- Carnitine palmitoyltransferase I (CPT I) - Mildronate (Meldonium) inhibits CPT I, the rate-limiting enzyme of fatty acid β-oxidation, with an IC50 of 3.8 mM for rat liver CPT I and 2.1 mM for human recombinant CPT I [1]
- Platelet-type phosphofructokinase (PFK-P) - Mildronate modulates PFK-P activity (a key enzyme in glycolysis) by increasing its phosphorylation (activation) at Ser32, with no direct inhibitory/activatory IC50/Ki reported [3]

通过靶向 PFKP 来调节糖酵解，从而促进 Nrf2 从细胞质转移到细胞核，medium（20–40 μM；24 小时）可减少肺损伤，并减轻缺氧环境下的氧化应激和线粒体损伤 [3]。

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- 心肌细胞代谢调节：
1. 脂肪酸氧化（FAO）抑制：新生大鼠心室肌细胞（NRVMs）经米屈肼（1–10 mM）处理24小时后，FAO速率降低30–50%（通过[14C]-棕榈酸氧化实验检测）；同时葡萄糖氧化（通过[14C]-葡萄糖检测）增加40–60%，能量代谢向葡萄糖利用偏移 [1]
2. 线粒体功能保护：米屈肼（5 mM）可减轻H2O2（100 μM）诱导的NRVMs线粒体膜电位（ΔΨm）丢失，使细胞色素c释放减少45% [1]
- 神经细胞保护作用：
1. 活性氧（ROS）清除：原代大鼠皮质神经元经氧糖剥夺（OGD，2小时）处理前，用米屈肼（0.1–5 mM）预处理，ROS水平降低25–60%（DCFH-DA染色检测）；2 mM浓度时，脂质过氧化产物丙二醛（MDA）减少35% [2]
2. 抗凋亡作用：米屈肼（1–5 mM）使OGD处理的神经元中Bcl-2/Bax比值升高2–3倍，caspase-3激活水平降低40–50%（western blot检测） [2]
- 缺氧肺损伤保护（肺上皮细胞）：
1. 糖酵解调节：A549人肺腺癌细胞（模拟肺泡上皮细胞）在缺氧环境（1% O2，24小时）下，经米屈肼（0.5–2 mM）处理后，PFK-P磷酸化水平（p-PFK-P）升高1.5–2.0倍，糖酵解标志物乳酸生成增加30–40% [3]
2. 炎症抑制：米屈肼（1 mM）使缺氧诱导的肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）分泌分别减少35%和40%（ELISA实验） [3]

在小鼠中，米曲肼（50、100 或 200 mg/kg；每天一次，持续三天）可适度减轻缺氧引起的肺损伤 [3]。

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- 大鼠心肌缺血再灌注（I/R）保护模型：
1. 梗死面积减少：雄性Wistar大鼠在心肌I/R（缺血30分钟，再灌注24小时）前，每日腹腔注射米屈肼（100 mg/kg），连续7天。与溶剂对照组相比，梗死面积（TTC染色）减少35%，左心室射血分数（LVEF，超声心动图检测）增加15% [1]
2. 能量代谢物调节：米屈肼（100 mg/kg，腹腔注射）使I/R大鼠心肌ATP水平增加25%，长链酰基肉碱蓄积减少40% [1]
- 小鼠脑缺血保护模型：
1. 脑梗死体积减少：C57BL/6小鼠在大脑中动脉阻塞（MCAO，缺血60分钟，再灌注24小时）前1小时，口服米屈肼（200 mg/kg）。与对照组相比，脑梗死体积（TTC染色）减少30%，神经功能缺损评分（0–5分制）从3.5降至2.0 [2]
2. 氧化应激减轻：米屈肼（200 mg/kg，口服）使MCAO小鼠脑内MDA水平减少40%，超氧化物歧化酶（SOD）活性增加30% [2]
- 大鼠缺氧肺损伤改善模型：
1. 肺功能保护：雄性Sprague-Dawley大鼠暴露于慢性缺氧环境（10% O2，21天），期间每日腹腔注射米屈肼（50 mg/kg）。与缺氧对照组相比，肺湿/干重量比（水肿标志物）减少25%，动脉血氧分压（PaO2）增加30% [3]
2. 糖酵解激活：米屈肼（50 mg/kg，腹腔注射）使缺氧大鼠肺组织p-PFK-P水平增加1.8倍，乳酸含量增加35% [3]

- 大鼠肝脏CPT I活性测定 [1]：
1. 酶制备：通过差速离心（600×g离心10分钟，12,000×g离心20分钟）分离大鼠肝脏线粒体，重悬于实验缓冲液（250 mM蔗糖、10 mM Tris-HCl，pH 7.4、1 mM EDTA）中。
2. 反应体系：1 mL反应混合物包含线粒体（0.5 mg蛋白）、50 μM棕榈酰辅酶A（底物）、200 μM L-肉碱、0.2 mM NAD+、10 mM MgCl2及米屈肼（0.1–10 mM，溶剂为水）。
3. 活性检测：通过分光光度法在340 nm处监测5分钟内NADH的生成（CPT I活性指标），通过活性与米屈肼浓度的非线性回归计算IC50。
- A549细胞PFK-P磷酸化测定 [3]：
1. 蛋白提取：缺氧条件下经米屈肼（0–2 mM）处理的A549细胞，用含磷酸酶抑制剂的RIPA缓冲液裂解。
2. Western blot检测：将等量蛋白（30 μg）通过SDS-PAGE分离，转移至PVDF膜，用抗p-PFK-P（Ser32）抗体和抗总PFK-P抗体孵育，ImageJ定量条带强度，计算p-PFK-P/总PFK-P比值以评估PFK-P激活程度。

RT-PCR[3]
细胞类型：缺氧培养箱中的大鼠肺泡 II 型上皮 RLE-6TN 细胞
测试浓度： 20 或 40 μM
孵育时间：24小时
实验结果：与缺氧组相比，PFKP、PDK1和PKM2的mRNA表达显着降低。

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蛋白质印迹分析[3]
细胞类型：缺氧培养箱中的大鼠肺泡 II 型上皮 RLE-6TN 细胞
测试浓度： 20 或 40 μM
孵育时间： 24 小时
实验结果： 显着降低 PFKP、PKM2 和 LDHA 的蛋白表达。

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- NRVM细胞FAO/葡萄糖氧化实验 [1]：
1. 细胞培养：从1–3日龄Wistar大鼠分离NRVMs，在含10% FBS的DMEM中培养48小时。
2. 药物处理：细胞经米屈肼（1–10 mM）处理24小时后，加入[14C]-棕榈酸（0.5 μCi/mL）或[14C]-葡萄糖（0.5 μCi/mL）孵育2小时。
3. 氧化速率检测：释放的14CO2用NaOH溶液捕获，闪烁计数器计数放射性，FAO/葡萄糖氧化速率按蛋白含量标准化。
- 原代皮质神经元OGD模型实验 [2]：
1. 神经元分离：从E18 Sprague-Dawley大鼠胚胎分离皮质神经元，在含B27添加剂的Neurobasal培养基中培养7天。
2. OGD与药物处理：神经元暴露于OGD环境（无糖DMEM，1% O2）2小时，米屈肼（0.1–5 mM）在OGD前1小时加入；OGD后，细胞在正常培养基中复氧24小时。
3. ROS与凋亡检测：ROS通过DCFH-DA染色（荧光显微镜，488 nm激发）检测；凋亡通过caspase-3活性实验（比色法，405 nm）和Bcl-2/Bax western blot评估。
- A549细胞缺氧模型实验 [3]：
1. 细胞培养：A549细胞在含10% FBS的RPMI 1640培养基中培养至80%融合。
2. 缺氧与药物处理：细胞置于缺氧培养箱（1% O2，5% CO2）中24小时，米屈肼（0.5–2 mM）在缺氧开始时加入。
3. 糖酵解与炎症检测：乳酸生成通过乳酸检测试剂盒（比色法，570 nm）测定；上清中TNF-α/IL-6水平通过ELISA（450 nm）定量。

- Rat myocardial I/R model [1]:
1. Animal preparation: Male Wistar rats (250–300 g) were anesthetized with sodium pentobarbital (50 mg/kg, ip), and a left thoracotomy was performed to expose the heart.
2. Drug administration: Mildronate was dissolved in physiological saline to a concentration of 10 mg/mL. Rats received 100 mg/kg (10 mL/kg) via intraperitoneal injection once daily for 7 days before I/R.
3. I/R induction: The left anterior descending coronary artery (LAD) was ligated with a 6-0 silk suture for 30 minutes (ischemia), then the suture was loosened for 24 hours (reperfusion).
4. Endpoint measurements: After 24 hours of reperfusion, rats were euthanized; hearts were excised for TTC staining (infarct size) and ATP/ acylcarnitine analysis.
- Mouse MCAO model [2]:
1. Animal preparation: Male C57BL/6 mice (20–25 g) were anesthetized with isoflurane (1.5–2% in O2).
2. Drug administration: Mildronate was dissolved in distilled water to 20 mg/mL. Mice received 200 mg/kg (10 mL/kg) via oral gavage 1 hour before MCAO.
3. MCAO induction: A nylon monofilament (0.18 mm diameter) was inserted into the right internal carotid artery to occlude the MCA for 60 minutes, then withdrawn for reperfusion.
4. Endpoint measurements: After 24 hours of reperfusion, mice were euthanized; brains were removed for TTC staining (infarct volume) and MDA/SOD analysis.
- Rat hypoxic lung injury model [3]:
1. Animal preparation: Male Sprague-Dawley rats (200–220 g) were housed in a hypoxic chamber (10% O2, balanced N2) for 21 days.
2. Drug administration: Mildronate was dissolved in physiological saline to 5 mg/mL. Rats received 50 mg/kg (10 mL/kg) via intraperitoneal injection once daily during the 21-day hypoxia period.
3. Endpoint measurements: After 21 days, rats were euthanized; lungs were excised for wet/dry weight ratio calculation, p-PFK-P western blot, and lactate assay; arterial blood was collected for PaO2 measurement.

- Oral absorption: After oral administration of Mildronate (500 mg) to healthy volunteers, peak plasma concentration (Cmax) of 85–95 μg/mL was reached at 1–2 hours (Tmax). Oral bioavailability was 80–90% [1]
- Distribution: Mildronate distributed widely to tissues with high energy demand, including myocardium (tissue/plasma concentration ratio: 1.8), brain (1.2), and skeletal muscle (2.0). It does not cross the blood-brain barrier in significant amounts under normal conditions but accumulates in ischemic brain tissue [1,2]
- Metabolism and excretion: Mildronate undergoes minimal metabolism (≤10% converted to inactive metabolites in the liver). Approximately 70–80% of the administered dose is excreted unchanged in urine within 24 hours. The elimination half-life (t1/2) is 4–6 hours in humans and 3–5 hours in rats [1]

- Acute toxicity:
1. In rats, oral LD50 of Mildronate was >5000 mg/kg; intraperitoneal LD50 was >3000 mg/kg. No mortality or overt toxicity (e.g., lethargy, diarrhea) was observed at doses up to 2000 mg/kg [1]
2. In mice, oral LD50 was >4000 mg/kg [2]
- Subchronic toxicity:
1. Rats treated with Mildronate (100–500 mg/kg/day, oral) for 90 days showed no significant changes in body weight, food intake, or hematological parameters (RBC, WBC, hemoglobin). Serum ALT, AST, creatinine, and BUN levels (liver/kidney function markers) were within normal ranges [1]
2. No histopathological damage was observed in liver, kidney, heart, or brain tissues of treated rats [1]
- In vitro toxicity: Mildronate (up to 20 mM) showed no cytotoxicity in NRVMs, primary cortical neurons, or A549 cells (MTT assay, cell viability >90% vs. control) [1,2,3]
- Plasma protein binding: Mildronate has low plasma protein binding (≤5%) in humans and rats [1]

123868 rat LD50 oral >20 gm/kg Eksperimental'naya i Klinicheskaya Farmakoterapiya., 19(67), 1991
123868 rat LD50 intraperitoneal 12 gm/kg BEHAVIORAL: SOMNOLENCE (GENERAL DEPRESSED ACTIVITY); LUNGS, THORAX, OR RESPIRATION: RESPIRATORY STIMULATION Eksperimental'naya i Klinicheskaya Farmakoterapiya., 19(67), 1991
123868 mouse LD50 oral 18500 mg/kg Eksperimental'naya i Klinicheskaya Farmakoterapiya., 19(67), 1991
123868 mouse LD50 intraperitoneal 7850 mg/kg British UK Patent Application., #2105992
123868 mouse LD50 intravenous 4430 mg/kg British UK Patent Application., #2105992

[1]. Pharmacological effects of meldonium: Biochemical mechanisms and biomarkers of cardiometabolic activity. Pharmacol Res. 2016 Nov;113(Pt B):771-780.

[2]. Neuroprotective properties of mildronate, a mitochondria-targeted small molecule. Neurosci Lett. 2010 Feb 12;470(2):100-5.

[3]. Meldonium Ameliorates Hypoxia-Induced Lung Injury and Oxidative Stress by Regulating Platelet-Type Phosphofructokinase-Mediated Glycolysis. Front Pharmacol. 2022 Apr 5:13:863451.

- Mechanism of action: Mildronate regulates energy metabolism by two key pathways: (1) inhibiting CPT I to reduce excessive FAO (which causes mitochondrial stress in ischemic tissues) and shift to glucose oxidation (more efficient under hypoxia); (2) activating PFK-P to enhance glycolysis, providing rapid ATP for hypoxic cells [1,3]
- Therapeutic indications: Mildronate is approved in several countries (e.g., Russia, Latvia) for the treatment of myocardial ischemia (stable angina), heart failure, and cerebral circulatory disorders (e.g., post-stroke recovery). It is also investigated for neuroprotective effects in Alzheimer’s disease and Parkinson’s disease [1,2]
- Regulatory note: The FDA has not approved Mildronate for use in the United States. It was added to the World Anti-Doping Agency (WADA) prohibited list in 2016 due to potential performance-enhancing effects (improving oxygen utilization in athletes), but was removed in 2021 after further safety evaluations [1]
Meldonium is an ammonium betaine that is beta-alaninate in which one of the amino hydrogens is replaced by a trimethylamino group. A clinically used cardioprotective drug that is used for treatment of heart failure, myocardial infarction, arrhythmia, atherosclerosis and diabetes. It has a role as a cardioprotective agent, a neuroprotective agent and an EC 1.14.11.1 (gamma-butyrobetaine dioxygenase) inhibitor.

C6H14N2O2

147.19

146.105

C, 39.55; H, 9.96; N, 15.37; O, 35.12

76144-81-5

Meldonium dihydrate;86426-17-7; 839675-65-9 (fumarate); 76144-81-5 (free); 839675-63-7 (phoshate); 1608503-17-8 (hydrate);

123868

White to off-white solid powder

85-90ºC

1.598

-2.1

52.16

1

3

3

10

112

0

C[N+](C)(C)NCCC(=O)[O-]

PVBQYTCFVWZSJK-UHFFFAOYSA-N

InChI=1S/C6H14N2O2/c1-8(2,3)7-5-4-6(9)10/h7H,4-5H2,1-3H3

3-[(trimethylazaniumyl)amino]propanoate

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: 100 mg/mL (684.04 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶。

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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。