EP0531342A4

EP0531342A4 -

Info

Publication number: EP0531342A4
Application number: EP19910909510
Authority: EP
Inventors: David H. Coy; Sun Hyuk Kim; Jacques-Pierre Moreau
Original assignee: Tulane University; Ipsen Bioscience Inc
Current assignee: Tulane University; Ipsen Bioscience Inc
Priority date: 1990-05-09
Filing date: 1991-05-09
Publication date: 1994-04-06
Also published as: HUT62604A; CA2081890A1; HU9203481D0; EP0531342A1; WO1991017181A1; JPH05506862A

Abstract

A therapeutic peptide comprising between seven and ten amino acid residues, inclusive, the peptide being an analog of one of the following naturally occuring peptides which terminate at the carboxy-terminus with a Met residue: (a) litorin; (b) the ten amino acid carboxy-terminal region of mammalian GRP, neuromedin B, or neuromedin C; and (c) the ten amino acid carboxy-terminal region of amphibian bombesin, said analog being an agonist of one of the naturally occurring peptides.

Description

CYCLIZED AND LINEAR THERAPEUTIC PEPTIDES Background of the Invention This invention relates to peptides useful for treatment of benign or malignant proliferation of tissue.

The amphibian peptide bombesin, pGlu-Gln-Arg- Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (Anastasi et al., Experientia 22:166-167 (1971)), is closely related to the mammalian gastrin-releasing peptides (GRP), e.g., the porcine GRP, H₂N- Ala-Pro-Val-Ser-Val-Gly-Gly-Gly-Thr- Val-Leu-Ala-Lys-Met-Tyr-Pro-Arg-Gly-Asn-His-Trp-Ala- Val-Gly-His-Leu-Met-(NH₂) (McDonald et al., Biochem. Biophys. Res. Commun. ϊH):227-233 (1979)) and human GRP, H₂N-Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr-Val-Leu-Thr-Lys-M e t-Tyr-Pro-Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met (NH₂) . Bombesin has been found to be a growth factor for a number of human cancer cell lines, including small-cell lung carcinoma (SCLC) , and has been detected in human breast and prostate cancer (Haveman et al., eds. Recent Results in Cancer Research - Peptide Hormones in Lung Cancer. Springer-Verlag, New York:1986). A number of these cancers are known to secrete peptide hormones related to GRP or bombesin. Consequently, antagonists to bombesin have been proposed as agents for the treatment of these cancers.

Cuttitta et al. demonstrated that a specific monoclonal antibody to bombesin inhibited in vivo the growth of a human small-cell lung cancer cell line xenografted to nude mice (Cuttitta et al., Cancer Survey 4.:707-727 (1985)). In 3T3 murine fibroblasts which are responsive to the itotic effect of bombesin, Zachary and Rozengurt observed that a substance P antagonist (Spantide) acted as a bombesin antagonist (Zachary et al. , Proc. Natl. Acad. Sci . (USA) , 82.: 7616-7620 (1985) ) . Heinz-Erian et al. replaced His at position 12 in bombesin with D-Phe and observed bombesin antagonist activity in dispersed acini from guinea pig pancreas (Heinz-Erian et al., Am. J. of Physiol. 252:G439-G442 (1987)). Rivier reported work directed toward restricting the conformational freedom of the bioactive C-terminal decapeptide of bombesin by incorporating intramolecular disulfide bridges; however, Rivier mentioned that, so far, bombesin analogs with this modification fail to exhibit any antagonist activity (Rivier et al. , "Competitive Antagonists of Peptide Hormones," in Abstracts of the International Symposium on Bombesin-Like Peptides in Health and Disease, Rome, Italy (October, 1987) . Bombesin exhibits both direct and indirect effects on the gastrointestinal tract, including the release of hormones and the stimulation of pancreatic, gastric, an intestinal secretion and of intestinal mobility. Gastrin and cholecystokinin (CCK) which are released by bombesin, have been shown to play a role in the maintenance of normal gastrointestinal mucosa as well a in augmenting growth of normal and neoplastic tissues. The growth of xenografted human colon and stomach carcinomas in nude mice has been stimulated by the administration of gastrin and later inhibited with the addition of secretin (Tanake et al., 1986, Tokaku J. Exp. Med. 148:459) and the growth of MC-26 murine colon carcinoma, which possesses gastrin receptors is stimulated by pentagastrin (Winsett et al., 1980, Surgery 99:302, and inhibited by proglumide, a gastrin-receptor antagonist, Beauchamp et al., 1985, Ann. Surg. 202:303. Bombesin has been found to act concurrently as both a trophic agent for normal host pancreas and a growth inhibitory agent in xenografted human pancreatic tumor tissue, Alexander et al., 1988, Pancreas 3:247. Abbreviations (uncommon) : cyclohexyl-Ala = CHx-Ala (cyclohexyl alanine) H2, C identifying group

NH₂~CH—C0₂H; pGlu = H₂C CH-COOH (pyroglutamic acid) ;

H₂C NH C O

Nle = H₂N-CH-C00H (norleucine)

(CH₂)₃-CH₃ Pal = 3-pyridyl-alanine D-Cpa = D-para-chloro-phenylalanine HyPro = hydroxyproline Nal = naphthylalanine Sar = sarcosine

F₅-Phe = penta-fluoro-phenylalanine

R = right (D) configuration; S = left (L) configuration; racemate = equal mix of R and S

1-methyl-His; 3-methyl-His = methyl (CH₃) group on nitrogen at positions 1 or 3 of Histidine:

Summary of the Invention The invention features a linear or a cyclic therapeutic peptide, which includes between seven and ten amino acid residues, inclusive, and which is an analog of one of the following naturally occurring peptides which terminate at the carboxy-terminus with a Met residue: (a) litorin; (b) the ten amino acid carboxy-terminal region of mammalian GRP, neuromedin B, or neuromedin C; and (c) the ten amino acid carboxy-terminal region of amphibian bombesin, which is an analog of the formula:

^Rι

_A0__A1__A2__A3__A __A5__A6__A7._A8__A9__R3

^R2

wherein A⁰ = Gly, D- or L- isomer of any of pGlu, Nle, a-aminobutyric acid, Ala, Val, Gin, Asn, Leu, lie, p-X-Phe (where X = H, F, Cl, Br, N0₂, OH, CH₃) , Trp, or j8-Nal, or is deleted; A¹ = the D- or L-isomer of any of pGlu, Nle, a-aminobutyric acid, Ala, Val, Gin, Asn, Leu, lie, p-X-Phe (where X = H, F, Cl, Br, N0₂, OH, or CH₃) , Asp, Glu, F₅-Phe, Trp, j8-Nal, Cys, Lys, or is deleted; A² = Gly, D- or L- isomer of any of pGlu, Ala, Val, Gin, Asn, Leu, lie, p-X-Phe (where X = H, F, Cl,

Br, N0₂, OH, or CH₃) , Trp, jS-Nal, Asp, Glu, His, 1-methyl-His 3-methyl-His, Cys, Lys, or is deleted; A = the D- or L-isomer of any of p-X-Phe (where X = H, F, Cl, Br, N0₂, OH, or CH₃) , j8-Nal, or Trp;

A⁴ = Ala, Val, Gin, Asn, Gly, Leu, lie, Nle, a-aminobutyric acid, p-X-phe (where X = H, F, Cl, Br, N0₂, OH, or CH₃) , Trp, or j8-Nal; A⁵ = Gin, Asn, Gly, Ala, Leu, lie, Nle, a-aminobutyric acid, Val, p-X-Phe (where X = H, F, Cl, Br, N0₂, OH, or CH₃) , Trp, Thr, or /3-Nal; A⁶ = Sar, Gly or the D-isomer of any Ala, N-methyl-Ala, Val, Gin, Asn, Leu, lie, p-X-Phe

(where X = H, F, Cl, Br, N0₂, OH, or CH₃) , Trp, Cys, or /3-Nal; A⁷ = 1-methyl-His, 3-methyl-His, His, Lys, Asp, or Glu; A⁸ = Leu, lie, Val, Nle, a-aminobutyric acid, p-X-Phe

(where X = H, F, Cl, Br, N0₂, OH, or CH₃) , Trp, Thr, 3-Nal, Lys, Asp, Glu, CHx-Ala, or Cys; A⁹ = L-isomer of any of Met, Met-oxide, Leu, lie, Nle, a-aminobutyric acid, p-X-Phe (where X = H, F, Cl, Br, N0₂, OH, or CH₃) , Trp, /3-Nal, CHx-Ala, or

Cys; each R_j^ and R₂, independently, is H, C₁_₁₂ alkyl, ^c ₇_₁₀ phenylalkyl, COE_j^ (where E_χ is C₁_₂₀ alkyl, ^c ₃_₂₀ alkenyl, ^C ₃-₂₀ aikiπy¹- phenyl, naphthyl, or C₇_₁₀ phenylalkyl), or ^cι~^ci₂ ^acy1' ^{and R}ι ^{and R} ₂ ^{are b}°nded to the N-terminal amino acid of the peptide; provided that when one of R_j or R₂ is COE_j, the other must be H; and R₃ is H, NH₂, ^Cι-₁₂ ₇-ιo phenylalkyl, or C₃_₂₀ naphthylalkyl; and further provided that, if A⁰ is present, A¹ cannot be pGlu; and, if A⁰ or A¹ is present, A² cannot be pGlu; and further provided that, when A is deleted and A is pGlu, R must be H and R₂ must be the portion of Glu that forms the imine ring in pGlu; and further provided that, where A⁰ is deleted and A¹ is not pGlu, A¹ may be bonded to A⁹, or where A⁰ and A¹ are deleted and A² is not pGlu, A² may be bonded to A , or where A⁰, A¹ and A are deleted. A³ can be bonded to A⁹ to form a cyclized peptide; and provided that where A⁰ is deleted and A¹ is Asp or Glu, or where A and A¹ are deleted and A is Asp or Glu, either A¹ or A² can be bonded with A⁷ or A⁸, where A⁷ or A⁸ is Lys, or where A⁰ is deleted and A¹ is

Lys or A⁰ and A¹ are deleted and A² is Lys, either A¹ or

A² can be bonded to A⁷ or A⁸, where A⁷ or A⁸ is Asp or Glu; and further provided that either one of A¹ or A² can be Cys and can be bonded through a disulfide bridge with either A⁸ or A⁹, provided that either one of A⁸ or A⁹ can be Cys and can be bonded through a disulfide bridge with either A¹ or A²; and further provided that where A⁰ and A¹ are deleted and A⁶ is D-Ala, A⁸-A⁹ cannot be

Leu-Met-NH₂; or a pharmaceutically acceptable salt thereof.

In the generic formula given above, when either of R_χ or R₂ is an aliphatic, aromatic, or lipophilic group, the .in vivo activity can be long lasting, and delivery of the compounds of the invention to the target tissue can be facilitated.

More preferably, the analog of the generic formula above is of the formula: A⁰ = pGlu, Gly, D-Phe, or is deleted;

A¹ = pGlu, D-Phe, D-Ala, D-/3-Nal, D-Cpa, D-Asn, Cys, or is deleted;

A² = pGlu, Asn, Gin, His, 1-methyl-His, 3-methyl-His, Cys, or is deleted; A³ = Trp;

A⁴ = Ala;

A⁵ = Val;

A⁶ = Sar, Gly, D-Phe, or D-Ala;

A⁷ = His; A⁸ = Leu, Phe, Chx-Ala, or Cys;

A⁹ = L-isomer of any of Met, Leu, lie, Nle, Phe, or Cys.

Examples of preferred peptide analogs are: pGlu-Gln-Trp-Ala-Val-Gly-His-Leu-Leu-NH₂; D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Leu-NH₂ ;

D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ ;

D-Cpa-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ ;

D-Cpa-Gln-Trp-Ala-Val-Gly-His-Leu-Leu-NH₂ ;

D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Leu-Leu-NH₂ ;

D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Leu-Met-NH₂ ;

D-Cpa-Gln-Trp-Ala-Val-D-Ala-His-Leu-Met-NH₂ ; pGlu-Gln-Trp-Ala-Val-Gly-His-Phe-Leu-NH₂ ;

D-Phe-Gln-Trp-Ala-Val-Gly-His-Phe-Leu-NH₂ ;

D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Phe-Met-NH₂ ;

D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Phe-Leu-NH₂ ;

D-Phe-Gln-Trp-Ala-Val-Gly-His-CHx-Ala-Leu-NH₂ ;

D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Nle-NH₂ ;

D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Leu-Nle-NH₂ ;

D-Phe-Gln-Trp-Ala-Val-Gly-His-Phe-Nle-NH₂ ;

D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Phe-Nle-NH₂ ;

Ac-His-Trp-Ala-Val-D-Ala-His-Leu

-Leu-NH,

cyclo-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Leu;

D-Cys-Asn-Trp-Ala-Val-Gly-His-Leu-Cys-NH₂ ;

cyclo-His-Trp-Ala-Val-Gly-His-Leu-Met;

Cys-Trp-Ala-Val-Gly-His-Leu-Cys-NH₂ ;

cyclo-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Met;

cyclo-D-Phe-His-Trp-Ala-Val-Gly-His-Leu-Met;

and cyclo-Trp-Ala-Val-Gly-His-Leu-Met; Modification of a natural amino acid or substitution of a synthetic amino acid, examples of which are (but are not limited to) pGlu, Nle, Pal, D-Cpa, Met-oxide, Nal, Sar, 1-methyl-His, or 3-methyl-His, at one or more of positions A⁰, A¹, A⁶, or A⁹ of an analog, or cyclization of the analog result in enhanced stability; i.e., protection of the amino terminus from exopeptidase degradation and of the internal portion of the peptide from endopeptidase degradation. The carboxy-terminal dipeptide sequence of some analogs of the invention corresponds to the natural C-terminal sequence of bombesin or bombesin-related analogs.

The invention also features a linear or a cyclic therapeutic peptide, which includes between seven and ten amino acid residues, inclusive, and which is an analog of one of the following naturally occurring peptides which terminate at the carboxy-terminus with a Met residue: (a) litorin; (b) the ten amino acid carboxy-terminal region of mammalian GRP, neuromedin B, or neuromedin C; and (c) the ten amino acid carboxy-terminal region of amphibian bombesin, and the analog is an agonist of one of these naturally occurring peptides. In preferred embodiments, the analog may be an agonist or a partial agonist of the naturally occurring biologically active peptide; preferably, the analog is at least 25%, more preferably 50% or 75%, homologous with a region of the naturally occurring peptide. As used herein, an "agonist" mimics or enhances the biological effect of the natural peptide on its target cell and a "partial agonist" mimics or enhances the biological effect of the natural peptide, but to a lesser extent than an agonist. Biological effect, as used herein, is measured by the effect of the natural peptide in one of two systems: an in vitro pancreatic amylase release assay and an in vitro 3T3 fibroblast cell division system, both of which are described in European Patent Application 88308916.6, hereby incorporated by reference. An agonist will stimulate the effect of the natural peptide on either amylase release from pancreatic cells or fibroblast cell division by 100%, whereas a partial agonist will have a lesser stimulatory effect, i.e., ranging between 0-99%.

Peptides of the invention are useful for treating non-malignant proliferative disease in a human patient, e.g., the proliferation of smooth muscle. Peptides of the invention are also useful for treating cancer in a human patient, particularly for the treatment of prostatic, colon, breast, pancreatic, or lung cancer. In addition, peptides of the invention may be used to suppress appetite, to stimulate pancreatic secretion, or to suppress a craving for alcohol.

Analogs of the invention can be provided in the form of pharmaceutically acceptable salts. Examples of preferred salts are those with therapeutically acceptable organic acids, e.g., acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, salicylic, methanesulfonic, toluene sulfonic, trifluoroacetic, or pa oic acid, as well as polymeric acids such as tannic acid or carboxymethyl cellulose, and salts with inorganic acids such as the hydrohalic acids,e.g., hydrochloric acid, sulfuric acid or phosphoric acid.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Description of the Preferred Embodiments Before describing preferred embodiments of the invention, the drawing will briefly be described. Drawing

Fig. 1 is the amino acid sequences of naturally occurring peptides of which peptides of the invention are analogs. We now describe the structure, synthesis, and use of the preferred embodiments of the invention. Structure

Peptides of the invention are derived from one of the sequences shown in Fig. 1, which represent the sequences, or portions thereof, of naturally-occurring peptides. Bombesin, neuromedin B, neuromedin C, litorin, and GRP analogs of the invention are described in Coy et al., U.S. Patent Application Serial No. 502,438, filed March 30, 1990, which is a continuation-in-part of U.S. Patent Application Serial No. 397,169, filed August 21, 1989, which is a continuation-in-part of U.S. Patent Application Serial No. 376,555, filed July 7, 1989, and U.S. Patent Application Serial Number 394,727, filed August 16, 1989, both of which are continuation-in-parts of U.S. Patent Application Serial No. 317,941, filed March 2, 1989, which is a continuation-in-part of U.S. Patent Application Serial Number 282,328, filed December 9, 1988, which in turn is a continuation-in-part of U.S. Patent Application Serial No. 257,998, filed October 14, 1988, which in turn is a continuation-in-part of U.S. Patent Application Serial No. 248,771, filed September 23, 1988, which in turn is a continuation-in-part of Coy et al., U.S. Patent Application Serial No. 207,759, filed June 16, 1988, which in turn is a continuation-in-part of Coy et al., U.S. Patent Application Serial No. 204,171, filed June 8, 1988, which in turn is a continuation-in-part of Coy et al., U.S. Patent Application Serial No. 173,311, filed March 25, 1988, which in turn is a continuation-in-part of Coy et al. U.S. Patent Application Serial No. 100,571, filed September 24, 1987; all of which are assigned to the same assignee and hereby incorporated by reference; or as described in Zachary et al., Proc. Nat. Aca. Sci. 82:7616, 1985; Heimbrook et al., "Synthetic Peptides: Approaches to Biological Problems", UCLA Symposium on Mol. and Cell. Biol. New Series, Vol. 86, ed. Tarn and Kaiser; Heinz-Erian et al., Am. J. Physiol. G439, 1986; Martinez et al., J. Med. Che . 28:1874, 1985; Gargosky et al., Biochem. J. 247:427, 1987; Dubreuil et al., Drug Design and Delivery, Vol 2:49, Harwood Academic Publishers, GB, 1987; Heikkila et al., J. Biol. Chem. 262:16456, 1987; Caranikas et al., J. Med. Chem. 25:1313, 1982; Saeed et al., 1989, Peptides 10:597; Rosell et al., Trends in Pharmacological Sciences 3:211, 1982; Lundberg et al., Proc. Nat. Aca. Sci. 80:1120, 1983; Engberg et al., Nature 293:222, 1984; Mizrahi et al.. Euro. J. Pharma. 82:101, 1982; Leander et al., Nature 294:467, 1981; Woll et al., Biochem. Biophys. Res. Comm. 155:359, 1988; Rivier et al. , Biochem.

17:1766, 1978; Cuttitta et al., Cancer Surveys 4:707, 1985; Aumelas et al., Int. J. Peptide Res. 30:596, 1987; all of which are hereby incorporated by reference. Synthesis of Analogs The synthesis of the bombesin agonist, BIM-26187,

D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Leu-NH₂ follows. Other bombesin or GRP agonists can be prepared by making appropriate modifications of the following synthetic method. 1) Incorporation of alpha-t-butoxycarbonyl (BOC)- leucine on 4-methyl benzhydrylamine.

4-methyl benzhydryla ine-polystyrene resin (Bachem, Inc.) (0.72 meq/g) in the chloride ion form is placed in the reaction vessel of an ACT200 peptide synthesizer (Advanced Chem Tech, Inc.) programmed to perform the following reaction cycle: (a) methylene chloride; (b) 10% triethylamine in chloroform; (c) methylene chloride; and (d) dimethylformide. The neutralized resin is mixed with alpha-t-butoxycarbonyl (BOC)-leucine and diisopropylcarbodiimide (3 molar eq each) in methylene chlrodie for 1 hour. The resulting amino acid resin is washed on the synthesizer with dimethylformamide and treated with 5% acetic anhydride in dimethylformamide for 5 min. Then it is washed with dimethylformamide and methylene chloride. 2) Couplings of the remaining amino acids.

The peptide synthesizer is programmed to perform the following reaction cycle: (a) methylene chloride; (b) 33% trifluoroacetic acid (TFA) in methylene chloride (2 times for 5 and 25 min. each) ; (c) methylene chloride; (d) isopropyl alcohol; (e) 10% triethylamine in chloroform; and (f) methylene chloride.

The following amino acids (3 molar eq.) are then coupled successively by the same procedure: BOC-Leu, BOC-His (tosyl) , BOC-Gly, BOC-Val, BOC-Ala, BOC-Trp, BOC-Gln (coupled in the presence of 1 eq. hydroxybenzotriazole) , BOC-D-Phe (coupled in the presence of 1 eq. hydroxybenzotriazole) . The completed resin is then washed with methanol and air dried.

The peptide resin described above (1.41 g) is mixed with anisole (5 ml) , dithioerythreitol (50mg) , and anhydrous hydrogen fluoride (25 ml) at 0°c for one hour. Excess hydrogen fluoride is evaporated rapidly under a stream of dry nitrogen, and the residue is washed in ether. Crude peptide is dissolved in 100ml of 4M acetic acid and the solution is then evaporated under reduced pressure. The crude peptide is dissolved in minimum volume of methanol/water and triturated with ethyl acetate. The triturated peptide is applied to a column (9.4mm I.D. x 50 cm) of octadecylcilane-silica (Whatman Partisil 10 ODS - 2M9) . The peptide is eluted with a linear gradient of 20-80% of 50/50 0.1% TFA/Acetronitrile i 0.1% TFA in water. Fractions are examined by analytical high performance liquid chromatography and appropriate fractions are evaporated to a small volume, which is further lyophilized, gives 65mg of the product as a colorless powder.

Other compounds including D-Cpa¹, CHx-Ala⁸, or Nle ⁹ can be prepared as above.

Peptides of the invention may be cyclized as follows.

Crude peptide acid obtained from peptide-resin ester by HF cleavage is dissolved in DMF (0.1%-1% concentration), treaed with condensing agent (e.g., BOP reagent, DEPC, DPPA, or any other condensing agent) followed by base (e.g., triethylamine, diisopropylethylamine) at room temperature for 1-3 days. Solvent is removed in vacuum to dryness. The residue is purified by HPLC, according to conventional procedures. The cyclization of, for example, cyclo[D-Phe¹, Leu⁸, Leu⁹]Litorin, in which D-Phe¹ is covalently linked to Leu⁹, is accomplished according to the above procedure using Benzotriazol-1-yloxytris(dimethylamine)phosphonium hexafluorophosphate a the BOP reagent, diethylcyanophosphonate as the DEPC reagent, and diphenylphosphoryalazide as the DPPA reagent. Mechanism of Action

Analogs of the invention may prevent or inhibit the growth of cancer cells, or may prevent the proliferation of non-malignant tissue, by acting as agonists or partial agonists; i.e., the analog may fully or partially mimic or enhance the biological effect of the natural peptide on a target cell. One possible mechanism of analog inhibition of growth of cancer cells is suggested in Bunn et al. (1990, Proc. Nat. Aca. Sci. 87:2162) , in which calcium ion flux was measured in CHO cells after administration of one or more neuropeptides. Bunn et al. observed a desensitization of cells to the neuropeptide after administration of the second dose, i.e., after administration of the first dose and the return of calcium concentration to resting values, administration of a second dose of an identical peptide resulted in no calcium flux. However, when the second dose was administered using a different peptide, a new calcium flux occurred. Thus different peptides may trigger different calcium flux pathways. The results of Bunn et al. show that desensitization to the neuropeptide may occur both in cancerous and normal tissue, thus suggesting that an agonist may suppress growth in a tumor cell by a similar mechanism. Use

Analogs of the invention are useful for treating colon, prostatic, breast, pancreatic, or lung cancer, for preventing the proliferation of smooth muscle, to suppress appetite, to stimulate pancreatic secretion, or to suppress a craving for alcohol. Analogs of the invention are administered to a mammal, particularly a human, in one of the traditional modes (e.g., orally, parenterally, transdermally, or trans ucosally) , in a sustained release formulation using a biodegradable biocompatible polymer, or by on-site delivery using micelles, gels and liposomes, or rectally (e.g., by suppository or enema) . The analogs can be administered to a human patient in a dosage of 0.25 mg/kg/day to 5 mg/kg/day.

Other embodiments are within the following claims.

Claims

1. A therapeutic peptide comprising between seven and ten amino acid residues, inclusive, said peptide being an analog of one of the following naturally occurring peptides which terminate at the carboxy-terminus with a Met residue: (a) litorin; (b) the ten amino acid carboxy-terminal region of mammalian GRP, neuromedin B, or neuromedin C; and (c) the ten amino acid carboxy-terminal region of amphibian bombesin, said analog being of the formula: R_χ A°-A¹-A²-A³-A -A⁵-A⁶-A⁷-A⁸-A⁹-R₃ R₂

wherein A = Gly, D- or L- isomer of any of pGlu, Nle, a-aminobutyric acid, Ala, Val, Gin, Asn, Leu, He, p-X-Phe (where X = H, F, Cl, Br, N0₂, OH, CH₃) , Trp, or 3-Nal, or is deleted; A¹ = the D- or L-isomer of any of pGlu, Nle, a-aminobutyric acid, Ala, Val, Gin, Asn, Leu, He, p-X-Phe (where X = H, F, Cl, Br, N0₂, OH, or CH₃) , Asp, Glu, F₅-Phe, Trp, /3-Nal, Cys, Lys, or is deleted; A² = Gly, D- or L- isomer of any of pGlu, Ala, Val, Gin, Asn, Leu, He, p-X-Phe (where X = H, F, Cl, Br, N0₂, OH, or CH₃) , Trp, /3-Nal, Asp, Glu, His, 1-methyl-His 3-methyl-His, Cys, Lys, or is deleted; A³ = the D- or L-isomer of any of p-X-Phe (where X = H, F, Cl, Br, N0₂, OH, or CH₃) , /3-Nal, or Trp; A⁴ = Ala, Val, Gin, Asn, Gly, Leu, He, Nle, a-aminobutyric acid, p-X-phe (where X = H, F, Cl, Br, N0₂, OH, or CH₃), Trp, or /3-Nal; A⁵ = Gin, Asn, Gly, Ala, Leu, He, Nle, a-aminobutyric acid, Val, p-X-Phe (where X = H, F, Cl, Br, N0₂, OH, or CH₃) , Trp, Thr, or /3-Nal; A⁶ = Sar, Gly or the D-isomer of any Ala, N-methyl-Ala, Val, Gin, Asn, Leu, He, p-X-Phe (where X = H, F, Cl, Br, N0₂, OH, or CH₃) , Trp, Cys, or /3-Nal; A⁷ = 1-methyl-His, 3-methyl-His, His, Lys, Asp, or Glu; A = Leu, He, Val, Nle, a-aminobutyric acid, p-X-Phe (where X = H, F, Cl, Br, N0₂, OH, or CH₃) , Trp, Thr, 3-Nal, Lys, Asp, Glu, CHx-Ala, or Cys; A⁹ = L-isomer of any of Met, Met-oxide, Leu, He, Nle, a-aminobutyric acid, p-X-Phe (where X = H, F, Cl, Br, N0₂, OH, or CH₃) , Trp, /3-Nal, CHx-Ala, or Cys; each R_χ and R₂, independently, is H, C_j_₁₂ alkyl, C_?_₁₀ phenylalkyl, COE^_^ (where E^_^ is C₁_₂₀ alkyl, C₃_₂₀ alkenyl, ^c ₃_₂₀ alkinyl, phenyl, naphthyl, or C₇_₁₀ phenylalkyl) , or ^cι~^ci₂ ^acy!' ^{and R}ι ^{and R} ₂ ^are bonded to the N-terminal amino acid of the peptide; provided that when one of R_χ or R₂ is COE_j^, the other must be H; and R₃ is H, NH₂, C_1-12 alkyl, C_?_₁₀ phenylalkyl, or C₃_₂₀ naphthylalkyl; and further provided that, if A is present, A cannot be pGlu; and, if A or A¹ is present, A² cannot be pGlu; and further provided that, when A⁰ is deleted and A¹ is pGlu, R_χ must be H and R₂ must be the portion of Glu that forms the imine ring in pGlu; and further provided that, where A is deleted and A¹ is not pGlu, A¹ may be bonded to A , or where A and A¹ are deleted and A is not pGlu, A² may be bonded to A⁹, or where A⁰, A¹ and A² are deleted. A³ may be bonded to A⁹; and provided that where A⁰ is deleted and A¹ is Asp or Glu, or where A⁰ and A¹ are deleted and A² is Asp or Glu, either A¹ or A² can be bonded with A⁷ or A⁸, where A⁷ or A⁸ is Lys, or where A⁰ is deleted and A¹ is Lys or A and A¹ are deleted and A² is Lys, either A¹ or A can be bonded to A⁷ or A⁸, where A⁷ or A⁸ is Asp or Glu; and further provided that either one of A¹ or A² can be Cys and can be bonded through a disulfide bridge with either A⁸ or A⁹, provided that either one of A⁸ or A⁹ can be Cys and can be bonded through a disulfide bridge with either A¹ or A²; and further provided that where A⁰ and A¹ are deleted and A⁶ is D-Ala, A⁸-A⁹ cannot be Leu-Met-NH₂; or a pharmaceutically acceptable salt thereof.

2. The therapeutic peptide of claim 1, comprising the formula: A⁰ = pGlu, Gly, D-Phe, or is deleted; A¹ = pGlu, D-Phe, D-Ala, D-/3-Nal, D-Cpa, D-Asn, Cys, or is deleted; A² = pGlu, Asn, Gin, His, 1-methyl-His, 3-methyl-His, Cys, or is deleted; A³ = Trp; A⁴ = Ala; A⁵ = Val; A⁶ = Sar, Gly, D-Phe, or D-Ala; A⁷ = His; A⁸ = Leu, Phe, Chx-Ala, or Cys; A⁹ = L-isomer of any of Met, Leu, He, Nle, Phe, or Cys.

3. The therapeutic peptide of claim 2, comprising the amino acid formula: pGlu-Gln-Trp-Ala-Val-Gly-His-Leu-Leu-NH₂. 4. The therapeutic peptide of claim 2, comprising the amino acid formula: D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Leu-NH₂.

5. The therapeutic peptide of claim 2, comprising the amino acid formula: D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂.

6. The therapeutic peptide of claim 2, comprising the amino acid formula: D-Cpa-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂.

7. The therapeutic peptide of claim 2, comprising the amino acid formula: D-Cpa-Gln-Trp-Ala-Val-Gly-His-Leu-Leu-NH₂.

8. The therapeutic peptide of claim 2, comprising the amino acid formula: D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Leu-Leu-NH₂.

9. The therapeutic peptide of claim 2, comprising the amino acid formula: D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Leu-Met-NH₂.

10. The therapeutic peptide of claim 2, comprising the amino acid formula: D-Cpa-Gln-Trp-Ala-Val-D-Ala-His-Leu-Met-NH₂.

11. The therapeutic peptide of claim 2, comprising the amino acid formula: pGlu-Gln-Trp-Ala-Val-Gly-His-Phe-Leu-NH₂.

12. The therapeutic peptide of claim 2, comprising the amino acid formula: D-Phe-Gln-Trp-Ala-Val-Gly-His-Phe-Leu-NH₂.

13. The therapeutic peptide of claim 2, comprising the amino acid formula: D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Phe-Met-NH₂.

14. The therapeutic peptide of claim 2, comprising the amino acid formula: D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Phe-Leu-NH₂.

15. The therapeutic peptide of claim 2, comprising the amino acid formula: D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Nle-NH₂.

16. The therapeutic peptide of claim 2, comprising the amino acid formula: D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Leu-Nle-NH₂.

17. The therapeutic peptide of claim 2, comprising the amino acid formula: D-Phe-Gln-Trp-Ala-Val-Gly-His-Phe-Nle-NH₂.

18. The therapeutic peptide of claim 2, comprising the amino acid formula: D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Phe-Nle-NH₂.

19. A therapeutic peptide comprising the amino acid formula: D-p-Cl-Phe-Gln-Trp-Ala-Val-Gly-His-Leuc[CH₂NH]Phe-NH₂.

20. A therapeutic peptide comprising the amino acid formula: D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-proplyamide. 21. The therapeutic peptide of claim 2, comprising the amino acid formula: Ac-His-Trp-Ala-Val-D-Ala-His-Leu-Leu-NH₂.

22. The therapeutic peptide of claim 2, comprising the amino acid formula: D-Phe-Gln-Trp-Ala-Val-Gly-His-CHx-Ala-Leu-NH₂.

23. The therapeutic peptide of claim 2, comprising the amino acid formula: cyclo-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Leu.

24. The therapeutic peptide of claim 2, comprising the amino acid formula: D-Cys-Asn-Trp-Ala-Val-Gly-His-Leu-Cys-NH₂.

25. The therapeutic peptide of claim 2, comprising the amino acid formula: cyclo-His-Trp-Ala-Val-Gly-His-Leu-Met.

26. The therapeutic peptide of claim 2, comprising the amino acid formula: Cys-Trp-Ala-Val-Gly-His-Leu-Cys-NH₂.

27. The therapeutic peptide of claim 2, comprising the amino acid formula: cyclo-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Met.

28. The therapeutic peptide of claim 2, comprising the amino acid formula: cyclo-D-Phe-His-Trp-Ala-Val-Gly-His-Leu-Met.

29. The therapeutic peptide of claim 2, comprising the amino acid formula: cyclo-Trp-Ala-Val-Gly-His-Leu-Met. 1 30. A therapeutic peptide comprising 2,between seven and ten amino acid residues, inclusive, 3said peptide being an analog of one of the following 4;naturally occurring peptides which terminate at the Scarboxy-terminus with a Met residue: (a) litorin; (b) 6the ten amino acid carboxy-terminal region of 7mammalian GRP, neuromedin B, or neuromedin C; and (c) 8the ten amino acid carboxy-terminal region of 9amphibian bombesin, said analog being an agonist of lOone of said naturally occurring peptides.

1 31. The therapeutic peptide of claim 30, 2wherein said peptide is a partial agonist of said 3naturally occurring biologically active peptide.

1 32. The therapeutic peptide of claim 30,

2said analog being at least 25% homologous with said 3naturally occurring peptide.

1 33. The therapeutic peptide of claim 32, 26aid analog being at least 50% homologous with said 3naturally occurring peptide.