Molidustat (BAY 85-3934)

In vitro, Molidustat induces the transcription of hypoxia-sensitive genes in HeLa, A549, and Hep3B cells. ^[1]

In healthy Wistar rats and cynomolgus monkeys, Molidustat (1.5 mg/kg, p.o.) induces dose-dependent production of EPO and erythropoiesis. In rats with gentamicin-induced renal anemiabe, Molidustat (5 mg/kg, p.o.) is also effective in raising hematocrit levels while stimulating endogenous EPO production without hypertensive effects. ^[1]

• Prolyl hydroxylase assay

  Biotinylated HIF-1α 556–574 (biotinyl-DLDLEMLAPYIPMDDDFQL) is bound to white 96-well NeutrAvidin high binding capacity plates, which are pre-blocked with Blocker Casein and subsequently blocked with 1 mM biotin. The immobilized peptide substrate is incubated with the appropriate amount of HIF-PH in buffer containing 20 mM Tris (pH 7.5), 5 mM KCl, 1.5 mM MgCl₂, 20 µM 2-oxoglutarate, 10 µM FeSO₄, 2 mM ascorbate, 4% protease inhibitors without EDTA in a final volume of 100 µl, with or without test compound added at appropriate concentrations. The reaction time is 60 min. To stop the reaction, plates are washed three times with wash buffer. Hydroxylated biotinyl-HIF-1α 556–574 is incubated with Eu-VBC in 100 µl binding buffer (50 mM Tris [pH 7.5], 120 mM NaCl) for 60 min at room temperature. After washing six times with DELFIA wash buffer and adding 100 µl enhancer solution, the amount of bound VBC is determined by measuring time-resolved fluorescence with a Tecan infinite M200 plate reader. Measurements are taken in triplicate or more, and results are expressed as means ± SEM. IC50 values are determined after curve fitting using GraphPad Prism software applying the four-parameter logistic equation to the data sets. When adjustment of the concentration of free Fe2+ is necessary, the reaction buffer is supplemented with appropriate amounts of ammonium iron(II) sulfate ((NH4)2Fe(SO4)2.6H2O).

• Cell lines

  Calu-3 cells

• Concentrations

  6 μM

• Incubation Time

  24 h

• Method

  Cells were treated with various concentrations of drug for 24 h.

• Animal Models

  Rats with gentamicin-induced renal anemia

• Dosages

  5 mg/kg

• Administration

  p.o.