Determinants for the drug release from T-0128, camptothecin analogue-carboxymethyl dextran conjugate

To improve pharmacological profiles of camptothecins (CPTs), a new macromolecular prodrug, denoted T-0128, was synthesized. This prodrug comprises a novel CPT analog (T-2513: 7-ethyl-10-aminopropyloxy-CPT) bound to carboxymethyl (CM) dextran through a Gly-Gly-Gly linker, with a molecular weight of 130 kDa. The present study was designed to elucidate the mechanisms that promote the release of linked T-2513. First, we compared the abilities of a rat liver homogenate, a cocktail of its lysosomal enzymes, and different types of pure enzymes, to liberate T-2513 from the conjugate. The releasing rate in the homogenate was very slow, but was accelerated with the lysosomes. Lysosomal cysteine proteinases, such as cathepsin B, were responsible, coupled with the results of in vitro and in vivo inhibition studies using proteinase inhibitors. The pH optimum for the cathepsin B-mediated drug release was approximately 4. This corresponds to the pH in lysosomes, suggesting lysosomotropic release. Second, to assess the effect of the length and composition of the peptidyl linker, we synthesized the conjugates with a different linker and compared the drug-releasing rates. We found that the insertion of Phe into Gly-Gly-Gly allowed various kinds of enzymes to produce a rapid cleavage, and the Gly-chain lengthening enhanced the lysosome-mediated drug release. The released T-2513 levels in the liver and tumor of the tumor-bearing rats dosed with each conjugate increased with the length of Gly linker, suggesting a good in vitro to in vivo relationship. Comparative efficacy studies of the conjugates with a different linker demonstrated that T-0128 showed the maximum efficacy against MX-1 human mammary xenograft tumors. Thus the Gly-Gly-Gly linker exploits lysosomal cathepsin B to liberate T-2513 slowly and steadily, resulting in improved therapeutic efficacy.