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| Targets |
NOS/nitric oxide synthase (Ki = 1.7 μM ~3.9 μM)
L-NIO dihydrochloride targets nitric oxide synthase (NOS) enzymes. NOS catalyzes the conversion of L-arginine to nitric oxide (NO), a key signaling molecule. By inhibiting NOS, L-NIO reduces NO production. It is a non-selective inhibitor, affecting all three isoforms, though some sources describe it as a selective nNOS inhibitor. |
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| ln Vitro |
L-NIO is a strong, non-selective inhibitor of NADPH-dependent nitric oxide synthase (NOS). The Ki values for neurons (nNOS), endothelial cells (eNOS) and inducible (iNOS) are correspondingly 1.7, 3.9, 3.9 μM. 1].
In vitro, L-NIO dihydrochloride inhibits the activity of all three NOS isoforms. Its potency is quantified by its Ki values against each isoform. By reducing NO production, it can be used to study the role of NO in various physiological and pathological processes. |
| ln Vivo |
In the adult rat brain, localized cerebral ischemia was caused by L-NIO (2.0 μmol, 3 days after ischemia) [2].
L-NIO infusion was associated with zero mortality, low surgical complexity and a reproducible infarct, providing advantages over established models of focal ischemia. The mean infarct volume of 8.5±5.3% of the volume of the contralateral striatum resulted in blood-brain barrier dysfunction, neuronal hypoxia and ongoing neurodegeneration. Further characteristics of ischemic stroke were exhibited, including robust microglia/macrophage and astroglial responses lasting at least 35 days post-ischemia, in addition to chronic motor function impairment. Comparison with existing methods: When compared to other models such as the MCAo models, the consistency in regions affected, high success rate, zero mortality, reduced surgical complexity and minimal welfare requirements of the L-NIO model make it ideal for initial high-throughput investigations into preclinical efficacy and proof of principle studies of acute ischemic stroke interventions. Conclusion: We propose that the L-NIO rat model of focal striatal ischemia does not replace the use of other ischemic stroke models. Rather it provides a new, complementary tool for initial preclinical investigations into the treatment of ischemic stroke[2]. In vivo, L-NIO dihydrochloride is used to induce a consistent focal ischemic infarct in rats. It reduces NO production in the central nervous system, which can protect against oxidative damage. It is used in research on neuroinflammation, pain, migraine, and epilepsy. |
| Enzyme Assay |
Nitric oxide synthase (NOS) catalyzes the NADPH- and O2-dependent conversion of L-arginine to nitric oxide (NO) and citrulline; three isoforms, the neuronal (nNOS), endothelial, and inducible, have been identified. Because overproduction of NO is known to contribute to several pathophysiological conditions, NOS inhibitors are of interest as potential therapeutic agents. Inhibitors that are potent, mechanism-based, and relatively selective for the NOS isoform causing pathology are of particular interest. In the present studies we report that vinyl-L-NIO (N5-(1-imino-3-butenyl)-L-ornithine; L-VNIO) binds to and inhibits nNOS in competition with L-arginine (Ki = 100 nM); binding is accompanied by a type I optical difference spectrum consistent with binding near the heme cofactor without interaction as a sixth axial heme ligand. Such binding is fully reversible. However, in the presence of NADPH and O2, L-VNIO irreversibly inactivates nNOS (kinact = 0.078 min-1; KI = 90 nM); inactivation is Ca2+/calmodulin-dependent. The cytochrome c reduction activity of the enzyme is not affected by such treatment, but the L-arginine-independent NADPH oxidase activity of nNOS is lost in parallel with the overall activity. Spectral analyses establish that the nNOS heme cofactor is lost or modified by L-VNIO-mediated mechanism-based inactivation of the enzyme. The inducible isoform of NOS is not inactivated by L-VNIO, and the endothelial isoform requires 20-fold higher concentrations to attain approximately 75% of the rate of inactivation seen with nNOS. Among the NOS inactivating L-arginine derivatives, L-VNIO is the most potent and nNOS-selective reported to date[1].
The inhibitory activity of L-NIO dihydrochloride on NOS is typically measured in a cell-free enzyme assay. Recombinant NOS is incubated with its substrate L-arginine and varying concentrations of the inhibitor. The production of NO or citrulline is measured, and the IC50 or Ki is calculated. |
| Cell Assay |
Cellular assays are performed using cells that express NOS, such as macrophages (for iNOS) or endothelial cells (for eNOS). Cells are stimulated to produce NO, and the production is measured in the presence of L-NIO dihydrochloride. The compound's ability to inhibit cellular NO production is quantified.
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| Animal Protocol |
Animal/Disease Models: Adult male Sprague Dawley rat (250-350 g) [2]
Doses: 0.04-2.0 μmol/(3.0-5.0 μL) per rat Route of Administration: Injection into the striatum 3 days after ischemia Experimental Results: The infarct volume produced by 2.0 μmol L-NIO is Dramatically different from that of sham-operated animals. The current study establishes a novel in vivo rat model of focal striatal ischemia using the vasoconstrictive agent N5-(1-iminoethyl)-L-ornithine (L-NIO). Adult male Sprague Dawley rats received a unilateral intrastriatal infusion of L-NIO in combination with jugular vein occlusion.[2] In vivo studies are conducted in animal models. For example, L-NIO dihydrochloride is used to induce focal ischemic infarcts in rats, making it a tool for studying stroke. It is also used in models of neuroinflammation and pain. The compound is typically administered via injection. |
| ADME/Pharmacokinetics |
L-NIO dihydrochloride has a molecular weight of 246.13 g/mol and a chemical formula of C7H17Cl2N3O2. As a small molecule, it is water-soluble and can be administered parenterally. Its pharmacokinetic properties, such as half-life and tissue distribution, would be determined in preclinical studies.
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| Toxicity/Toxicokinetics |
L-NIO dihydrochloride is generally well-tolerated at research doses. As a NOS inhibitor, its toxicity is related to its on-target effects on NO signaling. At high doses, it can cause significant physiological disturbances, such as hypertension. Its safety profile is evaluated in toxicology studies.
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| References | |
| Additional Infomation |
L-NIO dihydrochloride is a potent and non-selective inhibitor of nitric oxide synthase (NOS). It is a valuable research tool for studying the role of NO in various physiological and pathological processes, including neuroinflammation, pain, and stroke.
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| Molecular Formula |
C7H17CL2N3O2
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|---|---|
| Molecular Weight |
246.134779691696
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| Exact Mass |
245.069
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| CAS # |
159190-44-0
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| Related CAS # |
L-NIO;36889-13-1
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| PubChem CID |
2733507
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| Appearance |
Colorless to off-white solid-liquid Mixture
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
14
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| Complexity |
180
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| Defined Atom Stereocenter Count |
1
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| SMILES |
N[@@H](CCCNC(C)=N)C(O)=O.[H]Cl.[H]Cl
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| InChi Key |
RYCMAAFECCXGHI-ILKKLZGPSA-N
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| InChi Code |
InChI=1S/C7H15N3O2.2ClH/c1-5(8)10-4-2-3-6(9)7(11)12;;/h6H,2-4,9H2,1H3,(H2,8,10)(H,11,12);2*1H/t6-;;/m0../s1
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| Chemical Name |
(2S)-2-amino-5-(1-aminoethylideneamino)pentanoic acid;dihydrochloride
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| Synonyms |
L-NIO DIHYDROCHLORIDE; 159190-44-0; N5-(1-Iminoethyl)-L-ornithineDihydrochloride; N5-(1-Iminoethyl)-L-ornithine dihydrochloride; L-NIO (dihydrochloride); (2S)-2-amino-5-(1-aminoethylideneamino)pentanoic acid;dihydrochloride; L-NIO (hydrochloride); L-NIO2HCl;
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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 (e.g. under nitrogen), 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) |
H2O : ~125 mg/mL (~507.86 mM)
DMSO : ~33.33 mg/mL (~135.42 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (10.16 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 (10.16 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (10.16 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 100 mg/mL (406.29 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 4.0629 mL | 20.3145 mL | 40.6289 mL | |
| 5 mM | 0.8126 mL | 4.0629 mL | 8.1258 mL | |
| 10 mM | 0.4063 mL | 2.0314 mL | 4.0629 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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