For drug-targeting aimed at the treatment of LHRH receptor overexpressing cancers the LHRH receptor agonistic peptide D-Lys6-LHRH has been conjugated to the novel highly cytotoxic natural compound Disorazol Z. As shown previously by early proof of concept in an ovary cancer xenograft model, differentially linked conjugates possess a high potential regarding the treatment of LHRH-R positive tumors [1]. Here we present further characterization of these conjugates with respect to PK/PD parameter and provide evidence that LHRH receptor targeting significantly contributes to their mechanism of action.

Materials and Methods

LHRH-R competitive binding, calcium release and cytotoxic activity were measured by Tag-Lite technology (Cisbio), and Fluo-4 (Invitrogen) or Resazurin-based detection, respectively. PK parameter were assessed by standard procedures followed by LC-MS/MS analysis. For the xenograft studies, tumor fragments were transplanted subcutaneously in female nude mice and treatment was started at a tumor size of approx. 100 mm3.


Disorazol Z - D-Lys6-LHRH conjugates showed varying cytotoxic activity from single digit nanomolar to higher submicromolar EC50 values but comparable LHRH receptor binding and activation in the low nanomolar EC50 range. Comparison in ovarian and triple negative breast cancer xenograft models revealed potent inhibition of tumor growth for the conjugates, whereas equimolar dosing of Disorazol Z failed to reach statistical significance.

PK analysis showed substantial plasma levels for the conjugates with only minor release of Disorazol Z, pointing to stabilization by conjugation and demonstrating reasonable half-life of the intact conjugates as prerequisite for tumor targeting.

In the same tumor models, competition by previous administration of D-Lys6-LHRH provides evidence for LHRH receptor targeting as mechanism of action. Increased sensitivity of LHRH receptor overexpressing cells towards conjugate cytotoxicity, i.e. leading to about 30 fold decreased EC50 values for the conjugate AEZS-125, further supports the LHRH-R dependency of conjugate efficacy.


The presented LHRH receptor-dependent efficacies of Disorazol Z - D-Lys6-LHRH conjugates in vitro and in mouse xenograft models support the principle of tumor targeting by the LHRH receptor as already employed by the drug candidate AEZS-108, which is currently in phase II clinical studies. Preclinical development of Disorazol Z conjugates will be started in the first half of 2013.

Citation Format: Babette Aicher, Tilmann Schuster, Lars Blumenstein, Antje Schubert, Carsten Gründker, Joerg B. Engel, Olaf Ortmann, Rolf Mueller, Eckhard Guenther, Matthias Gerlach, Michael Teifel. LHRH receptor targeting as mechanism of anti-tumor activity for cytotoxic conjugates of Disorazol Z with the LHRH receptor agonistic peptide D-Lys6-LHRH. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5476. doi:10.1158/1538-7445.AM2013-5476

Aicher, et al ( 
), Highly potent cytotoxic conjugates of Disorazol Z linked to a LHRH receptor targeting peptide interfere with cell cycle progression in human cancer cell lines and suppress tumor growth in a LHRH receptor positive ovarian cancer xenograft model, EORTC-NCI-AACR, November 6–9, 
, Dublin.