Leuprolide is a synthetic nonapeptide, analogue of Luteinizing Hormone Releasing Hormone (LHRH or GnRH). Is a super agonist of LHRH and as an active pharmaceutical ingredient has been used in the treatment of many sex hormone-related disorders. Regulation of hypothalamic-gonadal-axis by LHRH analogues is used for the treatment of hormone-dependent diseases, such as endometriosis, benign prostate hyperplasia, fertility disorders, as well as prostate, ovarian and breast cancer to induce reversible chemical castration. The currently available LHRH analogues as drugs and their applications in the treatment of the above diseases lead the scientists to the development of new more potent LHRH analogues with better pharmacological profile. In recent years the biology of GnRH has been revised, due to accumulating evidence that peripheral, extrapituitary, normal and malignant tissues locally produce GnRH and express GnRH binding sites. An autocrine/paracrine role of GnRH at these normal and malignant tissues, including the mammary gland, has been suggested. The direct antiproliferative effect of the LHRH analogues in many malignant tissues inaugurates the field and leads to the design of effective LHRH analogues with new perspectives. To-date, only a few studies have investigated the effects of LHRH analogues in pituitary and cancer cells in parallel. Information concerning the impact of systematic single amino acid modifications of LHRH on pituitary and malignant cells is still lacking. The structural investigation of this field seems to be a very important issue. The aim of this study was the contribution in the field of the structure-activity relationships based in the rational design (bibliographic data, conformational analysis), development (modern synthesis) and biological characterization of the new analogues of Leuprolide. Particularly, with the biological characterization, we tried to compare the binding affinities of the new Leuprolide analogues in the LHRH hypophysial receptor with their direct antiproliferative activity on breast cancer cells. Furthermore, the development of a new synthetic protocol which leads to high yield and purities of the synthesized products was a requisition. Important structural data obtained by conformational analysis of LHRH and Leuprolide using NMR spectroscopy. This conformational study in combination with the bibliographic data led us to design and synthesize new analogues of Leuprolide with main modifications in positions 3 and 6. Trp3 of peptide sequence of Leuprolide is substituted by non natural aromatic amino/imino acids. D-Leu6 is substituted by α,α-dialkyl amino acids L, D-Gly(tBu), D-Lys and D-Dab. In the ε- and γ-amino group of Lysine and diaminoisobutyric acid in position 6 several modifications have been performed by conjugation of natural amino acids (Gly, Ala, Sar) and 2-amino-4-pyrrolidinothieno[2,3-d]pyrimidine-6-carboxylic acid (ATPC). ATPC was also incorporated in the N-terminal segment replacing the final dipeptide of LHRH and Leuprolide. Like in the peptide sequence of Leuprolide, most of the new LHRH analogues lack the carboxy-terminal Gly10-amide of LHRH and an ethylamide residue is added to Pro9 (Fujino modification). The new analogues of LHRH were assembled on a [3-((Ethyl-Fmoc-amino)-methyl)-1-indol-1-yl]-acetyl AM resin to provide the peptide amide using Fmoc/tBu methodology. The affinities for the LHRH receptor of the new analogues were determined by competition binding experiments in mouse anterior pituitary αT3-1 cells while their antiproliferative activity was performed with human breast cancer cell lines (MCF-7). Modern peptide synthesis afforded us peptides in high yields which could be easily purified. This study provides binding requirements which can be used in the design of new Leuprolide analogues with high pituitary binding affinity and high antiproliferative activity in breast cancer cells (MCF-7). Results show in many cases differential impact on pituitary binding affinity and direct antiproliferative effect on breast cancer cells which is in accordance with bibliographic reports that mainly support this dissimilarity to different signal transduction mechanisms in hypothalamic and extra hypothalamic malignant tissues. Final this study provides new Leuprolide analogues with higher binding affinity and antiproliferative activity than it has exhibited and they deserve further pharmaceutical/biological investigation. This structure-activity relationship study for LHRH analogues at pituitary and breast cancer cell lines contributes to the on-going research of LHRH binding requirements of breast cancer cells and provides new perspectives for the design of effective GnRH analogues.
Non-randomised comparison of efficacy and side effects of bicalutamide compared with LHRH analogues in combination with radiotherapy in the xxxxxx trial.
