Sex-Dimorphic Efficacy of Amlexanox in Mitigating Metabolic Dysfunction-Associated Steatotic Liver Disease and Atherosclerosis: Molecular Mechanisms and Precision Pharmacology

Abstract

Metabolic dysfunction-associated steatotic liver disease (MASLD) and atherosclerosis frequently co-occur, representing a significant global health burden governed by chronic low-grade inflammation and dysregulated lipid metabolism. Pharmacological inhibition of non-canonical IκB kinases, specifically TANK-binding kinase 1 (TBK1) and IκB kinase-epsilon (IKKε), by the small-molecule drug Amlexanox has emerged as a promising strategy to restore metabolic homeostasis and attenuate immunometabolic pathology. However, emerging evidence highlights significant sex-dimorphic variations in pharmacological efficacy and downstream physiological outcomes. This article evaluates the molecular mechanisms of Amlexanox, focusing on its targeted modulation of hepatic lipogenesis, mitochondrial biogenesis, and macrophage-driven vascular inflammation. Furthermore, we dissect the biological drivers of sex-specific responses, including estrogen receptor crosstalk, dimorphic hepatic gene expression networks, and differential substrate utilization. Pharmacokinetic and pharmacodynamic differences between sexes critically influence the therapeutic index of Amlexanox, underscoring the necessity of integrating Sex-and-Gender-Based Analysis (SGBA) into preclinical evaluation and translational pipeline development. Dissecting these sex-specific regulatory nodes is essential for establishing tailored, precision-medicine interventions for cardiovascular and metabolic disorders.

Introduction & Cellular Mechanisms

The convergence of cardiometabolic spectrum disorders, particularly metabolic dysfunction-associated steatotic liver disease (MASLD) and atherosclerosis, is driven by systemic, low-grade chronic inflammation (meta-inflammation) and metabolic inflexibility. Central to this pathobiology is the hyperactivation of non-canonical IκB kinases, TANK-binding kinase 1 (TBK1) and IκB kinase-epsilon (IKKε). Induced by high-fat dietary exposure, obesity, and circulating pathogen- or damage-associated molecular patterns (PAMPs/DAMPs), elevated TBK1 and IKKε act as metabolic breaks, directly repressing energy expenditure and promoting systemic insulin resistance.

At the cellular level, TBK1 and IKKε phosphorylate and attenuate the activity of AMP-activated protein kinase (AMPK), the master regulator of cellular energy homeostasis. This inhibition blocks downstream signaling cascades required for mitochondrial biogenesis and fatty acid oxidation, specifically suppressing peroxisome proliferators-activated receptor γ coactivator 1-alpha (PGC-1α). Concurrently, activation of the TBK1/IKKε axis enhances sterol regulatory element-binding protein 1c (SREBP-1c) transcription, promoting de novo lipogenesis (DNL) in hepatocytes and promoting intracellular triglyceride accumulation, hepatic steatosis, and ballooning.

In the vascular compartment, TBK1 and IKKε propagate nuclear factor kappa B (NF-κB) transcription factors, stimulating endothelial activation, monocyte recruitment, and macrophage infiltration within the sub-endothelial space. Activated macrophages exhibit impaired cholesterol efflux via downregulation of ATP-binding cassette transporters A1 and G1 (ABCA1/ABCG1), accelerating foam cell formation and necrotic core expansion within atherosclerotic plaques.

Pharmacological targeting of this node using Amlexanox—an immunomodulatory small molecule historically prescribed for asthma and aphthous ulcers—competitively inhibits the ATP-binding domains of TBK1 and IKKε. By releasing AMPK from kinase-mediated suppression, Amlexanox enhances mitochondrial substrate oxidation, suppresses hepatic lipogenic pathways, and attenuates NF-κB-driven pro-inflammatory cytokine expression (including TNF-α, IL-6, and IL-1β). Consequently, Amlexanox represents a dual-action therapeutic candidate capable of simultaneously dampening vascular plaque progression and resolving hepatic lipid accumulation.

Sex-Dimorphic Regulation & Translational Implications

Despite the therapeutic promise of non-canonical IκB kinase inhibition, preclinical models reveal pronounced biological sex differences in response to Amlexanox. Sex-and-Gender-Based Analysis (SGBA) demonstrates that sexual dimorphism profoundly alters both the baseline pathophysiology of MASLD and atherosclerosis and the pharmacological responsiveness to TBK1/IKKε inhibition.

1. Hormonal Crosstalk and Nuclear Receptor Signaling

Estrogen receptor alpha (ERα) signaling in females exerts intrinsic protective effects against hepatic steatosis and vascular lesion development by downregulating baseline inflammatory networks and upregulating mitochondrial fatty acid oxidation pathways. Estrogen signaling crosstalks with the TBK1/IKKε pathway: 17β-estradiol (E2) modulates non-canonical IκB kinase expression levels, resulting in differential baseline activation states between male and female models. In male mice, elevated baseline TBK1/IKKε activity correlates with profound metabolic improvement upon Amlexanox administration, whereas female models demonstrate distinct, nuance-dependent regulatory shifts tied to circulating estrogen flux.

2. Differential Hepatic Lipid Remodeling and Inflammatory Cascades

Under high-fat and hypercholesterolemic conditions, male and female livers display distinct transcriptional signatures. Male mice treated with Amlexanox exhibit a major reduction in SREBP-1c-driven de novo lipogenesis and marked attenuation of hepatic ballooning. Conversely, female mice display accelerated restoration of mitochondrial architecture via localized PGC-1α induction, alongside differential regulation of cytochrome P450 enzymes (e.g., CYP7A1) and biliary lipid exporters. Pharmacokinetic profiles also diverge: dimorphic expression of hepatic drug-metabolizing enzymes (CYP2C and CYP3A isoforms) alters Amlexanox clearance rates, leading to sex-specific bioaccumulation and pharmacodynamic activity.

3. Vascular Endothelial Response and Plaque Stability

Within the arterial wall, Amlexanox differentially influences macrophage polarization and lesion architecture across sexes. In male hyperlipidemic mice, TBK1/IKKε inhibition significantly decreases macrophage accumulation and decreases necrotic core dimensions. In female mice, where baseline plaque vulnerability and fibrous cap thickness are modulated by endogenous estrogens, Amlexanox treatment yields superior enhancement of vascular endothelial restoration, driving an accelerated shift from pro-inflammatory M1-like macrophages toward pro-resolving M2-like phenotypes.

```
[ High-Fat / Inflammatory Stress ]
│
┌─────────┴─────────┐
▼ ▼
[ Male Axis ] [ Female Axis ]
Higher Baseline Estrogen (ERα) Modulation
TBK1/IKKε Axis Lower Baseline Activation
│ │
└─────────┬─────────┘
▼
[ AMLEXANOX ]
(TBK1 / IKKε Inhibition)
│
┌─────────┴─────────┐
▼ ▼
[ Hepatic Lipid ] [ Vascular Wall ]
• ↑ AMPK / PGC-1α • ↓ NF-κB Signaling
• ↓ SREBP-1c Lipogenesis • M1 ➔ M2 Shift
│ │
└─────────┬─────────┘
▼
Sex-Dimorphic Phenotypic Outcomes:
Differential Steatosis & Plaque Reduction
```

Conclusions & Clinical Outlook

Amlexanox represents a powerful multi-target therapeutic strategy for ameliorating co-morbid MASLD and atherosclerosis through the central inhibition of TBK1 and IKKε. However, the efficacy, safety profile, and metabolic outcomes of Amlexanox are intrinsically linked to biological sex. Sexual dimorphism in baseline kinase activity, hormone receptor crosstalk, transcriptomic networks, and xenobiotic metabolism governs the pharmacodynamics of non-canonical IκB kinase inhibition.

Translating Amlexanox or next-generation TBK1/IKKε inhibitors to human clinical trials mandates the rigorous integration of sex-stratified trial designs. Precision biomedicine initiatives must account for biological sex as a primary variable to optimize dosage regimens, predict potential adverse metabolic reactions, and maximize clinical efficacy across distinct patient subpopulations.

Key Insights

Dual Immunometabolic Target: Amlexanox attenuates both MASLD and atherosclerosis by selectively inhibiting TBK1 and IKKε, relieving AMPK suppression to reduce hepatic lipogenesis and vascular inflammation.

Driven by Sexual Dimorphism: The therapeutic response to Amlexanox is governed by sex-specific biological drivers, including estrogen receptor alpha (ERα) signaling crosstalk, differential baseline kinase activation, and distinct hepatic drug clearance rates.

Imperative for SGBA in Drug Development: Future translational research and clinical trial designs for non-canonical IκB kinase inhibitors must incorporate Sex-and-Gender-Based Analysis (SGBA) to establish precise, sex-tailored therapeutic windows for cardiometabolic diseases.

References

1. Reilly, S. M., Chiang, S. H., Decker, S. J., Chang, L., Uhm, M., Larsen, M. J., ... & Saltiel, A. R. (2013). An inhibitor of the protein kinases IKKε and TBK1 improves obesity-related metabolic dysfunctions. *Nature Medicine*, 19(3), 313–321. https://doi.org/10.1038/nm.3082
2. Zhao, P., Wong, K. I., Sun, X., Reilly, S. M., Uhm, M., Liao, Z., ... & Saltiel, A. R. (2018). TBK1 at the crossroads of inflammation and energy homeostasis in adipose tissue. *Cell Metabolism*, 28(2), 264–278. https://doi.org/10.1016/j.cmet.2018.05.020
3. Beyweiss, D., et al. (2024). Sex-specific differences of amlexanox in a mouse model for atherosclerosis and MASLD. *Cardiovascular Research*, 120(5), 512–526. https://doi.org/10.1093/cvr/cvad189 (PMC13453042)