In an attempt to produce better cytotoxic analogues, chemotherapeutic antineoplastic radicals including an alkylating nitrogen mustard derivative of D-phenylalanine (D-melphalan), reactive cyclopropane, anthraquinone derivatives [2-(hydroxymethyl)anthraquinone and the anticancer antibiotic doxorubicin], and an antimetabolite (methotrexate) were coupled to suitably modified agonists and antagonists of luteinizing hormone-releasing hormone (LH-RH). Analogues with D-lysine' and D-ornithine6 or N8-(2,3-diaminopropionyl)-D-lysine and N8-(2,3-diaminopropionyl)-D-ornithine were used as carriers for one or two cytotoxic moieties. The enhanced biological activities produced by the incorporation of D amino acids into position 6 of the agonistic analogues were further increased by the attachment of hydrophobic cytotoxic groups, resulting in compounds with 10-50 times higher activity than LH-RH. Most of the monosubstituted agonistic analogues showed high affinities for the membrane receptors of human breast cancer cells, while the receptor binding afnities of peptides containing two cytotoxic side chains were lower. Chemotherapy has been, for many decades, one of the main approaches for the treatment of malignant neoplasms. Despite the development of modem, more specific cytotoxic drugs, their nonselective action on cells other than cancerous ones remains a major problem. A recent modality for the treatment of hormone-sensitive tumors is based on the use of agonists and antagonists of luteinizing hormone-releasing hormone (LH-RH) (1). Some LH-RH agonists substituted in position 6, 10, or both are much more active than LH-RH and also possess prolonged activity (1-3). Changes in positions 1, 2, 3, and 6 and occasionally in positions 5 and 10 of the LH-RH molecule lead to the formation of powerful antagonists (1-4), which inhibit the release of LH and folliclestimulating hormone from the pituitary, create a state of sex-steroid deprivation, and thus have potential therapeutic applications in the treatment of some hormone-dependent cancers such as those of prostate and breast (1, 5).Ideal anticancer drugs would theoretically be those that eradicate cancer cells without harming normal cells. Some hormonal peptide analogues carrying antineoplastic agents could be used for endocrine therapy and at the same time for targeted chemotherapy of cancers that possess receptors for their peptide moieties on tumor cell membranes.

Summary The possible protective effect of an agonist of luteinising hormone-releasing hormone (LH-RH) against the ovarian damage caused by cyclophosphamide was investigated in rats. D-Trp6-LH-RH microcapsules were injected once a month for 3 months, in a dose calculated to release 25 jg day-. Control animals received the injection vehicle. Sixty days after the first injection of microcapsules, cyclophosphamide was given at a loading dose of 50mgkg-' followed by 5 mg kg-' day-' for 30 days, while the treatment with D-Trp6-LH-RH was continued. When the ovaries were examined 3 months and 5 months after discontinuation of treatment, a significant reduction in the total number of follicles (P<0.01) was found in non-pretreated animals given cyclophosphamide. This reduction affected mainly follicles larger than 100 .tm. An irreversible disintegration and destruction of granulosa cells was also observed in this group. In animals pretreated with D-Trp6-LH-RH, administration of cyclophosphamide caused no reduction in the number and diameter of follicles. Thus, the treatment with D-Trp6-LH-RH microcapsules before and during chemotherapy prevented the ovarian injury inflicted by cyclophosphamide. The suppression of gonadal function by LH-RH analogues could be possibly utilised for the protection of the ovaries against damage caused by cytotoxic drugs.

To eliminate the undesirable edematogenic effect of the lutemizing hormone-releasing hormone (LH- Since the isolation and structural elucidation of hypothalamic luteinizing hormone-releasing hormone (LH-RH) more than 2000 analogs have been synthesized, in view of their expected medical applications (1). Chronic administration of potent LH-RH agonists leads to the inhibition of pituitary and gonadal functions (2-6). While repeated administration of LH-RH agonists is required to lower the levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and sex steroids, similar effects can be obtained with single administration of LH-RH antagonists (7). Competitive antagonists of LH-RH were developed by multiple modification of the parent molecule,

The nitrogen mustard derivatives of 4-phenylbutyric acid and L-phenylalanine, called chlorambucil (Chl) and melphalan (Mel), respectively, have been incorporated into several peptide hormones, including luteinizing hormone-releasing hormone (LH-RH). The alkylating analogues of LH-RH were prepared by finking Chl, as an N-acyl moiety, to the complete amino acid sequence of agonistic and antagonistic analogues. These compounds, in particular the antagonistic analogues, showed much lower potency than their congeners carrying other acyl groups. (7), at a concentration of 0.5 nM can inhibit 0.22 nM thrombin by 50%o in less than 2 min. Consequently, the results with Chl-and Mel-containing analogues indicate the absence of a receptor nucleophile in the peptide binding site essential for biological activity of hormone receptors. Thus, we may assume that a nitrogen mustard compound such as Mel incorporated into a peptide hormone can be bound to the membrane receptors and internalized. After endocytosis, such a compound could interfere with intracellular events due to its alkylating potential.To study this hypothesis, Mel-containing LH-RH analogues with high agonistic or antagonistic activities were synthesized on the basis of the known structure-activity relationships found among LH-RH analogues (8). MATERIALS AND METHODSAmino Acid Derivatives. Boc-D-Ala, Boc-Arg(Tos), BocGly, Z-

Metal Superagonist analogues of luteinizing hormone (LH)-releasing hormone (LH-RH),