University of Wisconsin Madison Madison, Wisconsin, United States
Purpose: To eliminate the need for toxic co-solvents and gain aqueous solubility of hydrophobic molecules for intravenous injection, nano-sized polymeric micelles have been proposed as safe, scalable drug delivery systems. Paclitaxel (PTX), a potent microtubule stabilizer, has been researched extensively as a candidate for micelle encapsulation. In order to improve the carrier stability following injection, we have developed a novel paclitaxel prodrug conjugated with 8 lactic acid units (o(LA)8-PTX) for enhanced compatibility with the core of poly(ethylene glycol)-b-poly(lactic acid) (PEG-b-PLA) micelles. Our goal was to develop a liquid chromatography mass spectrometry (LCMS) method for detection and quantitation of o(LA)8-PTX and its metabolites in biological matrices such as plasma and bile. We hypothesized that the AUC of the bioactive metabolites would be higher than that of the marketed PTX nano-formulation, Abraxane. Methods: Micelle solutions containing 50% w/w o(LA)8-PTX in PEG-b-PLA micelles were freeze-dried using PEG as a lyoprotectant. In collaboration with the NIH's Nanotechnology Characterization Laboratory, Sprague Dawley rats were intravenously administered a single 10 mg PTX-equivalent dose of o(LA)8-PTX-loaded micelles or Abraxane. Plasma and bile were collected and analyzed by LCMS equipped with a Q Exactive basic quadrupole Orbitrap MS analyzer and UHPLC with UV detection following extensive method development in vitro. Noncompartmental PK analysis was performed with Phoenix WinNonlin software. Results: Lyophilized micelle solutions were able to be rapidly reconstituted in aqueous solution and demonstrated consistent particle size before and after freezing. An LC gradient method using ACN and water was successfully developed to separate o(LA)8-PTX from its shorter-chained o(LA)n-PTX intermediate species. Following a single injection of prodrug-loaded micelles in SD rats, LCMS analysis of collected plasma and bile revealed o(LA)2-PTX and o(LA)1-PTX to be the primary metabolites in vivo. Surprisingly, the final step of the prodrug conversion from o(LA)2-PTX or o(LA)1-PTX to parent drug PTX was slow, presumably due to steric hindrance. Previous studies in our group have shown these 2 metabolites to be bioactive, meaning complete conversion back to PTX is not necessary for drug action. As a result, the AUC of PTX generated from o(LA)8-PTX was lower than that from Abraxane while the AUC of the bioactive metabolite o(LA)1-PTX was higher than Abraxane. Conclusion: A reproducible LC-Orbitrap method was successfully developed for the separation, identification and quantitation of o(LA)8-PTX prodrug and its metabolites. The most abundant bioactive metabolite detected in plasma, o(LA)1-PTX, displayed a higher AUC versus Abraxane, indicating higher plasma exposure for our formulation over the marketed product. This prolonged exposure may enhance the cell-cycle effects of PTX and warrants further preclinical evaluation.
Acknowledgments: The authors thank the Nanotechnology Characterization Laboratory (NCL) for characterization support of this project; the formulation described herein was characterized by the NCL’s free Assay Cascade characterization service for cancer nanomedicines. This project has been funded in part or in whole with federal funds from the NCI, NIH, under contract no 75N91019D00024. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the U.S. Government.
