogen receptor expression in breast cancer cells.

In women breast cancer is still the most commonly diagnosed cancer. This type of cancer is classified as a hormone-dependent tumor. Estrogen receptor (ER) expression and functional status contribute to breast cancer development and progression. Another important factor associated with cancer is hypoxia which is defined as the state of reduced oxygen availability in tissues. Intratumoral hypoxia results in the activation of the hypoxia inducible factors (HIFs). HIFs are heterodimeric transcription factors involved in the regulation of many cellular processes, such as angiogenesis, anaerobic metabolism, cell proliferation/survival, and promotion of metastasis. In this study we evaluated the interplay between hypoxia, HIF stabilization and the ER-α/ß-ratio in several ER-positive breast cancer cell lines. Hypoxia was shown to inhibit ER expression in ER-positive breast cancer cells. Further experiments using the hypoxia mimetic CoCl2 and HIF-1α knockdown cells indicated that the influence of hypoxia on breast cancer cells involves other pathways than the molecular oxygen sensing pathway. Moreover, we demonstrated that MCF-7 cells in long-term culture lost part of their ability to respond to hypoxic incubation. Understanding the relationships between HIF, ER-α and ER-ß expression holds the promise of the development of new therapeutic agents and may provide future advances in prognosis.

Investigational therapies for renal disease-induced anemia.

INTRODUCTION: The main pillars for the treatment of chronic kidney disease (CKD) associated anemia are peptidic erythropoiesis stimulating agents (ESAs) and iron preparations. Both approaches benefit from long-term efficacy and safety data but are surrounded by clinical and economic concerns, driving the search for novel anti-anemic drugs. AREAS COVERED: By answering pivotal questions, the authors describe the recent developments of next generation ESAs, introduce cutting-edge iron formulations and focus on investigational approaches that interact with pathways involved in erythropoietin (Epo) synthesis and myeloid hematopoiesis. Finally, the challenges encountered with these drug candidates are discussed. EXPERT OPINION: Current peptidic ESAs are effective and well-tolerated, but are costly, require parenteral application and iron supplementation. ESA resistance may develop calling for increased doses. Therefore, orally available hypoxia-inducible factor (HIF) stabilizing compounds are attractive alternatives, which may be approved in the near future. Prominent compounds are molidustat, daprodustat and roxadustat. HIF stabilizers suppress hepcidin production and improve iron balance as the present ESAs, but also raise safety concerns in association with their pleiotropic actions. Other investigational erythropoietic biologics are growth-differentiation factor-11 (GDF11) ligand traps (sotatercept, luspatercept), which are also well advanced in development. Possibly, they will provide an add-on for established therapies. However, immunogenicity of these compounds still needs to be carefully investigated.

Prolyl-4-Hydroxylase 2 Potentially Contributes to Hepatocellular Carcinoma-Associated Erythrocytosis by Maintaining Hepatocyte Nuclear Factor-4α Expression.

BACKGROUND: Increased red blood cell count (Erythrocytosis) is an important paraneoplastic syndrome of hepatocellular carcinoma (HCC) and is a significant risk factor for lethal lung artery thromboembolism. HCC-associated erythrocytosis is partially caused by the ability of several HCC cells to produce erythropoietin (EPO). Prolyl-4-hydroxylase 2 (PHD2) is an enzyme encoded by the gene EGLN1. The best-known function of PHD2 is to mediate the oxygen-dependent degradation of the labile α-subunit of hypoxia-inducible factor (HIF). However, there is increasing evidence that PHD2 also regulates HIF-independent pathways by interacting with other substrates. METHODS: In the EPO-producing human HCC cell line HepG2, the expression of PHD2 gene was silenced with siRNA. EPO production was estimated using quantitative PCR and ELISA. RESULTS: In HepG2 cells, PHD2 suppresses the activity of TGF-ß1 pathway and consequently maintains the expression of hepatocyte nuclear factor-4α (HNF-4α), an important transcription factor promoting the EPO expression in hepatocytes. PHD2 knockdown caused a marked reduction of EPO production. HIF seemed not to be involved in this biology. CONCLUSION: Our findings show that PHD2 represents a potential contributing factor for HCC-associated erythrocytosis. Selective inhibition of PHD2 in HCC cells might be considered as a new way to manage erythrocytosis in HCC patients.

Nuclear-cytoplasmatic shuttling of proteins in control of cellular oxygen sensing.

In order to pass through the nuclear pore complex, proteins larger than ∼40 kDa require specific nuclear transport receptors. Defects in nuclear-cytoplasmatic transport affect fundamental processes such as development, inflammation and oxygen sensing. The transcriptional response to O2 deficiency is controlled by hypoxia-inducible factors (HIFs). These are heterodimeric transcription factors of each ∼100-120 kDa proteins, consisting of one out of three different O2-labile α subunits (primarily HIF-1α) and a more constitutive 1ß subunit. In the presence of O2, the α subunits are hydroxylated by specific prolyl-4-hydroxylase domain proteins (PHD1, PHD2, and PHD3) and an asparaginyl hydroxylase (factor inhibiting HIF-1, FIH-1). The prolyl hydroxylation causes recognition by von Hippel-Lindau tumor suppressor protein (pVHL), ubiquitination, and proteasomal degradation. The activity of the oxygen sensing machinery depends on dynamic intracellular trafficking. Nuclear import of HIF-1α and HIF-1ß is mainly mediated by importins α and ß (α/ß). HIF-1α can shuttle between nucleus and cytoplasm, while HIF-1ß is permanently inside the nucleus. pVHL is localized to both compartments. Nuclear import of PHD1 relies on a nuclear localization signal (NLS) and uses the classical import pathway involving importin α/ß receptors. PHD2 shows an atypical NLS, and its nuclear import does not occur via the classical pathway. PHD2-mediated hydroxylation of HIF-1α occurs predominantly in the cell nucleus. Nuclear export of PHD2 involves a nuclear export signal (NES) in the N-terminus and depends on the export receptor chromosome region maintenance 1 (CRM1). Nuclear import of PHD3 is mediated by importin α/ß receptors and depends on a non-classical NLS. Specific modification of the nuclear translocation of the three PHD isoforms could provide a promising strategy for the development of new therapeutic substances to tackle major diseases.

The ESA scenario gets complex: from biosimilar epoetins to activin traps.

Recombinant human erythropoietin (rhEpo, epoetin) has proved beneficial in preventing transfusion-dependent anaemia in patients with chronic kidney disease. Apart from copied epoetins distributed in less regulated markets, 'biosimilar' epoetins have gained currency in many regions, where they compete with the originals and with rhEpo analogues with prolonged survival in circulation ('biobetter'). Recombinant erythropoiesis stimulating agents are potent and well tolerated. However, their production is costly, and they must be administered by the parenteral route. Hence, other anti-anaemia treatments are being evaluated. Clinical trials are being performed with stabilizers of the hypoxia-inducible transcription factors (HIFs), which increase endogenous Epo production. HIF stabilizers are chemical drugs and they are active on oral administration. However, there is fear that they may promote tumour growth. Epo mimetic peptides have also raised expectations. Yet the prototype peginesatide was recalled after just 1 year of its widespread use in the USA because of serious side-effects including cases of death. Most recently, clinical trials have been initiated with sotatercept, a recombinant soluble activin receptor type 2A IgG-Fc fusion protein. Sotatercept binds distinct members of the transforming growth factor-ß family, thereby preventing the inhibitory action of these factors in erythropoiesis. Taken together, rhEpo and its long-acting recombinant analogues will likely remain mainstay of anti-anaemia therapies in the near future.

Intolerability of cobalt salt as erythropoietic agent.

Unfair athletes seek ways to stimulate erythropoiesis, because the mass of haemoglobin is a critical factor in aerobic sports. Here, the potential misuse of cobalt deserves special attention. Cobalt ions (Co(2+) ) stabilize the hypoxia-inducible transcription factors (HIFs) that increase the expression of the erythropoietin (Epo) gene. Co(2+) is orally active, easy to obtain, and inexpensive. However, its intake can bear risks to health. To elaborate this issue, a review of the pertinent literature was retrieved by a search with the keywords 'anaemia', 'cobalt', 'cobalt chloride', 'erythropoiesis', 'erythropoietin', 'Epo', 'side-effects' and 'treatment', amongst others. In earlier years, cobalt chloride was administered at daily doses of 25 to 300 mg for use as an anti-anaemic agent. Co(2+) therapy proved effective in stimulating erythropoiesis in both non-renal and renal anaemia, yet there were also serious medical adverse effects. The intake of inorganic cobalt can cause severe organ damage, concerning primarily the gastrointestinal tract, the thyroid, the heart and the sensory systems. These insights should keep athletes off taking Co(2+) to stimulate erythropoiesis.

Progress in detecting cell-surface protein receptors: the erythropoietin receptor example.

Testing for the presence of specific cell-surface receptors (such as EGFR or HER2) on tumor cells is an integral part of cancer care in terms of treatment decisions and prognosis. Understanding the strengths and limitations of these tests is important because inaccurate results may occur if procedures designed to prevent false-negative or false-positive outcomes are not employed. This review discusses tests commonly used to identify and characterize cell-surface receptors, such as the erythropoietin receptor (EpoR). First, a summary is provided on the biology of the Epo/EpoR system, describing how EpoR is expressed on erythrocytic progenitors and precursors in the bone marrow where it mediates red blood cell production in response to Epo. Second, studies are described that investigated whether erythropoiesis-stimulating agents could stimulate tumor progression in cancer patients and whether EpoR is expressed and functional on tumor cells or on endothelial cells. The methods used in these studies included immunohistochemistry, Northern blotting, Western blotting, and binding assays. This review summarizes the strengths and limitations of these methods. Critically analyzing data from tests for cell-surface receptors such as EpoR requires understanding the techniques utilized and demonstrating that results are consistent with current knowledge about receptor biology.

Physiology and pharmacology of erythropoietin.

Human erythropoietin (Epo) is a 30.4 kDa glycoprotein hormone composed of a single 165 amino acid residues chain to which four glycans are attached. The kidneys are the primary sources of Epo, its synthesis is controlled by hypoxia-inducible transcription factors (HIFs). Epo is an essential factor for the viability and proliferation of erythrocytic progenitors. Whether Epo exerts cytoprotection outside the bone marrow still needs to be clarified. Epo deficiency is the primary cause of the anemia in chronic kidney disease (CKD). Treatment with recombinant human Epo (rhEpo, epoetin) can be beneficial not only in CKD but also for other indications, primarily anemia in cancer patients receiving chemotherapy. Considering unwanted events, the administration of rhEpo or its analogs may increase the incidence of thromboembolism. The expiry of the patents for the original epoetins has initiated the production of similar biological medicinal products ('biosimilars'). Furthermore, analogs (darbepoetin alfa, methoxy PEG-epoetin beta) with prolonged survival in circulation have been developed ('biobetter'). New erythropoiesis-stimulating agents are in clinical trials. These include compounds that augment erythropoiesis directly (e.g. Epo mimetic peptides or activin A binding protein) and chemicals that act indirectly by stimulating endogenous Epo synthesis (HIF stabilizers).

The Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT/HIF-1ß) is influenced by hypoxia and hypoxia-mimetics.

BACKGROUND: The Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT, HIF-1ß) is a member of the basic-Helix-Loop-Helix PER/ARNT/SIM (bHLH/PAS) protein family and a vital transcriptional regulator regarding development and physiological adaptation processes. ARNT is discussed to be linked with cancer, and other diseases. ARNT is known to be translocated into the cell nucleus, where accumulation of the protein takes place. ARNT is a heterodimerisation partner of the xenobiotic ligand activated Aryl Hydrocarbon Receptor (AhR), the Single Minded proteins (SIM), the cardiovascular helix-loop-helix factor 1 and the Hypoxia Inducible Factor proteins (HIF-α). ARNT is obligatory for HIF-1, HIF-2 and HIF-3 binding to DNA. Whereas degradation of the HIF-α subunits is suppressed by hypoxia, ARNT is generally regarded as constitutively expressed in excess within the cell, and stabilisation is commonly thought to be oxygen-independent. However, we provide evidence that the regulation of ARNT is far more complex. The aim of our study was to reevaluate the regulation of ARNT expression. METHODS: We examined cell lines of different origin like MCF-7 and T47D (human breast cancer), HeLa (human cervix carcinoma), Hep3B and HepG2 (human hepatoma), Kelly (human neuroblastoma), REPC (human kidney) and Cos7 (primary primate kidney) cells. We used immunoblot analysis, densitometry, RT-PCR and transient transfection. RESULTS AND CONCLUSION: Our results show that ARNT protein levels are influenced by hypoxia and hypoxia mimetics such as cobalt(II)-chloride (CoCl2) and dimethyloxalylglycine (DMOG) in a cell line specific manner. We demonstrate that this effect might be triggered by HIF-1α which plays an important role in the process of stabilizing ARNT in hypoxia.

Antibody-mediated pure red cell aplasia in chronic kidney disease patients receiving erythropoiesis-stimulating agents: new insights.

Antibody-mediated pure red cell aplasia is a very rare but devastating condition affecting patients receiving treatment with erythropoiesis-stimulating agents. New cases continue to emerge, generally in clusters, consistent with an 'environmental' trigger to its pathogenesis. Defining the causes of antibody-mediated pure red cell aplasia is clearly of importance for patients with chronic kidney disease, but any developments in this area may also have relevance to other disease areas as therapeutic delivery of endogenous proteins rapidly increases. This review focuses on the current knowledge regarding the etiology of antibody-mediated pure red cell aplasia and the current approach to therapy.

Stabilisation and knockdown of HIF--two distinct ways comparably important in radiotherapy.

BACKGROUND: Radiotherapy is one of the most widely used treatments for cancer. The benefit of radiation is known to be negatively affected by tumor hypoxia and the expression of hypoxia-inducible factors (HIF), respectively. HIF-1α/ ß and HIF-2α/ ß are transcriptional activators of oxygen-regulated genes. The aim of the study was to examine cell type-specific effects of HIF-1α and -2α knockdown or oxygen-independent HIF-stabilisation on radiosensitivity. METHODS: Herein, we treated four different wildtype and HIF-1α- or HIF-2α-deficient human cancer cell lines, cultured under normoxic or hypoxic conditions, with ionising radiation in doses from 2 to 6 Gy and examined clonogenic survival. Furthermore, the cells were partly preincubated with a HIF-stabiliser (di-tert-butyroyl-oxymethyl-2,4-pyridine-dicarboxylate, (t)Bu-2,4-PDC). RESULTS: The results show that both hypoxia exposure and treatment with (t)Bu-2,4-PDC increased the radioresistance of human cancer cells. The HIF-mediated decrease of radioresponsiveness induced by the chemical stabiliser emerged to be as strong as the one caused by hypoxia. Clonogenic survival assays furthermore revealed that HIF-1 expression enhanced resistance to radiation, whereas knocking-down HIF-1 increased the sensitivity to radiation under normoxic as well as under hypoxic conditions. CONCLUSION: These data extend previous observations of HIF-1α and broaden the view by showing HIF-2α inverse correlation between HIF expression and prognosis for the outcome of radiotherapy.

Blood doping and its detection.

Hemoglobin mass is a key factor for maximal exercise capacity. Some athletes apply prohibited techniques and substances with intent to increase hemoglobin mass and physical performance, and this is often difficult to prove directly. Autologous red blood cell transfusion cannot be traced on reinfusion, and also recombinant erythropoietic proteins are detectable only within a certain timeframe. Novel erythropoietic substances, such as mimetics of erythropoietin (Epo) and activators of the Epo gene, may soon enter the sports scene. In addition, Epo gene transfer maneuvers are imaginable. Effective since December 2009, the World Anti-Doping Agency has therefore implemented "Athlete Biologic Passport Operating Guidelines," which are based on the monitoring of several parameters for mature red blood cells and reticulocytes. Blood doping may be assumed, when these parameters change in a nonphysiologic way. Hematologists should be familiar with blood doping practices as they may play an important role in evaluating blood profiles of athletes with respect to manipulations, as contrasted with the established diagnosis of clinical disorders and genetic variations.

Curcumin decreases survival of Hep3B liver and MCF-7 breast cancer cells: the role of HIF.

BACKGROUND: Curcumin, a commonly used spice, affects the activities of cytokines, enzymes, and transcription factors that are linked to inflammation. Furthermore, curcumin has been assigned tumor growth inhibiting effects, possibly mediated by promoting hypoxia-inducible factor (HIF) degradation. HIFs are transcription factors that play a central role in the adaptation and response to low oxygen levels in metazoan cells. However, curcumin also exhibits properties of an iron chelator indicating its potential of inhibiting HIF-α prolyl hydroxylase (PHD) activity. METHODS: We were interested in clarifying these divergent actions of curcumin in due consideration of the effects on radio-therapy. Thus, concentration- and time-dependent effects of curcumin on HIF-α and -ß protein levels and activity in hepatoma and breast carcinoma cell cultures under normoxic and hypoxic conditions were studied. RESULTS: It was shown that HIF-1α accumulated in normoxia after the application of higher doses of the drug. Curcumin proved to lower HIF-1α and HIF-2α protein levels in hypoxia. HIF-1ß (ARNT; arylhydrocarbon nuclear translocator) protein levels and HIF transcriptional activity were reduced in normoxia and hypoxia after 4 h and 24 h incubation periods. Furthermore, curcumin treatment negatively impacted on clonogenic cell survival of Hep3B hepatoma and MCF-7 breast carcinoma cells. CONCLUSION: Effects of curcumin on cell growth and survival factor expression suggest its potential benefit in the treatment of cancer without a direct radiosensitizing influence of curcumin on these cells.

Regulation of erythropoietin production.

The hormone erythropoietin (Epo) maintains red blood cell mass by promoting the survival, proliferation and differentiation of erythrocytic progenitors. Circulating Epo originates mainly from fibroblasts in the renal cortex. Epo production is controlled at the transcriptional level. Hypoxia attenuates the inhibition of the Epo promoter by GATA-2. More importantly, hypoxia promotes the availability of heterodimeric (α/ß) hypoxia-inducible transcription factors (predominantly HIF-2) which stimulate the Epo enhancer. The HIFs are inactivated in normoxia by enzymatic hydroxylation of their α-subunits. Three HIF-α prolyl hydroxylases (PHD-1, -2 and -3) initiate proteasomal degradation of HIF-α, while an asparaginyl hydroxylase ('factor inhibiting HIF-1', FIH-1) inhibits the transactivation potential. The HIF-α hydroxylases contain Fe(2+) and require 2-oxoglutarate as co-factor. The in vivo response is dynamic, i.e. the concentration of circulating Epo increases initially greatly following an anaemic or hypoxaemic stimulus and then declines despite continued hypoxia. Epo and angiotensin II collaborate in the maintenance of the blood volume. Whether extra-renal sites (brain, skin) modulate renal Epo production is a matter of debate. Epo overproduction results in erythrocytosis. Epo deficiency is the primary cause of the anaemia in chronic kidney disease and a contributing factor in the anaemias of chronic inflammation and cancer. Here, recombinant analogues can substitute for the hormone.

Biosimilar epoetins and other "follow-on" biologics: update on the European experiences.

After the patents of biopharmaceuticals have expired, based on specific regulatory approval pathways copied products ("biosimilars" or "follow-on biologics") have been launched in the EU. This article summarizes experiences with hematopoietic medicines, namely the epoetins (two biosimilars traded under five different brand names) and the filgrastims (two biosimilars, six brand names). Physicians and pharmacists should be familiar with the legal and pharmacological specialities of biosimilars: The production process can differ from that of the original, clinical indications can be extrapolated, glycoproteins contain varying isoforms, the formulation may differ from the original, and biopharmaceuticals are potentially immunogenic. Only on proof of quality, efficacy and safety, biosimilars are a viable option because of their lower costs.

Erythropoietin and the skin: a role for epidermal oxygen sensing?

Erythropoietin (EPO), long appreciated as the chief endocrine regulator of red blood cell formation, is now recognized to exert many additional functions outside the bone marrow. Thus, the quest is on to define the full range of EPO functions in the physiology and pathology of non-hematopoietic tissues. Two recent studies in man and mice have highlighted the importance of the mammalian skin as one peripheral tissue with a previously unsuspected role in EPO biology; both, as a target and as a source of EPO. In addition, the skin has been proposed to function as an oxygen sensor. The present hypothesis essay critically reviews the currently available evidence for this and provides a unifying theoretical scenario for intracutaneous EPO functions and for a potential role of the skin in the control of EPO production. Mainly, we propose that the skin itself directly contributes to the up-regulation of EPO plasma levels in response to hypoxia.

Effects of chloroquine treatment on circulating erythropoietin and inflammatory cytokines in acute Plasmodium falciparum malaria.

Anemia is a common and serious complication of malaria due to Plasmodium falciparum infection, a major health problem in tropical areas. Herein, the relation was investigated between the levels of circulating erythropoietin (EPO) and immunomodulatory cytokines in response to chloroquine treatment. Thirty-seven healthy control subjects and 40 patients with acute P. falciparum infection were included in the study. All subjects were adult male Sudanese. Blood samples were collected before chloroquine administration (25 mg/kg body weight, orally on three consecutive days) and 3 and 30 days after start of the therapy. Measurements included routine hematological parameters and the concentrations of immunoreactive EPO, tumor necrosis factor-alpha (TNF-alpha), interleukin 1alpha (IL-1), IL-6, and interferon gamma (INF-gamma). Chloroquine treatment led to a decrease in EPO levels in the control subjects but an increase in malaria patients at day 30. The latter was likely due to the anti-inflammatory action of the drug because INF-gamma, IL-1, and IL-6 concentrations declined on chloroquine treatment. Based on these findings, we propose that an impaired EPO production in association with a prolonged elevation of certain inflammatory cytokines can contribute to the anemia in some malaria patients which can be reversed by chloroquine therapy.