Glucuronidase Immobilized in Nanoparticles for Use in Site Specific Activation of Anti-Cancer Glucuronide Prodrugs.

摘要

The site-specific treatment of cancer can reduce the toxic side effects of chemotherapy. This thesis reviews current techniques and describes a nanotechnology approach to investigate some of the obstacles in site-specific drug targeting and activation. One site-specific approach is antibody-directed enzyme prodrug therapy, ADEPT. For this strategy, a targeting antibody directed against a tumor antigen is connected to an activating enzyme. For this project, beta-glucuronidase was selected as the activating enzyme and glucuronide prodrugs, of known highly potent chemotherapeutic agents, were selected as enzyme substrates. Prodrug-activating enzymes localizing exclusively at a tumor site, with tumor-specific targeting nanoparticles, minimizes the exposure of active chemotherapeutic agents. Because of the inactivity of glucuronide prodrugs, this treatment does not kill healthy cells.;This thesis reviews current techniques on glucuronide production and is a description of a beta-glucuronidase immobilization in nanoparticles procedure that investigates some of the obstacles in site-specific drug activation. Chapter I is an introduction to glucuronides, the glucuronidation procedure, and enzyme immobilization. Chapter II is a description of the glucuronidation of 4-nitrophenol, epirubicin, and homoharringtonine. It begins with the synthesis of 4-nitrophenyl-glucuronide. 4-Nitrophenol is a classic substrate for glucuronidation, is easy to prepare, and was used to evaluate the conditions for glucuronide formation and cleavage with beta-glucuronidase in nanoparticles. Formation of free p-nitrophenol was determined by HPLC with UV detection.;Homoharringtonine (HHT, Omacetine, Synribo(TM)), a highly potent chemotherapy agent, was initially chosen for an anti-cancer glucuronide prodrug for activation with beta-glucuronidase embedded in nanoparticles. HHT's aliphatic alcohol may be conjugated with beta-D-glucuronic acid, either by chemical or biosynthetic methods, to produce the desired glucuronide. A glucuronide of Homoharringtonine has not been reported in literature and its production is of interest for researchers to pharmaceutically evaluate a new anti-cancer glucuronide prodrug. Since HHT is such a potent cancer drug, it would be of interest to compare the cleavage of HHT-glucuronide by beta-glucuronidase to a well-studied compound such as epirubicin glucuronide; that has been evaluated as ADEPT stragety.;Unfortunately, synthetic methods (the Koenig-Knorr reaction, failed to produce the desired HHT-glucuronide. Consequently, experiments with beta-glucuronidase entrapped-nanoparticles were conducted with p-nitrophenol glucuronide and epirubicin glucuronide. When preparations of a glucuronide of HHT fail, due to steric hindrance, epirubicin is chosen as an alternative. Epirubicin glucuronide is mostly not activated by beta-glucuronidase endogenous in microbial bio-flora within humans or naturally produced beta-glucuronidase within human liver and other tissues (Hasima, H.J.,et al. 1992). Lack of promiscuity in glucuronide cleavage is possible to be beneficial in retaining site-specific activation. The production of epirubicin glucuronide is catalyzed by the human enzyme UDP-Glucuronosyltransferase 2B7 (UGT 2B7), in the liver (Innocenti, F., et al 2001).;Toxic side effects of chemotherapeutic drugs are overcome with their glucuronides by localizing activity to a target tumor site with the activating enzyme encapsulated in a nanoparticle, in-vivo. After biosynthesis and HPLC purification of the anti-cancer glucuronide prodrug epirubicin glucuronide, cleavage by beta-Glucuronidase was tested in-vitro. A large amount of enzyme (100 U/ml of glucuronidase in 4mM phosphate buffer pH=6.8) is needed to activate the prodrug. An added benefit of protein encapsulation is to prevent proteins being recognized as foreign in-vivo and consequently degraded.;In Chapter III, a suitable polymer for encapsulation of glucuronidase is alginic acid cross-linked with the addition of calcium ions, displacing sodium, forming alginate nanoparticles. The materials produce nano-droplet sized emulsions and the denaturing of protein and reduction of enzyme activity is not significant (Nesamony, J., et al. 2012). Optimization of the polymerizing procedure and material concentrations produce a nanoparticle size range appropriate for protein drug delivery. Sodium alginate, polymerization by the displacement of sodium ions with cross-linking calcium ions, is effective for the entrapment of beta-glucuronidase that produces active microparticles (Burgess, D. J., and S. Ponsart. 1998). The strongly polar property of alginate is a suitable environment for activity during entrapment in nanoparticles. Active glucuronidase immobilization in nanoparticles is produced and an increase in activity, over standalone beta-glucuronidase, is shown in-vitro. Nanoparticle targeting strategies outlined, in Chapter IV, with the future directions sections of this paper complete the thesis.