IKKε (IC50 = 1-2 μM); TBK1 (IC50 = 1-2 μM)

Amlexanox has a dose-response relationship that inhibits IKK- and TBK1 activity by phosphorylating MBP, with an IC50 of roughly 1-2 μM. At roughly the same concentrations, amlexanox also inhibits the activity of TBK1, but it has no effect on IKK-α or IKK-β, and at these concentrations, it does not inhibit any other kinases from a large panel representing the majority of kinase families. Amlexanox competes with IKK-ɛ - or TBK1 for its substrate ATP, showing that it interacts with the enzymes in the ATP-binding site. Amlexanox increases the phosphorylation of TBK1 at Ser172 in 3T3-L1 adipocytes and inhibits the phosphorylation of interferon responsive factor-3 (IRF3), a presumed substrate of IKK-ɛ - and TBK1[1], when it is stimulated by polyinosinic:polycytidylic acid (poly I:C).

Amlexanox therapy reduces steatosis, improves insulin sensitivity, and causes weight loss in obese mice through an increase in thermogenesis[1].

The in vitro kinase assay is carried out by incubating purified kinase (IKK or TBK1) for 30 minutes at 30°C in the presence of 0.5 Ci µCi γ-[32P]--ATP and 1 g MBP per sample as a substrate in kinase buffer that contains 25 mM Tris (pH 7.5), 10 mM MgCl2, 1 mM DTT, and 10 µM ATP. When 4x sodium dodecyl sulfate (SDS) sample buffer is added, the kinase reaction is stopped by boiling for 5 minutes at 95°C. The Typhoon 9410 phosphorimager is used to conduct autoradiography analysis after supernatants have been resolved using SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose.

The manufacturer's instructions are followed when using a Cell Counting Kit-8 to look at cell proliferation. BMMs are seeded in 96-well plates at a density of 5×103 cells per well. After 24 hours, cells are given treatments with varying doses of AmLexanox (0, 1.5, 3, 6, 12, 25 μM) every 2 days while being exposed to M-CSF (30 ng/mL) for a total of 7 days. The culture medium is changed to one containing 10% CCK-8 after 1, 3, 5, and 7 days, and cells are then incubated at 37°C for an additional 2 hours. On an ELX800 absorbance microplate reader, the absorbance is then measured at a wavelength of 450 nm.

In contrast to ND C57BL/6 controls, wildtype male C57BL/6 mice are fed a HFD starting at eight weeks of age for 12–24 weeks that contains 45% of their calories from fat. ω-3 fatty acids are included in the diets fed to C57BL/6 mice. Mice that have been on the HFD for 16 weeks receive a three-week course of rosiglitazone treatment by ingesting the medication. The average daily dose of rosiglitazone consumed by each mouse is 3.5 mg/kg. Every day oral gavage is used to administer AmLexanox. AmLexanox (25 mg or 100 mg per kg) administration for the prevention groups starts at eight weeks of age, concurrent with HFD feeding. After 12 weeks of HFD, the treatment groups start receiving 25 mg per kg of amLexanox at 20 weeks old. After eight weeks of amLexanox treatment, mice in the treatment group are switched from receiving amLexanox gavage to receiving a vehicle control to test the effects of amLexanox withdrawal. At ten weeks of age, control and ob/ob mice receive a standard chow diet along with 100 mg per kg of amLexanox or a vehicle control. The animals are kept in a facility that is specifically pathogen-free, has a 12-hour light/12-hour dark cycle, and they have unrestricted access to food and water.

Absorption, Distribution and Excretion
No significant absorption directly through the active ulcer. Most of the systemic absorption is via the gastrointestinal tract.

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Metabolism / Metabolites
Metabolized to hydroxylated and conjugated metabolites.

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Biological Half-Life
Elimination half-life is 3.5 ± 1.1 hours.

[1].Nat Med . 2013 Mar;19(3):313-21.

Amlexanox is a pyridochromene-derived monocarboxylic acid having an amino substituent at the 2-position, an oxo substituent at the 5-position and an isopropyl substituent at the 7-position. It has a role as an anti-allergic agent, an anti-ulcer drug and a non-steroidal anti-inflammatory drug. It is a pyridochromene and a monocarboxylic acid.
Amlexanox is an antiallergic drug, clinically effective for atopic diseases, especially allergic asthma and rhinitis. Amlexanox as a topical paste is a well tolerated treatment of recurrent aphthous ulcers. Recurrent aphthous ulcer (RAU) is the most prevalent oral mucosal disease in humans, estimated to affect between 5% and 50% of the general population.
The physiologic effect of amlexanox is by means of Decreased Histamine Release.
Amlexanox is an anti-aphthous ulcer drug. Amlexanox inhibits the synthesis and release of inflammatory mediators, including leukotrienes and histamine, from mast cells, neutrophils, and mononuclear cells. Amlexanox also acts as a leukotriene D4 antagonist and a phosphodiesterase inhibitor. Amlexanox decreases the time ulcers take to heal as well as the pain associated with the ulcers.

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Drug Indication
Used as a paste in the mouth to treat aphthous ulcers (canker sores).
FDA Label

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Mechanism of Action
As a benzopyrano-bipyridine carboxylic acid derivative, amlexanox has anti-inflammatory and antiallergic properties. It inhibits chemical mediatory release of the slow-reacting substance of anaphylaxis (SRS-A) and may have antagonistic effects on interleukin-3. When cells are under stress, they release an inactive form of human fibroblast growth factor 1 (FGF-1), a potent mitogen (entity that causes mitosis). Amlexanox binds to FGF1, increasing its conformational stability, sterically blocking Cu(2+) induced oxidation which normally leads to activation of FGF-1.

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Pharmacodynamics
Amlexanox is a mucoadhesive oral paste which has been clinically proven to abort the onset, accelerate healing and resolve the pain of aphthous ulcers (canker sores). It decreases the time ulcers take to heal. Because amlexanox decreases the healing time, it also decreases the pain you feel. Recent studies have also shown that the majority of ulcers can be prevented by application of the paste during the prodromal (pre-ulcerative) phase of the disease. Recurrent Aphthous Ulcers (RAU) also known as Recurrent Aphthous Stomatitis (RAS) is recognized as the most common oral mucosal disease known to man. Estimates suggest that 20% - 25% of the general population suffer at least one incidence of aphthous ulcers each year. Amlexanox is also being investigated for its anti-allergenic and anti-inflammatory properties.

C16H14N2O4

298.2934

298.095

C, 64.42; H, 4.73; N, 9.39; O, 21.45

68302-57-8

68302-57-8

2161

Solid powder

1.4±0.1 g/cm3

570.0±50.0 °C at 760 mmHg

>3000C

298.5±30.1 °C

0.0±1.6 mmHg at 25°C

1.669

3.74

2

6

2

22

467

0

O1C2C(=C([H])C(C(=O)O[H])=C(N([H])[H])N=2)C(C2=C1C([H])=C([H])C(=C2[H])C([H])(C([H])([H])[H])C([H])([H])[H])=O

SGRYPYWGNKJSDL-UHFFFAOYSA-N

InChI=1S/C16H14N2O4/c1-7(2)8-3-4-12-9(5-8)13(19)10-6-11(16(20)21)14(17)18-15(10)22-12/h3-7H,1-2H3,(H2,17,18)(H,20,21)

2-amino-5-oxo-7-propan-2-ylchromeno[2,3-b]pyridine-3-carboxylic acid

CHX 3673 CHX-3673 CHX3673AA-673 AA673 AA 673 Amoxanox, Aphthasol.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO: 59~100 mg/mL (197.8~335.2 mM)
Ethanol: ~2 mg/mL(~6.7 mM)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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