A galanin antagonist which is a galanin receptor ligand is described. New peptides, Galanin (1-12)-Pro-Substance P(5-11), Galanin (1-12)-Pro-Bradykinin(2-9), Galanin (1-12)-Pro-Pro-Pro- (Leu.sup.5 -Enkephalin (5-1), Galanin (1-12)-Pro-Lys(.epsilon.-NH-)Pro-(Leu5-Enkephalin (5-1) and functional analogues and functional derivatives thereof are disclosed. The peptides are used in pharmaceutical preparations to treat a disorder in a mammal which depends on the physiological function of galanin at the galanin receptor.

The present invention relates to a galanin antagonist which is a galanin 
receptor ligand. Further, it relates to the peptides 
Galanin(1-12)-Pro-Substance P(5-11), Galanin (1-12)-Pro-Bradykinin (2-9), 
Galanin (1-12)-Pro-Pro-Pro-(Leu.sup.5 -Enkephalin (5-1)), Galanin 
(1-12)-Pro-Lys (.epsilon.--NH-- Pro-(Leu.sup.5 -Enkephalin (5-1)) and 
functional analogues and functional derivatives thereof which exhibit 
substantially the same galanin antagonistic effect as said peptides. Use 
of the galanin antagonist, a pharmaceutical preparation comprising it and 
a method of treating a disorder in a mammal which depends on the 
physiological function of galanin at the galanin receptor are also 
included. 
BACKGROUND 
Neuro-transmitters and hormones can induce their cellular effects by 
binding to and activating membrane bound receptors. The neuro-peptide 
galanin was isolated in 1983 from porcine upper intestine and was found to 
contain 29 amino acid residues (Tatemoto, K., et al, FEBS Lett., 164 
(1983) 124-128). The sequences of galanin from two other mammals, rat and 
cow, have been described (Vrontakis M. E., et al, J. Biol. Chem. 262(1987) 
16755-16758; Kaplan L. M. et al, Proc. Natl. Acad. Sci. U.S.A. 85(1988) 
1065-1069 and Rokaeus .ANG.. and Carlquist M., FEBS Lett. 234 (1988) 
400-406). A comparison of the peptide sequence of galanin from the mammals 
rat, porcine and bovine reveals that the N-terminal amino acids 1-15 are 
identical. Thus, it is most likely that said conserved region will be 
found in galanin from other mammals, including man. 
Galanin has a wide pattern of distribution, often correlating with multiple 
neuro-formula actions exerted in a variety of different systems. 
Hitherto no galanin antagonists, which are galanin receptor ligands, have 
been reported. A galanin antagonist would be a useful tool in determining 
the physiological significance of galanin and to develope pharmaceutical 
preparations for the regulation of the physiological function of galanin 
at the galanin receptor.

DESCRIPTION OF THE INVENTION 
One aspect of the invention is directed to a galanin antagonist which is a 
galanin receptor ligand. Thus, the antagonistic effect of the galanin 
antagonist according to the invention is exercised at galanin receptors. 
In an embodiment of this aspect of the invention the antagonist is 
selected from the group consisting of the peptides 
##STR1## 
wherein X represents --NH.sub.2 or --OH (amide or free acid), and 
functional analogues and functional derivatives thereof. 
In the specification and claims, it is intended that "functional analogues" 
of the peptides of the invention i.a. should comprise shorter of longer 
peptides, with or without substitution of one or several amino acid 
residues for other amino acid residues, as long as such analogues exhibit 
substantially the same pharmacological function as the peptides of the 
invention, i.e. are galanin antagonists at the galanin receptor. 
Further, it is intended that "functional derivatives" of the peptides 
according to the invention comprise any compound which exhibits 
substantially the same pharmacological function as the peptides of the 
invention, i.e. is a galanin antagonist at the galanin receptor. 
Specifically, such a compound could be one which is derived from one of 
the peptides of the invention, but wherein one or several amino acid 
residues are substituted for other chemical groups, i.e. organic as well 
as inorganic molecules or elements resulting in a "peptidomimetic". 
It is believed that it is the structural conformation at the galanin 
receptor of a peptide according to the invention which is responsible for 
its pharmacological function as a galanin antagonist and for its affinity 
to the galanin receptor (thus being a galanin receptor ligand). 
Thus, it is believed that the functional derivatives and the functional 
analogues comprised by the invention should have a similar structural 
conformation at the galanin receptor as the peptides of the invention. The 
surrounding of the galanin receptor could be mimicked by e.g. blood or 
physiological saline solution. 
Another aspect of the invention is directed to the peptides 
##STR2## 
wherein X represents --NH.sub.2 or --OH (amide or free acid), and 
functional analogues and functional derivatives thereof which exhibit 
substantially the same galanin antagonistic effect as said peptides. 
As mentioned above, the peptides may also be named 
Galanin-(1-12)-Pro-Substance P(5-11), Galanin(1-12)-Pro-Bradykinin(2-9), 
Galanin(1-12)-Pro-Pro-Pro-(Leu.sup.5 -Enkephalin(5-1)) and 
Galanin(1-12-Pro-Lys(.epsilon.-NH-)Pro(Leu.sup.5 -Enkephalin(5-1)), 
respectively. 
In the experimental part of this specification the above listed peptides, 
in amide form (i.e. X=--NH.sup.2), have been named M15, M35, M36, A and 
M34, A, respectively. 
Functional analogs may be peptides such as 
##STR3## 
wherein X represents --NH2 or --OH (amide or free acid). 
A further aspect of the invention is directed to the use of a galanin 
antagonist which is a galanin receptor ligand for the preparation of a 
pharmaceutical preparation. It can be mentioned that the above listed 
peptides are soluble in water, which facilitates the preparation of a 
pharmaceutical preparation for injection. 
Yet another aspect of the invention is directed to a galanin antagonist 
which is a galanin receptor ligand for use in a pharmaceutical 
preparation. 
Yet a further aspect of the invention is directed to a pharmaceutical 
preparation comprising a galanin antagonist which is a galanin receptor 
ligand as an active ingredient, together with pharmaceutically acceptable 
additive(s). Depending on the specific type of pharmaceutical preparation 
to be prepared, such additive(s) should be chosen to make up the desired 
preparation. Suitable additive(s) for the specific type of preparation to 
be prepared, such as solutions for injection, tablets or plasters, can be 
found in the U.S. Pharmacopoeia. 
Thus the present invention also provides compositions containing an 
effective amount of compounds of the present invention, including the 
nontoxic addition salts, amides and esters thereof, which may, alone, 
serve to provide the above-recited therapeutic benefits. Such compositions 
can also be provided together with physiologically tolerable liquid, gel 
or solid diluents, adjuvants and excipients. 
These compounds and compositions can be administered to mammals for 
veterinary use, such as with domestic animals, and clinical use in humans 
in a manner similar to other therapeutic agents. In general, the dosage 
required for therapeutic efficacy will range from about 0.01 to 1000 
mcg/kg, more usually 0.1 to 1000 mcg/kg of the host body weight. 
Alternatively, dosages within these ranges can be administered by constant 
infusion over an extended period of time until the desired therapeutic 
benefits have been obtained. 
Typically, such compositions are prepared as injectables, either as liquid 
solutions or suspensions; solid forms suitable for solution in, or 
suspension in, liquid prior to injection may also be prepared. The 
preparation may also be emulsified or incorporated in drug delivery 
systems like liposomes or microspheres. The active ingredient is often 
mixed with diluents or excipients which are physiologically tolerable and 
compatible with the active ingredient. Suitable diluents and excipients 
are, for example, water, saline, dextrose, glycerol, or the like, and 
combinations thereof. In addition, if desired the compositions may contain 
minor amounts of auxiliary substances such as wetting or emulsifying 
agents, stabilizing or pH-buffering agents, and the like. 
The compositions are conventionally administered parenterally, by 
injection, for example, either subcutaneously, intramuscularly, 
intraperitoneally or intravenously. Additional formulations which are 
suitable for other modes of administration include suppositories, 
vagitories, intranasal aerosols, buccal formulations like adhesive 
tablets, gels or patches and in some cases, oral formulations. For 
suppositories and vagitories, traditional binders and excipients may 
include, for example, polyalkylene glycols or triglycerides; such 
suppositories may be formed from mixtures containing the active ingredient 
in the range of 0.5% to 10% preferably 1%-2%. Oral formulations include 
such normally employed excipients as, for example, pharmaceutical grades 
of mannitol, lactose, starch, magnesium stearate, sodium saccharin, 
cellulose, magnesium carbonate, and the like. These compositions take the 
form of solutions, suspensions, tablets, pills, capsules, 
sustained-release formulations, or powders, and contain 10%-95% of active 
ingredient, preferably 25%-70%. 
The peptide compounds may be formulated into the compositions as neutral or 
salt forms. Pharmaceutically acceptable nontoxic salts include the acid 
addition salts (formed with the free amino groups) and which are formed 
with inorganic acids such as, for example, hydrochloric or phosphoric 
acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and 
the like. Salts formed with the free carboxyl groups may be derived from 
inorganic bases such as, for example, sodium, potassium, ammonium, 
calcium, or ferric hydroxides, and such organic bases as ispropylamine, 
trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. 
Still another aspect of the invention is directed to a method of treating a 
disorder in a mammal which depends on the physiological function of 
galanin at the galanin receptor, which comprises administering to said 
mammal a pharmacologically effective amount of a galanin antagonist which 
is a galanin receptor ligand. Since the physiological function of galanin 
at the galanin receptor is specific for each individual, the 
pharmacologically effective amount of a galanin antagonist which is to be 
administered to treat a disorder in a mammal has to be decided by a 
physician or veterinary on an individual bases. 
Galanin antagonists would be useful in the regulation of at least the 
following: 
insulin release, 
growth hormone release, 
acetylcholine release, 
dopamine release, 
Substance P release, 
Somatostatin release, 
Noradrenaline release. 
At present, the presumed fields of medical indication are endochrinology, 
food intake, neurology and psychiatry: senile dementia of Alzheimer's 
type, schizophrenia, analgesia, intestinal diseases. 
Preparation of a Galanin Antagonist According to the Invention 
Conventional methods of preparing peptides can be used to prepare the 
galanin antagonists of the invention, suitably modified if the antagonist 
is a peptidomimetic. In addition to liquid phase synthesis, conventional 
procedures for synthesizing the novel compounds of the present invention 
include any solid phase peptide synthesis method. In such a method the 
synthesis of the novel compounds can be carried out by sequentially 
incorporating the desired amino acid residues one at a time into the 
growing peptide chain according to the general principles of solid phase 
methods. 
Abbreviations used in the following: 
NMP=N-Methylpyrrolidone 
HOBt=N-hydroxybenzotriazole 
MBHA=p-Methylbenzhydrylamine 
PAM=p-Acetoxymethyl 
Boc=tert-Butyloxycarbonyl 
DMSO=Dimethylsulfoxide 
DIEA=diisopropylethylamine 
Tos=tosyl 
OcHex=cyclohexyl ester 
4--MeBzl=4-methylbenzyl 
OBzl=bensyl ester 
Bom=t-Benzyloxymethyl 
Clz=2-Chlorobenzyloxycarbonyl 
Bzl=O-benzyl 
For=formyl 
Brz=2-bromobenzyloxycarbonyl 
TFA=trifluoroacetic acid 
DMS=dimethyl sulfid 
DCM=dichloromethan 
DMF=N,N-dimethylformamide 
The peptides "M15", "M35" and "M36,A" of this invention were assembled in a 
step-wise manner on a solid support using an Applied Biosystems Model 431A 
Peptide Synthesizer using the standard NMP/HOBt Solvent-Activation 
strategy on a 0.1 mmole scale (small scale). 
The peptide "M34,A" was synthesized in the same manner, except for the 
protective group which was .epsilon.-Fmoc and Boc for Lys. Accordingly one 
part of the peptide was synthesized using Boc-chemistry, and the other 
part was synthesized using Fmoc chemistry. 
The functional derivatives of the invention were prepared in analogous 
manner. 
tert-Boc-amino acids were coupled to tert-Boc-amino acid-PAM (Nova Chemical 
Company Ltd., UK) resin for fee acid (X=--OH) or MBHA(Bachem 
Feinchemikalien AG, Switzerland) resin for amide (X=--NH.sub.2) as 
hydroxybenzotriazole(HOBt) esters. 
As individual cycle for each amino acid included deprotection of the 
tert-Boc-group with 50% trifluoroacetic acid in CH.sub.2 Cl.sub.2 (DCM) 
for 19 min, and acylation with 10-fold excess (compared to the amount of 
amino groups on a resin) of the protected amino acids in a mixture of 15% 
DMSO in N-methyl pyrrolidone (NMP) for 35 min. Between each operation 
several extensive washings were performed with CH.sub.2 Cl.sub.2, DIEA and 
NMP. After each coupling acetylation (capping) was carried out using 10% 
acetic anhydride and 5% DIEA in NMP for 5 min. 
With the excention of Boc-Lys(Fmoc) in the case of the peptide 34,A, all 
amino acids used were tert-Boc-protected at the N-terminal (obtained from 
Nova Chemical Company Ltd., UK), and their reactive side chains were 
protected with Tos in 
Arg, OcHex in Asp, 4-MeBzl in Cys, OBzl in Glu, Bom in His, ClZ in Lys, Bzl 
in Ser and Thr, For in Trp, and Brz in Tyr. 
All the solvents and other reagents for automatic peptide synthesis were 
from Applied Biosystems. 
The reagents used in deprotection and cleavage steps were of analytical 
grade and used without further purification: 
1,2-ethaneditiol, acetic anhydride, trifluoromethyl sulfonic acid (TFMSA), 
dimethyl sulfide (DMS) and p-cresol from Fluka, diethyl ether and 
acetonitrile from Merck and HF from AGA Gas. 
The fully assembled peptide-resins were dried under high vacuum overnight. 
Deprotection from formyl-group on Trp and benzyl-groups was performed 
using "low TFMSA" method. For that 100 mg of peptide-resin was treated 
with 2 ml of the mixture of TFMSA(10%), TFA(50%), DMS(30%), p-cresol(8%) 
and dimercaptoethan(2%) for 2 h at 0.degree. C. while shaking, the 
procedure was followed by washing with EtOH, DCM, DMF, DIEA/DCM, DMF and 
DCM. 
After drying under vacuum the peptide was cleaved from resin and 
deprotected by mixing of 10 ml liquid HF (containing 20% of 1:1 mixture of 
p-cresol and p-thiocresol) with 1 g of peptide-resin at 0.degree. C. for 
60 min. The resin was washed 2 times with 10 ml of Et.sub.2 O, peptide 
extracted with 20% acetonitrile/water and filtered. Lyophilization of the 
aqueous filtrate yielded the crude peptide. 
Preparative purifications were carried out on the crude product by HPLC on 
reversed phase C18 column. 10 mg of crude peptide was dissolved in 1 ml of 
20% acetonitrile/water and eluted with a gradient (45 min) of 20-60% 
mixture of 0.1% TFA/acetonitrile in 0.1 & TFA/water at a flow rate of 2.0 
ml/min. The fractions were collected according to the absorption detected 
at 238 nm. 
Purity of the individual peptides was checked by analytical HPLC and 
determined to be 99%. Molecular weights of the peptides were determined 
using Plasma Desorption Mass Spectrometer (PDMS) Model Bioion 20, Applied 
Biosystems, the calculated molecular weight values were obtained in each 
case for the purified peptide. 
TABLE 
__________________________________________________________________________ 
Purity of the individual peptides was checked by analytical HPLC and 
determined to be 99 %. Molecular 
weights of the peptides were determined using Plasma Desorption Mass 
Spectrometer (PDMS) Model 
Bioion 20, Applied Biosystems, the expected values were obtained in each 
case. 
__________________________________________________________________________ 
Galanin(1-12)-Pro-SP(5-11) amide (M15) (SEQ ID NO:1): (MW = 2199) 
IC.sub.50 = 0.1 nM 
##STR4## 
Galanin(1-12)-Pro-Spantide amide (C 7): (MW = 2827) IC.sub.50 = 0.2 nM 
##STR5## 
Galanin(1-12)-ProProPro-SP(5-11) amide (M37) (SEQ ID NO:11): (MW = 2392) 
IC.sub.50 = 40 nM 
##STR6## 
Galanin(1-13)-AlaLeuAlaLeuAlaLeuAla amide (M 38) (SEQ ID NO:5): (MW = 
1970) IC.sub.5o = 0.2 nM 
##STR7## 
Galanin(1-12)- -ProProProAlaLeuAlaLeuAla amide (M 40) (SEQ ID NO:6): (MW 
= 1980) IC.sub.50 = 13 nM 
##STR8## 
Galanin(1-13)-Bradykinin(3-9) amide (M20) (SEQ ID NO:7): (MW = 2135) 
IC.sub.50 = 1 nM 
##STR9## 
Galanin(1-13)-Bradykinin(2-9) amide (M 35) (SEQ ID NO:2): (MW = 2232) 
IC.sub.50 = 0.2 nM 
##STR10## 
Galanin(1-13)-NPY(25-36) amide (M 32) (SEQ ID NO:8): (MW = 2961) 
IC.sub.50 = 0.05 nM 
##STR11## 
Galanin(1-12)-Ala-NPY(25-36) amide (M 88) (SEQ ID NO:9): (MW = 2935) 
IC.sub.50 = 1 nM 
##STR12## 
Galanin(1-12)-ProProPro-Leuenkephalin(1-5) amide (M 36) (SEQ ID NO:10): 
(MW = 2078) IC.sub.50 = 40 nM 
##STR13## 
Galanin(1-12)-ProProPro-Leuenkephalin(5-1) amide (M36,A) (SEQ ID NO:3): 
(MW = 2078) (IC.sub.50 = 20 nM 
##STR14## 
Galanin(1-13)-Lys amide (Pro-Leuenkephalin(5-1)) (M34,A) (MW = 2223) 
IC.sub.50 = 10 nM 
##STR15## 
__________________________________________________________________________ 
IC.sub.50 has been determined in displacement experiments in rat 
hypothalamus 
PHARMACOLOGICAL EXPERIMENTS 
1) Galanin inhibits glucose-induced insulin release, measured according to 
Ahr en, B., et al (Biochemical and Biophysical Research Communications, 
Vol. 140. No. 3, 1986, 1059-1063). By using the same procedure as the 
cited Ahr en B., et al, a peptide according to the invention, M15, is now 
shown to block the galanin-inhibited insulin release in equimolar 
concentration. 
Animals and Preparation of Cells 
Adult obese hyperglycemic mice (ob/ob) of both sexes were taken from a 
local non-inbred colony and starved overnight. The animals were killed by 
decapitation and the islets were isolated by collagenase isolation 
technique. A cell suspension was prepared essentially as described by 
Lehrnmark, .ANG.., in Diabetologia 10, 1974, 431-438. Briefly, the islets 
were dissociated into single cells and small clusters by shaking in a 
Ca.sup.2+ -Mg.sup.2+ -deficient medium supplemented with EGTA (Ethylene 
glycol-bis(beta-aminoethyl ether)N,N,N',N',-tetra-acetic acid, SIGMA). 
Thereafter, the cells were incubated at 37.degree. C., pH 7.4, overnight 
in 12 ml RPMI 1640 medium (Tissue culture medium from SIGMA, which 
contains L-glutamine and does not contain sodium bicarbonate) supplemented 
with 10% NU-serum TM (Collaborative Research Inc., Lexington, Mass., 
U.S.A.), 100 IU/ml penicillin, 60 .mu.g/ml gentamycin and 100 .mu.g/ml 
streptomycin. To avoid attachment of the cells to the culture flasks 
during incubation, the suspension was shaken gently. 
Media 
The basal medium used was a HEPES buffer 
(N-[2Hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid], SIGMA), pH 7.4, 
physiologically balanced in cations with Cl.sup.- as the sole anion. In 
all cases the basal medium was supplemented with 1 mg/ml albumin. 
Measurements of Insulin Release 
The kinetics of insulin release were studied by perfusing pancreatic 
beta-cells (approx. 1.times.10.sup.6) with 20 mM or 5 mM glucose mixed 
with Bio-Gel P-4 polyacrylamide beads (200-400 mesh, Bio-Rad Lab., 
Richmond, Calif., U.S.A.) in a 0.5 ml column. The flow rate was 0.5 ml/min 
and 1-3 min fractions were collected and analyzed for insulin 
radioimmunologically, using crystalline rat insulin as a reference. 
Results 
After 60 min incubation in the presence of glucose (8.3 mM), galanin 
(10.sup.-7 M) completely abolished insulin release from 
34.+-.2 .mu.U/ml to 3.+-.1 .mu.U/ml (P &lt;0.001; N=32). The peptide of the 
invention, 
M 15, alone (10.sup.-6 M-10.sup.-9 M) dose-dependently counteracted the 
galanin-induced inhibition of insulin release. 
Insulin release in the presence of glucose, galanin and M15 at 10.sup.-6 M, 
10.sup.-7 or 10.sup.-8 was 28.+-.2 .mu.U/ml (N=16), 17.+-.4 .mu.U/ml 
(N=16), and 10.+-.3 .mu.U/ml (N=16), respectively. M15 at 10.sup.-9 M was 
without effect. 
As a result of the above experiments it can be concluded that the peptide 
of the invention, M15, counteracts the galanin-induced inhibition of 
insulin release in islets. 
2) Galanin inhibits acetylcholine release, measured according to Fisone G., 
et al (Proc. Natl. Acad. Sci. vol 84, 1987, 7339-7343). By using 
substantially the same procedure as the cited Fisone G., et al, a peptide 
according to the invention, M15, is now shown to counteract the inhibitory 
effects of galanin on the scopolamine-induced release of acetylcholine in 
vivo. 
Microdialysis Experiments 
Surgery and basic methodology. Experiments were carried out with female 
CD-COBS (Charles River, Como, Italy) rats, weighing 200-260 g. Animals 
were anesthetized with equitensin (1% pentobarbital, 4% chloral hydrate). 
Guide cannula was implanted stereotoxically into the ventral hippocampus 
according to the following coordinates from the atlas of Paxinos and 
Watson (Paxinos G. and Watson C. (1986) The Rat Brain in Stereotoxic 
Coordinates, 2nd edn. Academic Press, Sydney.): 5 mm posterior to the 
bregma, 4.0 mm lateral to the midline, and 6.8 mm below the surface of the 
dura mater. A styler was then inserted to keep the guide cannula patent 
until the next day. At the time the styler was removed and replaced with a 
microdialysis probe (CMA 10, Carnegie Medicine AB, Stockholm, Sweden) 
containing a membrane having an exposed area of 3.times.0.5 mm. The 
microdialysis probe extended 3 mm beyond the end of the guide cannula; it 
was perfused at a constant rate of 2 .mu.l (min with Ringer's solution 
(NaCl, 147 mM; CaCl.sub.2, 2.2 mM and KCl, 4.0 mM), containing 10 .mu.M 
physostigmine sulfate and adjusted to pH 7.0 with NaOH. The initial 40 min 
perfusate was discarded. Thereafter, perfusates were collected every 20 
min during a total perfusion period of 260 min. At the end of the 
collection period, the samples were immediately frozen on solid CO.sub.2 
and lyophilized. Acetylcholine (Ach) content was quantified by a specific 
radioenzymatic method described in detail by Consolo S., et al. (J. 
Neurochem. 48, (1987), 1459-1465.) and Wu C. F., et al (Neurobiol. Aging 
9, (1988), 357-361.) and involving (a) the conversion of choline to 
phosphorylcholine in the presence of choline phosphokinase and ATP, (b) 
the enzymatic hydrolysis of acetylcholine to choline and acetic acid, (c) 
the reacetylation of the resulting choline to [.sup.3 H]acetylcholine with 
the addition of [.sup.3 H]acetylcoenzyme-A, 99.9 GBq/mmol, and 
acetyl-coenzyme A:ChAT, and (d) the separation and scintillation counting 
of the resulting [.sup.3 H]acetylcholine by extraction into 
tetraphenylboron-containing ketone phase by liquid--liquid ion exchange 
chromatography. Phosphorylation, hydrolysis and acetylation reactions were 
validated routinely. 
The concentration of acetylcholine in each sample was calculated by linear 
regression based on the radioactivity of the standards (linear from 10 
fmol-25 pmol acetylcholine) with the slope equal to 7800 net dpm/pmol. The 
coefficient of variation of the replicate perfusate samples or 
acetylcholine standards was approximately 3%. 
In vitro recovery of acetylcholine through three dialysis tubings was 
determined as previously described (Wu et al., ibid). The average recovery 
was 17.6.+-.0.5%. 
At the termination of the release experiments, the placements of the 
dialysis probes were verified histologically by use of staining for Nissl 
substance. 
______________________________________ 
Effect of galanin and M 15 on the scopolamine-induced 
release of ACh, measured "in vivo" from the rat ventral 
hippocampus 
______________________________________ 
Saline 2.0 pmoles 
Scopolamine 13.1 pmoles 
Scopolamine + 6.9 pmoles 
Galanin 
Scopolamine + 9.4 pmoles 
M15 
______________________________________ 
Galanin (1.56 nmoles) and M15 (3.12 nmoles) were injected i.c.v. 2 min 
before scopolamine (0.3 mg/kg, s.c.). ACh release was measured in the 
perfusate collected during the following 80 min, in four 20 min-fractions. 
The values are expressed as pmoles of ACh measured during the 20 min 
fraction corresponding to the peak of the scopolamine-effect and 
corresponding to the second 20 min-fraction. The data are the means of 
experiments carried out in two rats (n=2). 
As a result of the above experiments it can be concluded that the peptide 
of the invention, M15, counteracts the galanin-induced inhibition of 
scopolamine-induced release of acetylcholine in hippocampus. 
3) Effects of intrathecal galanin and C-fiber stimulation on the flexor 
reflex has been described by Wiesenfeld-Hallin Z., et al (Brain Res. 486 
(1989)205-213). By using substantially the same procedure as the cited 
reference, a peptide according to the invention, M15, is shown to have an 
antagonistic effect on intrathecal galanin-induced facilitation of the 
flexor reflex. An other peptide of the invention, M35, has given similar 
results in preliminary experiments. 
Electrophysiological Study 
In acute experiments, the magnitude of the polysynaptic hamstring flexor 
reflex in response to activation of high threshold afferents was examined 
in decerebrated, spinalized, unanaesthetized rats by recording the 
electromyogram (EMG) from the posterior bicepts femoris/semitendinosus 
muscles. The animals were briefly anaesthetized with Brietal.RTM., and a 
tracheal cannula was inserted. The rats were mounted in a stereotaxic 
frame, decerebrated by aspiration of the forebrain and midbrain and then 
ventilated. The spinal cord was exposed by laminectomy at thoracic level 
and sectioned at Th.sub.8-9. An i.t. catheter (PE 10) was implanted 
caudally to the transection with its tip on the left side of the lumbar 
spinal cord (L.sub.4-5). The flexor reflex was elicited by test stimuli to 
the sural nerve or its innervation area with single electric shocks (0.5 
ms, 10 mA) of sufficient strength to activate C-fibres (Wall and Woolf, 
1984). In some experiments a CS (conditioning stimulus) (1 Hz, 20 s) of 
the same strength as the test stimuli was administered to the sural nerve. 
The flexor reflex was recorded as EMG activity via stainless steel needle 
electrodes inserted in the ipsilateral hamstring muscles. The number of 
action potentials elicited during the reflex was integrated over 2 s. The 
integrated reflex was recorded on a Gould chart recorder (Model 2400 S). 
During the experiments the heart rate and rectal temperature of the rat 
were monitored and maintained within normal limits. The proper location of 
the i.t. catheter was confirmed after each experiment by laminectomy. 
Galanin (Bachem, Bubendorff, Switzerland) and Somatostatin (Ferring, Malm 
o, Sweden) were dissolved in 0.9% saline. All peptides were injected i.t. 
in a volume of 10 .mu.l followed by 10 .mu.l saline to flush the catheter. 
Data Collection 
A stable baseline reflex magnitude was established for at least 20 min 
before each i.t. injection or nerve CS. The effect of i.t. peptides or the 
nerve CS on the flexor reflex was expressed as percent change in reflex 
magnitude compared to baseline which was defined as 100%. To test the 
interaction of galanin with other peptides or the sural CS, it was 
administered 15 min prior to the injection of the other peptides when the 
facilitatory effect of galanin had subsided or simultaneously with the 
sural CS. 
The antagonistic effect of intrathecal (i.t.) M-15 on the i.t. 
galanin-induced facilitation of the flexor reflex was measured. The peak 
facilitatory effect of 30 pmol galanin was 167.0.+-.42.8% over baseline 
reflex magnitude. The antagonism of M-15, injected 5-10 min after galanin 
was calculated as the percentage reduction of the peak facilitatory effect 
of galanin. Data is expressed as meant S.E.M. 
______________________________________ 
Dose of M-15 n % antagonism 
______________________________________ 
30 pmol 4 35.8 .+-. 20.7 
300 pmol 4 79.3 .+-. 8.4 
3 nmol 3 93.0 .+-. 3.4 
______________________________________ 
The experiments were repeated using the peptide of the invention, M35, and 
similar results were achieved. 
As a result of the above experiments it can be concluded that peptides of 
the invention counteract the intrathecal galanininduced facilitation of 
the flexor reflex in a dose-dependent manner. 
4) Ligand binding studies were conducted. Displacement of .sup.125 
I-galanin by galanin, galanin fragment (1-13), Substance P fragment 
(4-11), galanin fragment (1-13) + Substance P fragment (4-11) in equimolar 
concentrations and the peptides of the invention M15, M35 and M34, A was 
studied. The peptides of the invention proved to bind specifically to the 
galanin receptor. 
Preparation of .sup.125 I-monoiodo-Tyr.sup.26 -Porcine Galanin 
Synthetic porcine galanin(1-29) was iodinated by chloramine-T-method to 
yield .sup.125 I-monoiodo-Tyr.sup.26 -porcine galanin (specific activity 
1800-2000 Ci/mmol), (as described by Land T., et al in: Methods in 
Neurosciences, Ed. M. Conn, 5, 1991, 225-234), and employed in ligand 
binding studies carried out at equilibrium. 
Preparation of Membrane Fraction of Rin m 5F Cells 
The establishment and cell culture of Rin m 5F (a rat pancreatic 
.beta.-tumor cell-line) cells have been described earlier (Gazdav A. F., 
et. al. Proc. Natl. Acad. Sci. USA, 77 (1980) 3519-3523). Briefly, the 
cells were grown in RPMI-1640 (Gibco) medium containing 10% (v/v) fetal 
calf serum, 2.06 mM L-gluthamine, 100 IU/ml penicillin and 100 .mu.g/ml 
streptomycin in 10% CO.sub.2 -90% air 37.degree. C. They were passaged 
every 5 days. The cells were detached from the surface of the bottles 
using 0.25% trypsin containing 1 mM EDTA and centrifuged at 2000 .times. g 
for 5 min at 4.degree. C. The pellet was exposed for 15 min to hypoosmotic 
5 mM HEPES buffer (pH 7.4). The suspension was centrifuged at 20000 
.times. g for 15 min, the resulting pellet was resuspended in 
bacitracin-containing (1 mg/mg) 5 mM HEPES-buffered Krebs-Ringer solution 
(137 mM NaCl, 2.68 nM KCl, 1.8 mM CaCl.sub.2, 1 g/1 glucose), pH 7.4, and 
used immediately in equilibrium binding experiments. 
Displacement of .sup.125 I-galanin by Galanin and Galanin Receptor Ligands 
Displacement experiments were carried out in a final volume of 400 .mu.l of 
bacitracin-containing (1 mg/ml) HEPES-buffered (5 mM) Krebs-Ringer 
solution, 0.05% (w/v) of BSA (pH 7.4) in the presence of 0.1-0.2 nM 
.sup.125 I-galanin, the membrane preparation and the increasing 
concentrations (10.sup.-12 -10.sup.-6 M) of unlabeled porcine galanin or 
of other galanin receptor ligands. Samples were incubated for 30 min at 
37.degree. C. Incubation was terminated by the addition of 10 ml of 
ice-cold HEPES-buffered Krebs-Ringer solution, followed by rapid 
filtration over Whatman GF/C filters, precoated for 5-6 hours in 0.3% 
(v/v) of polyethyleneimine solution. Filters were washed with 10 ml of 
assay solution. Radioactivity retained on the filters was determined in a 
Packard gamma counter. Specific binding was defined as that displaceable 
by 1 .mu.M galanin (or appropriate concentration of a displacer). 
The IC.sub.50 values of the displacing ligands were calculated from the 
computer-generated IC.sub.50 values as described by Land, T., et. al. 
(ibid). 
Fitting of the experimental data was carried out on a Macintosh SE by means 
of a nonlinear least squares method using the Macintosh program 
"KaleidGraph". 
______________________________________ 
Displacement of .sup.125 I-galanin from membranes of Rin m 5F 
by galanin receptor ligands 
IC.sub.50 
______________________________________ 
Galanin (1-29) 1 nM 
M15 0.1 nM 
Galanin fragment (1-13) 100 nM 
Substance P fragment (4-11) 
no 
displacement &gt; 
10 .mu.m 
Galanin fragment (1-13) + 
Substance P fragment (4-11) 
100 nM 
in equimolar concentrations 
M35 0.1 nM 
M34,A 10 nM 
______________________________________ 
As is evident from the above experiments, the peptides of the invention are 
galanin receptor ligands. 
The pharmacological experiments 1), 2) and 3) thus show that the peptides 
of the invention are galanin antagonists, and the experiment 4) shows that 
they are galanin receptor ligands. Thus, the galanin antagonists of the 
invention are galanin receptor ligands, unlike other compounds which 
earlier have been shown to antagonize a specific effect of galanin in a 
specific tissue by virtue of interaction with a reaction step beyond the 
galanin receptor--which was involved in the biological action of galanin 
in that given cell type. Such antagonism is not specific for galanin 
action, and is not exerted at galanin receptor. Neither it is applicable 
to several tissues where galanin acts, whereas the antagonists according 
to the present invention are bona fide antagonists in the pharmacological 
meaning and exert their action at the receptor site on the outside of the 
cell by competing with the endogenous ligand. 
Preparation of Membranes from Rat Hypothalamus 
Adult male rats (Sprague-Dawley, 180-200 g) were decapitated, the 
hypothalami quickly dissected and homogenized (10% mass/vol.) in 0.05M 
TRIS-Cl buffer, pH 7.4. The homogenate was diluted tenfold and centrifuged 
at 1000 .times. g for 10 min. The supernatant was centrifuged at 10,000 
.times. g for 45 min and the pellet resuspended in 5 mM Hepes buffered 
Krebs-Ringer solution (137 mM NaCl, 2.68 mM KCl, 1.8 mM CaCl2 1 g/1 
glucose), pH 7.4 to yield a final protein concentration of 1.0-1.5 mg/ml. 
Ligand Binding Studies in Rat Hypothalamus 
Displacement experiments were carried out in a final volume of 400 .mu.l of 
Hepes-buffered (5 mM) Krebs-Ringer solution, 0.05% (w/v) of BSA (pH 7.4 ) 
supplemented with bacitracin (1 mg/ml ) in the presence of 0.1-0.2 nM 
1251-galanin, the membrane preparation from rat hypothalami and increasing 
concentrations (10-12, 10-6M) of unlabeled galanin or of other galanin 
recepto-ligands. Samples were incubated for 30 min at 37.degree. C. 
Incubation was terminated by the addition of 10 ml of ice-cold 
Hepes-buffered Krebs-Ringer solution, followed by rapid filtration over 
Whatman GF/C filters, precoated for 5-6 hours in 0.3% (v/v) of 
polyethylencimine solution. The filters were washed with 10 ml of assay 
solution. Radioactivity retained on the filters was determined in a 
Packard gamma counter. Specific binding was defined as that displaceable 
by 1 .mu.M galanin. Rat and porcine galanin resulted in indistriguishable 
displacement curves with the membranes from rat hypothalami. 
The 1C50 values of the displacing ligands were calculated by fitting of the 
experimental data on a Macintosh SE by means of a nonlincar least squares 
method using the program "KaleidaGraph". 
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SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 11 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
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(A) NAME/KEY: Modified-site 
(B) LOCATION: 20 
(D) OTHER INFORMATION: /note="amide or free acid" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GlyTrpThrLeuAsnSerAlaGlyTyrLeuLeuGlyProGlnGlnPhe 
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(2) INFORMATION FOR SEQ ID NO:2: 
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(A) LENGTH: 21 amino acids 
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(A) NAME/KEY: Modified-site 
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(D) OTHER INFORMATION: /note="amide or free acid" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
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(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino acids 
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GlyTrpThrLeuAsnSerAlaGlyTyrLeuLeuGlyProProProLeu 
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(A) LENGTH: 21 amino acids 
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(2) INFORMATION FOR SEQ ID NO:5: 
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(A) LENGTH: 20 amino acids 
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LeuAlaLeuAla 
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(A) LENGTH: 20 amino acids 
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(2) INFORMATION FOR SEQ ID NO:7: 
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(A) LENGTH: 20 amino acids 
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(A) LENGTH: 25 amino acids 
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IleAsnLeuIleThrArgGlnArgTyr 
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(A) LENGTH: 25 amino acids 
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IleAsnLeuIleThrArgGlnArgTyr 
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(A) LENGTH: 20 amino acids 
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GlyGlyPheLeu 
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(2) INFORMATION FOR SEQ ID NO:11: 
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(A) LENGTH: 22 amino acids 
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(D) OTHER INFORMATION: /note="amide" 
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GlyTrpThrLeuAsnSerAlaGlyTyrLeuLeuGlyProProProGln 
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GlnPhePheGlyLeuMet 
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