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Mimicking hypoxia to treat anemia: HIF-stabilizer BAY 85-3934 (Molidustat) stimulates erythropoietin production without hypertensive effects.

Flamme I, Oehme F, Ellinghaus P, Jeske M, Keldenich J, Thuss U - PLoS ONE (2014)

Bottom Line: Mimicking hypoxia by inhibiting HIF-PHs, thereby stabilizing HIF, is a novel treatment concept for restoring endogenous EPO production.Notably, unlike treatment with the antihypertensive enalapril, the blood pressure normalization was achieved without a compensatory activation of the renin-angiotensin system.Clinical studies are ongoing to investigate the effects of BAY 85-3934 therapy in patients with renal anemia.

View Article: PubMed Central - PubMed

Affiliation: Cardiology/Hematology, Acute Care Research, Global Drug Discovery, Bayer Pharma AG, Wuppertal, Germany.

ABSTRACT
Oxygen sensing by hypoxia-inducible factor prolyl hydroxylases (HIF-PHs) is the dominant regulatory mechanism of erythropoietin (EPO) expression. In chronic kidney disease (CKD), impaired EPO expression causes anemia, which can be treated by supplementation with recombinant human EPO (rhEPO). However, treatment can result in rhEPO levels greatly exceeding the normal physiological range for endogenous EPO, and there is evidence that this contributes to hypertension in patients with CKD. Mimicking hypoxia by inhibiting HIF-PHs, thereby stabilizing HIF, is a novel treatment concept for restoring endogenous EPO production. HIF stabilization by oral administration of the HIF-PH inhibitor BAY 85-3934 (molidustat) resulted in dose-dependent production of EPO in healthy Wistar rats and cynomolgus monkeys. In repeat oral dosing of BAY 85-3934, hemoglobin levels were increased compared with animals that received vehicle, while endogenous EPO remained within the normal physiological range. BAY 85-3934 therapy was also effective in the treatment of renal anemia in rats with impaired kidney function and, unlike treatment with rhEPO, resulted in normalization of hypertensive blood pressure in a rat model of CKD. Notably, unlike treatment with the antihypertensive enalapril, the blood pressure normalization was achieved without a compensatory activation of the renin-angiotensin system. Thus, BAY 85-3934 may provide an approach to the treatment of anemia in patients with CKD, without the increased risk of adverse cardiovascular effects seen for patients treated with rhEPO. Clinical studies are ongoing to investigate the effects of BAY 85-3934 therapy in patients with renal anemia.

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Characterization of the in vitro activity of BAY 85-3934.(A)–(C) Concentration–response curves of hypoxia-inducible factor prolyl hydroxylase (PHD2) activity for BAY 85–-934 after the addition of increasing concentrations of 2-oxoglutarate, Fe2+, and ascorbate. Data, presented as means ± SEM of 4 replicates, were normalized to basal activity without BAY 85-3934 (100%) and residual activity (0%). (D) Detection of HIF-1α and HIF-2α in HeLa, A549, and Hep3B cells by western blot analysis. (E) Time-course of induction of HIF-1α in HeLa cells after addition of serum-free medium containing BAY 85-3934 (5 µM). β-actin levels were measured as a loading control. (F) Time-course of disappearance of HIF-1α in A549 cells after induction with BAY 85-3934 (20 µM). Culture medium was withdrawn and replaced with medium containing cycloheximide (100 µM). β-actin levels were measured as a loading control (D–F show representative data of 3 independent experiments). (G) Concentration–response curve for luciferase activity of A549 HIF-RE2 reporter cells (relative luciferase units [RLUs]) after addition of BAY 85-3934 in the presence or absence of additional Fe2+. Data are presented as means ± SEM of 4 replicates. (H) Relative mRNA expression levels (means ± SD of 2 replicates) of a panel of HIF target genes (shown as fold-increase from baseline levels) after exposure to BAY 85-3934 in HeLa, A549, and Hep3B cells. For definition of gene symbols, see Table 1.
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pone-0111838-g001: Characterization of the in vitro activity of BAY 85-3934.(A)–(C) Concentration–response curves of hypoxia-inducible factor prolyl hydroxylase (PHD2) activity for BAY 85–-934 after the addition of increasing concentrations of 2-oxoglutarate, Fe2+, and ascorbate. Data, presented as means ± SEM of 4 replicates, were normalized to basal activity without BAY 85-3934 (100%) and residual activity (0%). (D) Detection of HIF-1α and HIF-2α in HeLa, A549, and Hep3B cells by western blot analysis. (E) Time-course of induction of HIF-1α in HeLa cells after addition of serum-free medium containing BAY 85-3934 (5 µM). β-actin levels were measured as a loading control. (F) Time-course of disappearance of HIF-1α in A549 cells after induction with BAY 85-3934 (20 µM). Culture medium was withdrawn and replaced with medium containing cycloheximide (100 µM). β-actin levels were measured as a loading control (D–F show representative data of 3 independent experiments). (G) Concentration–response curve for luciferase activity of A549 HIF-RE2 reporter cells (relative luciferase units [RLUs]) after addition of BAY 85-3934 in the presence or absence of additional Fe2+. Data are presented as means ± SEM of 4 replicates. (H) Relative mRNA expression levels (means ± SD of 2 replicates) of a panel of HIF target genes (shown as fold-increase from baseline levels) after exposure to BAY 85-3934 in HeLa, A549, and Hep3B cells. For definition of gene symbols, see Table 1.

Mentions: Under standard assay conditions, in the presence of 20 µM 2-oxoglutarate, 10 µM Fe2+, and 2 mM ascorbate, the mean IC50 values of BAY 85-3934 for PHD1, PHD2, and PHD3 were 480 nM, 280 nM, and 450 nM, respectively. The IC50 values were found to be dependent on the concentration of 2-oxoglutarate in the reaction buffer. By lowering the 2-oxoglutarate concentration from 20 µM to 0.3 µM, the potency of the test compound increased up to 10-fold (Fig. 1A). Variation of the concentrations of Fe2+ and ascorbate in the reaction buffer by factors of 30 and 200, respectively, did not alter the potency of the inhibitor by more than 2-fold (Fig. 1B, C). The IC50 values were always well below the concentration of Fe2+.


Mimicking hypoxia to treat anemia: HIF-stabilizer BAY 85-3934 (Molidustat) stimulates erythropoietin production without hypertensive effects.

Flamme I, Oehme F, Ellinghaus P, Jeske M, Keldenich J, Thuss U - PLoS ONE (2014)

Characterization of the in vitro activity of BAY 85-3934.(A)–(C) Concentration–response curves of hypoxia-inducible factor prolyl hydroxylase (PHD2) activity for BAY 85–-934 after the addition of increasing concentrations of 2-oxoglutarate, Fe2+, and ascorbate. Data, presented as means ± SEM of 4 replicates, were normalized to basal activity without BAY 85-3934 (100%) and residual activity (0%). (D) Detection of HIF-1α and HIF-2α in HeLa, A549, and Hep3B cells by western blot analysis. (E) Time-course of induction of HIF-1α in HeLa cells after addition of serum-free medium containing BAY 85-3934 (5 µM). β-actin levels were measured as a loading control. (F) Time-course of disappearance of HIF-1α in A549 cells after induction with BAY 85-3934 (20 µM). Culture medium was withdrawn and replaced with medium containing cycloheximide (100 µM). β-actin levels were measured as a loading control (D–F show representative data of 3 independent experiments). (G) Concentration–response curve for luciferase activity of A549 HIF-RE2 reporter cells (relative luciferase units [RLUs]) after addition of BAY 85-3934 in the presence or absence of additional Fe2+. Data are presented as means ± SEM of 4 replicates. (H) Relative mRNA expression levels (means ± SD of 2 replicates) of a panel of HIF target genes (shown as fold-increase from baseline levels) after exposure to BAY 85-3934 in HeLa, A549, and Hep3B cells. For definition of gene symbols, see Table 1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230943&req=5

pone-0111838-g001: Characterization of the in vitro activity of BAY 85-3934.(A)–(C) Concentration–response curves of hypoxia-inducible factor prolyl hydroxylase (PHD2) activity for BAY 85–-934 after the addition of increasing concentrations of 2-oxoglutarate, Fe2+, and ascorbate. Data, presented as means ± SEM of 4 replicates, were normalized to basal activity without BAY 85-3934 (100%) and residual activity (0%). (D) Detection of HIF-1α and HIF-2α in HeLa, A549, and Hep3B cells by western blot analysis. (E) Time-course of induction of HIF-1α in HeLa cells after addition of serum-free medium containing BAY 85-3934 (5 µM). β-actin levels were measured as a loading control. (F) Time-course of disappearance of HIF-1α in A549 cells after induction with BAY 85-3934 (20 µM). Culture medium was withdrawn and replaced with medium containing cycloheximide (100 µM). β-actin levels were measured as a loading control (D–F show representative data of 3 independent experiments). (G) Concentration–response curve for luciferase activity of A549 HIF-RE2 reporter cells (relative luciferase units [RLUs]) after addition of BAY 85-3934 in the presence or absence of additional Fe2+. Data are presented as means ± SEM of 4 replicates. (H) Relative mRNA expression levels (means ± SD of 2 replicates) of a panel of HIF target genes (shown as fold-increase from baseline levels) after exposure to BAY 85-3934 in HeLa, A549, and Hep3B cells. For definition of gene symbols, see Table 1.
Mentions: Under standard assay conditions, in the presence of 20 µM 2-oxoglutarate, 10 µM Fe2+, and 2 mM ascorbate, the mean IC50 values of BAY 85-3934 for PHD1, PHD2, and PHD3 were 480 nM, 280 nM, and 450 nM, respectively. The IC50 values were found to be dependent on the concentration of 2-oxoglutarate in the reaction buffer. By lowering the 2-oxoglutarate concentration from 20 µM to 0.3 µM, the potency of the test compound increased up to 10-fold (Fig. 1A). Variation of the concentrations of Fe2+ and ascorbate in the reaction buffer by factors of 30 and 200, respectively, did not alter the potency of the inhibitor by more than 2-fold (Fig. 1B, C). The IC50 values were always well below the concentration of Fe2+.

Bottom Line: Mimicking hypoxia by inhibiting HIF-PHs, thereby stabilizing HIF, is a novel treatment concept for restoring endogenous EPO production.Notably, unlike treatment with the antihypertensive enalapril, the blood pressure normalization was achieved without a compensatory activation of the renin-angiotensin system.Clinical studies are ongoing to investigate the effects of BAY 85-3934 therapy in patients with renal anemia.

View Article: PubMed Central - PubMed

Affiliation: Cardiology/Hematology, Acute Care Research, Global Drug Discovery, Bayer Pharma AG, Wuppertal, Germany.

ABSTRACT
Oxygen sensing by hypoxia-inducible factor prolyl hydroxylases (HIF-PHs) is the dominant regulatory mechanism of erythropoietin (EPO) expression. In chronic kidney disease (CKD), impaired EPO expression causes anemia, which can be treated by supplementation with recombinant human EPO (rhEPO). However, treatment can result in rhEPO levels greatly exceeding the normal physiological range for endogenous EPO, and there is evidence that this contributes to hypertension in patients with CKD. Mimicking hypoxia by inhibiting HIF-PHs, thereby stabilizing HIF, is a novel treatment concept for restoring endogenous EPO production. HIF stabilization by oral administration of the HIF-PH inhibitor BAY 85-3934 (molidustat) resulted in dose-dependent production of EPO in healthy Wistar rats and cynomolgus monkeys. In repeat oral dosing of BAY 85-3934, hemoglobin levels were increased compared with animals that received vehicle, while endogenous EPO remained within the normal physiological range. BAY 85-3934 therapy was also effective in the treatment of renal anemia in rats with impaired kidney function and, unlike treatment with rhEPO, resulted in normalization of hypertensive blood pressure in a rat model of CKD. Notably, unlike treatment with the antihypertensive enalapril, the blood pressure normalization was achieved without a compensatory activation of the renin-angiotensin system. Thus, BAY 85-3934 may provide an approach to the treatment of anemia in patients with CKD, without the increased risk of adverse cardiovascular effects seen for patients treated with rhEPO. Clinical studies are ongoing to investigate the effects of BAY 85-3934 therapy in patients with renal anemia.

Show MeSH
Related in: MedlinePlus