Rapid Determination Of The Pharmacokinetics, Metabolism, Elimination And Tissue Distribution Of A PROTACs Drug Using UHPLC-MS/MS, HRMS And DESI Imaging

Significance of the topic

Proteolysis targeting chimeras (PROTACs) represent an innovative therapeutic modality that harnesses the ubiquitin–proteasome system to selectively degrade disease-related proteins. Their large molecular weight (>800 Da) and bifunctional structure pose unique challenges for absorption, distribution, metabolism and excretion (ADME) profiling. Rapid and reliable analytical workflows are essential to characterize PROTAC pharmacokinetics, metabolic pathways and tissue distribution during preclinical development.

Objectives and study overview

This study aimed to assess the pharmacokinetic behavior, metabolic biotransformation, elimination profile and hepatic distribution of PROTAC-3-gefitinib (a PROTAC analogue of the tyrosine kinase inhibitor gefitinib) in male rats. Key goals included:

• Quantitative measurement of PROTAC-3-gefitinib and gefitinib in plasma and urine over a 24 h period.
• Identification and structural elucidation of major metabolites.
• Evaluation of urinary elimination pathways.
• Spatial imaging of drug and metabolites in liver tissue using DESI-MS.

Methodology and instrumentation

A combination of chromatographic and mass spectrometric approaches enabled comprehensive DMPK profiling:

• UHPLC-MS/MS assay (5 min RPLC run) on Waters ACQUITY Premier UPLC with Xevo TQ Absolute for quantitation (0.02–500 ng/mL).
• High-resolution MS (Waters Xevo MRT HRMS) for metabolite identification.
• DESI-MS imaging on a Xevo TQ Absolute in MRM mode for liver tissue distribution.
• Chromatographic separation on 2.1 × 100 mm, 1.7 µm HSS T3 C18 MaxPeak Premier column.
• Data analysis via Waters-connect metabolite application and MassMetaSite software.

Main results and discussion

Pharmacokinetics:

• PROTAC-3-gefitinib: half-life 7.2 h; Tmax 6 h; Cmax 67 ng/mL; AUC0–t 898 µM·h.
• Gefitinib reference: half-life 5.9 h; Tmax 6 h; Cmax 132 ng/mL; AUC0–t 1879 µM·h.

Metabolite profile:

• Nine major biotransformations detected, including N-dealkylation, ester hydrolysis of the linker, sulfation and glucuronidation.
• Predominant urinary elimination of ~22% of administered dose, with metabolites M1, M4, M7 and M9 accounting for most drug-related material.

DESI imaging:

• Unchanged PROTAC and metabolites M4 and M7 were visualized in liver at 1 h and 3 h post dose; parent compound was weakly detected at 8 h.
• Endogenous choline signal increased at 8 h, indicating metabolic perturbation.

Benefits and practical applications

The integrated UHPLC-MS/MS, HRMS and DESI-MS workflow enables rapid ADME screening of large bifunctional drug candidates. Microsampling reduces animal use and sample volumes while maintaining sensitivity. Spatial imaging adds a new dimension to tissue distribution studies, informing on local accumulation and potential toxicity.

Future trends and potential applications

Advancements may include:

• Integration of ion mobility separation for enhanced isomer resolution.
• Quantitative DESI imaging to correlate local concentration with pharmacodynamic markers.
• Automated data processing pipelines powered by machine learning for metabolite annotation.
• Extension to other PROTAC chemotypes and scaling to higher-throughput in vivo models.

Conclusion

This study demonstrates a robust analytical platform for rapid determination of PROTAC pharmacokinetics, metabolism and tissue localization in preclinical models. The workflow supports efficient drug candidate evaluation and provides critical insights for PROTAC optimization.

References

1. Békés M, Langley DR, Crews CM. PROTAC targeted protein degraders: the past is prologue. Nat Rev Drug Discov. 2022;21:181–200.
2. Goracci L, Desantis J, Valeri A, et al. Understanding the Metabolism of Proteolysis Targeting Chimeras (PROTACs): The Next Step toward Pharmaceutical Applications. J Med Chem. 2020;63(20):11615–11638.
3. McKillop D, et al. Pharmacokinetics of gefitinib in rat and dog. Xenobiotica. 2004;34(10):901–915.
4. Molloy BJ, King A, Mullin LG, et al. Rapid determination of the pharmacokinetics and metabolic fate of gefitinib in the mouse using UPLC/MS/MS, UPLC/QToF/MS, and ion mobility-enabled UPLC/QToF/MS. Xenobiotica. 2021;51(4):434–446.