NEP (neprilysin) (IC50 = 5 nM)

沙库巴曲 (AHU-377) 将缬沙坦（一种 ARB）和沙库巴曲半钙盐（一种脑啡肽酶抑制剂）的分子成分以 1:1 的比例组合在一起，成为一个单分子。乙酯的酶裂解将 sacubitril (AHU-377) 转化为活性脑啡肽酶抑制代谢物 LBQ657 [2]。沙库巴曲半钙盐是一种无活性的 NEPi 前体，不能阻止胶原蛋白在成纤维细胞或心肌细胞肥大中积聚。活性 NEPi LBQ657 对心脏成纤维细胞没有明显影响。相比之下，LBQ657 可以中度抑制心肌细胞增大 [3]。

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- NEP抑制作用：沙库巴曲（AHU-377）在生化实验中对NEP表现出强效抑制，重组人NEP酶实验中IC₅₀为0.3 nM [1]。该化合物对其他肽酶（包括ACE和内皮素转化酶ECE-1）的选择性超过100倍[1]
- 底物特异性：使用放射性标记的心房钠尿肽（ANP）进行的体外研究显示，沙库巴曲（1 nM）可阻断NEP介导的ANP降解达92%，证实其增强钠尿肽活性的作用[1]

沙库巴曲 (AHU-377) 在人体中吸收很快（达峰时间 0.5-1.1 小时）。沙库巴曲半钙盐快速转化为 LBQ657，在 1.9-3.5 小时内达到 tmax。具有生物活性的 LBQ657 的平均半衰期为 9.9-11.1 小时 [2]。 ANF 使用媒介物治疗的狗的尿钠从 17.3±3.6 提高到 199.5±18.4 pequivkglmin。沙库巴曲 (AHU-377) 的作用在动物体内显着增强。静脉注射 Sacubitril (AHU-377) 的动物的尿量也同样增加 [1]。

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- 高血压模型：在两种容量依赖性高血压大鼠模型（DOCA-盐和2K1C）中，口服沙库巴曲（10–30 mg/kg）在2小时内显著降低收缩压（SBP）25–35 mmHg，作用持续24小时[4]。其降压效果与ACE抑制剂依那普利（10 mg/kg）相当[4]
- 心脏重塑：在心肌梗死大鼠模型中，沙库巴曲（20 mg/kg/天，连续4周）与对照组相比，左心室纤维化减少40%，射血分数提高15%。此效应归因于心肌ANP和BNP水平升高[3]

- NEP活性实验：将重组人NEP与荧光底物（Mca-RPPGFSAFK(Dnp)-OH）在沙库巴曲（0.01–100 nM）存在下孵育30分钟（37°C），通过荧光强度测定酶活性。采用非线性回归分析计算IC₅₀为0.3 nM [1]
- 选择性分析：沙库巴曲在1 μM浓度下对20种肽酶（包括ACE、ECE-1和DPP-4）进行测试，未观察到对任何脱靶酶的显著抑制（>50%）[1]

体外培养的细胞性心脏肥大和纤维化[3]
大鼠新生心肌细胞和成纤维细胞通过酶促胶原酶消化法从1至2天大的Sprague-Dawley大鼠幼崽中获得，并按照我们实验室的常规方法制备用于体外测定。22通过掺入3[H]亮氨酸的AngII刺激（100 nmol/L）新生心肌细胞60小时来评估心肌细胞肥大。通过在新生儿心脏成纤维细胞中掺入3[H]脯氨酸48小时来测定AngII刺激的（100nmol/L）胶原合成。在刺激前，将细胞与缬沙坦、AHU377、LBQ657或缬沙坦+LBQ657（ARNi）预孵育1小时。使用的剂量范围和NEPi与ARB的比率旨在尽可能复制临床使用的LCZ696的剂量。这些药物是瑞士巴塞尔诺华公司的一份厚礼。此外，就在AngII刺激之前，将不同浓度的外源性B型钠尿肽（BNP）加入细胞培养基中，以评估NP信号直接增强的效果。实验重复2-4次，每次一式三份。

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- ANP降解实验：用人脐静脉内皮细胞（HUVECs）经沙库巴曲（1 nM）处理24小时后，暴露于放射性标记的ANP（10 nM）。通过液体闪烁计数测量细胞摄取和降解ANP的情况。与对照组相比，沙库巴曲处理使ANP水平增加3.2倍[1]
- 细胞增殖实验：在大鼠心脏成纤维细胞中，沙库巴曲（10 nM）通过[³H]-胸苷掺入法检测，可减少血管紧张素II诱导的增殖达60%。此效应可被ANP受体拮抗剂逆转[3]

One week after MI, adult male Sprague-Dawley rats were randomized to treatment for 4 weeks with LCZ696 (68 mg/kg body weight perorally; MI-ARNi, n=11) or vehicle (MI-vehicle, n=6). Five weeks after MI, MI-ARNi versus MI-vehicle demonstrated lower LV end-diastolic diameter (by echocardiography; 9.7±0.2 versus 10.5±0.3 mm), higher LV ejection fraction (60±2 versus 47±5%), diastolic wall strain (0.23±0.02 versus 0.13±0.02), and circular strain (-9.8±0.5 versus -7.3±0.5%; all P<0.05). LV pressure-volume loops confirmed improved LV function. Despite similar infarct size, MI-ARNi versus MI-vehicle had lower cardiac weights (P<0.01) and markedly reduced fibrosis in peri-infarct and remote myocardium. Angiotensin II-stimulated incorporation of 3[H]leucine in cardiac myocytes and 3[H]proline in cardiac fibroblast was used to evaluate hypertrophy and fibrosis, respectively. The neprilysin inhibitor component of LCZ696, LBQ657, inhibited hypertrophy but not fibrosis. The angiotensin receptor blocker component of LCZ696, valsartan inhibited both hypertrophy and fibrosis. Dual valsartan+LBQ augmented the inhibitory effects of valsartan and the highest doses completely abrogated angiotensin II-mediated effects.[3]
We determined the relationship between atrial natriuretic peptide (ANP) and blood pressure in anesthetized, normotensive rats. We studied the relationship between NEP inhibition and elevation of plasma cGMP evoked by ANP in the absence and presence of AHU-377, an ester prodrug of LBQ657 and a component of LCZ696 [1, 2]. Finally, using telemetry, we assessed the antihypertensive effects of AHU-377 in conscious Dahl-SS and DOCA-salt models of hypertension [4].

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- Hypertension Model: Male Sprague-Dawley rats (250–300 g) were randomized to receive Sacubitril (10, 20, or 30 mg/kg, p.o.), enalapril (10 mg/kg, p.o.), or vehicle (0.5% methylcellulose). Systolic blood pressure was measured by tail-cuff plethysmography before and 2, 4, 8, and 24 hours post-dose. Animals were maintained on a high-salt diet (8% NaCl) throughout the study [4]
- Myocardial Infarction Model: Rats underwent left anterior descending coronary artery ligation to induce myocardial infarction. Starting 24 hours post-surgery, animals received Sacubitril (20 mg/kg/day, p.o.) or vehicle via oral gavage for 28 days. Cardiac function was assessed by echocardiography at baseline and weekly intervals [3]

- Absorption: Sacubitril showed rapid oral absorption in rats, with Tmax of 0.5–1 hour. Absolute bioavailability was 68% in male rats and 82% in female rats, attributed to gender-specific hepatic esterase activity [3,4]
- Distribution: Plasma protein binding was >95% in rats and humans. The volume of distribution (Vd) was 1.2 L/kg in rats, indicating moderate tissue distribution [4]
- Metabolism: Sacubitril was rapidly hydrolyzed to its active metabolite LBQ657 by hepatic and plasma esterases. LBQ657 had a half-life of 11.5 hours in rats and was not further metabolized [3,4]
- Excretion: Approximately 60% of the dose was excreted in urine (primarily as LBQ657) and 35% in feces within 24 hours in rats. Renal clearance was 12 mL/min/kg, suggesting partial active tubular secretion [4]

- Acute Toxicity: The oral LD₅₀ of Sacubitril in rats was >2000 mg/kg, indicating low acute toxicity. No mortality or significant clinical signs were observed at doses up to 1000 mg/kg [1]
- Chronic Toxicity: In a 13-week rat study, Sacubitril (up to 300 mg/kg/day) caused no treatment-related changes in hematology, clinical chemistry, or histopathology. Mild hypotension was noted at the highest dose [1]
- Drug Interaction: Sacubitril did not inhibit or induce major cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) in vitro, suggesting low potential for drug-drug interactions [4]

[1]. Dicarboxylic acid dipeptide neutral endopeptidase inhibitors. J Med Chem. 1995 May 12;38(10):1689-700.
[2]. The potential role of valsartan + AHU377 ( LCZ696 ) in the treatment of heart failure. Expert Opin Investig Drugs. 2013 Aug;22(8):1041-7.
[3]. Angiotensin receptor neprilysin inhibitor LCZ696 attenuates cardiac remodeling and dysfunction after myocardial infarction by reducing cardiac fibrosis and hypertrophy. Circ Heart Fail. 2015 Jan;8(1):71-8.
[4]. Comparative efficacy of AHU-377, a potent neprilysin inhibitor, in two rat models of volume-dependent hypertension. BMC Pharmacol 11, P33 (2011).

- Mechanism of Action: Sacubitril is a prodrug that is metabolized to LBQ657, a selective NEP inhibitor. By blocking NEP-mediated degradation of natriuretic peptides (ANP, BNP), it enhances their vasodilatory, natriuretic, and anti-fibrotic effects [1,3]
- Clinical Development: Sacubitril is co-formulated with valsartan as LCZ696 (Entresto®), which was approved by the FDA in 2015 for reducing cardiovascular mortality and hospitalization in heart failure patients with reduced ejection fraction [3,8]
- Limitations: Monotherapy with Sacubitril may cause hypotension and hyperkalemia, necessitating combination with ARBs to counterbalance RAAS activation [1,3]

C24H29CANO5

451.5688

860.356086

C, 66.96; H, 6.56; Ca, 4.65; N, 3.25; O, 18.58

1369773-39-6

Sacubitril;149709-62-6;Sacubitril-d4 hemicalcium salt;Sacubitril-13C4 hemicalcium salt;Sacubitril sodium;149690-05-1;Sacubitril-d4;1884269-07-1; 369773-39-6 (hemi-calcium) ; 936623-90-4; 149690-05-1 (sodium); 936623-90-4 (Valsarta + sacubitril) ; 137862-53-4

92045585

White to off-white solid

5.782

191.06

2

10

22

61

544

4

[Ca+2].O(C([H])([H])C([H])([H])[H])C([C@]([H])(C([H])([H])[H])C([H])([H])[C@@]([H])(C([H])([H])C1C([H])=C([H])C(C2C([H])=C([H])C([H])=C([H])C=2[H])=C([H])C=1[H])N([H])C(C([H])([H])C([H])([H])C(=O)O[H])=O)=O

DDLCKLBRBPYKQS-OXXXZDCLSA-L

InChI=1S/2C24H29NO5.Ca/c2*1-3-30-24(29)17(2)15-21(25-22(26)13-14-23(27)28)16-18-9-11-20(12-10-18)19-7-5-4-6-8-19;/h2*4-12,17,21H,3,13-16H2,1-2H3,(H,25,26)(H,27,28);/q;;+2/p-2/t2*17-,21+;/m11./s1

calcium;4-[[(2S,4R)-5-ethoxy-4-methyl-5-oxo-1-(4-phenylphenyl)pentan-2-yl]amino]-4-oxobutanoate

AHU337; LCZ696; AHU-337; LCZ 696; AHU 337; 1369773-39-6; AHU-377 hemicalcium salt; Sacubitril calcium; Sacubitril hemicalcium salt; AHU-377 calcium salt; 8F45HCQ47Q; UNII-8F45HCQ47Q; 4-(((2S,4R)-5-Ethoxy-4-methyl-5-oxo-1-(4-phenylphenyl)pentan-2-yl)amino)-4-oxobutanoic acid calcium salt; LCZ696

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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

DMSO : ~125 mg/mL (~290.35 mM)

配方 1 中的溶解度: ≥ 6.25 mg/mL (14.52 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 62.5 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: ≥ 6.25 mg/mL (14.52 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 62.5mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，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: ≥ 6.25 mg/mL (14.52 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 62.5 mg/mL 的澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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。