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

对于 PHD1、PHD2 和 PHD3，BAY 85-3934 的平均 IC50 值分别为 480 nM、280 nM 和 450 nM。 HeLa 细胞只需暴露于 5 μM BAY 85-3934 20 分钟即可产生可检测水平的 HIF-1α。使用缺氧反应元件启动子作为对照，BAY 85-3934 在细胞报告基因测定中诱导萤火虫荧光素酶报告基因的表达，平均 (± SD) EC50 为 8.4±0.7 μM (n=4) [1] 。

当健康 Wistar 大鼠和食蟹猴口服 HIF-PH 抑制剂 BAY 85-3934 (Molidustat) 时，它可以稳定 HIF 并导致 EPO 的剂量依赖性产生。与 rhEPO 治疗相比，除了使 CKD 大鼠模型的高血压血压正常化外，莫度司他治疗还可有效控制肾功能受损大鼠的肾性贫血 [1]。

脯氨酰羟化酶测定[1]
脯氨酰羟化酶测定如前所述，稍作修改。生物素化HIF-1α556–574（生物素DLDLDLELMLAPYIPMDDDFQL）与白色96孔NeutrAvidin高结合能力板结合，该板用阻断剂酪蛋白预阻断，随后用1 mM生物素阻断。将固定的肽底物与适量的HIF-PH在含有20 mM Tris（PH 7.5）、5 mM KCl、1.5 mM MgCl2、20µM 2-酮戊二酸、10µM FeSO4、2 mM抗坏血酸、4%蛋白酶抑制剂（不含EDTA，罗氏诊断公司）的缓冲液中孵育，最终体积为100µl，加入或不加入适当浓度的测试化合物。反应时间为60分钟。为了停止反应，用洗涤缓冲液洗涤板三次。[1]
羟基化生物素-HHIF-1α556–574与Eu-VBC在100µl结合缓冲液（50 mM Tris[pH 7.5]，120 mM NaCl）中在室温下孵育60分钟。用DELFIA洗涤缓冲液洗涤六次并加入100µl增强剂溶液后，通过Tecan无限M200平板读数器测量时间分辨荧光来确定结合的VBC的量。测量重复三次或更多次，结果以平均值±SEM表示。使用GraphPad Prism软件对数据集应用四参数逻辑方程进行曲线拟合后，确定IC50值。当需要调节游离Fe2+的浓度时，向反应缓冲液中补充适量的硫酸铁（II）铵（（NH4）2Fe（SO4）2.6H2O，莫尔盐）。

细胞系、细胞培养基和萤光素酶报告测定[1]
A549和HeLa癌细胞系（美国典型培养物保藏中心）在DMEM/F-12中培养，Hep3B细胞在RPMI培养基中培养，两者均添加了抗生素、L-谷氨酰胺和10%胎牛血清。用HIF-RE2-luc HIF报告构建体（在pGL3中构建）稳定转染的A549细胞以2500个细胞/孔的密度接种在384孔板上，体积为25µl的完整细胞培养基中，并在测试前重新孵育16-24小时。以10µl的体积加入适当稀释的试验化合物，并在测量前将细胞重新孵育6小时。在加入细胞裂解/萤光素酶缓冲液后，在光度计中测定萤光素酶活性。通过STR DNA分型验证细胞系身份。

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蛋白质印迹分析[1]
对于蛋白质印迹分析，细胞裂解物在4-12%SDS-聚丙烯酰胺梯度凝胶上分离。蛋白质被印迹到聚偏二氟乙烯（PVDF）膜上。使用HIF-1α特异性单克隆抗体以1∶250的稀释度检测HIF-1α蛋白。使用稀释度为1∶1000的HIF-2α特异性多克隆抗体检测HIF-2α蛋白。抗β-肌动蛋白抗体作为负载对照。根据制造商的说明，通过结合辣根过氧化物酶偶联的抗小鼠IgG抗体来观察抗体的结合，随后使用化学发光增强。Novex Sharp预染色蛋白标准品用作分子量标记。

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).

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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.

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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.

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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.

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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.

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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

106.75

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)

配方 1 中的溶解度: ≥ 0.5 mg/mL (1.59 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 5.0 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 0.5 mg/mL (1.59 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 5.0 mg/mL 澄清 DMSO 储备液加入 900 μL 20% SBE-β-CD 生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 0.5 mg/mL (1.59 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 5.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 10 mg/mL (31.82 mM) in 0.5% CMC-Na/saline water (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。