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DIM350 (Ver 2.1 Jan 2001) 2999001/0
1011716
CORRECTED EUROPEAN PATENT SPECIFICATION
B9
B1
20040303
W1
DE
Beschreibung
EN
Description
FR
Description
DE
Ansprüche
EN
Claims
FR
Revendications
EP
95943375.6
19951207
19970702
20020226
en
en
en
352392
19941208
US
20040303
200410
20000628
200026
20030723
200330
20040303
200410
7
7A 61K 38/31 A
7C 07K 7/00 B
7C 07K 14/655 B
de
ANTAGONISTEN DES NEUROMEDIN B REZEPTORS
en
NEUROMEDIN B RECEPTOR ANTAGONISTS
fr
ANTAGONISTES DES RECEPTEURS DE LA NEUROMEDINE B
EP-A- 0 215 171
EP-A- 0 298 732
EP-A- 0 389 180
WO-A-94/02163
US-A- 5 462 926
MOLECULAR PHARMACOLOGY, Volume 44, issued 1993, ORBUCH et al., "Discovery of a Novel Class of Neuromedin B Receptor Antagonists, Substituted Somatostatin Analogues", pages 841-850.
SMITH et al., "Principles of Biochemistry: General Aspects", Seventh Edition, Published 1983, by MAcGRAW-HILL (N.Y.), pages 32-33 and 617.
19991001
COY, David, H.
1529 Fourth Street
New Orleans, LA 70130
US
TAYLOR, John, E.
74 Fiske Mill Road
Upton, MA 01568
US
THE ADMINISTRATORS OF THE TULANE EDUCATIONAL FUND
00762742
P70410EP
ADMINISTRATORS OF THE TULANE EDUCATIONAL FUND, THE
TULANE EDUCATIONAL FUND, THE ADMINISTRATORS OF THE
1430 Tulane Avenue
New Orleans, LA 70118-5679
US
SOCIETE DE CONSEILS DE RECHERCHES ET D'APPLICATIONS SCIENTIFIQUES S.A.S.
00687533
P70410EP
scras
CONSEILS DE RECHERCHES ET D'APPLICATIONS SCIENTIFIQUES S.A.S., SOCIETE DE
51-53 rue du Docteur Blanche
75016 Paris
FR
Lunt, Mark George Francis
et al
00050466
Harrison Goddard Foote, Fountain Precinct, Leopold Street
Sheffield S1 2QD
GB
AT
BE
CH
DE
DK
ES
FR
GB
GR
IE
IT
LI
LU
MC
NL
PT
SE
LT
19970715
SI
19970715
US1995015808
19951207
en
WO1996017617
19960613
199627

BACKGROUND OF THE INVENTION
The mammalian bombesin (Bn)-related peptides, gastrin-releasing peptide (GRP.), neuromedin B (NMB), and neuromedin C (NMC) have a wide range of biological effects. These include chemotaxis, contraction of smooth muscle stimulation, and the release of numerous gastrointestinal hormones. GRP and NMB are also active in the central nervous system, affecting thermoregulation, behavioral effects, satiety, maintenance of circadian rhythm, and inhibition of TSH release. Bn-related peptides function as a growth factor in numerous normal cells (e.g., stomal, epithelial, and neuroendocrine cells) as well as neoplastic cells such as human small cell lung cancer cells, non-small cell lung cancer cells, rat hepatocellular tumor cells, prostatic cells and breast adenocarcinoma cells.
Recent structure and cloning studies demonstrate that Bn-related peptides mediate the actions of two distinct receptor classes. GRP has a high affinity, and NMB has a low affinity, for the GRP-preferring class or subtype (GRP receptor or GRP-R). In contrast, GRP has a low affinity, and NMB has a high affinity, for the other class, the NMB-preferring subtype (NMB receptor or NMB-R). Both receptor classes are present throughout the central nervous system and the gastrointestinal tract.
Native somatostatin, somatostatin-14 (SS-14), has been shown to inhibit the cross-linking of ^{125}I-GRP to a 120 kD protein in Triton® extracts of 3T3 cells and human small cell lung cancer cells which are known to possess bombesin receptors. Recently, somatostatin octapeptide analogs have also demonstrated binding affinity to NMB-R in Orbuch, et al., Mol. Pharmacol., 44:841 (1993). These analogs, however, also maintain a substantial activity for somatostatin receptors.
WO94/02163 describes a method of selectivity inhibiting biochemical activity of cells induced by neuromedin B. The method includes the step of contacting cells which contain neuromedin B receptor with a cyclic octapeptide, having lysinc or ornithine at position 5.
EP0389180 describes octapeptides exhibiting GH-releasing-inhibiting activity. The octapeptides have lysine at position 5.
EP0215171 describes octapeptides active in inhibiting the secretion of GH, insulin and glucagon. The octapeptides have lysine at position 5.
EP0298732 describes a peptide comprising the sequence - Cys-X-D-Trp-Lys-Val-Cys wherein X represents any amino acid residue.

SUMMARY OF THE INVENTION

Abbreviations
- Nal = 3- (2-naphtyhl)-alanine or 3- (1-naphthyl) -alanine
- Bpa = 3-(4-biphenyl)-alanine
- X-Phe = phenylalanine with a p-, o- or m-substituent, such as -OH, CH_3, NO_2, and halogen, on the phenyl ring, e.g., 3-(4-chloropheny,1)-alanine
- F_5Phe = 3-(pentafluorophenyl)-alanine
- Nle=norleucine
- Me-Trp = Trp with a methyl-substituted indolyl nitrogen
Dab = 2,4-diamino butyric acid
Aub = 2-amino butyric acid
The present invention relates to cyclic octapeptides which have both high affinity and high selectivity for the NMB receptor and are encompassed by the following formula (I): A^1 is the D-isomer of Nal or Trp, and is preferably D-Nal. A^3 is F_5-Phe or ortho-, para-, or meta-substituted X-Phe wherein X is halogen, NO_2, CH_3 or OH. A^3 is preferably Phe or para-substituted X-Phe, where X is C1, F, or OH. A^5 is -NH-CH(Y)-CO- wherein Y is (CH_2)_m-R_4-N(R_5) (R_6) or (CH_2)_n-R_4-NH-C(R_7)-N(R_5)(R_6). In one aspect, A^5 is -NH-CH(Y)-CO- where Y is (CH_2)_m-R_4-N(R_5) (R_6), and preferably Dab, 7-amino-phenylalanine, and 2,3-diamino propionic acid, provided that A^5 is not Orn. In another aspect, A^5 is -NH-CH(Y)-CO- where Y is (CH_2)_n-R_4-NH-C-(R_7)-N(R_5) (R_6), and is preferably Arg or 7-guanidinyl-phenylalanine. A^6 is the D- or L- isomer of Thr, Leu, Ile, Nle, , Val, Nal, Trp, Me-Trp, Abu, Bpa, Phe, F_5-Phe, or X-Phe wherein X is a halogen, NO_2, CH_3, or OH. A^6 is preferably the D- or L-isomer of Thr, Leu, Ile, Nle, Trp, Val, and Abu. A^8 is Nal or Trp, and is preferably Nal. Subscript m is 1, 2, or 3, and preferably 2 or 3; n is 1, 2, 3, 4 or 5, and preferably 2, 3, or 4. Each of R_1 and R_2, independently, is H, E, COE, or COOE. E is a hydrocarbon of between 1 and 25 carbon atoms; substituted or unsubstituted; saturated or unsaturated; straight chain or branched; cyclic, acyclic, or polycyclic. Examples of E include C_{1-12} alkyl, C_{2-12} alkenyl, C_{2-12} alkynyl, phenyl, naphthyl, C_{7-12} phenylalkyl or alkylphenyl, C_{8-12} phenylalkenyl or alkenylphenyl, C_{8-12} phenylalkynyl or alkynylphenyl, C_{11-20} naphthylalkyl or alkylnaphthyl, C_{12-20} naphthylalkenyl or alkenylnaphthyl, or C_{12-20} naphthylalkynyl or alkynylnaphthyl, provided that when one of R_1 or R_2 is COE or COOE, the other must be H. R_3 is -NH_2 and R_5, R_6, R_8 are each independently H or E. Each of R_5, R_6 and R_8 is preferably H or a C_{1-10} hydrocarbon, such as alkyl, alkenyl, alkylphenyl, phenyl, and phenylalkyl, including C_{1-5} alkyl. Each of R_5 and R_6 is more preferably H. R_4 is C_6H_4 or absent, and preferably is absent. R_7 is =NR_8, =S, or =O, and preferably =NR_8, and more preferably R_7 is =NH. Preferred octapeptides encompassed by the above formula (I) of the invention include H_2-D-Nal-Cys-Tyr-D-Trp-Arg-Val-Cys-Nal-NH_2 (peptide Arg^5); and H_2-D-Nal-Cys-Tyr-D-Trp-Dab-Val-Cys-Nal-NH_2 (peptide Dab^5).
In formula (I), the N-terminus is at the left and the C-terminus at the right- in accordance with the conventional representation of a polypeptide chain. The symbol A^1, A^2, or the like in a peptide sequence stands for an amino acid residue, i.e. =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 of that amino acid residue. Thus, R is -CH(CH_3)_2 for Val. Also, when the amino acid residue is optically active, it is the L-form configuration that is intended unless D-form is expressly designated. Note that the two Cys residues (i.e., A^2 and A^7) in formula (I) are linked together via a disulfide bond. However, for convenience a line which is used conventionally to denote a disulfide bond between two Cys residues is omitted herein. COE refers to -(C=O)-E and COOE refers to -(C=O)-O-E.
Administration of a pharmaceutically acceptable salt of an octapeptide covered by formula (I) into a patient whose disorder arises from biochemical activity mediated by NMB is also within the present invention. In other words, the octapeptides can be provided in the form of pharmaceutically acceptable salts such as acid addition salts, or metal complexes such as with zinc or iron. Examples of acid addition salts are (i) those made with organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartric, methanesulfonic or toluenesulfonic acid; (ii) those made with polymeric acids such as tannic acid or carboxymethyl cellulose; and (iii) those made with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid.
Other features and advantages of the present invention will be apparent from the following description of the preferred embodiments, and also from the appending claims.

BRIEF DESCRIPTION OF THE DRAWINGS
We first briefly describe the drawings.
- Fig. 1 is a graph showing the suppression of NMB stimulated food intake by an analog of the invention.
- Fig. 2 is a graph showing suppression of NMC stimulated food intake by an analog of the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS
Octapeptides of the invention are synthesized on methylbenzhydrylamine resin using standard solid phase procedures and cleaved with hydrogen fluoride/anisol mixtures. The peptides are cyclized in dilute 90% acetic acid solution by titration with I_2 and purified by gel filtration on SEPHADEX™ G-25 (Aldrich, Milwaukee, WI) in 50% acetic acid and gradient elution on C18 silica using acetonitrile/0.1% trifluoroacetic acid buffers. See, e.g., Sasaki, Y. et al. J. Med. Chem., 30:1162 (1987); Stewart, J.M. et al. Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, IL (1984); and Coy, D.H. et al. Tetrahedron, 44:835 (1988). Homogeneity is assessed by thin layer chromatography ("TLC"), analytical HPLC, amino acid analysis and mass spectrometry.
Preferably, homogeneity should be determined to be >96% for each peptide. Example 1 below is a detailed description regarding the synthesis of peptide Dab^5. Other peptides of the invention can be prepared by making appropriate modifications, within the ability of someone of ordinary skill in the art, of the synthetic methods disclosed herein.
The NMB analogs of the invention are screened in binding assays to determine their respective affinities for the NMB, GRP, and somatostatin receptors. See Examples 2, 3, and 4, respectively. Agonists of the NMB receptor have been shown to stimulate the generation of inositol phosphates. Wang, et al, J. Biochem., 286:641-648 (1992). In Example 5 below, an inositol phosphate turnover assay measured the ability of the NMB analogs to antagonize the NMB receptor activation. In Example 6 below, an in vivo assay demonstrated the ability of the NMB analogs of the invention to block suppression of food intake produced by NMB.
The NMB analogs of this invention behave as potent antagonists of the NMB receptor. NMB has been shown to stimulate the growth of cancer cell lines (Moody, et al. J. Pharmacol., 263:1 (1992); Wang, et al., Biochem. J., 286:641 (1992)). As NMB antagonists the analogs of this invention can be used to treat cancers such as small cell lung tumors and glioblastomas. In addition, NMB has been shown to suppress food intake (Kirkman, et al., Society for the Study of Ingestive Behavior, Toronto, Canada). The analogs of the invention can be used to stimulate food intake to treat eating disorders such as anorexia or those resulting from cancer or AIDS. Furthermore, NMB has also been shown to decrease gastrin release, Kawai, et al., Endocrinol. Japan, 37(6):857 (1990). The analogs of the invention, thus, can be used to stimulate gastrin release in patients who are producing insufficient amounts of gastrin.
The analogs of the invention are also highly selective for the NMB receptor. The analogs of the invention will, therefore, have reduced cross-reactivity with both of these receptors. For example, other agonists of the somatostatin receptors may inhibit growth hormone release or disturb carbohydrate metabolism by the agonists' inhibition of insulin release.
The dose of the compound of the present invention for treating the above-mentioned diseases varies depending upon the manner of administration, the age and the body weight of the subject and the condition of the subject to be treated, and ultimately will be decided by the attending physician or veterinarian. Such amount of the active compound as determined by the attending physician or veterinarian is referred to herein as a "therapeutically effective amount".
The formulations are presented in unit dosage form and are prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient(s) into association with the carrier which constitutes one or more accessory ingredients. In general, the formulations for tablets or powders are prepared by uniformly and intimately blending the active ingredient with finely divided solid carriers, and then, if necessary as in the case of tablets, forming the product into the desired shape and size.
Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construted merely as illustrative, and not limitative of the remainder of the disclosure in any way. All publications cited herein are incorporated by reference.

Example 1
Synthesis of Boc-D-Nal-S-methylbenzyl-Cys-O-bromobenzyl-oxycarbonyl-Tyr-D-Trp-N-benzyloxycarbonyl-Dab-Val-S-methylbenzyl-Cys-Nal-benzhydrylamine resin was as follows. Benzhydrylamine-polystyrene resin (Advanced Chem Tech, Inc., Louisville, KY) (0.7 g, 0.3 mmole) in the chloride ion form was placed in the reaction vessel of an Advanced Chem Tech™ peptide synthesizer programmed to perform the following reaction cycle: (a) methylene chloride; (b) 33% trifluoroacetic acid in methylene chloride (2 time for 1 and 25 min each); (c) methylene chloride;
(d) ethanol; (e) methylene chloride; (f) 10% triethylamine in chloroform.
The neutralized resin was stirred with t-butyloxy-carbonyl("Boc")-Nal and diisopropylcarbodiimide (1.5 mmole each) in methylene chloride for 1 hr and the resulting amino acid resin was then cycled through steps (a) to (f) in the above wash program. The following amino acids (0.9 mmole) were then coupled successively by the same procedure: Boc-S-methylbenzyl-Cys, Val, Boc-N-benzyloxycarbonyl-Dab, Boc-D-Trp, Boc-O-bromobenzyloxycarbonyl-Tyr, and Boc-S-methyl-benzyl-Cys and Boc-D-Nal. After washing and drying, the completed resin weighed 1.13 g.
Using the completed resin, H-D-Nal-Cys-Tyr-D-Trp-Lys-Dab-Cys-Nal-NH_2 was prepared. The peptide resin obtained above (1.13 g, 0.5 mmole) was mixed with anisole (5 ml), dithiothreitol (100 mg) and anhydrous hydrogen fluoride (35 ml) at 0°C and stirred for 45 min. Excess hydrogen fluoride was evaporated rapidly under a stream of dry nitrogen. Free peptide was precipitated and washed with ether. The crude peptide was then dissolved in 250 ml of 90% acetic acid to which was added a concentrated solution of I_2/MeOH until a permanent brown color was observed. Excess I_2 was removed by addition of ascorbic acid and the solution was reduced to a small volume by evaporation. The crude peptide solution was applied to a column (2.5 x 90 cm) of SEPHADEX™ G-25 and eluted with 50% acetic acid. Fractions containing a major component by UV absorption and TLC were then pooled, reduced to a small volume by evaporation and applied to a column (1.5 x 70 cm) of VYDAC® octadecylsilane silica (10-15 µ) (Vydac, Hesperia, CA) followed by elution with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid in water. Fractions were examined by TLC and analytical high performance liquid chromatography and pooled to give maximum purity.
Repeated lyophilization of the solution from water gave 97 mg of the product as a white, fluffy powder. The product was found to be homogeneous by HPLC and TLC. Amino acid analysis of an acid hydrolysate and FAB MS confirmed the composition of the octapeptide.

Example 2

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 NMB receptor binding assay were obtained by homogenizing BALB-3T3 fibroblasts, transfected with the rat NMB receptor, with a POLYTRON™ tissue homogenizer (setting 6, 15 sec) (Brinkman, Westbury, NY) in ice-cold 50 mM Tris-HCl (Buffer A) (Sigma Chemicals, St. Louis, MO) and centrifuging twice at 39,000 x g (10 min), with an intermediate resuspension in fresh Buffer A. The final pellets were resuspended in the 50 mM Tris-HCl, containing 0.1 mg/ml bacitracin (Sigma Chemicals, St. Louis, MO), and 0.1% bovine serum albumin (BSA) (Buffer B) (Sigma Chemicals, St. Louis, MO), and held on ice for the receptor binding assay. Aliquots (0.4 ml) were incubated with 0.05 ml [^{125}I-Tyr^4]bombesin (-2200 Ci/mmole) (New England Nuclear, Boston, MA) in Buffer B, with and without 0.05 ml of unlabeled NMB analogs. After a 30 min incubation (4°C), 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 (Brandel, Gaithersberg, MD). 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]bombesin bound minus that bound in the presence of 1 µM unlabeled NMB. Analogs of the invention had a high binding affinity for the NMB receptor. Examples of the NMB receptor binding assay results for three analogs of the invention were (K_i values in nM) 47.4 ± 10.3 (peptide Arg^5), and 85.1 ± 2.7 (peptide Dab^5).

Example 3

GRP Receptor Binding Assay
Membranes for the GRP receptor binding assay were obtained by homogenizing cultured AR42J cells with a Polytron™ tissue homogenizer (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 intermediate resuspension in fresh Buffer A. The final pellets were resuspended in the 50 mM Tris-HCl containing 0.1 mg/ml bacitracin and 0.1% bovine serum albumin (BSA) (Buffer B) and held on ice for the GRP receptor binding assay. Aliquots (0.4 ml) were incubated with 0.05 ml of [^{125}I-Tyr^4]bombesin (-2200 Ci/mmole) in Buffer B, with and without 0.05 ml of unlabeled NMB analogs. After a 30 min incubation (4°C), 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 binding was defined as the tatal [^{125}I-Tyr^4]bombesin bound minus that bound in the presence of 1 µM unlabeled GRP. Analogs of the invention had a weak binding affinity for the GRP receptor. Examples of the GRP receptor binding assay results for analogs of the invention were (K_i values in nM) 2921 ± 250 (peptide Arg^5) and 2632 ± 216 (peptide Dab^5).

Example 4

Somatostatin Receptor Binding Assay
Membranes for the somatostatin receptor binding assay were obtained by homogenizing cultured AR42J acinar pancreas cells with a Polytron™ tissue homogenizer (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 intermediate resuspension in fresh Buffer A. The final pellets were resuspended in 10 mM Tris-HCl for the receptor binding assay. For determination of the K_i values, the various concentrations of NMB analogs were incubated for 90 min at 25°C with approximately 0.05 nM [^{125}I]MK-678 (University of Arizona, School of Medicine, Tucson, AZ) in 50 mM HEPES (pH 7.4) (Sigma Chemicals, St. Louis, MO) containing BSA (fraction V) (10 mg/ml) (Sigma Chemicals, St. Louis, MO), MgCl_2 (5 mM) (Sigma Chemicals, St. Louis, MO), aprotinin (200 KIU/ml) (Sigma Chemicals, St. Louis, MO) bacitracin (0.02 mg/ml), and phenylmethylsulphonyl fluoride (0.02 mg/ml) (Sigma Chemicals, St. Louis, MO). The final assay volume was 0.3 ml. The incubations were terminated by rapid filtration through GF/C filters (presoaked in 0.3% polyethylenimine) using a BRANDEL™ filtration manifold. Each tube and filter were then washed three times with 5 ml aliquots of ice-cold buffer. Specific binding was defined as the total [^{125}I]MK-678 bound minus that bound in the presence of 200 nM MK-678. A known cyclic octapeptide D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Nal-NH_2 (Lys^5) was disclosed in Orbuch et al., Mol. Pharmacol. 44:841 (1993), and had an extremely high affinity for the somatostatin receptor (K_i = 0.84 ± 0.53). In contrast, analogs of the invention had a much lower affinity, in a range of about one hundredth or one thousandth the K_i value of Lys^5, For example, K_i values (nM) for analogs of the invention were 407 ± 82 (peptide Dab^5) and, notably, 1032 ± 113 (peptide Arg^5).

Example 5

Inositol Phosphate Turnover Assay
For the measurement of inositol phosphate turnover, BALB-3T3 fibroblasts, transfected with the rat NMB receptor were harvested and resuspended in a phosphate-buffered saline solution containing 25 mM glucose (Sigma Chemicals, St. Louis, MO) and 75 mM sucrose (PBS+GS) and pre-incubated with 25 µCi/ml myo-[^3H]inositol (New England Nuclear, Boston, MA) for 60 min at 37°C. The cells were washed, resuspended in PBS+GS, and incubated with LiCl (100 mM) (Sigma Chemicals, St. Louis, MO) and the NMB analogs in a final volume of 0.30 ml. The reaction was terminated by the addition of chloroform/methanol (1:2) (Burdick & Johnson, Muskegeon, Michigan; Mallinckrodt, Paris, Kentucky), and the total [^3H] inositol phosphates were isolated as described in Snider et al., J. Neurochem., 47:1214 (1986). Peptide Dab^5 is a potent agonist of the NMB receptor, with a K_i (µM) of 78.1 ± 25.9 in the inositol phosphate turnover assay.
The following example describes a method for the determination of in vivo suppression of food intake using a cyclic octapeptide.

Example 6
Individually housed male Sprague-Dawley rats (Charles River, n=8) weighing 450-500 g. were maintained in a temperature-controlled room on a 12:12 hr. light: dark cycle. Rats were adapted to a 5 hr. food deprivation schedule followed by 60 min. access to a 0.5 kcal/ml glucose solution. Rats were injected intraperitoneally with either 0.9% saline (1.0 ml/kg) or 100 nmole/kg of peptide Orn^5. One minute later, rats were injected intraperitoneally with either saline, 32.0 nmole/kg NMB, or 3.2 nmole/kg NMC (GRP18-27), the biologically active portion of GRP. These agonist doses have previously been determined to reliably suppress intake in this experimental paradigm, Ladenheim, et al., Amer. Physiol. Soc. R263-R266 (1991). Five minutes after the second injection, the glucose solution was presented and intake was monitored at 15, 30, 45 and 60 min. Each rat received all four conditions for both NMB and NMC. Administration of either the agonists or antagonist was separated by at least 48 hr. Data were statistically analysed using a 4 (injection) x 4 (time) analysis of the variance followed by planned t-test comparisons for each agonist. Because intake following the baseline condition (Saline+Saline) for both sets of experiments was not significantly different (p>0.5) these were averaged and used to compare with effects of the agonists and peptide Orn^5.
The results showed that both NMB (Fig. 1) and NMC (Fig.2) significantly suppressed food intake compared to baseline intake at all time points (p<.01). Prior administration of 100 nmole/kg of peptide Orn^5 completely blocked the suppression of glucose intake produced by NMB. Intake for peptide Orn^5+NMB and peptide Orn^5+Saline conditions was greater than in the Saline+Saline condition (p<.01). In contrast to NMB, prior administration of peptide Orn^5 had no effect on suppression of intake produced by NMC, in that suppression of intake in the Saline+NMC condition was not significantly different from intake in the peptide Orn^5+NMC condition (p>0.5).

Other Embodiments
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.
1. A cyclic octapeptide of the formula: wherein:
A^1 is D-Nal or D-Trp;
A^3 is Phe, F_5 -phe, or X-Phe wherein X is a halogen, NO_2, CH_3, or OH;
A^5 is -NH-CH(Y)-CO- wherein Y is (CH_2)_m-R_4-N(R_5)(R_6) or (CH_2)_n-R_4-NH-C(R_7)-N(R_5)(R_6), provided that A^5 is not Orn;
A^6 is the D- or L- isomer of an amino acid selected from the group consisting of Thr, Leu, Ile, Nle, Val, Abu, Nal, Trp, Me-Trp, Bpa, F_5-Phe, Phe and X-Phe where X is a halogen, NO_2, CH_3, or OH;
A^8 is Nal or Trp;
m is 1, 2, or 3;
n is 1, 2, 3, 4 or 5;
each of R_1 and R_2, independently, is H, E, COE, or COOE wherein E is C_{1-12} alkyl, C_{2-12} alkeny, C_{2-12} alkynyl, phenyl, naphthyl, C_{7-12} phenylalkyl or alkylphenyl, C_{8-12} phenylalkenyl or alkenylphenyl, C_{8-12} phenylalkynyl or alkynylphenyl, C_{11-20} naphthylalkyl or alkylnaphthyl, C_{12-20} naphthylalkenyl or alkenylnaphthyl, or C_{12-20} naphthylalkynyl or alkynylnaphthyl, provided that when one of R_1 or R_2 is COE or COOE, the other must be H;
R_3 is -NH_2;
R_4 is C_6H_4 or absent;
R_7 is =NR_8, =S, or =O;
each of R_5, R_6, and R_8, independently, is H or E; and the two Cys residues are linked together via a disulfide bond
2. A cyclic octapeptide of claim 1, wherein Y is
a) (CH_2)_m-R_4-N(R_5) (R_6); or
b) (CH_2)_n-R_4-NH-C(R_7)-N(R_5) (R_6), wherein R, is absent, and, in the case of b), R_7 is = NR_8.
3. A cyclic octapeptide of claim 2, wherein m is 2 or 3, n is 2, 3 or 4, and each of R_5, R_6 and R_8, where present, independently is H or C_1-C_5 alkyl.
4. A cyclic octapeptide of claim 3, wherein A^6 is the D-or L- isomer of an amino acid selected from the group consisting of Thr, Leu, Ile, Nle, Trp, Val, and Abu.
5. A cyclic octapeptide of claim 4, wherein A^3 is Phe or para-substituted X-Phe where x is Cl, F, or OH.
6. A cyclic octapeptide of claim 5, wherein A^1 is D-Nal and A^8 is Nal.
7. A cyclic octapeptide of claim 6, wherein A^3 is Tyr and A^6 is Val.
8. A cyclic octapeptide of claim 3, wherein A^5, in the case of a), is Dab and, in the case of b), is Arg.
9. A cyclic octapeptide of claim 8, wherein A^6 is a D-or L- isomer of an amino acid selected from the group consisting of Thr, Leu, Ile, Nle, Trp, Val, and Abu.
10. A cyclic octapeptide of claim 8, wherein A^3 is Phe or para-substituted X-Phe where X is Cl, F, or OH; or
wherein A^1 is D-Nal and A^8 is Nal; or
wherein A^3 is Tyr and A^6 is Val.
11. A cyclic octapeptide of claim 10, having the formula:
H_2-D-Nal-Cys-Tyr-D-Trp-Dab-Val-Cys-Nal-NH_2;
or having the formula:
H_2-D-Nal-Cys-Tyr-D-Trp-Arg-Val-Cys-Nal-NH_2.
12. A cyclic octapeptide of claim 9, wherein A^1 is D-Nal and A^8 is Nal.
13. A cyclic octapeptide of claim 9, wherein A^3 is Tyr and A^6 is Val.
14. A cyclic octapeptide of claim 13, having the formula:
H_2-D-Nal-Cys-Tyr-D-Trp-Dab-Val-Cys-Nal-NH_2.
15. A cyclic octapeptide of claim 1, wherein Y is (CH_2)_n-R_4-NH-C(R_7)-N(R_5)(R_6).
16. A cylic octapeptide of claim 15, wherein R_4 is absent, and R_7 is =NR_8.
17. A cyclic octapeptide of claim 16, wherein n is 2, 3, or 4; and each of R_5, R_6, and R_8, independently, is H or C_1-C_5 alkyl.
18. A cyclic octapeptide of claim 17, wherein A^6 is a D-or L- isomer of an amino acid selected from the group consisting of Thr, Leu, Ile, Nle, Trp, Val and Abu.
19. A cyclic octapeptide of claim 18, wherein A^3 is Phe, or para-substituted X-Phe where X is Cl, F, or OH.
20. A cyclic octapeptide of claim 19, wherein A^1 is D-Nal and A^8 is Nal.
21. A cyclic octapeptide of claim 20, wherein A^3 is Tyr and A^6 is Val.
22. A cyclic octapeptide of claim 19, wherein A^5 is Arg.
23. A cyclic octapeptide of claim 22, wherein A^6 is a D-or L- isomer of an amino acid selected from the group consisting of Leu, Ile, Nle, Trp, Val, and Abu.
24. A cyclic octapeptide of claim 23, wherein A^3 is Phe, or para-substituted X-Phe where X is Cl, F, or OH.
25. A cylic octapeptide of claim 24, wherein A^1 is D-Nal and A^8 is Nal.
26. A cyclic octapeptide of claim 25, wherein A^3 is Tyr and A^6 is Val.
27. A cyclic octapeptide of claim 26, wherein said octapeptide is of the formula:
H_2-D-Nal-Cys-Tyr-D-Trp-Arg-Val-Cys-Nal-NH_2.
1. Cyclisches Octapeptid mit der Formel: in der
A^1 D-Nal oder D-Trp ist;
A^3 Phe, F_5-Phe oder X-Phe ist, wobei X Halogen, NO_2, CH_3 oder OH ist;
A^5 -NH-CH(Y)-CO- ist, wobei Y (CH_2)_m-R_4-N(R_5)(R_6) oder (CH_2)_n-R_4-NH-C(R_7)-N(R_5) (R_6) ist, mit der Maßgabe, dass A^5 nicht Orn ist;
A^6 das D- oder L-Isomer einer Aminosäure ausgewählt aus der Gruppe bestehend aus Thr, Leu, Ile, Nle, Val, Abu, Nal, Trp, Me-Trp, Bpa, F_5-Phe, Phe und X-Phe ist, wobei X Halogen, NO_2, CH_3 oder OH ist;
A^8 Nal oder Trp ist;
m 1, 2 oder 3 ist;
n 1, 2, 3, 4 oder 5 ist;
jedes von R_1 und R_2 unabhängig H, E, COE oder COOE ist, wobei E C_{1-12}-Alkyl, C_{2-12}-Alkenyl, C_{2-12}-Alkinyl, Phenyl, Naphthyl, C_{7-12}-Phenylalkyl oder -Alkylphenyl, C_{8-12}-Phenylalkenyl oder -Alkenylphenyl, C_{8-12}-Phenylalkinyl oder -Alkinylphenyl, C_{11-20}-Naphthylalkyl oder -Alkylnaphthyl, C_{12-20}-Naphthylalkenyl oder -Alkenylnaphthyl oder C_{12-20}-Naphthylalkinyl oder -Alkinylnaphthyl ist, mit der Maßgabe, dass, wenn eines von R_1 oder R_2 COE oder COOE ist, dea andere H sein muss;
R_3 -NH_2 ist;
R_4 C_6H_4 ist oder fehlt;
R_7 =NR_8, =S oder =O ist;
jedes von R_5, R_6 und R_8 unabhängig H oder E ist; und die beiden Cys-Reste über eine Disulfidbrücke miteinander verbanden sind.
2. Cyclisches Octapeptid nach Anspruch 1, bei dem Y
a) (CH_2)_m-R_4-N(R_5) (R_6) oder
b) (CH_2)_n-R_4-NH-C(R_7)-N-(R_5) (R_6) ist, wobei R_4 fehlt und im Fall von b) R_7 =NR_8 ist.
3. Cyclisches Octapeptid nach Anspruch 2, bei dem m 2 oder 3 ist, n 2, 3 oder 4 ist, und jedes von R_5, R_6 und R_8, wo vorhanden, unabhängig H oder C_1- bis C_5-Alkyl ist.
4. Cyclisches Octapeptid nach Anspruch 3, bei dem A^6 das D-oder L-Isomer einer Aminosäure ausgewählt aus der Gruppe bestehend aus Thr, Leu, Ile, Nle, Trp, Val und Abu ist.
5. Cyclisches Octapeptid nach Anspruch 4, bei dem A^3 Phe oder para-substituiertes X-Phe ist, wobei x Cl, F oder OH ist.
6. Cyclisches Octapeptid nach Anspruch 5, bei dem A^1 D-Nal ist und A^8 Nal ist.
7. Cyclisches Octapeptid nach Anspruch 6, bei dem A^3 Tyr ist und A^6 Val ist.
8. Cyclisches Octapeptid nach Anspruch 3, bei dem A^5 im Fall von a) Dab ist und im Fall von b) Arg ist.
9. Cyclisches Octapeptid nach Anspruch 8, bei dem A^6 ein D-oder L-Isomer einer Aminosäure ausgewählt aus der Gruppe bestehend aus Thr, Leu, Ile, Nle, Trp, Val und Abu ist.
10. Cyclisches Octapeptid nach Anspruch 8, bei dem A^3 Phe oder para-substituiertes X-Phe ist, wobei X Cl, F oder OH ist; oder
bei dem A^1 D-Nal ist und A^8 Nal ist; oder
bei dem A^3 Tyr ist und A^6 Val ist.
11. Cyclisches Octapeptid nach Anspruch 10 mit der Formel:
H_2-D-Nal-Cys-Tyr-D-Trp-Dab-Val-Cys-Nal-NH_2
oder mit der Formel
H_2-D-Nal-Cys-Tyr-D-Trp-Arg-Val-Cys-Nal-NH_2.
12. Cyclisches Octapeptid nach Anspruch 9, bei dem A^1 D-Nal ist und A^8 Nal ist.
13. Cyclisches Octapeptid nach Anspruch 9, bei dem A^3 Tyr ist und A^6 Val ist.
14. Cyclisches Octapeptid nach Anspruch 13 mit der Formel:
H_2-D-Nal-Cys-Tyr-D-Trp-Dab-Val-Cys-Nal-NH_2.
15. Cyclisches Octapeptid nach Anspruch 1, bei dem Y (CH_2)_n-R_4-NH-C(R_7)-N(R_5) (R_6) ist.
16. Cyclisches Octapeptid nach Anspruch 15, bei dem R_4 fehlt und R_7 =NR_8 ist.
17. Cyclisches Octapeptid nach Anspruch 16, bei dem n 2, 3 oder 4 ist, und jedes von R_5, R_6 und R_8 unabhängig H oder C_1bis C_5-Alkyl ist.
18. Cyclisches Octapeptid nach Anspruch 17, bei dem A^6 ein D-oder L-Isomer einer Aminosäure ausgewählt aus der Gruppe bestehend aus Thr, Leu, Ile, Nle, Trp, Val und Abu ist.
19. Cyclisches Octapeptid nach Anspruch 18, bei dem A^3 Phe oder para-substituiertes X-Phe ist, wobei X Cl, F oder OH ist.
20. Cyclisches Octapeptid nach Anspruch 19, bei dem A^1 D-Nal ist und A^8 Nal ist.
21. Cyclisches Octapeptid nach Anspruch 20, bei dem A^3 Tyr ist und A^6 Val ist.
22. Cyclisches Octapeptid nach Anspruch 19, bei dem A^5 Arg ist.
23. Cyclisches Octapeptid nach Anspruch 22, bei dem A^6 ein D-oder L-Isomer einer Aminosäure ausgewählt aus der Gruppe bestehend aus Leu, Ile, Nle, Trp, Val und Abu ist.
24. Cyclisches Octapeptid nach Anspruch 23, bei dem A^3 Phe oder para-substituiertes X-Phe ist, wobei X Cl, F oder OH ist.
25. Cyclisches Octapeptid nach Anspruch 24, bei dem A^1 D-Nal ist und A^8 Nal ist.
26. Cyclisches Octapeptid nach Anspruch 25, bei dem A^3 Tyr ist und A^6 Val ist.
27. Cyclisches Octapeptid nach Anspruch 26, bei dem das Octapeptid die Formel
H_2-D-Nal-Cys-Tyr-D-Trp-Arg-Val-Cys-Nal-NH_2
hat.
1. Octapeptide cyclique de la formule : dans laquelle :
- A^1 représente D-Nal ou D-Trp ;
- A^3 représente Phe, F_5-Phe ou X-Phe, où X représente un halogène, NO_2, CH_3 ou OH ;
- A^5 représente -NH-CH(Y)-CO-, où Y représente (CH_2)_m-R_4-N(R_5)(R_6) ou (CH_2)_n-R_4-NH-C(R_7)-N(R_5)(R_6), à la condition que A^5 ne représente pas Orn ;
- A^6 est l'isomère D ou L d'un acide aminé choisi dans le groupe constitué par Thr, Leu, Ile, Nle, Val, Abu, Nal, Trp, Me-Trp, Bpa, F_5-Phe, Phe et X-Phe, où X représente un halogène, NO_2, CH_3 ou OH ;
- A^8 représente Nal ou Trp ;
- m vaut 1, 2 ou 3 ;
- n vaut 1, 2, 3, 4 ou 5 ;
- R_1 et R_2 représentent chacun indépendamment H, E, COE ou CODE, où E représente alkyle en C_{1-12}, alcényle en C_{2-12}, alcynyle en C_{2-12}, phényle, naphtyle, phénylalkyle ou alkylphényle en C_{7-12}, phénylalcényle ou alcénylphényle en C_{8-12}, phénylalcynyle ou alcynylphényle en C_{8-12}, naphtylalkyle ou alkylnaphtyle en C_{11-20}, naphtylalcényle ou alcénylnaphtyle en C_{12-20}, ou naphtylalcynyle ou alcynylnaphtyle en C_{12-20}, à la condition que, lorsque l'un parmi R_1 ou R_2 représente COE ou CODE, l'autre doit être H ;
- R_3 représente -NH_2 ;
- R_4 représente C_6H_4 ou est absent ;
- R_7 représente =NR_8, =S ou =O ;
- R_5, R_6 et R_8 représentent chacun indépendamment H ou E ; et
- les deux résidus Cys sont liés ensemble par l'intermédiaire d'une liaison disulfure.
2. Octapeptide cyclique selon la revendication 1, dans lequel Y représente :
(a) (CH_2)_m-R_4-N(R_5) (R_6) ; ou
(b) (CH_2)_n-R_4-NH-C(R_7)-N(R_5) (R_6), où R_4 est absent et, dans le cas de (b), R_7 représente =NR_8.
3. Octapeptide cyclique selon la revendication 2, dans lequel m vaut 2 ou 3, n vaut 2, 3 ou 4, et R_5, R_6 et R_8, s'ils sont présents, représentent chacun indépendamment H ou alkyle en C_1-C_5.
4. Octapeptide cyclique selon la revendication 3, dans lequel A^6 est l'isomère D ou L d'un acide aminé choisi dans le groupe constitué par Thr, Leu, Ile, Nle, Trp, Val et Abu.
5. Octapeptide cyclique selon la revendication 4, dans lequel A^3 représente Phe ou X-Phe substitué en para, où X représente Cl, F ou OH.
6. Octapeptide cyclique selon la revendication 5, dans lequel A^1 représente D-Nal et A^8 représente Nal.
7. Octapeptide cyclique selon la revendication 6, dans lequel A^3 représente Tyr et A^6 représente Val.
8. Octapeptide cyclique selon la revendication 3, dans lequel A^5, dans le cas de (a), représente Dab et, dans le cas de (b), représente Arg.
9. Octapeptide cyclique selon la revendication 8, dans lequel A^6 est un isomère D ou L d'un acide aminé choisi dans le groupe constitué par Thr, Leu, Ile, Nle, Trp, Val et Abu.
10. Octapeptide cyclique selon la revendication 8, dans lequel A^3 représente Phe ou X-Phe substitué en para, où X représente Cl, F ou OH ; ou
dans lequel A^1 représente D-Nal et A^8 représente Nal ; ou
dans lequel A^3 représente Tur et A^6 représente Val.
11. Octapeptide cyclique selon la revendication 10, ayant la formule :
H_2-D-Nal-Cys-Tyr-D-Trp-Dab-Val-Cys-Nal-NH_2 ;
ou ayant la formule :
H_2-D-Nal-Cys-Tyr-D-Trp-Arg-Val-Cys-Nal-NH_2.
12. Octapeptide cyclique selon la revendication 9, dans lequel A^1 représente D-Nal et A^8 représente Nal.
13. Octapeptide cyclique selon la revendication 9, dans lequel A^3 représente Tyr et A^6 représente Val.
14. Octapeptide cyclique selon la revendication 13, ayant la formule :
H_2-D-Nal-Cys-Tyr-D-Trp-Dab-Val-Cys-Nal-NH_2.
15. Octapeptide cyclique selon la revendication 1, dans lequel Y représente (CH_2)_n-R_4-NH-C(R_7)-N(R_5) (R_6).
16. Octapeptide cyclique selon la revendication 15, dans lequel R_4 est absent, et R_7 représente =NR_8.
17. Octapeptide cyclique selon la revendication 16, dans lequel n vaut 2, 3 ou 4 ; et R_5, R_6 et R_8 représentent chacun indépendamment H ou alkyle en C_1-C_5.
18. Octapeptide cyclique selon la revendication 17, dans lequel A^6 représente un isomère D ou L d'un acide aminé choisi dans le groupe constitué par Thr, Leu, Ile, Nle, Trp, Val et Abu.
19. Octapeptide cyclique selon la revendication 18, dans lequel A^3 représente Phe ou X-Phe substitué en para, où X représente Cl, F ou OH.
20. Octapeptide cyclique selon la revendication 19, dans lequel A^1 représente D-Nal et A^8 représente Nal.
21. Octapeptide cyclique selon la revendication 20, dans lequel A^3 représente Tyr et A^6 représente Val.
22. Octapeptide cyclique selon la revendication 19, dans lequel A^5 représente Arg.
23. Octapeptide cyclique selon la revendication 22, dans lequel A^6 représente un isomère D ou L d'un acide aminé choisi dans le groupe constitué par Leu, Ile, Nle, Trp, Val et Abu.
24. Octapeptide cyclique selon la revendication 23, dans lequel A^3 représente Phe ou X-Phe substitué en para, où X représente Cl, F ou OH.
25. Octapeptide cyclique selon la revendication 24, dans lequel A^1 représente D-Nal et A^8 représente Nal.
26. Octapeptide cyclique selon la revendication 25, dans lequel A^3 représente Tyr et A^6 représente Val.
27. Octapeptide cyclique selon la revendication 26, dans lequel ledit octapeptide est représenté par la formule :
H_2-D-Nal-Cys-Tyr-D-Trp-Arg-Val-Cys-Nal-NH_2.