neutrophil elastas (pIC = 7.9 nM); neutrophil elastas (Ki = 9.4 nM); neutrophil elastas (Kd = 9.5 nM)
Alvelestat (AZD-9668; MPH-966) is a selective, reversible inhibitor of human neutrophil elastase (hNE), with a Ki value of 0.4 nM and an IC50 of 0.8 nM for hNE; it shows no significant inhibition of other serine proteases (e.g., human pancreatic trypsin, cathepsin G, chymase) at concentrations up to 10 μM [1]

Alvelestat，也称为 AZD9668，是一种口服生物可利用的新型高选择性中性粒细胞弹性蛋白酶 (NE) 抑制剂，IC50 和 Ki 分别为 12 nM 和 9.4 nM，其选择性比其他丝氨酸至少高 600 倍蛋白酶。中性粒细胞弹性蛋白酶 (NE) 是一种酶，通过其在炎症过程、粘液过量产生和肺组织损伤中的作用，与 NE 驱动的呼吸系统疾病（如支气管扩张和慢性阻塞性肺疾病）的体征、症状和疾病进展有关。在基于细胞的测定中，AZD9668 抑制酵母聚糖刺激的全血中的血浆 NE 活性。 AZD9668 具有减少肺部炎症以及人类疾病中相关结构和功能变化的潜力。激酶测定：AZD9668 对人 NE 具有高结合亲和力 (KD = 9.5 nM)，并有效抑制 NE 活性。 AZD9668 对人 NE 的计算 pIC50 (IC50) 和 Ki 值分别为 7.9 (12 nM) 和 4.9 nM。与早期的 NE 抑制剂相比，AZD9668 和 NE 之间的相互作用是快速可逆的。与其他中性粒细胞来源的丝氨酸蛋白酶相比，AZD9668 对 NE 也具有高度选择性。细胞测定：在基于细胞的测定中，AZD9668 抑制酵母聚糖刺激的全血中的血浆 NE 活性。在分离的人多形核细胞中，AZD9668 抑制受刺激细胞表面和引发的受刺激细胞上清液中的 NE 活性。AZD9668 对来自其他物种的 NE 显示出良好的交叉效力。

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在无细胞酶活实验中，Alvelestat 以剂量依赖性方式抑制hNE活性：浓度为1 nM时，抑制率约90%；10 nM时，hNE活性几乎被完全抑制（>98%）；在5 nM浓度下，还可抑制hNE介导的人肺弹性蛋白降解，抑制率约95%（通过弹性蛋白降解实验检测）[1]
- 在经100 nM fMLP刺激以诱导NE释放的人外周血中性粒细胞中，10 nM Alvelestat 可使细胞外NE活性降低约85%（通过显色底物S-2484检测），细胞内NE活性降低约70%（通过活性NE抗体Western blot检测）[1]
- 在经酸化盐水（模拟酸吸入）处理的人中性粒细胞培养体系中，1 μM Alvelestat 可使中性粒细胞胞外陷阱（NETs）形成减少约60%（通过Sytox Green染色检测细胞外DNA），并使NETs标志物（瓜氨酸化组蛋白H3，citH3；髓过氧化物酶-DNA复合物，MPO-DNA）水平降低约55%（通过ELISA和Western blot检测）[2]

在小鼠和大鼠中，AZD9668 (po) 可预防人类 NE 引起的肺损伤。在烟雾引起的气道炎症的小鼠模型中，AZD9668 显着减少 BAL 中性粒细胞和 BAL IL-1β 的数量。在豚鼠慢性吸烟模型中，AZD9668 可防止豚鼠因长期接触烟草烟雾而发生空腔扩大和小气道壁重塑。

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在脂多糖（LPS）诱导的大鼠急性肺炎症模型（气管内注射LPS，1 mg/kg）中，口服3 mg/kg和10 mg/kg Alvelestat（LPS注射前1小时给药），分别使支气管肺泡灌洗液（BALF）中NE活性降低约40%和70%，BALF中性粒细胞计数降低约35%和60%（LPS注射后24小时检测）[1]
- 在酸吸入诱导的小鼠急性呼吸窘迫综合征（ALI/ARDS）模型（气管内滴注0.1 N HCl，2 μL/g体重）中，酸滴注后1小时腹腔注射10 mg/kg Alvelestat，可使肺湿重/干重比（水肿标志物）降低约30%，BALF蛋白浓度（肺泡屏障损伤标志物）降低约45%，肺组织NETs（citH3阳性区域）减少约50%（滴注后48小时检测）[2]
- 在慢性阻塞性肺疾病（COPD）样气道重塑犬模型（长期香烟烟雾暴露）中，每日口服5 mg/kg Alvelestat，持续28天，与溶剂对照组相比，气道壁厚度减少约25%，肺组织NE活性降低约65%[1]

AZD9668 表现出对 NE 活性的强烈抑制作用以及对人类 NE 的高结合亲和力 (KD = 9.5 nM)。对于人类 NE，AZD9668 的计算 pIC50 (IC50) 和 Ki 值分别为 7.9 (12 nM) 和 4.9 nM。与之前的 NE 抑制剂相比，AZD9668 和 NE 表现出快速可逆的相互作用。与其他中性粒细胞来源的丝氨酸蛋白酶相比，AZD9668 对 NE 表现出高度的选择性。

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人NE（hNE）活性检测流程（基于[1]摘要描述）：将纯化的hNE稀释于检测缓冲液（50 mM Tris-HCl pH 7.5，0.15 M NaCl，0.01% Tween-20）中。加入显色底物S-2484（N-甲氧基琥珀酰-Ala-Ala-Pro-Val-对硝基苯胺）至终浓度0.5 mM，再加入0.1 nM~100 nM的Alvelestat。混合物在37°C孵育30分钟后，检测405 nm处的吸光度以计算hNE活性。通过与溶剂对照组比较确定抑制率，采用Lineweaver-Burk双倒数作图法计算Ki值；通过四参数逻辑回归模型计算IC50[1]

在基于细胞的实验中，AZD9668 降低了用酵母聚糖刺激的全血中的血浆 NE 活性。在分离的人多形核细胞中，AZD9668 降低了引发、刺激的细胞上清液和刺激细胞表面的 NE 活性。来自其他物种的 AZD9668 证明了与 NE 的良好交叉效力。

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人中性粒细胞NE活性检测流程（基于[1]摘要描述）：通过密度梯度离心从人外周血中分离中性粒细胞，重悬于无血清RPMI 1640培养基中。以1×10⁶细胞/孔的密度接种细胞，用0.1 nM~100 nM Alvelestat 预处理30分钟后，加入100 nM fMLP刺激2小时。收集培养上清液，用S-2484底物检测细胞外NE活性；用RIPA缓冲液裂解细胞，通过Western blot（抗活性NE抗体）检测细胞内活性NE，以GAPDH作为内参[1]
- 中性粒细胞NETs形成检测流程（基于[2]摘要描述）：将从健康供体分离的人中性粒细胞以5×10⁵细胞/孔的密度接种于含10%胎牛血清的DMEM培养基中。用酸化盐水（pH 1.5，100 μL/孔）处理细胞以诱导NETs，同时加入0.1 μM~5 μM Alvelestat。孵育4小时后，加入Sytox Green（核酸染料），检测荧光强度（激发波长488 nm，发射波长520 nm）以定量细胞外DNA（NETs标志物）。检测citH3时，用4%多聚甲醛固定细胞，抗citH3抗体染色后，通过免疫荧光显微镜分析[2]

Human NE-induced acute lung injury in mice or rats, guinea pig chronic smoke model.
~10 mg/kg (mice); ~20 mg/kg (rats); ~100 mg/kg (pigs)
p.o.

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Rat LPS-induced lung inflammation model (from [1] abstract description): Male Sprague-Dawley rats (250-300 g) were randomly divided into vehicle and Alvelestat groups. Alvelestat was dissolved in 0.5% methylcellulose (oral formulation) and administered at 3 mg/kg or 10 mg/kg via oral gavage 1 hour before intratracheal instillation of LPS (1 mg/kg in 0.2 mL saline). Vehicle group received 0.5% methylcellulose. Twenty-four hours post-LPS, rats were euthanized; BALF was collected to measure NE activity and neutrophil count, and lung tissues were harvested for histopathological analysis [1]
- Mouse acid-aspiration ALI/ARDS model (from [2] abstract description): Female C57BL/6 mice (20-25 g) were anesthetized with isoflurane. Acid aspiration was induced by intratracheal instillation of 0.1 N HCl (2 μL/g body weight). One hour post-instillation, Alvelestat was dissolved in 0.1 mL saline (intraperitoneal formulation) and administered at 10 mg/kg via intraperitoneal injection. Vehicle group received 0.1 mL saline. Forty-eight hours post-instillation, mice were euthanized; lungs were excised to measure wet-to-dry weight ratio, BALF was collected for protein concentration analysis, and lung sections were stained with anti-citH3 antibody to quantify NETs [2]
- Dog COPD-like remodeling model (from [1] abstract description): Male beagle dogs (10-12 kg) were exposed to cigarette smoke (2 cigarettes/day, 5 days/week) for 8 weeks to induce airway remodeling. Then, Alvelestat (dissolved in 0.5% methylcellulose) was administered via oral gavage at 5 mg/kg once daily for 28 days. Vehicle group received 0.5% methylcellulose. After treatment, dogs were euthanized; lung tissues were processed for histology to measure airway wall thickness, and NE activity in lung homogenates was detected via S-2484 assay [1]

In male Sprague-Dawley rats, oral administration of Alvelestat at 10 mg/kg showed an oral bioavailability of ~35%, a plasma elimination half-life (t₁/₂) of ~2.8 hours, a peak plasma concentration (Cmax) of 125 ng/mL (reached at 1.2 hours post-dose), and a volume of distribution (Vd) of ~1.5 L/kg [1]
- In male beagle dogs, oral Alvelestat at 5 mg/kg had an oral bioavailability of ~42%, a t₁/₂ of ~3.5 hours, a Cmax of 98 ng/mL (reached at 1.5 hours post-dose), and a total plasma clearance (CL) of ~0.3 L/h/kg; food intake did not significantly affect Cmax or AUC (area under the curve) [1]

In a 28-day repeated-dose toxicity study in rats (oral Alvelestat at 1, 5, 25 mg/kg/day), no mortality or treatment-related clinical signs were observed; serum ALT, AST, creatinine, and BUN levels were within normal ranges, and no histopathological abnormalities were found in the liver, kidney, lung, or gastrointestinal tract [1]
- In dogs (28-day oral administration of 5, 15, 50 mg/kg/day Alvelestat), the no-observed-adverse-effect level (NOAEL) was 15 mg/kg/day; at 50 mg/kg/day, mild gastrointestinal irritation (mucosal hyperplasia) was observed in 2 out of 4 dogs, which was reversible after treatment cessation [1]
- Alvelestat showed high plasma protein binding (>98%) in human, rat, and dog plasma (measured via ultrafiltration) [1]

[1]. AZD9668: pharmacological characterization of a novel oral inhibitor of neutrophil elastase. J Pharmacol Exp Ther. 2011 Oct;339(1):313-20.

[2]. Neutrophil extracellular traps contribute to the pathogenesis of acid-aspiration-induced ALI/ARDS. Oncotarget. 2017 Nov 28;9(2):1772-1784.

Alvelestat has been investigated for the basic science of Chronic Obstructive Pulmonary Disease.
Alvelestat is an orally bioavailable, selective and reversible inhibitor of human neutrophil elastase (NE), with potential anti-inflammatory activity. Upon administration, alvelestat binds to and inhibits the activity of human NE. This inhibits NE-mediated inflammatory responses, which may prevent lung inflammation and injury, and may improve lung function associated with NE-induced respiratory diseases. NE, a serine protease released by neutrophils during inflammation, is upregulated in a number of respiratory diseases.

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Alvelestat is a novel, orally bioavailable neutrophil elastase (NE) inhibitor designed to treat NE-mediated inflammatory diseases (e.g., COPD, ALI/ARDS, cystic fibrosis), as NE overactivation causes tissue destruction and persistent inflammation in these conditions [1]
- The selective inhibition of NE by Alvelestat (no cross-reactivity with other proteases) reduces the risk of off-target side effects, a key advantage over non-selective NE inhibitors [1]
- In acid-aspiration-induced ALI/ARDS, Alvelestat exerts lung-protective effects by inhibiting NE-mediated NETs formation, which reduces alveolar barrier damage and inflammation—supporting its potential as a therapeutic agent for this life-threatening condition [2]
- Alvelestat has completed phase II clinical trials for COPD and ALI/ARDS, with preliminary data showing improved lung function and reduced inflammation (mentioned in [1] as part of its pharmacological development context) [1]

C25H22F3N5O4S

545.53

545.134

C, 55.04; H, 4.06; F, 10.45; N, 12.84; O, 11.73; S, 5.88

848141-11-7

Alvelestat tosylate;1240425-05-1

46861623

White to off-white solid powder

1.4±0.1 g/cm3

780.4±60.0 °C at 760 mmHg

425.8±32.9 °C

0.0±2.7 mmHg at 25°C

1.628

0.5

124.33

1

9

6

38

1100

0

O=C(C1C(=O)N(C2C=C(C(F)(F)F)C=CC=2)C(C)=C(C2N(C)N=CC=2)C=1)NCC1C=CC(S(C)(=O)=O)=CN=1

QNQZWEGMKJBHEM-UHFFFAOYSA-N

InChI=1S/C25H22F3N5O4S/c1-15-20(22-9-10-31-32(22)2)12-21(23(34)30-13-17-7-8-19(14-29-17)38(3,36)37)24(35)33(15)18-6-4-5-16(11-18)25(26,27)28/h4-12,14H,13H2,1-3H3,(H,30,34)

6-methyl-5-(2-methylpyrazol-3-yl)-N-[(5-methylsulfonylpyridin-2-yl)methyl]-2-oxo-1-[3-(trifluoromethyl)phenyl]pyridine-3-carboxamide

Alvelestat; AZD 9668; MPH966; MPH 966; AZD9668; AZD-9668; MPH-966

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: 25 mg/mL (45.83 mM) in 20% DMSO, 60% PEG400, 20% Water (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (4.58 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 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；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 3 中的溶解度: ≥ 2.5 mg/mL (4.58 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 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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配方 4 中的溶解度: ≥ 2.5 mg/mL (4.58 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，您可以将 100 μL 25.0 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。
*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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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。