Cyclic octapeptide neuromedin B receptor antagonists

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 .sup.125 I-GRP to a 120 kD protein in Triton.RTM. 
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. 
SUMMARY OF THE INVENTION 
Abbreviations 
Nal =3-(2-naphthyl)-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.sub.3, NO.sub.2, and halogen, on the phenyl ring, e.g., 
3-(4-chloropheny, l)-alanine 
F.sub.5 Phe =3-(pentafluorophenyl)-alanine 
Nle =norleucine 
Me-Trp =Trp with a methyl-substituted indolyl nitrogen 
Dab =2,4-diamino butyric acid 
Abu =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): 
##STR2## 
A.sup.1 is the D-isomer of Nal or Trp, and is preferably D-Nal. A.sup.3 is 
F.sub.5 -Phe or ortho-, para-, or meta-substituted X-Phe wherein X is 
halogen, NO.sub.2, CH.sub.3 or OH. A.sup.3 is preferably Phe or 
para-substituted X-Phe, where X is Cl, F, or OH. A.sup.5 is 
--NH--CH(Y)--CO-- wherein Y is (CH.sub.2).sub.m --R.sub.4 
--N(R.sub.5)(R.sub.6) or (CH.sub.2).sub.n --R.sub.4 
--NH--C(R.sub.7)--N(R.sub.5) (R.sub.6). In one aspect, A.sup.5 is 
--NH--CH(Y)--CO-- where Y is (CH.sub.2).sub.m --R.sub.4 
--N(R.sub.5)(R.sub.6), and preferably Orn, Dab, 7-amino-phenylalanine, and 
2,3-diamino propionic acid. In another aspect, A.sup.5 is 
--NH--CH(Y)--CO-- where Y is (CH.sub.2).sub.n --R.sub.4 
--NH--C(R.sub.7)--N(R.sub.5)(R.sub.6), and is preferably Arg or 
7-guandindylphenylalanine. A.sup.6 is the D-- or L-- isomer of Thr, Leu, 
Ile, Nle, , Val, Nal, Trp, Me-Trp, Abu, Bpa, Phe, F.sub.5 -Phe, or X-Phe 
wherein X is a halogen, NO.sub.2, CH.sub.3, or OH. A.sup.6 is preferably 
the D-- or L-- isomer of Thr, Leu, Ile, Nle, Trp, Val, and Abu. A.sup.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.sub.1 and R.sub.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.sub.1-12 alkyl, 
C.sub.2-12 alkenyl, C.sub.2-12 alkynyl, phenyl, naphthyl, C.sub.7-12 
phenylalkyl or alkylphenyl, C.sub.8-12 phenylalkenyl or alkenylphenyl, 
C.sub.8-12 phenylalkynyl or alkynylphenyl, C.sub.11-20 naphthylalkyl or 
alkylnaphthyl, C.sub.12-20 naphthylalkenyl or alkenylnaphthyl, or 
C.sub.12-20 naphthylalkynyl or naphthylalkynyl, provided that when one of 
R.sub.1 or R.sub.2 is COE or COOE, the other must be H. R.sub.3, R.sub.5, 
R.sub.6, R.sub.8 are each independently H or E. Each of R.sub.3, R.sub.5, 
R.sub.6, and R.sub.8 is preferably H or a C.sub.1-10 hydrocarbon, such as 
alkyl, alkenyl, alkylphenyl, phenyl, and phenylalkyl, including C.sub.1-5 
alkyl. Each of R.sub.5 and R.sub.6 is more preferably H. R.sub.4 is 
C.sub.6 H.sub.4 or absent, and preferably is absent. R.sub.7 is 
.dbd.NR.sub.8, .dbd.S, or .dbd.O, and preferably .dbd.NR.sub.8, and more 
preferably R.sub.7 is .dbd.NH. Preferred octapeptides encompassed by the 
above formula (I) of the invention include H.sub.2 
-D-Nal-Cys-Tyr-D-Trp-Arg-Val-Cys-Nal-NH.sub.2 (peptide Arg.sup.5); H.sub.2 
-D-Nal-Cys-Tyr-D-Trp-Dab-Val-Cys-Nal-NH.sub.2 (peptide Dab.sup.5); and 
H.sub.2 -D-Nal-Cys-Tyr-D-Trp-Orn-Val-Cys-Nal-NH.sub.2 (peptide Orn.sup.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.sup.1, A.sup.2, or the like in a peptide sequence 
stands for an amino acid residue, i.e., .dbd.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.sub.3).sub.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.sup.2 and A.sup.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.dbd.O)--E and COOE refers to --(C.dbd.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 carboxyethyl 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.

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.sub.2 and purified by gel filtration on 
SEPHADEX.TM. G-25 (Aldrich, Milwaukee, Wis.) 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, Ill. (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 &gt;96% for each peptide. 
Example 1 below is a detailed description regarding the synthesis of 
peptide Dab.sup.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-benzylo 
xycarbonyl-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.TM. 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.sub.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.degree. 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.sub.2 /MeOH until a permanent brown color was observed. 
Excess I.sub.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.times.90 cm) of SEPHADEX.TM. 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.times.70 cm) of VYDAC.RTM. 
octadecylsilane silica (10-15.mu.) (Vydac, Hesperia, Calif.) 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.TM. tissue 
homogenizer (setting 6, 15 sec) (Brinkman, Westbury, N.Y.) in ice-cold 50 
mM Tris-HCl (Buffer A) (Sigma Chemicals, St. Louis, Mo.) and centrifuging 
twice at 39,000.times.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 [.sup.125 I-Tyr.sup.4 ] bombesin (.about.2200 
Ci/mmole) (New England Nuclear, Boston, Mass.) in Buffer B, with and 
without 0.05 ml of unlabeled NMB analogs. After a 30 min incubation 
(4.degree. C.), the bound [.sup.125 I-Tyr.sup.4 ] bombesin was separated 
from the free by rapid filtration through WHATMAN.TM. 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 [.sup.125 I] bombesin bound minus that 
bound in the presence of 1 .mu.M 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.sub.i values in nM) 47.4.+-.10.3 (peptide Arg.sup.5), 85.1.+-.2.7 
(peptide Dab.sup.5), and 69.9.+-.7.1 (peptide Orn.sup.5). 
EXAMPLE 3 
GRP Receptor Binding Assay 
Membranes for the GRP receptor binding assay were obtained by homogenizing 
cultured AR42J cells with a Polytron.TM. tissue homogenizer (setting 6, 15 
sec) in ice-cold 50 mM Tris-HCl (Buffer A) and centrifuging twice at 
39,000.times.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 [.sup.125 I-Tyr.sup.4 ] bombesin (.about.2200 
Ci/mmole) in Buffer B, with and without 0.05 ml of unlabeled NMB analogs. 
After a 30 min incubation (4.degree. C.), the bound [.sup.125 I]-Tyr.sup.4 
] bombesin was separated from the free by rapid filtration through 
WHATMAN.TM. GF/B filters which had been previously soaked in 0.3% 
polyethyleneimine using a Brandel.TM. 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 [.sup.125 I-Tyr.sup.4 ] bombesin 
bound minus that bound in the presence of 1 .mu.M 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.sub.i values in nM) 2921.+-.250 (peptide Arg.sup.5) and 
2632.+-.216 (peptide Dab.sup.5). Orn had a particularly weak affinity with 
a K.sub.i value &gt;10,000. 
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.TM. 
tissue homogenizer (setting 6, 15 sec), in ice-cold 50 mM Tris-HCl (Buffer 
A) and centrifuging twice at 39,000.times.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.sub.i values, the various concentrations of NMB analogs were incubated 
for 90 min at 25.degree. C. with approximately 0.05 nM [.sup.125 I]MK-678 
(University of Arizona, School of Medicine, Tucson, Ariz.) 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.sub.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 (pre-soaked in 0.3% polyethylenimine) using a BRANDEL.TM. 
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 [.sup.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.sub.2 (Lys.sup.5) was disclosed in 
Orbuch et al., Mol. Pharmacol. 44:841 (1993), and had an extremely high 
affinity for the somatostatin receptor (K.sub.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.sub.i value of Lys.sup.5. 
For example, K.sub.i values (nM) for analogs of the invention were 
54.2.+-.9.6 (peptide Orn.sup.5), 407.+-.82 (peptide Dab.sup.5) and, 
notably, 1032.+-.113 (peptide Arg.sup.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 .mu.Ci/ml myo-[.sup.3 H] inositol (New England Nuclear, Boston, 
Mass.) for 60 min at 37.degree. 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, Mich.; Mallinckrodt, Paris, Ky.), and the total [.sup.3 H] 
inositol phosphates were isolated as described in Snider et al., J. 
Neurochem., 47:1214 (1986). Peptide Dab.sup.5 is a potent agonist of the 
NMB receptor, with a K.sub.i (.mu.M) of 78.1.+-.25.9 in the inositol 
phosphate turnover assay. 
EXAMPLE 6 
In vivo Suppression of Food Intake 
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.sup.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 analyzed using a 4 (injection).times.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&gt;0.5) these were 
averaged and used to compare with effects of the agonists and peptide 
Orn.sup.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&lt;0.01). Prior administration of 100 nmole/kg of peptide Orn.sup.5 
completely blocked the suppression of glucose intake produced by NMB. 
Intake for peptide Orn.sup.5 +NMB and peptide Orn.sup.5 +Saline conditions 
was greater than in the Saline+Saline condition (p&lt;0.01). In contrast to 
NMB, prior administration of peptide Orn.sup.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.sup.5 +NMC condition (p&gt;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 spirit and 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.