Diagnostic exogenous probe strategy using ezetimibe-glucuronide effectively identifies metabolic dysfunction-associated steatohepatitis.

Abstract

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease with an urgent need for reliable, noninvasive biomarkers that can serve as reasonably likely surrogate endpoints, as diagnosis remains limited by the invasiveness of liver biopsy and the inaccuracy of existing noninvasive tests. MASH disrupts hepatic and renal transporter expression and function, leading to increased systemic retention of drug substrates. Because ezetimibe-glucuronide (EZEG) is a substrate of transporters altered in MASH, we evaluated whether EZEG could serve as a noninvasive, mechanism-based exogenous diagnostic function test. Plasma and urine EZEG concentrations were quantified by liquid chromatography-tandem mass spectrometry in biopsy-confirmed patients with nonalcoholic fatty liver disease activity scores (NAS) of 0-5 after administration of a 1 mg subtherapeutic dose of ezetimibe. Patients with MASH (NAS = 5, n = 8) exhibited approximately 4.5-fold higher plasma EZEG concentrations than non-MASH individuals (NAS 0-4, n = 45), demonstrating excellent discrimination with an area under the receiver operating characteristic curve of 0.92 (95% CI, 0.79-1.00) using the 60-minute postdose concentration. Plasma EZEG was strongly associated with hepatocyte ballooning, the U.S. Food and Drug Administration-defining and most difficult-to-detect histologic feature of MASH, supporting mechanistic specificity. In contrast, urinary EZEG excretion showed only modest increases and poor diagnostic discrimination. These findings indicate that MASH-associated transporter dysfunction leads to systemic EZEG accumulation, supporting its use as a mechanism-based diagnostic function test and a promising noninvasive strategy for early detection and disease stratification. SIGNIFICANCE STATEMENT: Metabolic dysfunction-associated steatohepatitis (MASH) lacks reliable noninvasive tests for diagnosis and therapeutic development. This study demonstrates that plasma ezetimibe-glucuronide, measured after a subtherapeutic ezetimibe dose, sensitively detects MASH-associated transporter dysfunction and strongly correlates with hepatocyte ballooning, the defining histologic feature of MASH. This mechanism-based probe-drug approach provides a highly discriminative, noninvasive strategy to support early disease detection and patient stratification.