Cytokeratin-18 is a sensitive biomarker of alanine transaminase increase in a placebo-controlled, randomized, crossover trial of therapeutic paracetamol dosing (PATH-BP biomarker substudy)

Abstract

Drug-induced liver injury (DILI) is a challenge in clinical medicine and drug development. Highly sensitive novel biomarkers have been identified for detecting DILI following a paracetamol overdose. The study objective was to evaluate biomarker performance in a 14-day trial of therapeutic dose paracetamol. The PATH-BP trial was a double-blind, placebo-controlled, crossover study. Individuals (n = 110) were randomized to receive 1 g paracetamol 4× daily or matched placebo for 2 weeks followed by a 2-week washout before crossing over to the alternate treatment. Blood was collected on days 0 (baseline), 4, 7, and 14 in both arms. Alanine transaminase (ALT) activity was measured in all patients. MicroRNA-122 (miR-122), cytokeratin-18 (K18), and glutamate dehydrogenase (GLDH) were measured in patients who had an elevated ALT on paracetamol treatment (≥50% from baseline). ALT increased in 49 individuals (45%). All 3 biomarkers were increased at the time of peak ALT (K18 paracetamol arm: 18.9 ± 9.7 ng/ml, placebo arm: 11.1 ± 5.4 ng/ml, ROC-AUC = 0.80, 95% CI 0.71-0.89; miR-122: 15.1 ± 12.9fM V 4.9 ± 4.7fM, ROC-AUC = 0.83, 0.75-0.91; and GLDH: 24.6 ± 31.1U/l V 12.0 ± 11.8U/l, ROC-AUC = 0.66, 0.49-0.83). All biomarkers were correlated with ALT (K18 r = 0.68, miR-122 r = 0.67, GLDH r = 0.60). To assess sensitivity, biomarker performance was analyzed on the visit preceding peak ALT (mean 3 days earlier). K18 identified the subsequent ALT increase (K18 ROC-AUC = 0.70, 0.59-0.80; miR-122 ROC-AUC = 0.60, 0.49-0.72, ALT ROC-AUC = 0.59, 0.48-0.70; GLDH ROC-AUC = 0.70, 0.50-0.90). Variability was lowest for ALT and K18. In conclusion, K18 was more sensitive than ALT, miR-122, or GLDH and has potential significant utility in the early identification of DILI in trials and clinical practice.

Keywords: DILI; diagnosis; glutamate dehydrogenase; keratin-18; liver; microRNA.

Figure 1.

Change in serum biomarkers over 14 days of treatment with paracetamol (1 g 4 times per day) (A–C) or matched placebo (double-blind randomized trial) (D–F). Each line is a patient. The solid line is the population median. Population presented are patients from PATH-BP Trial with a 50% or more increase in peak ALT from baseline on paracetamol treatment. The concentration of GLDH was below the lower limit of detection (<5 U/l) in 261 out of 392 samples and these samples were excluded from the analysis.

Figure 2.

Biomarker performance at the trial visit at time of peak ALT in paracetamol treatment arm. A–C, biomarker concentration in placebo and paracetamol treatment arms. Significance determined by Wilcoxon test. Each dot is a participant. Bars show median and IQR. D–F, ROC curve analysis for biomarkers. AUC = area under curve. G–H, Biomarker correlation with ALT. Dots are placebo treatment, triangles are paracetamol treatment. Pearson r values are reported. The concentration of GLDH was below the lower limit of detection (<5 U/l) in 261 out of 392 samples and these samples were excluded from the analysis.

Figure 3.

Biomarker performance at visit preceding the visit with measurement of peak ALT in paracetamol treatment arm. A–D, Biomarker concentration in placebo and paracetamol treatment arms. Significance determined by Wilcoxon test. Each dot is a participant. Bars show median and IQR. E–H, ROC curve analysis for biomarkers. AUC = area under curve. The concentration of GLDH was below the lower limit of detection (<5 U/L) in 261 out of 392 samples and these samples were excluded from the analysis.