Design, synthesis, and biological evaluation of a dual tumor-specific motive containing integrin-targeted plasmin-cleavable doxorubicin prodrug.

Franciscus M H de Groot, Henk J Broxterman, Hans P H M Adams, Alexandra van Vliet, Godefridus I Tesser, Yvonne W Elderkamp, Astrid J Schraa, Robert Jan Kok, Grietje Molema, Herbert M Pinedo, Hans W Scheeren

Index: Mol. Cancer Ther. 1(11) , 901-11, (2002)

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Abstract

The design, synthesis, and initial biological evaluation of a doxorubicin prodrug that contains a dual tumor specific moiety, which allows enhanced tumor recognition potential, is reported. Both a tumor-specific recognition site and a tumor selective enzymatic activation sequence are incorporated in the prodrug. The first tumor-specific sequence is the bicyclic CDCRGDCFC (RGD-4C) peptide that selectively binds alpha v beta 3 and alpha v beta 5 integrins. These integrins are highly overexpressed on invading tumor endothelial cells. The second tumor-specific sequence is a D-Ala-Phe-Lys tripeptide that is selectively recognized by the tumor-associated protease plasmin, which is involved in tumor invasion and metastasis. An aminocaproyl residue was incorporated as a spacer between the two peptide sequences, whereas a self-eliminating 4-aminobenzyl alcohol spacer was inserted between the plasmin substrate and doxorubicin. Although the prodrug showed a decreased binding affinity as compared with the unconjugated reference peptide, it was still a potent ligand for alpha v beta 3 and alpha v beta 5 integrin receptors. The synthesized construct also possessed plasmin substrate properties as demonstrated by doxorubicin release from 1 upon incubation with plasmin. The release of doxorubicin from 1 was not complete, possibly related to low prodrug solubility. In vitro prodrug 1 showed plasmin-dependent cytotoxicity for endothelial cells and HT1080 fibrosarcoma cells. On the basis of these in vitro results, derivatives of 1 with improved water solubility are considered good candidates for additional development and in vivo evaluation of this dual targeting concept.