Title of Invention

"A CYTOTOXIC PEPTIDE HORMONE CONJUGATES"

Abstract

A cytotoxic peptide hormone conjugates of the formula Where R is a single bond H or- C(0)-(CH2) n-C(O) and n is 0-7 R'is selected from the group consisting of NH2, an aromatic or hydrogenated 5 or 6 membered heterocycle having at least one ring nitrogen and such a heterocycle having a butadiene moiety bonded to adjacent carbon atoms of said ring to form a bicyctic system and P is H or a peptide, provided that where R' is NH2 then R - P is other than H and where R-P is H, then R' is other than NH2.

Full Text

-1A- FIELD OF THE INVENTION This invention relates to cytotoxic peptide hormone conjugates. This invention is in the field of the chemistry of targeting anticancer anthracycline derivatives. More particularly, it concerns doxorubicin (DOX) or its daunosamine modified derivatives (DM-DOX) finked covatently to analogs of peptide hormones such as Lh-RH, bombesin and somatostatin. These covalent conjugates!, are^jtargeted to various tumors bearing receptors for the peptide "hormone analogs. Discussion of the Prior Art LH-RH Analogs which have cytotoxic moieties at the sixth position are shown tn Schally, Janaky and Bajusz, EP 0 450 461 61, grant publication September 6, 1995. GnRH(lH-RH) analogs for destroying gonadofrops are described in Nett and Glode, WO 90/09799, published on September 7, 1990. This application describes toxins, tike rtein, linked to analogs of LH-RH for destroying gonadotrophs and thus curing sex hormone dependent cancers. LH-RH doxorubicin derivative is aiso mentioned without specification of the chemistry of linking. Cytotoxic somatostatin analogs are described by Schally et al. in U.S. Pal. application filed on April 6, 199G and refiled in July 15, 1993 under Serial No. 08/076,846 A review by A. V. Schally in Anti-Cancer Drugs 5, 115-130 (1994) gives details about the presence of receptors on the cell membranes of a wide variety of tumors for analogs of LH-RH, bombesin or somatostatin. G. Weckbecker lists several references that show the presence of receptors and receptor subtypes for somatostatin analogs on several normal and tumorous tissues in his review in Farmac. Ther. 60, 245-264 (1993). Bombesin-like peptides and the presence of bombesin/GRP receptors on various normal and tumorous tissues are discussed in the review by N. Bunnett in Gut Peptides: Biochemistry and Physiology 423-445 (1994) Ed.: J. Walsh and G. J. Dbckray, Raven Press, New York and by E. Spindell in Recent Progress in Hormone Research 48, (1993) (Academic Press) Doxorubicin (DOX) is, at this time, the most widely used, and very potent anticancer agent. However, certain tumors do not respond to it at alt and its use is also limited by multidrug resistance (MDR) and cardiotoxicity as well as neutropenia, which are the results of chronic treatment. In order to overcome these drawbacks and to further exploit the enormous tumoricida! potential inherent in the structure of anthracycline antibiotics, thousands of synthetic derivatives have been described, including their targeted analogs linked to various carrier macromolecules. Most of the history of DOX and its analogs is described in "Adriamycin", David W. Henry, ACS Symposium Series, No. 30, Cancer Chemotherapy, American Chemical Society, pp. 15-57 (1976) and in the book Doxorubicin. Federico Arcamone, Academic Press, (1981). 3 Highly active, alkylating, non-cross resistant 3'-deamino-3'-(3"-cyano-4"-morpholinyO-DOX and derivatives thereof which have antitumor activity are described in Mosher, et al., U.S. Pat. 4,464,529, August 7, 1984. The synthesis and biological evaluation of these "Intensely Potent Morpholinyl Anthracyclines" are also described in J. Med. Chem. 1984, 27, 638-645. In Proc. Natl. Acad. Sci. USA Vol. 88, pp. 4845-4849, June 199). Gao et al. describe formaldehyde-mediated alkylation of a DNA sequence by a daunorubicin derivative. Ari'thracycline analogues bearing latent alkylating substituents are described in J. Med. Chem. 35, 3208-3214 (1992). The use of an d.oi-diiodo compound for the alkylation of the daunosamine, nrtrogen of DOX and thus the formation of a new morpholinyl DOX derivative is described in European Patent EP 434 960, filed by Pharmacia Carlo Erba on December 12, 1989. N-Trifluoroacetyladriamycin14-0-hemiglutarate and -hemiadipate are disclosed as analogs of N-trifluoroacetyladriamicyn14-0-valerate (AD-32) with improved water solubility in Israel, et al., U.S. Patent 4,299,822, November. 10, 1981. Horton and Priebe (J. Antibiotics, XXXVI, 1211-1215.) describe several 14-O-esters of different anthracycline analogs with no dramatic changes in anticancer activity as compared to the 14-OH parent analogs. In the art of designing targeted chemotherapeutic agents, the following objectives are sought: 4 1. Stable linkage between the carrier molecule and the chemo therapeutic agent until the target is reached, 2. Retained biological characteristics of the carrier molecule within the conjugate, such as retained binding properties. 3. Retained pharmacological activity of the chemotherapeutic agent within the conjugate, such as retained cytotoxic activity. 4. As a result of conjugation, the production of analogs of more intense activity and/or lower peripheral toxicity relative to the unconjugated moieties. Conjugation of DOX by Nal04 oxidation of the daunosamine moiety of DOX followed by reductive alkylation involving a primary amine of a carrier molecule is described in Seta, et at., U.S. Patent 4,263,279, April 21, 198.1, A cis-acbnttic acid spacer was used to link the daunosamine nitrogen to macromolecular carriers with a pH-sensitive bond, as described in Biocherni Biophys. Res. Commun. 1981 102, 1048-1054. The formation of ester bonds and C-N linkages between 14-bromodauhorubicin and proteins or poty-L-amino acids is described by Zunino et.al. (1981) Tumori 67, 521-524 and (1984) Eur. J. Cancer Clin. Oncol. 20, 421-425. MorphoIino-DOX (a highly active, daunosamine modified analog of DOX) was conjugated to antibody via a hydrolyzable (lysosomotrop, pH sensitive) hydrazone linkage, involving the C-13 oxo function of the cytotoxic agent, as described in Bioconjugate Chemistry 1990 1(5), 325-330 Sensitivity of the carboxamide bond of a leucine residue to enzymatic degradation was used successfully in conjugates of DOX containing a "spacer arm" peptide, preferentially Ala-Leu-Ala-Leu, where the carboxy terminal Leu acylates the daunosamine nitrogen in DOX and the amino 5 terminal Ala is linked to the carrier through dicarboxylic acid spacer as described in Proc. Natf. Acad. Sci USA 1982 79, 626-629. The daunosamine nitrogen of DOX was acylated by a glutaric acid spacer and linked to LH-RH analogs with a severe loss of cytotoxic activity as described in Proc. Natl. Acad. Sci. USA 1992 89, 972-976. Further references related to the use of the compounds accordipg to the present invention for the treatment of various human tumors: 1. Schally et.al. (1996) in Treatment with GnRH Analogs: Controversies and Perspectives, eds. Filicori, M. & Flamigni, C. (Parthenon,Camforjh. U.K.}, pp 33-44. 2. Nagy e».al. 3.Yano«t.al. {1994} Proc. Natt. Acad. Sci. U.S.A. 91, 7090-7094. 4. Rekasi ei.al. (1993) Endocrinology 132(5) 1991-2000. 5. Srkatovic et.al. (1990) Cancer Res. 50, 1841-1846. 6. Emons et.al (1993) Cancer Res 53, 5439-5446 7. Emons et.al. (1993) Journal of Clin. Ertfccrin. and Metabol. 77(6) 1458-) 8. Schally,-A. V. (1988) Oncological applications of somatostatin analogs. Cancer Res. 48, 6977-6985. 9. Schally et.al. (1994) International Journal of Pancreatoiogy 16, 277-280. 10. Srkalovic et.al. (1990) Journal of Clinical Endocrinology and Metabolism 70(3), 661-669. 4 Pinski et.al. (1994) Int. J. Cancer 57. 574-580. 11. Radulovic et.al. (1992) Cancer Letters 62, 263-271. 12. Qin et.al. (1995) Int. J. Cancer 60, 694-700. 13. Radulovic et.al. (1992) P.S.E.B.M. 200, 394^01. 14. Radulovic et.al. (1994) Acta Oncologica 33(6) 693-701. 15. Pinski et.al. (1993) Cancer Letters 71, 189-196. 16. O'Byrne et.al. (1994) Eur. J. of Cancer 30A(11) 1682-1687 17. Pinski et.al. (1994) Br. J. of Cancer 70, 886-892. 18. Pinski et. al (1994) Cancer Res. 54, 5895-5901. 6 19. Pinski et.al. (1996) Int. J. Cancer 65, 870-874. 20. Banks et.al. (1992) Anticancer Drugs. 3, 519-523. 21. Reubi and Kvols (1992) Cancer Res. 52, 6074-6078.) 22.Schally et.al. (1994) International Journal of Pancreatology 16, 277-280. 23Halmos et.al. (1995) Cancer Res. 55, 280-287. 24. Halmos et.al. (1994) Cancer Letters 85, 111-118. 25. Qin et.al. (1994) J. Cancer Res. Clin. Oncol. 120, 519-528 26. Qin et.al. (19940 Cancer Res. 54, 1035-1041. 27. Qin et.al. (1995) Int. J. Cancer 63, 257-262. 28. Reile et.al. (1994) The Prostate 25, 29-38. 29. Pinski et.al. (1934) Int. J. Cancer 57, 574-580. 30. Radulovic et.al. (1992) P.S.E.8.M. 200, 394^401 31. Radulovic et.al (1994) Acta Oncologica 33(6) 693-701. 32. Pinski et.al. (1993) Cancer Letters 71, 189-196. 33. Pinski et.al. (1994) Br. J. of Cancer 70, 886-892. 34. Pinski et. al. (1994) Cancer Res. 54, 5895-5901.) All the citations referred to are incorporated herein by reference. Summary of the Invention The compounds of the invention are novel, targeted cytotoxic peptide hormones comprising an anthracycline cytotoxic agent, such as DOX or DM-DOX, conjugated to a peptide hormone, such as analogs of LH-RH, bombesin, and somatostatin. These cytotoxic peptide hormone conjugates are designed for the treatment of tumors bearing specific receptorsToT the conjugate, such as breast cancer, ovarian, cancer, endometfiaTcancer, prostate cancer, pancreatic cancer, colon cancer, gastric cancer, and lung cancer. Certain of these (unconjugated) anthracycline cytotoxic agents utilized herein are per se novel, and are highly potent, their level of toxicity however is too high for them to be used in unconjugated form. -7- Daunosamine modified DOX analogs presented in this invention were developed during a search for new, highly active, non-cross resistant analogs of DOX suitable for the formation of covalent conjugates with peptide carriers. The formation of stable, covalently jinked conjugates with fully retained biological activities of their components was achieved by using a dicarboxylic acid spacer, like glutaric acid. One carboxyl group of the spacer forms an ester bond with the 14-OH group of DOX or DM-OOX and the other carboxyl group of tfie spacer forms a carboxmide bond with a well chosen free amino group of the peptide carrier. According to this invention there is provided a cytotoxic peptide hormone conjugates of the formula wherein Q has the detailed chemical structure: where -8- R is a single bond or -C(0)-(CH2)n-C(0)- and n = 0-7, Rf is NH2 or an aromatic, saturated or partially saturated 5 or 6 membered heterocyclic compounds having at least one ring nitrogen and optionally having a butadiene moiety bonded to adjacent carbon atoms of said ring to form a bicydie system, P is H or a peptide moiety, suitably an LHRH, somatostatin bombesin analog, but not excluding other physiologically active peptides. Particularly desirble are those LHRH analogs having affinity for neoplastic cell receptors, expecially those analogs having a D-Lys moiety at the 6 position, as well as shortened somatostatin and bombesin analogs. Nevertheless where R' is NH2 then R-P is other than H. When R-P is H, then R' is other than NH2. A novel synthetic reaction has been discovered in the course of this work. Not only was it found that doxorubicin and its derivatives can be coupled via a dicarboxylic moiety at the 14 position to yield novel pharmacologically effective conjugates but a novel way was provided to form partially saturated heterocyclic moieties from vicinal and disjunct i.e,Cr:.p- or CF/y-hydroxy primary amines. The particular application in the present invention was the formation of 2*-pyrrolinyl and l"3*-tetrahydropyridinyl moieties on the daunosamine sugar. However, this reaction has broader applicability. 5 and 6 membered partially saturated heterocyclic moieties may be formed when a vicinal or disjunct hydroxy amine is reacted with a halo-substituted aldehyde having 2 or 3 moieties between the aldehyde carbon and the carbon atoms having the halo group. These moieties may all be methylene, or a hetero atom such as oxygen may be involved. The reaction takes place in three stages. A very large excess of the haioaldehyde is reacted with the acid salt of the hydroxy amine, suitably in a polar inert anhydrous organic solvent. There is thus formed a five membered oxsxolidine ring (or a six-membered 1,3-tetrahydrooxazine ring) by condensation of the aldehyde group with the hydroxy! and the amine groups. This product is treated with an organic base, -. -.. s suitably a tertiary amine, whereby (he elements of hydro-halic acid are eliminated between the halo moiety of the former halo aldehyde and the secondary amino group of the oxazolidine or 1,3-tetrahydrooxazine ring to form a fused ring structure by the addition of a 5 or 6 membered ring The base is then neutralized with a weak acid suitably an organic acid such as glacial acetic acid. Treatment with aqueous acid, suitably an organic acid opens the oxazolidine or 1,3-tetrahydrooxazine portion of the fused ring. It will be understood by those skilled in the art that depending on {he starting aldehyde, the final nitrogen containg ring may contain at least one additional hetero atom as mentioned above. The general reaction may be illustrated as follows: (III) (large Excess) in Solvent, anhydrous aprotic solvent (IV) Base (tertiary anhydrous amine) 10 10 Wherein X' is halo, suitably bromo or iodo, preferably iodo, Y is CH2, OCH2l CH2-CH2, 2 is nil or CH2 When 21s nil, the aldehyde moiety forms a 5-membered oxazolidine ring as the first'step of the reaction. When 2 is CH2, the aldehyde moiety forms a 6-membered'1,3-tetrahydrooxazine ring. While such ring formations are well known, in combination with the ring closure effected by the haloalkane side chain in a basic medium such as a tertiary amine in an anhydrous medium, the reaction is new and surprising. Brief Description of theAcompanying Drawings FIGURE 1 is plot of volume changes of estrogen independent MXT mouse mammary cancers for different dosage levels of compounds of the present invention and DOX. FIGURE 2 is plot of volume changes of estrogen independent MXT mouse mammary cancers for different dosage levels of a certain compound of the present invention, a prior art compound, DOX and a control. FIGURE 3 is plot of the effect of certain cytotoxic LHRH analogs on the survival of mice with estrogen independent MXT mouse mammary cancers. 11 FIGURE A is a plot of tumor volume in male Copenhagen rats bearing rat Dunning R-3327-H prostate carcinoma transplants during the treatment with a prior art agonist and a certain compound of the present invention. FIGURE 5 is a plot showing the effect of treatment with a certain compound of the present invention and the corresponding cytotoxic LH-RH analog on the tumor volume in rats with Dunning R-3327-H prostate cancer. FIGURE 6 is a plot showing the effect of treatment with with a certain compound of thepresent invention and the corresponding cytotoxic LH-RH analog on the body weight of Copenhagen rats bearing Dunning R-3327-H. prostate cancer. FIGURE 7 is:a plot showing inhibition of tumor growth achieved by treatment, with a certain compound of the present invention and DOX. Description of the Preferred Embodiments The;moiety Q, when substituted at R' by certain preferred groups, has submoiety designations of Q, through Q8l of which Q2 through Q8 are novel cytotoxic moieties. R' has the preferred values, leading to the desired Qx moieties listed in parentheses as follows: NH2 (Qi), pyrrolidine-1-yl (Q2), isoindoline-2-yl (Q3), 3-pyrroline-1-yl (Q4), 3-pyrrolidone-1-yl (Qs), 2-pyrroline-1-yl (Q6), 3-piperidone-1-yl (Q7), or 1,3-tetrahydropyridine-1-yl(Qa). Thus if R-P is H and -R' is -NH2, Qi is DOX, if R-P is H and -R' is pyrrolidine- 1-yl, Q2 is 3'-deamino-3'-(pyrrolidine-1"-yl)-doxorubicin (Q2); if R-P is H and - R* is isoindotine-2-yi, Q3 is S'-deamino-SXisoindoiine-Z'-yO-doxorubicin (Q3); if R-P is H and -R' is 3-pyrroline-1-yl. Q -12-doxorub/rin (Q*); if R-P is H and -R' is 3-pyrrolidone-l-yl, Q5 is 3'-deamino-3'-(S'pyrrolidone-l'-ylJ-doxorubicin (Q5; if R-P is H and R'is 2-pyrroline-i-yi, Q6 is 3,-deamino-3'-(2"-pyrroline-lff-yl)-doxorubicin (Q6); if R-P is H and -R' is 3-piperidone-1-yl, Q7 is r' The compounds incorporating the daunosamine nitrogen in a five membered ring with alkylating function are 10-50 times more active in vitro than their homolog counterparts,incorporating the daunosamine nitrogen in a six membered ring. (Such pairs are Q5 and Q7 as well as Q6 and Q$). In the present embodiments of the present invention, in the substance of formula QM-O-R-P, R-P is other than hydrogen. Where P is other than hydrogen, that is whwQ it is PA, P2 and P3, suitab}y where Pi is an LH-RH agonist carrier, an LH-RH antagonist carrier or a shortened LH-RH analog carrier, P2 is a shortened somafcostain analog and P3 is a bombesin antagonist. Suitably, Pi is Aaa-Bbb-Cee-Ser-Tyr-D-Lys (Xxx)-Leu-Arg-Pro-Ddd, wherein (Xxx) is hydrogen or a diamino substituted such as A2Bu or A2Pr wherein where: Aaa is Glp, then Bbb is His, Ccc is Trp, and Odd is G!y-NH2, Aaa is Ac-D-Nal(2),Ac-D-Phe or AcD-Phe(4Cl), then Bbb is D-Phe(4Cl) or D-Phe, Ccc is D-Pal(3) and D-Trp and Odd is D-Ala—NH2; andf where Aaa-Bbb-Ccc is Ac, then Ddd is -NH-CH2~CH3; P2 is Aaa-Cys-Bbb-D-Trp-Lys-Cec-Cys-Ddd-NH2 wherein: where Aaa is D-Phe, then Bbb is Tyr, Ccc is Val and Ddd is Thr or Trp; and 13 where Aaa is D-Trp, then Bbb is Phe, and Ccc and Ddd are Thr; and P3 is Aaa-Gln-Trp-Ala-Val-G!y-His-Leu Bbb-NH2 wherein: Aaa is nil, D-Tpi or D-Phe and Bbb is (CH2-NH)Leu, (CH2-NH)Phe, (CH2-NH)Trp, (CH2-N)Tac or (CH2-N)DMTac. In the novel compounds of the present invention incorporating analogs of LH-RH, the cytotoxic radical Q is attached to the D-Lys side chain on the LH-RH analogs or the (Xxx) group attached thereto, through a dicarboxylic acid spacer as formulated in Formula VII: Aaa-Bbb-Ccc-Ser-Tyr-D-Lys(Xxx)m(Q14-0-R)0-Leu-Arg-Pro-Ddd (VII) where m is 1 or 0 and n is 1 or 2 provided that when m is 1 i.e. (Xxx) is A2Bu or A2Pr, n is 1 or 2, when m is 0 i.e. (Xxx) is H, n is 1. In the novel compounds of the present invention incorporating analogs of somatostatin the cytotoxic radical Q is attached to the amino terminal of the somatostatin analogs through a dtcarboxylic acid spacer as formulated in Formula VIII: QM-O-R-Aaa-Cys-Bbb-D-Trp-Lys-Ccc-Cys-0dd-NH2 (VIII) In the novel compounds of the present invention incorporating analogs of bombesin antagonists, the cytotoxic radical Q is linked to the amino terminal of the bombesin antagonists as formulated in Formula IX: Q14-0-R-Aaa-Gln-Trp-Ala-Val-Gly-His-Leu Bbb-NH2 (IX) Especially preferred.embodiments of this invention are those peptide conjugates that contain Qt and Q6 as the cytotoxic radicals and glutaric acid (n=3) as the dicarboxylic acid spacer forming a 14-O-ester bond with Q, 14 (doxorubicin) or Q6 (2-pyrrolino-doxorubictn) and a carboxamide bond with the peptide carrier. The most preferred embodiments of this invention are cytotoxic j LH-RH analogs of the following formulae: 1. Glp-His-Trp-Ser-Tyr-D-Lys(Qi1"-0-glt)-Leu-Arg-Pro-Gly-NH2; 2. Glp-His-Trp-Ser-Tyr-D-Lys(Q61"-0-glt)-Leu-Arg-Pro-Gly-NH2; cytotoxic somatostatin analogs of the following formulae: 3. Q,u-0-glt-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2; 4. Q614-0-glt-D-Phe-Cys-Tyr-D-Trp-Lys-Va{-Cys-Thr-NHr; h5. Q/'-O-glt-O-Trp-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NHj; 6. Q6M-0-git-D-Trp-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH3; 7. Q/^O-glt-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NHz; and 8. Q6lJ,-0-glt-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH3; 15 and cytotoxic bombesin antagonist analogs of the following formulae: 9. Qi14-0-git-Gln-Trp-Ala-VaI-Gly-His-Leu(CH2-NH)Leu-NH2; 10. Q614-0-glt-Gln-Trp-Ala-Val-Gly-His-Leu(CH2-NH)Leu-NH2 11. Qi14-0-g!t-D-Tpi-Gln-Trp-Ala-Val-Gly-His-Leu(CH2-NH)Leu-NH2:and 12. Qe14-0-glt-D-Tpi-Gln-Trp-Ala-Va!-Gly-His-Leu (CH2-NH)l_eu-NH2. In the novel process of forming a partially saturated heterocyclic ring with the nitrogen of a vicinal or disjunct i.e., a,|3~ or a,y-hydroxy amine the first step of the reaction is carried out in an anhydrous inert organic polar non-hydroxylic (aprotic) solvent, suitably dimethyl formamide using substantial excess, suitably a 30 fold excess of the halo aldehyde, 4-iodobutyraldehyde and 5-iodovaleraldehyde are especially effective, The Invention is not limited to these however, bromo may be used in place of iodo. This reaction as well as the subsequent steps may be carried out at ambient temperature. The basification step is carried out with an excess, suitably a 2-4 fold excess of an organic base. Tertiary amines such as trialkylamines are suitable for this purpose. The thus formed bicyclic ring is opened to release the vicinal or disjunct hydroxy! group by treatment with an organic acid in the presence of water. Dilute aqueous trifuoracetic acid, suitably in an inert organic solvent such as acetonitrile may be used. The product is purified by removal ofthe volatiles under reduced pressure, excess halo compound extracted with hexane. and the residue purified on HPLC. Abbreviations For the description of the peptides and their derivatives of this invention, the conventional abbreviations for the amino acids are used as generally accepted in the peptide chemistry art and as recommended by the IUPAC- 16 IUB Commission on Biochemical Nomenclature (European J. Biochem., 138, 9-37(1984). The abbreviations for the individual amino acid residues are based on the trivia! name of the amino acid, e.g. Glp is pyroglutamic acid, His is histidine, Trp is tryptophan, etc The abbreviations indicate the L isomeric form of the amino acids, unless expressed otherwise, e. g., Ser is L-serine, and D-Lys is D-lysine. Abbreviations of the uncommon amino acids in this invention are as follows: D-Nal(2)' is D-3-(2-naphthyi)alanine, and 0-Pal(3) is D-3-(3-pyridyl)alanine, • D-Phe(4CI) is D-4-chlorophenylalanine. Peptide sequences are written according to the convention whereby the N- terminal amino acid is on the left and the C-terminal amino acid is on the right e.g., Gtp-His-Trp. The formula, Leu (CH2-NH)Leu-NH2 describes a reduced peptide bond between B leucine and leucine amide residue at the C-terminal of a peptide sequence. Other abbreviations used are: AjBu: diaminobutyric acid A2Pr: diaminopropionic acid BN: bombesin BOP reagent. benzotriazole-1-yloxitris(dimethylamino)phosphonium hexa- fluorophosphate DIPEA: N,N-diisopropylethylamine DM-DOX: daunosamine modified doxorubicin OMF: N,N-dimethylformamide DMTac: 5,5-dimethyl-thiazolidine-4-carboxylic acid 1 7 DOX doxorubicin Fmoc: 9-fluorenyfmethyioxycarbonyl git : -C(0)-CH2-CH2-CH2-C(0)-, giutaryi Glt20: glutaric anhydride HOBt; 1-hydroxibenzotriazoie HO-glt-OH: glutaric acid HOSu : N-hydroxysuccinimide HPLC: high performance liquid chromatography TFA: trifluoroacetic acid Tac; thiazolidine-4-carboxylic acid Tpi: 2,3,4,9-tetrahydro-1 H-pyrido[3,4-b]indole-3-carboxylic acid A Beckman analytical HPLC system equipped with model 168 diode array detector and System Gold chromatography software (Beckman) was used to monitor :the chemical reactions and to check the purity of the compounds of this invention. The column used was Dynamax C-18(250x4 6 mm; pore size. 300A; particle size: 12 urn. The solvent system consisted of two components: (i) 0.1% TFA in water, and (it) 0.1% TFA in 70% aqueous acetonitrile and usedin linear gradient mode, growing 1% (ii) in 1 min., for monitoring the chemical reactions. The system was used in isocratic mode for purity control. A Beckman model 342 semipreparative HPLC system was used for isolation and purification of the compounds of this invention. The column was Aquapore Octyl {250x10mm; pore size: 300A; particle size: 15 urn). The solvent system was the same described for the analytical HPLC above. Analysis Bruker ARX300 NMR spectrometer (300MHz 1H frequency,. 75MHz 13C frequency) and electrospray mass spectrometer Finnigan-MAT TSQ 7C00 were used for the structure identification of the doxorubicin derivatives 18 Synthesis of peptide carriers The peptides of the invention are often administered in the form of pharmaceutical acceptable, nontoxic salts, such as acid additional salts. Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, fumarate, glyconate, (annate, maleate, acetate, tri-fluoroacetate, citrate, benzoate, succinate, alginate, pamoate, malate, ascorbate, tartrate, and the like. If the active ingredient is to be administered in tablet form, the tablet may contain a pharmaceutical^ acceptable diluent whicn includes a binder, such as tragacanth, corn starch or gelatin, a disintegrating agent, such as alginic acid and a lubricant, such as magnesium stearate. If administration in liquid form is desired, sweetening and/or flavoring may be used as part of the pharmaceutically-acceptable diluent, an intravenous administration in isotonic saline, phosphate buffer solutions or the like may be effected, The pharmaceutical compositions will usually contain the peptide in conjunction with a conventional, pharmaceutically-acceptable carrier. Usually, the dosage will be from about 1 to about 100 micrograms of the peptide per kilogram of the body weight of the host when given intravenously; oral dosages will be much higher. Overall, treatment of subjects with these peptides is generally carried out in the same manner as the clinical treatment using other analogs of LHRH, somatostatin and analogs of doxorubicin. These peptides can be administered to mammals intravenously, subcutaneously, intramuscularly, orally, intranasally or intravaginally to achieve biological hormonal effects through binding to specific receptors. In the case of LHRH analogs, these effects may include reversible suppression 19 of gonadal activity, and in (he case of somatostatin analogs inhibition of gastrointentinal function. Effective dosages will vary with the form of administration and the particular species of mammal being treated An example of one typical dosage form is a physiological saline solution containing the peptide which solution is administered to provide a dose in the range of about 0.1 to 2 5 mg/kg of body weight. Oral administration of the peptide may be given in either solid form or liquid form The synthesis of the peptide carriers of the present invention can be performed by any techniques that are known to those skilled in the art of peptide chemistry. A summary of the suitable techniques can be found in M Bodanszky Principles of Peptide Synthesis. Spnnger-Verlag. Heidelberg. 1984 Techniques for solid phase peptide synthesis can be found in the textbook of J M Stewart and J D Young. Solid Phase Peptide Synthesis, Pierce Chem. Co.. Rockford, IL. 1984 (2nd ed ) and in the review of C Barany et al;. Int. J Peptide and Protein Res 30, 705-739 (1987) The synthesis of the LH-RH analog carriers used in this invention is detailed in the examples of US Patent 5.258,492, Sandor Bajusz and Andrew V Schally, November 2, 1993 and in the articles of Bajusz et al . Proc Natl Acad Sci USA 85, 1637-1641 (1988) and 86, 6318-6322 (1989) and Janaky et al., Proc Natl Acad Sci. USA, 89. 1023-1027 and 972-976 (1992) The synthesis of the somatostatin analog carriers used in this invention is detailed in the examples of U.S. Patent 4,650,787, March 17, 1987, Andrew V Schally and Ren 2 Car A description of the synthesis can also be found in the articles by Cai et al.. Proc. Natl. Acad Sci USA 83, 1896-1900 (1986) and Proc Natl Acad. Sci USA 84, 2502-2506 (1987). The synthesis of the bombesin antagonist earners used in this invention is detailed in the anicles by Coy et al.. J Biol Chem. 263.-5056-5060 (1988) 20 and 264, 14691-14697 (1989) and by Cai et a!., Peptides 13, 267-271 (1992) andProc. Nat!. Acad. Sci. USA 91, 12664-12668(1994). The synthesis of the doxorubicin derivatives used in this invention and the formation of their conjugates with different peptide carriers is detailed in the following examples which are intended to be illustrative and not limiting: EXAMPLE 1 Preparation and isolation of N-Fmoc-DOX14-0-hemiglutarate DOX HO salt, 50 mg (86 pmol). was dissolved in 1 ml_ DMF and 30 mg(90 pmol) Fmoc-OSu was added followed by the addition of 31 pL (180 pmol) DIPEA. After stirring for three hours, the reaction was complete as assessea by analytical HPLC. The solvent was evaporated to dryness in Speed Vac high vacuum evaporatpr and the residue was crystallized by rubbing with 0.1% TFA in H20. The crystals were filtered and washed once by cold ether to remove traces of excess Fmoc-OSu. After drying in a desiccator, m=62mg, of 98% pure N-Fmoc-QOX was obtained. Yield:94% This intermediate was reacted overnight with 11.4 mg(100 pmol) Glt20 in 1 mL anhydrous DMF in the presence of 26.1 pL (150 pmol) DIPEA. The solvent was evaporated in Speed Vac and the residual oil was solidified by rubbing with 0.1% aqueous TFA (v/v) The crude material thus obtained contains 70% N-Fmoc-DOX14-0-hemiglutarate, 20% unreacted N-Fmoc-DOX and 10% other impurities as assessed by analytical HPLC- This crude product can be used for the preparation of peptide DOX conjugates without further purification When this crude material was dissolved in 20 mL 60% aqueous acetonitrile containing 0.1% TFA and applied on semipreparative HPLC, 45.7 mgT of 98% pure N-Fmoc-DOX14-0-herniglutarate end product was obtained. (Yield: 64%.) 21 EXAMPLE 2 Preparation and isolation of 3'-deamino-3*-(pyrro!idine-r*-yl)-doxorubicin TFA salt (Q2) and its 14-O-hemiglutarate (AN-193) TFA salt DOX HCI salt. 50 mg{86 umol), was dissolved in 1 mL DMF and 171 yL (1.3 mmol) 15 fold excess 1,4-diiodobutane was added followed by the addition of 45 (JL {260 umol) 3 fold excess DIPEA. The reaction mixture was stirred overnight at room temperature. After 16 hours the reaction was complete as assessed by analytical HPLC, The solvent is evaporated in Speed Vac and the residual oil is dissolved in 3 mL 0 1% TFA in*H20 and extracted with ether to remove excess 1,4-diiodobutane The aqueous extract was then applied on HPLC and m:416 mg, of 98% pure DOX derivative was obtained. (Yield 68%) The 416 mg (58 umol) 3'-dearnino-3'-(pyrrolidine-r-yl)-doxorubicin TFA salt (Q2) thus obtained was reacted with 1.2 equivalent Glt20 in dry DMF exactly as described in Example 1. The yield was 35% (16.9 mg) and the purity was 98%. EXAMPLE 3 Preparation and isolation of 3'-deamino-3'-(isoindoline-2"-yl)doxorubicin TFA salt(Q3) DOX HCI salt, 50 mg(86 umol), was dissolved in 1 mL DMF and 226 mg (13 mmol) 15 fold excess a.a'-dichloro-ortho-xylene was added followed by the addition of 45 uL (260 umol) 3 fold excess DIPEA and catalytica! amount of Nal. After 16 hours the solvents were removed with Speed Vac and the residue was dissolved in 3 mL 0 1% aqueous TFA and extracted with 3 mL 22 ether to remove the'excess of the ha!ogen compound. The--crude material thus obtained was applied on HPLC. After purification 36 mgT 98% pure end product was obtained. (Yield: 55%) EXAMPLE 4 Preparation and isolation of 3'-deamino-3'-(3"-pyrroline-1,,-yl)-doxorubicin TFA salt (Q4) DOX HCI salt, 50 mg(86 umol), was dissolved in 1 mL DMF and 136.8 pL (1.3 mmol) 15 fold excess cis-1,4-dichloro-2-butene (Aldrich) was added followed by the addition of 45 pL (260 umol) 3 fold excess DIPEA. After 16 hours the solvents were removed in Speed Vac and the residue was dissolved iri 3 mL 0.1% aqueous TFA and extracted with 3 mL hexane to remove the excess of the halogen compound The crude material thus obtained was applied on HPLC. After purification 22.6 mg, 98% pure end product was obtained. (Yield:37%) EXAMPLE 5 Preparation and isolation of 1-chloro-4-bromo-2-butanone (C4H6CIBrO) and 1 -chloro~5-bromo-2-pentanone (C5H8CIBrO) 3-Bromopropionyi chloride, 100.8 uL (1 mmol), (Aldrich) was reacted with excess diazomethane in ether. After 1 hr the ethereal solution was eluted and spot tested on TLC. Thin layer chromatography aluminum sheets pre-coated with silica gel 60 F254 by Merck Art No. 5554 was used as the stationary phase and CHCI3:MeOH 95:5 (v/v) as the mobile phase. For the spot test 2,4-dinitrophenylhydrazine reagent (Vogel: A textbook of Practical Organic Chemistry, page 1061. Third Edition, Longmans, New York.) was sprayed on the TLC sheet after elution. The diazomethylketcne derivative 23 thus formed showed a yellow spot with Rf:0.3 The ethereal solution was then reacted with anhydrous HCI in ether converting the diazomethylketone to the desired end product, 1-chloro-4-bromo-2-butanone This product showed a yellow spot characteristic of oxo compounds, with Rf:0.8 in the same solvent system and with the spot test reagent described above. After evaporation of the solvent, the crude product was applied on a column (15 cm long, 2.5 cm in diameter) packed with 15 g silica gel, Merck, grade 9385, 230-400 mesh, pore size 60A. The liquid, mobile phase was neat CHCI3. Fractions containing the desired end product (characterized by the spot test detailed above) were mixed and evaporated to dryness. M=1.5 g, clear oil was obtained Yield: 80% 1-chlorb-5-bromo-2-pentanone was prepared from 4-bromobutyryl chloride exactly the same way as described for 1-ch!oro-4-bromo-2-pentanone, except that 4-bromobutyryl chloride was used Instead of 3-bromopropionyl chloride. 1.6 g. clear oil was obtained. Yield: 80%. EXAMPLE 6 Preparation and isolation of 3'-deamino-3'-(3"-pyrrolidone-1 "-yl)-doxorubicin TFA salt (Q5) DOX HCI salt, 50 mg(86 umol), was dissolved in 1 mL DMF and 241 mg (1.3 mmol) 15 fold excess 1-ch!oro-4-bromo-2-butanone was added followed by the addition of 45 uL (260 umol) 3-fold excess DIPEA. After 16 hours the solvents were removed in a Speed Vac and the residue was dissolved in 3 mL 0.1 % aqueous TFA and extracted with 3 mL hexane to remove the excess halogen compound. The crude material thus obtained was applied on HPLC. After purification, 20.6 mg, 98% pure end product was obtained. (Yield:33%) 24 EXAMPLE7 Preparation and isolation of 3'-deamino-3'-(3"-piperidone-1 "-yl)-doxorubicin TFA salt (Q7) DOX HCI salt, 50 mg(86 umol), was dissolved in 1 mL DMF and 260 mg (1.3 mmol) 15 fold excess 1-chloro-5-bromo-2-pentanone was added followed by the addition of 45 uL (260 umol) 3 fold excess DIPEA After 16 hours the solvents were removed in a Speed Vac and the residue was dissolved in 3 mL 0.1% aqueous TFA and extracted with 3 mL hexane to remove the excess of the halogen compound. The crude material thus obtained was applied on HPLC. After purification, 18 mg, 95% pure end product was obtained. (Yield:28%) EXAMPLE 8 Preparation and isolation of 4-iodobutyrafdehyde and 5-iodovaleraldehyde 2-(3-Chloropropyl)-1,3-dioxolane (4-chloro-n-butyraldehyde ethylene acetal), 1.3 mL (10 mmol), (Fluka) was dissolved in 200 mL acetone containing 30 g {200 mmol, 20-fold excess) Nal. The solution was refluxed for 24 hours followed by evaporation to dryness. 100 mL ether was used to extract the organic material from the inorganic solid residue/The ethereal solution was then washed with 50 mL H20, 50 mL 5% aqueous Na2S203 solution and 3 times with 50 mL H20. The ether was removed in vacuo and the remaining oil was dissolved in 3 mL 50% aqueous acetic acid. After 1 hr 100 mL ether was added to this solution and the acetic acid as well as the ethylene glycol was removed by washing with 50 mL H20 3 times. The main product was eluted at Rf: 0.8 on TLC in neat CHCI3. The spot test used for the aldehyde function was the same described for the ketones in Example 5. The ether was then removed and the black oil was applied on a column (15 cm long, 2.5 cm in 25 diameter) packed with 15 g silica gel. Merck, grade 9385, 230-400 mesh, pore size 60A. The liquid, mobile phase was CHCI3. Fractions containing the desired end product (characterized by the spot test detailed above) were mixed and evaporated to dryness. 1.6 g yellow oil was obtained. Yield: 80%. 5-lodovaleraldehyde was obtained exactly the same way starting from 2-(4-chlorobutyl)-1,3-dioxo!ane (5-chloro-n-vaieraldehyde ethylene acetal) (Fluka). 1.65 g yellow oil was obtained. Yield: 80%. EXAMPLE 9 Preparation and isolation of 3'-deamino-3'-(2"-pyrroline-1"-yl)-doxorubicin TFA salt (Q6) DOX HCI salt, 50 HPLC system). This solution was diluted with 2 mL 0.1 % aqueous TFA solution followed by the removal of the acetonitrile in a Speed Vac. The resulting solution was extracted with hexane to remove the excess of the halogen compound. The material thus obtained was applied on HPLC. After purification 52 mg, 98% pure end product was obtained. (Yield:85%) EXAMPLE 10 Preparation and isolation of 3,-deamino-3'-(1"l3"-tetrahydropyhdine-1"-yl)-doxorubicin TFA salt (Q8) 26 DOX HCt salt, 50 mg{86 umol), was dissolved in ImL DMF and 552 mg (2.6 mmol) 30-fold excess 5-iodovaleraldehyde was added followed by the addition of 45 uL (260 umol) 3-foid excess DIPEA. After 1 hour 100 uL glacial acetic acid was added to the reaction mixture which was then added dropwise to 5 ml_ of 0.1 % TFA in 70% aqueous acetonitriie (solvent ii of the HPLC system). This solution was diluted with 2 mL 0.1% aqueous TFA solution followed by the removal of the acetonitriie in a Speed Vac. The resulting solution was extracted with hexane to remove the exce'ss halogen compound. The material thus obtained was applied on HPLC. After purification, 46 mg, 98% pure end product was obtained. (Yieid:75%) EXAMPLE 11 Preparation and isolation of cytotoxic LH-RH agonist analog containing DOX ([D-Lys6(DOX14-0-glt)jLH-RH, Q,14gL) [D-Lys6]LH-RH, 60 mg (37.5 umol), and 52 mg (64% pure, 37.5 umol) N-Fmoc-DOX14-0-hemiglutarate, (see Example 1), was dissolved in 1mL DMF and 22 mg (50 umol) BOP reagent (Aldrich), 13.5 mg (100 Mmol) HOBt as well as 52 uL (300 umol) DIPEA was added. After stirring for 1 hr at room temperature the reaction is complete. The solvents were evaporated and the residual oil was crystallized by 3 mL ethyl acetate and then washed twice with 3 mL ethyl acetate. The 90 mg crude solid material was then dissolved in 3 mL DMF and 300 uL piperidine was added. After 5 minutes, the reaction was placed into an ice bath and was acidified by the addition of a mixture of 300 uL TFA, 700 uL pyridine and 2 mL DMF. After evaporation of the solvents, the residual oil was solidified by ethyl acetate. The crude solid thus obtained, was dissolved in 1 mL 70% aqueous acetonitriie containing 0.1% TFA (i) and diluted with 3 mL 0.1% aqueous TFA (ii) and applied on semipreparative HPLC. 40 mg (14,8 umol) 98% pure end product was obtained. Yield: 48%. 27 EXAMPLE 12 Preparation of cytotoxic LH-RH agonist analog containing 2-pyrrolino-DOX ([D-Lys6{2-pyrrolino-DOX1"-0-glt)]LH-RH, Q6"gL) Q,,4gL, 11.2 mg(5pmol), (see Example 11) was dissolved in 200 pL DMF and 30 mg (150 pmol, 30-fold excess) 4-iodobutyraldehyde (Example 8) was added followed by the addition of 3 pL (17 pmol) DIPEA. After 1 hour, the reaction was complete (see Example 9) and 10 pL glacial acetic acid was added to the reaction mixture which was then added dropwise to 1 mL 0.1% TFA in 70% aqueous acetonitrile. This solution was then diluted with 1 mL 0.1% aqueous TFA and the acetonitrile was removed in vacuo. The remaining aqueous solution was then extracted with 1 mL hexane and applied on HPLC. m:7.6 mg, 99% pure end product was obtained. (Yield: 66%) EXAMPLE 13 Preparation and isolation of a cytotoxic somatostatin analog containing DOX (DOX,4-0-glt-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2l Q,14gS) D-Phe-Cys-Tyr-D-Trp-Lys(Fmoc)-Val-Cys-Thr-NH2, 20 mg (14.5 pmol) (Proc. Natl. Acad. Set USA 1986, pp. 1986-1990) and 20 mg (64% pure, 14.5 prrtol) N-Fmoc-DOX14-0-hemiglutarate (Example 1) was dissolved in 200 pL DMF and 8.8 mg (20pmol) BOP reagent (Aldrich), 5.4 mg (40 pmol) HOBt as well as 17 pL (100 pmol) DIPEA was added. After stirring for 1 hour at room temperature, the reaction was complete. After removal of the solvents in vacuo, the residue was crystallized by ethyl acetate. This solid material was 26 then dissolved in 1 mL DMF and 100 pL piperidine was added. After.7 min the reaction was placed into an ice bath and was acidified by the addition of a mixture of 100 pL TFA, 300 pL pyridine and 2 mL DMF. After evaporation of the solvents, the residual oi! was solidified by ethyl acetate. The crude solid thus obtained was dissolved in 1 mL 70% aqueous acetonithle containing 0.1% TFA (i) and diluted with 3 mL 0.1% aqueous TFA (ii) and applied on semipreparative HPLC. 9.7 mg (5.1 pmol) 95% pure end product was obtained. Yield: 35%. EXAMPLE 14 Preparation of cytotoxic somatostatin analog containing 2-pyrrolino-DOX (2-pyrrolino-DOX14-0-git-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2, Q614gS) D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 (6.4 mg, 5pmol) was dissolved in 100 pL DMF and 2-pyrrolino-DOX14-0-hemigiutarate (4.1mg, 5pmol) was added, followed by BOP reagent (4.4 mg, 10pmol) HOBt (100pmol) and DIPEA (50pmol). After stirring for 2 hr at room temperature, the reaction mixture was acidified by 20pL AcOH and diluted with 500pL 70% aqueous acetonithle containing 0.1'% TFA and further diluted with 700pL 0.1% aqueous TFA and applied on HPLC. 3.9 mg (Yield:40%) of 99% pure end product was obtained. 2-Pyrrolino-DOX14-0-hemiglutarate was prepared by reacting DOX14-0-hemiglutarate with 4-iodobutyraldehyde as described in EXAMPLE 9. 29 DOX'^-O-hemiglutarate was prepared from N-Fmoc-DOX14-0-hernig!utarate by cleaving the Fmoc protecting group as described in EXAMPLE 11. (Yield: 40%) EXAMPLE 15 Preparation and isolation of a cytotoxic bombesin antagonist containing DOX (DOXt4-0-glt-Gln-Trp-Ala-Val-GIy-His-Leu (CH2-NH)Leu-NH2) 6/4gB) Gln-Trp-Ala-Val-Gly-His-Leu (CH2-NH)Leu-NH2l 20 mg (15.8 pmol) (Int. J. Peptide Protein Res. 38, 1991, pp. 593-600) and 22 mg (64% pure, 15.8 umol) N-Fmoc-DOX14-O-hemigiutarate (Example 1) was dissolved in 200 uL DMF^and 8.8 mg (20 umol) BOP reagent (Aldrich), 5.4 mg (40 umol) HOBt as well'as -17 pL (100 pmol) DIPEA was added. After stirring for 1 hour at room temperature the reaction was complete. After removal of the solvents in vacuo, the residue was crystallized by ethyl acetate. This solid material was then dissolved in 1 mL DMF and 100 uL piperidine was added. After 5 min the reaction was placed into an ice bath and was acidified by the addition of a mixture of 100 uL TFA, 300 uL pyridine and 2 mL DMF. After evaporation of the solvents, the residual oil was solidified by ethyl acetate. The crude solid thus obtained was dissolved in 1 mL 70% aqueous acetonitrile containing 0.1% TFA (i) and diluted with 3 mL 0.1% aqueous TFA (ii) and applied on semipreparative HPLC. 13.5 mg (7.1 pmol) 98% pure end product was obtained. Yield: 45%. EXAMPLE 16 Preparation and isolation of a cytotoxic bombesin antagonistic analog containing 2-pyrrolino-DOX 2-pyrrolino-DOX14-0-glt-Gln-Trp-Ala-Val-Gly-His-Leu(CH2-NH)Leu-NH2, Q614gB 30 (VgB, 9.5 mg (5 umol), (Example 15) was dissolved in 200 uL DMF and 30 mg (150 pmol, 30-fold excess) 4-iodobutyraldehyde (Example 8) was added followed by the addition of 3 uL (17 pmol) DIPEA. After 1 hour the reaction was complete (Example 9) and 10 pL glacial acetic acid was added to the reaction mixture which was then added dropwise to 1 mL 0.1% TFA in 70% aqueous acetonitrile. This solution was then diluted with 1 mL 0.1% aqueous TFA and the acetonitrile was removed in vacuo. The remaining aqueous solution was then extracted with 1 mL hexane and applied on HPLC. 6 mg 98% pure end product was obtained. (Yield:60%.) Determination of in vitro cytotoxic activity MXTestrogen-independent mouse mammary carcinoma cell line was obtained from Dr. Gunter Berndhardt, University of Regensburg, Germany. All the other cell lines used in the determination of the antiproliferative activity'of the compounds of this invention were obtained from the American Type Culture Collection (ATCC). For the evaluation of the activity of the analogs, a colorimetric cytotoxicity assay in microtitration plates was used based on quantification of biomass by staining cells with crystal violet, which correlates very well with determination of cell numbers. (Reileeta!.; Anal. Biochem. 187, 262-267, 1990; Bernhardt G. et al, J. Cancer Res. Clin. Oncol. (1992), 118, 35-43; Spruss Th. et al, J Cancer Res. Clin. Oncol 117, 435^43, 1991; Gillies, R. J., Anal. Biochem. 159, 109-113, 1986; Kueng, W. eta); Anal. Biochem., 182 16-19, 1989.) Assay Protocol One to two days after seeding cells in 96-well plates the culture medium is exchanged with fresh medium containing the compounds to be tested and 31 fresh medium only for the control cultures. After varying time of incubation, cells are fixed with glutaric dialdehyde and stored under fetal bovine serum (FBS) at 4oC until the end of the experiment. Cells are stained with crystal violet and bound stain is extracted with 70% aqueous EtOH Optical denstiy is measured with EIA Reader (Bio-Tek Instruments) or Biomek 1000 (Beckman) at 590 nm or 600 nm, respectively. Each data point represents the mean value of eight culture wells. T/C values are calculated as T/C= (T-C0)/(C-C0) where T= optical density of treated cultures, C= optical density of control (untreated) cultures, C0= optical density of cultures at the start of incubation (t=0). EXAMPLE 17 In vitro ^cytotoxic activity of daunosamine modified derivatives of DOX Table 17-1 demonstrates the effects of doxorubicin and its daunosamine modified derivatives on MCF-7 human mammary carcinoma cell line in vitro. Cytotoxic radicals having their daunosamine N incorporated into a five- membered ring with a reactive function are 5 to 50 times more active than their homolog counterpart with a six-membered ring as the examples of 3-pyrroiidono-DOX (Q5) and 3-piperidono-DOX (Q7) as well as 2-pyrrolino-DOX (Q6) and 1,3-tetrahydro-pyridino-DOX (Qe) 32 Tabfe 17-1. Effects of Doxorubicin and its Daunosamine modified derivatives on MCF-7 Human Mammary Carcinoma CelL Line in vitro Compound Incubation T/C Value at (M) Time (hr.) 3x10-,c 10'9 3x10"9 10^ 3x10"8 10'7 Doxorubicin 70 98 82 54 (DOX) 120 95 66 33 Pyrrolidino- 70 97 25 -26 DOX (Q2) 120 94 17 -19 Piperidino- 70 114 70 4 DOX(AN-183) 120 109 67 0 Isoindoiino- 70 118 86 -11 DOX 3-Pyrrolino- 70 106 72 -3 DOX (Q,) 120 97 65 -5 3-Pyrrolidono- 70 87 30 -28 DOX 3-Piperidono- 70 96 80 59 DOX (Q7) 120 97 70 43 2-Pyrrolino- 70 50 -3 -18 DOX(Q6) 120 26 2 -9 1,3-Tetrahydro 70 96 88 69 pyridino-DOX 120 99 93 62 Cells were incubated in iMEM media containing 5% HI-DCC-FBS (heat inactivated dextran coated charcoal treated fetal bovine serum) on 96 well plates. Relative cell number in treated and control plates was determined by 33 the crystal violet staining method and was expressed as T/C vaiues where T/C=(T-Cc/C-Co) x 100 [T= absorbance of treated cultures, O absorbance c-control cultures, C0= absorbance of cultures at the start of incubation (t=0). The measured absorbance is proportionate to the cell number] Lower T/C values indicate a decrease in the survival of cancerous cells due to treatment. That is to say, 75 would indicate 75% survival of cells as compared to 100% for control or 25% inhibition. EXAMPLE 18 Full retaining of in vitro cytotoxic activity of DOX in LH-RH agonist peptide conjugate Qi14gL and superactive 2-pyrrolino-DOX (Q6) in LH-RH agonist peptide conjugate (VgL. Table 18-1 demonstrates the effects doxorubicin and its daunosamine modified derivative, 2-pyrrolinodoxorubicin (Q6) in comparison with their conjugates with LH-RH agonistic analog, |D-Lys€ JLH-RH (Qi14gL and Q614gL. respectively) on the growth of MCF-7 human mammary carcinoma cell line -and MXT estrogen independent mouse mammary carcinoma cell line in vitro. 34 Table 18-1: Compound Incubation Time (hr.) T/C Value on MCF-7 Ceil Line at Conc.(M) 3x10'*1 10"10 3x1010 109 |3x10"9 10"* |3x10"6 10"7 Doxorubicin* 70 120 98 95 82 66 54 33 QiMgl 70 120 111 78 89 55 63 28 Q6 70 120 50 26 -3 -2 -18 -9 Q614gl 70 74 28 -24 120 60 1 16.. -14 Compound Incubation Time (hr.) T/C value on MXT celt fine at Conc.(IVI) 3x10" 10-10 3x10"10 10"9 3X10"9 10"8 3X10*8 10"7 Doxorubicin 26 50 85 74 90 60 59 43 Qi14gL 26 50 87 71 91 59 73 50 Q6 28 69 90 52 78 6 56 -13 Q614gL 28 69 . 91 59 78 15 64 -11 35 MCF-7 ceiis were incubated in IMEM medta containing 5% HI-DCC-FBS on 96 well plates. MXT cells were incubated in RPMI 1640 media containing 0.6 g/L L-glutamine and 10% FBS. "Determined as in Table 17-1. EXAMPLE 19 Table 19-1 demonstrates that the in vitro cytotoxic activity of the somatostatin analogs containing DOX of the invention is fully retained. Table 19-1: Effects of Cytotoxic Analogs of Somatostatin Containing Doxorubicin on the Growth of Ml IA PaCa-2 Human Pancreatic Cancer Ceil Line in Vitro Compound 28 76 32 -3 96 98 95 11 Incubation Time (hr.) T/C Value at Concentration^M). 10"8 io-7 10"6 93 103 DOX14-0-glt- 28 76 S-98*(Q,14gS9a) Carrier Analog S-98* DOX14-0-glt-S-12l-(Q^gS12') Carrier Analog S-121** Doxorubicin 28 76 28 76 "28 76 93 97 95 71 82 10 64 10 35 -4 76" 96 ^28 -7 36 Cells were incubated in RPMI 1640 media containing 10% fetal bovine serum on 96 well plates. *D-Trp-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2; *D-Phe-Cys-Tyr-D-Trp-Lys-Va1-Cys-Thr-NH2; EXAMPLE 20 Effects of Cytotoxic Analogs of Bombesin Antagonists Containing Doxorubicin on the Growth of CFPAC-1 Human Pancreatic Cancer Cell In Vitro Table 20-1 demonstrates that the in vitro cytotoxic activity of bombesin antagonistic analogs containing DOX of the invention is fully retained. 37 Table 20-1 Compound Incubation T/C Value at Concentration (M) Time (hr.) 3x10* 107 3x10"7 10"6 D0X14-0-glt 66 95 81 44 9 B-94 95 95 57 28 4 (Qi14gB) 137 94 28 19 / 0 B-94* 66 99 106 104 100 95 97 99 99 96 137 98 98 100 96 DOX'4-O-g!t-B-50 66 Y02 78 39 5 95 97 55 24 -1 137 92 28 19 -2 B-50" 66 100 93 99 93 95 98 100 102 98 137 97 98 99 98 DOX 66 88 52 15 -7 95 73 32 10 -6 137 49 20 7 -4 Cells were incubated in IMDM media containing 10% fetal bovine serum on 24 well plates. *Gln-Trp-Ala-Val-Gly-His-Leu-v(CH2-N)-Leu-NH2 **D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-v/(CH2-N)-Tac-NH2 Preserved Binding Properties of Hormone Derivatives 38 EXAMPLE 21 Hormonal activities and receptor binding potencies of cytotoxic LH-RH agonist analogs Q-, 14gL ([D-Lys6]LH-RH carrying DOX) and Qe14gL ([D- Lys6]LH-RH carrying 2-pyrrolino-DOX) in comparison with the carrier peptide, [D-Lys6]LH-RH Table 21-1 Compound Hormonal activity* IC50" vatue IC50** value (LH-response ret for rat pituitary for breast cancer toLH~RH=1) receptors (nM) receptors (nM) Q,"gL - 15 2.29 7.24 Q614gL 10 5.59 6.70 (D-Lys6]LH-RH 8 2.26 1.80 10 In Table 21-1 *LH responses to the analogs were determined in dispersed rat pituitary cell superfusion system as described in S. Vigh and A. V. Senary, Peptides 5, 241-247 (1984). "Binding affinities of the analogs to rat pituitary LH-RH receptors and human breast cancer receptors were determined in competitive binding experiments using [1251] labeled [D-Trp6]LH-RH as radio iigand as described in B. Szoke et al., Peptides, 15(2), 359-366 (1994). The binding affinities were expressed by IC50 values, the concentration of unlabeled analog required to inhibit 50% of the specific binding of the radio Iigand. 39 EXAMPLE 22 Somatostatin analogs inhibit the secretion of growth hormone (GH) from perfused rat pituitary as it is described by Carlson et al., Thyrotropin- releasing hormone stimulation and somatostatin inhibition of growth hormone secretion from perfused rat adenohypophyses Endocrinology, 94, 1709-(1974). Accordingly, this method was used to compare the cytotoxic somatostatin analogs of the present invention to their parent carrier molecules with respect to their hormonal activities. Inhibition of human growth hormone-releasing hormone (hGH-RH(1-29)NH2) induced growth hormone release from superfused rat pituitary cells by somatostatin analogs S-98-I D-Trp-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2; and S-121 D-Phe-Cys-Tyr-D-Trp-Lys-VaI-Cys-Thr-NH2; in comparison with their cytotoxic derivative, Q114gS98",(DOX14-0-glt-S-98-l) and Q!14gS121 (DOX14-0-glt-S-121), respectively. In rat pituitary superfusion system, the somatostatin analogs were administered for 3 min at 1 nM dose simultaneously with 1 nM hGH-RH(1-29)NH2. The infusion of the somatostatin analogs was maintained for another 6 min. GH responses to 3 min administration of 1 nM hGH-RH(1-29)NH2 were determined during the perfusion of the somatostatin analogs (0 min) and 30, 60 and 90 min after the administration stopped. The data are presented in Table 22-1. 40 Table 22-1 Somatostatin GH release** induced by 3 min administration of 1 nM analogs hGH-RH(1-29)NH2 at different time points after infusion of the somatostatin analogs 0 min 30 min 60 min 90 min S-98-I 2.9 94.7 117.6 - CVgS98 0 90 89.7 - S-121 7.8 62.2 57.3 77.9 Q,,4gS121 8.8 58.5 54.3 67.7 Expressed as percentage of GH release induced by 3 min infusion of 1 nM hGH-RH (T-29)NH2 prior to the administration of the somatostatin analogs. EXAMPLE 23 Receptor binding studies with cytotoxic bombesin antagonists Radio iodination of [Tyr^BN (Sigma) using a Bio-Rad Enzymobead Radio lodination kit and isolation of mono-iodinated [125l-Tyr4]BN was performed as described earlier (1). Binding of labeled [Tyr^BN and displacement by cytotoxic bombesin antagonist analog, Gs14gB was conducted using confluent Swiss 3T3 cells (obtained from the American Type Culture Collection) in 24-well plates in a modification (2) of the method of Kris et al (3). Three to five days after seeding, the confluent cells were washed twice with Hanks' Balanced Salt Solution (HBSS) and incubated for 30 min at 37oC with 50 pM [125l-Tyr4]BN in the absence or presence of several concentrations of unlabeled competitors (Q614gB or BN) in a total volume of 0.5 ml binding buffer (DMEM with 50 mM HEPES, 0.1% bovine serum albumin (BSAK 5 mM MgCI2 and 100 ug/ml bacitracin, pH: 7.4). Nonspecific binding was 41 determined in the presence of 1 u!Vi unlabeled iigand. After three washings with ice-cold HBSS containing 0.1% BSA (pH: 7.4) the cells were detached with 0.05% Trypsin/ 0.53 mM EDTA solution and transferred to tubes. Radioactivity was measured with a gamma-counter {Micromedic Systems Inc. Huntsville, AL). Binding data were evaluated using radio Iigand binding analysis programs by McPherson (4). Kj values presented in Table 23-1, were calculated according to the formula of Cheng and Prusoff (5). 1. Halmos, etal., Cancer Letters, 85, 111-118 (1994) 2. Cai, et al., Proc. Natl. Acad. Sci., USA 91:12664 -12668, (1994.) 3. Kris, etal., J. Biol. Chem, 262: 11215-11220, (1987.) 4. McPherson, G.A., J.Pharmaco Methods, 14: 213-228, (1985) 5. Cheng and Prusoff, Biochem. Pharmacol* 22:3099-3108, (1973) Table 23-1 Characterization of the specific binding of cytotoxic bombesin antagonist a6MgB (2-pyrrolino-DOX1'-0-glt-Gln-Trp-Ala-Val-Gly-His-Leu-v/-(CH2-N)Leu-NH2 to bombesin receptors on Swiss 3T3 cell line in comparison with bombesin Compound Ki (nM) Bombesin Qi14gB 1.2 1.0 Comparative Effectiveness and Toxicity of Hormone Conjugates vs. Cytotoxic Radical Alone 42 EXAMPLE 24 Treatment with 2-pyrrotino-DOX (Q6), cytotoxic LH-RH agonist analog Q614gL ([D-Lys6]LH-RH linked to Q614-0-hemiglutarate) and (DOX) on estrogen independent MXT mouse mammary cancers (KS-49) In order to compare the tumor inhibitory activity of cytotoxic doxorubicin derivative! Q6 and its targeted cytotoxic peptide conjugate, Q614gl- as well as the well known antineoplastic agent, DOX and to determine the optimal way of administration and the nontoxic doses, LH-RH receptor positive MXT (3.2) ovex tumor pieces (1 mm3) were implanted s. c. in female BQD2F1 mice. One day after-transplantation the mice were randomly divided intogrpups of five animals and the treatment started. The compounds were dissolved in 0:1 % -(flcioroacetie'acid (pH 2) and given intraperitoneal^. Groups, treatment schedules and doses as well as average survival times are shown in Table 24-1 Results are summarized in Table 24-2 and Figure 1. Table 24-2 shows the effect of treatment with Q6 and cytotoxic LH-RH analog Q614gL on tumor volumes and survival of mice with estrogen-independent breast cancers. As is shown in Table 24-2, 1.25 nmol Q6 administered on day 1, 2, 7,8,14 and 15, (Group 2) exerted strong toxicity characterized with an average survival of 17.4 days, which is significantly shorter than that of the untreated control group. In comparison, the same dosage of G614gL (Group 6) resulted in an average survival of 30.8 days, which is significantly longer than that of the untreated control group. Higher efficacy of Q6MgL over Q6 can also be demonstrated by comparing the average final tumor volumes in Group 2 (1065 mm3 at day 16) and in Group 6 (863 mm3 at day 31). Similar conclusions can be demonstrated by comparing Q6 and Q614gL in a different treatment schedule where 0.5 nmol of the drugs were administered five days a week for three consecutive weeks. 43 Doxorubicin at a toxic dose (total amount: 1560 nmol, average survival: 20 days) could not eradicate the tumor, while treatment with Q614gL at nontoxic dose (total amount: 7 nmol, average survival: >31 days) led to the survival of 2 out of 5 animals, without development of the tumor. Table 24-1 No of group I Admin. I Dose/ Inj. (nmol) Dose/ Inj. (M9) Inj. /week I Days between Injection Weeks Admin. i 'Total Amt. Reed Aver. surviv.* day 1 t Control 22 2 125 0.92 2 5 3 7.5 17.5 3 ,, 0 5 0.37 5 2 19.6 4 0.25 * 0.19 9,5.. 14.6 5 0.2 0.15 21 130 6 (VV 1.25 2.9 2 5 7.5 30.8 7 0.5 1.16 5 2 26.8 8 0.25 * 0.58 9.5 18.4 9 0.2 0.46 21 13.6 10 3.5 8.12 1 6 2 7 >31 11 4 9.28 8 12 5 11.6 10 13.4 13 DOX 520 340 3 1560 20.0 * From day 9 to day 12, dose was raised to 2.5 nmol From day 9 to day 12, dose was raised to 5.0 nmol 'Survival Table -24-2! No Group M ode of Final Day of Number of a d m i n i 8 t r lion - Tumor Volume (mm*) Measurement Average survival (days) surviving mice without tumor from 5 mice per group Dose/ No of Pause Duration Tout on on inj. (nmol) injections per week between injections (days) of treatment (weeks) amount injected (nmol) day 18 day 31 1 Control 7322 21 22.0±1.6 0 0 2 Q. 1.25 2 5 3 7.5 1065 16 17.4±0.2 0 0 6 Q."«L 1.25 2 5 3 7,5 863 31 • 30.8±0.4 •• — 2 0 3 Q. 0.5 5 2 3 7.5 2531 18 I9.6±0.7 0 0 7 Q."gL 0.5 5 2 3 7.5 3978 31 26.8 ±2.6 •• 1 0 10 Q,"«L 3.5 1 6 2 7 669 31 >31 - •• 4 2 12 0."«L 5.0 1 6 2 10 0 10 13.4 0 0 13 DOX 520 1 6 3 1560 e ...I., .L 1560 28 20 e<.> 1 J. '.1 0 Duncan's test. 45 EXAMPLE 25 Effects of a single treatment with (DOX), cytotoxic LH-RH analogs T-107 and Qi^gL on estrogen independent MXT mouse mammary cancers (KS-55) Test Compounds: Qi^gL: Doxorubicin14-0-hemiglutarate linked to [D-Lys6]LH-RH T-107: N-glutaryl-doxorubicin linked to [O-Lys^LH-RH PFOC, Natl. Acad. Sci. Vol. 89. Pp. 972-976 (1992)); and DOX The assays were run as follows: In order to determine the maximum tolerated doses and compare the effeqts, MXT (3.2) ovex tumor pieces (1 mrr>3) were implanted s.c in female 86D2F1 mice. One day after transplantation the mice were randomly divided into groups of five animals and they were treated with a single injection i.p. The groups and doses are shown in the Table 25-1. The table also shows the numbers of mice that had tumors when volume was measured and the average survival times for groups. Tumor volume changes are .shown in Figure 2. The compounds were dissolved in 0.1% TFA (pH: 2.0). Tumor volume was measured on days 10, 13, 17, and 20. As shown in Table 25-1 and Figure 2, T-107, ([D-Lys6]LH-RH linked to N-glutaryl-DOX) is completely ineffective in inhibiting the growth of this turner at a dose of 850 nmo!/20 g mouse. In contrast, Qi,4gL, ([D-Lys6]LH-RH linkec to 14-O-glutaryl-DOX) exerted strong suppression of tumor growth (Figure) ai a nontoxic dose of 650 nmol/20 g mouse. DOX alone was highly toxic (average survival time: 13.6 days) at a single dose of 650 nmol/20 g mcuse and signrficantly less effective, than Qi14gL (Figure 2) Ab Table 25 No. Group Dose Number of tumorous mice/number of surviving Average survival mice nmol/ M9/ umof/kg Day Day Day Day days 20 g 20 g 10 13 17 20 1 Control 5/5 5/5 5/5 5/5 21.2±0 3 2 Q114gl 680 1520 34 1/4 2/4 2/4 3/4 28.6±693 53±25" 31 Q,,4gl 710 1587 35.5 2/4 3/4 3/4- 3/4 26.0+663 2.0±34* 4 Qi,4gL 760 1698 38 3/5 4/5 4/5 (Sacr.) (Sacr) 5 DOX 650 427 32.5 3/3 2/2 1/1 1/1 13.6±25 6 ., DOX 700 460 35 2/3 2/3 2/2 15.2±24 7 DOX 750 493 37.5 1/1 7.8+1.3 8 T-107 750 1676 37.5 5/5 5/5 5/5 4/4 21.8±05 9 T-107 850 1900 44.4 5/5 5/5 5/5 4/4 21 6±07 "Survival is significantly shorter (p 47 EXAMPLE 26 Effect of cytotoxic LH-RH analogs on estrogen independent MXT mouse mammary cancers (KS-47) Substances used for treatment In an earlier experiment, Q2 at 20 nmol daily dose for 17 days had only a moderate inhibitory effect on tumor growth, and it was toxic at 4D nmol dose (mean survival was 14.6 days). A daily dose of 30 nmol was chosen for the present experiment, which compared the efficacy and toxicity of Q214gL MXT (3.2) ovex tumor pieces (1 mm3) were transplanted in female B6D2F1 mice. The treatment started one day after transplantation and was continued for 12 days by i.p. injections once a day. All groups received equimolar amounts fbY'thecompounds as shown in Table 26-1. Tumors were measured on days i0, 14 and 18, and tumor votume was calculated. The data are shown in Table 26-1 and in Figure 3. Treatment with a daily dose of 30 nmol of daunosamine modified doxorubicin analog Q2 (pyrrolidino-DOX) resulted in strong inhibitory effect on tumor growth (tumor volume: 144 mm3 at day 14 vs. 1391 mm3 for the control group), but exerted severe toxicity killing all the animals before the end of the experiment (mean survival 17.9 days). Similarly, Q2 combined (mixture) with [D-Lys^LH-RH resulted in strong tumor inhibitory effect (tumor volume: 80 mm3 at day 14) but the mean survival (18.5 days) was significantly shorter than that of the untreated control group (23.1 days). As a result of the treatment with Q214gL, (Q2 covalently linked to [D-Lys6]LH-RH) two animals died, one at day 16 and another at day 26. From the 8 surviving animals only one developed tumors at the last measurement at day 18 and they all looked 48 healthy, but later on all of them started to develop the tumors The mean survival for this group was significantly longer (28.3 days), than that of the control group. Treatment with [D-Lys6]LH-RH aione did not affect tumor growth. This experiment demonstrates that the higher efficacy and the lower peripheral toxicity of Q214gL over the cytotoxic radical Q2 is attributable to the covalent conjugation of the cytotoxic radical to the targeting carrier LH-RH analog. Table 26-1 Effect of cytotoxic LH-RH analogs on growth of estrogen independent MXT mouse mammary cancers and survival of mice with Tumors No Treatment Dose No. of Mean tumor Mean survival after (ug/day) mice volume in mm3 on days transplantation (days) 10 14 18 1 Control 15 253 1391 4794 23.1 2 Q2,4gL 687 10 33 16 23 28.3* 3 Q2 21.3 10 153 144 137 17.9 4 [D-Lys6}LH-RH 48.0 10 165 1348 4003 23.5 5 [D-l_ys6]LH-RH 48.0 + 10 121 80 27 18.5 + Q2 21 3 All daily doses are 30 nmol equimolar amounts. Significantly shorter than control (p 49 EXAMPLE 27 Effects of 2-pyrrolino-DOX (Q6) and cytotoxic LH-RH agonist analog Q614gL ([D-Lys6]LH-RH linked to Q614-0-hemiglutarate) on the growth of androgen dependent rat Dunning R-3327-H prostate carcinomas Male Copenhagen rats bearing hormone-dependent Dunning R-3327-H prostate carcinomas were treated with Qe14gL, a new cytotoxic analog of luteinizing hormone-releasing hormone (LH-RH) consisting of the agonist fD-Lys6]LH-RH linked to 2-pyrrolinodoxorubicin. In the first experiment, 2-pyrroiihodoxorubicin was administered at a concentration of 50 nmol/kg. "as a single drug:(Q6) and as an unconjugated mixture with [D-Lys6]LH-RH or Conjugated to the carrier [D-Lys6]LH-RH (Q614gL). Following the second administration of 50 nmol/kg of radical Q6 alone or mixed with [D-Lys^LH-RH, all rats died with signs of general toxicity, whereas all animals, treated with the cytotoxic LH-RH conjugate Q614gL, survived. After 5 weeks of treatment with a total dose of 150 nmol/kg G614gL, the tumors regressed from an originalvolume of 8.35 ±1.7 cm3 at the beginning of the experiment to 4.47 ± 0.8 cm3, while tumors in the control group continued to grow and measured 17.84 ± 2.2 cm3. The therapy with G614gL also significantly reduced tumor weight and tumor burden. In the second experiment, designed for comparing the efficacy and toxicity of Q6 and Q614gL the therapeutic regimen consisted of 3 applications of 25 nmol/kg Q6 or 25 nmol/kg and 50 nmol/kg Q614gL. When the treatment was started, tumor volume in all groups was between 3.9 and 4.5 cm3. After 5 weeks of therapy, the tumors in rats treated with 50 nmol/kg Q614gL regressed to 2.3 ±0.51 cm3, whereas 25 nmol/kg Q6 was still toxic and could only produce a reduction in final tumor volume to 6.76 ± 1.4 cm3, similar to that obtained with 25 nmol/kg Q614gL (6.74 ± 1 cm3), as compared to 15.6 ± 2.2 cm3 for untreated animals. Histological evaluation of the specimens showed a significant decrease of mitotic cells in the Q; ~cL 50 treated groups only. LH-RH receptors with high binding capacity were detected in the membranes of untreated Dunning tumor specimens, but after treatment with Q6,4gL, no binding sites for LH-RH coutd be found. Inhibition of tumor growth by AN-201 and Q614gL was also associated with a significant decrease in binding capacity of EGF receptors. As is demonstrated by Figures 4-6, targeted cytotoxic LH-RH analog Q614gL is an effective antitumor agent causing regression of rat Dunning R-3327-H prostate carcinomas. Our studies also show that the cytotoxic LH-RH analog GV4gL is mdch less toxic than the antineoplastic radical (Q6) incorporated, and significantly more active in inhibiting tumor growth Figure legends for EXAMPLE 27: Fig.4. Experiment I: Tumor volume in male Copenhagen rats bearing rat Dunning R-3327-H prostate carcinoma transplants during the treatment consisting of 3 applications of 50 nmol/kg agonist [D-Lys6]LH-RH and 50 nmol/kg of cytotoxic LH-RH analog Q614gL. Vertical lines indicate the SEM. p treatment indicated by arrows was applied on days 1,8 and 29. f The animals treated with Q6 as a single drug or an unconjugated mixture with [D-Lys6]LH-RH died in the second week. In these two groups the volume of tumors recorded on day 8 is shown. Fig. 5. Experiment II: Effect of treatment with 25 nmol/kg 2- pyrrolinodoxorubicin 51 Fig 6.: Experiment IJ: Effect of treatment with 25 nmol/kg 2-pyrrolinodoxorubicin (Q6), 25 nmol/kg and 50 nmol/kg cytotoxic LH-RH analog Q614gL on the body weight of Copenhagen rats bearing Dunning R-3327-H prostate cancer. Vertical lines indicate the SEM. * p EXAMPLE 28 Comparative study on the effects of doxorubicin (DOX) and targeted cytotoxic LH-RH agonist analog Q,14gL ([D-Lys^LH-RH finked to D0X14-O-hemiglutarate) oh the growth of OV-1063 human* ovarian carcinoma in nude mice Human epithelial oyarian cancer cell line OV-1063 originated from a metastatic papillan/'cystadenocarcinoma of the ovary of a 57-year old womarj ( Horowitz et.al. (1985) Oncology 42, 332-337). Ten million cells of OV-1063 were injected subcutaneously into three nude mice to grow tumors Pieces of 1 mm3 of these tumors were transplanted into sixty animals for in vivo growth inhibition studies. The aim of the experiment was to demonstrate that, as a result of the presence of receptors for LH-RH on OV-1063, the cytotoxic conjugate of LH-RH was more effective and less toxic, than DOX, the cytotoxic radical it contained. Thus, the effects of cytotoxic LH-RH conjugate was compared to those of DOX, the mixture of DOX with the carrier molecule, the carrier alone and the untreated control groups. All injections were administered intra peritoneally The compounds were dissolved in 0.9 % sodium chloride in water (saline). Mice with an average tumor size of about 15 mm3 were divided into six groups of nine animals and received the following treatment seven days after tumor transplantation: group 1, saline; group 2. Qi14gL at a dose of 700 52 nmol/20g animal; group 3, Q^gL at a dose of 413 nmol/20g animal (maximum tolerated dose, MTD for DOX); group 4, DOX at 413 nmoi/20g animal (MTD); group 5, mixture of 700 nmol/20g of DOX and 700 nmol/20g of [D-Lys6]LH-RH; group 6, carrier agonist analog [D-Lys6]LH-RH at a dose of 700 nmol/20g animal. Receptor analysis of OV-1063 showed the presence of high affinity binding sites for LH-RH. Results: as shown on Fig. 7, strong inhibition of tumor growth was achieved by treatment with Q,14gL at 413nmol/20g dose (group 3). The animals did not show'signs of severe toxicity In comparison, treatment with DOX. administered at the same dose of 413 nmol/20g (12 mg/kg, MTD, group 4) did riot result in significant inhibition of tumor growth in the three animals surviving at the end of the expriment. Three animals died by dav five .and six animsrts were dead by day nine due to toxicity. At a higher dose, (7.00 nmo(/20g, group 2), Qi14gL showed very strong inhibition of tumor growth (Fig. 7). Two out of nine animals died due to toxicity and one animal died accidentaly. The six surviving animals were recovering from a weight loss of about 20%at the end of the experiment. In group 6, the same high dose (700 nmol/20g) DOX was mixed with 700 nmol of [D-Lys6]LH-RH. By day 5, all animals died in this group as a result of severe toxicity. Conclusions: Our results clearly demonstrate that due to the presence of receptors for LH-RH on the cells of epithelial ovarian cancer OV-1063, targeted cytotoxic LH-RH conjugate Qi14gL shows lower toxicity and higher antitumoral activity than doxorubicin (Q,), the cytotoxic radical it contains. -53- WE CLAIM i. A cytotoxic peptide hormone conjugates of the formula Q^O-R-P 'Wherein 0 has the detailed chemical structure; Where-R-is a single boncferpr -C(0)-(CH5) n-C{0)-and n is 0-7 R' is selected from the group consisting of NH 2, an aromatic or hydrogenated 5 or 6 membered heterocycle having at least one ring nitrogen and such a heterocycle having a butadiene moiety bonded to adjacent carbon atoms of said ring to form a bicyclic system and — P is H or a peptide, provided that where R' is NH2 then(R-P is other than HI and wherefR-P is H then R'is other than NH2, 2.JThe compound of claim 1 wherein R' is selected from the group consisting of NF12 pyrrolidine-1, isoindoline-2-yl, 3-pyrroline-l-yl, 3-pyrrolidone-t-yl,2- pyrroline-1-yl, 3-piperidone-l-yl, 1.3-feetrahydro-pyridine-l-yl, ^nd P is Pi,P2,and P3, where Pi is selected from the group consisting of an LH-RH analog of the formula Aaa-Bbb-Ccc-Ser-Tyr-D-Lys(Xxx)-Leu-Arg-Pro-Ddd wherein (Xxx) is hydrogen, A2BU or A?Pr wherein where: Aaa is Glp, then Bbb is His Ccc is Trp and Ddd is Gly-Nhb, Where AA is Ac-D-Nal(2), then Bbb is D-Phe(4Cl) , Ccc is -54-D-Pal(3), D-Trp and Ddd is D-Ala-NHz, and where Aaa-Bbb-Ccc is Ac, then Ddd is -NH-CH2-CH3 wherein the group Q"-0-R- forms a carboxamido link with the free amino group of the D-Lys moiety or with at feast one of the free amino groups of A28u or A2Pr when present (Xxx), P2 is an analog of somatostatin of the formula Aaa-Cys-Bbb-D-Trp-Lys-CeC"Cys-Ddd-NH2 wherein where: Aaa is D-Phe,then Bbb is Tyr, Ccc is Vai and Ddd is Thr or Trp, where Aaa is D- Trp, then Bbb is Phe, Ccc and Ddd are Thr, wherein the Group Q"-0-R forms a carboxamido link with the terminal amono group of the AA moiety, P3 is a bombesin antagonist analog of the formula Aaa-Gln-Trp-A!a-Val-Gly-his-Leu Bbb-NH2 Where Aaa is nil, D-Tpi or D-Phe, Bbb is (CH2-NH)Leu, (CH2-NH)Phe or (CH2~ NH)Trpor(CH2-N)Tac wherein the group AM -O-R-forms a carboxamido link with the terminal amino group of the Aaa moiety where present or with that of Gin where the Aaa moiety is absent. 3.The compound of claim 1 where n =3. 4.The compound of claim 3 where Rf is NH2. 5. The compound of claim 3 wherein Rris 2-pyrroline-l-yl. 6. The compound of Claim 4 where P is Pi, 7. The compound of Claim 5 where P is Pi 8. The compound of Claim 4 where P is Pz 9. The compound of Claim 5 where P is P2, lO.The compound of Claim 4 where P is ?3. ILThe compound of Claim 5 where P is P3 12.The compound of claim 1 where -R-P is H and R'is other than NH2. 13.The compound of Claim 12 where -R'is pyrrolidine-1-yl. -55- 14. The compound of Claim 12 where -R'is isoindoline-2-yl. lS.The compound of Claim 12 where -R'- is 3-pyrroline-l-yl. !6,The compound of Claim 12where-R'is 3-pyrrolidone-l-yl. 17.The compound of Claim 12 where -R'is 2-pyrroline-l-yl. 56 18. The compound of claim 12 where -R' is 3-piperidone-1-yl. 19. The compound of claim 12 where -R' is 1,3-tetrahydropyridine- 1-yl. 20. The compound of claim 1 of the formula 1 4 Gllp-His-Trp-Ser-Tyr-D-LystQ. -O-glt )-Arg-Leu-Pro-Gly-NH_, 14 wherein Q. is doxorubicin-1 4-yl. 21. The compound of claim 1 of the formula Glp-His-Trp-Ser-Tyr-D-Lys(Q,1 4 -O-glt)-Arg-Leu-Pro-Gly-NH_, 14 wherein Q_ is 3'-deamino-3'-(2"-pyrroline-1"-yl)-doxorubicin- 14-yl. 22. The compound of claim 1 of the formula Q. -O-glt-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH., 14 wherein Q. is doxorubicin-14-yl. 23. The compound of claim 1 of the formula Q, -0-glt-a-Pjie-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH9 wherein; Q, @fi4£ is 3 ' -deamino-3 ' -(2"-pyrroline-1 "-yl) -doxorubicin-1 4-yl. fcJ 24. The compound of claim 1 of the formula 14 Q -0-glt-D-Trp-€ys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH„; 14 wherein Q1 is doxorubicin-14-yl. 25. The compound of claim 1 of the formula 14 Qfi ,-O-gltjD-Trp-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH- wfierein Qr - .isv3.V-deanunc—3" -(2"-pyrroline--1-"-yl0-d^o3^icin-14-yl. b 26. The compound of claim 1 of the formula 14 Q -0-glt-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 wherein Q. is doxorubicin-14-yl. 27. The compound of claim 1 of the formula Q,14 -o-glt-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH- wherein Qc is 3'-deamino-3'-(2"-pyrroline-1"-yl)-doxorubicin-b 14-yl. -57- 28. The compound of claim 1 of the formula 1 4 Q. -0-glt-Gln-Trp-Ala-Val-Gly-His-Leu(CHo-NH)Leu-NH., 14 wherein; Q.. is doxorubicin-1 4-yl. 29. The compound of claim 1 of the formula 14 Qfi -0-glt-Gln-Trp-Ala-Va!-Gly-His-Leu(CH -NH)Leu-NH 14 wherein Qfir is 3'-deamino-3'- 14-yl, 30..The compound of claim 1 of the formula Q1 ' -O-glt-D-Tpi-Gln Trp-Ala-Val-Gly-His-Leu(CH2-NH)Leu-NH2 '1 4 wherein Q1 is doxorubicin-14-yl. 31. The compound of claim 1 of the formula Q,.1.4 -0-glt-D-Tpi-Gln-Trp-Ala-Val-Gly-His-Leu(CH„-NH)Leu-NH, wherein Qfi is 3'-deamino-3'-{2"-pyrroline-1"-yl)-doxorubicin- 1 4~yl. 32. A composition comprising a compound as claimed in claim 1 and a pharmaceutically acceptable carrier thereof. A cytotoxic peptide hormone conjugates of the formula Where R is a single bond H or- C(0)-(CH2) n-C(O) and n is 0-7 R'is selected from the group consisting of NH2, an aromatic or hydrogenated 5 or 6 membered heterocycle having at least one ring nitrogen and such a heterocycle having a butadiene moiety bonded to adjacent carbon atoms of said ring to form a bicyctic system and P is H or a peptide, provided that where R' is NH2 then R - P is other than H and where R-P is H, then R' is other than NH2.

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