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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Anthracycline-Gonadotropin Releasing Hormone-IIIBioconjugates: Synthesis, Antitumor Activity and in vitroDrug ReleaseGábor Mező 1 , Ulrike Leurs 2 , Pascal Schlage 2 , Erika Orbán 1 ,Ildikó Szabó 1 , Szilvia Bősze 1 , and Marilena Manea 2,31 Research Group of Peptide Chemistry, Eötvös L University, Budapest, Hungary;2 Laboratory of Analytical Chemistry, University of Konstanz, Konstanz, Germany;3 Zukunftkolleg, University of Konstanz, Konstanz, GermanyIntroductionChemotherapy is still the main approach for the treatment of advanced or metastatic cancer.However, due to the lack of selectivity for tumor tissues, the application of free anticancerdrugs can lead to severe side effects and low cure rates. Therefore, targeted delivery ofanticancer drugs is one of the most actively pursued goals in cancer chemotherapy.Bioconjugates with receptor mediated tumor-targeting functions and carrying cytotoxicagents should enable the specific delivery of chemotherapeutics to malignant tissues, thusincreasing their local efficacy while limiting the peripheral toxicity [1]. It was found thatreceptors for peptide hormones, such as gonadotropin-releasing hormone (GnRH), areexpressed in higher amount on cancer cells compared to normal cells. Consequently, GnRHand its derivatives can be used as targeting moieties to deliver cytotoxic agents directly totumor cells [2,3]. One of the most promising natural GnRH analogs is lamprey GnRH-III(Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH 2 ) which specifically binds to GnRHreceptors on cancer cells and has lower hormonal effect in mammals than the human GnRH[4,5]. It was shown that the modification of the side chain of 8 Lys or the replacement of4 Ser by Lys or Lys(Ac) did not result in a significant change of the biological activity ofGnRH-III [6,7]. In our previous work, the antineoplastic agent daunorubicin (Dau) wasattached to GnRH-III via oxime linkage resulting in bioconjugates (Glp-His-Trp-Ser-His-Asp-Trp-Lys(Dau=Aoa-X)-Pro-Gly-NH 2 , where X is 0 or Gly-Phe-Leu-Gly spacer) thathad both in vitro and in vivo antitumor activity and no significant toxic side effects [8].The major goal of the present work was to increase the antitumor activity ofDaunorubicin – GnRH-III bioconjugates by modifying: (i) the GnRH-III in position 4;(ii) the number of drug molecules attached to the carrier; (iii) the linkage between the drugand GnRH-III hormone peptide.Results and DiscussionSerine in position 4 of GnRH-III(Dau=Aoa) was replaced by N-MeSer or acetylated lysine(Lys(Ac), see Figure 1B). Both compounds showed not only increased enzymatic stabilitytoward chymotrypsin, but also increased in vitro cytostatic effect on MCF-7 human breastand HT-29 human colon cancer cell lines (Table 1). The enzymatic stability of [N- 4 MeSer]-GnRH-III(Dau=Aoa) was higher than that of [ 4 Lys(Ac)]-GnRH-III(Dau=Aoa). In case ofGnRH-III(Dau=Aoa), the peptide bond Trp 3 -Ser 4 was almost completely cleaved bychymotrypsin within 6 hrs, while only 20% of [N- 4 MeSer]-GnRH-III(Dau=Aoa) wasdegraded during this time.The incorporation of an additional oxime-linked Dau to the side chain of 4 Lys (Glp-His-Trp-Lys(Dau=Aoa)-His-Asp-Trp-Lys(Dau=Aoa)-Pro-Gly-NH 2 , Figure 1C) did notincrease the in vitro antitumor activity further (Table 1). The reason might be the differentmetabolic pathway of the two Dau containing lysine residues, process which will be furtherinvestigated.A bioconjugate containing the self-imolative PABC linker (Figure 1A) was alsoprepared according to Dubowchik, et al. [9]. Although the degradation of this compound bycathepsin B or in rat liver lysosomal homogenate showed free Dau release, its in vitrocytostatic effect was not higher than that observed in the case of oxime bond-linkedbioconjugates (that do not provide free Dau under the same conditions). The determinationof the in vivo toxicity and antitumor activity of the bioconjugates is in progress.524