Exploration of Biomarkers for Amoxicillin/Clavulanate-Induced Liver Injury: Multi-Omics Approaches

Abstract

To explore potential biomarkers for amoxicillin/clavulanate-induced liver injury (AC-DILI), we conducted a clinical trial in 32 healthy subjects based on multi-omics approaches. Every subject was administered amoxicillin/clavulanate for 14 days. The liver-specific microRNA-122 (miR-122) level increased prior to and correlated well with the observed alanine aminotransferase (ALT) level increase. This result indicates its potential as a sensitive early marker for AC-DILI. We also identified urinary metabolites, such as azelaic acid and 7-methylxanthine, with levels that significantly differed among the groups classified by ALT elevation level on day 8 after drug administration (P < 0.05). Lymphocyte proliferation in response to the drug was also observed. These findings demonstrate sequential changes in the process of AC-DILI, including metabolic changes, increased miR-122 level, increased liver enzyme activity, and enhanced lymphocyte proliferation after drug administration. In conclusion, this study provides potential biomarkers for AC-DILI based on currently known mechanisms using comprehensive multi-omics approaches.

Figure 1

Study design. GSTT1/M1, blood collection for genotyping of glutathione s‐transferase mu‐1/theta‐1; LTT, blood collection for lymphocyte transformation test; HLA, blood collection for genotyping human leukocyte antigen (HLA); PK, blood collection for pharmacokinetic analyses of amoxicillin and clavulanate; Metabolomics, 12‐h interval urine collection for metabolomics analysis; miRNA, blood collection for microRNA analyses.

Figure 2

ALT fold changes relative to baseline levels (IU/L) in the subjects over the course of the 60‐day study in the (a) Responder group (left: ALT fold change, right: ALT levels) and the (b) Nonresponder group (left: ALT fold change, right: ALT levels). The baseline ALT levels were defined as the value of the first day predose sample.

Figure 3

Time courses depicting the serum miR‐122 and ALT fold changes in the (a) Responder and (b) Nonresponder groups. The data represent the mean ± standard deviation.

Figure 4

Box‐whisker plots for significantly regulated endogenous metabolites. (a) Azelaic acid, (b) 7‐methyluric acid, (c) 3‐methylxanthine, (d) 7‐methylxanthine, and (e) acetylcarnitine on days 1, 8, and 14 (white box: Responder; gray box: Nonresponder). The box plot shows the median (line) and 25–75% interquartile range for endogenous metabolites. Circles represent outliers.

Figure 5

Time courses depicting changes in (a) urinary metabolites, (b) miR‐122, (c) ALT and AST, (d) lymphocyte proliferation, and (e) INF‐γ release in subject AN004.