Preparation, Analysis And PEGylation Of Epothilone B

Epothilones （Epo） are16-membered macrolides. They were originally found in themyxobacterium Sorangium cellulosum. Their activity, like that of paclitaxel, involvesinduction of tubulin polymerization and stabilization of microtubule assembly.Epothilones showed broad activities against a variety of human malignances in cellculture and xenograft models, moreover, they also showed dramatically potency againstmultiple drug resistant （MDR） tumor cell lines which even resistant to Taxol. The firstdrug in this class, ixabepilone, has been approved by FDA for the treatment of breastcancer and ovarian cancer in2007. Several other epothilones have entered humanclinical trials.In our study, the extraction and preparation of epothilone B were optimized. Thefed-batch broth of500L fomenter was adsorbed using adsorber resin. Then, theadsorber resin was extracted with defferent concentrations of ethanol solution.Epothilone B was separated by chromatography on a Sephadex LH-20column andC18reversed-phase column. The structure of epothilone B was confirmed by NMR.In order to analysis of Epothilone B in purified products and the fermentation brothof Sorangium cellulosum, we developed an ultra performance liquid chromatographycoupled with time-of-flight mass spectrometry （UPLC-Q-TOF/MS） method. EpothiloneB was determinate with an ACQUITY UPLC HSS T3（2.1×50mm，1.8μm）by gradientelution using methanol and0.1%formic acid as mobile phase. ESI source was used anddata were acquired in negative mode. The UPLC-Q-TOF/MS method is simple and fastfor the rapid determination of Epothilone B.PEGylation of antitumor drug can improve the therapeutic effects. In our study,PEGylation was introduced at the7-OH position of Epothilone B with theMPEG5000-COOH and MPEG20000-COOH. Structures were confirmed using TLC, 1H-NMR.The molecular mass of the conjugates were measured by MALDI/TOF-MS.The result showed that one Epothilone B molecule was conjugated with one PEGpolymer molecule. The new epothilone B-PEG conjugation products were tested forinhibiting the growth of tumor cells. The results showed that the IC50of MPEG5000-EpoB and MPEG20000-Epo B are0.57μM and8.4×10^-3μM respectively against MCF-7cellsline in vitro.