Amlexanox is a therapeutic agent primarily recognized for its anti-inflammatory and anti-allergic properties. It has been effectively used in treating aphthous ulcers, also known as canker sores. Its molecular mechanism provides insights into its potential uses and pharmacological significance, extending its utility beyond this narrow application.
Primary Mechanism of Action: Inhibition of IKK and TBK1
Amlexanox functions primarily by inhibiting two key enzymes: IκB kinase (IKK) and TBK1. These enzymes are crucial components of the NF-κB signaling pathway and the IRF3/IRF7 pathway, respectively, both of which play significant roles in inflammation and immune responses.
Inhibition of the NF-κB Pathway
The NF-κB pathway is a well-established mediator of inflammatory responses. Under normal conditions, NF-κB is sequestered in the cytoplasm in an inactive state, bound to IκB proteins. Upon activation by stimuli such as stress, cytokines, or bacterial and viral infections, IκB kinases (IKKs) phosphorylate IκB proteins, marking them for degradation. This process releases NF-κB, allowing it to translocate to the nucleus where it can activate the transcription of various inflammatory genes.
Amlexanox inhibits IKKs, preventing the phosphorylation and subsequent degradation of IκB proteins. This keeps NF-κB in its inactive form, reducing inflammation.
Inhibition of the IRF3/IRF7 Pathway
Amlexanox also inhibits TBK1, a kinase involved in the IRF3/IRF7 pathway, which is critical for the production of type I interferons important for antiviral responses. TBK1 phosphorylates and activates IRF3 and IRF7 transcription factors, leading to the production of interferons and other inflammatory cytokines.
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By inhibiting TBK1, Amlexanox reduces the activity of this pathway, diminishing the production of pro-inflammatory cytokines. This dual inhibition results in reduced inflammation and immune responses, providing a basis for its use in treating inflammatory conditions.
Additional Potential Benefits
Recent research has suggested that Amlexanox can improve metabolic profiles in obese mice, indicating potential benefits for metabolic disorders such as obesity and type 2 diabetes. The proposed mechanism for these effects involves the inhibition of TBK1 and IKKε, kinases that are upregulated in obesity and insulin resistance. By inhibiting these kinases, Amlexanox enhances insulin sensitivity and promotes weight loss in experimental models.
Amlexanox has also shown promise in oncology. Certain cancers exploit the NF-κB pathway to promote survival, proliferation, and resistance to apoptosis. By inhibiting IKK and blocking NF-κB activation, Amlexanox could potentially hinder cancer cell growth and sensitize tumors to chemotherapy.
Clinical Efficacy and Safety
The clinical efficacy and safety of Amlexanox for conditions beyond aphthous ulcers are still under investigation. Preclinical studies are promising, but further research and clinical trials are necessary to fully understand the therapeutic potential and limitations of this compound.
Amlexanox in Treating Asthma and Allergic Reactions
Amlexanox has been successfully used in the treatment of asthma and atopic conditions and is administered as an oral tablet for treating asthma and allergic reactions, including allergic rhinitis. A study conducted by Imokawa et al. included non-aspirin induced and aspirin-induced asthmatics, where patients were given either amlexanox or a placebo. Administration of the drug showed an effect as a bronchodilator and may prevent “exacerbations,” due to its histamine-inhibiting properties. The drug can improve symptoms of asthma.
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Amlexanox and Metabolic Dysfunction
Amlexanox has shown promise in multiple settings, including diabetes, obesity, and liver disease, and its effects on cytokine signaling, specifically in IL-6 production, are linked to diabetes, obesity, and inflammation. Zhao et al. found that amlexanox stimulates the translocation of the glucose transporter to the cell membrane in Zucker rats. It also appears to modulate inflammatory conditions and have an impact on ameliorating obesity-related metabolic dysfunctions. Consequences of obesity include the development of diabetes and fatty liver disease.
In patients with type 2 diabetes and NAFLD, daily dosing of amlexanox at 12 weeks improved insulin sensitivity, with responders showing a distinct profile in serum and gene expression profiles. In a study, one subject had a comorbidity of asthma/emphysema and experienced better control of their diabetes compared to other subjects.
Furthermore, Takeuchi et al. reported that amlexanox increased the expression of the gene encoding the B3-adrenergic receptor (ADRB3) in Japanese men, leading to lipolysis, which makes this finding notable.
Amlexanox and Neuroinflammation
Neuroinflammation is associated with the onset and progression of several neurodegenerative disorders. Studies have shown that signal transducers and activators of transcription 3 (STAT3) mediate inflammatory responses and are associated with several neurodegenerative diseases. Activation of STAT3 induces neuronal apoptosis through increased TNF-α expression in the diabetic hippocampus.
Both TANK-binding kinase 1 (TBK1) and its homolog, IκB kinase epsilon (IKKε), are activated by phorbol esters (PMA), LPS, and cytokines, and their activity might promote the activation of various signaling pathways and has been linked to the pathology of inflammatory diseases.
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In the nervous system, over-expression of IKKε has been found in the spinal cord, as well as in neurons and glial cells after inflammatory stimulation in mice. Knockout or pharmacological inhibition of IKKε attenuates pain-like behavior in the spared nerve injury model of neuropathic pain, and TBK1 depletion suppresses nociceptive effects in inflammation models. These studies indicate that TBK1/IKKε plays a certain role in the formation or progression of neurological diseases.
Amlexanox Alleviates LPS-Induced Neuroinflammation In Vivo
To investigate the effects of amlexanox on LPS-induced neuroinflammation in vivo, wild-type (WT) mice were given amlexanox (50 mg/kg, i.p.) for 3 days before being given LPS (10 mg/kg, i.p.) or phosphate-buffered saline (PBS). Following amlexanox treatment, the mRNA expression level of IKKε but not TBK1 was significantly reduced in LPS-induced injured brain. LPS-injected WT mice also had higher levels of TNF-α, IL-1β, and IL-6 than vehicle-injected WT mice. Amlexanox treatment significantly attenuated LPS-induced cytokine production, including TNF-α, IL-1β, and IL-6.
The amount of IKKε expression in the brain increased significantly following LPS injection. However, the regulation of TBK1 was shown to be minimal. Following treatment with amlexanox, the mRNA expression level of IKKε was markedly reduced in injured brains induced by LPS.
Furthermore, activated microglia cells are also important contributors to neuroinflammation. The expression of the ionized calcium-binding adaptor molecule (Iba-1) was determined by immunohistochemistry. The number of Iba1-reactive cells increased significantly in LPS-injected animals. Amlexanox treatment considerably reduced the activating impact of LPS on microglia.
Amlexanox Regulates Proinflammatory Enzymes
To investigate the effects of amlexanox in vitro, its cytotoxicity towards BV2 microglial cells was measured. BV2 microglial cells were treated for 24 h with increasing concentrations of amlexanox or vehicle in the presence or absence of LPS, and cell viability was assessed using CCK-8 assays. In both normal and LPS-stimulated conditions, amlexanox did not induce BV2 microglial cell toxicity up to 6 μM.
The mRNA expression and protein levels of IKKε were significantly reduced in LPS-activated microglia after treatment with amlexanox. Next, activated glial cells produce inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which are directly toxic to neurons. To examine the effects of amlexanox on LPS-elicited expression of iNOS and COX-2, BV2 microglial cells were pre-treated with amlexanox for 1 h, and then treated with LPS. Treatment with amlexanox markedly suppressed LPS-induced iNOS and COX-2 mRNA expression levels in both microglia and BMDM and significantly inhibited iNOS protein expression levels in a dose-dependent manner, but not COX-2 in microglia cells.
Amlexanox Suppresses Proinflammatory Mediators
To investigate the potential regulatory effects of amlexanox on proinflammatory factors, BV2 microglial cells were treated with vehicle or amlexanox for 1 h, followed by LPS or PBS treatment for 6 h. TNF-α, CCL2, and CXCL10 mRNA levels were significantly reduced by amlexanox treatment, but there were no significant changes in IL-1β or IL-6 levels in LPS-induced BV2 microglial cells. Similarly, amlexanox significantly reduced proinflammatory cytokines and chemokines in BMDM and HMC3 cells treated with LPS.
Amlexanox Suppresses LPS-Induced AKT and p38 MAPK Phosphorylation
Inhibition of TBK1/IKKε by amlexanox leads to the downregulation of AKT and MAPKs. Treatment with amlexanox significantly inhibited LPS-stimulated phosphorylation of p38 MAPK and AKT activation in a concentration-dependent manner. However, phosphorylation of ERK and JNK was not affected by amlexanox pre-treatment, suggesting that inhibition IKKε by amlexanox differentially alters LPS-stimulated AKT and p38 MAPK signaling in microglia.
Amlexanox Inhibits NF-κB Activity
To further investigate the underlying molecular mechanisms of the anti-inflammatory effects of amlexanox, the levels of NF-κB, the main transcription factor modulating proinflammatory gene expression in nuclei, were examined. Amlexanox significantly suppressed the phosphorylation of both IκBα and NF-κB p65 during LPS-stimulated microglia. Immunostaining showed that LPS induced the nuclear translocation of p65 to the nucleus, and the translocation was prevented by amlexanox treatment. These results demonstrate that amlexanox inhibits NF-κB activity, which may further attenuate proinflammatory mediator expression in LPS-evoked microglial activation.
Amlexanox Improves Atherosclerosis
Amlexanox administration to obese rodents or humans improves energy and glucose metabolism. Studies have assessed the effects of amlexanox on Western diet-induced (WD-induced) atherosclerosis in LDL receptor-knockout (Ldlr-/-) mice, examining its effects on lipid metabolism, inflammation, and vascular dysfunction. Amlexanox systemically ameliorated 3 major pathogenic mechanisms that promote atherogenesis.
Ldlr-/- mice were fed WD for 3 weeks, then orally gavaged with vehicle or amlexanox for 8 weeks with the continuation of WD feeding. En face staining demonstrated that amlexanox substantially reduced the area of aortic lesions, and staining of aortic roots also showed that amlexanox substantially reduced the size of lesions.
Mice gavaged with amlexanox had clear serum, while serum from mice in the vehicle group was milky, indicating a robust reduction in serum lipid content in response to the drug. Measurement of circulating levels of triglycerides and cholesterol demonstrated that 8-week gavage of amlexanox significantly reduced both triglycerides and cholesterol in WD-fed Ldlr-/- mice.
FPLC analysis indicated that amlexanox reduced both VLDL-cholesterol and LDL-cholesterol and slightly increased HDL-cholesterol. VLDL-triglycerides were also substantially reduced by amlexanox. Moreover, amlexanox significantly reduced liver weight, and H&E staining indicated an improvement of hepatic steatosis. Measurement of hepatic lipid content confirmed a dramatic reduction of both triglycerides and cholesterol by amlexanox in WD-fed Ldlr-/- mice. In normal chow diet-fed Ldlr-/- mice, amlexanox showed no effects on serum cholesterol and triglycerides.
Amlexanox also mildly increased the levels of unsaturated fatty acids. Among the saturated fatty acids, amlexanox increased the levels of 17:0, 18:0, 20:0, while reducing 12:0, 14:0, 22:0, 23:0, 24:0, and 26:0. Among the unsaturated fatty acids, amlexanox upregulated the levels of 18:2, 18:3 N3, 18:3 N6, 20:2, 20:5, 22:4, and 22:5 N6, while downregulating the levels of 16:1, 17:1, 18:1, 20:1, 20:3 N3, 20:4, 22:5 N3, 22:6, and 24:1.
Amlexanox and Cholesterol Metabolism
Mice were fed WD for 8 weeks with 4 additional weeks of feeding along with vehicle or amlexanox administration. The 4 weeks of amlexanox treatment significantly reduced circulating levels of cholesterol and triglycerides in WD-fed Ldlr-/- mice. Amlexanox did not affect cholesterol absorption, and the hepatic cholesterol synthesis rate was also examined in response to amlexanox administration. Amlexanox does not affect cholesterol absorption or synthesis.
WD-fed Ldlr-/- mice received vehicle or amlexanox, followed by oral gavage with cold cholesterol mixed with 14C-cholesterol. After 21 hours, radioactivity was measured in serum, feces, and liver lysates. Amlexanox induced transcriptional changes in the livers of WD-fed Ldlr-/- mice. Ontological analysis of genes upregulated by amlexanox showed significant enrichment of pathways that mediate bile acid synthesis and metabolism. A notable example is the higher expression of Cyp7a1, the rate-limiting enzyme of bile acid synthesis. Amlexanox significantly increased the amount of fecal bile acids.
Amlexanox and Inflammation
Staining of plaques with the macrophage marker macrophage antigen-3 (Mac-3) showed a significant attenuation of macrophages in aortic lesions. Amlexanox significantly reduced the numbers of monocytes and eosinophils, while neutrophil, lymphocyte, and basophil numbers were unaffected. Amlexanox attenuates monocytosis, which in turn would lead to decreased numbers of monocytes recruited to the artery wall, contributing to the decrease of atherosclerosis. In contrast, amlexanox had no effect on the number of blood monocytes in mice fed normal chow diet.
Amlexanox and Vascular Dysfunction
Endothelial cells that are dysregulated in atherosclerosis trigger monocyte adhesion and thus promote macrophage infiltration into the aortic vessel. TNF-α increased monocyte-endothelial cell adhesion. Gene expression analysis on HAECs demonstrated that TNF-α and serum from WD-fed Ldlr-/- mice induced the expression of the inflammatory markers Ccl2 and Vcam1 in endothelial cells. These effects were significantly attenuated by the pretreatment of cells with amlexanox.
Chemical Structure and Properties
Amlexanox has a similar chemical structure to sodium cromoglycate (SCG) and is a stable compound as a powder stored at -20 °C with a shelf life of 3 years.
Dosage and Safety
The dose of oral amlexanox at which no side effect is demonstrated is 10 mg/kg/day, approximately 12 mg/day of amlexanox. Oral Solfa 25 mg describes an eruptive skin rash during treatment. There were no serious significant adverse events to 3 times daily oral amlexanox.