title: GnRH-III based multifunctional drug delivery systems containing daunorubicin and methotrexate.
abstract: Here we report on the design, synthesis and biochemical characterization of multifunctional bioconjugates containing two chemotherapeutic agents, daunorubicin and methotrexate, coupled to the GnRH-III decapeptide, which served as a targeting moiety. This represents a possible approach to increase the receptor mediated tumor targeting and consequently the cytostatic effect of anticancer drug-peptide bioconjugates. The multifunctional bioconjugates were prepared according to two drug design approaches recently developed by our group. Both bifunctional GnRH-III derivatives, [(4)Lys]-GnRH-III (Glp-His-Trp-Lys-His-Asp-Trp-Lys-Pro-Gly-NH(2)) and [(8)Lys(Lys)]-GnRH-III (Glp-His-Trp-Ser-His-Asp-Trp-Lys(Lys)-Pro-Gly-NH(2)), contain two free amino groups suitable for the attachment of two anticancer drugs, such as methotrexate and daunorubicin. The drugs were chosen with respect to their different mechanisms of action, with the goal of increasing the antitumor effect of the bioconjugates. The in vitro cytostatic effect of the bioconjugates was determined on MCF-7 human breast, HT-29 human colon and LNCaP human prostate cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their in vitro stability/degradation in human serum and in the presence of rat liver lysosomal homogenate was investigated by liquid chromatography in combination with mass spectrometry. The influence of the multifunctional bioconjugates on the cell adhesion and cell proliferation was studied on Mono Mac 6 human leukemic monocytes. It was found that (1) all synthesized bioconjugates had in vitro cytostatic effect; (2) they were stable in human serum for at least 24 h; (3) they were hydrolyzed in the presence of lysosomal homogenate and (4) they exerted a moderate cell-cell adhesion inducing effect. These results demonstrate that multifunctional bioconjugates containing two different anticancer drugs attached to the same GnRH-III targeting moiety could be successfully prepared and resulted in higher in vitro cytostatic effect than the monofunctional bioconjugates containing either methotrexate or daunorubicin, in particular on HT-29 human colon cancer cells.
authors.affiliation: Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry, University of Konstanz, 78457 Konstanz, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Leurs
authors.forename: Eszter
authors.initials: E
authors.lastname: Lajkó
authors.forename: Gábor
authors.initials: G
authors.lastname: Mező
authors.forename: Erika
authors.initials: E
authors.lastname: Orbán
authors.forename: Peter
authors.initials: P
authors.lastname: Öhlschläger
authors.forename: Andreas
authors.initials: A
authors.lastname: Marquardt
authors.forename: László
authors.initials: L
authors.lastname: Kőhidai
authors.forename: Marilena
authors.initials: M
authors.lastname: Manea
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000097:blood / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002448:Cell Adhesion; D049109:Cell Proliferation / Q000187:drug effects; D003630:Daunorubicin / Q000097:blood / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D004337:Drug Carriers / Q000737:chemistry*; D004355:Drug Stability; D007987:Gonadotropin-Releasing Hormone / Q000737:chemistry*; D006801:Humans; D008099:Liver / Q000166:cytology; D008247:Lysosomes / Q000378:metabolism; D008727:Methotrexate / Q000097:blood / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry*; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D051381:Rats
chemical_list: D000970:Antineoplastic Agents; D004337:Drug Carriers; D010446:Peptide Fragments; C448022:gonadotropin-releasing hormone-III; D007987:Gonadotropin-Releasing Hormone; D011761:Pyrrolidonecarboxylic Acid; D008727:Methotrexate; D003630:Daunorubicin