AU703865B2

AU703865B2 - Bombesin analogs

Info

Publication number: AU703865B2
Application number: AU14808/95A
Authority: AU
Inventors: Sun Hyuk Kim; Jacques-Pierre Moreau
Original assignee: Ipsen Bioscience Inc
Current assignee: Ipsen Pharma SAS
Priority date: 1995-03-13
Filing date: 1995-03-13
Publication date: 1999-04-01
Anticipated expiration: 2015-03-13
Also published as: AU1480895A

Description

I

1 ATTORNEY DOCKET NO: 00537/004002 BOM SIN ANALOGS BACKGROUND OF THE INVENTION This invention relates to bombesin analogs useful for treatment of benign or malignant proliferation of tissue.

The amphibian peptide bombesin (see Anastasi, et al., Experientia 27:166-167, 1971) is closely related to the mammalian homolog gastrin-releasing peptide (GRP), 10 neuromedin B (NMB), neuromedin C (NMC), and litorin. See Minamino, et al. Ann. N.Y. Acad. Sci. 547: 373-390 (1988). Both GRP and NMB receptors have been identified and characterized on human tumors. See Taylor, et al., Growth Factors, Peptides, and Receptors (ed. Moody, T.

1993). Bombesin has been found to be a growth factor for a number of human cancer cell lines, including smill-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 mitotic 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.

I I I--r I-r I I 2 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. Synthetic analogs of bombesin have also been 15 reported in PCT Application WO 91/17181 (1991); Coy, et al. J. Biol. Chem. 266(25):16441 (1991); and Siegfried, Anat. Record 236: 241-247 (1993). Bombesin exhibits both direct and indirect effects on the gastrointestinal tract, including the release of hormones and the stimulation of pancreatic, gastric, and intestinal secretion and of intestinal mobility. GRP and cholecystokinin, which are released by bombesin, have been shown to play a role in the maintenance of normal gastrointestinal mucosa as well as 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., Tokaku, J. Exp. Med. 148:459, 1986) and the growth of MC-26 murine colon carcinoma which possesses gastrin receptors is stimulated by pentagastrin (Winsett, et al., Surgery 99:302 1980), and inhibited by proglumide, a gastrin-receptor antagonist (Beauchamp, et al., Ann. Surg. 202:303, 1985). Bombesin has been found to act concurrently as both a trophic agent for normal i -1 i I~IL LM host pancreas and a growth inhibitory agent in xenografted human liancreatic tumor tissue. Alexander, et al., Pancreas 3:247, 1988. NMB has been shown to effect the growth of cancer cells (Moody, et J. Pharmacol. 261:1 (1992)), suppress food intake, and decrease gastrin release (Kawai, et al., Endocrinol. Japan 37(6):857 (1990)).

Summary of the Invention 0 0 0*0**0 0 0 0* 0 0 Abbreviations: Chx-Ala cyclohexyl-Ala (3-cycylohexylalanine) pGiu =pyroglutamic acid Nie =norleucine lo D-Cpa D-p-chlorophenylalanine HyPro hydroxyproline Nal =3-(x4-naphthyl)-alanine, or 3-(P3-naphthyl)-alanine DOPA 3,4-dihydroxyphenylalanin~z Tcc 1, 2, 3, 4-tetrahydro-2-carboline-3-carboxylic acid 15 Tic 1, 2, 3, 4-tetrahiydroisoquinoline-3-carboxylic acid Aza-Tyrosine 3-(5-hydroxy-2-pyridy 1)-alani ne Sar sarcosine 1- or 3-methyl-His His with a methyl group on its position I or w-alkyl-Lvs Lys with its N* substituted by an alkyl group [-Ala 3-aminopropionic acid According to a first embodiment of the invention, there is peptide of the following tormula

A

1 I -A 2

-A

3

-A

4

-A

5

-A

6

-A

7

-A

8

-A

9

-R

3 3 heterocyclic nitrogen provided a therapeutic (1) 25 1\(2 in which: Al is a D-cL-aromatic amino acid or a D-a-tethered amino acid;

A

2 is Gln, His, 1-methyl-His, or 3-mnethyl-His;

A

3 is the D- or L-isomer selected from Nal, Trp, Phie, and p-X-Phle, where X is F, Cl, Br-, NO 2 OH- or CL- 3 Al 4 is Ala, Val, Leu. lie, Nle, or a-amninobutyric acid;

A

5 is Val, Ala, Leu, Ie, Nie, Thr, or c-amninobutyric acid;

A

6 is [3-Ala;

A

7 is His, 1-methyl-His, 3-m-ethyl-His, Lys, or alkyl-Lys;

A

8 is Leu, Ile, Val, Nle, ax-aminobutyric acid, Trp. Pro, Nal, Chx-Ala, Pie, or p- X-Phe, where X is F, Cl, Br, NO 2 OH or Cl- 3

A

9 is Met, Met-oxide, Leu, Ilie, Nle, oc-arninobutyric acid, or Cys; each R, and R 2 independently, is H, CI.- 1 2 alkyl, C 7 1 0 phienylalkyl, or COE,, where El is C 1 20 alkyl, C 3 20 alkenyl, C 3 20 alkynyl, phenyl, 3,4-dihydroxyphenylalkyl, naphthyl, 4.i, In: \ibaa 10 1470: M CC or C 7 -1 0 phenylalkyl; provided that when either R 1 or R 2 is COE 1 the other must be H; and

R

3 is OH, NH 2

C

1 -1 2 alkoxy, C 7 1 0 phenylalkoxy, C 11 20 naphthylalkoxy, C 1 1 2 alkylamino, C 7 1 0 phenylalkylamino, Cll-20 naphthylalkylamino; or a pharmaceutically acceptable salt of such peptides.

According to a second embodiment of the invention, there is provided a pharmaceutical formulation comprising an effective amount of at least one peptide in accordance with the first embodiment of the invention, together with a pharmaceutically acceptable carrier diluent or adjuvant therefor.

According to a third embodiment of the invention, there is provided a method for the treatment and/or prophylaxis of tumors in a mammal requiring said treatment and/or prophylaxis, which method comprises administering to said mammal an effective amount of at least one compound in accordance with the first embodiment of the invention, or of a composition in accordance with the second embodiment of the invention.

According to a fourth embodiment of the invention, there is provided a compound in accordance with the first embodiment of the invention, or composition in accordance with e: the second embodiment of the inventio;', when used in the treatment and/or prophylaxis of tumours in a mammal requiring said treatment or prophylaxis.

According to a fifth embodiment of the invention, there is provided use of a 20 compound in accordance with the first embodiment of the invention, in the preparation of a medicament for the treatment and/or prophylaxis of tumours in a mammal requiring said S treatment and/or prophylaxis.

t According to a sixth embodiment of the invention, there is provided a method for the treatment and/or prophylaxis of proliferation of smooth muscle in a mammal requiring 25 said treatment and/or prophylaxis, which method comprises administering to said mammal an effective amount of at least one compound according in accordance with the first embodiment of the invention, or of a composition in accordance with the second embodiment of the invention.

According to a seventh embodiment of the invention, there is provided a compound in accordance with the first embodiment of the invention, or a composition in accordance with the second embodiment of the invention, when used in the treatment and/or prophylaxis of proliferation of smooth muscle in a mammal requiring said treatment and/or prophylaxis.

According to an eighth embodiment of the invention, there is provided use of a compound in accordance with the first embodiment of the invention, in the preparation of a medicament for the treatment and/or prophylaxis of proliferation of smooth muscle in a mammal requiring said treatment and/or prophylaxis.

According to a ninth embodiment of the invention, there is provided a method for the suppression of appetite in a mammal requiring said suppression, which method comprises administering to said mammal an effective amount of at least one compound in i: H\A 1i:ii:\li aa1()147 0:MC accordance with the first embodiment of the invention, or a compositioh in accordance with the second embodiment of the invention.

According to a tenth embodiment of the invention, there is provided a compound in accordance with the first embodiment of the invention, or a composition in accordance with the second embodiment of the invention, when used in the suppression of appetite in a mammal requiring said suppression.

According to an eleventh embodiment of the invention, there is provided use of a compound in accordance with the first embodiment of the invention, in the preparation in a medicament for the suppression of appetite in a mammal requiring said suppression.

1o According to a twelfth embodiment of the invention, there is provided a method for the stimulation of pancreatic secretion in a mammal requiring said stimulation, which method comprises administering to said mammal an effective amount of at least one compound in accordance with the first embodiment of the invention, or a composition in accordance with the second embodiment of the invention.

According to a thirteenth embodiment of the invention, there is provided a compound in accordance with the first embodiment of the invention, or a composition in iaccordance with the second embodiment of the invention, when used in the stimulation of pancreatic secretion in a mammal requiring said stimulation.

According to a fourteenth embodiment of the invention, there is provided use of 20 compound in accordance with the first embodiment of the invention, in the preparation of a medicament for the stimulation of pancreatic secretion in a mammal requiring said stimulation.

According to a fifteenth embodiment of the invention, there is provided a method for the suppression of a craving for alcohol in a mammal requiring said suppression, 25 which method comprises administering to said mammal an effective amount of at least one :II: compound in accordance with the first embodiment of the invention, or a composition in accordance with the second embodiment of the invention.

According to a sixteenth embodiment of the invention, there is provided a compound in accordance with the first embodiment of the invention, or a composition in accordance with the second embodimeat of the invention, when used in the suppression of a craving for alcohol in a mammal requiring said suppression.

According to a seventeenth embodiment of the invention, there is provided use of a compound in accordance with the first embodiment of the invention, in the preparation of a medicament for the suppression of a craving for alcohol in a mammal requiring said suppression.

What is meant by "aromatic ac-amino acid" is an amino acid residue of the formula

NH

2

-CH(CH

2 -Z)-COOH where Z is a moiety containing an aromatic ring. Examples of Z include, but are not limited to, phenyl, 1-naphthyl, 2-naphthyl, 3-indolyl, l-Me-3indolyl, biphenyl, and imidazolyl, with or without one or more substituent X on the aromatic ring(s) of Z (where X is halogen, NO 2

CH

3 or OH).

S\ 470: Q L [ll:\llha|O1470: MCC 4b What is meant by "tethered a-ainino acid" is an a-amino acid with A carbon atom of its side chain tethered to the N atom of the a-amino group. Examples include, but are not limited to, Pro, HyPro, Tic, and Tec.

e a *e o a s Sn:llhbaa101470:MCC

I--

5 The symbol A 1

A

2 or the like herein stands for the residue of an a-amino acid. Except for tethered amino acids Pro, HyPro, Tcc, or Tic) and Sar, such symbols represent the general structure, -NH-CH(R)-CO- or =N-CH(R)-CO- when it is at the N-terminus or -NH-CH(R)- CO- when it is not at the N-terminus, where R denotes the side chain (or identifying group) of the a-amino acid, R is -CH 2 COOH for Asp. Note that the N-terminus is at the left and the C-terminus at the right in accordance with the conventional representation of a polypeptide chain. When A 6 is Sar, it has the structure of -N(CH 3

CH

2 The residue of a tethered amino acid, on the other hand, is of the structure where N, C and R together form a ring. HyPro herein refers to any 15 of 2-hydroxy-Pro, 3-hydroxy-Pro, 4-hydroxy-Pro, and hydroxy-Pro; 4-hydroxy-Pro is preferred.

Furthermore, where the amino acid residue is optically active, it is the L-form configuration that is intended unless the D-form is expressly designated. An 20 alkyl group, if not specified, contains 1-12 carbon 0 atoms. COE 1 stands for C-E 1 In formula given above, when either of R1 or Rd is an aliphatic, aromatic, or lipophilic group, the in S 25 vivo activity can be long lasting, and delivery of the compounds of the invention to the target tissue can be facilitated.

Preferably, in formula A 1 is the D-isomer selected from Nal, DOPA, Trp, Tcc, Tic, Aza-Tyr, Phe, and p--X-Phe, where X is F, Cl, Br, NO 2 OH or CH 3 It is particularly preferred that A 3 be the D-isomer selected from Phe, Trp, and p-X-Phe, where X is F, Cl, Br, NO 2

OH

or CH 3 and A 7 be His, 1-methyl-His, or 3-methyl-His.

Also preferably, in formula A 1 be D-Phe, D- Trp, or D-Tyr; A 2 be Gln; A 3 be Trp; A 4 be Ala; A 5 be Val;

A

7 be His; A 8 be Leu or Phe; and A 9 be Met, Leu, and Nle.

Particularly preferred peptides of the invention include the following: -Y P3 II JL I 6 H-D-Phe-Gln-Trp-Ala-Val-P-Ala-His-Phe-Nle-NH 2 Phe-Gln-Trp-Ala-Val-P-Ala-His-Leu-Leu-NH 2 H. yr-Gln-Trp-Ala-Val-p-Ala-His-Phe-Nle-NH2; H-D-Trp-Gln-Trp-Ala-Val-P-Ala-His-Phe-Nle-NH 2 and H-D-Phe-Gln-Trp-Ala-Val-P-Ala-His-Leu-Nle-NH 2 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, acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, salicylic, methanesulfonic, toluene sulfonic, trifluoroacetic, or pamoic acid, as well as polymeric acids such as tannic o. acid or carboxymethyl cellulose, and salts with inorganic acids such as the hydrohalic acids, hydrochloric 15 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.

BRIEF DESCRIPTION OF THE DRAWINGS The drawings will be first described.

Fig. 1 is a graph showing the growth inhibitory effect of a compound of the present invention on prostate S* tumors.

DESCRIPTION OF THE PREFERRED EMBODIMENTS We now describe the structure, synthesis, biological assays, and use of the preferred embodiments of the present invention.

Structure Peptides of the invention are derived from litorin, neuromedin B (NMB), neuromedin C (NMC), bombesin (last ten amino acids), and human GRP (last ten amino acids).

Synthesis of Analogs r _ly I II 7 The synthesis of one of the compounds of the invention, Analog #1 H-D-Phe-Gln-Trp-Ala-Val-- Ala-His-Phe-Nle-NH 2 follows.

4-methyl benzhydrylamine-polystyrene resin (Bachem, Inc.) (0.72 meq/g), in the chloride ion form, was placed in the reaction vessel of an ACT200 peptide synthesizer (Advanced Chem Tech, Inc.) programmed to perform the following reaction cycle: methylene chloride; 10% triethylamine in chloroform; (c) methylene chloride; and dimethylformide. The neutralized resin was mixed with BOC-Norleucine and diisopropylcarbodiimide (3 molar eq each) in methylene chloride for 1 hour. The resulting amino acid resin was washed on the synthesizer with dimethylformamide and 15 treated with 5% acetic anhydride in dimethylformamide for 5 min. Then it was washed with dimethylformamide and methylene chloride.

The peptide synthesizer was programmed to perform the following reaction cycle: methylene chloride; (b) 20 33% trifluoroacetic acid in methylene chloride (2 times for 5 and 25 min. each); methylene chloride; isopropyl alcohol; 10% triethylamine in chloroform; and methylene chloride.

The following amino acids (3 molar eq.) are then 25 coupled successively by the same procedure: BOC-Phe, BOC-His (CBZ), BOC-P-Ala, BOC-Val, BOC-Ala, BOC-Trp, BOC-Gin (coupled in the presence of 1 eq.

hydroxybenzotriazole), and BOC-D-Phe (coupled in the presence of 1 eq. hydroxybenzotriazole). The completed resin was then washed with methanol and air dried.

The peptide resin described above (3.5076 g) was mixed with anisole (10 ml), dithiothreitol (100 mg), and anhydrous hydrogen fluoride (50 ml) at 0 0 c for one hour.

Excess hydrogen fluoride was evaporated rapidly under a stream of dry nitrogen, and the residue was washed in ether. Crude peptide was dissolved in 100 ml of 4 M acetic acid and the solution is then evaporated under e 131i I ~s L- I I u 8 reduced pressure. The crude peptide was dissolved in minimum volume of methanol/water and triturated with ethyl acetate. The triturated peptide was applied to a column (9.4 mm I.D. x 50 cm) of octadecylcilane-silica (Whatman Partisil 10 ODS 2M9). The peptide was eluted with a linear gradient of 20-80% of 50/50 0.1% TFA/Acetronitrile i 0.1% TFA in water. Fractions were examined by analytical high performance liquid chromatography and appropriate fractions were evaporated to a small volume, which was further lyophilized, giving mg of the product as a colorless powder.

Other compounds of the present invention, e.g., .Analog #2 H-D-Phe-Gln-Trp-Ala-Val-p-Ala-His-Leu- Leu-NH 2 can be prepared in an analogous manner by 15 making appropriate modifications of the above-described synthetic method.

Biological Assays GRP Receptor Binding Assay Rat AR42J pancreatic acinar cells were cultured in 20 Dulbecco's modified Eagle's medium without antibiotics e and supplemented with 10% (vol/vol) fetal calf serum.

The incubation atmosphere consisted of 10% C0 2 humidified air at 37 0

C.

Membranes for the bombesin receptor binding assay were obtained by homogenizing AR42J cells (Polytron, setting 6, 15 sec)(Brinkman, Westbury, NY) in ice-cold mM Tris-HCl (Buffer A) and centrifuging twice at 39,000 x g (10 min), with an intermediate resuspension in fresh Buffer A. The final pellets were resuspended in 50 mM Tris-HCl, containing 0.1 mg/ml bacitracin, and 0.1% BSA (Buffer and held on ice for the receptor binding assay.

For assay, aliquots (0.4 ml) were incubated with 0.05 ml [125 I-Tyr 4 bombesin (-2200 Ci/mmol, New England Nuclear) and Buffer B, with and without 0.05 ml of unlabeled competing analogs. After a 30 min incubation ~lllll~e"l' BIPP~ C-L~ L- I_ 9 (4 0 the bound [125 I-Tyr 4 bombesin was separated from the free by rapid filtration through Whatman m

GF/B

filters which had been previously soaked in 0.1% polyethyleneimine using a Brandel filtration manifold.

The filters were then washed three times with 5 ml aliquots of ice-cold Buffer A. Specific binding was defined as the total [125 I-Tyr 4 bombesin bound minus that bound in the presence of 1 .M unlabeled bombesin.

The results (expressed as TC 5 0 in nM) and the structures of the tested compounds are shown in Table 1.

Replacement of Gly at position A 6 in a prior art compound 'torin, Leu-litorin, and [D-Phe 1 Leu 8 9 litorin) o. with led unexpectedly to a great increase in their affinity for the GRP receptor. For example, replacement 15 of Gly with P-Ala resulted in a 2.8 to 23 time increase in the affinity (compare Analog #2 with [D-Phel, Leu 8 9 litorin or Leu-litorin). Modifications at position A 1

A

8 or A 9 further increases the affinity. For example, Analog #3 has an IC 50 being as low as 0.03 nM, compared to 20 2.4 nM for litorin, 23 nM for Leu-litorin, or 2.8 nM for [D-Phe 1 Leu 8 9 litorin-- an increase of 80 to 766 times in the affinity for the GRP receptor.

LI

L1 I I- L- L- 10 TABLE 1 AFFINITY BINDING DATA FOR THE GRP RECEPTOR OF LINEAR BOMBESIN ANALOGS WITH p-Ala REPLACEMENT AT POSITION A 6 r r a e os a r r r a r s 15 Code Name Structure IC 50 (nM) Litorin p-Glu-Gln-Trp-Ala-Val-Gly-His-Phe-Met-NH 2 2.4 Lcu.Litorin p-Glu-Gln-Trp-Ala-Val-Gly-His-Leu-Leu-NH 2 23 [D-Phe 1 Leu 8 9 litorin H-D- Phe-Gln-Trp-Ala-Val-Gly-His-Leu-Leu-NH 2 2.8 Analog #1 H-D-Phe-Gln-Trp-Ala-Val-3-Ala-His-Phe-Nle-NH 2 0.2 Analog #2 H-D-Phe-Gln-Trp-Ala-Val-p-Ala- s-Leu-Leu-NH 2 Analog #3 H-D-Tyr-Gln-Trp-Ala-Val-p-Ala-His-Phe-Nle-NH 2 0.03 Analog #4 H-D-Trp-Gln-Trp-Ala-Val-3-Ala-His-Phe-Nle-NH 2 0.1 Analog #5 H-D-Phe-Gln-Trp-Ala-Val-p-Ala-His-Leu-Nle-NH 2 2.1 See formula in "SUMMARY OF THE INVENTION" above.

NMB Receptor Binding Assay The procedure for transfecting the rat NMB receptor into BALB-3T3 fibroblasts is discussed in Wada, et al., Neuron, 6:4221-430 (1991) and Benya, et al., Mol.

Pharmacol., 42:1058 (1992).

Membranes for the bombesin receptor binding assay were obtained by homogenizing BALB-373 fibroblasts, transfected with the rat NMB receptor (Polytron, setting 6, 15 sec) in ice-cold 50 mM Tris-HC1 (Buffer A) and centrifuging twice at 39,000 x g (10 min), with an intermediate resuspension in fresh Buffer A. The final pellets were resuspended in 50 mM Tris-HCl, containing 0.1 mg/ml bacitracin, and 0.1% BSA (Buffer Pr, and held on ice for the receptor binding assay.

For assay, aliquots (0.4 ml) were incubated with 0.05 ml [125 I-Tyr 4 bombesin (-2200 Ci/mmol, New England Nuclear) and Buffer B, with and without 0.05 ml of L- ~r I I 11 unlabeled competing analogs. After a 30 min incubation (4 0 the bound [125 I-Tyr 4 bombesin was separated from the free by rapid filtration through Whatman GF/B filters which had been previously soaked in 0.3% polyethyleneimine using a Brandel filtration manifold.

The filters were then washed three times with 5 ml aliquots of ice-cold Buffer A. Specific biiiding was defined as the total [125 I-Tyr 4 neuromedin-B bound minus that bound in thei presence of 1 gM unlabeled neuromedin-

B.

The results (expressed as IC 50 in nM) and the structures of the tested compounds are shown in Table 1.

Replacement of Gly at position A 6 in a prior art compound litorin, leu-litorin, and [D-Phel, Leu 8 9 litorin) 15 with p-Ala led unexpectedly to a great increase in their affinity for the NMB receptor. For example, replacement of Gly with p-Ala resulted in a 11.7 time increase in the affinity (compare Analog #2 with [D-Phe 1 Leu 8 9 litorin). Modifications at position Al, A 8 or A 9 further 20 increases the affinity. For example, Analog #4 has an

IC

50 being as low as 0.04 nM, compared 1.66 nM for litorin or 480 nM for [D-Phel, Leu 8 9 1 litorin-- an increase of 41.5 to .'2,000 times in the affinity for the NMB receptor. Leu-litorin was found to have almost no S 25 binding affinity on the NMB receptor.

II i LI I 12

I

s r b s

D

TABLE 2 AFFINITY BINDING DATA FOR THE NMB RECEPTOR OF LINEAR* BOMBESIN ANALOGS WITH P-Ala REPLACEMENT AT POSITION A Code Name Structure IC50 (nM) Litorin p-Glu-Gln-Trp-Ala-Val-Gly-His-Phe-Met-NH2 1.7 Leu-Litorin p-Glu-Gln-Trp-Ala-Val-Gly-His-Leu-Leu-NH 2 >10,000 [D-Phel Leu8,9] litorin H-D-Phc-Gln-Trp-Ala-Val-Gly-His-Leu-Leu-NH, 480 Analog #1 H-D-Phe-Gln-Trp-Ala-Val--Ala-His-Phe-Nle-NHI Analog #2 H-D-Phe-Gln-Trp-Ala-Val-P-Ala-His-Leu-Leu-NF 2 41 Analog #3 H-D-Tyr-Gln-Trp-Ala-Val--Ala-Hlis-Phe-Nlc-NHF 2 Analog#4 H-D-Trp-Giln-Trp-Ala-Val-.-Ala-His-Phe-Nlc-Nf 2 0.04 Analog #5 H-D-Phe-Gln-Trp-Aa-Val-p-Ala-His-Leu-Nl-NH 2 8.9 See formula in "SUMMARY OF THE INVENTION" above.

Assay on in vivo prostate tumor growth The androgen responsive R-3327/H prostate tumor line was implanted into 38 testes-intact, syngeneic Copenhagen male rats. The tumored animals were individually staged for castration when their tumors reached a size approximating 235 mgs. Tumors were then staged for bombesin analogue treatment when two sequential tumor measurements indicated an "escape from castration inhibition" condition, as evidenced by an increase in tumor growth rate. This transition of the prostate tumor tissue from androgen sensitive to androgen insensitive occurred without surgical perturbation of the tumor, thus providing a clinically realistic model for evaluating the anti-prostate tumor activity of the bombesin analogues.

o r o II-- I-

I-

13 The tumored animals were separated into two groups of 19 animals. Treatment was only initiated when tumors reached the "escape from castration inhibition" condition. Group 1 received 0.2 ml/inj. of 2.6% glycerol/water vehicle, Group 2 received a 200 mg/inj. of Analog #5 Both injections were administered in the flank opposite from the tumor. The treatment period lasted for 24 days.

Tumor measurements were taken at day 3, day 7, day day 14, day 17, day 21, and day 24. Tumors were measured using Vernier calipers and the volume was calculated using the following formula: (0.5)(length)(width) 2 Fig. 1 illustrates the tumor growth inhibitory effect of the treatments in Group 1 and Group 2.

15 Relative tumor volume was calculated by the following formula: Tumor Volume (measuring day) x 100 Tumor Volume (staging day) The data is provided by the mean relative tumor volume 20 standard error. There was a steadily increasing divergence in the size of vehicle treated control tumors (Group 1) and tumors treated with Analog #5 (Group 2) during the first 21 days of treatment. Rate of tumor growth was significantly (p 0.05) inhibited by Analog 25 #5 treatment after day 17.

9 Use Analogs of the invention are useful for treating colon, prostatic, breast, pancreatic, liver cancer or lung cancer, for preventing the proliferation of smooth muscle, for suppressing appetite, for stimulating pancreatic secretion, or for suppressing a craving for alcohol. Analogs of the invention are administered to a mammal, particularly a human, in one of the traditional modes orally, parenterally, transdermally, transmucosally, or via drug-releasing implants), in a sustained release formulation using a biodegradable biocompatible polymer, or by on-site delivery using

-IMM

I I I- 14 micelles, gels and liposomes, or rectally by suppository or enema). The analogs can be administered to a human patient in a dosage to be determined by the attending physician ranging from 0.25 mg/kg/day to mg/kg/day.

Furthermore, compounds of the present invention, particularly those with Tyr at the N-terminus, can be used for diagnostic purposes and for the tumor targeting of radioisotopes such as 131Iodine.

OTHER EMBODIMENTS The foregoing description has been limited to *specific embodiments of this invention. It will be apparent, however, that variations and modifications may be made to the invention, with the attainment of some or 15 all of the advantages of the invention. Such embodiments are also within the scope of the following claims.

4 4 e III I I

Claims

1. A therapeutic peptide of the formula: R 1 A 1 -A 2 -A 3 -A 4 -A 5 -A 6 -A 7 -A 8 -A 9 -R 3 R wherein: A' is a D-a-aromatic amino acid or a D-cz-tethered amino acid; A 2 is Gin, His, 1-methyl-His, or 3-methyl-His; A 3 is the D- or L-isomer selected from Nal, Trp, Phe, and p-X-Phe, where X is F, Cl, Br, NO 2 OH or CH 3 A 4 is Ala, Val, Leu, Ile, Nle, or ca-aminobutyric acid; A is Val, Ala, Leu, Ile, Nle, Thr, or a- 15 aminobutyric acid; A6 is P-Ala; A 7 is His, 1-methyl-His, 3-methyl-His, Lys, or e- alkyl-Lys; A 8 i Leu, Ile, Val, Nie, c-aminobutyric acid, z0 Trp, Pro, HyPro, Nal, Chx-Ala, Phe, or p-X-Phe, where X is F, Cl, Br, NO 2 OH or CH 3 A 9 is Met, Met-oxide, Leu, Ile, Nle, a- aminobutyric acid, or Cys; each R 1 and R 2 independently, is H, Cl-. 12 alkyl, C 7 10 phenylalkyl, or COE,, where El is C 1 20 alkyl, C 3 20 alkenyl, C 3 20 alkynyl, phenyl, 3,4-dihydroxyphenylalkyl, naphthyl, or C 7 10 phenylalkyl; provided that when either Ror R 2 is COE 1 the other must be H; and R 3 is OH, NH 2 Cl-. 12 alkoxy, C 7 10 phenylalkoxy, Cll- 2 0 naphthylalkoxy, Cl.. 12 alkylamino, C 7 10 phenylalkylamino, C 11 20 naphthylalkylamino; or a pharmaceutically acceptable salt thereof.

2. The therapeutic peptide of claim 1, wherein Al is the D-isomer selected from Nal, DOPA, Trp, Tcc, Tic, I I 16 Aza-Tyr, Phe, and p-X-Phe, where X is F, Cl, Br, NO 2 OH or CH 3

3. The therapeutic peptide of claim 2, wherein A 3 is the D-isomer selected from Trp, Phe, and p-X-Phe, where X is F, Cl, Br, NO 2 OH or CH 3 and A 7 is His, 1- methyl-His, or 3-methyl-His.

4. The therapeutic peptide of claim 3, wherein A 3 is Trp. The therapeutic peptide of claim 4, wherein A 7 a. a .0*0 a 10 is His.

6. The therapeutic peptide of claim 5, wherein A 2 is Gln.

7. The therapeutic peptide of claim 6, wherein A 4 is Ala. a a

8. The therapeutic peptide of claim 7, wherein A is Val.

9. The therapeut"- peptide of claim 8, wherein A 8 is Leu or Phe. The therapeutic peptide of claim 9, wherein A 9 is Met, Leu, or Nle.

11. The therapeutic peptide of claim 10, wherein A 1 is D-Phe, D-Tyr, or D-Trp.

12. The therapeutic peptide of claim 11 of the formula: H-D-Phe-Gln-Trp-Ala-Val-p-Ala-His-Phe-Nle-NH 2 I II L *0* as 00 0** 0@ 0 09 0 0 0 A 17

13. The therapeutic peptide of claim 11 of the formula: H-D-Phe-Gln-Trp-Ala-Val-P-Ala-His-Leu-Leu-NH 2

14. The therapeutic peptide of claim 11 of the formula: H-D-Tyr-Gln-Trp-Ala-Val-P-Ala-His-Phe-Nle-NH 2

15. The therapeutic peptide of claim 11 of the formula: H-D-Trp-Gln-Trp-Ala-Val-P-Ala-His-Phe-Nle-NH 2

16. The therapeutic peptide of claim 11 of the formula: H-D-Phe-Gln-Trp-Ala-Val-p-Ala-His-Leu-Nle-NH2.

17. A therapeutic peptide substantially as hereinbefore described with reference to Tables 1 or 2.

18. A pharmaceutical formulation comprising an effective amount of at least one peptide according to any one of claims 1 to 17 together with a pharmaceutically acceptable carrier diluent or adjuvant therefor.

19. A method for the treatment and/or prophylaxis of tumors in a mammal 15 requiring said treatment and/or prophylaxis, which method comprises administering to said mammal an effective amount of at least one compound according to any one of claims 1 to 17, or of a composition according to claim 18. A compound according to any one of claims 1-17, or composition according to claim 18, when used in the treatment and/or prophylaxis of tumours in a mammal 20 requiring said treatment and/or prophylaxis.

21. Use of a compound according to any one of claims 1-17, in the preparation of a medicament for the treatment and/or prophylaxis of tumours in a mammal requiring said treatment and/or prophylaxis.

22. A method for the treatment and/or prophylaxis of proliferation of smooth 25 muscle in a mammal requiring said treatment and/or prophylaxis, which method comprises administering to said mammal an effective amount of at least one compound according to any one of claims 1 to 17, or of a composition according to claim 18.

23. A compound according to any one of claims 1-17, or a composition according to claim 18, when used in the treatment and/or prophylaxis of proliferation of smooth muscle in a mammal requiring said treatment and/or prophylaxis.

24. Use of a compound according to any one of claims 1-17, in the preparation of a medicament for the treatment and/or prophylaxis of proliferation of smooth muscle in a mammal requiring said treatment and/or prophylaxis.

25. A method for the suppression of appetite in a mammal requiring said suppression, which method comprises administering to said mammal an effective amount of at least one compound accordirng to any one of claims 1 to 17, or a composition according to claim 18.

26. The compound according to any one of claims 1-17, or a composition according to claim 18, when used in the suppression of appetite in a mammal requiring said suppression. i (r I n:\hba1i101470:MCC b 18

27. Use of a compound according to any one of claims 1-17, in the preparation in a medicament for the suppression of appetite in a mammal requiring said suppression.

28. A method for the stimulation of pancreatic secretion in a mammal requiring said stimulation, which method comprises administering to said mammal an effective amount of at least one compound according to any one of claims 1 to 17, or of a composition according to claim 18.

29. The compound according to any one of claims 1-17, or a composition according to claim 18, when used in the stimulation of pancreatic secretion in a mammal requiring said stimulation.

30. Use of compound according to any one of claims 1-17, in the preparation of a medicament for the stimulation of pancreatic secretion in a mammal requiring said stimulation.

31. A method for the suppression of a craving for alcohol in a mammal requiring said suppression, which method comprises administering to said mammal an effective amount of at least one compound according to any one of claims 1 to 17, or of a composition according to claim 18.

32. The compound according to any one of claims 1-17, or a composition according to claim 18, when used in the suppression of a craving for alcohol in a mammal requiring said suppression. 20 33. Use of a compound according to any one of claims 1-17, in the preparation of a medicament for the suppression of a craving for alcohol in a mammal requiring said suppression. Dated 22 January, 1999 Biomesure, Inc. 25 Patent Attorneys for the Applicant/Nominated Person "SPRUSON FERGUSON [Ii:\libaa101470:MCC v 1 I-- ABSTRACT OF THE DISCLOSURE The invention features linear therapeutic peptides of the following formula: R 1 A 1 -A 2 -A 3 -A 4 -A 5 -A 6 -A 7 -A 8 -A 9 -R 3 in which Al is a D-a-aromatic amino acid or a D-a- tethered amino acid; A 2 is Gln, His, 1-methyl-His, or 3- me'thyl-His; A 3 is the D- or L-isomer selected from Nal, Trp, Phe, and p-X-Phe, where X is F, Cl, Br, NO 2 OH or CH 3 A 4 is Ala, Val, Leu, Ile, Nle, or a-aminobutyric acid; A 5 is Val, Ala, Leu, Ile, Nle, Thr, or a- aiinobutyric acid; A 6 is P-Ala; A 7 is His, l-mtethyl-His, 3-methyl-His, Lys, or c-alkyl-Lys; A8 is Leu, Ile, Val, Nle, a-aminobutyric acid, Trp, Pro,. Nal, Chx-Ala, Phe, or p-X-Phe, where X is F, Cl, Br, NO 2 OH or CH 3 A 9 is Met, Met-oxide, Leu, Ile, Nle, a-aminobutyric acid, or Cys; each R, and R 2 independently, is H, Cl-. 12 alkyl, C 7 1 C. phenylalkyl, or COEJ, where El is c 1 20 alkyl, C3-. 20 alkenyl, C 3 20 alkynyl, phenyl, 3,4-dihydroxyphenylalkyl, naphthyl, or C 7 10 phenylalkyl; provided that when either i or R 2 is COE,, the other must be H; and R 3 is OH, NH, C 1 12 alkoxy, C 7 10 phenylalkoxy, Cll-. 20 naphthylalkoxy, C.. 12 alkylamino, C 7 10 phenylalkylamino, Cll.. 20 25 naphthylalkylamino; or a pharmaceutically acceptable salt &fee of such peptides. No Figure.