Glucuronidase immobilized in nanoparticles for use in site specific activation of anti-cancer glucuronide prodrugs

Abstract

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, -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 -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 -glucuronidase in nanoparticles. Formation of free p-nitrophenol was determined by HPLC with UV detection. Homoharringtonine (HHT, Omacetine, Synribo), a highly potent chemotherapy agent, was initially chosen for an anti-cancer glucuronide prodrug for activation with -glucuronidase embedded in nanoparticles. HHT's aliphatic alcohol may be conjugated with -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 -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 -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 -glucuronidase endogenous in microbial bio-flora within humans or naturally produced -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, invivo. After biosynthesis and HPLC purification of the anti-cancer glucuronide prodrug epirubicin glucuronide, cleavage by β-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 invivo 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 β-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 -glucuronidase, is shown in-vitro. Nanoparticle targeting strategies outlined, in Chapter IV, with the future directions sections of this paper complete the thesis.