IC50: 480 nM (PHD1), 280 nM (PHD2), 450 nM (PHD3)[1]

莫利司他钠 (5 μM；20 分钟) 足以在 HeLa 细胞中诱导可检测水平的 HIF-1α。在基于细胞的报告基因检测中，莫利司他钠在缺氧反应元件启动子的控制下诱导萤火虫荧光素酶报告基因的表达，平均 (± SD) EC50 为 8.4 μM (n=4)[1]。

莫利司他钠（0.5-5 mg/kg；口服；每天一次，共26天）在Wistar大鼠中给药，莫利司他钠（0.5-1.5 mg/kg；每天一次，共5天）在食蟹猴中给药，可通过稳定缺氧诱导因子（HIF）在健康Wistar大鼠和食蟹猴中诱导剂量依赖性促红细胞生成素（EPO）产生[1]。莫利司他钠（0.5-10 mg/kg；口服；每周五次）可使慢性肾病（CKD）大鼠模型的高血压血压正常化，并有效缓解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]

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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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[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.

See also: Molidustat (has active moiety).

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Drug Indication
Treatment of anaemia due to chronic disorders

C13H13N8NAO2

336.28

336.105

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

1375799-59-9

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

69669724

Typically exists as White to off-white solid at room temperature

1.558 at 20℃

110

0

8

3

24

402

0

VYRQLKYGGSWDNH-UHFFFAOYSA-M

InChI=1S/C13H14N8O2.Na/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;/h1-2,7-9,22H,3-6H2;/q;+1/p-1

sodium;2-(6-morpholin-4-ylpyrimidin-4-yl)-4-(triazol-1-yl)pyrazol-3-olate

Molidustat; 1154028-82-6; BAY 85-3934; Molidustat sodium; Molidustat (sodium); 1375799-59-9; BAY-1053048; BAY-85-3934 SODIUM; CI0NE7C96T; sodium;2-(6-morpholin-4-ylpyrimidin-4-yl)-4-(triazol-1-yl)pyrazol-3-olate; 1-(6-(MORPHOLIN-4-YL)PYRIMIDIN-4-YL)-4-(1H-1,2,3-TRIAZOL-1-YL)-1H-PYRAZOL-5-OL, SODIUM SALT (1:1); Molidustat [INN]; UNII-9JH486CZ13; BAY85-3934;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。