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Integrins, especially integrin alpha vbeta3, are attractive receptors for vascular targeting strategies. Recently, a divalent RGD peptidomimetic, E-[c(RGDfK)2], has been described that demonstrates increased uptake in human ovarian carcinoma OVCAR-3 xenograft tumors. Inspired by these results, we set out to develop doxorubicin conjugates with E-[c(RGDfK)2] by binding two different maleimide derivatives of doxorubicin to E-[c(RGDfK)2] that was thiolated with iminothiolane. In this way, two water-soluble derivatives were obtained, E-[c(RGDfK)2]-DOXO-1 and E-[c(RGDfK)2]-DOXO-2. In E-[c(RGDfK)2]-DOXO-1, doxorubicin was bound to the peptide through a stable amide bond, and in E-[c(RGDfK)2]-DOXO-2, a MMP-2/MMP-9 cleavable octapeptide was introduced between doxorubicin and the peptide. The rationale for a MMP-2/MMP-9-cleavable linker was that MMP-2 and MMP-9 bind to integrin alpha vbeta3 and both are overexpressed in tumor vasculature. In addition, analogous control doxorubicin-containing peptides bearing c(RADfK) that does not bind to integrin alpha vbeta3 were synthesized, i.e., c(RADfK)-DOXO-1 and c(RADfK)-DOXO-2. Whereas E-[c(RGDfK) 2]-DOXO-2 was cleaved effectively by MMP-2 and in OVCAR-3 tumor homogenates releasing a doxorubicin-tetrapeptide or doxorubicin as the final cleavage product, no release of doxorubicin was observed for E-[c(RGDfK)2]-DOXO-1. Proliferation of HUVEC in the presence of MMP-2-cleavable doxorubicin-containing peptides exhibited 6- to 10-fold increased inhibition compared to the amide-linked doxorubicin-containing peptides. In addition, inhibition of HUVEC sprouting during a 24 h exposure was approximately 3-fold stronger for E-[c(RGDfK) 2]-DOXO-2 and 20-fold stronger for the reference peptide conjugate c(RADfK)-DOXO-2 than for doxorubicin alone. In vivo studies in an OVCAR-3 xenograft model demonstrated no or only moderate antitumor efficacy for either E-[c(RGDfK)2], E-[c(RGDfK)2]-DOXO-1, E-[c(RGDfK)2]-DOXO-2, or c(RADfK)-DOXO-2, even at doses of 3 x 24 mg/kg doxorubicin equivalents, compared to an improved antitumor effect for doxorubicin at 2 x 8 mg/kg.

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