Assessing metabolic profiles in chimeric PXB mouse with humanized livers following oral dosing of troglitazone

Importance of the Topic

Chimeric mice carrying human hepatocytes offer a powerful platform to predict human-specific drug metabolism and potential toxic metabolites. Troglitazone, an antidiabetic compound with known interspecies metabolic differences, serves as a model to evaluate the relevance of the PhoenixBio (PXB) mouse in replicating human liver biotransformation pathways.

Objectives and Study Overview

The study compared the metabolic fate of troglitazone and endogenous lipid profiles in PXB mice versus severe combined immunodeficient (SCID) control mice. Key aims included:

• Oral administration of troglitazone at 300 and 600 mg/kg daily for seven days.
• Identification and quantification of phase I and II metabolites in liver extracts.
• Profiling of endogenous metabolites to detect phenotype-driven differences.

Methodology and Instrumentation

Sample Preparation and Analysis:

• Aqueous and organic liver extracts were prepared post-dosing.
• Chromatographic separation using Shimadzu Nexera LC with C18 Kinetex column at 30 °C and flow rates of 0.6 mL/min (aqueous) or 0.5 mL/min (organic).
• Gradient elution with water/formic acid/ammonium acetate and acetonitrile for aqueous samples; water/acetonitrile/ammonium formate and isopropanol/acetonitrile for organic extracts.

Mass Spectrometry and Data Processing:

• LCMS-IT-TOF system (Shimadzu) in polarity‐switching mode (m/z 150–1250), acquiring MS and MS/MS data.
• MetID Solution software for targeted metabolite identification via accurate mass and isotope scoring.
• Profiling Solution and Simca-P for data alignment, principal component analysis (PCA) and OPLS-DA.

Main Results and Discussion

Troglitazone Metabolism:

• Four primary phase II metabolites detected: sulfate and glucuronide conjugates plus di-hydroxy derivatives.
• PXB mice exhibited a metabolic profile closer to human data, with sulfate conjugates predominating and lower glucuronidation relative to SCID mice.

Endogenous Metabolite Profiling:

• Over 80 lipid ions showed significant differences between PXB and SCID livers.
• Glycerophosphocholine species displayed consistent regulation independent of dosing, highlighted by S-plot analysis.
• PCA separated samples by host phenotype rather than dose, indicating intrinsic metabolic distinctions.

Benefits and Practical Applications

Using PXB mice allows early prediction of human-specific drug metabolites and potential bioactivation pathways, reducing late-stage failures. Concurrent profiling of endogenous lipids aids biomarker discovery for liver function and xenobiotic response in preclinical safety and efficacy studies.

Future Trends and Opportunities

• Expansion of humanized models to include multi-tissue chimeras and immune components.
• Integration of high-throughput LC-MS metabolomics with genomics and proteomics for systems-level insight.
• Advances in software algorithms for automated metabolite annotation and deeper pathway analysis.
• Application to personalized medicine through donor-specific humanized animals.

Conclusion

The PXB mouse model effectively reproduces human troglitazone metabolism and reveals intrinsic lipidomic differences, demonstrating its value for preclinical drug metabolism studies. High-resolution LC-MS combined with targeted and untargeted data analysis provides a robust workflow for comprehensive metabolite characterization.

References

1. Barnes A., Loftus N.J., Hobby K., Wilson I., Morikawa Y. Assessing metabolic profiles in chimeric PXB mouse with humanized livers following oral dosing of troglitazone. Shimadzu Technical Bulletin; PhoenixBio Co. Ltd.
2. Castro-Perez J. et al. Development of a high-throughput lipidomics method for biofluids and tissues. Analytical Chemistry, 2010.