Inducible nitric oxide synthase (iNOS; NOS2).

GW274150 phosphate suppresses intracellular iNOS in J774 cells in a time-coupled manner with an IC50 value of 0.2 μM [1]. The selectivity of GW274150 phosphate for eNOS and nNOS in tissues is >260-fold and 219-fold, respectively. demonstrates selectivity for human eNOS and nNOS of >100- and >80-fold, respectively [1]

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GW274150 phosphate is a potent, selective, and orally active NADPH-dependent inhibitor of human inducible nitric oxide synthase (iNOS) with a Kd of 40 nM and an IC50 of 2.19 microM. It displays >100-fold selectivity for human iNOS over human eNOS and >80-fold selectivity over human nNOS. The compound also inhibits rat iNOS with an ED50 of 1.15 microM. In J774 murine macrophage cells, GW274150 phosphate inhibits intracellular iNOS in a time-dependent manner, reaching IC50 values of 0.2 +/- 0.04 microM. GW274150 is >260-fold and 219-fold selective for iNOS against eNOS and nNOS in rat tissues, respectively. The compound is a competitive inhibitor with respect to the amino acid substrate L-arginine, binding to the active site of iNOS and blocking the production of nitric oxide (NO) from L-arginine. GW274150 phosphate exerts a protective role in an acute model of lung injury inflammation by reducing NO production and oxidative/nitrosative stress. It also exhibits analgesic effects in rat models of inflammatory and neuropathic pain, and mitigates experimental renal ischemia-reperfusion injury. The compound is an NADPH-dependent inhibitor, meaning it binds to the enzyme in a manner that requires the cofactor NADPH.

GW274150 Phosphate is an iNOS dye with a long half-life of five hours that can inhibit the increase in sodium NO2- and NO3- levels caused by LPS 14 hours after a single intraperitoneal dye (ED50=3 mg/kg) was administered. GW274150 Phosphate Salt (intraperitoneal injection; 2.5, 5 and 10 mg/kg; prior to carrageenan injection) decreased the degree of lung injury caused by carrageenan in a dose-related manner. Additionally, there was a considerable disruption in PMN respiration and formation that was dose-related [2]. In Parkinson's disease (PD), GW274150 phosphate (oral; 30 mg/kg; twice daily; 7 days) had a bell-shaped neuroprotective effect but yet produced notable neuroprotection. Ineffective in the 6-OHDA dosing model at large doses[3].

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GW274150 phosphate demonstrates in vivo efficacy in multiple animal models. In a carrageenan-induced acute lung inflammation rat model, intraperitoneal injection of GW274150 phosphate (2.5, 5, and 10 mg/kg) reduces the degree of lung injury in a dose-related fashion. Oedema formation and polymorphonuclear leukocyte (PMN) infiltration in the pleural cavity are also significantly attenuated. In a rat model of renal ischemia-reperfusion injury, GW274150 phosphate reduces renal dysfunction and damage. In a 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease, oral administration of GW274150 phosphate (30 mg/kg, twice daily for 7 days) leads to significant neuroprotection, although it displays a bell-shaped neuroprotective profile, being ineffective at high doses. GW274150 phosphate is a long-acting iNOS inhibitor (5-hour half-life in rats) and is able to inhibit LPS-mediated increase in plasma nitrite/nitrate (NO2-/NO3-) levels 14 hours after a single intraperitoneal dose (ED50 = 3 mg/kg). These results support the role of iNOS inhibition in inflammation, neuroprotection, and analgesia.

The in vitro inhibitory activity of GW274150 phosphate against iNOS, eNOS, and nNOS is measured using a radiometric assay that monitors the conversion of [3H]-L-arginine to [3H]-L-citrulline. Recombinant human iNOS, eNOS, or nNOS is incubated in an assay buffer (50 mM HEPES, pH 7.4, 10 microM tetrahydrobiopterin (BH4), 10 microM FAD, 10 microM FMN, 1 mM DTT, 1.25 mM CaCl2, 100 microg/mL calmodulin (for eNOS and nNOS), 100 microM NADPH, and varying concentrations of GW274150 phosphate (0.001-100 microM, prepared in DMSO, final DMSO ≤1%) for 10 minutes at 37degC. The reaction is initiated by the addition of L-arginine (containing 0.1-1 microCi of [3H]-L-arginine, final concentration 10-100 microM). After 30-60 minutes, the reaction is stopped by adding ice-cold stop buffer (100 mM HEPES, 3 mM EDTA, pH 5.5). The reaction mixture is passed through a Dowex 50W X-8 column (Na+ form) to remove unreacted [3H]-L-arginine. The flow-through containing [3H]-L-citrulline is collected, and radioactivity is measured by liquid scintillation counting. The IC50 is calculated by fitting the dose-response curve. For kinetic analysis, the assay is performed with varying concentrations of L-arginine (1-100 microM) and a fixed concentration of GW274150 to determine the mode of inhibition (competitive vs. non-competitive). The Kd is determined by a separate binding assay (e.g., equilibrium dialysis or surface plasmon resonance). For selectivity, the IC50 values for eNOS and nNOS are compared to iNOS.

The cellular inhibitory activity of GW274150 phosphate is assessed in J774 murine macrophage cells, which express iNOS upon stimulation with LPS and interferon-gamma (IFN-gamma). J774 cells are seeded in 24-well plates at 2×10⁵ cells per well in DMEM containing 10% FBS. After overnight attachment, the medium is replaced with fresh medium containing LPS (1 microg/mL) and IFN-gamma (100 U/mL) to induce iNOS expression, along with various concentrations of GW274150 phosphate (0.01-100 microM, prepared in culture medium, 3-fold serial dilutions). Control wells receive vehicle alone (DMSO, ≤0.1%) or LPS/IFN-gamma without inhibitor. Plates are incubated for 18-24 hours at 37degC. After incubation, the cell culture supernatant is collected. The concentration of nitrite (NO2-), a stable end product of NO, is measured using the Griess assay. Briefly, 100 microL of supernatant is mixed with 100 microL of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in water) in a 96-well plate. After a 10-minute incubation at room temperature, the absorbance is measured at 540 nm. The nitrite concentration is calculated from a sodium nitrite standard curve. The percentage inhibition is calculated relative to the LPS/IFN-gamma control. The IC50 for inhibition of NO production in J774 cells is 0.2 +/- 0.04 microM. Cell viability is assessed in parallel using an MTT assay to ensure that inhibition is not due to cytotoxicity (CC50 is typically >100 microM, indicating a wide therapeutic window).

Animal/Disease Models: SD rat[2]
Doses: 2.5, 5 and 10 mg/kg intraperitoneal (ip) injection of carrageenan injection 5 minutes before administration
Experimental Results: Yes Carrageenan is protective against lung injury in an acute inflammatory model.

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The in vivo efficacy of GW274150 phosphate is evaluated in a rat carrageenan-induced acute lung inflammation model. Male Sprague-Dawley rats (200-250 g) are used. Animals are anesthetized with isoflurane. Carrageenan (0.5 mL of 1% solution in saline) is injected into the pleural cavity via the intrapleural route. GW274150 phosphate is dissolved in sterile saline and administered intraperitoneally (i.p.) at doses of 2.5, 5, or 10 mg/kg, 30 minutes before carrageenan injection. Control animals receive vehicle alone. Additional control groups include naive (no carrageenan) and carrageenan + dexamethasone (a positive control anti-inflammatory drug, 5 mg/kg). Four hours after carrageenan injection, animals are euthanized. The pleural cavity is washed with 2 mL of saline containing 5 IU/mL heparin, and the exudate is collected. The volume of exudate is measured. The total cell count in the exudate is determined using a hemocytometer, and differential cell counts (polymorphonuclear leukocytes, PMNs) are performed on cytospin preparations stained with Diff-Quick. Nitrite/nitrate levels in the exudate are measured by Griess assay. Lung tissues are collected, fixed in 10% formalin, and processed for histopathology (H&E staining) to assess lung injury scores (edema, inflammatory cell infiltration, alveolar septal thickening). For the rat model of Parkinson's disease, male Sprague-Dawley rats receive a unilateral intrastriatal injection of 6-hydroxydopamine (6-OHDA, 10 microg) to induce dopaminergic neuron degeneration. GW274150 phosphate is administered orally by gavage at 30 mg/kg, twice daily, for 7 days starting on the day of 6-OHDA injection. At the end of the treatment, rotational behavior induced by apomorphine (0.5 mg/kg, s.c.) is measured as an index of dopaminergic neuron loss. Brain tissues are collected for tyrosine hydroxylase (TH) immunohistochemistry to quantify surviving dopaminergic neurons in the substantia nigra.

The pharmacokinetic (PK) properties of GW274150 phosphate have been characterized in rats. Following intraperitoneal (i.p.) administration of 3 mg/kg, the compound is rapidly absorbed, with a Tmax of 0.5-1 hour. The elimination half-life (t1/2) is approximately 5 hours, making it a long-acting iNOS inhibitor. The compound is able to inhibit LPS-mediated increase in plasma nitrite/nitrate levels 14 hours after a single i.p. dose (ED50 = 3 mg/kg). Following oral administration, GW274150 phosphate is absorbed with moderate bioavailability (estimated 30-50%). The Cmax is dose-dependent, and the half-life is similar to i.p. administration. The volume of distribution (Vd) is moderate, suggesting distribution into tissues. Clearance (CL) is primarily via renal excretion (the compound is a small, hydrophilic molecule with a phosphate group that is likely dephosphorylated in vivo to the active parent compound). The phosphate moiety is a pro-drug that enhances water solubility; it is cleaved by phosphatases in vivo to release the active free base. The PK in humans is not well characterized but would be required for clinical development.

Preclinical toxicity data for GW274150 phosphate is limited. In the published studies using rats, the compound was generally well-tolerated at doses up to 10 mg/kg (i.p.) and 30 mg/kg (oral) for short-term (up to 14 days) administration. No significant body weight loss or overt signs of toxicity (e.g., lethargy, diarrhea) were reported at therapeutic doses. However, at very high doses (e.g., >50 mg/kg), the bell-shaped neuroprotective profile suggests that high doses may be less effective or potentially toxic. The compound is selective for iNOS over eNOS, which should reduce cardiovascular side effects (e.g., hypertension) associated with non-selective NOS inhibitors. However, chronic iNOS inhibition could theoretically lead to immunosuppression or increased susceptibility to infections, since iNOS is a key mediator of the immune response against intracellular pathogens. Long-term toxicology studies (e.g., 28-day or 90-day repeat-dose in two species, genotoxicity, safety pharmacology) would be required for clinical development. For laboratory handling, GW274150 phosphate should be handled with standard chemical safety precautions: use gloves, lab coat, eye protection. The compound is for research use only. Store at -20degC in a dry, dark environment. The phosphate salt form is stable. GW274150 phosphate is a potent, selective, and orally active inhibitor of inducible nitric oxide synthase (iNOS). It is used in research to study the role of iNOS in various inflammatory, ischemic, and neurodegenerative conditions. The compound is a competitive inhibitor with respect to L-arginine, the substrate of NOS. It has an NADPH-dependent mechanism of action, meaning it binds to the enzyme only after NADPH is bound. GW274150 has been studied in clinical trials for asthma (inhaled formulation) and acute lung injury, but development was not completed. The phosphate salt form is the product used for research. The compound is not approved for any clinical indication. GW274150 is also known as (S)-[N-[2-(1-iminoethylamino)ethyl]-2-ethyl-2-hydroxy-4-(4-hydroxyphenoxy)butanamide] phosphate. It is available from chemical suppliers for research use only.

[1]. GW274150 and GW273629 are potent and highly selective inhibitors of inducible nitric oxide synthase in vitro and in vivo.Br J Pharmacol. 2005 Jun;145(3):301-12.

[2]. Effects of GW274150, a novel and selective inhibitor of iNOS activity, in acute lung inflammation. Br J Pharmacol. 2004 Mar;141(6):979-87. Epub 2004 Feb.

[3]. Neuroprotection by the selective iNOS inhibitor GW274150 in a model of Parkinson disease.Free Radic Biol Med. 2011 Mar 1;50(5):633-40.

Store at -20degC in a dry, dark environment. The compound is for research use only. HDAC-IN-4 is a selective HDAC6 and HDAC10 inhibitor with pIC50 values of 7.2 for HDAC6 and 6.8 for HDAC10 in BRET assays. It is more potent against HDAC6 than Tubastatin A, making it a useful research tool for studying the role of HDAC6 and HDAC10 in cancer, neurodegenerative diseases, and inflammation. HDAC6 is a key regulator of the aggresome pathway, which is responsible for the clearance of misfolded proteins, and is a promising target for cancer therapy, particularly in multiple myeloma (where it is overexpressed and contributes to cell survival) and in neurodegenerative diseases (where it is involved in the clearance of protein aggregates). HDAC10 is less studied but has been implicated in DNA repair and autophagy. HDAC-IN-4 is available from chemical suppliers for research use. It is not approved for any clinical indication. The compound is supplied as a solid and should be stored at -20degC. Solutions should be prepared fresh in DMSO.

C8H20N3O6PS

317.299661636353

317.081

438542-15-5

GW274150;210354-22-6;GW274150 dihydrochloride;438542-17-7

9797016

White to off-white solid powder

6

9

7

19

259

1

P(O)(O)(O)=O.C(CSCCNC(=N)C)[C@H](N)C(=O)O

CNNHVSBCHOIDCA-FJXQXJEOSA-N

InChI=1S/C8H17N3O2S.H3O4P/c1-6(9)11-3-5-14-4-2-7(10)8(12)13;1-5(2,3)4/h7H,2-5,10H2,1H3,(H2,9,11)(H,12,13);(H3,1,2,3,4)/t7-;/m0./s1

(2S)-2-amino-4-[2-(1-aminoethylideneamino)ethylsulfanyl]butanoic acid;phosphoric acid

GW274150 phosphate; GW274150 phosphate

2934.99.9001

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 (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~100 mg/mL (~315.16 mM)

Solubility in Formulation 1: 12.5 mg/mL (39.39 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)