PHD1 (IC50 = 480 nM); PHD2 (IC50 = 280 nM); PHD3 (IC50 =450 nM)[1]

For PHD1, PHD2, and PHD3, the average IC50 values of BAY 85-3934 are 480 nM, 280 nM, and 450 nM, respectively. It only takes 20 minutes of exposure to 5 μM BAY 85-3934 for HeLa cells to produce detectable levels of HIF-1α. Using the hypoxia response element promoter as the control, BAY 85-3934 induced the expression of the firefly luciferase reporter gene in a cell reporter assay, with a mean (± SD) EC50 of 8.4±0.7 μM (n=4) [1].

When healthy Wistar rats and cynomolgus monkeys were given the HIF-PH inhibitor BAY 85-3934 (Molidustat) orally, it stabilized HIF and caused dose-dependent production of EPO. In addition to normalizing hypertensive blood pressure in a CKD rat model, molidustat therapy, in contrast to rhEPO therapy, is effective in managing renal anemia in rats with impaired renal function [1].

Prolyl hydroxylase assay[1]
The prolyl hydroxylase assay was performed as described previously with minor modifications. Biotinylated HIF-1α 556–574 (biotinyl-DLDLEMLAPYIPMDDDFQL) was bound to white 96-well NeutrAvidin high binding capacity plates, which were pre-blocked with Blocker Casein and subsequently blocked with 1 mM biotin. The immobilized peptide substrate was 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 was 60 min. To stop the reaction, plates were washed three times with wash buffer.[1]
Hydroxylated biotinyl-HIF-1α 556–574 was 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 was determined by measuring time-resolved fluorescence with a Tecan infinite M200 plate reader. Measurements were taken in triplicate or more, and results were expressed as means ± SEM. IC50 values were 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+ was necessary, the reaction buffer was supplemented with appropriate amounts of ammonium iron(II) sulfate ((NH4)2Fe(SO4)2.6H2O, Mohr’s salt).

Cell lines, cell culture media, and luciferase reporter assay[1]
A549 and HeLa carcinoma cell lines (American Type Culture Collection) were cultured in DMEM/F-12, and Hep3B cells in RPMI medium, both supplemented with antibiotics, L-glutamine and 10% fetal calf serum. A549 cells stably transfected with the HIF-RE2-luc HIF reporter construct (constructed in pGL3) were seeded on 384-well plates at a density of 2500 cells/well in a volume of 25 µl complete cell culture medium, and re-incubated for 16–24 h before the test. Test compounds were added at appropriate dilutions in a volume of 10 µl, and cells were re-incubated for 6 h before measurement. Luciferase activity was determined in a luminometer after addition of cell lysis/luciferase buffer. Cell line identities were verified by STR DNA typing.

---

Western blot analysis[1]
For western blot analysis, cell lysates were separated on 4–12% SDS polyacrylamide gradient gels. Proteins were blotted onto polyvinylidene difluoride (PVDF) membranes. HIF-1α protein was detected using a HIF-1α specific monoclonal antibody at a dilution of 1∶250. HIF-2α protein was detected using a HIF-2α specific polyclonal antibody at a dilution of 1∶1000. Anti-β-actin antibody served as a loading control. Binding of the antibodies was visualized by binding of a horseradish peroxidase-conjugated anti-mouse IgG antibody, and subsequently enhanced using chemiluminescence, according to the manufactureŕs instructions. Novex Sharp Pre-stained Protein Standard was used as molecular weight marker.

Studies in rats[1]
Male Wistar rats (240–340 g in body weight) were housed with five animals per cage for at least 1 week before experimentation. Blood samples from rats were collected under anesthesia (2% isoflurane in air) by puncturing the retro-orbital vein plexus with a glass capillary. In a repeat-dose, 26-day experiment, animals were administered vehicle or Molidustat (BAY 85-3934) at doses of 0.5 mg/kg, 1.25 mg/kg, 2.5 mg/kg, and 5 mg/kg. PCV was determined at baseline and at weekly intervals after centrifugation in a hematocrit capillary tube (Brand) for 10 min at full speed in a Haemofuge centrifuge (Heraeus). The number of reticulocytes in 5 µl blood was counted after staining with thiazol orange (Becton Dickinson) according to the manufacturer’s instructions by FACS analysis on a BD FACSCalibur system (Becton Dickinson). The efficacy of BAY 85-3934 (2.5 mg/kg, once-daily, oral) was also compared with that of rhEPO (25 IU/kg, 50 IU/kg, and 100 IU/kg, twice-weekly, s.c. injection). The time-course of induction of EPO mRNA expression and plasma EPO was determined at baseline and 0.5 h, 1 h, 2 h, 4 h, 6 h, and 8 h after oral administration of a single dose of BAY 85-3934 (5 mg/kg).

---

Studies in cynomolgus monkeys[1]
Male and female cynomolgus monkeys (2.8–5.6 kg in body weight) were used, which were housed two per cage. Blood samples from conscious cynomolgus monkeys were taken by puncturing a superficial vein. In a 5-day, repeat-dose study of plasma EPO response, Molidustat (BAY 85-3934) was administered at doses of 0.5 mg/kg and 1.5 mg/kg at 0 h, 24 h, 48 h, 72 h, and 96 h. Blood samples were taken at 7 h, 31 h, 55 h, 79 h, 103 h, and 168 h. Erythropoietic parameters were also evaluated after a 2-week treatment period with s.c. administration of rhEPO (100 IU/kg twice weekly at days 1, 4, 8, and 11) and BAY 85-3934 (1.5 mg/kg) once daily.

---

Gentamicin-induced kidney failure model[1]
Male Wistar rats were treated once daily with gentamicin (Gibco/Invitrogen) at a dose of 100 mg/kg body weight via i.p. injection on 14 consecutive days. Control animals received injections of an equal volume of 0.9% saline. After gentamicin treatment, PCV was determined and animals were distributed to the vehicle or treatment groups with respect to equal mean PCV. On day 15, Molidustat (BAY 85-3934) was given orally once daily at doses of 1 mg/kg, 2.5 mg/kg, 5.0 mg/kg, and 10.0 mg/kg, five times weekly.

---

PG-PS-induced inflammatory anemia model[1]
Female Lewis rats, with a body weight of 155–181 g were used. Body weight, ankle diameter, hematocrit, and blood cell count were determined at baseline and thereafter at regular intervals. PG-PS from Streptococcus pyogenes was dissolved in sterile saline and administered via i.p. injection at 15 mg/kg. Animals that did not show an inflammatory response were not studied further. Two weeks after injection, animals were distributed into treatment groups in equal proportions based on their hematocrit levels. On day 15, Molidustat (BAY 85-3934) was given orally once daily at doses of 2.5 mg/kg and 5.0 mg/kg. At the end of the study, animals were sacrificed and kidney and liver samples were processed for qRT-PCR analysis.

---

Subtotal nephrectomy model[1]
Subtotal nephrectomy was conducted in adult male Wistar rats. Body weight, blood pressure, hematocrit, and blood cell counts were determined at baseline and thereafter at weekly intervals. At baseline, rats were randomly distributed into two groups: those that underwent subtotal nephrectomy and those that underwent a sham procedure without reduction of renal mass. Surgery was performed in deeply anesthetized (2% isoflurane in air) animals. Kidneys were accessed via a dorsolateral incision of the body wall of about 2 cm in length. The right kidney was removed after ligature of the renal peduncle, and subsequently the upper and lower pole of the left kidney were removed, followed by careful hemostasis. Approximately one third of the initial kidney mass remained (removed tissue was weighed to check this was achieved). In the sham-treated animals, both kidneys were exposed before closure of the wound. Three weeks after surgery, animals were allocated to each group in equal proportions with respect to systolic blood pressure and hematocrit values. For 5 weeks, animals were treated twice weekly with rhEPO (100 IU/kg), or once daily with BAY 85–3936 sodium (2.5 mg/kg or 5.0 mg/kg) or vehicle. In experiments using enalapril or a combination of Molidustat (BAY 85-3934) sodium and enalapril, study drugs were administered with drinking water. BAY 85-3934 sodium and enalapril were administered in drinking water at concentrations of 80 ppm and 30 ppm, respectively. This was equivalent to approximately 2 mg/kg/day for enalapril and 5 mg/kg/day for BAY 85-3934. Systolic blood pressure and heart rate were determined using the tail-cuff method (a semi-automatic, non-invasive blood pressure monitor; TSE Systems), with three repeated measurements per animal.

---

Rats: 0.5 mg/kg, 1.25 mg/kg, 2.5 mg/kg, and 5 mg/kg; oral

Rats: BAY 85-3934 is prepared as a solution in ethanol:Solutol HS 15:water (10:20:70). In a repeat-dose, 26-day experiment, male Wistar rats (240–340 g in body weight) are administered vehicle or BAY 85-3934 at doses of 0.5 mg/kg, 1.25 mg/kg, 2.5 mg/kg, and 5 mg/kg. The efficacy of BAY 85-3934 (2.5 mg/kg, once-daily, oral) is also compared with that of rhEPO (25 IU/kg, 50 IU/kg, and 100 IU/kg, twice-weekly, s.c. injection). The time-course of induction of EPO mRNA expression and plasma EPO is determined at baseline and 0.5 h, 1 h, 2 h, 4 h, 6 h, and 8 h after oral administration of a single dose of BAY 85-3934 (5 mg/kg); Monkey: BAY 85-3934 is prepared as a solution in 0.5% tylose. Male and female cynomolgus monkeys (2.8–5.6 kg in body weight) are administered at doses of 0.5 mg/kg and 1.5 mg/kg at 0 h, 24 h, 48 h, 72 h, and 96 h. Blood samples are taken at 7 h, 31 h, 55 h, 79 h, 103 h, and 168 h. Erythropoietic parameters are also evaluated after a 2-week treatment period with s.c. administration of rhEPO (100 IU/kg twice weekly at days 1, 4, 8, and 11) and BAY 85-3934 (1.5 mg/kg) once daily

Overall, 94.5% of patients experienced at least 1 TEAE during the study: 92.7% of patients in the molidustat group and 96.3% in the darbepoetin group (Table 2). The most commonly reported TEAEs were nasopharyngitis (34.1% and 40.2% in the molidustat and darbepoetin groups, respectively), worsening of CKD (18.3% and 9.8%, respectively), and diarrhea (8.5% and 12.2%, respectively) (Table 2). TEAEs leading to death were reported in 2 patients (2.4%) in the molidustat group and none in the darbepoetin group, and serious TEAEs were reported in 32.9% and 26.8% of patients, respectively. MACEs that occurred after the start of the study drug were reported in 3.7% of patients treated with molidustat and 1.2% of patients receiving darbepoetin (online suppl. Table 3). Additionally, 3.7% of patients in the molidustat group and 1.2% in the darbepoetin group developed diabetic retinopathy, and 3.7% in the molidustat group and 4.9% in the darbepoetin group developed neoplasms (benign, malignant, or unspecified) (online suppl. Table 4). The mean serum eGFR appeared to remain stable in the molidustat group (online suppl. Fig. 7). Subgroup analyses of TEAEs by age group (<65 and ≥65 years old) and by sex are presented in online supplementary Table 5. The proportion of serious TEAEs was similar between the 2 groups in female patients but higher for males in the molidustat group than in the darbepoetin group.[2]

[1]. Mimicking hypoxia to treat anemia: HIF-stabilizer BAY 85-3934 (Molidustat) stimulates erythropoietin production without hypertensive effects. PLoS One. 2014 Nov 13;9(11):e111838.

[2]. Molidustat for Renal Anemia in Nondialysis Patients Previously Treated with Erythropoiesis-Stimulating Agents: A Randomized, Open-Label, Phase 3 Study. Am J Nephrol. 2021;52(10-11):884-893.

Molidustat is under investigation in clinical trial NCT03350321 (A Study of Molidustat for Correction of Renal Anemia in Non-dialysis Subjects).
See also: Molidustat Sodium (active moiety of).

C13H14N8O2

314.3

314.123

C, 49.68; H, 4.49; N, 35.65; O, 10.18

1154028-82-6

1375799-59-9 (Sodium);1154028-82-6;

59603622

White to off-white solid powder

1.7±0.1 g/cm3

589.2±60.0 °C at 760 mmHg

310.2±32.9 °C

0.0±1.7 mmHg at 25°C

1.820

-1.77

1

8

3

23

481

0

IJMBOKOTALXLKS-UHFFFAOYSA-N

InChI=1S/C13H14N8O2/c22-13-10(20-2-1-16-18-20)8-17-21(13)12-7-11(14-9-15-12)19-3-5-23-6-4-19/h1-2,7-9,17H,3-6H2

2-(6-morpholin-4-ylpyrimidin-4-yl)-4-(triazol-1-yl)-1H-pyrazol-3-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 0.5 mg/mL (1.59 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 0.5 mg/mL (1.59 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 0.5 mg/mL (1.59 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 10 mg/mL (31.82 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

 (Please use freshly prepared in vivo formulations for optimal results.)