HMG-CoA reductase (Ki = 0.2 nM)

在用硫酸苯甲醇处理的 hCM 细胞中，辛伐他汀酸（0.1–20 μM；24 小时）可显着减少 ROS 生成 8.9% 至 43% [2]。 hCM- 被辛伐他汀酸（0.1–20 μM；24 小时）改变。

阿尔茨海默病（AD）是一种神经退行性疾病，其特征是β-淀粉样蛋白斑块和乙酰胆碱消耗导致神经行为缺陷。AD还与TGF-β1/SMAD2和GSK3β/β-catenin通路下调有关。辛伐他汀（SMV）改善记忆功能的实验和临床。因此，本研究旨在探讨SMV对氯化铝（AlCl3）诱导的神经行为损伤的机制作用。AlCl3 （50 mg/kg）诱导AD 6周。小鼠给予辛伐他汀（10、20 mg/kg）或多奈哌齐（3 mg/kg）治疗6周后进行组织病理学、免疫组化和生化检查。SMV治疗可改善AlCl3诱导的记忆衰退，组织病理学改变明显恢复。同时伴有乙酰胆碱酯酶（AChE）和β (a -42)的降低。SMV通过上调β-catenin、TGF-β1蛋白的表达，下调GSK3β、TLR4、p- smad2的表达，发挥神经保护作用https://pubmed.ncbi.nlm.nih.gov/37454825/

OATP3A1功能测定[2]
采用荧光底物荧光素钠检测转染hCMs、HEK293-NEO（空载体）和hek293 -OATP3A1的细胞中OATP3A1的功能。细胞（500,000个细胞/皿）在100 mm培养皿上生长。荧光素钠是OATP3A1的一般底物（Patik et al., 2015）。将细胞与pH值为7.4和5.5的Dulbecco磷酸盐缓冲盐水（DPBS）在37°C、5% CO2的气氛中预孵育10分钟，因为有几项研究报告了酸性pH下OATP转运活性的增加（Kobayashi等人，2003年，Nozawa等人，2004年，Varma等人，2011年）。然后用荧光素钠（2 μM）处理细胞30分钟（摄取期）（Wen et al., 2014）。仅hcm在存在或不存在环孢素（疑似转运抑制剂）（10 μM）或辛伐他汀酸 (10 μM)的条件下于37°C、5% CO2气氛下孵育30分钟。用DPBS pH 7.4或5.5洗涤后，收集所有细胞，用0.1 N NaOH裂解，在BioTek Synergy™4 Hybrid micromicroplate Reader上使用Gen5 1.10软件在激发/发射460/515 nm处定量荧光强度。

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[3H]表达OATP3A1细胞的辛伐他汀酸摄取实验[2]
在pH 7.4和pH 5.5、37℃、5% CO2气氛下进行吸收实验。在24孔板培养的HEK293-NEO（空载体）和HEK293-OATP3A1转染细胞（100,000个细胞/孔）的单层培养中，测定了放射性标记的辛伐他汀酸的细胞摄取。培养两天后（Chiba et al., 2013），细胞洗涤一次，在pH 7.4和pH 5.5的DPBS中预孵育10分钟，37°C， 5% CO2气氛。然后通过添加放射性标记的辛伐他汀酸 (0.05 μM)评估DPBS的摄取情况。在指定时间（1、2、5、15、30、45分钟），用冰冷的DPBS替代传输缓冲液，终止摄取。在冷水DPBS中洗涤2次后，细胞在0.1 N NaOH中裂解。细胞中的放射性是通过贝克曼ls6000ic闪烁计数器上的液体闪烁计数来测定的。 为了进行动力学分析，将细胞与未标记的辛伐他汀酸 (0.01, 0.05, 0.1, 0.5, 1 μM)在DPBS中预孵卵30分钟（摄取周期），然后评估放射性标记的辛伐他汀酸 (0.05 μM)的摄取情况。用非线性回归法绘制数据，确定Vmax和Km。

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药物-药物相互作用筛选[2]
在24孔板培养的HEK293-NEO（空载体）和HEK293-OATP3A1转染细胞（100,000个细胞/孔）的单层培养中，测定了放射性标记的辛伐他汀酸的细胞摄取。培养两天后（Chiba et al., 2013），细胞洗涤一次，在pH 7.4或pH 5.5的DPBS环境中预孵育10分钟，37°C， 5% CO2气氛。为了评估竞争抑制的潜力，在DPBS中，细胞在存在或不存在原型底物[苄青霉素（10和100 μM），环孢素（10和100 μM），雌酮-3-硫酸（10和100 μM）和吲哚基硫酸（10和100 μM）]的情况下处理30分钟（摄取期）。然后通过添加放射性标记的辛伐他汀酸 (0.05 μM)评估DPBS的摄取情况。1分钟后，用冰冷的DPBS代替摄取液终止摄取。在冷水DPBS中洗涤2次后，细胞在0.1 N NaOH中裂解。细胞中的放射性是通过贝克曼ls6000ic闪烁计数器上的液体闪烁计数来测定的

蛋白质印迹分析[2]
细胞类型： hCM 和 HEK293（转染 OATP3A1）
测试浓度： 0.1、1、10 和 20 μM
孵育时间： 24 小时
实验结果： hCM 和 OATP3A1 表达细胞中 OATP3A1 表达以剂量依赖性方式减少 1.5% 至 90% 。

Simvastatin/SMV was dissolved in 0.5% carboxymethylcellulose (CMC), while AlCl3, and DPZ were dissolved in water.
Mice were assigned randomly to five groups (6 mice per group): Group (1); normal untreated group received water daily for 6 weeks, Group (2–5); positive control group received AlCl3 (50 mg/kg/day, p.o.) for 6 weeks (Al-Amin et al., 2019, Li et al., 2018, Singh and Goel, 2015), Group (3−4); mice received SMV (10 or 20 mg/kg/day, p.o.), respectively (Jin et al., 2016), 45 min before AlCl3 (Nampoothiri et al., 2015), Group (5); mice received donepezil (3 mg/kg/day, p.o.) (Shin et al., 2018), 45 min before AlCl3.
After six weeks, mice were tested for the assessment of memory using Barnes Maze test (a test for spatial learning & memory), T Maze Spontaneous Alternation test (a test for spatial memory), and Novel Object Recognition Test (a test for different phases of learning and memory). After the behavior tests, the rodents were anesthetized using I.P. anaesthetic dose of 100 mg/kg ketamine and 10 mg/kg xylazine, then sacrificed by cervical displacement. The brains were removed immediately and cut into halves. Each mouse's right cerebral hemisphere is dissected, fixed with a 10% neutral buffer formalin, and used for histological and immunohistochemical dyeing. Hippocampus extracted from the left cerebral hemisphere was refrigerated and saved at − 80 °C, then used later for biochemical analysis.
https://pubmed.ncbi.nlm.nih.gov/37454825/

[1]. HMG-CoA Reductase inhibitors: an updated review of patents of novel compounds and formulations (2011-2015). Expert Opin Ther Pat. 2016 Nov;26(11):1257-1272.

[2]. Characterization of simvastatin acid uptake by organic anion transporting polypeptide 3A1 (OATP3A1) and influence of drug-drug interaction. Toxicol In Vitro. 2017 Dec;45(Pt 1):158-165.

Statins are remarkably safe and efficient medications that are the mainstay of hypercholesterolemia treatment and have proven to be an invaluable tool to lower the risk of acute cardiovascular events. These compounds are inhibitors of 3-hydroxy-methylglutaryl CoA reductase (HMG-R), the rate-limiting enzyme in cholesterol biosynthesis. In spite of their success, they present undesirable side effects and are now loosing patent protection, which provides a great opportunity for the development of new and improved statins. Areas covered: This review summarizes the new patents for HMG-R inhibitors for the 2011-2015 period. Combinations of existing statins with other drugs are also addressed, as well as novel applications of existing statins. Expert opinion: Recent efforts for the discovery of HMG-CoA-R inhibitors has resulted in several new molecules. Most of these are based on commercially available statins, including sterol and terpenoid derivatives. A few peptides have also been patented. However, the origin of the side effects caused by previous statins continues to be, to a large extent, unknown. Although the patents published in the past 5 years are promising, and might result in new drugs, there is still no way to know if they will present reduced toxicity. Only future clinical trials will answer this question.[1]

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Human organic anion transporting polypeptide 3A1 (OATP3A1) is predominately expressed in the heart. The ability of OATP3A1 to transport statins into cardiomyocytes is unknown, although other OATPs are known to mediate the uptake of statin drugs in liver. The pleiotropic effects and uptake of simvastatin acid were analyzed in primary human cardiomyocytes and HEK293 cells transfected with the OATP3A1 gene. Treatment with simvastatin acid reduced indoxyl sulfate-mediated reactive oxygen species and modulated OATP3A1 expression in cardiomyocytes and HEK293 cells transfected with the OATP3A1 gene. We observed a pH-dependent effect on OATP3A1 uptake, with more efficient simvastatin acid uptake at pH5.5 in HEK293 cells transfected with the OATP3A1 gene. The Michaelis-Menten constant (Km) for simvastatin acid uptake by OATP3A1 was 0.017±0.002μM and the Vmax was 0.995±0.027fmol/min/105 cells. Uptake of simvastatin acid was significantly increased by known (benzylpenicillin and estrone-3-sulfate) and potential (indoxyl sulfate and cyclosporine) substrates of OATP3A1. In conclusion, the presence of OATP3A1 in cardiomyocytes suggests that this transporter may modulate the exposure of cardiac tissue to simvastatin acid due to its enrichment in cardiomyocytes. Increases in uptake of simvastatin acid by OATP3A1 when combined with OATP substrates suggest the potential for drug-drug interactions that could influence clinical outcomes.[2]

C25H39O6-.H4N+

453.61202

453.309

C, 66.20; H, 9.56; N, 3.09; O, 21.16

139893-43-9

Simvastatin hydroxy acid sodium;101314-97-0;Simvastatin acid;121009-77-6;Simvastatin acid-d6 ammonium

10961424

White to off-white solid powder

4.429

107.3

3

6

10

32

689

7

CCC(C)(C)C(=O)O[C@H]1C[C@H](C=C2[C@H]1[C@H]([C@H](C=C2)C)CC[C@H](C[C@H](CC(=O)[O-])O)O)C.[NH4+]

FFPDWNBTEIXJJF-OKDJMAGBSA-N

InChI=1S/C25H40O6.H3N/c1-6-25(4,5)24(30)31-21-12-15(2)11-17-8-7-16(3)20(23(17)21)10-9-18(26)13-19(27)14-22(28)29;/h7-8,11,15-16,18-21,23,26-27H,6,9-10,12-14H2,1-5H3,(H,28,29);1H3/t15-,16-,18+,19+,20-,21-,23-;/m0./s1

Ammonium (3R,5R)-7-[(1S,2S,6R,8S,8aR)-8-(2,2-dimethylbutanoyloxy)-2,6-dimethyl-1,2,6,7,8,8a-hexahydronaphthalen-1-yl]-3,5-dihydroxyheptanoate

MK-733; Synvinolin; MK-733; Simvastatin ammonium salt; 139893-43-9; Tenivastatin ammonium; UNII-76RD797JAX; 76RD797JAX; Ammonium simvastatin; EC 604-165-5; Simvastatin Carboxylic Acid Ammonium Salt; Sinvacor; MK 733; MK733;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~11.11 mg/mL (~24.49 mM)

配方 1 中的溶解度: ≥ 1.11 mg/mL (2.45 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 11.1 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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: ≥ 1.11 mg/mL (2.45 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 11.1 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: ≥ 1.11 mg/mL (2.45 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 11.1 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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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。