an-207 and Breast-Neoplasms

Targeted chemotherapy is a modern approach aimed at increasing the efficacy of systemic chemotherapy and reducing its side effects. The peptide receptors expressed primarily on cancerous cells can serve as targets for a selective destruction of malignant tumors. Binding sites for LHRH (now known in genome and microarray databases as GNRH1), were found on 52% of human breast cancers, about 80% of human ovarian and endometrial cancers, and 86% of human prostatic carcinoma specimens. Because LHRH receptors are not expressed on most normal tissues, they represent a specific target for cancer chemotherapy with antineoplastic agents linked to an LHRH vector molecule. To test the efficacy of targeted chemotherapy based on LHRH analogs, we recently developed a cytotoxic analog of LHRH, designated AN-152, which consists of [D-Lys6]LHRH covalently linked to one of the most widely used chemotherapeutic agents, doxorubicin (DOX). In addition, we designed and synthesized a highly active derivative of DOX, 2-pyrrolino-DOX (AN-201), which is 500-1000 times more potent than DOX in vitro. AN-201 is active against tumors resistant to DOX, and noncardiotoxic. As in the case of DOX, AN-201 was coupled to carrier peptide [D-Lys6]LHRH to form a superactive targeted cytotoxic LHRH analog, AN-207. Both AN-152 and AN-207 can effectively inhibit the growth of LHRH receptor-positive human breast, ovarian, endometrial, and prostate cancers xenografted into nude mice. DOX-containing cytotoxic LHRH analog AN-152 is scheduled for clinical phase I/IIa trials in patients with advanced ovarian and breast cancers in 2005.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Doxorubicin; Endometrial Neoplasms; Epithelial Cells; Female; Gonadotropin-Releasing Hormone; Humans; Male; Ovarian Neoplasms; Prostatic Neoplasms; Pyrroles; Receptors, LHRH

Receptor targeted chemotherapy is less toxic and more effective than conventional chemotherapy. Receptors for luteinizing hormone-releasing hormone (LH-RH) are found in about 50% of human breast cancers. Highly potent cytotoxic radical 2-pyrrolinodoxorubicin (AN-201) was linked to the agonistic analog [D-Lys6]LH-RH to form cytotoxic LH-RH analog AN-207. We evaluated whether AN-207 could be targeted to the hormone-independent MDA-MB-231 human breast cancers. Nude mice bearing MDA-MB-231 tumors were injected i.v. with 250 nmol/kg doses of cytotoxic radical AN-201, cytotoxic LH-RH analog AN-207, the unconjugated mixture of AN-201 and carrier [D-Lys6]LH-RH, [D-Lys6]LH-RH alone and vehicle (control). The growth of MDA-MB-231 tumors in animals given a single dose of AN-207 was inhibited significantly (p = 0.01) for 3 weeks after injection, whereas tumors in all the other groups grew steadily. All cytotoxic compounds produced leukopenia, but the strongest lymphocyte suppression was caused by cytotoxic radical AN-201. Three weeks after treatment, the presence of mRNA for LH-RH receptors was demonstrated by RT-PCR in all the groups and radioreceptor assays demonstrated high-affinity binding sites for LH-RH on tumor cell membranes of control animals and those treated with AN-201, the carrier peptide alone or in combination with AN-201. At this time point binding assays did not reveal the expression of membrane proteins in tumors treated with AN-207, but 60 days after administration of AN-207, high affinity LH-RH binding sites were found again in MDA-MB-231 tumors. These results indicate that cytotoxic LH-RH analog AN-207 could be utilized for receptor targeted chemotherapy of breast cancers expressing receptors for LH-RH.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Breast Neoplasms; Disease Models, Animal; DNA Primers; Doxorubicin; Drug Delivery Systems; Female; Gonadotropin-Releasing Hormone; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Pyrroles; Radioligand Assay; Receptors, LHRH; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Specific Pathogen-Free Organisms; Tumor Cells, Cultured

A highly potent derivative of doxorubicin, 2-pyrrolinodoxorubicin (AN-201), was linked to [D-Lys6]luteinizing hormone-releasing hormone (LH-RH) to form a cytotoxic analogue, AN-207, that can be targeted to LH-RH receptors. The effects of AN-207 were investigated in MDA-MB-435 human estrogen-independent breast carcinomas, which express LH-RH receptors. In experiment 1, nude mice bearing orthotopically implanted tumors received a single i.v. injection of AN-207, AN-201, or the carrier at 250 nmol/kg. Five weeks after administration of AN-207, tumor volume was significantly decreased by 66% (P < 0.001) and tumor burden by 71% (P < 0.05) as compared with controls, but no significant effects occurred in other groups. Six of eight (75%) control animals and 37.5% of mice treated with AN-201 developed metastases in the lymph nodes, whereas no lymphatic spread was found in any of the mice that received injections of AN-207. The antitumor effect of AN-207 could be partially blocked by pretreatment of the tumor-bearing mice with high doses of agonist [D-Trp6]LH-RH, which suggests that AN-207 acts on LH-RH receptors on tumors. The mortality due to toxicity was 25% in the group receiving AN-201 and 12.5% in the AN-207-treated group. Radioligand binding assays revealed the presence of high-affinity binding sites for LH-RH on tumor membranes, and mRNA for LH-RH receptors was demonstrated by reverse transcription-PCR. In experiment 2, two i.v. injections of AN-207 or AN-201 at 150 nmol/kg were given on days 0 and 28 to mice bearing orthotopic xenografts of MDA-MB-435. The outcome of the treatment was similar to that observed in experiment 1, but without any toxicity-related deaths. Tumor growth inhibition and prevention of metastatic disease suggest that cytotoxic LH-RH analogue AN-207 could be considered for the treatment of human estrogen-independent breast cancers expressing receptors for LH-RH.

Topics: Animals; Breast Neoplasms; Doxorubicin; Estrogens; Female; Gonadotropin-Releasing Hormone; Humans; Luteinizing Hormone; Lymphatic Metastasis; Mice; Mice, Nude; Neoplasm Transplantation; Receptors, LHRH; Reverse Transcriptase Polymerase Chain Reaction; RNA; Time Factors; Tumor Cells, Cultured

Recently, we developed two new cytotoxic analogs of luteinizing hormone-releasing hormone (LH-RH), AN-152 in which doxorubicin (DOX) is linked to [D-Lys6]LH-RH, and AN-207 which consists of 2-pyrrolino-DOX coupled to [D-Lys6]LH-RH. In this study, we examined binding of AN-152 and AN-207 to membranes of human breast cancer specimens and MCF-7 and MDA-MB-231 human breast cancer lines. Both cytotoxic analogs displayed IC50 values in the nanomolar concentration range (IC50 = 2-13 nM). Using radioligand binding studies, we characterized the receptors for LH-RH on membranes of breast cancers. In addition, the expression of mRNA for LH-RH receptors in MCF-7 and MDA-MB-231 cell lines was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR). These highly active cytotoxic analogs of LH-RH have been designed as targeted chemotherapeutic agents for the treatment of various cancers expressing receptors for LH-RH.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Binding, Competitive; Breast Neoplasms; Cell Membrane; Doxorubicin; Female; Gonadotropin-Releasing Hormone; Humans; Middle Aged; Radioligand Assay; Receptors, LHRH; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

Receptors for luteinizing hormone-releasing hormone (LH-RH) are found in about 50% of human breast carcinomas. A highly potent cytotoxic agent, 2-pyrrolinodoxorubicin (AN-201), was linked to the agonist [D-Lys6]LH-RH to form a cytotoxic LH-RH analog, AN-207, that can be targeted to LH-RH receptors on breast carcinomas.. Nude mice bearing MX-1 hormone-independent, doxorubicin-resistant human breast carcinomas were injected intravenously with vehicle (control), 250 nmol/kg doses of AN-201, AN-207, or an unconjugated mixture of AN-201 and [D-Lys6]LH-RH. Tumor growth and changes in hematologic parameters were evaluated. Receptors for LH-RH were investigated by radioreceptor assays, and the expression of their mRNA was determined by reverse transcriptase-polymerase chain reaction.. AN-207 caused complete regression of MX-1 tumors in all 10 animals, and they were still tumor free 60 days after treatment. In contrast, after therapy with AN-201 or the mixture of AN-201 and [D-Lys6]LH-RH, the regression of most MX-1 tumors was only transitory. AN-201 caused the death of 1 of the 10 animals and significantly greater leukopenia than AN-207, which produced no toxic deaths. Radioreceptor assays revealed high affinity binding sites for LH-RH on tumor cell membranes. The expression of mRNA for LH-RH receptors also was found in tumors.. The results of this study indicate that powerful, targeted cytotoxic LH-RH analogs such as AN-207 could be considered for the treatment of human breast carcinomas that possesses receptors for LH-RH.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Doxorubicin; Female; Gonadotropin-Releasing Hormone; Humans; Male; Mice; Mice, Nude; Neoplasm Transplantation; Receptors, LHRH; RNA, Messenger; Transplantation, Heterologous