title: In vitro and in vivo evaluation of doxorubicin conjugates with the divalent peptide E-[c(RGDfK)2] that targets integrin alphavbeta3.
abstract: 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.
authors.affiliation: Tumor Biology Center, Breisacher Strasse 117, 79106 Freiburg, Federal Republic of Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Ryppa
authors.forename: Hagit
authors.initials: H
authors.lastname: Mann-Steinberg
authors.forename: Iduna
authors.initials: I
authors.lastname: Fichtner
authors.forename: Holger
authors.initials: H
authors.lastname: Weber
authors.forename: Ronit
authors.initials: R
authors.lastname: Satchi-Fainaro
authors.forename: Martin L
authors.initials: ML
authors.lastname: Biniossek
authors.forename: Felix
authors.initials: F
authors.lastname: Kratz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004317:Doxorubicin / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D005260:Female; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D051379:Mice; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D011355:Prodrugs / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
chemical_list: D039302:Integrin alphaVbeta3; D009842:Oligopeptides; D011355:Prodrugs; C047981:arginyl-glycyl-aspartic acid; D004317:Doxorubicin