Peptide, a bronchus-expanding agent, and a blood-flow-improving agent

The present invention relates to a peptide represented by the general formula (1): ##STR1## wherein A is Ala or Gly; B is Ile or Val; C is Asn or Ser; D is Thr or Ser; E is Leu or Tyr; each of F, I and J is Lys or Arg, and at least one of F, I and J is Arg; G is Met, Leu or Nle; K is Asn or Ala; L is Ser or Ala; M is Ile or Val; and N is --NH.sub.2 or Asn--NH.sub.2, excluding the combination where A is Ala, B is Val, C is Asn, D is Thr, E is Leu, K is Asn, L is Ser, M is Ile, and N is Asn--NH.sub.2, and pharmaceutically acceptable salts thereof, as well as to a bronchus-expanding agent or a blood-flow-improving agent comprising said peptide or a pharmaceutically acceptable salt thereof as an active ingredient.

This application is a 371 of PCT/JP96/01543 filed Jun. 6, 1996. 
FIELD OF THE INVENTION 
The present invention relates to a novel peptide, a bronchus-expanding 
agent and a blood-flow-improving agent. 
BACKGROUND OF THE INVENTION 
VIP (vasoactive intestinal peptide) is one kind of physiologically active 
peptide called brain-gut peptide, the action of which is to promote blood 
flow and depress blood pressure. VIP was extracted from the intestine of a 
pig in 1970 and it consists of 28 amino acid residues (S. I. Said, V. Mutt 
Science, 169, 1217 (1970)). 
AP (pituitary adenylate cyclase activating polypeptide) was isolated 
from the hypothalamus of a sheep in 1989 in a bioassay system using as an 
indicator the activation of adenylate cyclase from cultured pituitary 
cells, and it is a structurally determined peptide consisting of 38 amino 
acid residues (A. Miyata, A. Arimura et al., Biochem. Biophys. Res. 
Commun.,164, 567 (1989)). 27 amino acid residues from the N-terminal side 
of AP has the AP activity and the amino acid sequence of said 27 
amino acids has a considerably similar structure to that of VIP. 
VIP and AP are similar in amino acid sequence to secretin, glucagon etc. 
and are thus classified as peptides belonging to the glucagon family. By 
virtue of their strong action on vessel expansion and blood-flow 
promotion, VIP and AP are expected for use in the treatment of an 
ulcer, frostbite, bedsore, impotence etc. and in the restoration and 
growth of hair. In addition, VIP and AP have considerably strong 
relaxation action on the bronchial smooth muscle in the respiratory 
system, thus alleviating the smooth muscle contraction induced by 
stimulators such as acetylcholine, histamine, serotonin etc. Such 
relaxation action of VIP and AP differ from the relaxation action 
caused via adrenalin .beta..sub.2 receptor by general bronchial expansion, 
so VIP and AP are expected to have an treatment effect on asthmatic 
spasm difficult to cure on which a stimulator of .beta..sub.2 receptor 
does not effectively act. 
Besides VIP and AP, a peptide consisting of 28 to 31 amino acid residues 
with bronchus expanding action, blood-pressure reducing action and hair 
restoring action is known (see Japanese Patent Application Laid-Open 
Publication Nos. 246,595/87, 83,012/89 and 297,498/92). 
It is however desired to develop a bronchus-expanding agent and a 
blood-flow-improving agent superior to VIP, AP and other peptides in 
the durability of bronchus-expanding action and blood-flow increasing 
action. 
DISCLOSURE OF THE INVENTION 
The object of the present invention is to provide a novel peptide excellent 
in the durability of bronchus-expanding action and further excellent in 
blood-flow increasing action, a bronchus-expanding agent and a 
blood-flow-improving agent comprising said peptide as an active 
ingredient. 
The present invention provides a peptide represented by the general formula 
(1): 
##STR2## 
wherein A is Ala or Gly; B is Ile or Val; C is Asn or Ser; D is Thr or 
Ser; E is Leu or Tyr; each of F, I and J is Lys or Arg, and at least one 
of F, I and J is Arg; G is Met, Leu or nLeu; K is Asn or Ala; L is Ser or 
Ala; M is Ile or Val; and N is --NH.sub.2 or Asn--NH.sub.2, excluding the 
combination where A is Ala, B is Val, C is Asn, D is Thr, E is Leu, K is 
Asn, L is Ser, M is Ile, and N is Asn--NH.sub.2, or the general formula 
(2): 
##STR3## 
wherein A, B, C, D, E, F, G, I, J, K, L and M possess the same meanings 
defined as above; at least one of F, I and J is Arg; P is Asn or a 
chemical bond; Q is Lys, Arg, Lys-Arg, Arg--Arg or a chemical bond; and R 
is --OH or --NH.sub.2, or pharmaceutically acceptable salts thereof. 
Further, the present invention provides a bronchus-expanding agent 
comprising said peptide or a pharmaceutically acceptable salt thereof as 
an active ingredient. 
Further, the present invention provides a blood-flow-improving agent 
comprising said peptide or a pharmaceutically acceptable salt thereof as 
an active ingredient. 
Further, the present invention provides a pharmaceutical composition 
comprising said peptide or a pharmaceutically acceptable salt thereof and 
a pharmaceutically acceptable carrier. 
Further, the present invention provides a method of expanding a bronchus, 
which comprises administrating an effective amount of said peptide or a 
pharmaceutically acceptable salt thereof into humans. 
Further, the present invention provides a method of improving a blood flow, 
which comprises administrating an effective amount of said peptide or a 
pharmaceutically acceptable salt thereof into humans. 
Further, the present invention provides use of said peptide or a 
pharmaceutically acceptable salt thereof for expanding a bronchus. 
Further, the present invention provides use of said peptide or 
pharmaceutically acceptable salt thereof for improving a blood flow. 
The peptide of the invention or a salt thereof is excellent in the 
durability of bronchus-expanding action resulting from the relaxation 
action of smooth muscle. Hence, the peptide of the invention and a salt 
thereof is useful as a bronchus-expanding agent. 
In addition, the peptide of the invention or a salt thereof is excellent in 
blood-flow-increasing action and in its durability. Therefore, the peptide 
of the invention or a salt thereof is also useful as a 
blood-flow-improving agent and it can exert an improvement effect on a 
blood flow at a low dosage. 
The abbreviations of amino acids, peptides, protective groups, solvents 
etc., in the present specification follow those according to International 
Union of Pure and Applied Chemistry (IU) or International Union of 
Biochemistry (IUB) or conventional abbreviations in this field. Examples 
are shown below. If there can be optical isomers for amino acids etc., 
L-isomers are meant unless otherwise specified. 
______________________________________ 
His: histidine residue 
Ser: serine residue 
Asp: aspartic acid residue 
Ala: alanine residue 
Val: valine residue 
Phe: phenylalanine 
residue 
Thr: threonine residue 
Tyr: tyrosine residue 
Asn: asparagine residue 
Leu: leucine residue 
Arg: arginine residue 
Lys: lysine residue 
Gln: glutamine residue 
Met: methionine 
Ile: isoleucine residue 
residue 
Gly: glycine residue 
Nle: norleucine residue 
Boc: t-butyloxycarbonyl group 
Aoc: t-amyloxycarbonyl group 
Bzl: benzyl group Z: benzyloxycarbonyl group 
Tos: p-toluenesulfonyl group 
OBut: t-butylester 
OMe: methylester OBz: benzylester 
ONP: p-nitrophenylester 
Bom: benzyloxymethyl group 
TFA: trifluoroacetic acid 
THF: tetrahydrofuran 
DCM: dichloromethane 
DMF: dimethylforamide 
DCC: dicyclohexylcarbodiimide 
WSC: N-ethyl-N'-dimethylamino- 
propyl-carbodimimide 
OSu: N-hydroxysuccinimide ester 
HOSu: N-hydroxysuccinimide 
HOBt: 1-hydroxybenzotriazole 
DIEA: dissopropylethylamine. 
______________________________________ 
The peptides represented by the above formula (1) or (2) can be produced by 
conventional means for synthesis of known peptides. For example, these 
peptides can be produced in e.g. an azide method, acid chloride method, 
acid anhydride method, mixed acid anhydrides method, DCC method, active 
ester method (p-nitrophenylester method, N-hydroxysuccinic acid imide 
ester method, cyanomethylester method etc.), a method using Woodward 
reagent K, carboimidazole method, oxidation-reduction method, DCC-additive 
(HONB, HOBt, HOSu) method etc. according to methods described in e.g. "The 
Peptides" Vol. 1 (1966) Schreder and Luhke: Academic Press, New York, 
U.S.A.! or "Peputido Gosei" (Peptide Synthesis) Izumiya et al., Maruzen 
K. K. (1975), Japan!. These methods can be applied to both solid-phase 
synthesis and liquid-phase synthesis. According to the above-described 
methods for synthesis of conventional polypeptides, a target peptide can 
be produced in a stepwise elongation method where each of the amino acids 
in said peptide is linked sequentially to the C-terminal amino acid or in 
a fragment condensation method where partial fragments in said peptide are 
synthesized and then coupled to one another. 
For example, the solid-phase synthesis in the stepwise elongation method 
can be carried out in accordance with the method of Merrifield, R. B. 
(Solid Phase Peptide Synthesis, J. Amer. Chem. Soc.,85, 2149-2159 (1963)) 
as follows: The C-terminal amino acid of a target peptide (that is, in the 
present invention, the amino acid sequence of the above general formula 
(1) or (2)) is protected on its amino group and then coupled to insoluble 
resin having functional groups capable of binding to carboxyl groups. The 
amino group is then deprotected, and to this free amino group is bound the 
next amino acid with its amino group protected. These steps are repeated 
many times, thus elongating the amino acid chain until the histidine 
residue at the 1-position of the formula (1) or (2). The peptide thus 
obtained is then cleaved off from the resin. 
The above method requires the protection (that is, linking a protective 
group to the amino group) and the deprotection (that is, releasing the 
protective group from the amino group) for the amino group participating 
in peptide bonding between amino acids as well as the activation of the 
carboxyl group participating in peptide bonding between amino acids. If 
necessary, a protective group is further linked to a functional group in a 
side chain of the amino acid. 
The protective group used in protecting the amino group includes those 
conventionally used. Examples are benzyloxycarbonyl (Z), 
t-butyloxycarbonyl (Boc), t-amyloxycarbonyl (Aoc), isobornyloxycarbonyl, 
p-methoxybenzyloxycarbonyl, 2-chloro-benzyloxycarbonyl, 
amadantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 
o-nitrophenylsulphenyl, and diphenylphosphinothioyl. 
The protective group used in protecting the carboxyl group includes those 
conventionally used. Examples are alkyl esters such as methyl ester, ethyl 
ester, propyl ester, butyl ester and tert-butyl ester, benzyl ester, 
p-nitrobenzyl ester, methylbenzyl ester, p-chlorobenzyl ester, benzhydryl 
ester, benzyloxycarbonylhydrazide, tert-butyloxycarbonylhydrazide and 
tritylhydrazide. 
The activation of the carboxyl group participating in peptide bonding can 
be carried out in any of the conventionally known methods described above, 
and the reagents etc. used in the activation can be selected from known 
ones. The activation of the carboxyl group involves reacting various 
reagents with said carboxyl group to form e.g. their corresponding acid 
chlorides, acid anhydrides or mixed acid anhydrides, azides, and active 
esters (for example, esters such as pentachlorophenol, p-nitrophenol, 
N-hydroxysuccinimide, N-hydroxybenztriazole, 
N-hydroy-5-norbornen-2,3-dicarboxyimide etc.). 
For the amino acid with a functional group in its side chain, the 
functional group is preferably protected during the reaction of forming a 
peptide bond. In particular, His, Tyr, Thr, Lys, Asp, Arg and Ser are 
preferably protected at the functional group in their side chain. The 
protection of the functional group is carried out by linking the 
protective group to it in a conventional method. After the completion of 
peptide synthesis, the protective group is released. 
The protective group used in protecting the imino group in His includes 
e.g. benzyloxymethyl (Bom), tosyl (Tos), benzyl (Bzl), benzyloxycarbonyl 
(z) and trityl. 
The hydroxyl group in Ser and Thr can be protected by esterification or 
etherification, but its protection is not necessarily required. The 
protective group to be introduced by esterification includes e.g. lower 
alkanoyl groups such as acetyl etc., aroyl groups such as benzoyl etc., 
and carbonic acid-derived groups such as benzoyloxycarbonyl, 
ethyloxycarbonyl etc. Preferable examples of protective groups introduced 
by etherification are benzyl (Bzl), tetrahydropyranyl and tert-butyl. 
The protective group used in protecting the hydroxyl group in Tyr includes 
e.g. benzyl (Bzl), bromobenzyloxycarbonyl (BrZ), dichlorobenzyl (Cl.sub.2 
-Bzl), benzyloxycarbonyl (z), acetyl, and tosyl (Tos). 
The protective group used in protecting the amino group in Lys includes 
e.g. benzyloxycarbonyl (Z), chlorobenzyloxycarbonyl (Cl-Z), dichlorobenzyl 
(Cl.sub.2 -Bzl), t-butyloxycarbonyl (Boc), and tosyl (Tos). 
The protective group used in protecting the guanidino group in Arg includes 
e.g. tosyl (Tos), nitro, benzyloxycarbonyl (z), and t-amyloxycarbonyl 
(Aoc). 
The protection of the carboxyl group in Asp is carried out by 
esterification with e.g. benzyl alcohol, methanol, ethanol or 
tert-butanol. 
The reaction of forming a peptide bond can sometimes be carried out in the 
presence of a condensation reagent e.g. a carbodiimide reagent such as 
dicyclohexylcarbodiimide (DCC), carbodiimidazole etc., or tetraethyl 
pyrophosphate, benzotriazole-N-hydroxytrisdimethylaminophosphonium 
hexafluorophosphate (Bop reagent). 
The insoluble resin used in the above solid-phase synthesis may be any 
resin having functional groups capable of binding to carboxyl groups. 
Examples are benzhydrylamine resin (BHA resin), chloromethyl resin, 
oxymethyl resin, aminomethyl resin, p-methylbenzhydrylamine resin (MBHA 
resin), 4-aminomethylphenoxymethyl resin, 4-hydroxymethylphenoxymethyl 
resin, 4-oxymethylphenylacetamidem ethyl resin (PAM resin) etc. The 
linking of the amino acid to the resin and cleavage of the synthesized 
peptide from the resin can be carried out in any of the conventionally 
known methods. 
The solvent used in the above solid-phase synthesis may be any solvents 
known to be usable in forming a peptide bond, and examples are water-free 
or water-containing dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 
pyridine, chloroform, dioxane, dichloromethane (DCM), tetrahydrofuran 
(THF), ethyl acetate, N-methylpyrrolidone, and hexamethyl phosphate 
triamide (HMPA). These solvents can be used singly or in combination. 
The peptide thus prepared can be desalted and purified in a usual manner. 
For example, it can be desalted and purified by ion-exchange 
chromatography on DEAE-cellulose etc., partition chromatography on 
Sephadex LH-20, Sephadex G-25 etc., normal phase chromatography on silica 
gel etc., reverse phase chromatography on ODS-silica gel etc., or in high 
performance liquid chromatography (HPLC), etc. 
The pharmaceutically acceptable salts of the peptides represented by the 
above formula (1) or (2) include e.g. acetate, hydrochloride, phosphate 
etc. 
The peptide of the invention and salts thereof are useful as a 
bronchus-expanding agent by virtue of their bronchus-expanding action. 
Hence, the bronchus-expanding agent containing the peptide of the 
invention or a salt thereof as an active ingredient is useful in 
suppression and improvement of asthma etc. 
As the bronchus-expanding agent, the peptide of the invention or a salt 
thereof can be administered as such, but usually it is mixed in a usual 
manner with various pharmaceutically acceptable carriers to be formed into 
preparations in the form of liquid, gel or solid, for oral or parenteral 
purposes. The administration method includes inhalation (e.g. with an 
aerosol agent), injection, application etc. 
The dosage of the present peptide or a salt thereof when used as a 
bronchus-expanding agent is preferably in the range of about 1 ng/kg to 1 
mg/kg (body weight)/day/person, although the dosage is determined 
depending on the purpose for use, symptom, the age and body weight etc. of 
a patient, method of administration, etc. 
Further, the peptide of the invention or a salt thereof is excellent in 
blood-flow increasing action which is superior in durability. Hence, a 
blood-flow improving agent comprising the peptide of the invention or a 
salt thereof is effective for treatment of an ulcer, frostbite, bedsore, 
impotence etc. as well as for restoration and growth of hair. 
The peptide of the invention or a salt thereof when used as a blood-flow 
improving agent is mixed with a conventional pharmaceutically acceptable 
carrier to be formed into pharmaceutical preparations, preferably in the 
form of an injection. 
For preparation of such an injection, the resulting preparation is 
preferably sterilized and isotonic relative to blood. Any conventionally 
used diluents can be used for manufacture of a preparation in the form of 
an injection. Example of diluents are water, physiological saline etc. In 
this case, a sufficient amount of common salt or glycerin to prepare an 
isotonic solution can be contained in a preparation in the form of an 
injection. This preparation can contain a conventional buffer, soothing 
agent, preservative etc. and if necessary a coloring agent, preservative, 
flavoring agent, sweetener, etc., as well as other pharmaceuticals. The 
above active ingredient for injection purposes can be dissolved before use 
in distilled water. The method of administering the resulting 
pharmaceutical preparation is varied depending on the form of the 
preparation. For example, the preparation in the form of an injection is 
intravenously administered singly or after being mixed with conventional 
complements such as amino acids etc. 
The present peptide or a salt thereof when used as a blood-flow-improving 
agent is administered preferably in 2 to 4 portions daily in the range of 
about 1 ng/kg to 1 mg/kg (body weight)/day/person, although the dosage is 
determined depending on the purpose for use, symptom conditions, etc.

BEST EMBODIMENTS FOR CARRYING OUT THE INVENTION 
Hereinafter, the present invention is described in detail by reference to 
Examples, which however are not intended to limit the scope of the present 
invention. 
In the following examples, the identification of the resulting purified 
peptide was carried out by measurement of retention time in high 
performance liquid chromatography (HPLC), measurement of degree of optical 
rotation, and amino acid analysis, as shown below. 
High performance liquid chromatography (HPLC) 
For analysis in high performance liquid chromatography, LC-Module-1 (Waters 
Ltd. Japan) was used. 
(Analysis conditions in HPLC) 
Column : TSK Gel ODS-120T (4.6.times.250 mm) 
Solvent: 0.1% TFA-acetonitrile (linear gradient of from 20% to 50% 
acetonitrile for 30 min). 
Flow rate: 1 ml/min. 
Detection wavelength: 220 nm. 
Degree of optical rotation 
For measurement of degree of optical rotation, DIC-370 (Nippon Bunko Kogyo 
K. K., Japan) was used. 
(Conditions for measurement of degree of optical rotation) 
Light source: Na lamp, 589 nm. 
Temperature: 20.degree. C. 
Layer length: 100 mm. 
Concentration: 1% (0.1M in acetic acid). 
Amino acid analysis 
The amino acid analysis was carried out by hydrolyzing a peptide at 
110.degree. C. for 20 hours in 6N HCl containing 0.1% phenol and analyzing 
the hydrolysate in Hitachi Amino Acid Analyzer L-8500 (Hitachi, Ltd., 
Japan). 
EXAMPLE 1 
Production of peptide 1 
The solid-phase synthesizer used was Peptide Synthesizer 9600 (Milligen 
Bioresearch). Peptide 1 shown in SEQ ID NO:1 whose C-terminal group was 
amidated was produced by the following solid-phase synthesis and 
purification. 
First, 694 mg MBHA resin (0.72 mmol/g amino group, manufactured by Peptide 
Kenkyuzyo K. K., Japan) was introduced into a reactor for peptide 
solid-phase synthesis and treated with 8 ml DCM (4 times, 1 minute each), 
8 ml DCM containing 60% TFA (20 minutes), 4 ml DCM (3 times, 15 seconds 
each), 3 ml DMF solution containing 1 ml DIEA (2 times, 1 minute each), 
and 8 ml DMF (6 times, 40 seconds each) in this order with stirring in an 
argon stream. After each treatment, filtration was carried out. 
Separately, 2 mmol amino-group-protected amino acid Boc-Leu--OH 
corresponding to the amino acid residue at the 27-position in SEQ ID NO:1 
was dissolved in 4 ml DCM. This solution was introduced into a vessel for 
amino acid activation, and after 3 ml DCC (0.5M in DCM) and 4 ml HOBt 
(0.5M in DCM) were added to it, and they were reacted for 30 minutes. 
Thereafter, the reaction solution was filtered off and the filtrate was 
transferred to a concentration vessel. 3 ml DMF was added to it and the 
DCM was distilled off in an argon stream. Then, 3 ml DMF was added to the 
concentrate, and it was transferred to the above reactor for peptide 
solid-phase synthesis and reacted for 30 minutes. Then, the reaction 
mixture was washed with 8 ml DCM (6 times, 20 seconds each). Separately, 2 
mmol Boc-Leu--OH was dissolved in 4 ml DCM and the solution was introduced 
into the reactor for amino acid activation and the same procedure as 
described above was repeated (double-coupling) and filtered. Boc-Leu-MBHA 
resin was thus obtained. 
Then, the Boc-Leu-MBHA resin was washed with 8 ml DCM (4 times, 1 minute 
each) and filtered. This resin was treated with 8 ml DCM containing 60% 
TFA (20 minutes), 4 ml DCM (3 times, 15 seconds each) , 3 ml DMF solution 
containing 1 ml DIEA (2 times, 1 minute each), and 8 ml DMF (6 times, 40 
seconds each) in this order with stirring in an argon stream. After each 
treatment, filtration was carried out. 
Separately, 2 mmol amino-group-protected amino acid Boc-Val--OH 
corresponding to the amino acid residue at the 26-position in SEQ ID NO:1 
was dissolved in 4 ml DCM and introduced into a vessel for amino acid 
activation, and after 1.5 ml DCC (0.5M in DCM) was added to it, and they 
were reacted for 7 minutes. Thereafter, the reaction solution was filtered 
off and the filtrate was transferred to a concentration vessel. 3 ml DMF 
was added to it and the DCM was distilled off in an argon stream. Then, 3 
ml DMF was added to the concentrate, and it was transferred to the above 
reactor for peptide solid-phase synthesis and reaction was carried out for 
30 minutes. Then, the reaction mixture was washed with 8 ml DCM (6 times, 
20 seconds each) and filtered. Boc-Val-Leu-MBHA resin was thus obtained. 
Then, the following amino-group-protected amino acids, corresponding to the 
25th to 1st amino acids in SEQ ID NO:1 respectively, were coupled 
successively to it. 
______________________________________ 
Amino Acid Amount 
No. Protected Amino Acid 
(mmol) 
______________________________________ 
25 Boc-Ala-OH 2 
24 Boc-Ala-OH 2 .times. 2 
23 Boc-Leu-OH 2 
22 Boc-Tyr(Bz1)-OH 
2 
21 Boc-Arg(Tos)-OH 
2 .times. 2 
20 Boc-Arg(Tos)-OH 
2 .times. 2 
19 Boc-Val-OH 2 
18 Boc-Ala-OH 2 
17 Boc-Leu-OH 2 
16 Boc-Gln-OH 2 .times. 2 
15 Boc-Arg(Tos)-OH 
2 .times. 2 
14 Boc-Arg(Tos)-OH 
2 .times. 2 
13 Boc-Tyr(Bzl)-OH 
2 
12 Boc-Arg(Tos)-OH 
2 .times. 2 
11 Boc-Ser(Bzl)OH 
2 
10 Boc-Tyr(Bzl)-OH 
2 
9 Boc-Ser(Bzl)-OH 
2 
8 Boc-Asp(OBz)-OH 
2 
7 Boc-Thr(Bzl)-OH 
2 
6 Boc-Phe-OH 2 
5 Boc-Ile-OH 2 
4 Boc-Gly-OH 2 
3 Boc-Asp(OBz)-OH 
2 
2 Boc-Ser(Bzl)-OH 
2 
1 Boc-His(Bom)-OH 
2 .times. 2 
______________________________________ 
In the above solid-phase synthesis, double coupling was carried out in the 
case of Asn, Arg, Gln and His. 
2.76 g of the following protected peptide-HBHA resin was thus obtained: 
Boc-His(Bom)-Ser(Bzl)-Asp(OBz)-Gly-Ile-Phe-Thr(Bzl)-Asp(OBz)-Ser(Bzl)-Tyr 
(Bzl)-Ser(Bzl)-Arg(Tos)-Tyr(Bzl)-Arg(Tos)-Arg(Tos)-Gln-Leu-Ala-Val-Arg 
(Tos)-Arg(Tos)-Tyr(Bzl)-Leu-Ala-Ala-Val-Leu-MBHA resin. 
5 ml anisole was added to 2.76 g of the above protected peptide-MBHA resin, 
and 25 ml anhydrous hydrogen fluoride was added to it and the mixture was 
reacted with stirring at 0.degree. C. for 1 hour. Thereafter, the 
anhydrous hydrogen fluoride was distilled off under reduced pressure. The 
residue was washed with ether and then the peptide was extracted from it 
with 100 ml of 0.1M acetic acid. 
The extract was stirred for 15 minutes together with 20 ml anion-exhange 
resin Amberlite IR-410 and the insoluble resin was removed by filtration. 
The suspension thus obtained was filtered through 0.22.mu. Millipore 
filter and the filtrate was lyophilized. 823 mg white powder was thus 
obtained. Then, this sample was applied to a column of CM-cellulose 
(2.5.times.30 cm) and eluted with a linear gradient of from 0.05M to 0.5M 
AcONH.sub.4 (pH 7.0), and fraction Nos. 69 to 82 (10 ml/fraction) were 
combined and gave 382 mg of the desired peptide in partially purified 
form. This peptide was further purified in preparative high performance 
liquid chromatography. 
Column: TSK Gel ODS-120T (21.5.times.300 mm). 
Solvent: 0.1% TFA-acetonitrile (linear gradient of from 20% to 40% 
acetonitrile). 
Flow rate: 10 ml/min. 
The eluate corresponding to a peak of peptide 1 as the desired substance 
was lyophilized. The peptide in purified form was obtained in said HPLC in 
an amount of 63 mg per 100 mg of the partially purified peptide. The 
purified peptide 1 was determined for retention time in HPLC and for 
degree of optical rotation, and its amino acids were analyzed. The results 
are shown below: 
Retention time in HPLC: 27.2 min. 
.alpha.!.sub.D : -55.8.degree. 
Amino acid analysis: 
Asp(2) 2.17, Thr(1) 0.98, Ser(3) 2.45, Glu(1) 1.09, Gly(1) 1.03, Ala(3) 
3.18, Val(2) 2.14, Ile(1) 0.96, Leu(3) 3.07, Tyr(3) 2.73, Phe(1) 0.92, 
His(1) 1.12, Arg(5) 5.16. 
EXAMPLE 2 
Peptide 2 shown in SEQ ID NO:2 whose C-terminal carboxyl group was amidated 
was obtained in purified form in the same manner as in Example 1. The 
yield of this purified peptide is shown below. Its retention time in HPLC 
and its degree of optical rotation were determined, and its amino acids 
were analyzed. The results are shown below: 
Yield: 73 mg 
Retention time in HPLC: 27.6 min. 
.alpha.!.sub.D : -53.2.degree. 
Amino acid analysis: 
Asp(2) 2.10, Thr(1) 0.97, Ser(3) 2.46, Glu(1) 0.97, Gly(2) 1.97, Ala(3) 
3.15, Val(2) 2.13, Ile(1) 1.00, Leu(3) 3.08, Tyr(3) 2.75, Phe(1) 0.98, 
His(1) 0.99, Arg(5) 4.54. 
EXAMPLE 3 
Peptide 3 shown in SEQ ID NO:3 whose C-terminal carboxyl group was amidated 
was obtained in purified form in the same manner as in Example 1. The 
yield of this purified peptide is shown below. Its retention time in HPLC 
and its degree of optical rotation were determined, and its amino acids 
were analyzed. The results are shown below: 
Yield: 88 mg 
Retention time in HPLC: 27.0 min. 
.alpha.!.sub.D : -52.8.degree. 
Amino acid analysis: 
Asp(2) 2.11, Thr(1) 0.95, Ser(3) 2.56, Glu(1) 0.97, Gly(2) 2.00, Ala(3) 
3.15, Val(2) 2.10, Ile(1) 1.00, Leu(3) 3.08, Tyr(3) 2.75, Phe(1) 0.98, 
His(1) 1.03, Arg(5) 5.14, Lys(1) 1.01. 
EXAMPLE 4 
Peptide 4 shown in SEQ ID NO:4 whose C-terminal carboxyl group was amidated 
was obtained in purified form in the same manner as in Example 1. The 
yield of this purified peptide is shown below. Its retention time in HPLC 
and its degree of optical rotation were determined, and its amino acids 
were analyzed. The results are shown below: 
Yield: 84 mg 
Retention time in HPLC: 25.1 min. 
.alpha.!.sub.D : -54.3.degree. 
Amino acid analysis: 
Asp(2) 2.09, Thr(1) 0.96, Ser(3) 2.44, Glu(1) 0.97, Gly(2) 2.01, Ala(3) 
3.11, Val(2) 2.03, Ile(1) 1.02, Leu(3) 3.08, Tyr(3) 2.74, Phe(1) 0.97, 
His(1) 1.05, Arg(6) 6.10 
EXAMPLE 5 
Peptide 5 shown in SEQ ID NO:5 whose C-terminal carboxyl group was amidated 
was obtained in purified form in the same manner as in Example 1. The 
yield of this purified peptide is shown below. Its retention time in HPLC 
and its degree of optical rotation were determined, and its amino acids 
were analyzed. The results are shown below: 
Yield: 76 mg 
Retention time in HPLC: 24.4 min. 
.alpha.!.sub.D : -52.2.degree. 
Amino acid analysis: 
Asp(2) 2.11, Thr(1) 0.96, Ser(3) 2.47, Glu(1) 1.01, Gly(2) 2.00, Ala(3) 
3.03, Val(2) 2.03, Ile(1) 1.02, Leu(3) 3.03, Tyr(3) 2.70, Phe(1) 0.97, 
His(1) 1.07, Arg(6) 6.15, Lys(1) 1.02. 
EXAMPLE 6 
Peptide 6 shown in SEQ ID NO:6 whose C-terminal carboxyl group was amidated 
was obtained in purified form in the same manner as in Example 1. The 
yield of this purified peptide is shown below. Its retention time in HPLC 
and its degree of optical rotation were determined, and its amino acids 
were analyzed. The results are shown below: 
Yield: 85 mg 
Retention time in HPLC: 23.6 min. 
.alpha.!.sub.D : -53.7.degree. 
Amino acid analysis: 
Asp(2) 2.09, Thr(1) 0.94, Ser(3) 2.51, Glu(1) 1.05, Gly(2) 2.00, Ala(3) 
3.03, Val(2) 2.01, Ile(1) 1.02, Leu(3) 3.03, Tyr(3) 2.77, Phe(1) 0.97, 
His(1) 1.07, Arg(7) 7.11. 
EXAMPLE 7 
Peptide 7 shown in SEQ ID NO:7 was obtained in purified form in the same 
manner as in Example 1 except that 704 mg of Boc-Arg (Tos)-PAM resin (0.68 
mmol/g amino group, produced by Peptide Kenkyuzyo K. K.), i.e. a resin 
having the C-terminal amino acid residue of the target peptide, was used. 
The yield of this purified peptide is shown below. Its retention time in 
HPLC and its degree of optical rotation were determined, and its amino 
acids were analyzed. The results are shown below: 
Yield: 84 mg 
Retention time in HPLC: 23.8 min. 
.alpha.!.sub.D : -56.7.degree. 
Amino acid analysis: 
Asp(2) 2.12, Thr(1) 0.96, Ser(3) 2.47, Glu(1) 1.01, Gly(2) 2.00, Ala(3) 
3.03, Val(2) 2.03, Ile(1) 1.02, Leu(3) 3.03, Tyr(3) 2.70, Phe(1) 0.97, 
His(1) 1.03, Arg(6) 6.10, Lys(1) 1.02. 
EXAMPLE 8 
Peptide 8 shown in SEQ ID NO:8 whose C-terminal carboxyl group was amidated 
was obtained in purified form in the same manner as in Example 1. The 
yield of this purified peptide is shown below. Its retention time in HPLC 
and its degree of optical rotation were determined, and its amino acids 
were analyzed. The results are shown below: 
Yield: 68 mg 
Retention time in HPLC: 26.9 min. 
.alpha.!.sub.D : -58.7.degree. 
Amino acid analysis: 
Asp(5) 5.11, Thr(2) 1.72, Ser(2) 1.61, Glu(1) 1.10, Ala(2) 2.00, Val(2) 
2.00, Ile(1) 1.02, Leu(4) 4.15, Tyr(2) 1.86, Phe(1) 0.99, His(1) 0.95, 
Arg(5) 4.75. 
EXAMPLE 9 
Peptide 9 shown in SEQ ID NO:9 whose C-terminal carboxyl group was amidated 
was obtained in purified form in the same manner as in Example 1. The 
yield of this purified peptide is shown below. Its retention time in HPLC 
and its degree of optical rotation were determined, and its amino acids 
were analyzed. The results are shown below: 
Yield: 76 mg 
Retention time in HPLC: 25.4 min. 
.alpha.!.sub.D : -52.9.degree. 
Amino acid analysis: 
Asp(5) 5.11, Thr(2) 1.83, Ser(2) 1.49, Glu(1) 1.09, Gly(1) 1.04, Ala(2) 
2.01, Val(2) 2.01, Ile(1) 0.96, Leu(4) 4.19, Tyr(2) 2.03, Phe(1) 1.01, 
His(1) 1.07, Arg(6) 6.10, Lys(1) 1.05. 
EXAMPLE 10 
Peptide 10 shown in SEQ ID NO:10 whose C-terminal carboxyl group was 
amidated was obtained in purified form in the same manner as in Example 1. 
The yield of this purified peptide is shown below. Its retention time in 
HPLC and its degree of optical rotation were determined, and its amino 
acids were analyzed. The results are shown below: 
Yield: 77 mg 
Retention time in HPLC: 24.9 min. 
.alpha.!.sub.D : -52.5.degree. 
Amino acid analysis: 
Asp(5) 5.12, Thr(2) 1.83, Ser(2) 1.55, Glu(1) 1.08, Gly(1) 1.03, Ala(2) 
2.01, Val(2) 2.01, Ile(1) 0.96, Leu(4) 4.17, Tyr(2) 2.03, Phe(1) 1.01, 
His(1) 1.03, Arg(6) 6.15. 
EXAMPLE 11 
Peptide 11 shown in SEQ ID NO:11 whose C-terminal carboxyl group was 
amidated was obtained in purified form in the same manner as in Example 1. 
The yield of this purified peptide is shown below. Its retention time in 
HPLC and its degree of optical rotation were determined, and its amino 
acids were analyzed. The results are shown below: 
Yield: 87 mg 
Retention time in HPLC: 23.5 min. 
.alpha.!.sub.D : -55.5.degree. 
Amino acid analysis: 
Asp(5) 5.11, Thr(2) 1.83, Ser(2) 1.49, Glu(1) 1.09, Gly(1) 1.04, Ala(2) 
2.01, Val(2) 2.01, Ile(1) 0.96, Leu(4) 4.19, Tyr(2) 2.03, Phe(1) 1.01, 
His(1) 1.07, Arg(6) 6.10, Lys(1) 1.05. 
EXAMPLE 12 
Peptide 12 shown in SEQ ID NO:12 whose C-terminal carboxyl group was 
amidated was obtained in purified form in the same manner as in Example 1. 
The yield of this purified peptide is shown below. Its retention time in 
HPLC and its degree of optical rotation were determined, and its amino 
acids were analyzed. The results are shown below: 
Yield: 78 mg 
Retention time in HPLC: 23.0 min. 
.alpha.!.sub.D : -56.2.degree. 
Amino acid analysis: 
Asp(5) 5.13, Thr(2) 1.84, Ser(2) 1.49, Glu(1) 1.08, Gly(1) 1.03, Ala(2) 
2.01, Val(2) 2.01, Ile(1) 0.96, Leu(4) 4.19, Tyr(2) 2.03, Phe(1) 1.01, 
His(1) 1.02, Arg(7) 7.11. 
EXAMPLE 13 
Peptide 13 shown in SEQ ID NO:13 was obtained in purified form in the same 
manner as in Example 1 except that 704 mg of Boc-Arg(Tos)-PAM resin, i.e. 
a resin having the C-terminal amino acid residue of the target peptide, 
was used. The yield of this purified peptide is shown below. Its retention 
time in HPLC and its degree of optical rotation were determined, and its 
amino acids were analyzed. The results are shown below: 
Yield: 83 mg 
Retention time in HPLC: 22.8 min. 
.alpha.!.sub.D : -58.6.degree. 
Amino acid analysis: 
Asp(5) 5.15, Thr(2) 1.83, Ser(2) 1.53, Glu(1) 1.09, Gly(1) 1.01, Ala(2) 
2.01, Val(2) 2.01, Ile(1) 1.02, Leu(4) 4.10, Tyr(2) 1.86, Phe(1) 1.00, 
His(1) 0.95, Arg(6) 6.15. 
EXAMPLE 14 
Peptide 14 shown in SEQ ID NO:14 whose C-terminal carboxyl group was 
amidated was obtained in purified form in the same manner as in Example 1. 
The yield of this purified peptide is shown below. Its retention time in 
HPLC and its degree of optical rotation were determined, and its amino 
acids were analyzed. The results are shown below: 
Yield: 64 mg 
Retention time in HPLC: 25.1 min. 
.alpha.!.sub.D : -52.7.degree. 
Amino acid analysis: 
Asp(2) 2.17, Thr(1) 0.98, Ser(3) 2.55, Glu(1) 1.03, Gly(1) 1.03, Ala(3) 
3.18, Met(1) 0.96, Val(2) 2.14, Ile(1) 0.96, Leu(2) 2.07, Tyr(3) 2.73, 
Phe(1) 1.00, His(1) 1.01, Arg(5) 5.15. 
EXAMPLE 15 
Peptide 15 shown in SEQ ID NO:15 whose C-terminal carboxyl group was 
amidated was obtained in purified form in the same manner as in Example 1. 
The yield of this purified peptide is shown below. Its retention time in 
HPLC and its degree of optical rotation were determined, and its amino 
acids were analyzed. The results are shown below: 
Yield: 62 mg 
Retention time in HPLC: 26.2 min. 
.alpha.!.sub.D : -52.5.degree. 
Amino acid analysis: 
Asp(2) 2.11, Thr(1) 0.95, Ser(3) 2.45, Glu(1) 1.04, Gly(1) 1.03, Ala(3) 
3.18, Val(2) 2.14, Ile(1) 0.96, Leu(2) 2.10, Tyr(3) 2.73, Phe(1) 0.96, 
His(1) 1.02, Arg(5) 5.16, nLeu(1) 1.02. 
EXAMPLE 16 
Purified peptides 1 to 15 obtained in Examples 1 to 15 were examined for 
their bronchus-expanding action in the following manner and compared with 
that of VIP and AP. 
Male guinea pigs each weighing 450 to 500 g were cut at the jugular veins 
to open the chests, and immediately the tracheae were immediately removed. 
Their tracheae were immediately placed in a Krebs solution and cut into 
loops and then formed into a chain by linking the cartilage parts with one 
another via a steel string. Then, the cartilage parts in the opposite side 
to the smooth muscular parts were cut such that the smooth muscular parts 
were attached to one another. The resulting sample was placed in an about 
10-ml isothermic organ bath and perpendicularly suspended. The lower part 
was fixed and the upper part was connected to a grass force transducer 
with a load of 1.4 g. A gas of 94% oxygen and 6% carbon dioxide was 
sufficiently blown into this sample while a Krebs solution containing 
0.1.mu. mol/l carbachol was dropped to it at a rate of 0.33 ml/min. to 
determine relaxation action. Because the relaxation reaction occurs 
depending on the content of a test sample in this drop solution, this can 
be used to compare the relaxation action of VIP and AP and that of the 
peptides of the invention on the bronchial smooth muscle. 
A solution of each of VIA, AP and the peptides of the invention was 
prepared in a concentration as high as 100-fold than the final 
concentration so that its concentration in the organ bath could be 
adjusted. 30 minutes after the carbachol-containing Krebs solution was 
dropped, 100 .mu.l of the sample solution was dropped. 
It was assumed that the degree of contraction of the smooth muscle is 0 in 
the absence of carbachol and the degree is 100 in the presence of 
carbachol. Minimum contraction degree A (i.e. maximum relaxation value) in 
the presence of the sample was determined, and maximum relaxation degree B 
was calculated as follows: 
Maximum relaxation degree B (%)=100-A 
The time required to reach half of the maximum relaxation value (referred 
to hereinafter as "half-time T") after the addition of the sample was 
determined. 
A change in the relaxation degree of the bronchial smooth muscle with time 
was determined using each peptide at 3 dosages of 0.3 .mu.M, 1 .mu.M, and 
3 .mu.M respectively. The results are shown in the drawings: AP in FIG. 
1; peptide 5 obtained in Example 5, in FIG. 2; VIP in FIG. 3; and peptide 
11 obtained in Example 11, in FIG. 4. The maximum relaxation degree B at a 
dosage of 3 .mu.M is 75% for peptide 5 and 80% for peptide 11, and 
half-time T is 360 minutes or more in both the cases. From these results, 
the peptides of the invention, while possessing a similar relaxation 
effect to those of VIP and AP, exhibit superior durability, i.e. a 
longer working period than those of VIP and AP. 
Table 1 shows maximum relaxation degree B and half-life T for each peptide 
at each dosage. 
TABLE 1 
______________________________________ 
0.3 .mu.M 
1 .mu.M 3 .mu.M 
B T B T B T 
(%) (min) (%) (min) 
(%) (min) 
______________________________________ 
AP 40 60 90 60 90 100 
VIP 45 130 80 150 85 240 
Peptide 1 in Example 1 
50 100 80 240 85 &gt;360 
Peptide 2 in Example 2 
55 120 80 240 85 &gt;360 
Peptide 3 in Example 3 
55 120 80 240 85 &gt;360 
Peptide 4 in Example 4 
55 120 80 240 85 &gt;360 
Peptide 5 in Example 5 
60 120 80 &gt;360 75 &gt;360 
Peptide 6 in Example 6 
65 150 80 &gt;360 75 &gt;360 
Peptide 7 in Example 7 
60 120 80 &gt;360 75 &gt;360 
Peptide 8 in Example 8 
50 150 80 300 80 &gt;360 
Peptide 9 in Example 9 
55 180 80 300 80 &gt;360 
Peptide 10 in Example 10 
55 200 80 300 80 &gt;360 
Peptide 11 in Example 11 
65 300 85 &gt;360 85 &gt;360 
Peptide 12 in Example 12 
65 300 90 &gt;360 85 &gt;360 
Peptide 13 in Example 13 
65 300 90 &gt;360 85 &gt;360 
Peptide 14 in Example 14 
40 100 80 240 85 &gt;360 
Peptide 15 in Example 15 
40 100 80 240 85 &gt;360 
______________________________________ 
EXAMPLE 17 
Purified peptides 1 to 15 obtained in Examples 1 to 15 were examined for 
their blood-flow increasing action and compared with those of VIP and 
AP. 
Wistar male rats each weighing 220 to 250 g were anesthetized by 
intraperitoneal administration of pentobarbital (25 mg/kg) and fixed on 
their back. An arterial canula was inserted into the carotid and then 
connected to a blood-pressure transducer (DX-300, Nippon Koden, Japan) via 
a doom kit (SCK-512, Nippon Koden) filled with heparin-containing 
physiological saline (10 U/ml) to measure blood pressure with a strain 
blood-pressure amplifier (AP-601G, Nippon Koden). Further, a blood-flow 
measurement probe (FR-030T, Nippon Koden) was attached to the right 
femoral artery and then connected to an electromagnetic blood-flow meter 
(MFV-3200, Nippon Koden) to measure blood flow. The dosage of each peptide 
intraarterially administrated was 100 pmol/kg. 
Table 2 shows an increase in blood flow in rats given each peptide. 
TABLE 2 
______________________________________ 
Blood Flow (ml/min) 
______________________________________ 
AP 16.04 .+-. 5.92 
VIP 10.18 .+-. 3.38 
Peptide 1 in Example 1 
50.02 .+-. 42.34 
Peptide 2 in Example 2 
52.63 .+-. 39.58 
Peptide 3 in Example 3 
52.13 .+-. 37.85 
Peptide 4 in Example 4 
51.87 .+-. 37.24 
Peptide 5 in Example 5 
54.60 .+-. 41.05 
Peptide 6 in Example 6 
53.71 .+-. 46.46 
Peptide 7 in Example 7 
55.05 .+-. 46.71 
Peptide 8 in Example 8 
53.75 .+-. 37.31 
Peptide 9 in Example 9 
54.26 .+-. 35.95 
Peptide 10 in Example 10 
55.86 .+-. 34.69 
Peptide 11 in Example 11 
57.04 .+-. 38.67 
Peptide 12 in Example 12 
57.34 .+-. 41.88 
Peptide 13 in Example 13 
56.76 .+-. 36.15 
Peptide 14 in Example 14 
52.92 .+-. 32.12 
Peptide 15 in Example 15 
52.63 .+-. 31.05 
______________________________________ 
No particular change was observed in the blood pressure of rats given 100 
pmol/kg of each of peptides 1 to 15. 
EXAMPLE 18 
Acute Toxicity Test 
Peptides 1 to 15 obtained in Examples 1 to 15 were dissolved in 
physiological saline and administered intravenously into mice at a dosage 
of 10 mg/kg. The result indicated that every mouse survived. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 15 
(2) INFORMATION FOR SEQ ID NO: 1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 
HisSerAspGlyIlePheThrAspSerTyrSerArgTyrArgArgGln 
151015 
LeuAlaValArgArgTyrLeuAlaAlaValLeu 
2025 
(2) INFORMATION FOR SEQ ID NO: 2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 
HisSerAspGlyIlePheThrAspSerTyrSerArgTyrArgArgGln 
151015 
LeuAlaValArgArgTyrLeuAlaAlaValLeuGly 
2025 
(2) INFORMATION FOR SEQ ID NO: 3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 
HisSerAspGlyIlePheThrAspSerTyrSerArgTyrArgArgGln 
151015 
LeuAlaValArgArgTyrLeuAlaAlaValLeuGlyLys 
2025 
(2) INFORMATION FOR SEQ ID NO: 4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 
HisSerAspGlyIlePheThrAspSerTyrSerArgTyrArgArgGln 
151015 
LeuAlaValArgArgTyrLeuAlaAlaValLeuGlyArg 
2025 
(2) INFORMATION FOR SEQ ID NO: 5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 
HisSerAspGlyIlePheThrAspSerTyrSerArgTyrArgArgGln 
151015 
LeuAlaValArgArgTyrLeuAlaAlaValLeuGlyLysArg 
202530 
(2) INFORMATION FOR SEQ ID NO: 6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 
HisSerAspGlyIlePheThrAspSerTyrSerArgTyrArgArgGln 
151015 
LeuAlaValArgArgTyrLeuAlaAlaValLeuGlyArgArg 
202530 
(2) INFORMATION FOR SEQ ID NO: 7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 
HisSerAspGlyIlePheThrAspSerTyrSerArgTyrArgArgGln 
151015 
LeuAlaValArgArgTyrLeuAlaAlaValLeuGlyLysArg 
202530 
(2) INFORMATION FOR SEQ ID NO: 8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 
HisSerAspAlaValPheThrAspAsnTyrThrArgLeuArgArgGln 
151015 
LeuAlaValArgArgTyrLeuAsnSerIleLeuAsnGly 
2025 
(2) INFORMATION FOR SEQ ID NO: 9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 
HisSerAspAlaValPheThrAspAsnTyrThrArgLeuArgArgGln 
151015 
LeuAlaValArgArgTyrLeuAsnSerIleLeuAsnGlyLys 
202530 
(2) INFORMATION FOR SEQ ID NO: 10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: 
HisSerAspAlaValPheThrAspAsnTyrThrArgLeuArgArgGln 
151015 
LeuAlaValArgArgTyrLeuAsnSerIleLeuAsnGlyArg 
202530 
(2) INFORMATION FOR SEQ ID NO: 11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: 
HisSerAspAlaValPheThrAspAsnTyrThrArgLeuArgArgGln 
151015 
LeuAlaValArgArgTyrLeuAsnSerIleLeuAsnGlyLysArg 
202530 
(2) INFORMATION FOR SEQ ID NO: 12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: 
HisSerAspAlaValPheThrAspAsnTyrThrArgLeuArgArgGln 
151015 
LeuAlaValArgArgTyrLeuAsnSerIleLeuAsnGlyArgArg 
202530 
(2) INFORMATION FOR SEQ ID NO: 13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: 
HisSerAspAlaValPheThrAspAsnTyrThrArgLeuArgArgGln 
151015 
LeuAlaValArgArgTyrLeuAsnSerIleLeuAsnGlyLysArg 
202530 
(2) INFORMATION FOR SEQ ID NO: 14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: 
HisSerAspGlyIlePheThrAspSerTyrSerArgTyrArgArgGln 
151015 
MetAlaValArgArgTyrLeuAlaAlaValLeu 
2025 
(2) INFORMATION FOR SEQ ID NO: 15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Norleucine (Nle) 
(B) LOCATION: 17 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: 
HisSerAspGlyIlePheThrAspSerTyrSerArgTyrArgArgGln 
151015 
XaaAlaValArgArgTyrLeuAlaAlaValLeu 
2025 
__________________________________________________________________________