Octreotat-Benzamid-Zytostatika-Konjugate für die zielgerichtete Therapie von Somatostatinrezeptor exprimierenden Tumoren
Final Report Abstract
The aim was the development of alkylating benzamide-octreotate conjugates for anticancer drug targeting. In addition to tissue specificity (octreotate) intracellular transport should be envisioned (benzamide). Due to their enhanced DNA affinity the alkylating benzamide part is supposed to show high DNA affinity after cleavage from octreotate. Even though the benzamides show affinity to isolated DNA, they do not reach the DNA in the cell. The targeting moiety responsible for the DNA affinity of the alkylating benzamides is the N-(2- diethylaminoethyl)aminocarbamoyl pharmacophore. This pharmacophore mediates affinity to acidic cell organelles as well. After intracellular uptake the benzamides are entrapped in lysosomes and acidic cell organelles where they are protonated. The alkylating benzamides are lysosomotropic probes containing besides the A^-(2-dialkylaminoethyl)amine side chain a hydrophobic residue. After protonation they have surface-active properties. Only in acidic cell organelles they can behave as detergents, but not in neutral media. Such lysosomotropic detergents disrupt the lysosomes resulting in cell death. The disruption was proven by fluorescence microscopy. For this reason the benzamides are not suitable for intracellular DNA targeting. However, by exploiting the physiological transport and trafficking process of tumor affine peptides such as melanocortin and octreotate the peptide-conjugates of lysosomotropic detergents are targeted to the lysosomes, the compartment where lysosomotropic detergents are activated. Conjugation of a lysosomotropic detergent bearing a Ar-(2-diethylaminoethyl)aminocarbamoyl pharmacophor to tumor affine peptides (octreotate and a melanocortin analogous) resulted in receptor mediated uptake of the peptide conjugates into tumor cells expressing the peptide receptors. By coupling lysosomotropic detergents to tumor affine peptides, they became more water-soluble. As the peptide derivatives of lysosomotropic detergents are more hydrophilic and do not diffuse passively through the lipid bilayer, involvement of the peptide receptor is essential for the cellular uptake of the conjugates. During intracellular trafficking the lysosomotropic detergent was cleaved from the peptide and mediated disrupture of lysosomes as shown by confocal laser scanning microscopy. However, the conjugates showed hydrolysis in the cell culture medium of about 40% with liberation of the lysosomotropic detergent which could diffuse into the lysosomes. For this reason the compounds were cytotoxic to the control cells as well, but to a lesser extend. Conjugation of the lysosomotropic detergent to the peptides resulted in a more selective and enhanced cytotoxicity for cells expressing the receptors.
Publications
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Wolf M, Bauder-Wüst U, Eskerski H, Bauer C, Eisenhut M. Role of acidic cell organelles in the higher nonmelanoma retention of melanoma markers based on N-(2-dialkylaminoethy)benzamides and the cytotoxicity of alkylating benzamides. Melanoma Res. 2007 Apr; 17(2):61-73.
