A doxorubicin-CNGRC-peptide conjugate with prodrug properties

Yvette Van Hensbergen, Henk J. Broxterman, Yvonne W. Elderkamp, Jan Lankelma, Judith C C Beers, Marc Heijn, Epie Boven, Klaas Hoekman, Herbert M. Pinedo

Research output: Contribution to journalArticlepeer-review

71 Scopus citations


There is increasing interest in the exploitation of molecular addresses for the targeting of tumor imaging or therapeutic agents. A recent study demonstrated anticancer activity in human xenografts of doxorubicin (DOX)-peptide conjugates targeted to the tumor vascular endothelium, among them DOX coupled to the cyclic pentapeptide CNGRC [Science 279 (1998) 377]. In order to learn more about the mechanism of action of this type of DOX-peptide conjugates, we have studied the interaction of DOX-CNGRC with primary human umbilical cord vein endothelial cells (HUVEC) and tumor cells under defined in vitro conditions. We used a DOX conjugate, in which the cyclic CNGRC peptide, for which an in vivo endothelial address has recently been identified as aminopeptidase N (APN)/CD13, has been coupled via a hydrolysable spacer to the C-14 anthracycline-side chain. First we determined that the t1/2 of DOX-CNGRC conjugate in human blood was 442min (at 37°) allowing sufficient time for endothelial targeting when administered i.v. When cultured cells were exposed for 30min to DOX-CNGRC a more cytoplasmic localization of fluorescent drug was seen when compared to DOX exposure and intracellular DOX-CNGRC was identified after extraction from the cells. This revealed differences in the cellular uptake process of the conjugate compared to DOX. The antiproliferative effect of DOX-CNGRC was determined by 30min exposure in medium with a high protein content in order to mimick the in vivo targeting situation. In this medium, the IC50 was 1.1μM for highly CD13 expressing HT-1080, 1.45μM for CD13 negative SK-UT-1 sarcoma cells and 6.5μM for CD13 positive HUVEC. The IC50 of DOX for these cells were 1.0, 2.0 and 7.3μM, respectively. Although DOX-CNGRC inhibited the peptidase activity of CD13 up to 50%, our data do not favor an important role for the enzyme inhibition in the cytotoxic effect of the conjugate. The antitumor activity was tested in nude mice bearing human ovarian cancer xenografts (OVCAR-3). A weekly i.v. administration (3mg/kg DOX-equivalent, 3×) showed a minor (40%) growth delay, which does not indicate efficacy better than that expected for free DOX. In conclusion, this study indicates that the antiproliferative and anti-angiogenic effects of DOX-CNGRC as reported before, are likely caused by the cytostatic effects of intracellularly released parent drug DOX, independent of CD13 expression/activity. More research is needed to identify the optimal specific chemical configuration of DOX-peptide conjugates for in vivo targeting and receptor-mediated cellular uptake.

Original languageEnglish (US)
Pages (from-to)897-908
Number of pages12
JournalBiochemical Pharmacology
Issue number5
StatePublished - Mar 1 2002
Externally publishedYes


  • Aminopeptidase N
  • CD13
  • Doxorubicin
  • Doxorubicin-peptide conjugate
  • Prodrug
  • Tumor vasculature

ASJC Scopus subject areas

  • Pharmacology


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