NOS/nitric oxide synthase; iNOS

1400W is a human inducible nitric-oxide synthase (iNOS) inhibitor that binds slowly and tightly. Saturation kinetics are evident in the gradual onset of inhibition by 1400W, with a maximal rate constant of 0.028 s-1 and a binding constant of 2.0 μM. NADPH is required as a cofactor for inhibition. For iNOS compared to eNOS, 1400W is at least 5000 times more selective. By comparison, the inhibition of endothelial NOS (eNOS) and human neuronal NOS (Ki values of 2 μM and 50 μM, respectively], is competitive with L-arginine, quickly reversible, and relatively weaker[1]. Without influencing nNOS or eNOS, 1400W treatment inhibits the expression of iNOS. In the cerebral cortex, 1400W also inhibits the production of NO, 3-NT, and MDA and stops the death of neural cells[2].

In rats exposed to LPS-induced iNOS, 1400W potently (ED50=0.3 mg/kg) decreases the delayed vascular injury, but when administered in conjunction with LPS, it does not worsen acute vascular leakage[1]. Every experimental group's NOx levels are reduced by the administration of 1400W. Furthermore, the late post-hypoxia period (48 hours and 5 days) is marked by lipid peroxidation, the proportion of apoptotic cells, and nitrated protein expression[3].

---

Nitric oxide (NO(*)) from inducible NO(*) synthase (iNOS) has been reported to either protect against, or contribute to, hypoxia/re-oxygenation lung injury. The present work aimed to clarify this double role in the hypoxic lung. With this objective, a follow-up study was made in Wistar rats submitted to hypoxia/re-oxygenation (hypoxia for 30 min; re-oxygenation of 0 h, 48 h, and 5 days), with or without prior treatment with the selective iNOS inhibitor 1400W (10 mg/kg). NO(*) levels (NOx), lipid peroxidation, apoptosis, and protein nitration were analysed. This is the first time-course study which investigates the effects of 1400W during hypoxia/re-oxygenation in the rat lung. The results showed that the administration of 1400W lowered NOx levels in all the experimental groups. In addition, lipid peroxidation, the percentage of apoptotic cells, and nitrated protein expression fell in the late post-hypoxia period (48 h and 5 days). Our results reveal that the inhibition of iNOS in the hypoxic lung reduced the damage observed before the treatment with 1400W, suggesting that iNOS-derived NO(*) may exert a negative effect on this organ during hypoxia/re-oxygenation. These findings are notable, since they indicate that any therapeutic strategy aimed at controlling excess generation of NO(*) from iNOS may be useful in alleviating NO(*)-mediated adverse effects in hypoxic lungs[3].

Reverse Phase Chromatography of [14C]1400 W Incubated with iNOS[1]
[14C]1400 W (15 μM) was incubated with iNOS (at a concentration that would convert 2 μM/min of 10 μML-arginine), and the reaction was analyzed by HPLC at 10, 20, and 40 min. Reactions were as described above for NOS except L-arginine was not included. Control reactions were without enzyme or without NADPH. 50-μl aliquots were filtered through Ultrafree MC filters and applied to a Waters Symmetry C18 HPLC column. The column was developed isocratically with 5 mM 1-octanesulfonic acid in 22% acetonitrile at a flow rate of 1 ml/min. 1400W was eluted from the column at 15 min.

---

NO production assay[2]
The nitrate/nitrite concentration was considered an indicator of NO production and was measured as previously described using a commercially available Nitric Oxide Fluorometric Assay Kit according to the manufacturer's instructions. Fluorescence was measured at 360 nm excitation/450 nm emission using the Thermo Scientific Varioskan Flash fluorescence reader. The fluorescence was an indicator of the concentration of sodium nitrite in the solution, and sodium nitrite concentrations were used to draw a standard curve, from which the concentration of nitrite was calculated. Microglia culture medium and cerebral cortex tissues homogenate were used to assess NO production. The values of NO production were expressed in nmol/mg protein.

Cytotoxicity assay[2]
Cell viability was evaluated using an MTT assay as previously described. Cells were seeded into 96 well plates and maintained at 37 °C for 24 h. The cells were exposed to various concentrations of 1400 W (20, 40, 60, 80, and 100 μM). After 24 h exposure, 0.5 mg/ml MTT in DPBS was added to each well and incubated for further 4 h. Then 150 μl of DMSO was added to the wells to dissolve the formazan crystals, and absorbance was measured at 490 nm using the Thermo Scientific Varioskan Flash microplate reader. The cellular viability was determined from the absorbance value and compared with that of the untreated control group.

---

Detection of apoptosis using flow cytometry[2]
Cells were seeded into 96-well plates at a density of 4 × 104 cells/cm2 and maintained at 37 °C for 24 h. Cells were then cultured in complete DMEM/F12 medium supplemented with 500 μM arginine, and placed in a hypoxic humidified incubator (1% O2). After 12 h hypoxia, cells were cultured in normoxic conditions for reoxygenation for 0, 6, or 24 h 1400 W (60 μM) dissolved in PBS was added to cell cultures 1 h before H/R, and control cultures received only vehicle (PBS). After H/R, cells were harvested and washed three times with ice-cold PBS. Cells were resuspended at a concentration of 4 × 105 cells per 500 μl binding buffer, and incubated with Annexin V-FITC and propidium iodide (PI) in the dark for 15 min at room temperature. The samples were analyzed using BD FACSCanto II flow cytometer. Apoptosis ratio was defined as the ratio between Annexin V positive/PI negative cells (right lower quadrant) and total cells.

Endotoxin-induced Vascular Leakage in Rats[1]
The effects of 1400 W on plasma leakage were assessed in rats by determining the leakage of [125I]human serum albumin from plasma into organs essentially as described. 1400 W (0.1-10 mg/kg, subcutaneous) was dissolved in isotonic saline and administered either concurrently with endotoxin or 3 h following LPS administration (E. coli LPS, 3 mg/kg intravenously). Plasma leakage was then assessed 1 or 5 h after delivery of 1400W. The intravascular volumes were subtracted, and the results were expressed as Δμl g−1 tissue.

---

Animals were randomly assigned to one of four experimental groups: vehicle-treated normoxia group, 1400 W-treated normoxia group, vehicle-treated hypoxia group, and 1400 W-treated hypoxia group. The 1400 W-treated groups were pretreated with ip injections of 1400 W (20 mg/kg, optimum dose) at 12 h intervals as previously described. 1400 W was dissolved in sterile distilled water at a concentration of 20 mg/ml. Vehicle-treated groups were pretreated with ip injections of an equal volume of sterile distilled water. Two hours after administration of vehicle or 1400 W, normoxia groups were maintained in a normoxic environment while hypoxia groups were exposed to simulated hypobaric hypoxia (HH) and reoxygenation as previously described. In brief, rats were exposed to simulated HH for 12 h at 8000 m (267 Torr) in an animal decompression chamber with the temperature and humidity maintained at 22 ± 2 °C and 30 ± 5%, and animals were provided with food and water ad libitum. After 12 h of HH, the hypoxia groups were brought down to sea level. Subjects from each experimental group were assessed at 0, 1 or 3 days post-HH with behavioral experiments or by resection of the cerebral cortex for embedding in paraffin and preparing tissue homogenate. Treatment of all 1400 W treated animals was stopped prior to spatial memory retention trial or resection of the cerebral cortex.

---

isotonic saline; 0.1-10 mg/kg; s.c.

Rats

[1]. 1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo. J Biol Chem. 1997 Feb 21;272(8):4959-63.

[2]. 1400W ameliorates acute hypobaric hypoxia/reoxygenation-induced cognitive deficits by suppressing the induction of inducible nitric oxide synthase in rat cerebral cortex microglia. Behav Brain Res. 2017 Feb 15;319:188-199.

[3]. Inducible NOS inhibitor 1400W reduces hypoxia/re-oxygenation injury in rat lung. Redox Rep. 2010;15(4):169-78.

N-(3-(Aminomethyl)benzyl)acetamidine (1400W) was a slow, tight binding inhibitor of human inducible nitric- oxide synthase (iNOS). The slow onset of inhibition by 1400W showed saturation kinetics with a maximal rate constant of 0.028 s-1 and a binding constant of 2.0 microM. Inhibition was dependent on the cofactor NADPH. L-Arginine was a competitive inhibitor of 1400W binding with a Ks value of 3.0 microM. Inhibited enzyme did not recover activity after 2 h. Thus, 1400W was either an irreversible inhibitor or an extremely slowly reversible inhibitor of human iNOS with a Kd value Nitric oxide (NO) is involved in neuronal modifications, and overproduction of NO contributes to memory deficits after acute hypobaric hypoxia-reoxygenation. This study investigated the ability of the iNOS inhibitor 1400W to counteract spatial memory deficits following acute hypobaric hypoxia-reoxygenation, and to affect expression of NOS, NO, 3-NT and MDA production, and apoptosis in rat cerebral cortex. We also used primary rat microglia to investigate the effect of 1400W on expression of NOS, NO, 3-NT and MDA production, and apoptosis. Acute hypobaric hypoxia-reoxygenation impaired spatial memory, and was accompanied by activated microglia, increased iNOS expression, NO, 3-NT and MDA production, and neuronal cell apoptosis in rat cerebral cortex one day post-reoxygenation. 1400W treatment inhibited iNOS expression without affecting nNOS or eNOS. 1400W also reduced NO, 3-NT and MDA production, and prevented neuronal cell apoptosis in cerebral cortex, in addition to reversing spatial memory impairment after acute hypobaric hypoxia-reoxygenation. Hypoxia-reoxygenation activated primary microglia, and increased iNOS and nNOS expression, NO, 3-NT, and MDA production, and apoptosis. Treatment with 1400W inhibited iNOS expression without affecting nNOS, reduced NO, 3-NT and MDA production, and prevented apoptosis in primary microglia. Based on the above findings, we concluded that the highly selective iNOS inhibitor 1400W inhibited iNOS induction in microglial cells, and reduced generation of NO, thereby mitigating oxidative stress and neuronal cell apoptosis in the rat cerebral cortex, and improving the spatial memory dysfunction caused by acute hypobaric hypoxia-reoxygenation.[2]

C10H15N3.2HCL

250.17

249.079

C, 48.01; H, 6.85; Cl, 28.34; N, 16.80

214358-33-5

180001-34-7;214358-33-5 (HCl);

2733515

White to pink solid powder

329ºC at 760 mmHg

152.7ºC

0.000183mmHg at 25°C

4.027

4

2

3

15

177

0

WDJHSQZCZGPGAA-UHFFFAOYSA-N

InChI=1S/C10H15N3.2ClH/c1-8(12)13-7-10-4-2-3-9(5-10)6-11;;/h2-5H,6-7,11H2,1H3,(H2,12,13);2*1H

N'-[[3-(aminomethyl)phenyl]methyl]ethanimidamide;dihydrochloride

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, 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)

Solubility in Formulation 1: ≥ 2 mg/mL (7.99 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 20.0 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 mg/mL (7.99 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 20.0 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.

---

View More

Solubility in Formulation 3: ≥ 2 mg/mL (7.99 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 20.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 100 mg/mL (399.73 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

---

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