HUT62604A - Process for producing peptides for treating tissue proliferation and pharmaceutical compositions comprising same - Google Patents

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

The present invention relates to peptides for the treatment of benign and malignant tissue proliferations. The amphibian peptide bombesin, which is pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (Anastasi et al., Experientia 27, 166-167). 1971)] is closely related to mammalian gastrin-releasing peptide (GRP), such as porcine GRP, which is H ₂ N-AlaPro-Val-Ser-Val-Gly-Gly-Thr-Val-Leu-Ala-Lys-Met -TyrPro-Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met (NH ₂ ) [McDonald et al., Biochem. Biophys. Gap. Commun. 90, 227-233 (1979)] and human GRP, which is H ₂ N-Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr-Val-Leu-Thr-Lys-Met-Tyr -Pro-Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met- (NH ₂ ). Bombesin has been found to act as a growth factor in a number of human cancer cell lines, including small cell lung carcinoma (SCLC) and has also been shown in human breast and prostate cancers. in Lung Cancer, Springer-Verlag, New York, 1986]. Many of these cancers are known to secrete peptide hormones such as GRP or bombesin. Consequently, bombesin antagonists 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 proliferation of human small cell lung cancer cell lines implanted in bald mice (Cuttitta et al., Cancer Survey 4: 707-727 (1985)). In a 3T3 mouse fibroblast responsible for the mitotic action of bombesin, Zachary and Rozengurt were observed to have a P antagonist substance (Spantide) acting as a bombesin antagonist [Zachary et al., Proc. Natl. Acad. Sci. (USA) 82: 7616-7620 (1985)]. Heinz-Erian et al., Introduced D-Phe in place of His at position 12 of bombesin and observed bombesin antagonist activity in guinea pig pancreatic acines (HeinzErian et al., Am. J. of Physiol. 252. G439-G442 (1987)]. _V Rivier reported a work, which was intended to conformational freedom of the bioactive C-terminal decapeptide of bombesin restricts intramolecular disulfide bridges incorporating; however, Rivier mentioned that, to date, modified bombesin analogues have failed to show antagonistic activity [Rivier et al., Competitive Antagonists of Peptide Homones, Abstracts of the Bombesin-Like Peptides in Health and Disease, Rome, 1987 October)].

Bombesin has both direct and indirect effects on the gastrointestinal tract, including effects on hormone release, stimulation of pancreas, stomach and small intestine secretion, and small bowel mobility. Gastrin and cholecystokinin (CCK), which are released by bombesin, play a role in maintaining normal gastrointestinal mucosa and accelerating the growth of normal and cancerous tissues. Growth of human intestinal and gastric cancers implanted in bald mice was enhanced by administration of gastrin and subsequently inhibited by administration of secretin (Tanake et al., Tokaku J. Exp. Med. reproduction was stimulated with pentagastrin (Winsett et al., Surgery 99, 302 (1980)) and inhibited by proglum, a gastrin receptor antagonist [Beauchamp et al., Ann. Surg. 202, 303 (1985)]. Bombesin has been found to act simultaneously as a tissue nourishing agent for pancreas normal hosts and as a growth inhibitor in human pancreatic tumor tissue transplanted into a foreign body (Alexander et al., 1988, Pancreas 2: 247 (1988)).

Below are the (unusual) abbreviations used:

cyclohexyl-Ala - CHx-Ala (cyclohexyl-alanine) «2 c

H ₂ C CH ₂ '

H ₂ C ch ₂

CH

CH ₂

NH ₂ —CH — CO ₂ H v

pGlu = H ₂ C --- CH-COOH (pyroglutamic acid);

H ₂ C NH

C

Nle = H ₂ N-CH-COOH (norleucine)

I (CH ₂ ) ₃ -CH ₃

Paul - 3-pyridylalanine

D-Cpa = D-para-chlorophenylalanine

HyPro = hydroxyproline

Nal = naphthylalanine

Mud = sarcosine

F5 ~ Phe = pentafluorophenylalanine

R = better configuration (D); S = left (L) configuration; racemate = 1: 1 mixture of R and S

1-methyl-His; 3-methyl-His = methyl (CH3) on the histidine or 3-position nitrogen;

H

C-N ¹ H ⁺ _ CH C-N ³ H

HCH

NH ₂ - CH - COOH

The present invention relates to a linear or cyclic therapeutic peptide consisting of seven to ten amino acid units which is analogous to one of the following naturally occurring peptides which terminate with one Met unit at the carboxyl terminus: (a) litorin; (b) a ten-amino acid carboxyl terminal region of mammalian GRP, neuromedin B or neuromedin C; and (c) a ten-amino acid carboxyl-terminal region of amphibian bombesin, analogous to the following formula:

R j

A ° -A ¹ -A ² -A ³ -A ⁴ -A ⁵ -A ⁶ -A ⁷ -A ⁸ -A ⁹ -R 3, /

R2 where

A ° is Gly, pGlu, Nle, α-aminobutyric acid, Ala, Val, Gin,

Asn, Leu, Ile, pX-Phe (where X = H, F, Cl, Br, NO ₂ , OH,

CH3), any D or L isomer of Trp or β-Nal, or is absent,

A ¹ is pGlu, Nle, α-aminobutyric acid, Al, Val, Gln, Asn,

Leu, Ile, pX-Phe (where X = H, F, Cl, Br, NO ₂ , OH or

CH ₃ ), any D or L isomer of any of Asp, Glu, F ₅ -Phe, Trp, β-Nal, Cys or Lys;

A ² is Gly or pGlu, Al, Val, Gln, Asn, Leu, Ile, pX-Phe (where X is H, F, Cl, Br, NO ₂ , OH or CH ₃ ), Trp, β-Nal, Asp , The D or L isomer of any of Glu, His, 1-methyl-His, 3-methyl-His, Cys or Lys, or is absent,

³ is pX-Phe (where X = H, F, Cl, Br, NO ₂ , OH or

CH ₃ ), D- or L-isomer of any of β-Nal or Trp;

A ⁴ is Ala, Val, Gln, Asn, Gly, Leu, Ile, Nle, α-aminobutyric acid, pX-Phe (where X = H, F, Cl, Br, NO ₂ , OH or CH ₃ ), Trp or β-Nal;

⁵ is Gln, Asn, Gly, Ala, Leu, Ile, Nle, α-aminobutyric acid, Val, pX-Phe (where X = H, F, Cl, Br, NO ₂ , OH or CH ₃ ), Trp, Thr or β-Nal;

A ⁶ is Sar, Gly, or Ala, N-methyl-Ala, Val, Gln, Asn, Leu, Ile, pX-Phe (where X = H, F, Cl, Br, NO ₂ , OH or CH ₃ ), The D isomer of any of Trp, Cys, or β-Nal;

⁷ is 1-methyl-His, 3-methyl-His, His, Lys, Asp or Glu;

A ⁸ is Leu, Ile, Val, Nle, α-aminobutyric acid, pX-Phe (where X = H, F, Cl, Br, NO ₂ , OH or CH ₃ ), Trp, Thr, β-Nal, Lys, Asp , Glu, CHx-Ala or Cys;

⁹ is Met, Methoxide, Leu, Ile, Nle, α-amino butyric acid, pX-Phe (where X = H, F, Cl, Br, NO ₂ , OH or CH ₃ ), Trp, β-Nal, CH x The L-isomer of any of Ala or Cys;

R 1 and R ₂ are each, independently hydrogen,

C 1 -C 12 alkyl, C 7 -C 10 phenylalkyl, COE 1 - wherein E 1 is C 1 -C 20 alkyl,

C 3 -C 20 alkenyl, C 3 -C 20 alkynyl, phenyl, naphthyl or C 7 -C 10 phenylalkyl, or C 1 -C 12 acyl, and R f and R _{2 are} attached to the N-terminal amino acid of the peptide. ; with the proviso that if R ₂ is one or Rf Coef, the other must be H; and R ₃ is hydrogen, amino, C 1 -C 12 alkyl, C 7 -C 10 phenylalkyl, or C 3 -C 20 naphthylalkyl; with the further proviso that when A 0 is present, A ¹ is other than ρ-Glu; and when A 0 or A ^{1 is} present, A ² is different from pGlu, with the proviso that when A 0 is absent and A ¹ is pGlu, R f must be hydrogen and R ₂ is a portion of Glu forms an imine ring in pGlu; with the proviso that if Αθ is missing and A ¹ is different from pGlu, then A ^{1 may} be linked to A ⁹ , or if A 0 and A ^{1 are} absent and A ² is different from pGlu, it may be linked to A ² A ⁹ , or when A 0, A ¹ and A ^{2 are} absent, A ³ may bind to A ^{9 to} form a cyclic peptide; and with the further proviso that if A ° is missing and

A ¹ is Asp or Glu, or when A 0 and A ^{1 are} absent and A ² is Asp or Glu, it can be linked to either A ² A ⁷ or A ⁸ , where A ⁷ or A ⁸ is Lys, or when A 0 is absent and A ¹ is Lys or A 0 and A ¹ is absent and A ² is Lys, A ¹ or A ² may be linked to A ⁷ or A ⁸ , wherein A ⁷ or A ⁸ is Asp or Glu; further provided that any of A ¹ or A ² may be Cys and may be linked to A ⁸ or A ⁹ via a disulfide bridge, provided that any of A ⁸ or A ⁹ may be Cys and a disulfide bridge may be linked to A ¹ or A ² ; with the proviso that when A 0 and A ^{1 are} absent and A ⁶ is D-Ala then A ⁸ -A ⁹ cannot be Leu-Met-NH 2;

and pharmaceutically acceptable salts thereof.

When any of R 1 or R _{2 in} the above general formula is an aliphatic, aromatic or lipophilic group, the in vivo activity of the compound is prolonged and the delivery of the compounds of the invention to target tissues can be facilitated.

More preferably, in the above general formula the substituents have the following meanings:

A °

A ¹ is pGlu, Gly, D-Phe or A 0 is absent, pGlu, D-Phe, D-Ala, DB-Nal, D-Cpa, D-Asn, Cys or A ^{1 are} absent,

In ^{Figure 2} pGlu, Asn, Gln, His, 1-methyl Cys or A ^{2 are} absent, In ^{Figure 3} meaning Trp, In ^{Figure 4} represents Ala; A5 is Val; ⁶ is Sar, Gly, D-Phe or D-Ala; ⁷ is His; ⁸ is Leu, Phe, CHx-Ala, or Cys; ⁹ is Met, Leu, Ile, Nle, Phe or

L-isomer.

To any Cys

-His, 3-methyl-His,

Examples of preferred peptide analogs include the following two:

pGlu-Gln-Trp-Ala-Val-Gly-His-Leu-Leu-NH2;

D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Leu-NH ₂ ;

D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH ₂ ;

D-Cpa-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH ₂ ;

• ·

- 9 D-Cpa-Gln-Trp-Ala-Val-Gly-His-Leu-Leu-NH ₂ ;

D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Leu-Leu-NH ₂ ;

D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Leu-Met-NH ₂ ;

D-Cpa-Gln-Trp-Ala-Val-D-Ala-His-Leu-Met-NH ₂ ; pGlu-Gln-Trp-Ala-Val-Gly-His-Phe-Leu-NH ₂ ; D-Phe-Gln-Trp-Ala-Val-Gly-His-Phe-Leu-NH ₂ ;

D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Phe-Met-NH ₂ ;

D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Phe-Leu-NH ₂ ;

D-Phe-Gln-Trp-Ala-Val-Gly-His-CHx-Ala-Leu-NH ₂ ;

D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Nle-NH ₂ ;

D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Leu-Nle-NH ₂ ;

D-Phe-Gln-Trp-Ala-Val-Gly-His-Phe-Nle-NH ₂ ;

D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Phe-Nle-NH ₂ ;

Ac-His-Trp-Ala-Val-D-Ala-His-Leu-Leu-NH ₂ ; cyclo-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Leu; D-Cys-Asn-Trp-Ala-Val-Gly-His-Leu-Cys-NH ₂ ; cyclo-His-Trp-Ala-Val-Gly-His-Leu-Met; Cys-Trp-Ala-Val-Gly-His-Leu-Cys-NH ₂ ; 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, such as, but not limited to, pGlu, Nle, Paul, D-Cpa, Methoxide, Nal, Sar, 1-methylHis or 3-methyl-His, is an analog of A °, A ¹ , A ⁶ or A ⁹ , or the ring closure of the analogue, increased stability of the analogue * ·

- Protects the amino terminal from degradation by exopeptidase, and protects the interior of the peptide from degradation by endopeptidase. The carboxyl-terminal dipeptide sequence of some analogs of the invention corresponds to the natural C-terminal sequence of bombesin or bombesin-related analogs.

The present invention provides a linear or cyclic therapeutic peptide consisting of seven to ten amino acid units, which is an analog of the following naturally occurring peptides terminated at the carboxyl terminus with a Met moiety: (a) litorin; (b) a ten-amino acid carboxyl-terminal region of mammalian GRP, neuromedin B or neuromedin-C; and (c) a ten-amino acid carboxyl-terminal region of amphibian bombesin, and is an agonist of one of the above naturally occurring peptides.

In a preferred embodiment of the invention, the analog is an agonist or partial agonist of the naturally occurring biologically active peptide, preferably at least 25%, more preferably 50 or 75% homologous to a region of the naturally occurring peptide. The term agonist is used in the sense that it mimics or enhances the biological activity of the natural peptide on its target cell, while the partial agonist mimics or enhances the biological activity of the natural peptide, but to a lesser extent than the agonist. The biological effect, as used herein, is measured by the action of the natural peptide in one or two of the following systems: an in vitro pancreatic amylase release assay and an in vitro 3T3 fibroblast cell secretion system.

11 of both systems are described in European Patent Application 88308916.6, which is incorporated herein by reference. An agonist increases the amylase release or fibroblast cell division of a natural peptide from pancreatic cells by 100%, while a partial agonist has a lower stimulatory effect of 0 to 99%.

The peptides of the invention are useful in the treatment of non-malignant cell proliferative diseases in humans, such as smooth muscle proliferation. The peptides of the invention are also useful in the treatment of human cancers, particularly prostate, bowel, breast, pancreatic or lung cancers. In addition, the peptides of the invention are useful for reducing appetite, enhancing pancreatic secretion, or decreasing craving for alcohol.

The analogs of the invention may also be used in the form of their pharmaceutically acceptable salts. Examples of preferred salts include the therapeutically acceptable organic acids such as acetic acid, lactic acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzoic acid, salicylic acid, methanesulfonic acid, polysulfonic acid, trifluoroacetic acid, salts with inorganic acids such as hydrochloric acids such as hydrochloric acid, sulfuric acid or phosphoric acid.

Further advantages of the invention will become apparent from the following description of preferred embodiments.

Before describing preferred embodiments of the invention, the drawing will be briefly described:

• · · · · · * * · · · · · · · · · · · · · · · · · · · · · · · · · ·

Figure 1 depicts the amino acid sequences of naturally occurring peptides having analogs of the peptides of the invention.

The following describes the structure, synthesis and use of the preferred embodiments of the invention.

Structure

The peptides of the invention are derived from one of the sequences shown in Figure 1, which correspond to the naturally occurring peptides or fragments thereof. Bombesin, Neuromedin Bt, Neuromedin Ct, Litorin, and GRP analogues of the compounds of the invention are disclosed in U.S. Patent No. 502,438 (filed March 30, 1990), U.S. Patent No. 397,169, issued August 21, 1989. CIP Application (continuation in part) filed in U.S. Patent Application Serial No. 376,555 (filed July 7, 1989) and U.S. Patent No. 394,727 (Aug. 16, 1989). US Patent Application No. 317,941 (filed March 2, 1989), CIP Application, filed December 9, 1988, U.S. Patent No. 282,328, filed December 9, 1988. US Patent Application CIP, filing U.S. Pat. No. 257,998, filed October 14, 1988. U.S. Patent Application Serial No. 248,771 (1988); United States Patent Application, filed September 23, CIP, filing U.S. Patent No. 207,759, filed June 16, 1988. · · · · · · · · · · · · · · · · ·

- CIP filing of U.S. Patent Application Publication No. 13,331, filed March 25, 1988, filed June 8, 1988, filed under U.S. Patent No. 204,171, filed June 8, 1988. U.S. Patent Application CIP Application, which is CIP Application No. 100,571, filed September 24, 1987, each of which is hereby incorporated by reference in its entirety, by Coy et al. and the compounds described above are 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, Volume 86, editors: Tam and Kaiser; Heinz-Erian et al., Am. J. Physiol. G439 (1986);

Martinez et al., J. Med. Chem., 28, 1874 (1985); Gargosky et al., Biochem. J. 247, 427 (1987); Dubreuil et al., Drug Design and Delivery, Volume 2, 49, Harwood Academic Publishers, GB, 1987; Heikkila et al., J. Bioi. Chem. 262, 16456 (1987); Caranikas et al., J. Med. Chem. 25, 1313 (1982); Saed et al., Peptides 10: 597 (1989);

Rosell et al., Trends in Pharmacological Sciences 3, 211 (1982); Lundberg et al., Proc. Nat. Aca. Sci. 80, 1120 (1983); Engberg et al., Natura 293: 222 (1984); Mizrahi et al., Euro. J. Pharma. 82, 101 (1982); Leander et al., Natura 294, 467 (1981); Woll et al., Biochem. Biophys. Gap. Comm., 155, 359 (1988); Rivier et al., Biochem. 17, 1766 (1978); Cuttitta et al., Cancer.

- 14 Surveys 4, 707 (1985); and Aumelas et al., Int. J. Peptide Sl. 30, 596 (1987), which are also incorporated herein by reference.

Synthesis of analogs

The following describes the synthesis of the bombesin agonist DIM-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Leu-NH2, BIM-26187. Other bombesin or GRP agonists may be prepared by carrying out a modified version of the following synthesis procedure.

1) incorporation of alpha-tert-butoxycarbonyl (BOC) leucine

4-methyl-benzhydrylamine

The chloride ion-form 4-methylbenzhydrylamine polystyrene resin (Bachem, Inc.) (0.72 milliequivalents / g) is charged to a reaction vessel of an ACT200 peptide synthesizer (Advanced Chem. Tech, Inc.) to carry out the following reaction cycle. programmed with: (a) methylene chloride; (b) 10% triethylamine in chloroform; (c) methylene chloride; and (d) dimethylformamide.

The neutralized resin was stirred with alpha-tert-butoxycarbonyl (BOC) leucine and diisopropylcarbodiimide in 3 molar equivalents each in methylene chloride for 1 hour. The resulting amino acid resin was washed in the synthesizer with dimethylformamide and reacted with 5% dimethylformamide acetic anhydride for 5 minutes. It is then washed with dimethylformamide and methylene chloride.

2) Coupling of the remaining amino acids

The peptide synthesizer is programmed to carry out the following reaction cycle: (a) methylene chloride; (b) 33% me ♦ · »· *« · 9 ·

Λ 9 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Trifluoroacetic acid (TFA) (15 times, 5 and 25 minutes each), (c) methylene chloride; (d) isopropyl alcohol; (e) 10% chloroform triethylamine; and (f) methylene chloride.

The following amino acids (3 molar equivalents) are then sequentially coupled using the same procedure: BOC-Leu, BOC-His (tosyl), BOC-Gly, BOC-Val, BOC-Ala, BOC-Trp, BOC-Gln In the presence of 1 equivalent of hydroxybenzotriazole), BOC-D-Phe (coupling is carried out in the presence of 1 equivalent of hydroxybenzotriazole). The finished resin is then washed with methanol and air dried.

The peptide resin (1.41 g) described above was treated with 5 ml of anisole, 50 mg of dithioerythritol and 25 ml of anhydrous hydrogen fluoride at 0 ° C for 1 hour. The excess hydrogen fluoride was evaporated rapidly in a stream of dry nitrogen and the residue was washed with ether. The crude peptide was dissolved in 100 ml of 4 M acetic acid and the solution was concentrated in vacuo. The crude peptide was then dissolved in a minimum volume of methanol / water and triturated with ethyl acetate. The triturated peptide was loaded onto a 9.4 mm internal diameter 50 cm high octadecylsilane silica column (Whatman Partisil 10 ODS - 2M9). Elution was performed with a linear gradient of 50/50 / 0.1% TFA / acetonitrile in 20% to 80% 0.1% aqueous TFA. The fractions were analyzed by HPLC and the appropriate fractions were concentrated to low volume and lyophilized. 65 mg of product are obtained in the form of a colorless powder.

Other compounds, including D-Cpa ¹ , CHx-Ala ⁸ or Nle ^9, may be prepared as described above.

·· «« · ···

4 «·« 4 ··· • · 4 4 · «·» · «» ··· ♦ · «

The peptides of the invention can be cyclized as follows.

The crude peptide acid cleaved from the peptide resin ester with hydrogen fluoride is dissolved in DMF (0.1 to 1%), treated with a condensing agent (e.g. BOP reagent, DEPC, DPPA or any other condensing agent) and then with a base (e.g. triethylamine or diisopropylethylamine) at room temperature for 1-3 days. The material is then dried by removing the solvent in vacuo. The residue was purified by high pressure liquid chromatography in the usual manner. Ring closure, for example, the preparation of cyclofD-Phe ¹ , Leu ⁸ , Leu ⁹ ] Litorin, in which D-Phe ^{1 is} covalently bound to Leu ⁹ , is carried out according to the above procedure, as the BOP reagent benzotriazol-1-yloxy. tris (dimethylamine) phosphonium hexafluorophosphate, DEPC reagent diethyl cyanophosphonate and DPPA reagent diphenylphosphoryl azide.

The following describes the mechanism of action of the compounds of the invention.

The analogs of the present invention are useful for inhibiting the proliferation of cancer cells or preventing proliferation of non-malignant tissues by acting as agonists or partial agonists; that is, the analogue wholly or partially mimics or enhances the biological activity of the natural peptide on the target cell. A possible mechanism for inhibiting the growth of cancer cells by analogues is described by Bunn et al., Proc. Nat. Aca. Sci., 87, 2162 (1990)], who, after administration of one or more neuropeptides, produce calcium ions in CHO cells. · «« «

- 17 flux was measured. Bunn et al. Observed a loss of cellular sensitivity to the neuropeptide after a second dose, that is, after the first dose, and a second dose of the same peptide did not induce calcium flux following the return of calcium concentration to rest. However, when another peptide was used as the second dose, calcium flux was reappeared. This indicates that different peptides can induce different calcium flux pathways. The results of Bunn et al. Suggest that neuropeptide insensitivity can occur in both cancerous and normal tissues, suggesting that an agonist may suppress the growth of tumor cells through a similar mechanism.

The analogs of the invention are useful for treating bowel, prostate, breast, pancreatic, or lung cancer, preventing cell proliferation in smooth muscle, suppressing appetite, enhancing pancreatic secretion, or suppressing craving for alcohol. The analogs of the invention may be administered to a mammal, especially a human, by conventional routes of administration (e.g., orally, parenterally, transdermally or transmucosally) using a biodegradable, biocompatible polymer, or by administration of micelles, gels and liposomes. on-site delivery) and rectally (e.g. in the form of a suppository or enema). Analogs may be administered to a human in a dosage range of 0.25 mg / kg / day to 5 mg / kg / day.

Claims (33)

Claims Claims 1. A therapeutic peptide having from seven to ten amino acid units which are naturally occurring peptides terminated with a carboxyl terminus methionine, namely (a) litorin; (b) the 10 amino acid carboxyl terminal region of mammalian GRP, neuromedin B or neuromedin C; and (c) an analogue of one of the ten amino acid carboxyl terminal regions of amphibian bombesin, and ^R A is -A ¹ -A ² -A ³ -A ⁴ -A ⁵ -A ⁶ -A ⁷ -A ⁸ -A ⁹ -R ₃ , * 2, wherein A ° is Gly, pGlu, Nle, α-aminobutyric acid, Ala, Val, Gln, Asn, Leu, Ile, pX-Phe (where X = H, F, Cl, Br, NO ₂ , OH, CH ₃ ), D or L isomer of either Trp or B-Nal, or is absent, A ¹ is pGlu, Nle, α-aminobutyric acid, Al, Val, Gln, Asn, Leu, Ile, pX-Phe (where X = H, F, Cl, Br, NO ₂ , OH or CH ₃ ), Asp, Glu, F 5 ~ Phe, Trp, β-Nal, Cys or Lys are any of D- or L -isomer or absent; A ² is Gly or pGlu, Ala, Val, Gln, Asn, Leu, Ile, pX-Phe (where X = H, F, Cl, Br, NO ₂ , OH or CH ₃ ), Trp, B-Nal, Asp , The D or L isomer of any of Glu, His, 1-methyl-His, 3-methyl-His, Cys or Lys, or is absent, ³ is ρ-Phe (where X = H, F, Cl, Br, NO ₂ , OH or CH ₃ ), D- or L-isomer of any of B-Nal or Trp; ⁴ represents Al, Val, Gin, Asn, Gly, Leu, Ile, Nle, α-amino ¹ · «« ·· 4 4 · * • 4 · ··· • · · »·· 4 • 44» 4 * 4 · »· • · 19-butyric acid, pX-Phe (where X = H, F, Cl, Br, NO ₂ , OH or CH ₃ ), Trp or β-Nal; ⁵ is Gln, Asn, Gly, Ala, Leu, Ile, Nle, α-aminobutyric acid, Val, pX-Phe (where X = H, F, Cl, Br, NO ₂ , OH or CH ₃ ), Trp, Thr or β-Nal; A ⁶ is Sar, Gly, or Ala, N-methyl-Ala, Val, Gln, Asn, Leu, Ile, pX-Phe (where X = H, F, Cl, Br, NO ₂ , OH or CH ₃ ), The D isomer of any of Trp, Cys, or β-Nal; ⁷ is 1-methyl-His, 3-methyl-His, His, Lys, Asp or Glu; A ⁸ is Leu, Ile, Val, Nle, α-aminobutyric acid, pX-Phe (where X = H, F, Cl, Br, NO ₂ , OH or CH ₃ ), Trp, Thr, β-Nal, Lys, Asp , Glu, CHx-Ala or Cys; ⁹ is Met, Me.t.-oxide, Leu, Ile, Nle, .alpha.-aminobutyric acid, pX-Phe (where X = H, F, Cl, Br, NO ₂ , OH or CH ₃ ), Trp, β-Nal. , The L-isomer of any of CHx-Ala or Cys; R 1 and R ₂ are each independently hydrogen, C 1 -C 12 alkyl, C 7 -C 10 phenylalkyl, COEi - where Εχ is C 1 -C 20 alkyl, C 3 -C 20 alkenyl, C 3 -C 20 alkynyl, phenyl, naphthyl or C 7 -C 10 phenylalkyl, or C 1 -C 12 acyl, and R _{2 is} attached to the N-terminal amino acid of the peptide; with the proviso that if one of R 1 or R ₂ is COE ₁ the other must be hydrogen; and R ₃ is hydrogen, amino, C 1 -C 12 alkyl, C 7 -C 10 phenylalkyl, or C 3 -C 20 naphthylalkyl; with the further proviso that when A 0 is present, A ¹ is different from p-Glu; and when A 0 or A 4 is present, A ² is different from pGlu, with the proviso that when A 0 is absent and A ¹ is pGlu, R ¹ must be hydrogen and R ₂ is a portion of Glu forms an imine ring in pGlu; and with the proviso that if A 0 is absent and A ¹ is different from pGlu, then A ^{1 may} be linked to A ⁹ , or if A 0 and A ^{1 are} absent and A ² is different from pGlu, it may be linked to A ² A ⁹ . , or when A 0, A ¹ and A ^{2 are} absent, A ³ may bind to A ⁹ ; and with the further proviso that if A ° is missing and ^A1 is Asp or Glu, or when A ° and A ¹ are deleted and ^A2 is Asp or Glu, A ¹ or A ² bonded to the ⁷ or ⁸ -tel genes in which A ⁷ or A ⁸ is Lys, or when A 0 is absent and A ¹ is Lys or A 0 and A ^{1 is} absent and A ² is Lys, A ¹ or A ² may be linked to A ⁷ or A ⁸ , wherein A ⁷ or A ⁸ is Asp or Glu; further provided that any of A ³ or A ² may be Cys and may be linked to A ⁸ or A ⁹ via a disulfide bridge, provided that any of A ⁸ or A ⁹ may be Cys and a disulfide bridge can be linked via A ¹ or A ² ; with the proviso that when A 0 and A ^{1 are} absent and A ⁶ is D-Ala then A ⁸ -A ⁹ cannot be Leu-Met-NH ₂ ; and pharmaceutically acceptable salts thereof. Therapeutic peptides according to claim 1, characterized in that they have the general formula A ° is pGlu, Gly, D-Phe or A ° is absent, A ¹ is pGlu, D-Phe, D-Ala, DB-Nal, D-Cpa, D-Asn, Cys or A ^{1 is} absent, ² represents pGlu, Asn, Gln, His, 1-methyl-His, 3-methyl-His, Cys or A ^{2 is} missing, In ^{Figure 3} meaning Trp, In ^{Figure 4} meaning Down; A5 meaning With; ⁶ meaning Mud, Gly, D-Phe or D-Ala; ⁷ meaning His; ⁸ meaning Leu, Phe, CHx-Ala, or Cys; ⁹ meaning Met, Leu, Ile, Nle, Phe or Cys L-isomer. The therapeutic peptide of claim 2, wherein the peptide comprises amino acids corresponding to the formula pGlu-Gln-Trp-Ala-Val-Gly-His-Leu-Leu-NH2. The therapeutic peptide of claim 2, wherein: a Contains amino acids corresponding to the formula D-Phe-Gln-Trp-Ala-Gly-His-Leu-Leu-NH2. The therapeutic peptide of claim 2, wherein: a Contains amino acids corresponding to the formula D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2. The therapeutic peptide of claim 2, wherein: a It contains the amino acids D-Cpa-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH ₂ . The therapeutic peptide of claim 2, wherein: a Contains amino acids corresponding to the formula D-Cpa-Gln-Trp-Ala-Gly-His-Leu-Leu-NH2. The therapeutic peptide of claim 2, wherein: a Contains amino acids corresponding to the formula D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Leu-Leu-NH ₂ . The therapeutic peptide of claim 2, wherein: a D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Leu-Met-NH ₂ contains the amino acids corresponding to the formula. 10. The therapeutic peptide of claim 2, wherein: a It contains the amino acids D-Cpa-Gln-Trp-Ala-Val-D-Ala-His-Leu-Met-NH ₂ . 11. A therapeutic peptide according to claim 2, characterized in that it contains amino acids corresponding to the formula pGlu-Gln-Trp-Ala-Val-Gly-His-Phe-Leu-NH ₂ . 12. The therapeutic peptide of claim 2, wherein: a Contains amino acids corresponding to the formula D-Phe-Gln-Trp-Ala-Val-Gly-His-Phe-Leu-NH ₂ . 13. The therapeutic peptide of claim 2, wherein: a Contains amino acids corresponding to the formula D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Phe-Met-NH ₂ . 14. The therapeutic peptide of claim 2, wherein: a Contains amino acids corresponding to the formula D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Phe-Leu-NH ₂ . 15. The therapeutic peptide of claim 2, wherein: a Contains amino acids corresponding to the formula D-Phe-Glyl-Trp-Ala-Val-Gly-His-Leu-Nle-NH2. 16. The therapeutic peptide of claim 2, wherein: a Contains amino acids corresponding to the formula D-Phe-Gln-Trp-Ala-Val-D-Ala-His-Leu-Nle-NH ₂ . 17. The therapeutic peptide of claim 2, wherein: a It contains the amino acids D-Phe-Gln-Trp-Ala-Val-Gly-His-Phe-Nle-NH2. 18. The therapeutic peptide of claim 2, wherein: a It contains the amino acids D-Phe-G1n-Trp-A1a-Va1-D-A1a-Hi s-Phe-Νe-NH2. 19. The therapeutic peptide of claim 2 wherein: a Dp-Cl-Phe-Gln-Trp-Ala-Val-Gly-His-Leuc _[CH2 NH] _Phe-NH2 corresponding formula contains amino acids. 20. The therapeutic peptide of claim 2, wherein: a Contains amino acids corresponding to the formula D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-propylamide. 21. The therapeutic peptide of claim 2, wherein: a _{Ac-His-Trp-Ala-Val-D-Ala-His-Leu-Leu-NH2} <· corresponding to formula contains amino acids. 22. The therapeutic peptide of claim 2, wherein: a Contains D-Phe-Gln-Trp-Ala-Val-Gly-His-CHx-Ala-Leu-NH2. 23. The therapeutic peptide of claim 2, wherein the therapeutic peptide comprises amino acids corresponding to cyclo-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Leu. 24. The therapeutic peptide of claim 2, wherein: a Contains amino acids corresponding to the formula D-Cys-Asn-Trp-Ala-Gly-His-Leu-Cys-NH2. 25. The therapeutic peptide of claim 2, wherein the therapeutic peptide comprises amino acids corresponding to the cyclo-His-Trp-Ala-Val-Gly-His-Leu-Met formula. 26. The therapeutic peptide of claim 2 wherein: a Contains amino acids corresponding to the formula Cys-Trp-Ala-Val-Gly-His-Leu-Cys-NH2. 27. The therapeutic peptide of claim 2, wherein the therapeutic peptide comprises amino acids corresponding to cyclo-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Met. 28. The therapeutic peptide of claim 2, wherein the therapeutic peptide comprises amino acids corresponding to the cyclo-D-Phe-His-Trp-Ala-Val-Gly-His-Leu-Met formula. 29. The therapeutic peptide of claim 2, wherein the therapeutic peptide comprises amino acids corresponding to cyclo-Trp-Ala-Val-Gly-His-Leu-Met. 30. A therapeutic peptide containing from seven to ten amino acid units which are naturally occurring peptides terminated at the carboxyl terminus with a methionine moiety, namely (a) litorin; (b) the 10 amino acid carboxyl terminal region of mammalian GRP, neuromedin B or neuromedin C; and (c) an analogue to one of the ten amino acid carboxyl terminal regions of amphibian bombesin, characterized in that the analogue is an agonist of one of the naturally occurring peptides mentioned herein. 31. The therapeutic peptide of claim 30, wherein said peptide is a partial agonist of one of the naturally occurring biologically active peptides of claim 30. 32. The therapeutic peptide of claim 30, wherein said peptide is at least 25% homologous to the naturally occurring peptide of claim 30. 33. The therapeutic peptide of claim 32, which is at least 50% homologous to the naturally occurring peptide.