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

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

Penetration of moxifloxacin into rat mandibular bone and soft tissue

Objective. Based on its in vitro activity and spectrum of activity, the new 8-methoxyquinolone antibiotic moxifloxacin (MXF) seems suited for the antibiotic therapy of odontogenic infections. Penetration into the relevant tissue is another prerequisite for clinical efficacy. For this reason, the levels of MXF in plasma, soft tissue, and mandibular bone were determined in an animal model with Wistar rats. Material and methods. Samples of 49 rats were analyzed. Tissue samples were homogenized and proteins were precipitated. The pharmacokinetic evaluation was conducted based on non-compartmental analysis. Results. The concentration-time courses of tissues show a more plateau-shaped curve compared to plasma. Calculated AUC (area under the curve) ratios tissue:plasma were M. masseter:plasma = 2.64 and mandibles:plasma = 1.13. Conclusions. Administration of antibiotics is considered an important part of therapy during and/or after surgical procedures in the maxillofacial area. Because of the good penetration into bone and muscle tissues demonstrated in Wistar rats, MXF might be an option for clinical application in this indication

Effects of BAY 85-3934 administration in male Wistar rats treated with gentamicin to induce renal anemia.

<p>Data are presented as means ± SEM. (A) Plasma EPO levels, and (B) kidney and (C) liver relative expression of erythropoietin (EPO) mRNA 4 h after oral administration of BAY 85-3934. Before administration, rats had been treated with vehicle or gentamicin. (<i>n</i> = 5 animals per group). *<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001 compared with vehicle group,^§<i>p</i><0.05 and ^§§<i>p</i><0.001 compared with control group, t-test. (D) Kidney and (E) liver mRNA expression levels of hypoxia-inducible factor target genes relative to mean of vehicle treated animals after oral administration of BAY 85-3934 (<i>n</i> = 4 to 5 animals per group, error bars not shown for clarity of presentation). For definition of gene symbols, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111838#pone-0111838-t001" target="_blank">Table 1</a>. (F) Time-course of changes in packed cell volume (PCV) following treatment with BAY 85-3934 or vehicle (once daily, five times per week, number of animals as indicated). (G) Hemoglobin levels 7 days after start of treatment with BAY 85-3934 or vehicle at day 22 of experiment shown in (F). *<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001 compared with vehicle group; ^#<i>p</i><0.05 compared with sham group; t-test.</p

Characterization of the <i>in vivo</i> activity of BAY 85-3934 (in male Wistar rats).

<p>Data are presented as means ± SEM. (A) Increase in plasma erythropoietin (EPO) at 4 h and (B) reticulocytes (as a proportion of red blood cells [RBCs]) at 72 h following single oral dosing of BAY 85-3934. Data were pooled from two sequential experiments (<i>n</i> = 2×5 animals per group). *<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001; unpaired t-test, sequentially pairwise-applied to dose groups and corresponding vehicle group. (C) Change in packed cell volume (PCV) during once-daily dosing with BAY 85-3934 (<i>n</i> = 12 animals per group). **<i>p</i><0.01 and ***<i>p</i><0.001; two-way ANOVA with Dunnett’s multiple comparison test versus vehicle group. (D) Induction of erythropoiesis after subcutaneous administration of recombinant human EPO (rhEPO) twice weekly or BAY 85-3934 (2.5 mg/kg) once daily (<i>n</i> = 10 animals per group). *<i>p</i><0.001 compared with control (t-test) at day 30. (E) BAY 85-3934 plasma levels, kidney EPO relative mRNA expression, and plasma EPO levels after oral administration of BAY 85-3934 (5 mg/kg) (<i>n</i> = 5 animals per group). (F) Relative mRNA expression levels of HIF target genes in rat kidney after administration of BAY 85-3934 (5 mg/kg). Baseline expression was set at 1 (<i>n</i> = 5 animals per group; error bars not show for clarity of presentation). For definition of gene symbols, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111838#pone-0111838-t001" target="_blank">Table 1</a>.</p

Characterization of the <i>in vitro</i> activity of BAY 85-3934.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111838#pone-0111838-t001" target="_blank">Table 1</a>.</p

Effects of BAY 85-3934 or recombinant human erythropoietin (rhEPO) on erythropoietic parameters in cynomolgus monkeys.

<p>Data are presented as means ± SEM. (A) Plasma erythropoietin (EPO) concentrations after repeat oral administration of BAY 85-3934 (<i>n</i> = 6 animals per group). (B) Plasma EPO concentrations after a single s.c. administration of rhEPO (100 IU/kg) or a single oral dose of BAY 85-3934 (1.5 mg/kg) (<i>n</i> = 3 animals per group). (C) Erythropoietic parameters (hemoglobin [HGB], red blood cells [RBCs], and reticulocytes) after s.c. administration of rhEPO twice weekly (100 IU/kg) for 2 weeks or BAY 85-3934 (1.5 mg/kg) once daily for 2 weeks (<i>n</i> = 3 animals per group).</p

Effect of BAY 85-3934, erythropoietin (EPO), and enalapril in the rat subtotal nephrectomy model.

<p>Data are presented as means ± SEM. (A) Packed cell volume (PCV), (B) systolic blood pressure (SBP), and (C) prorenin following oral administration of BAY 85-3934 (2.5 mg/kg or 5 mg/kg once daily) or rhEPO (100 IU/kg s.c. twice weekly) for 5 weeks, compared with control and sham-operated animals (<i>n</i> = 4–6 animals per group). Efficacy of BAY 85-3934 sodium (80 ppm), enalapril (30 ppm), and a combination of both, administered in drinking water for 5 weeks, on (D) PCV, (E) SBP (at 4 weeks), and (F) prorenin (<i>n</i> = 9–10 animals per group). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001; one-way ANOVA followed by Dunnett’s multiple comparison test to corresponding sham or control group for (A), (C), (D), and (E), and Bonferroni’s multiple comparison test to corresponding sham or control group for (B) and (F).</p

Oligonucleotide primers and probes used for quantitative RT-PCR analysis of samples from rat tissues, and human cell lines (in italics).

<p>Oligonucleotide primers and probes used for quantitative RT-PCR analysis of samples from rat tissues, and human cell lines (in italics).</p

Effect of BAY 85-3934 administration on peptidoglycan-polysaccharide (PG-PS)-induced inflammatory anemia in female Lewis rats.

<p>Data are presented as means ± SEM. (A) Packed cell volume (PCV) in PG-PS-treated animals administered BAY 85-3934 or vehicle (<i>n</i> = 11–12 animals per group), compared with control animals treated with vehicle alone (<i>n</i> = 5 animals). (B) Relative expression of erythropoietin (EPO) and monocyte chemotactic protein-1 (MCP-1) mRNA in kidney, and hepcidin mRNA in liver at the end of the study. *<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001; t-test.</p