Zoptarelin Doxorubicin

A characteristic of cancer cells including triple‐negative breast cancers (TNBC) is an increased aerobic glycolysis for ATP production representing a selective therapeutic target. More than 70% of TNBC express gonadotropin‐releasing hormone receptors (GnRH‐R). These receptors can be used for targeted chemotherapy with cytotoxic GnRH agonists such as Zoptarelin Doxorubicin, in which doxorubicin is covalently linked to [D‐Lys6]GnRH. In this study, we have analyzed whether inhibition of aerobic glycolysis can enhance the antitumor efficacy of GnRH‐R‐targeted chemotherapy using Zoptarelin Doxorubicin.

Triple‐negative breast cancers cell lines MDA‐MB‐231 and HCC1806 were treated with Zoptarelin Doxorubicin, glycolysis inhibitor 2‐deoxy‐D‐glucose (2DG) or the combination of both agents. Cell viability was measured using Alamar blue. Induction of apoptosis was quantified by measurement of loss of mitochondrial membrane potential. In vivo experiments were performed using nude mice bearing xenografted MDA‐MB‐231 tumors.

Treatment of TNBC cells with Zoptarelin Doxorubicin or with 2DG resulted in a significant decrease of cell viability and a significant increase of apoptosis. Treatment with Zoptarelin Doxorubicin in combination with 2DG resulted in significantly reduced viability and enhanced apoptosis compared with single‐agent treatments. Combinational index (CI) analysis revealed the co‐treatment effect as a synergistic. The antitumor effects of Zoptarelin Doxorubicin or 2DG were confirmed in nude mice. The tumor reducing effects of Zoptarelin Doxorubicin were enhanced by combination with 2DG.

The glycolytic phenotype of TNBC can be used to improve antitumor therapies. Co‐treatment of Zoptarelin Doxorubicin with glycolysis inhibitor 2DG might be a suitable therapeutic option for GnRH receptor‐positive TNBC.

Overexpression of gonadotropin‐releasing hormone (GnRH) receptor in many tumors but not in normal tissues makes it possible to use GnRH analogs as targeting peptides for selective delivery of cytotoxic agents, which may help to enhance the uptake of anticancer drugs by cancer cells and reduce toxicity to normal cells. The GnRH analogs [d‐Cys6, desGly10, Pro9‐NH2]‐GnRH, [d‐Cys6, desGly10, Pro9‐NHEt]‐GnRH, and [d‐Cys6, α‐aza‐Gly10‐NH2]‐GnRH were conjugated with doxorubicin (Dox), respectively, through N‐succinimidyl‐3‐maleimidopropionate as a linker to afford three new GnRH‐Dox conjugates. The metabolic stability of these conjugates in human serum was determined by RP‐HPLC. The antiproliferative activity of the conjugates was examined in GnRH receptor‐positive MCF‐7 human breast cancer cell line by MTT assay. The three GnRH‐Dox conjugates showed improved metabolic stability in human serum in comparison with AN‐152. The antiproliferative effect of conjugate II ([d‐Cys6, desGly10, Pro9‐NHEt]‐GnRH‐Dox) on MCF‐7 cells was higher than that of conjugate I ([d‐Cys6, desGly10, Pro9‐NH2]‐GnRH‐Dox) and conjugate III ([d‐Cys6, α‐aza‐Gly10‐NH2]‐GnRH‐Dox), and the cytotoxicity of conjugate II against GnRH receptor‐negative 3T3 mouse embryo fibroblast cells was decreased in comparison with free Dox. GnRH receptor inhibition test suggested that the antiproliferative activity of conjugate II might be due to the cellular uptake mediated by the targeting binding of [d‐Cys6‐des‐Gly10‐Pro9‐NHEt]‐GnRH to GnRH receptors. Our study indicates that targeting delivery of conjugate II mediated by [d‐Cys6‐des‐Gly10‐Pro9‐NHEt]‐GnRH is a promising strategy for chemotherapy of tumors that overexpress GnRH receptors.