UC San Diego

This thesis describes the development of acid-sensitive N- ethoxybenzylimidazoles (NEBIs) for use as linkers in drug delivery systems (DDSs). A common characteristic of many DDSs is the rapid internalization and intracellular localization of the DDS into the acidic endosomes and lysosomes of cells upon arrival to the targeted tissue. NEBIs, like other acid-sensitive moieties, can serve as crosslinkers for DDSs to conjugate small molecule drugs to macromolecules. While these conjugates are stable at physiological conditions, they hydrolyze in mildly acidic environments for the triggered release of small molecule drugs. Chapter 2 describes the initial studies performed to characterize the NEBI platform. We found that hydrolysis of the NEBI at pH 5.5 exhibited a > 10-fold acceleration in rate of hydrolysis compared to in solutions at normal, physiological pH. Chapter 3 describes the incorporation of an amine containing chemotherapeutic, doxorubicin, to the NEBI, and its hydrolytic properties and cytotoxicity. Chapter 4 describes the design of an acid-sensitive bifunctional NEBI cross-linker with a carboxylic acid functionality on one side and an azide functionality on the other side. This bifunctional crosslinker was used to construct a model DDS, by conjugating the anticancer drug doxorubicin to a human serum album model carrier. This model DDS was used to evaluate the utility of a NEBI as an acid-sensitive linker by analyzing the uptake and activity of the DDS on human ovarian cancer cells. Our most difficult challenge in Chapter 4, the inability to release native drug, was addressed in Chapter 5. An ortho-NEBI was designed to undergo two sequential hydrolysis steps, hydrolysis of the NEBI followed by the hydrolysis of an ester, to release native alcohol containing drugs. Studies with 5-fluoro-2'- deoxyuridine (5-FU) conjugated to the ortho-NEBI via an ester bond found that native 5-FU was released from this ortho-NEBI system. The cytotoxicity of the ortho-NEBI-5-FU exhibited IC50 values equal in the same order of magnitude of native 5-FU. Chapters 6 and 7 describe attempts in releasing amine containing drugs from the ortho-NEBI platform and studies towards the development of methods for site selective conjugation of a drug to a breast cancer targeting antibody, trastuzumab