Conjugation of antimitotic colchicine derivatives with proprietary lipophilic molecules (Zyn-Linkers™) via acid cleavable linkages (hydrazone or imine) produced prodrugs with enhanced antitumor activity. The pharmacodynamic properties (half-lives) of the pH-sensitive linkages were determined around values which might be expected in intracellular, especially lysosomal, environments. In buffered solutions at pH 4.1, 5.6, and 7.2, conjugates released 50% of the drug at rates from 0.1 h at pH 4.1 to over 3 months at neutral pH. In a tumor cell cytotoxicity assay, the slowest releasing molecules were up to 100-fold less active in vitro than the unlinked drug, which demonstrated that the linkages were stable and that a true prodrug had been generated. Binding of conjugate to cells, release of active drug and action on an intracellular target (tubulin) were demonstrated by the ability of the conjugates to block cells in the G2/M phase of the cell cycle 20 h after a 10 min exposure and was consistent with the rate of drug released. In vivo, a single injection of the Zyn-Linked conjugated colchicines enhanced the survival time of the mice to a greater extent than single doses of the unlinked colchicine analogues in a murine tumor model. Activity of the slowest releasing conjugate (ZYN 162) was shown to be comparable to a single LD 10 dose of doxorubicin. These data suggest that the Zyn-Linker conjugates described containing acid cleavable bonds form a body depot of prodrug which release an anti-mitotic agent and can significantly inhibit tumor growth.
