CA2144264A1 - Bombesin analogs - Google Patents

Abstract

The invention features linear therapeutic peptides of the following formula:

A1-A-A3-A4-A5-A6-A7-A8-A9-R3, in which A1 is a D-.alpha.-aromatic amino acid or a D-.alpha.-tethered amino acid; A is Gln, His, 1-methyl-His, or 3-methyl-His; A3 is the D- or L-isomer selected from Nal, Trp, Phe, and p-X-Phe, where X is F, Cl, Br, NO2, OH or CH3; A4 is Ala, Val, Leu, Ile, Nle, or .alpha.-aminobutyric acid; A5 is Val, Ala, Leu, Ile, Nle, Thr, or .alpha.-aminobutyric acid; A6 is .beta.-Ala; A7 is His, 1-methyl-His, 3-methyl-His, Lys, or .epsilon.-alkyl-Lys; A8 is Leu, Ile, Val, Nle, .alpha.-aminobutyric acid, Trp, Pro, Nal, Chx-Ala, Phe, or p-X-Phe, where X is F, Cl, Br, NO2, OH or CH3; A9 is Met, Met-oxide, Leu, Ile, Nle, .alpha.-aminobutyric acid, or Cys;
each R1 and R2, independently, is H, C1-12 alkyl, C7-10 phenylalkyl, or COE1, where E1 is C1-20 alkyl, C3-20 alkenyl, C3-20 alkynyl, phenyl, 3,4-dihydroxyphenylalkyl, naphthyl, or C7-l0 phenylalkyl; provided that when either R1 or R2 is COE1, the other must be H; and R3 is OH, NH2, C1-12 alkoxy, C7-10 phenylalkoxy, C11-20 naphthylalkoxy, C1-12 alkylamino, C7-10 phenylalkylamino, C11-20 naphthylalkylamino; or a pharmaceutically acceptable salt of such peptides.

Description

~ 2 G ~

Ei. I I ~.A I N ~N~r.naA
BA~ UNL~ OF TT~ INYENTION
This invention relates to bombesin analogs u5eful 5 for treatment of benign or r~ nqnt proliferation of tissue.
The ~ _~ihiqn peptide bombesin (see Anastasi, et al., Experientia ~7:166-167, 1971) is closely related to the r-r-q l i qn homolog gastrin-releasing peptide (GRP), 10 neuL, -~;n B (NMB), nc:UL -~in C (NMC), and litorin. See Min Im;no, 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 .
15 1993). 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. ~aveman, et al-, eds- B~Ç~
Re~--lts ;n cqnc er ~F~CearCh - Pe~tide H ~ in T~lnr 5~;, Springer-Verlag, New York, 1986. A number of these cancers are known to secrete peptide h -~.
related to GRP or borh~c;n. Consequently, antagonists to bombesin have been proposed as agents for the ~Laal -nt of these cancers.
Cuttitta, et al. demonstrated that a specific monoclonal antibody to bombesin inhibited .ia v Vo 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 30 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.

42G~
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, 5 1987. Rivier reported work directed toward restricting the conformational freedom of the bioactive C-t~~rn{nAl decapeptide of bombesin by incorporating intramolecular disulfide bridges; however, Rivier mentioned that, so far, bombesin analogs with this modification fail to 10 exhibit any antagonist activity. Rivier, et al., "Competitive Antagonists of Peptide Hormones, " in Abstracts of the International Symposium on B~ ci n-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, J., Anat. Record 236: 241-247 (1993). Bombesin exhibits both direct and indirect effects on the gastrointestinal tract, including the release of h' ^-. and the 20 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 25 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 . ~ed . 148: 459, 198 6 ) and 30 the growth of ~C-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 (B~RI-rh , et al., Ann. Surg. 202: 303, 1985) . Bombesin has been found 35 to act concurrently as both a trophic agent for normal ~' ~14~26 i host pancreas and a growth inhibitory agent in xenografted human pancreatic tumor tissue . Al f-Y~n~?t~r, et al., Pancreas 3:247, 1988. NMB has been shown to effect the growth of cancer cells (Moody, et al., J. Pharmacol.
5 261:1 (1992) ), ~u~r~ss ~ood intake, and decrease gastrin release (Kawai, et al., Endocrinol. Japan 37(6):857 (1990) ), SU~RY OF THB TNV~N~ION
AhhreviatiOnS:
10 Chx-Ala = cyclohexyl-Ala (3-cyclohexy~Aliqni pGlu = pyroglutamic acid Nle = norleucine D -Cpa = D-p -chlorophenyl A l i~ n i n HyPro = ILy~lL~>xyyLoline 15 Nal = 3- (~-naphthyl) -alanine, or 3- (~-naphthyl) -alanine DOPA = 3, 4 -dihyd ~ y~henyl ~ l An i ne Tcc = 1, 2, 3, 4-tetrahydro-2-carboline-3-carboxylic acid Tic = 1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid Aza-Tyrosine = 3- (5-hydroxy-2-pyridyl) -alanine 20 Sar = sarcosine 1- or 3-methyl-His = His with a methyl group on its position 1 or 3 heterocyclic nitrogen ~-alkyl-Lys = Lys with its N~ substituted by an alkyl group 25 ,~-Ala = 3-aminopropionic acid The invention features linear therapeutic peptides of the following formula:
R1 Al_A2 -A3 - A4-As-A6 - A7 - A8 - A9 - ( I ) in which:
Al is a D-~-aromatic amino acid or a D-~r-tethered amino acid;
A2 is Gln, His, 1-methyl-His, or 3-methyl-His;

21~264 A3 i8 the D-- or L-i60mer selected from Nal, Trp, Phe, and p-X-Phe, where X is F, Cl, Br, N02, OH or CH3;
A4 is Ala, Val, Leu, Ile, Nle, or ~-aminol,uLyLlc acid;
A5 i5 Val, Ala, Leu, Ile, Nle, Thr, or c~-aminobutyric acid;
A6 is ~-Ala;
A7 is His, 1-methyl-His, 3-methyl-8is, Lys, or 6-alkyl-Lys;
A8 is Leu, Ile, Val, Nle, ~r-aminobutyric acid, Trp, Pro, Nal, Chx-Ala, Phe, or p-X-Phe, where X is F, Cl, Br, N02, OH or CH3;
A9 is Met, Met-oxide, Leu, Ile, Nle, -aminobutyric acid, or Cys;
each Rl and R2, ;n~lep.on~ntly, is H, C1_12 alkyl, C7_10 phenylalkyl, or COE1, where E1 is C1-20 alkyl, C3_20 alkenyl, C3_20 alkynyl, phenyl, 3, 4-dihydroxyphenylalkyl, naphthyl, or C7_10 phenylalkyl; provided that when either R1 or R2 is COE1, the other must be H; and R3 is OH, NH2, C1_12 alkoxy, C7_10 phenylalkoxy, C11_20 naphthylalkoXy, C1_12 alkylamino~ C7_10 phenylalkylamino, C11_20 naphthylalkylamino; or a rh~nr~^Putically acceptable salt of such peptides.
What is meant by "aromatic ~-amino acid" is an 25 amino acid residue of the formula NH2-CH(CH2-Z)-COOH where Z is a moiety containing an aromatic ring. Examples of Z
include, but are not limited to, phenyl, 1-napthyl, 2-napthyl, 3-indolyl, 1-Me-3-indolyl, biphenyl, and imidazolyl, with or without one or more substituent X on 30 the aromatic ring(s) of Z (where X is halogen, N02, CH3, or OH).
What is meant by "tethered ~-amino acid" is an ~-amino acid with a carbon atom of its side chain tethered to the N atom of the ~-amino group. Examples 35 include, but are not limited to, Pro, HyPro, Tic, and Tcc .

2~2~
The 23ymbol Al, A2, or the like herein stdnds for the residue of an -amino acid. Except for tethered a~ino acids (e.g., Pro, HyPro, Tcc, or Tic) and Sar, such symbols represent the general ::~LLU-;~ULa~ -NH-CH(R)-CO- or 5 N-CH(R) -CO- when it is at the N-terminus or -NH-CH(R) -CO- when it is not at the N-~rm;nllC, where R denotes the side chain (or identifying group) of the ~-amino acid, e.g., R is -CH2COOH for Asp. Note that the N-t~;m~a is at the left and the C-t~rm; nllC at the right in accordance 10 with the conventional representation of a polypeptide chain. When A6 is Sar, it has the structure of -N(CH3)-CH2-CO-. The residue of a tethered amino acid, on the other hand, is of the structure -N-CH(R)-CO-, where N, C
and R together form a ring. HyPro herein refers to any 15 oP 2-hydroxy-Pro, 3-hydroxy-Pro, 4-hydroxy-Pro, and 5-hydroxy-Pro; 4 -hydroxy-Pro is pref erred .
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 atoms. COEl stands for C-El.
In formula (I) given above, when either of Rl or R2 is an aliphatic, aromatic, or lipophilic group, the in 25 ViVQ activity can be long lasting, and delivery of the compounds of the invention to the target tissue can be facilitated .
Preferably, in formula (I), A1 is the D-isomer selected from Nal, DOPA, Trp, Tcc, Tic, Aza-Tyr, Phe, and 30 p-X-Phe, where X is F, Cl, Br, N02, OH or CH3. It is particularly preferred that A3 be the D-isomer selected f rom Phe, Trp, and p-X-Phe, where X is F, Cl, Br, NO2, OH
or CH3; and A7 be His, l-methyl-His, or 3-methyl-His.
Also preferably, in formula (I), A1 be D-Phe, D-35 Trp, or D-Tyr; A2 be Gln; A3 be Trp; A4 be Ala; A5 be Val;
A7 be His; A8 be Leu or Phe; and A9 be Met, Leu, and Nle.
Particularly preferred peptides of the invention include the f ollowing:

-26~

}I-D-Phe-Gln-Trp -Ala -Val -~-Ala-E~is -Phç-Nle-NH2;
H-D-Phe-Gln-Trp-Ala-Val -~-Ala-His-Leu-Leu-NH2;
H-D-Tyr-Gln-Trp-Ala -Val -~l-Ala-His-Phe-Nle-NH2;
H-D-Trp-Gln-Trp-Ala-Val-~-Ala-His-Phe-Nle-NH2; and H-D-Phe-Gln-Trp-Ala-Val-~-Ala-His-Leu-Nle-NH2.
Analogs of the invention can be provided in the form of rh~ tically acceptable salts. Examples of preferred salts are those with therapeutically acceptable organic acids, e.g., acetic, lactic, maleic, citric, lO malic, ascorbic, succinic, benzoic, salicylic, methanesulfonic, toluene sulfonic, trifluoroacetic, or pamoic acid, as well as polymeric acids such as tannic acid or caLI,u,Ly thyl cPlllllose, and salts with inorganic acids such as the hydrohalic acids, e.g., 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.
RRT~F DES~RTPTION OF THE nRAwTNG~
The drawings will be first described.
Fig. l is a graph showing the growth inhibitory effect of a c~ ln-l of the present invention on prostate tumors .
DESGRTPTION OF T~ ) EMP~t~DTMFNTS
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 30 litorin, neuromedin B (NMB), n~UL- -~l;n C (NMC), b~h~cin (last ten amino acids), and human GRP (last ten amino acids ) .
SYnthçsis of Analoqs The synthesis of one o~ the, _-lnrlc~ Of the lnvention, Analog ~1 (i.e., H-D-Phe-Gln-Trp-Ala-Val-~l-Ala -His -Phe-N l e -NH2 ), f o l l ows .
4-methyl benzhydrylamine-polya-yLG.-e resin 5 (Bachem, Inc. ) t0 . 72 meq/g), in the chloride ion ~orm, was placed in the reaction vessel of an ACT200 peptide synthesizer (Advanced Chem Tech, Inc.) ~LUyL -' to perform the following reaction cycle: (a) methylene chloride; (b) 10% triethylamine in chloroform; (c) 10 methylene chloride; and (d) dimethylformide. The neutralized resin was mixed with BOC-Norleucine and diisopropylcarbodiimide (3 molar eq each) in methylene chloride for l hour. The resulting amino acid resin was washed on the synthf~; 7~-r 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 ~L~IyL --' to perform the following reaction cycle: (a) methylene chloride; (b) 20 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 25 coupled successively by the same procedure: BOC-Phe, BOC -H is ( CB Z ), BOC -~ -A l a , BOC -Va l , BOC -Ala , BOC-Trp , BOC-Gln (coupled in the presence of 1 eq.
hy-lLoxybenzotriazole), and BOC-D-Phe (coupled in the presence of 1 eq. hydroxybenzotriazole). The completed 30 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 0c for one hour.
Excess hydrogen f luoride was evaporated rapidly under a 35 stream of dry nitrogen, and the residue was washed in ether. Crude peptide was dissolved in 100 ml of 4 N
acetic acid and the solution is then evaporated under ~ 21~Z64 reduced pressure. The crude peptide was dissolved in minimum volume of methanol/water and tr$turated with ethyl acetate. The triturated peptide was applied to a column (9.4 mm I.D. x 50 cm) of octadecylcilane-silica 5 (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 ~-YAn'li n-~ by analytical high performance liguid chromatography and appropriate f ractions were evaporated 10 to a small volume, which was further lyophilized, giving 80 mg of the product as a colorless powder.
other compounds of the present invention, e.g., Analog ~2 ( i . e., H-D-Phe-Gln-Trp-Ala-Val-~-Ala-His-Leu-Leu-NH2), can be prepared in an analogous manner by 15 making appropriate modif ications of the above-described synthetic method.
Bioloqical Assavs (1) GRP Receptor Binding Assay Rat AR42J pancreatic acinar cells were cultured in 20 Dulbecco's modified Eagle's medium without antibiotics and supplemented with 10% (vol/vol) fetal calf serum.
The incubation ~ re consisted of 10% CO2-90%
humidif ied air at 37C.
Membranes for the bombesin receptor binding assay 25 were obtained by homogenizing AR42J cells (Polytron, setting 6, 15 sec) (Brinkman, Westbury, NY) in ice-cold 50 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
30 Tris-HCl, containing 0.1 mg/ml bacitracin, and 0.1% BSA
(8uffer B), and held on ice for the receptor binding assay .
For assay, aliquots (0 . 4 ml) were incubated with 0.05 ml [125 I-Tyr4] bombesin (-2200 Ci/mmol, New England 35 Nuclear) and Buffer B, with and without 0 . 05 ml of unlabeled c _ ; ng analogs. After a 30 min incubation ~ 21~26~
(4c), the bound [125 I-Tyr4] bombesin was sep~r~ted ~rom the ~ree by rapid riltration through Whatmana' GF/B
f ilters which had been previously soaked in 0 .1%
polyethyl~n~im;ne using a Brandel filtration manifold.
5 The f ilters were then washed three times with 5 ml aliquots of ice-cold Buffer A. Specific binding was defined as the total [125 I-Tyr4] bombesin bound minus that bound in the presence of 1 ,uM unlabeled bombesin.
The results (expressed as IC50 in nM) and the 10 structures of the tested _ _.lds are shown in Table 1.
Replacement of Gly at position A6 in a prior art ~ _ ' (i.e., litorin, Leu-litorin, and [D-Phel, Leu8l9] litorin) with l?-Ala led unexpectedly to a great increase in their affinity for the GRP receptor. For example, rep~
15 of Gly with ~-Ala resulted in a 2 . 8 to 23 time increase in the affinity (compare Analog ~2 with [D-Phel, Leu8'9]
litorin or Leu-litorin). Modifications at position Al, A8, or A9 further increases the affinity. For example, Analog ~3 has an IC50 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-Phel, Leu8~9] litorin-- an increase of 80 to 766 times in the affinity for the GRP receptor.

21~264 AFFINITY BINDING DATA FOR THE CRP REOEPTOR OF LINEAR
BOMBESIN ANALOGS WITH ,13-Ala REPLACEMENT AT POSITION A6 Codo Namc Structure IC~o ~nM) litorill p-Glu-Gln--Trp-Ala-Val-Gly-E;is-Phe--Met-NEz 2.4 ~ Litonn p-Glu-Gln--Trp-Ala-Val-Gly-Els-Leu-Leu-NE2 23 lO[D-Phel, Leu8~9] litorin E-D-Phe--Gln--Trp--Ala-Val-Gly--His--Leu--Leu--NE2 2 . 8 Analog ~1 E-D--Phe-Gln--Trp--Ala-Val-,B--Ala--Eis--Phe--Nle--NE2 0.2 Analog t2 ~-D--Phe--Gln--Trp--Ala--Val--,3--Ala--Ei3--Leu--Leu--NE2 1.0 Analog ~3 E-D-Tyr-Gln-Trp-Ala-Val-~-Ala-Eis-Phe-Nle-NE2 0 . 03 15Analoq ~4 E--D--Trp--Gln--Trp--Ala--Val--,B-Ala--Eis-Phe--Nle--NE2 O. l An~loq ~5 E--D--Phe-Gln-Trp--Ala-Val--,3-Ala--Eis--Leu--Nle--NE2 2 .1 See formula (I) in "SUMMARY OF THE lNVI!. ~lUN'- abo~e.
20 (2) NMB Receptor Binding Assay The uroceduL-3 for transfecting the rat NMB
receptor into BALB-3T3 fibroblasts is fli~clles~cl in Wada, et al., Neuron, :4221-430 (1991) and Benya, et al., l~ol.
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-HCl (Buffer A) and centrifuging twice at 39, 000 x g (10 min), with an 30 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 B), and held on ice for the receptor binding assay.
For assay, ali~uots (0.4 ml) were incubated with 35 0.05 ml tl25 I-Tyr4] bombesin (-2200 Ci/mmol, New England Nuclear) and Buffer B, with and without 0. 05 ml of ~ 21~264 lnl AhOl Pd competing analogs . After a 30 min incubation (4C), the bound [125 I-Tyr4~ bombesin was separated from the free by rapid filtration through Whatman" GF/B
filters which had been previously soaked in 0. 3%
5 polyethylPnoi~ine using a Brandel filtration maniold.
The f ilters were then washed three times with 5 ml aliquots of ice-cold Buffer A. Specific binding was defined as the total tl25 I-Tyr4] n~uL, -';n-B bound minus that bound in the presence of l ~lM unlabeled neu,~ -'in-10 B.
The results (expressed as IC50 in nM) and thestructures of the tested ~ are shown in Table 1.
Replacement of Gly at position A6 in a prior art c (i.e., litorin, leu-litorin, and tD-Phe1, Leu8~9] litorin) 15 with ,B-Ala led unexpectedly to a great increase in their affinity for the NMB receptor. For example, replAc t of Gly with ,~-Ala resulted in a 11. 7 time increase in the affinity (compare Analog #2 with [D-Phe1, Leu8~9]
litorin). Modifications at position A1, A8, or A9 further 20 increases the affinity. For example, Analog ~74 has an IC50 being as low as 0. 04 nM, compared to 1. 66 nM or litorin or 480 nM for [D-Phe1, Leu8~9] litorin-- an increase of 41.5 to 12,000 times in the affinity for the NMB receptor. Leu-litorin was found to have almost no 25 binding affinity on the NMB receptor.

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r~ ~D r~ r n i ~ ~ z r 1~
r a~ ~n r ~
r f 1~ f 1~ ~r 5~
4 ~ n ~ ~ c ~ ~ a ~ rl ~ ~ t V ~!1 rv r rV ~ J
~ n V ~ Q Q a ~ a v n ra ~ V r~ r.~l r~
z; c ~ ~ 11 x a 'S- c~n c~ ~ ctn r~ r-l r~ r-l r-l 2~
- 12a -( 3 ) Assay on in vivo prostate tumor growth The androgen responsive R-3327/H prostate tumor line was implanted into 38 testes-intact, syngeneic 5 Copenhagen male rats. The tumored animalc were individually staged f or castration when their tumors reached a size approximating 235 mgs. Tumors were then staged f or ~ in analogue treatment when two sequential tumor mea~ul~ Ls in-iirAted an "escape from 10 castration inhibition" condition, as evidenced by an increase in tumor growth rate. This transition of the prostate tumor tissue from androgen ~ensitive to androgen insensitive oc~ uLL_d without surgical peLLuLLation of the tumor, thus providing a ~.l;n;c:~lly realistic model for 15 evaluating the anti-prostate tumor activity of the b ' ~- 8; n analogue~ .

~ 214~2~4 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%
5 glycerol/water vehicle, (s.c., B.I.D.). Group 2 received a 200 mg/inj. of Analog ~5 (s.c., B.I.D.). 80th injections were administered in the flank opposite from the tumor. The treatment period lasted for 24 days.
Tumor mea~ur~ ls were taken at day 3, day 7, day 10, 10 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 (measurinq 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.
Use Analogs of the invention are useful for treating colon, prostatic, breast, pancreatic, liver cancer or lung cancer, for preventing the proliferation of smooth 30 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 (e.g., orally, parenterally, tr~ncd~ ly, 35 transmucosally, or via drug-releasing implants), in a sustained release formulation using a biodegradable biocompatible polymer, or by on-site delivery using 214~264 micelles, gels and ~ , or rectally (e.g., by suppository or enema). The analogs can be administered to a human patient in a dosage to be det~rm;nGd by the attending physician ranging from O . 25 mg/kg/day to 5 5 mg/kg/day .
Furthermore, ~ u~.~s of the present invention, particularly those with Tyr at the N-t~-rm;mlc~ can be used for diagnostic purposes and for the tumor targeting of radioisotope~i such as 131Iodine.
OTHER ~MR~ TM~N~S
~ he foregoing description has been limited to specific ~mho~ nts of this invention. It will be apparent, however, that variations and modifications may be made to the invention, with the att~; l of some or 15 all of the advantages of the invention. Such ~mh~ I.S
are also within the scope of the following claims.

Claims (16)

1. A therapeutic peptide of the formula:

wherein:
A1 is a D-.alpha.-aromatic amino acid or a D-.alpha.-tethered amino acid;
A2 is Gln, His, 1-methyl-His, or 3-methyl-His;
A3 is the D- or L-isomer selected from Nal, Trp, Phe, and p-X-Phe, where X is F, Cl, Br, NO2, OH or CH3;
A4 is Ala, Val, Leu, Ile, Nle, or .alpha.-aminobutyric acid;
A5 is Val, Ala, Leu, Ile, Nle, Thr, or .alpha.-aminobutyric acid;
A6 is .beta.-Ala;
A7 is His, 1-methyl-His, 3-methyl-His, Lys, or .epsilon.-alkyl-Lys;
A8 is Leu, Ile, Val, Nle, .alpha.-aminobutyric acid, Trp, Pro, HyPro, Nal, Chx-Ala, Phe, or p-X-Phe, where X
is F, Cl, Br, NO2, OH or CH3;
A9 is Met, Met-oxide, Leu, Ile, Nle, .alpha.-aminobutyric acid, or Cys;
each R1 and R2, independently, is H, C1-12 alkyl, C7-10 phenylalkyl, or COE1, where E1 is C1-20 alkyl, C3-20 alkenyl, C3-20 alkynyl, phenyl, 3,4-dihydroxyphenylalkyl, naphthyl, or C7-l0 phenylalkyl; provided that when either R1 or R2 is COE1, the other must be H; and R3 is OH, NH2, C1-12 alkoxy, C7-10 phenylalkoxy, C11-20 naphthylalkoXy, C1-12 alkylamino, C7-10 phenylalkylamino, C11-20 naphthylalkylamino; or a pharmaceutically acceptable salt thereof.

2. The therapeutic peptide of claim 1, wherein A1 is the D-isomer selected from Nal, DOPA, Trp, Tcc, Tic, Aza-Tyr, Phe, and p-X-Phe, where X is F, Cl, Br, NO2, OH
or CH3.

3. The therapeutic peptide of claim 2, wherein A3 is the D-isomer selected from Trp, Phe, and p-X-Phe, where X is F, Cl, Br, NO2, OH or CH3; and A7 is His, 1-methyl-His, or 3-methyl-His.

4. The therapeutic peptide of claim 3, wherein A3 is Trp.

5. The therapeutic peptide of claim 4, wherein A7 is His.

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

7. The therapeutic peptide of claim 6, wherein A4 is Ala.

8. The therapeutic peptide of claim 7, wherein A5 is Val.

9. The therapeutic peptide of claim 8, wherein A8 is Leu or Phe.

10. The therapeutic peptide of claim 9, wherein A9 is Met, Leu, or Nle.

11. The therapeutic peptide of claim 10, wherein A1 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-.beta.-Ala-His-Phe-Nle-NH2.

13. The therapeutic peptide of claim 11 or the formula:
H-D-Phe-Gln-Trp-Ala-Val-.beta.-Ala-His-Leu-Leu-NH2.

14. The therapeutic peptide of claim 11 of the formula:
H-D-Tyr-Gln-Trp-Ala-Val-.beta.-Ala-His-Phe-Nle-NH2.

15. The therapeutic peptide oî claim 11 of the formula:
H-D-Trp-Gln-Trp-Ala-Val-.beta.-Ala-His-Phe-Nle-NH2.

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