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| 25mg |
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GSK256066 Trifluoroacetate (GSK256066; GSK-256066), the trifluoroacetic acid salt form of GSK256066, is a novel selective
inhibitor of PDE4B (phosphodiesterase 4B) with important biological
activity (e.g. inhibiting pulmonary neutrophilia) and thus has the potential to be used for treatment for chronic obstructive pulmonary disease. It inhibits PDE4B with an IC50 of 3.2 pM, and shows >380,000-fold
selectivity for PDE4B over PDE1/2/3/5/6.
| Targets |
Phosphodiesterase 4 (PDE4), specifically PDE4B (apparent IC₅₀ 3.2 pM; steady-state IC₅₀ <0.5 pM), PDE4A, PDE4C, and PDE4D with equal high affinity (pIC₅₀ ≥11.3-11.94).
Selective against other PDEs: >380,000-fold vs. PDE1, PDE2, PDE3, PDE5, PDE6; >2,500-fold vs. PDE7. High-affinity rolipram binding site (HARBS) ratio (HARBS IC₅₀ / PDE4B IC₅₀) >17 (pIC₅₀ 10.27). [1] |
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| ln Vitro |
GSK256066 trifluoroacetate has a very high affinity PDE4 inhibitor that is intended for inhalation [1]. GSK256066 trifluoroacetate inhibits PDE4 with the same affinity Subtype AD and is highly selective for PDE4, with >380,000-fold selectivity relative to PDE1/2/3/5/6 and >2500-fold selectivity relative to PDE7[1]. With an IC50 of 0.01 nM, GSK256066 Trifluacetate prevents human peripheral blood mononuclear cells stimulated by lipopolysaccharide (LPS) from producing tumor necrosis factor α[1].
GSK256066 is an exceptionally high-affinity, slow and tight-binding inhibitor of PDE4B. The overall dissociation constant (Kd) for the final enzyme-inhibitor complex is ≤0.5 pM (pKd ≥12.3). It potently inhibits lipopolysaccharide (LPS)-stimulated tumor necrosis factor α (TNFα) production in human peripheral blood mononuclear cells (PBMCs) with an IC₅₀ of 10 pM (pIC₅₀ 11.01). In human whole blood stimulated with LPS, it inhibits TNFα production with an IC₅₀ of 126 pM (pIC₅₀ 9.90). The compound shows no activity (pIC₅₀ <5) against a panel of protein kinases, the hERG channel, and a broad receptor screen, indicating high selectivity. [1] |
| ln Vivo |
GSK256066 trifluoroacetate (0.3-100 μg/kg; intratracheal) in a rat model of acute lung inflammation generated by lipopolysaccharide (LPS) and ovalbumin (OVA) decreases bronchoalveolar perfusion in a dose-dependent manner. An increase in the washout's (BAL) eosinophil count. [2]. LPS-induced pulmonary neutrophilia is inhibited by GSK256066 trifluoroacetate, and ferrets did not exhibit any episodes of emesis [2].
When administered intratracheally to rats as an aqueous suspension 2 hours before an LPS challenge, GSK256066 dose-dependently inhibited LPS-induced pulmonary neutrophilia with an ED₅₀ of 1.1 μg/kg, achieving maximal inhibition of 72%. When administered as a dry powder formulation blended with lactose, it showed an ED₅₀ of 2.9 μg/kg, with maximal inhibition of 62%. In the same model, it was approximately 7-fold more potent than the corticosteroid fluticasone propionate (aqueous suspension ED₅₀ 9.3 μg/kg). The compound was well-tolerated at doses up to 100 μg/kg with no overt behavioral changes. [1] |
| Enzyme Assay |
PDE activity was measured using a scintillation proximity assay (SPA). Enzyme in assay buffer was preincubated with inhibitor or vehicle for 30 minutes. The reaction was initiated by adding tritiated cAMP (for PDE4, PDE7) or cGMP (for PDE1, PDE2, PDE5, PDE6). After a 1-hour incubation, the reaction was terminated by adding SPA bead suspension. Bound radioactivity was measured by liquid scintillation counting.
For detailed kinetics of PDE4B inhibition by GSK256066, assays were conducted at 25°C. Inhibitor and tritiated cAMP were mixed, and the reaction was started by adding PDE4B enzyme. Samples were taken at intervals over 90 min, mixed with SPA beads, and radioactivity was counted. Progress curves showing time-dependent inhibition were analyzed using an integrated equation for slow, tight-binding inhibition. For selectivity profiling, similar SPA assays were used for other PDE families (PDE1, 2, 3, 5, 6, 7). [1] |
| Cell Assay |
For the human peripheral blood mononuclear cell (PBMC) TNFα assay, PBMCs were isolated from heparinized human blood. Cells were incubated with inhibitor or vehicle and LPS (1 ng/mL final) for 20 hours. Supernatants were collected, and TNFα concentration was measured by an electrochemiluminescence assay.
For the human whole blood TNFα assay, heparinized whole blood was pre-incubated with inhibitor or vehicle for 1 hour and then stimulated with LPS (50 ng/mL final) for 20 hours. Plasma was collected, and TNFα concentration was measured by electrochemiluminescence. [1] |
| Animal Protocol |
Animal/Disease Models: Male brown Norway rat (180-200 g) [2]
Doses: 0.3-100 μg/kg Route of Administration: intratracheal; 30 minutes before and 6 hrs (hrs (hours)) after ovalbumin challenge Experimental Results: Dose-dependent way to inhibit the increase in the number of eosinophils in BAL. For the rat LPS-induced pulmonary neutrophilia model, male CD rats (220-250 g) were used. Animals were anesthetized with isoflurane. GSK256066 or fluticasone propionate (FP) was administered intratracheally either as an aqueous suspension (0.2% Tween 80 in saline) or as a dry powder blended with lactose, 2 hours before LPS challenge. For aqueous dosing, a blunt needle was inserted into the trachea, and 200 μL of suspension was delivered. For dry powder dosing, a known quantity of drug/lactose blend in a three-way tap attached to a needle and syringe was delivered into the trachea by expelling 4 mL of air. Two hours after compound administration, rats were exposed for 15 minutes to an aerosol of LPS (150 μg/mL in nebulizer). Four hours after LPS exposure, animals were euthanized. Lungs were lavaged, and bronchoalveolar lavage fluid was collected. Cells were counted, and differential counts were performed to determine neutrophil influx. [1] |
| ADME/Pharmacokinetics |
In male CD rats, plasma clearance of GSK256066 (1 mg/kg) after intravenous bolus administration was 39 mL/min/kg, volume of distribution was 0.8 L/kg, and half-life was 1.1 hours. Oral bioavailability was low (≤1% when administered in solution). Based on portal vein sample analysis after oral administration, absorption was also low (3.8%). [1]
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| Toxicity/Toxicokinetics |
In human plasma, GSK256066 had a plasma protein binding rate of 98.4% (at a concentration of 1 μg/mL). When administered intratracheally to rats at doses up to 100 μg/kg, the compound was well tolerated, and no significant behavioral effects were observed. [1]
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| References |
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| Additional Infomation |
GSK256066 is a novel quinoline carboxamide derivative designed for inhalation administration to treat respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD). It was developed to improve the therapeutic index, making it superior to oral PDE4 inhibitors, while minimizing systemic exposure and associated side effects such as nausea and vomiting. GSK256066 exerts a potent anti-inflammatory effect by inhibiting PDE4, thereby increasing intracellular cAMP levels in immune cells and inhibiting the production of pro-inflammatory cytokines such as TNFα. As of the time of this publication, the compound is undergoing clinical trials for asthma and COPD. [1]
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| Molecular Formula |
C₂₉H₂₇F₃N₄O₇S
|
|---|---|
| Molecular Weight |
632.61
|
| Exact Mass |
632.155
|
| CAS # |
1415560-64-3
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| Related CAS # |
GSK256066;801312-28-7
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| PubChem CID |
71576691
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
5.949
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
12
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
44
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| Complexity |
1010
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
PTTKEASHQDSBJR-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C27H26N4O5S.C2HF3O2/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;3-2(4,5)1(6)7/h5-15H,1-4H3,(H2,28,32)(H,29,30);(H,6,7)
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| Chemical Name |
3-Quinolinecarboxamide, 6-[[3-[(dimethylamino)carbonyl]phenyl]sulfonyl]-4-[(3-methoxyphenyl)amino]-8-methyl-, 2,2,2-trifluoroacetate (1
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| Synonyms |
GSK256066 GSK-256066 GSK 256066 2,2,2-trifluoroacetic acid
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~25 mg/mL (~39.52 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (3.95 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (3.95 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.5808 mL | 7.9038 mL | 15.8075 mL | |
| 5 mM | 0.3162 mL | 1.5808 mL | 3.1615 mL | |
| 10 mM | 0.1581 mL | 0.7904 mL | 1.5808 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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