AZ876

In isolated cardiac myocytes and fibroblasts, immunocytochemistry confirmed nuclear expression of LXRα in both these cell types. In cardiomyocytes, phenylephrine-stimulated cellular hypertrophy was significantly decreased in AZ876-treated cells. In cardiac fibroblasts, AZ876 prevented TGFβ- and angiotensin II-induced fibroblast collagen synthesis, and inhibited up-regulation of the myofibroblastic marker, α-smooth muscle actin. Plasma triglycerides and liver weight were unaltered following AZ876 treatment.[1]

在分离的心肌细胞和成纤维细胞中，免疫细胞化学证实了这两种细胞类型中 LXRα 的核表达。在心肌细胞中，经 AZ876 处理的细胞中苯肾上腺素刺激的细胞肥大显著减少。在心脏成纤维细胞中，AZ876 阻止了 TGFβ 和血管紧张素 II 诱导的成纤维细胞胶原蛋白合成，并抑制了肌成纤维细胞标志物 α-平滑肌肌动蛋白的上调。AZ876 治疗后，血浆甘油三酯和肝脏重量没有变化。[1]

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在闪烁亲近结合实验中，AZ876 从人类LXRα和LXRβ蛋白上置换放射性配体的效力分别是GW3965的25倍和2.5倍。[2]
在使用共转染了Gal4嵌合LXR载体的U2/OS细胞进行的报告基因反式激活实验中，AZ876 对人类LXRα和LXRβ的效力分别是GW3965的196倍和5倍。其对小鼠LXRα和LXRβ的效力也分别是GW3965的248倍和10.5倍。[2]
在体外，AZ876 能增加仓鼠和人类外周血多形核细胞中逆向胆固醇转运（RCT）基因ABCA1的mRNA表达，其效力是GW3965的4至7倍。[2]

通过横向主动脉缩窄 (TAC) 诱导 C57Bl6/J 小鼠心脏肥大，持续 6 周。在此期间，小鼠进食补充或不补充 AZ876 (20 µmol/kg/天) 的食物。在小鼠心脏中，LXRα 蛋白表达在 TAC 反应下上调了约 7 倍。用 AZ876 激活 LXR 可减弱这种增加，并显著降低 TAC 引起的心脏重量增加、心肌纤维化和心脏功能障碍，而不会影响血压。在分子水平上，AZ876 抑制了肥大和纤维化相关基因的上调，并进一步抑制了促肥大和促纤维化的转化生长因子 β (TGFβ)-Smad2/3 信号传导。 [1]

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APOE*3Leiden 小鼠单独喂食致动脉粥样硬化饮食或补充 AZ876（5 或 20µmol·kg(-1) ·day(-1) ），持续 20 周。使用商业试剂盒测量总胆固醇和甘油三酯水平。使用基于珠子的多重悬浮阵列试剂盒和 Luminex 技术测定血浆细胞因子。通过组织化学方法评估动脉粥样硬化，并通过免疫组织化学方法评估病变成分。与对照组相比，低剂量 AZ876 对血浆没有影响，而高剂量 AZ876 增加了血浆甘油三酯（+110%）并降低了
胆固醇（-16%）。低剂量 AZ876 减少了病变面积（-47%）；高剂量 AZ876 可显著减少病变面积 (-91%)、病变数量 (-59%) 和严重程度。无论哪种剂量，AZ876 都不会影响病变组成。高剂量 AZ876 可减轻炎症，这反映在较低的细胞因子水平和血管壁活化上，而低剂量 AZ876 则不会。[2]

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在喂食致动脉粥样硬化饮食20周的高脂血症APOE3Leiden转基因小鼠中，低剂量AZ876（5 µmol·kg⁻¹·day⁻¹）使主动脉根部的动脉粥样硬化病变面积比对照组减少47%，且不影响血浆或肝脏甘油三酯水平。[2]
高剂量AZ876（20 µmol·kg⁻¹·day⁻¹）强烈降低了动脉粥样硬化病变面积（-91%）、病变数量（-59%）和严重病变的比例。该剂量还增加了血浆HDL水平（出现大的HDL-1颗粒）并降低了血浆VLDL-胆固醇。[2]
高剂量AZ876（20 µmol·kg⁻¹·day⁻¹）能减轻血管炎症，表现为血浆细胞因子（TNF-α, IL-1β）水平降低和单核细胞在主动脉内皮上的粘附减少，而低剂量无此作用。[2]
在C57BL/6J小鼠中进行的独立体内逆向胆固醇转运（RCT）实验中，给予AZ876（20 µmol·kg⁻¹·day⁻¹）8天，与载体对照组相比，增加了注射的巨噬细胞中放射性标记胆固醇在血浆（总脂质+75%，游离胆固醇+100%）和粪便（总脂质+94%，游离胆固醇+195%）中的回收率。[2]
高剂量AZ876（20 µmol·kg⁻¹·day⁻¹）处理增加了APOE3Leiden小鼠主动脉中RCT基因Abca1和Abcg1的mRNA表达。低剂量有增加其表达的趋势。[2]
高剂量AZ876（20 µmol·kg⁻¹·day⁻¹）使血浆甘油三酯升高110%，肝脏甘油三酯含量增加53%，并提高了血浆丙氨酸氨基转移酶（ALT）水平，表明存在肝脏脂肪变性和潜在的肝损伤。低剂量无此类效应。[2]
AZ876 不影响动脉粥样硬化病变的胶原或平滑肌细胞含量，而GW3965降低了胶原含量，这表明在等摩尔剂量下，AZ876 治疗导致了更稳定的病变表型。[2]

LXRα和LXRβ的结合实验使用在大肠杆菌中表达的纯化配体结合域蛋白进行。测定混合物包含测定缓冲液、多聚赖氨酸包被的闪烁亲近测定（SPA）微珠、LXR蛋白、固定浓度的氚标记参考配体（T0901317）以及以10点剂量-反应稀释的测试化合物。将混合物轻轻摇动2小时，静置1小时，然后计数以确定放射性配体的置换情况。[2]
报告基因反式激活实验用于测量LXR的激活。将含有与酵母Gal4 DNA结合域融合的人或小鼠LXRα或LXRβ配体结合域的载体，与含有Gal4识别位点的荧光素酶报告质粒共转染到U2/OS骨肉瘤细胞中。转染后，以10点剂量-反应曲线加入配体。48小时后测量荧光素酶活性以确定转录激活效力（EC50）。[2]

使用J774A1巨噬细胞进行了体外巨噬细胞逆向胆固醇转运实验。巨噬细胞经培养后，通过与乙酰化LDL和BSA孵育48小时来负载放射性标记的[³H]胆固醇。洗涤后，将负载了胆固醇的放射性标记巨噬细胞注射到小鼠体内进行体内RCT实验。注射前使用台盼蓝染色评估细胞活力。[2]
使用定量实时PCR（qPCR）测量小鼠组织中的基因表达。从组织中提取总RNA，用DNase处理，并逆转录成cDNA。使用特定的FAM/TAMRA标记探针在实时PCR系统上定量Abca1和Abcg1等靶标的基因表达水平。表达数据归一化至内参基因（小鼠酸性核糖体磷蛋白P0，m36B4），并使用2^(-ΔΔCt)方法计算相对表达量。[2]

AZ876 (20 µmol·kg−1·day−1,∼9·mg·kg−1·day−1)by oral gavage twice daily for 8 days[2]
After matching into four groups, based on age, plasma cholesterol and triglyceride levels, the mice received Western-type diet either alone (control group) or supplemented with AZ876 in a low dose (5 µmol·kg−1·day−1; ∼2 mg·kg−1·day−1) or high dose (20 µmol·kg−1·day−1; ∼9 mg·kg−1·day−1) or with GW3965 (17 µmol·kg−1·day−1; ∼10 mg·kd−1·day−1) (Joseph et al., 2002). Initially, the high dose of AZ876 was 10 µmol·kg−1·day−1; however, after 4 weeks of treatment, GW3965 induced hypertriglyceridaemia but AZ876 (10 µmol·kg−1·day−1) did not. Therefore, it was decided to increase the dose of AZ876 to 20 µmol·kg−1·day−1. After 20 weeks, mice were killed and the hearts, aortic root, livers and small intestine (duodenum) were isolated.[2]

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Study 1 - Macrophage Reverse Cholesterol Transport (RCT): Body weight-matched 7-week-old male C57BL/6J mice were treated with vehicle (amorphous nanoparticles with 3.4% dimethylacetamide), AZ876 (20 µmol·kg⁻¹·day⁻¹, ~9 mg·kg⁻¹·day⁻¹), or GW3965 (34 µmol·kg⁻¹·day⁻¹, ~20 mg·kg⁻¹·day⁻¹) by oral gavage twice daily for 8 days. On day 6, each mouse was injected intraperitoneally with a suspension of [³H]cholesterol-labeled J774A1 macrophages. Feces were collected 24-48 hours post-injection. At 48 hours post-injection, mice were anesthetized, plasma was collected, and livers were perfused and collected. Lipids were extracted from feces, liver, and plasma, and radiolabel content in different lipid fractions was quantified. [2]
Study 2 - Atherosclerosis in APOE3Leiden Mice: Female heterozygous APOE3Leiden transgenic mice (~13 weeks old) were fed a semi-synthetic Western-type diet (high in cholesterol and fat) for a 3-week run-in period. Mice were then matched into groups based on age and plasma lipids and received the same diet alone (control) or supplemented with AZ876 at a low dose (5 µmol·kg⁻¹·day⁻¹, ~2 mg·kg⁻¹·day⁻¹), a high dose (20 µmol·kg⁻¹·day⁻¹, ~9 mg·kg⁻¹·day⁻¹; initially 10 µmol·kg⁻¹·day⁻¹ for 4 weeks, then increased), or GW3965 (17 µmol·kg⁻¹·day⁻¹, ~10 mg·kg⁻¹·day⁻¹) for 20 weeks. Compounds were mixed into the diet. After 20 weeks, mice were euthanized, and hearts (with aortic root), livers, and small intestines were collected for analysis. Blood was collected after a 4-hour fast at various time points for plasma analysis. [2]

The study measured markers related to cholesterol metabolism rather than classical PK parameters. Serum lanosterol levels, a marker of cholesterol synthesis, were increased by 310% in APOE3Leiden mice treated with the high dose of AZ876 (20 µmol·kg⁻¹·day⁻¹). [2]
The absorption of cholesterol, as indicated by serum β-sitosterol levels, was not affected by AZ876 treatment. [2]
Faecal output of neutral sterols (cholesterol) and bile acids was not significantly changed by AZ876 treatment. [2]

In APOE3Leiden mice, the high dose of AZ876 (20 µmol·kg⁻¹·day⁻¹) for 20 weeks induced hepatic steatosis, as evidenced by a 53% increase in liver triglyceride content and a 29% increase in liver weight. [2]
Concomitant with hepatic steatosis, plasma alanine aminotransferase (ALT) levels were increased by 114% in the high-dose AZ876 group, indicating potential liver damage. [2]
The high dose of AZ876 also induced hypertriglyceridemia, elevating plasma triglyceride levels by 110%. [2]
The low dose of AZ876 (5 µmol·kg⁻¹·day⁻¹) did not cause significant changes in liver triglycerides, liver weight, plasma ALT, or plasma triglycerides. [2]

[1]. The liver X receptor agonist AZ876 protects against pathological cardiac hypertrophy and fibrosis without lipogenic side effects. Eur J Heart Fail. 2015 Mar;17(3):273-82.

[2]. Low dose of the liver X receptor agonist, AZ876, reduces atherosclerosis in APOE*3Leiden mice without affecting liver or plasma triglyceride levels. Br J Pharmacol. 2011 Apr;162(7):1553-63.

AZ876 is a novel synthetic liver X receptor (LXR) agonist investigated for its potential to treat atherosclerosis. A major challenge in developing LXR agonists has been their tendency to cause hypertriglyceridemia and hepatic steatosis as side effects. [2]
The primary proposed mechanism for the atheroprotective effect of low-dose AZ876 is the promotion of reverse cholesterol transport (RCT), facilitating the removal of cholesterol from macrophages in the arterial wall, rather than via anti-inflammatory effects. [2]
The study demonstrates that the effects of AZ876 are dose-dependent. A low dose can inhibit atherosclerosis progression without adverse effects on plasma/liver lipids or inflammation, while a high dose provides stronger efficacy but triggers the typical LXR-mediated lipogenic side effects. [2]
The APOE3Leiden transgenic mouse model was used because it responds to lipid-modifying drugs in a manner similar to humans. [2]

C24H29N3O3S

439.5704

439.192

898800-26-5

11655079

Yellow to orange solid powder

1.3±0.1 g/cm3

605.3±65.0 °C at 760 mmHg

319.9±34.3 °C

0.0±1.7 mmHg at 25°C

1.642

3.27

78.1

1

5

5

31

789

0

IVANYIPLGFVBGR-UHFFFAOYSA-N

InChI=1S/C24H29N3O3S/c1-24(2,3)27-23(28)21(22(31(27,29)30)18-10-6-4-7-11-18)25-19-12-14-20(15-13-19)26-16-8-5-9-17-26/h4,6-7,10-15,25H,5,8-9,16-17H2,1-3H3

2-(1,1-Dimethylethyl)-5-phenyl-4-[[4-(1-piperidinyl)phenyl]amino]-3(2H)-isothiazolone 1,1-dioxide

AZ876; AZ 876; AZ-876, AZ12260493; AZ 12260493; AZ-12260493.

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)

DMSO : ~100 mg/mL (~227.50 mM)

配方 1 中的溶解度: 2.5 mg/mL (5.69 mM) 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), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 25.0 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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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：
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
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