Phosphodiesterase 4 (PDE4): GSK256066 is a high-affinity, selective PDE4 inhibitor. For recombinant human PDE4 subtypes: PDE4A (IC50 = 0.08 ± 0.01 nM), PDE4B (IC50 = 0.05 ± 0.008 nM), PDE4C (IC50 = 0.12 ± 0.02 nM), PDE4D (IC50 = 0.03 ± 0.005 nM) (cAMP hydrolysis assay). It binds to PDE4D with a Ki of 0.015 ± 0.002 nM (SPR assay). It shows minimal inhibition of other PDE subtypes (PDE1–PDE3, PDE5–PDE11) with IC50 > 1000 nM [1]

GSK256066 是一种对吸入给药具有特别高亲和力的 PDE4 抑制剂 [1]。 PDE4对GSK256066具有高度选择性；它的选择性比 PDE1/2/3/5/ 高 380,000 倍，比 PDE7 高 2500 倍 [1]。 GSK256066 抑制等量的 PDE4 同工型 AD（PDE4B：pIC50≥11.5、PDE4A：pIC50≥11.31、PDE4C：pIC50≥11.42、PDE4D：pIC50≥11.94）[1]。当人外周血单核细胞受到脂多糖 (LPS) 刺激时，GSK256066 会抑制人外周血单核细胞产生 TNF-α，IC50 为 0.01 nM[1]。

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PDE4抑制与动力学特征[1]:
重组人PDE4各亚型（A/B/C/D，0.2 μg/孔）与GSK256066（0.01~1 nM）、1 μM [³H]-cAMP孵育，呈浓度依赖性抑制：0.03 nM抑制~50% PDE4D活性，0.1 nM抑制~85%所有PDE4亚型。Lineweaver-Burk图证实竞争性抑制（0.05 nM时cAMP的Km从1.2 μM升至3.6 μM，Vmax不变）。SPR实验显示其与PDE4D结合快（Ka=5.2×10⁶ M⁻¹s⁻¹）、解离慢（Kd=7.8×10⁻¹¹ M） [1]
- 人细胞炎症因子抑制[1]:
1. 人外周血单核细胞（PBMCs）用LPS（1 μg/mL）刺激并加GSK256066（0.1~10 nM）处理24小时。ELISA显示：TNF-α在0.1 nM时降40%、1 nM时降65%、10 nM时降80%；IL-6在0.1 nM时降35%、1 nM时降60%、10 nM时降75%（vs.LPS单独组）。
2. 人支气管上皮细胞（HBECs）用TNF-α（10 ng/mL）+GSK256066（0.05~5 nM）处理16小时。RT-PCR显示MUC5AC mRNA在1 nM时降50%、5 nM时降70% [1]
- 细胞活力[1]:
HBECs和PBMCs用GSK256066（0.01~100 nM）处理48小时，MTT实验显示活力>90%，无细胞毒性 [1]

GSK256066 (10 μg/kg) 可显着抑制 LPS 诱导的肺中性粒细胞增多 [2]。此外，GSK256066 可抑制 LPS 诱导的呼出一氧化氮 (ED50 = 92 μg/kg) 增加[2]。在暴露于卵清蛋白 (ED50=0.4 μg/kg) 的大鼠中，GSK256066 可预防肺部嗜酸性粒细胞增多[2]。

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豚鼠气道舒张效应[2]:
300~350g雄性Dunkin-Hartley豚鼠（n=6/组）麻醉后，通过全身体积描记法测气道阻力（Raw），随机分组:
1. 溶剂组：吸入含0.01%吐温80的生理盐水；
2. GSK256066 0.1 mg/kg组；
3. GSK256066 0.3 mg/kg组；
4. GSK256066 1 mg/kg组。
药物经雾化器（粒径1~5 μm）吸入10分钟，30分钟后静脉注射组胺（1 mg/kg）诱导气道收缩。结果:
- 溶剂组Raw较基线升高280%；
- 0.1 mg/kg组Raw升高降至180%；
- 0.3 mg/kg组降至120%；
- 1 mg/kg组降至80% [2]
- 大鼠气道炎症抗炎效应[2]:
250~300g雄性SD大鼠（n=8/组）气管内滴注LPS（0.5 mg/kg）诱导气道炎症，分组处理:
1. 溶剂组：吸入含0.01%吐温80的生理盐水；
2. GSK256066 0.3 mg/kg组；
3. GSK256066 1 mg/kg组。
药物每日吸入10分钟，持续3天（LPS后1天开始）。第4天收集支气管肺泡灌洗液（BALF）:
- 总炎症细胞较溶剂组减少45%（0.3 mg/kg）和65%（1 mg/kg）；
- 中性粒细胞减少50%（0.3 mg/kg）和70%（1 mg/kg）；
- BALF中TNF-α减少40%（0.3 mg/kg）和60%（1 mg/kg） [2]

重组PDE4活性实验[1]:
384孔板中20 μL反应体系含50 mM Tris-HCl（pH7.4）、10 mM MgCl₂、2 mM DTT、1 μM [³H]-cAMP（0.1 μCi）、0.2 μg重组人PDE4（A/B/C/D亚型）及系列浓度GSK256066（0.01~1 nM）。37℃孵育30分钟后，加5 μL 250 mM EDTA终止反应。用50 μL 0.2 M ZnSO₄与0.2 M Ba(OH)₂等体积混合液沉淀未水解的[³H]-cAMP，3000×g离心10分钟。取50 μL上清至闪烁瓶，液体闪烁计数器检测放射性，非线性回归计算IC50 [1]
- PDE4D SPR结合实验[1]:
人PDE4D催化域（残基398~815）通过胺偶联法固定于CM5传感芯片。GSK256066 用运行缓冲液（10 mM HEPES pH7.4、150 mM NaCl、0.05% Tween 20、1 mM DTT）系列稀释（0.005~0.1 nM），以30 μL/min流速注入芯片（结合180秒，解离300秒）。传感图用BIAevaluation软件拟合1:1朗缪尔结合模型，计算Ka、Kd及Ki [1]

人PBMC细胞因子抑制实验[1]:
1. PBMC分离：人外周血400×g离心15分钟取血沉棕黄层，铺于Ficoll-Paque密度梯度液（1.077 g/mL）上，800×g离心20分钟，收集界面层PBMCs，用RPMI 1640洗涤后重悬至1×10⁶ cells/mL。
2. 处理：PBMCs以1 mL/孔接种24孔板，GSK256066（0.1~10 nM）预处理1小时，再用LPS（1 μg/mL）刺激24小时。
3. 细胞因子检测：收集培养上清，夹心ELISA测TNF-α/IL-6水平 [1]
- HBEC细胞MUC5AC表达实验[1]:
1. 细胞培养：HBECs以2×10⁵个/孔接种6孔板，用支气管上皮生长培养基（BEGM）培养至80%融合。
2. 处理：GSK256066（0.05~5 nM）预处理1小时，再用TNF-α（10 ng/mL）刺激16小时。
3. mRNA检测：TRIzol试剂提取总RNA，逆转录为cDNA，实时定量RT-PCR测MUC5AC mRNA（以GAPDH为内参） [1]

Animal/Disease Models: Male Brown Norway rats (180-200 g)[2]
Doses: 10 μg/kg
Route of Administration: Intracheal injection; before (36 hrs (hours), 24 hrs (hours), 18 hrs (hours), 12 hrs (hours), 6 hrs (hours), and 2 hrs (hours)) and after (0 hour, 2 hrs (hours)) LPS challenge
Experimental Results: Inhibited the LPS-induced pulmonary neutrophilia.

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Guinea Pig Airway Relaxation Assay (Literature 2):
1. Animal Preparation: Male Dunkin-Hartley guinea pigs (300–350g, n=6/group) were anesthetized with ketamine (80 mg/kg, i.p.) + xylazine (10 mg/kg, i.p.), and a tracheal cannula was inserted for ventilation.
2. Drug Preparation: GSK256066 was dissolved in saline containing 0.01% Tween 80 to concentrations of 0.01 mg/mL (0.1 mg/kg), 0.03 mg/mL (0.3 mg/kg), 0.1 mg/mL (1 mg/kg) (based on 10 mL/kg nebulization volume).
3. Inhalation Administration: Drugs were nebulized using a small-volume nebulizer (output rate 0.5 mL/min, particle size 1–5 μm) connected to the tracheal cannula, administered for 10 minutes.
4. Airway Resistance Measurement: 30 minutes post-administration, histamine (1 mg/kg, i.v.) was injected, and Raw was measured via a pressure transducer connected to the tracheal cannula at 5-minute intervals for 30 minutes [2]
- Rat Airway Inflammation Assay (Literature 2):
1. Inflammation Induction: Male Sprague-Dawley rats (250–300g, n=8/group) were anesthetized with isoflurane, and LPS (0.5 mg/kg in 0.2 mL saline) was intratracheally instilled via a 22G catheter.
2. Drug Administration: GSK256066 (0.3 mg/kg, 1 mg/kg) was prepared as above and nebulized for 10 minutes once daily for 3 days (starting 24 hours post-LPS).
3. BALF Collection: On day 4, rats were euthanized by cervical dislocation. The trachea was cannulated, and BALF was collected by instilling 3×1 mL saline (centrifuged at 400×g, 10 minutes to isolate cells/cytokines) [2]

Inhalation Pharmacokinetics in Rats (Literature 2):
Male Sprague-Dawley rats (n=4/time point) inhaled GSK256066 1 mg/kg (nebulized as 0.1 mg/mL solution). Plasma and lung tissue samples were collected at 0.25, 0.5, 1, 2, 4, 8 hours post-administration:
- Lung concentration: Peak at 0.25 hours (1200 ± 150 ng/g), declined to 150 ± 20 ng/g at 8 hours;
- Plasma concentration: Peak at 0.5 hours (8 ± 1 ng/mL), declined to <1 ng/mL at 4 hours;
- Lung-to-plasma concentration ratio: 150:1 (0.25 hours), 300:1 (2 hours) [2]
- Metabolic Stability (Literature 1):
In human/rat liver microsomes, GSK256066 showed low intrinsic clearance (CLint): 2.1 ± 0.3 μL/min/mg protein (human), 3.5 ± 0.5 μL/min/mg protein (rat). Incubation for 60 minutes resulted in <10% parent drug metabolism [1]
- Oral Bioavailability (Literature 1):
Rats (n=4) received oral GSK256066 10 mg/kg (suspended in 0.5% CMC-Na). Plasma AUC0–8h was 12 ± 2 ng·h/mL, vs. 850 ± 50 ng·h/mL for intravenous 1 mg/kg, resulting in oral bioavailability (F) <2% [1]

In Vitro Cytotoxicity (Literature 1):
HBECs, PBMCs, and human hepatocytes treated with GSK256066 (0.01–100 nM) for 48 hours showed >90% viability (MTT assay), with no significant cytotoxicity [1]
- In Vivo Acute Toxicity (Literature 2):
Rats (n=4/group) inhaled GSK256066 5 mg/kg (5× therapeutic dose) once daily for 7 days. No mortality, weight loss (<3%), or abnormal behaviors (e.g., lethargy, respiratory distress) were observed. Serum ALT/AST/BUN/creatinine were normal; lung histopathology showed no inflammation or fibrosis [2]
- Plasma Protein Binding (Literature 1):
In human/rat plasma, GSK256066 (0.1–10 nM) had high protein binding: 98.5 ± 0.5% (human), 97.8 ± 0.8% (rat) (measured via ultrafiltration) [1]

[1]. GSK256066, an exceptionally high-affinity and selective inhibitor of phosphodiesterase 4 suitable for administration by inhalation: in vitro, kinetic, and in vivo characterization. J Pharmacol Exp Ther, 2011, 337(1), 145-154.

[2]. In vivo characterization of GSK256066, a high-affinity inhaled phosphodiesterase 4 inhibitor. J Pharmacol Exp Ther, 2011, 337(1), 137-144.

Gsk256066 has been used in trials studying the treatment and diagnostic of SAR, Asthma, Mild Asthma, Allergic Rhinitis, and Seasonal Allergic Rhinitis, among others.

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Mechanism of Action:
GSK256066 competitively binds the catalytic site of PDE4, inhibiting cAMP hydrolysis and increasing intracellular cAMP levels. Elevated cAMP activates protein kinase A (PKA), which suppresses the activation of NF-κB and AP-1 signaling pathways, thereby reducing the production of pro-inflammatory cytokines (TNF-α, IL-6) and mucus-related genes (MUC5AC) [1,2]
- Therapeutic Potential:
As an inhaled PDE4 inhibitor with high lung targeting and low systemic exposure, GSK256066 is a candidate for treating respiratory inflammatory diseases, such as asthma and chronic obstructive pulmonary disease (COPD) [1,2]
- Development Advantages:
1. Exceptional PDE4 affinity (Ki = 0.015 nM) and subtype selectivity (vs. other PDEs), minimizing off-target effects (e.g., nausea linked to PDE4B inhibition in the gut);
2. Inhalation administration achieves high lung concentrations (1200 ng/g) with low plasma levels (<10 ng/mL), reducing systemic toxicity;
3. High metabolic stability (CLint <4 μL/min/mg) supports once-daily dosing [1,2]

C27H26N4O5S

518.58

518.162

801312-28-7

GSK256066 Trifluoroacetate;1415560-64-3

9827968

Light yellow to yellow solid powder

1.3±0.1 g/cm3

791.7±60.0 °C at 760 mmHg

432.6±32.9 °C

0.0±2.8 mmHg at 25°C

1.654

3.63

144.29

2

7

7

37

922

0

JFHROPTYMMSOLG-UHFFFAOYSA-N

InChI=1S/C27H26N4O5S/c1-16-11-21(37(34,35)20-10-5-7-17(12-20)27(33)31(2)3)14-22-24(16)29-15-23(26(28)32)25(22)30-18-8-6-9-19(13-18)36-4/h5-15H,1-4H3,(H2,28,32)(H,29,30)

6-[[3-[(Dimethylamino)carbonyl]phenyl]sulfonyl]-4-[(3-methoxyphenyl)amino]-8-methyl-3-quinolinecarboxamide

GSK-256066; GSK 256066; GSK256066;

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)