Amlexanox

AmLexanox is a specific inhibitor of IKKε and TBK1, and inhibits the IKKε and TBK1 activity determined by MBP phosphorylation with an IC50 of approximately 1-2 μM.

In Vitro:AmLexanox increases phosphorylation of TBK1 on Ser172 in 3T3-L1 adipocytes, and blocks polyinosinic:polycytidylic acid (poly I:C)-stimulated phosphorylation of interferon responsive factor-3 (IRF3), a presumed substrate of IKKε and TBK1[1]. AmLexanox potently inhibits the release of histamine and leukotrienes from mast cells, basophils and neutrophils in in vitro settings, possibly through increasing intracellular cyclic AMP content in inflammatory cells, a mem-brane-stabilising effect or inhibition of calcium influx[2]. In primary bone marrow derived macrophages (BMMs), amLexanox inhibits osteoclast formation and bone resorption. At the molecular level, amLexanox suppresses RANKL-induced activation of nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPKs), c-Fos and NFATc1. AmLexanox decreases the expression of osteoclast-specific genes, including TRAP, MMP9, Cathepsin K and NFATc1[3].

In Vivo:AmLexanox (100 mg/kg, p.o.) prevents and reverses diet-induced or genetic obesity, and produces reversible weight loss in obese mice. AmLexanox also causes a significant decrease in adipose tissue mass in these mice, and an increase in circulating adiponectin. AmLexanox (25 mg/kg) significantly improves insulin sensitivity in mice with established DIO,and after four weeks of treatment, amLexanox produces marked improvements in glucose[1]. AmLexanox before the first application of the paste and at each has been shown to suppress both immediate and evaluation thereafter. A categorical scale is also delayed-type hypersensitivity reactions[2]. AmLexanox (20 mg/kg) enhances osteoblast differentiation of BMSCs. In ovariectomized (OVX) mouse model, amLexanox prevents OVX-induced bone loss by suppressing osteoclast activity[3].

References:
[1]. Reilly SM, et al. An inhibitor of the protein kinases TBK1 and IKK-e improves obesity-related metabolic dysfunctions in mice. Nat Med. 2013 Mar;19(3):313-21. [2]. Bell, J. AmLexanox for the treatment of recurrent aphthous ulcers. Clin Drug Investig, 2005. 25(9): p. 555-66. [3]. Zhang Y, et al. AmLexanox Suppresses Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss. Sci Rep. 2015 Sep 4;5:13575.

Amlexanox attenuates experimental autoimmune encephalomyelitis by inhibiting dendritic cell maturation and reprogramming effector and regulatory T cell responses.[Pubmed:30823934]

J Neuroinflammation. 2019 Mar 1;16(1):52.

BACKGROUND: Amlexanox (ALX), a TBK1 inhibitor, can modulate immune responses and has anti-inflammatory properties. To investigate its role in regulating the progression of experimental autoimmune encephalomyelitis (EAE), we studied the effect of ALX on the maturation of dendritic cells (DCs) and the responses of effector and regulatory T cells (Tregs). METHODS: In vitro, bone marrow-derived DCs (BMDCs) were cultured and treated with ALX. Their proliferation, maturation, and their stimulatory function to induce T cells responses were detected. In vivo, the development of EAE from different groups was recorded. At the peak stage of disease, HE, LFB, and electronic microscope (EM) were used to evaluate inflammation and demyelination. Maturation of splenic DC and Th1/Th17/Treg response in the CNS and peripheral were also detected. To further explore the mechanism underlying the action of ALX in DC maturation, the activation of TBK1, IRF3, and AKT was analyzed. RESULTS: Our data indicated that ALX significantly inhibited the proliferation and maturation of BMDCs, characterized by the reduced MHCII, a co-stimulatory molecule, IL12, and IL-23 expression, along with morphological alterations. Co-culture of ALX-treated BMDCs inhibited allogeneic T cell proliferation and MOG-specific T cell response. In EAE mice, ALX significantly attenuated the EAE development by decreasing inflammatory infiltration and demyelination in the spinal cords, accompanied by reduced frequency of splenic pathogenic Th1 and Th17 cells and increased Tregs. Moreover, ALX treatment decreased Th1 and Th17 cytokines, but increased Treg cytokines in the CNS and spleen. Notably, ALX treatment reduced the frequency and expression of CD80 and CD86 on splenic DCs and lowered IL-12 and IL-23 secretion, further supporting an impaired maturation of splenic DCs. In addition, ALX potently reduced the phosphorylation of IRF3 and AKT in BMDC and splenic DCs, both of which are substrates of TBK1 and associated with DC maturation. CONCLUSIONS: ALX, a TBK1 inhibitor, mitigated EAE development by inhibiting DC maturation and subsequent pathogenic Th1 and Th17 responses while increasing Treg responses through attenuating the TBK1/AKT and TBK1/IRF3 signaling.

Pharmacological inhibition of the IKKepsilon/TBK-1 axis potentiates the anti-tumour and anti-metastatic effects of Docetaxel in mouse models of breast cancer.[Pubmed:30790681]

Cancer Lett. 2019 May 28;450:76-87.

IkappaB kinase subunit epsilon (IKKepsilon), a key component of NFkappaB and interferon signalling, has been identified as a breast cancer oncogene. Here we report that the IKKepsilon/TBK1 axis plays a role in the initiation and progression of breast cancer osteolytic metastasis. Cancer-specific knockdown of IKKepsilon in the human MDA-MB-231-BT cells and treatment with the verified IKKepsilon/TBK1 inhibitor Amlexanox reduced skeletal tumour growth and osteolysis in mice. In addition, combined administration of Amlexanox with Docetaxel reduced mammary tumour growth of syngeneic 4T1 cells, inhibited metastases and improved survival in mice after removal of the primary tumour. Functional and mechanistic studies in breast cancer cells, osteoclasts and osteoblasts revealed that IKKepsilon inhibition reduces the ability of breast cancer cells to grow, move and enhance osteoclastogenesis by engaging both IRF and NFkappaB signalling pathways. Thus, therapeutic targeting of the IKKepsilon/TBK1 axis may be of value in the treatment of advanced triple negative breast cancer.

Amlexanox and UPF1 Modulate Wnt Signaling and Apoptosis in HCT-116 Colorectal Cancer Cells.[Pubmed:30719122]

J Cancer. 2019 Jan 1;10(2):287-292.

Deregulated Wnt signaling initiates most cases of colorectal cancer (CRC). Butyrate, a product of dietary fiber, hyperactivates Wnt signaling, resulting in induction of CRC cell apoptosis, which may in part explain the protective action of fiber. Nonsense mediated decay (NMD) of mRNAs containing premature stop codons (PTCs) affects tumorigenesis and upregulates Wnt signaling in human embryonic stem cells. However, it is unknown how NMD affects Wnt activity in CRC cells that exhibit constitutively activated Wnt signaling. We hypothesize that expression of genes that contain PTCs modulates Wnt signaling and response to butyrate in CRC cells. Amlexanox is a clinically utilized anti-allergic and anti-inflammatory drug that inhibits NMD and promotes PTC read-through. Therefore, Amlexanox is a relevant agent for assessing the role of NMD and PTC read-through in the response of CRC cells to butyrate. To test our hypothesis, we treated HCT-116 CRC cells with Amlexanox and determined effects on Wnt signaling levels, apoptosis, and response to butyrate. Amlexanox enhanced basal Wnt signaling levels; however, it repressed butyrate-induced Wnt signaling hyperactivation and suppressed apoptosis. To evaluate the contribution of NMD and altered expression of PTC-containing genes to these effects, we upregulated NMD by overexpression of up-frameshift protein 1 (UPF1), and observed effects opposite to these of Amlexanox (i.e., Wnt signaling hyperactivation by butyrate was enhanced and levels of apoptosis were increased). Our results support the possibility that altered expression of PTC-containing genes affects butyrate sensitivity of CRC cells.

A Randomized, Double-Blind, Placebo-Controlled Trial on Clinical Efficacy of Topical Agents in Reducing Pain and Frequency of Recurrent Aphthous Ulcers.[Pubmed:30369980]

Open Dent J. 2018 Sep 28;12:700-713.

Introduction: Recurrent Aphthous Ulcers (RAU) is one of the most common oral ulcerative Disease of the oral mucosa with high recurrence rate. Standard topical treatment options provide symptomatic relief with few have been found to be effective in treating or relieving the symptoms. Aim: The study aimed to evaluate the clinical efficacy of various topical agents in order to find the better treatment modality so as to decrease the number, size, exudate level and discomfort associated with pain with RAU. Materials and methods: The patients diagnosed with minor recurrent aphthous ulcers fulfilling the inclusion and exclusion criteria were enrolled. All the baseline parameters were measured by the principal investigator. The treatment modality was assigned by generating a randomization list by computer software, double-blinded in consecutively numbered sealed envelopes. The topical treatment modalities that were included: 5% Amlexanox, 0.1% Triamcinolone Acetonide, 20% Benzocaine gel, 100 mg Doxycycline hyclatemixed with denture adhesive and normal saline (20:2:1); The study was placebo controlled in which placebo gel 10 gm was used. The size, no of ulcers, pain, erythema and exudate level were measured by the principal investigator at days 1, 4, 8 and 10. All quantitative variables were estimated using measures of central tendency (mean, median) and measures of dispersion (standard deviation). Qualitative or categorical variables were described as frequencies or proportions. Proportions were compared using Post Hoc Test and N Par Tests. Effectiveness was checked using p-value (< 0.005). Results: It was observed that 0.1% Triamcinolone Acetonide and 5% Amlexanox proven to be more efficacious in the reduction of size, Number, Pain, Erythema and Exudate Levels at day 8, (p = .000*) and at day 10 (p =. 000*) as compared to single application of 100 mg Doxycycline Hyclate, 20% Benzocaine gel and the placebo, which was statistically significant. VAS scale was significant for 100 mg Doxycycline Hyclate and 20% Benzocaine gel. Conclusion: The selected topical treatment modality can deliver cheap, effective and safe drug therapy which benefits the patient in refining their regular activities and everyday events of life.

Aphthous ulcer drug inhibits prostate tumor metastasis by targeting IKKvarepsilon/TBK1/NF-kappaB signaling.[Pubmed:30279728]

Theranostics. 2018 Sep 9;8(17):4633-4648.

Tumor metastasis is the major cause of death for prostate cancer (PCa) patients. However, the treatment options for metastatic PCa are very limited. Epithelial-mesenchymal transition (EMT) has been reported to be an indispensable step for tumor metastasis and is suggested to associate with acquisition of cancer stem cell (CSC) attributes. We propose that small-molecule compounds that can reverse EMT or induce mesenchymal-epithelial transition (MET) of PCa cells may serve as drug candidates for anti-metastasis therapy. Methods: The promoters of CDH1 and VIM genes were sub-cloned to drive the expression of firefly and renilla luciferase reporter in a lentiviral vector. Mesenchymal-like PCa cells were infected with the luciferase reporter lentivirus and subjected to drug screening from a 1274 approved small-molecule drug library for the identification of agents to reverse EMT. The dosage-dependent effect of candidate compounds was confirmed by luciferase reporter assay and immunoblotting. Wound-healing assay, sphere formation, transwell migration assay, and in vivo intracardiac and orthotopic tumor xenograft experiments were used to evaluate the mobility, metastasis and tumor initiating capacity of PCa cells upon treatment. Possible downstream signaling pathways affected by the candidate compound treatment were analyzed by RNA sequencing and immunoblotting. Results: Drug screening identified Amlexanox, a drug used for recurrent aphthous ulcers, as a strong agent to reverse EMT. Amlexanox induced significant suppression of cell mobility, invasion, serial sphere formation and in vivo metastasis and tumor initiating capacity of PCa cells. Amlexanox treatment led to downregulation of the IKK-varepsilon/ TBK1/ NF-kappaB signaling pathway. The effect of Amlexanox on EMT reversion and cell mobility inhibition can be mimicked by other IKK-varepsilon/TBK1 inhibitors and rescued by reconstitution of dominant active NF-kappaB. Conclusions: Amlexanox can sufficiently suppress PCa metastasis by reversing EMT through downregulating the IKK-varepsilon/TBK1/NF-kappaB signaling axis.

Carboxylic Acid Derivatives of Amlexanox Display Enhanced Potency toward TBK1 and IKKepsilon and Reveal Mechanisms for Selective Inhibition.[Pubmed:30082428]

Mol Pharmacol. 2018 Oct;94(4):1210-1219.

Chronic low-grade inflammation is a hallmark of obesity, which is a risk factor for the development of type 2 diabetes. The drug Amlexanox inhibits IkappaB kinase epsilon (IKKepsilon) and TANK binding kinase 1 (TBK1) to promote energy expenditure and improve insulin sensitivity. Clinical studies have demonstrated efficacy in a subset of diabetic patients with underlying adipose tissue inflammation, albeit with moderate potency, necessitating the need for improved analogs. Herein we report crystal structures of TBK1 in complex with Amlexanox and a series of analogs that modify its carboxylic acid moiety. Removal of the carboxylic acid or mutation of the adjacent Thr156 residue significantly reduces potency toward TBK1, whereas conversion to a short amide or ester nearly abolishes the inhibitory effects. IKKepsilon is less affected by these modifications, possibly due to variation in its hinge that allows for increased conformational plasticity. Installation of a tetrazole carboxylic acid bioisostere improved potency to 200 and 400 nM toward IKKepsilon and TBK1, respectively. Despite improvements in the in vitro potency, no analog produced a greater response in adipocytes than Amlexanox, perhaps because of altered absorption and distribution. The structure-activity relationships and cocrystal structures described herein will aid in future structure-guided inhibitor development using the Amlexanox pharmacophore for the treatment of obesity and type 2 diabetes.

Loss of Tbk1 kinase activity protects mice from diet-induced metabolic dysfunction.[Pubmed:29935921]

Mol Metab. 2018 Oct;16:139-149.

OBJECTIVE: TANK Binding Kinase 1 (TBK1) has been implicated in the regulation of metabolism through studies with the drug Amlexanox, an inhibitor of the IkappaB kinase (IKK)-related kinases. Amlexanox induced weight loss, reduced fatty liver and insulin resistance in high fat diet (HFD) fed mice and has now progressed into clinical testing for the treatment and prevention of obesity and type 2 diabetes. However, since Amlexanox is a dual IKKepsilon/TBK1 inhibitor, the specific metabolic contribution of TBK1 is not clear. METHODS: To distinguish metabolic functions unique to TBK1, we examined the metabolic profile of global Tbk1 mutant mice challenged with an obesogenic diet and investigated potential mechanisms for the improved metabolic phenotype. RESULTS AND CONCLUSION: We report that systemic loss of TBK1 kinase function has an overall protective effect on metabolic readouts in mice on an obesogenic diet, which is mediated by loss of an inhibitory interaction between TBK1 and the insulin receptor.

Targeting IRF3 as a YAP agonist therapy against gastric cancer.[Pubmed:29339449]

J Exp Med. 2018 Feb 5;215(2):699-718.

The Hippo pathway plays a vital role in tissue homeostasis and tumorigenesis. The transcription factor IRF3 is essential for innate antiviral immunity. In this study, we discovered IRF3 as an agonist of Yes-associated protein (YAP). The expression of IRF3 is positively correlated with that of YAP and its target genes in gastric cancer; the expression of both IRF3 and YAP is up-regulated and prognosticates patient survival. IRF3 interacts with both YAP and TEAD4 in the nucleus to enhance their interaction, promoting nuclear translocation and activation of YAP. IRF3 and YAP-TEAD4 are associated genome-wide to cobind and coregulate many target genes of the Hippo pathway. Overexpression of active IRF3 increased, but depletion of IRF3 reduced, the occupancy of YAP on the target genes. Knockdown or pharmacological targeting of IRF3 by Amlexanox, a drug used clinically for antiinflammatory treatment, inhibits gastric tumor growth in a YAP-dependent manner. Collectively, our study identifies IRF3 as a positive regulator for YAP, highlighting a new therapeutic target against YAP-driven cancers.

Amlexanox Inhibits Cerebral Ischemia-Induced Delayed Astrocytic High-Mobility Group Box 1 Release and Subsequent Brain Damage.[Pubmed:29330155]

J Pharmacol Exp Ther. 2018 Apr;365(1):27-36.

High-mobility group box 1 (HMGB1) is increased in the cerebrospinal fluid (CSF) and serum during the early and late phases of brain ischemia and is known to contribute to brain damage. However, detailed characterization underlying cell type-specific HMGB1 release and pathophysiological roles of extracellularly released HMGB1 in ischemic brain remain unclear. Here, we examined cell type-specific HMGB1 release and the therapeutic potential of Amlexanox, an inhibitor of nonclassical release, and of an anti-HMGB1 antibody against ischemic brain damage. HMGB1 depletion from neuronal nuclei was observed within 3 hours after transient middle cerebral artery occlusion (tMCAO), whereas the intracerebroventricular (i.c.v.) pretreatment with Amlexanox blocked HMGB1 release from neurons, resulting in HMGB1 redistribution in the nuclei and cytoplasm. HMGB1 was selectively released from astrocytes 27 hours after tMCAO and this HMGB1 release was blocked by late treatment with Amlexanox (i.c.v.) 24 hours after tMCAO. Proximity extension assay revealed that the HMGB1 level was elevated in the CSF at 3 and 27 hours after tMCAO. This late treatment with Amlexanox significantly protected the brain from ischemic damage, but its pretreatment 30 minutes before tMCAO failed to show any protection. The late treatment (i.c.v.) with anti-HMGB1 antibody 24 hours after tMCAO also ameliorated ischemic brain damage 48 hours after tMCAO. Thus, the inhibition of brain damage by late treatment with Amlexanox or anti-HMGB1 antibody indicates that late HMGB1 release plays a role in the maintenance of stroke-induced brain damage, and the inhibition of this release would be a novel therapeutic target for protection of ischemic brain damage.

An inhibitor of the protein kinases TBK1 and IKK-varepsilon improves obesity-related metabolic dysfunctions in mice.[Pubmed:23396211]

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

Emerging evidence suggests that inflammation provides a link between obesity and insulin resistance. The noncanonical IkappaB kinases IKK-varepsilon and TANK-binding kinase 1 (TBK1) are induced in liver and fat by NF-kappaB activation upon high-fat diet feeding and in turn initiate a program of counterinflammation that preserves energy storage. Here we report that Amlexanox, an approved small-molecule therapeutic presently used in the clinic to treat aphthous ulcers and asthma, is an inhibitor of these kinases. Treatment of obese mice with Amlexanox elevates energy expenditure through increased thermogenesis, producing weight loss, improved insulin sensitivity and decreased steatosis. Because of its record of safety in patients, Amlexanox may be an interesting candidate for clinical evaluation in the treatment of obesity and related disorders.

Hsp90 is a direct target of the anti-allergic drugs disodium cromoglycate and amlexanox.[Pubmed:12803546]

Biochem J. 2003 Sep 1;374(Pt 2):433-41.

Hsp90 (heat-shock protein 90) alone can act to prevent protein aggregation and promote refolding in vitro, but in vivo it operates as a part of a multichaperone complex, which includes Hsp70 and cohort proteins. Since the physiological function of Hsp90 is not yet fully understood, the development of specific antagonists might open new lines of investigation on the role of Hsp90. In an effort to discover Hsp90 antagonists, we screened many drugs and found that the anti-allergic drugs DSCG (disodium cromoglycate) and Amlexanox target Hsp90. Both drugs were found to bind directly wild-type Hsp90 via the N- and C-terminal domains. Both drugs strongly suppressed the in vitro chaperone activity of native Hsp90 towards citrate synthase at 1.5-3.0 microM. Amlexanox suppressed C-terminal chaperone activity in vitro, but not N-terminal chaperone activity, and inhibited the association of cohort proteins, such as cyclophilin 40 and Hsp-organizing protein, to the C-terminal domain of Hsp90. These data suggest that Amlexanox might disrupt the multichaperone complex, including Hsp70 and cohort proteins, both in vitro and in vivo. Although DSCG inhibited the in vitro chaperone activity of the N-terminal domain, the drug had no effect either on the C-terminal chaperone activity or on the association of the cohort proteins with the C-terminus of Hsp90. The physiological significance of these interactions in vivo remains to be investigated further, but undoubtedly must be taken into account when considering the pharmacology of anti-allergic drugs. DSCG and Amlexanox may serve as useful tools for evaluating the physiological significance of Hsp90.

Mechanism of action of an antiallergic agent, amlexanox (AA-673), in inhibiting histamine release from mast cells. Acceleration of cAMP generation and inhibition of phosphodiesterase.[Pubmed:2433225]

Int Arch Allergy Appl Immunol. 1987;82(1):66-71.

Amlexanox markedly inhibits histamine release from rat mast cells. To clarify the mechanism of this inhibition, we investigated the effect of Amlexanox on cAMP content, which, when increased, inhibits histamine release in rat peritoneal mast cells. At concentrations of 10(-8)-10(-6)M, Amlexanox or isoproterenol increased the cAMP content of mast cells over that of control cells about 2-fold. When the mast cells were incubated with 10(-8), 10(-7) and 10(-6) M of Amlexanox combined with 10(-7) M isoproterenol, the cAMP contents were synergistically increased 15-, 60- and 88-fold, respectively. 3-Isobutyl-1-methylxanthine (IBMX) at 10(-6)-10(-4) M increased the cAMP content 1.7-3.8-fold, and a combination of 10(-4) M IBMX and 10(-7) M isoproterenol synergistically increased the cAMP content 41-fold. A combination of Amlexanox and IBMX synergistically increased the cAMP content 19-fold. The increase in cAMP content, when Amlexanox and isoproterenol were combined, was transient; it peaked at 0.5 min after the drugs were administered, then decreased to 20-30% of the peak value about 2 min later. Pretreatment of mast cells with Amlexanox reduced the effect of the combination of Amlexanox and isoproterenol, indicating tachyphylaxis; pretreatment with IBMX had no such effect. The cAMP content of macrophages was also increased by Amlexanox, but when combined with isoproterenol or PGE2, the effect was additive. Amlexanox inhibited cAMP phosphodiesterase in rat mast cells; its IC50 value was 1.4 X 10(-5) M, and its inhibitory activity was half that of IBMX.(ABSTRACT TRUNCATED AT 250 WORDS)

Amlexanox (AA673; Amoxanox; CHX3673) is a specific inhibitor of IKKε and TBK1, and inhibits the IKKε and TBK1 activity determined by MBP phosphorylation with an IC50 of approximately 1-2 μM.

Amlexanox,68302-57-8,AA673; Amoxanox; CHX3673,Natural Products, buy Amlexanox , Amlexanox supplier , purchase Amlexanox , Amlexanox cost , Amlexanox manufacturer , order Amlexanox , high purity Amlexanox