Cytotoxic luteinizing hormone-releasing hormone conjugates and their use in gynecological cancer therapy.

Endometrial cancer is the most frequent malignant gynecological tumor in the western world. In most cases, it is diagnosed at an early stage when surgery alone or in combination with radiotherapy can achieve high cure rates (1). However, steroid receptor-negative tumors in elderly women or at advanced stages are rarely cured (2±4). Ovarian cancer is less frequent than endometrial cancer, but it is the most common cause of death from gynecological neoplasms (5). Effective regimens for surgery and first line cytotoxic chemotherapy are established, but in advanced or relapsed cases curative or palliative treatments with long term efficacy remain to be established (6±8). In both endometrial and ovarian cancer, new therapeutic strategies are required that are well tolerated and more efficacious. Cytotoxic drugs are used as single agents or as combinations of several antitumor agents with different mechanisms of action. This chemotherapy is limited by its toxicity to normal cells. A more selective delivery of the cytotoxic agents to the tumor cells would allow the use of increased doses and would reduce the toxicity to normal cells. In earlier studies it was shown that breast, ovarian, endometrial, pancreatic, and prostatic cancers express specific binding sites for luteinizing hormone-releasing hormone (LHRH) (9±16). Although these LHRH binding sites had a molecular mass comparable with that of pituitary LHRH receptor, their binding characteristics were of the low-affinity/high-capacity type (11, 15±17). Later, it became evident that in breast, ovarian, endometrial and prostate cancer cell lines, as well as in respective biopsy samples two types of LHRH binding sites exist, one of low affinity and high capacity, the other of high affinity and low capacity. The latter is comparable to the pituitary LHRH receptor (11, 15, 16). In 1992, the cloning, sequencing and expression of the human pituitary LHRH receptor was reported (18). In the same publication, the authors reported the expression of mRNA for the human LHRH receptor in the breast cancer cell line, MCF-7. These findings stimulated intensive research leading to the demonstration of LHRH receptor gene transcripts in ovarian and endometrial cancer cell lines and in about 80% of the respective primary tumors (16, 19±21). In ovarian and endometrial cancer specimens and cell lines expressing mRNA for the pituitary LHRH receptor, highaffinity/low-capacity binding sites were found closely related to the pituitary LHRH receptor (19±23). Kakar et al. (24) demonstrated that the nucleotide sequence of LHRH receptors in human breast and ovarian tumors is identical to that found in pituitary. Data available today suggest that about 50% of breast cancers (25) and approximately 80% of ovarian and endometrial cancers express high-affinity binding sites for LHRH. For prostate cancer fewer findings have been published (15), but systematic investigations might lead to comparable results. Since over 80% of human ovarian and endometrial cancers express receptors for LHRH, but most of human normal tissues do not express LHRH receptors (P VoÈlker, C GruÈ ndker & G Emans, unpublished results), LHRH receptors on tumor cells might be used for targeted chemotherapy. This should improve antitumor effects and reduce side effects compared with systemic conventional cytotoxic chemotherapy (26). LHRH analogs covalently linked to cytotoxic radicals could bind specifically to LHRH receptors with their peptide moiety and act as chemotherapeutic agents after internalization of the ligand-receptor complex into cancer cells or by acting at the membrane level. In this fashion, these conjugates can selectively affect those cells that possess LHRH receptors and thus exert fewer side effects than unconjugated cytotoxic agents (27). A great variety of cytotoxic analogs containing different LHRH agonists and antagonists and different cytotoxic compounds including melphalan, cisplatinum, methotrexate and cyclophosphamide derivatives have been synthesised in the past years (28±31). Most of these compounds preserved their LHRH analog action and were internalized into cells expressing receptors. Major problems, however, were caused by the instability of these compounds and the lack of preservation of their cytotoxic action (31±35). New cytotoxic analogs made of LHRH analogs having a d-Lys moiety in position 6, seem to solve these problems. This amino acid offers an amino side chain for convenient attachment of various cytotoxic compounds. It turns out that even bulky molecules could be linked to the e-amino group of the d-Lys moiety, without significant loss of binding affinity of the peptide portion to the receptors for LHRH (35±36). Doxorubicin is the most widely used cytotoxic agent with a broad spectrum of antitumor activity (37). The strong antiproliferative effect of doxorubicin is mainly due to its ability to induce apoptosis. In some early ISSN 0804-4643 European Journal of Endocrinology (2000) 143 569±572