Abstract
We have previously shown that the bacterial enzyme thiaminase 1 has antitumor activity. In an attempt to make thiaminase I a more effective pharmaceutical agent, we have modified it by adding polyethylene glycol (PEG) chains of various lengths. We were surprised to find that 5k-PEGylation eliminated thiaminase cytotoxic activity in all cell lines tested. Both native thiaminase and 5k-PEGylated thiaminase efficiently depleted thiamine from cell culture medium, and both could use intracellular phosphorylated thiamine as substrates. However, native enzyme more effectively depleted thiamine and thiamine diphosphate in RS4 leukemia cell cytosol, and native thiaminase depressed cellular respiration, whereas PEGylated thiaminase did not. Despite the lack of in vitro cytotoxicity, PEGylation markedly increased the in vivo toxicity of the enzyme. Pharmacokinetic studies revealed that the half-life of native thiaminase was 1.5 h compared with 34.4 h for the 5k-PEGylated enzyme. Serum thiamine levels were depleted by both native and 5k-PEGylated enzyme. Despite superior pharmacokinetics, 5k-PEGylated thiaminase showed no antitumor effect against an RS4 leukemia xenograft, in contrast to native thiaminase, which showed antitumor activity. PEGylation of thiaminase I has demonstrated that depression of mitochondrial function contributes, at least in part, to its anticancer activity. PEGylation also enhances plasma retention time, which increased its vivo toxicity and decreased its activity against a leukemia xenograft, the opposite of the desired effects. These studies suggest that the mechanism of anticancer cytotoxicity of thiaminase requires acute depression of cellular respiration, whereas systemic toxicity is related to the duration of extracellular thiamine depletion.
Footnotes
This work was supported by the William Lawrence and Blanche Hughes Foundation and DanceBlue, an effort of the University of Kentucky student community to support pediatric oncology care and research.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS:
- PEG
- polyethylene glycol
- LCPTE
- linear chain PEGylated thiaminase enzyme
- OCR
- oxygen consumption rate
- ECAR
- extracellular acidification rate
- EFS
- event-free survival
- MTD
- maximum tolerated dose
- H&E
- hematoxylin and eosin
- HPLC
- high-performance liquid chromatography
- ATCC
- American Type Culture Collection
- AUC
- area under the curve
- STR
- short tandem repeat.
- Received January 20, 2012.
- Accepted March 16, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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