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Published on: November 27, 2016
Mechanism-guided untargeted-to-targeted lipidomics identifies phosphatidylcholine 38:4 in rat bile as an Abcb4/Mdr2
Renmeng Liu1, Zachary Rabow1, Tingyuan Yang2
1Drug Metabolism, Gilead Sciences Inc, Foster City, California.
Abstract:
Drug-induced liver injury can occur when the canalicular phospholipid floppase multidrug resistance protein 3 (Mdr2 in rodents) is inhibited, but there is still a lack of early biomarkers to detect this risk. In this study, bile duct-cannulated rats were dosed with multidrug resistance protein 3/Mdr2 inhibitor itraconazole (ITZ; 100 mg/kg/d for 3 days) to assess phospholipid changes via an untargeted-to-targeted lipidomics workflow. Untargeted profiling of bile and liver samples identified 1347 and 2475 tentative lipids, of which 221 and 404 were phosphatidylcholines (PCs) in bile and the liver, respectively. Unsupervised principal component analysis revealed strong treatment effects on bile PCs. A volcano plot indicated a selective, but not global, reduction in biliary PCs after ITZ treatment. Among these, PC 38:4 stood out as the most consistently decreased bile species. Structural elucidation using multistage collision-induced dissociation/mass spectrometry3 fragmentations confirmed its identity as arachidonyl PC 18:0/20:4. Subsequent absolute quantitation showed that bile PC 38:4 remained stable in controls (10.5 ± 1.02 μM; 5.6% CV) but declined rapidly after the first dose of ITZ (6.89 ± 1.50 μM at 0-4 hours) and continued to decrease to 4.22 ± 0.958 μM by day 3, a 2.7-fold decrease. Conversely, hepatic PC 38:4 showed a modest, yet significant increase (∼1.2-fold). Plasma bile acids remained unaffected, supporting a mechanism involving Mdr2 rather than the bile salt export pump. These findings identify PC 38:4 (18:0/20:4) as a sensitive and mechanistically relevant marker of Mdr2 inhibition. Monitoring PC 38:4 in nonclinical species may enable early, transporter-specific drug-induced liver injury risk assessment during drug development. SIGNIFICANCE STATEMENT: Untargeted-to-targeted lipidomics workflows identified phosphatidylcholine 38:4 as a sensitive, specific, and mechanistically linked biomarker of Mdr2 inhibition in rats. Multistage collision-induced dissociation/mass spectrometry3 fragmentation further confirmed the identity as arachidonyl phosphatidylcholine 18:0/20:4. Its rapid and specific decline in the presence of the Mdr2 inhibitor itraconazole offers a potential new tool for early detection of human multidrug resistance protein 3-related liver injury risk during drug development.
Insights
Researchers identified phosphatidylcholine 38:4 as a key biomarker for detecting drug-induced liver injury risk. This specific phospholipid
Area of Science:
- Biochemistry
- Pharmacology
- Toxicology
Background:
- Drug-induced liver injury (DILI) is a significant concern in drug development.
- Inhibition of the phospholipid transporter multidrug resistance protein 3 (Mdr2) can lead to DILI.
- Early detection biomarkers for Mdr2-related DILI are currently lacking.
Purpose of the Study:
- To identify early biomarkers for Mdr2 inhibition-induced liver injury.
- To investigate the effects of Mdr2 inhibition on phospholipid profiles in bile and liver.
- To assess the potential of specific phosphatidylcholines as predictive markers for DILI.
Main Methods:
- Utilized an untargeted-to-targeted lipidomics workflow in bile duct-cannulated rats.
- Administered the Mdr2 inhibitor itraconazole (ITZ) to assess phospholipid changes.
- Employed principal component analysis and volcano plots for data interpretation.
- Confirmed lipid identity using multistage collision-induced dissociation/mass spectrometry.
- Quantified specific phosphatidylcholine species in bile, liver, and plasma.
Main Results:
- Identified 221 biliary and 404 hepatic phosphatidylcholines (PCs).
- Demonstrated a selective reduction in biliary PCs, particularly PC 38:4 (arachidonyl PC 18:0/20:4), following ITZ treatment.
- Observed a rapid, significant decrease in bile PC 38:4 levels post-ITZ administration.
- Noted a modest increase in hepatic PC 38:4 and unaffected plasma bile acids.
- Confirmed the mechanism involves Mdr2 inhibition, not bile salt export pump dysfunction.
Conclusions:
- Phosphatidylcholine 38:4 is a sensitive and specific biomarker for Mdr2 inhibition.
- The rapid decline of bile PC 38:4 offers a potential early warning sign for Mdr2-related DILI.
- This finding may facilitate early, transporter-specific DILI risk assessment in drug development.
- Monitoring PC 38:4 could improve the safety profile of new drug candidates.
