Radwan Kiwan

Antitumor activity of new liposomal prodrug of mitomycin C in multidrug resistant solid tumor: Insights of the mechanism of action

The antitumor activity of a novel thiolytically cleavable lipid-based prodrug of mitomycin C (MMC) delivered by STEALTH® liposomes (SL) was studied in drug resistant human ovarian carcinoma A2780/AD model and compared with free MMC and both free and SL forms of an established anticancer drug—doxorubicin (DOX). It was found that SL-prodrug (SL-pMMC) possessed enhanced antitumor activity when compared with the parent MMC, free DOX, and SL-DOX. An observance of the high antitumor efficiency of SL-pMMC was a result of its preferential accumulation in the tumor by the enhanced permeability and retention (EPR) effect, suppression of multidrug resistance (MDR) associated with P-glycoprotein and MRP drug efflux pumps, activation of caspase-dependent apoptosis signaling pathways and suppression of antiapoptotic cellular defense by increasing the BAX/BCL2 ratio. Consequently, the described SL-pMMC formulations can be considered good candidates for the chemotherapy of multidrug resistant tumors.

Optimization, Production, and Characterization of a CpG-Oligonucleotide-Ficoll Conjugate Nanoparticle Adjuvant for Enhanced Immunogenicity of Anthrax Protective Antigen

We have synthesized and characterized a novel phosphorothioate CpG oligodeoxynucleotide (CpG ODN)-Ficoll conjugated nanoparticulate adjuvant, termed DV230-Ficoll. This adjuvant was constructed from an amine-functionalized-Ficoll, a heterobifunctional linker (succinimidyl-[(N-maleimidopropionamido)-hexaethylene glycol] ester) and the CpG-ODN DV230. Herein, we describe the evaluation of the purity and reactivity of linkers of different lengths for CpG-ODN-Ficoll conjugation, optimization of linker coupling, and conjugation of thiol-functionalized CpG to maleimide-functionalized Ficoll and process scale-up. Physicochemical characterization of independently produced lots of DV230-Ficoll reveal a bioconjugate with a particle size of approximately 50 nm and covalent attachment of more than 100 molecules of CpG per Ficoll. Solutions of purified DV230-Ficoll were stable for at least 12 months at frozen and refrigerated temperatures and stability was further enhanced in lyophilized form. Compared to nonconjugated monomeric DV230, the DV230-Ficoll conjugate demonstrated improved in vitro potency for induction of IFN-α from human peripheral blood mononuclear cells and induced higher titer neutralizing antibody responses against coadministered anthrax recombinant protective antigen in mice. The processes described here establish a reproducible and robust process for the synthesis of a novel, size-controlled, and stable CpG-ODN nanoparticle adjuvant suitable for manufacture and use in vaccines.

Reversible PEGylation: Thiolytic Regeneration of Active Protein from Its Polymer Conjugates

Polyethylene glycol (PEG) is known to convey to its protein conjugates a number of useful properties, including prolonged plasma circulation time, reduced immunogenicity, improved solubility, and resistance to proteolytic degradation [1]. Usually there is a direct relationship between the amount of the linked polymer and the gain in these characteristics. On the other hand, attachment of multiple chains of PEG to a protein, a process known as PEGylation, is often accompanied by a substantial loss of biological activity. This is a particularly severe problem for proteins acting on large substrates, e.g. receptor-binding proteins [1]. To circumvent this problem we introduced new linking chemistry (Figure 1) designed to produce gradual in vivo loss of PEG chains from its conjugates. Just as a promoiety of a prodrug, PEG would be present in these reversible conjugates only temporarily, improving such characteristics as pharmacokinetics and biodistribution. Here we communicate some of our initial results using lysozyme and cysteine as models of a protein and a cleaving agent respectively. Lysozyme is similar in size (14.5 kDa) to various cytokines and chemokines; it has six lysines available for PEGylation; and its activity directed toward a large substrate, peptidomurein of bacterial cell wall.