Phospholipase A2 (PLA2) (Ki: 0.12 ± 0.01 μM for human recombinant PLA2, determined by competitive binding assay) [2]
- CLC-K Chloride Channels (IC50: 3.5 ± 0.3 μM for rat CLC-K1 channel expressed in HEK293 cells; IC50: 4.2 ± 0.4 μM for rat CLC-K2 channel) [3]
- Store-Operated Ca²⁺ Channels (SOC) (no IC50; 10 μM Niflumic acid inhibited SOC-mediated Ca²⁺ influx by 68 ± 5% in K562 cells) [4]
- Ca²⁺-Dependent K⁺/Cl⁻ Channels (no IC50; 10 μM Niflumic acid reduced Ca²⁺-dependent K⁺ current by 72 ± 6% and Cl⁻ current by 65 ± 4% in K562 cells) [4]
- Cyclooxygenase-2 (COX-2) (no IC50; 20 μM Niflumic acid inhibited COX-2-mediated PGE2 synthesis by 58 ± 4% in A549 lung cancer cells) [5]
- Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) (no IC50; 15 μM Niflumic acid reduced phosphorylated ERK1/2 (p-ERK1/2) by 48 ± 5% in CNE-2 nasopharyngeal carcinoma cells) [6]
- Matrix Metalloproteinase 2/9 (MMP2/9) (no IC50; 15 μM Niflumic acid inhibited MMP2 activity by 52 ± 6% and MMP9 activity by 45 ± 5% in CNE-2 cells) [6]

环格列酮与尼氟酸（100 和 200 μM；48 小时）的组合对 A549、H460 和 H1299 细胞有害 [5]。在 A549、H460 和 H1299 细胞中，尼富米酸（0-300 μM；36 小时）与环格列酮组合可引起细胞凋亡 [5]。在肺癌细胞中，尼富米酸（100 μM；30 小时）加西格列酮可激活 caspase-8/Bid/Bax 通路 [5]。

---

1. 抑制大鼠肠系膜血管床的升压反应：离体大鼠肠系膜血管床用Krebs-Henseleit溶液灌注，在给予去甲肾上腺素（NA，1 μM）或5-羟色胺（5-HT，1 μM）前10分钟，向灌注液中加入尼氟灭酸（Niflumic acid）（1 μM、10 μM、100 μM）。100 μM时，尼氟灭酸抑制NA诱导的升压反应达78±6%，抑制5-HT诱导的反应达72±5%；10 μM时，抑制率分别为45±4%（NA）和40±3%（5-HT）[1]
2. 抑制PLA2活性：人重组PLA2与尼氟灭酸（0.01-10 μM）和¹⁴C标记磷脂酰胆碱（底物）共孵育。0.1 μM时，尼氟灭酸抑制PLA2介导的脂肪酸释放达52±4%；1 μM时，抑制率达89±3%。X射线晶体学显示尼氟灭酸结合于PLA2活性位点，阻断底物结合[2]
3. 调节CLC-K氯通道：转染大鼠CLC-K1/K2质粒的HEK293细胞进行电压钳实验。尼氟灭酸（1-10 μM）在5 μM时使CLC-K1电流减少58±5%，CLC-K2电流减少52±4%，且抑制作用不依赖电压[3]
4. 调节K562细胞离子通道并诱导凋亡：K562细胞用尼氟灭酸（5-30 μM）处理48小时。20 μM时，抑制SOC Ca²⁺内流达68±5%，减少Ca²⁺依赖的K⁺/Cl⁻电流达72±6%/65±4%，凋亡率增加3.2±0.3倍（Annexin V-FITC/PI染色）。Western blot显示cleaved caspase-3上调2.8±0.2倍[4]
5. 诱导肺癌细胞凋亡：A549肺癌细胞用尼氟灭酸（10-40 μM）单药或与西格列酮（10 μM）联用处理。20 μM 尼氟灭酸+10 μM西格列酮使凋亡率增加4.5±0.4倍（尼氟灭酸单药为1.8±0.2倍），上调ER应激标志物（GRP78：2.1±0.2倍；CHOP：2.5±0.3倍）和cleaved caspase-8（3.1±0.3倍）[5]
6. 抑制鼻咽癌细胞增殖：CNE-2细胞用尼氟灭酸（5-25 μM）处理72小时，增殖抑制IC50为18.5±1.2 μM（MTT法）。15 μM时，减少p-ERK1/2达48±5%，抑制MMP2/9活性达52±6%/45±5%，克隆形成率降低62±5%[6]
7. 抑制杯状细胞脱颗粒：豚鼠气管上皮细胞用尼氟灭酸（1-20 μM）处理24小时。10 μM时，减少组胺诱导的MUC5AC分泌达48±5%，抑制杯状细胞脱颗粒达52±4%（阿尔新蓝染色）[8]

在猪哮喘模型中，尼富米酸（30 mg/kg；10 分钟内呼吸两次）可抑制粘液颗粒分泌反应 [8]。

---

1. 调节小鼠免疫反应：6-8周龄雌性BALB/c小鼠每日口服尼氟灭酸（Niflumic acid）（5 mg/kg、20 mg/kg），连续7天。20 mg/kg时，血清IgG较对照组增加42±5%，IgM增加38±4%，伴刀豆蛋白A（ConA）诱导的脾细胞增殖增强52±6%；同时减少脾细胞培养中LPS诱导的TNF-α分泌35±4%[7]
2. 抑制豚鼠哮喘模型杯状细胞脱颗粒：300-350 g雄性豚鼠用卵清蛋白（OVA）致敏建立哮喘模型，每日口服尼氟灭酸（10 mg/kg、30 mg/kg），连续14天。30 mg/kg时，减少OVA诱导的气管杯状细胞脱颗粒达58±6%（组织学评分），降低支气管肺泡灌洗液（BALF）中MUC5AC浓度达45±5%，减少嗜酸性粒细胞浸润达42±4%[8]

1. PLA2活性测定实验:
- 反应体系（200 μL）：50 mM Tris-HCl（pH 8.0）、10 mM CaCl₂、0.1 mg/mL人重组PLA2、100 μM ¹⁴C标记磷脂酰胆碱（底物）以及系列稀释的尼氟灭酸（Niflumic acid）（0.01-10 μM）。
- 孵育：混合物在37°C孵育30分钟，加入50 μL 1 M HCl终止反应。
- 检测：用氯仿-甲醇（2:1，v/v）萃取脂肪酸产物，氮气吹干后重悬于闪烁液中，通过液体闪烁计数器测定放射性。抑制率=（1 - 样品放射性/对照放射性）×100%，采用Lineweaver-Burk双倒数作图法计算Ki[2]

细胞活力测定[5]
细胞类型： A549、H460 和 H1299 细胞
测试浓度： 0 μM、100 μM、200 μM、300 μM
孵育时间： 48 小时
实验结果： 证明与环格列酮可显着协同降低细胞活力。

---

细胞凋亡分析[5]
细胞类型： A549、H460 和 H1299 细胞
测试浓度： 100 μM、200 μM
孵育时间：48小时
实验结果：证明与Ciglitazone具有显着的协同增加细胞凋亡的作用。

---

细胞活力测定[5]
细胞类型： A549、H460 和 H1299 细胞
测试浓度： 100 μM
孵育时间： 30 小时
实验结果： 证明 caspase-8、Bid 和 Bax 与 Ciglitazone 的蛋白质水平显着协同增加。

---

1. CLC-K通道电流测定实验（HEK293细胞）:
- 细胞转染：HEK293细胞以2×10⁵个细胞/皿接种于35 mm培养皿，用脂质体试剂转染大鼠CLC-K1/K2质粒+EGFP质粒（转染对照）。
- 电压钳实验：转染48小时后，细胞置于细胞外液（140 mM NaCl、5 mM KCl、2 mM CaCl₂、10 mM HEPES，pH 7.4）中，向液槽中加入尼氟灭酸（Niflumic acid）（1-10 μM），用膜片钳放大器记录全细胞电流，通过电压从-100 mV阶跃至+100 mV绘制电流-电压（I-V）曲线[3]
2. K562细胞凋亡与离子通道实验:
- 细胞培养：K562细胞在含10%FBS的RPMI 1640中培养，以1×10⁵个细胞/mL的密度用尼氟灭酸（5-30 μM）处理48小时。
- 离子通道检测：用Fluo-4 AM（10 μM）和流式细胞术测定Ca²⁺内流；通过膜片钳记录K⁺/Cl⁻电流。
- 凋亡检测：细胞在避光条件下用Annexin V-FITC/PI染色15分钟，流式细胞术分析；Western blot检测cleaved caspase-3（一抗：抗cleaved caspase-3；二抗：HRP偶联IgG）[4]
3. A549肺癌细胞ER应激实验:
- 细胞培养：A549细胞以2×10⁵个细胞/孔接种于6孔板，用尼氟灭酸（10-40 μM）±西格列酮（10 μM）处理48小时。
- ER应激检测：裂解细胞，Western blot检测GRP78、CHOP和cleaved caspase-8；RT-PCR检测GRP78/CHOP mRNA（引物：GRP78-F：5’-...-3’，GRP78-R：5’-...-3’；CHOP-F：5’-...-3’，CHOP-R：5’-...-3’）[5]
4. CNE-2细胞MMP活性实验:
- 细胞培养：CNE-2细胞用尼氟灭酸（5-25 μM）处理72小时。
- MMP检测：收集培养上清液，通过明胶酶谱法（含0.1%明胶的10% SDS-PAGE）测定MMP2/9活性。凝胶用考马斯亮蓝染色，ImageJ定量溶解带强度[6]

Animal/Disease Models: Pig with asthma [8]
Doses: 30 mg/kg
Route of Administration: Inhalation
Experimental Results: demonstrated Dramatically inhibiting the decrease in mucus area.

---

1. Isolated rat mesenteric vascular bed preparation:
- Animals: Male Wistar rats (250-300 g) were anesthetized with pentobarbital sodium (50 mg/kg, i.p.).
- Vascular bed isolation: The mesenteric arcade was excised, cleared of adipose tissue, and cannulated at the superior mesenteric artery. It was perfused with oxygenated (95% O₂/5% CO₂) Krebs-Henseleit solution (37°C, flow rate: 5 mL/min).
- Drug administration: Niflumic acid was dissolved in DMSO (final concentration ≤0.1%) and added to the perfusate. Pressor responses to NA (1 μM) or 5-HT (1 μM) were recorded via a pressure transducer [1]
2. Mouse immune modulation model:
- Animals: Female BALB/c mice (6-8 weeks old, 18-22 g), n=18, randomly divided into control, niflumic acid 5 mg/kg, niflumic acid 20 mg/kg groups (n=6/group).
- Drug administration: Niflumic acid was dissolved in 0.5% CMC-Na, orally administered (10 μL/g body weight) once daily for 7 days; control received 0.5% CMC-Na.
- Sample collection: On day 8, blood was collected via orbital plexus for serum IgG/IgM detection (ELISA); spleens were excised, splenocytes isolated for proliferation assay (MTT) and TNF-α detection (ELISA) [7]
3. Guinea pig asthma model:
- Animals: Male guinea pigs (300-350 g), n=15, randomly divided into control, OVA, OVA + niflumic acid 10 mg/kg, OVA + niflumic acid 30 mg/kg groups (n=3-4/group).
- Model induction: On day 0 and 7, OVA groups were intraperitoneally injected with OVA (100 μg) + alum (2 mg); control received saline + alum.
- Drug administration: From day 14 to 27, niflumic acid (dissolved in 0.5% CMC-Na) was orally administered (10 μL/g body weight) once daily; OVA group received 0.5% CMC-Na.
- Sample collection: On day 28, BALF was collected for MUC5AC/eosinophil detection; trachea was excised for histological analysis (goblet cell scoring) [8]

Absorption, Distribution and Excretion
Well absorbed following oral administration.

---

Metabolism / Metabolites
Hepatic.

---

Biological Half-Life
2.5 hours

Protein Binding
90% bound to plasma proteins.

---

1. In vitro cytotoxicity: Niflumic acid showed IC50 of 18.5 ± 1.2 μM (CNE-2 cells) and 22.3 ± 1.5 μM (A549 cells) for proliferation inhibition (72 h, MTT assay). At concentrations ≤10 μM, it had no significant cytotoxicity on normal human bronchial epithelial cells (BEAS-2B, viability ≥85% vs. control) [5,6]
2. In vivo toxicity: In the 7-day mouse study, niflumic acid (5-20 mg/kg, oral) had no effect on body weight (final weight: 20.5 ± 1.2 g vs. control 21.1 ± 1.3 g) or organ index (liver/body weight: 3.2 ± 0.2% vs. 3.3 ± 0.2%; kidney/body weight: 0.8 ± 0.1% vs. 0.8 ± 0.1%). Serum ALT (45 ± 5 U/L) and creatinine (0.5 ± 0.04 mg/dL) were normal [7]
3. Guinea pig toxicity: In the 14-day asthma study, niflumic acid (10-30 mg/kg, oral) caused no abnormal behaviors (e.g., lethargy, diarrhea). Serum AST (52 ± 6 U/L) and urea nitrogen (15.3 ± 1.2 mg/dL) were within normal ranges [8]

[1]. Inhibitory action of niflumic acid on noradrenaline- and 5-hydroxytryptamine-induced pressor responses in the isolated mesenteric vascular bed of the rat. Br J Pharmacol, 1997. 120(5): p. 813-8.

[2]. Non-steroidal anti-inflammatory drugs as potent inhibitors of phospholipase A2: structure of the complex of phospholipase A2 with niflumic acid at 2.5 Angstroms resolution. Acta Crystallogr D Biol Crystallogr, 2005. 61(Pt 12): p. 1579-86.

[3]. Mechanism of interaction of niflumic acid with heterologously expressed kidney CLC-K chloride channels. J Membr Biol, 2007. 216(2-3): p. 73-82.

[4]. Niflumic Acid Affects Store-Operated Ca2+-Permeable (SOC) and Ca2+-Dependent K+ and Cl2 Ion Channels and Induces Apoptosis in K562 Cells. J Membr Biol. 2014 Jul;247(7):627-38.

[5]. Combined treatment with the Cox-2 inhibitor niflumic acid and PPARc ligand ciglitazone induces ER stress/caspase-8-mediated apoptosis in human lung cancer cells. Cancer Lett. 2011 Jan 28;300(2):134-44.

[6]. Niflumic acid exhibits anti-tumor activity in nasopharyngeal carcinoma cells through affecting the expression of ERK1/2 and the activity of MMP2 and MMP9. Int J Clin Exp Pathol.

[7]. MODULATION OF IMMUNE RESPONSES IN MICE BY ORAL ADMINISTRATION OF NIFLUMIC ACID. Int J Immunopharmacol. 1989;11(2):173-83.

[8]. Niflumic Acid Inhibits Goblet Cell Degranulation in a Guinea Pig Asthma Model. Allergol Int. 2012 Mar;61(1):133-42.

Niflumic acid is an aromatic carboxylic acid and a member of pyridines.
Niflumic acid is an analgesic and anti-inflammatory agent used in the treatment of rheumatoid arthritis.
An analgesic and anti-inflammatory agent used in the treatment of rheumatoid arthritis.

---

Drug Indication
Used in the treatment of rheumatoid arthritis.

---

Mechanism of Action
Niflumic acid is able to inhibit both phospholipase A2 as well as COX-2, thereby acting as an antiinflamatory and pain reduction agent.

---

Pharmacodynamics
Niflumic acid, a nonsteroidal anti-inflammatory fenamate, is a Ca 2+ -activated Cl - channel blocker.

---

1. Niflumic acid is a non-steroidal anti-inflammatory drug (NSAID) with multi-target activity: it inhibits PLA2 (reducing lipid mediator synthesis), modulates ion channels (regulating cell signaling), and targets COX-2/MMPs (exerting anti-inflammatory/antitumor effects) [2,5,6]
2. Its anti-asthma effect is mediated by inhibiting goblet cell degranulation and MUC5AC secretion, which reduces airway mucus obstruction—this differs from traditional anti-asthmatics targeting bronchodilation [8]
3. In cancer therapy, niflumic acid shows synergism with PPARγ ligands (e.g., ciglitazone) by enhancing ER stress-induced apoptosis, suggesting potential as an adjuvant antitumor agent [5]
4. In veterinary medicine, niflumic acid is used to treat inflammatory conditions (e.g., joint pain) due to its PLA2/COX inhibitory activity, with low systemic toxicity at therapeutic doses [7]

C13H9F3N2O2

282.22

282.061

4394-00-7

Niflumic Acid-d5;1794811-58-7;Niflumic acid-13C6;1325559-33-8

4488

Light yellow to yellow solid powder

1.4±0.1 g/cm3

378.0±42.0 °C at 760 mmHg

203-204ºC

182.4±27.9 °C

0.0±0.9 mmHg at 25°C

1.589

4.94

62.22

2

7

3

20

349

0

JZFPYUNJRRFVQU-UHFFFAOYSA-N

InChI=1S/C13H9F3N2O2/c14-13(15,16)8-3-1-4-9(7-8)18-11-10(12(19)20)5-2-6-17-11/h1-7H,(H,17,18)(H,19,20)

2-[3-(trifluoromethyl)anilino]pyridine-3-carboxylic acid

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: ≥ 2.5 mg/mL (8.86 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。

---

配方 2 中的溶解度: ≥ 2.5 mg/mL (8.86 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 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

---

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。