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Purity: ≥98%
GW-274150 is a novel, potent, arginine-competitive,highly selective, NADPH-dependent, and long-acting inhibitor of inducible nitric oxide synthase (iNOS) activity in rat and mouse models of renal I/R. GW274150 was identified from a series of acetamide amino acids that have a high selectivity for iNOS vs both eNOS (> 260-fold) and nNOS (> 219-fold) and high bioavailability (> 90%) after oral administration. GW274150 reduced histologic evidence of tubular injury and markedly reduced immunohistochemical evidence of nitrotyrosine and PAR formation, indicating reduced peroxynitrite formation and poly (ADP-ribose) polymerase (PARP) activation, respectively. It may be helpful in treating renal dysfunction and injury associated with I/R of the kidney.
| Targets |
Inducible nitric oxide synthase (iNOS) (Steady-state Kd < 40 nM; IC50 vs human iNOS: 0.69 ± 0.09 µM after 15 min preincubation; Selectivity: >5800-fold vs human eNOS, >114-fold vs human nNOS; In rat tissues: ED50 = 1.15 ± 0.6 µM, selectivity >260-fold vs eNOS and 219-fold vs nNOS)
Endothelial nitric oxide synthase (eNOS) (Ki = 185 ± 32 µM) Neuronal nitric oxide synthase (nNOS) (Ki = 4.57 ± 0.23 µM)[1] |
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| ln Vitro |
J774 cells' intracellular iNOS is inhibited by GW274150 in a time-dependent manner, with an IC50 value of 0.2 μM [1]. In rat tissues, GW274150 exhibits 260- and 219-fold selectivity for iNOS relative to eNOS and nNOS, respectively. In comparison to human eNOS and nNOS, human iNOS exhibits >100- and >80-fold increases, respectively [1].
GW274150 is a potent, time-dependent, and highly selective inhibitor of human inducible nitric oxide synthase (iNOS). It demonstrated an IC50 of 0.69 ± 0.09 µM against recombinant human iNOS after a 15-minute preincubation. The compound is competitive with L-arginine for binding to iNOS, with an apparent Ks of 1.12 ± 0.1 µM. Inhibition was time-dependent and essentially irreversible in the presence of NADPH, with a calculated steady-state Kd of <40 nM. Selectivity over human endothelial NOS (eNOS) and neuronal NOS (nNOS) was >5800-fold and >114-fold, respectively, at steady state. In murine J774 macrophage cells stimulated with IFNγ and LPS to express iNOS, GW274150 inhibited intracellular NO production in a time-dependent manner, reaching a steady-state IC50 of 0.2 ± 0.04 µM over 24 hours.[1] |
| ln Vivo |
One intraperitoneal injection of GW274150, a long-acting (rat half-life of 5 hours) iNOS inhibitor, can prevent LPS-mediated increases in plasma NO2- and NO3-levels 14 hours later (ED50=3 mg/kg) [2]. In a dose-related manner, GW274150 (intraperitoneal injection; 2.5, 5, and 10 mg/kg) lessens the extent of lung injury caused by carrageenan. Edema development and neutrophil infiltration in the pleural space were also markedly and dose-dependently reduced in rats [2]. When taken orally (30 mg/kg, twice daily, for seven days), GW274150 significantly protects neurons; however, in Parkinson's disease (PD), it has a neuroprotective effect that resembles a bell when combined with 6-OHDA. In mouse models, it is ineffective at high doses [3].
In a mouse model of LPS-induced endotoxemia, intraperitoneal (i.p.) administration of GW274150 (30 or 100 mg/kg) at 4 hours post-LPS effectively inhibited the elevation of plasma nitrate/nitrite (NOx) levels. The ED50 for inhibition of plasma NOx at 14 hours (18 hours post-LPS) was 3.2 ± 0.7 mg/kg i.p. Oral administration showed similar efficacy with an ED50 of 3.8 ± 1.5 mg/kg, indicating high oral bioavailability. GW274150 (up to 50 mg/kg i.v.) did not significantly affect blood pressure in normal, chronically instrumented mice, consistent with its high selectivity for iNOS over eNOS. In endotoxin-shocked mice, it increased mean arterial blood pressure selectively. Inhibition of nNOS in the rat cerebellum in vivo was weak, with significant inhibition (47%) only observed at a high dose of 100 mg/kg i.v.[1] |
| Enzyme Assay |
NOS enzyme activity was measured using an oxyhaemoglobin assay. The assay mixture contained HEPES buffer (100 mM, pH 7.4), dithiothreitol (100 µM), MgCl2 (0.8 mM), oxyhaemoglobin (5 µM), L-arginine (30 µM), NADPH (100 µM), FAD (1 µM), FMN (1 µM), calmodulin (100 nM), and tetrahydrobiopterin (BH4, 10 µM). The conversion of oxyhaemoglobin to methaemoglobin by NO was monitored spectrophotometrically. IC50 values were determined at 37°C over specified time periods (e.g., 0-10 min, 15-30 min). Human NOS isoforms were expressed using a baculovirus system in insect cells. For detailed kinetics, purified human iNOS (expressed in E. coli) was used. Progress curves for time-dependent inhibition by GW274150 were analyzed by fitting to an integrated rate equation. Reversibility of inhibition was tested by pre-incubating iNOS with GW274150 and NADPH, followed by extensive dilution into an assay mixture containing excess L-arginine.[1]
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| Cell Assay |
Inhibition of intracellular iNOS was assessed in murine J774 monocytic leukemia cells. Cells were plated and stimulated with murine interferon-γ (5 U/mL) and LPS (1 µg/mL) for 24 hours to induce iNOS expression. After washing, cells were incubated with test compounds and a fluorescent NO indicator (DAF-2DA, 20 µM) in medium containing L-arginine (200 µM), interferon-γ, and LPS. NO production was monitored by measuring fluorescence (excitation 492 nm, emission 515 nm) at various time points over 24 hours. The IC50 values for GW274150 were determined from the time-dependent inhibition curves.[1]
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| Animal Protocol |
Animal/Disease Models: SD-rat [2]
Doses: 2.5, 5 and 10 mg/kg; single dose Route of Administration: intraperitoneal (ip) injection 5 minutes before carrageenan injection Experimental Results: Protection against carrageenan-induced acute inflammation model of lung injury effect. For in vivo efficacy studies, male CD-1 mice were injected intravenously with LPS (E. coli, 3 mg/kg). Four hours later, GW274150 (dissolved in saline) or vehicle was administered intraperitoneally (i.p.) or orally (p.o.) at specified doses (e.g., 3.2 to 100 mg/kg). Plasma was collected at various time points (e.g., 18 hours post-LPS for ED50 determination) via cardiac puncture under anesthesia for measurement of nitrate/nitrite (NOx) levels. For blood pressure studies, conscious mice were chronically cannulated. Endotoxin shock was induced by i.v. bolus of E. coli LPS. GW274150 or vehicle was administered i.v., and mean arterial blood pressure was monitored. For nNOS inhibition in the brain, rats were injected i.v. with GW274150 (20-200 mg/kg) or saline. After 30 minutes, cerebellums were collected, frozen, and analyzed for NOx content.[1] For pharmacokinetic studies, healthy rats and mice were dosed intravenously or orally with GW274150 (1 or 10 mg/kg). Blood samples were collected at various times, and plasma concentrations of the parent compound were determined using HPLC with mass spectrometric detection.[1] |
| ADME/Pharmacokinetics |
In healthy rats, GW274150 exhibited biphasic pharmacokinetic characteristics after intravenous injection of a dose of 10 mg/kg, with a terminal half-life (T1/2) of approximately 6.5 hours, a clearance (CL) of 0.14 L/h/kg, and a steady-state volume of distribution (Vss) of 1.04 L/kg. In mice, the terminal T1/2 was approximately 5.7 hours after intravenous injection of a dose of 1 mg/kg. Oral bioavailability was high in both rats and mice (>90%). Compared with the analog GW273629, GW274150 had a lower maximum plasma concentration (Cmax) and a higher systemic exposure (AUC), suggesting a longer duration of action in vivo. [1]
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| References |
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| Additional Infomation |
GW274150 has been used in trials for the treatment and prevention of asthma, migraine, migraine-related disorders, and arthritis and rheumatoid arthritis. GW274150 is a novel acetamide amino acid derivative that has been identified as a highly selective inhibitor of inducible nitric oxide synthase (iNOS). Its high selectivity for constitutive nitric oxide synthases (eNOS and nNOS) is crucial for avoiding adverse effects on blood pressure regulation and neurological function. The compound is a time-dependent, mechanistic inhibitor that requires NADPH to achieve complete and almost irreversible inhibition. It has shown potential as a therapeutic agent for the treatment of diseases involving pathological iNOS activation, such as inflammatory diseases (e.g., arthritis, asthma, postoperative intestinal obstruction) and septic shock. GW274150 is characterized by its long duration of action in vivo, which is attributed to its pharmacokinetic properties and the irreversibility of its enzyme inhibition. [1]
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| Molecular Formula |
C8H16N2O3S
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|---|---|
| Molecular Weight |
220.28924
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| Exact Mass |
219.104
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| CAS # |
210354-22-6
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| Related CAS # |
GW274150 phosphate;438542-15-5;GW274150 dihydrochloride;438542-17-7
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| PubChem CID |
9797017
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| Appearance |
White to off-white solid powder
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| LogP |
1.198
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
14
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| Complexity |
209
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| Defined Atom Stereocenter Count |
1
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| SMILES |
CC(=NCCSCC[C@@H](C(=O)O)N)N
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| InChi Key |
MOLOJNHYNHBPCW-ZETCQYMHSA-N
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| InChi Code |
InChI=1S/C8H17N3O2S/c1-6(9)11-3-5-14-4-2-7(10)8(12)13/h7H,2-5,10H2,1H3,(H2,9,11)(H,12,13)/t7-/m0/s1
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| Chemical Name |
L-Homocysteine, S-(2-((1-iminoethyl)amino)ethyl)-
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| Synonyms |
GW-274150; GW 274150; GW274150.
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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 |
| 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 : ~100 mg/mL (~456.00 mM)
H2O : ≥ 62 mg/mL (~282.72 mM) |
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 4.5395 mL | 22.6974 mL | 45.3947 mL | |
| 5 mM | 0.9079 mL | 4.5395 mL | 9.0789 mL | |
| 10 mM | 0.4539 mL | 2.2697 mL | 4.5395 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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