Doxorubicin (DOX) is a widely used anti-cancer drug. It is hypothesized that the efficacy and toxicity of DOX is related to its distribution and metabolism. Capillary electrophoresis (CE) including its variants such as micellar electrokinetic chromatography (MEKC) is becoming a popular method in bioanalysis due to its high sensitivity and separation efficiency, small sample size requirements, simple sample preparation procedures, versatility in sampling and short separation times.
The goal of this thesis is to take advantage of these features and develop CE-based methods to investigate the metabolism, subcellular distribution and localization of DOX in biological samples after DOX treatments.
A direct sampling technique was developed to quantify DOX at or near the tumor site in hepatocellular carcinoma tissues after chemoembolization. This technique allows for sampling small volume of tissues (<10 picoliters) selectively from adjacent tumor and non-tumor regions with high spatial resolution (100 micrometers) and reproducibility. Using this technique coupled with MEKC-LIF (laser-induced fluorescence detection), DOX was detected and quantified, in both tumor and non-tumor regions in resected human livers.
A MEKC method was developed to monitor the incorporation, transformation and subcellular metabolism of a DOX prodrug, N-L-leucyl-doxorubicin (LeuDOX), which is expected to have higher efficacy and lower toxicity. It successfully separated LeuDOX from DOX and its major metabolite, doxorubicinol (DOXol). The metabolism of LeuDOX in four subcellular fractions of human uterine sarcoma cells suggested that LeuDOX is mainly activated to DOX in the lysosome-enriched fraction which contains hydrolytic enzymes.
Metabolism of DOX in isolated subcellular fractions from young and old rat livers was investigated by MEKC-LIF, suggesting that this technique is adequate to investigate the effect of aging on the metabolism of DOX at the subcellular level. The study showed that the young rat liver is more metabolic active than that of the old rat.
In subcellular drug analysis, the purity of the subcellular fractions limits the determination of the subcellular localization of metabolites. Organelles with high purity and biological function are necessary to refine the understanding of DOX metabolism in a specific organelle type. An immunoisolation method based on the use of an antibody specific to a peroxisomal membrane protein was developed to isolate peroxisomes with low levels of contaminating mitochondria and lysosomes. The metabolism of DOX and a model compound, BODIPY fatty acid analog, showed possible peroxisomal biotransformation of these xenobiotics.
The techniques and methodologies developed in the dissertation work would be the basis of future developments including assessing the function of new prodrugs at the cellular and subcellular levels, profiling subcellular specific metabolism and monitoring drug distribution and metabolism in tissue cross-sections. These measurements are necessary to understand the efficacy and toxicity of chemotherapy drug treatments in preclinical and clinical studies.
University of Minnesota Ph.D. dissertation. January 2011. Major: Chemistry. Advisor: Edgar A. Arriaga. 1 computer file (PDF); xiv, 182 pages, appendices A-D.
Development of capillary electrophoretic-based techniques to analyze doxorubicin in tissues, cells and subcellular fractions..
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