U.S. patent application number 15/307262 was filed with the patent office on 2017-02-23 for lipidated peptides as neuroprotective agents.
This patent application is currently assigned to USTAV ORGANICKE CHEMIE A BIOCHEMIE AV CR, V.V.I.. The applicant listed for this patent is USTAV ORGANICKE CHEMIE A BIOCHEMIE AV CR, V.V.I.. Invention is credited to Miroslava BLECHOVA, Jaroslav KUNES, Lenka MALETINSKA, Barbora MIKULASKOVA, Andrea SPOLCOVA, Blanka ZELEZNA.
Application Number | 20170051031 15/307262 |
Document ID | / |
Family ID | 53432908 |
Filed Date | 2017-02-23 |
United States Patent
Application |
20170051031 |
Kind Code |
A1 |
MALETINSKA; Lenka ; et
al. |
February 23, 2017 |
LIPIDATED PEPTIDES AS NEUROPROTECTIVE AGENTS
Abstract
Lipidated neuropeptides based on PrRP31, PrRP20, containing C14
and/or C16 fatty acid, in which sequence of IRPVGRF-NH.sub.2 at the
C-terminus is variable in the site of isoleucine, valine and
phenylalanine; the fatty acid is bound in position 1 or 11 for
PrRP31 or its analog and in position 1 or 7 for PrRP20 or its
analog; the fatty acid is bound directly or through a hydrophilic
linker X.sup.2, for use in the treatment and prevention of
neurodegenerative diseases such as Alzheimer's disease (AD),
Parkinson's disease (PD), cognitive impairment no dementia (CIND),
brain trauma, and neurodegenerative changes and disorders.
Inventors: |
MALETINSKA; Lenka; (Praha 6,
CZ) ; ZELEZNA; Blanka; (Praha 6, CZ) ;
BLECHOVA; Miroslava; (Praha 10, CZ) ; SPOLCOVA;
Andrea; (Ceske Budejovice, CZ) ; MIKULASKOVA;
Barbora; (Mnisek pod Brdy, CZ) ; KUNES; Jaroslav;
(Praha 4, CZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
USTAV ORGANICKE CHEMIE A BIOCHEMIE AV CR, V.V.I. |
Praha 6 |
|
CZ |
|
|
Assignee: |
USTAV ORGANICKE CHEMIE A BIOCHEMIE
AV CR, V.V.I.
Praha 6
CZ
|
Family ID: |
53432908 |
Appl. No.: |
15/307262 |
Filed: |
May 20, 2015 |
PCT Filed: |
May 20, 2015 |
PCT NO: |
PCT/CZ2015/000047 |
371 Date: |
October 27, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/57554 20130101;
A61P 25/28 20180101; A61K 38/22 20130101; C07K 14/575 20130101 |
International
Class: |
C07K 14/575 20060101
C07K014/575 |
Foreign Application Data
Date |
Code |
Application Number |
May 27, 2014 |
CZ |
PV 2014-364 |
Claims
1. Lipidated neuropeptides based on prolactin-releasing peptide,
selected from prolactin-releasing peptide 20 (PrRP20),
prolactin-releasing peptide 31 (PrRP31) and their analogs, wherein
in the C-terminal sequence IRPVGRF-NH.sub.2, one or more of
isoleucine, valine and phenylalanine can be replaced by another
amino acid; said PrRP-based neuropeptide containing C14 and/or C16
fatty acid chain, said fatty acid is bound in position 1 or 11 for
PrRP31 or its analogs and in position 1 or 7 for PrRP20 or its
analogs; said fatty acid being bound by a bond between an amino
acid having at least one free NH.sub.2, OH or SH group and the
carboxylic group of the fatty acid or through a hydrophilic linker
X.sup.2 selected from the group comprising polyoxyethylene moiety,
arylalkyl moiety, or a saturated or unsaturated, linear or branched
C.sub.3-C8 hydrocarbon chain, wherein some carbon atoms may be
replaced by heteroatoms selected from a group comprising N, S, and
O; said chain carrying at least one and preferably two amino groups
or carboxylic acid groups, one of which may be substituted to form
a group selected from: CONH.sub.2; NH-polyoxyethylene; COOM.sup.1
wherein M.sup.1 is alkali metal, preferably Na or K; CN;
COOR.sup.1, COR.sup.1, or CONHR.sup.1 wherein R.sup.1 is selected
from a group comprising lower alkyl, arylalkyl, polyoxyethylene,
methylpolyoxyethylene, and aminoethylpolyoxyethylene;
(CHOH).sub.nR.sup.2 wherein R.sup.2 is H or COOH and n is an
integer from 2 to 10; or (CH).sub.nN.sup.+R.sub.3, wherein R.sup.3
is the same or different, selected from H and C.sub.1-C.sub.4
alkyl; and the PrRP31 or its analogs may optionally have the amino
acid in position 11 replaced by an amino acid having a free
NH.sub.2, OH or SH group, particularly when the fatty acid is bound
in position 11 for PrRP31 or its analogs; and the PrRP20 or its
analogs may optionally have the amino acid in position 7 replaced
by an amino acid having a free NH.sub.2, OH or SH group in position
7, particularly when the fatty acid is bound in position 7 for
PrRP20 or its analogs; for use in a method of treatment and
prevention of neurodegenerative diseases, such as Alzheimer's
disease (AD), Parkinson's disease (PD), cognitive impairment no
dementia (CIND), brain trauma, and neurodegenerative changes and
disorders.
2. Lipidated neuropeptides according to claim 1, wherein isoleucine
can be replaced by phenylglycine or alanine, valine can be replaced
by phenylglycine and/or terminal phenylalanine can be replaced by
dichlorophenylalanine, pentafluorophenylalanine, nitrophenyalanine,
histidine, benzylhistidine, naphtylalanine, tryptofane,
pyroglutamic acid, benzylcysteine, benzyl-O-glutamate,
tetrachlorophenylalanine, methyl-O-phenylalanine or
methyl-NH-phenylalanine, in the sequence of the C-terminal
heptapeptide.
3. Lipidated neuropeptides according to claim 1, wherein X.sup.2 is
a hydrophilic linker selected from the group comprising
.beta.-alanine, .gamma.-aminobutyric acid and .gamma.-glutamic
acid.
4. Lipidated neuropeptides according to claim 1 having general
formulae selected from: TABLE-US-00005 (1)
(X)SRTHRHSMEIRTPDINPAWYASRGIRPVGRF-NH.sub.2, (2)
(X)SRAHQHSMETRTPDINPAWYTGRGIRPVGRF-NH.sub.2, (3)
(X)TPDINPAWYASRGIRPVGRF-NH.sub.2 (4)
(X)TPDINPAWYTGRGIRPVGRF-NH.sub.2,
wherein X.dbd.X.sup.1 or X.sup.1X.sup.2; X.sup.1 being
tetradecanoic or hexadecanoic acid, which is bound in a position 1
to an amino acid of the above mentioned peptide chain either
directly or through X.sup.2, X.sup.2 being a hydrophilic linker as
defined in claim 1, preferably selected from the group consisting
of .beta.-alanine, .quadrature.-amino butyric acid and
.quadrature.-glutamic acid, and wherein in the C-terminal sequence
IRPVGRF-NH.sub.2, one or more of isoleucine, valine and
phenylalanine can be replaced by another amino acid; for use in the
treatment and prevention, preferably by peripheral administration,
of neurodegenerative diseases, such as Alzheimer's disease (AD),
Parkinson's disease (PD), cognitive impairment no dementia (CIND),
brain trauma, and neurodegenerative changes and disorders.
5. Lipidated neuropeptides according to the claim 1 having formulae
selected from: TABLE-US-00006 (5)
(palm)SRTHRHSMEIRTPDINPAWYASRGIRPVGRF-NH.sub.2 and (6)
(palm)TPDINPKWYASRGIRPVGRF-NH.sub.2;
wherein palm is hexadecanoic acid, and wherein in the C-terminal
sequence IRPVGRF-NH.sub.2, one or more of isoleucine, valine and
phenylalanine can be replaced by another amino acid; for use in the
treatment and prevention, preferably by peripheral administration,
of neurodegenerative diseases, such as Alzheimer's disease (AD),
Parkinson's disease (PD), cognitive impairment no dementia (CIND),
brain trauma, and neurodegenerative changes and disorders.
6. Lipidated analogs according to claim 1 having formulae selected
from: TABLE-US-00007 (7)
SRTHRHSMEIK(palm)TPDINPAWYASRGIRPVGRF-NH.sub.2, (8)
TPDINPK(palm)WYASRGIRPVGRF-NH.sub.2, (9)
SRTHRHSMEIKTPDINPAWYASRGIRPVGRF-NH.sub.2, and | X.sup.2(palm) (10)
TPDINPKWYASRGIRPVGRF-NH.sub.2; | X.sup.2(palm)
wherein palm is hexadecanoic acid and X.sup.2 is
.quadrature.-glutamic acid, and wherein in the C-terminal sequence
IRPVGRF-NH.sub.2, one or more of isoleucine, valine and
phenylalanine can be replaced by another amino acid; for use in the
treatment and prevention, preferably by peripheral administration,
of diseases, such as Alzheimer's disease (AD), Parkinson's disease
(PD), cognitive impairment no dementia (CIND), brain trauma, and
neurodegenerative changes and disorders.
Description
FIELD OF THE INVENTION
[0001] New analogs of prolactin releasing peptide (PrRP) represent
neuroprotective agents for peripheral treatment and prevention of
neurodegenerative diseases, such as Alzheimer's disease (AD),
Parkinson's disease (PD), cognitive impairment no dementia (CIND),
brain trauma, and neurodegenerative changes and disorders.
BACKGROUND OF THE INVENTION
[0002] AD is a serious neurodegenerative brain disease affecting
mainly older people. The disease starts to manifest with memory
decline, learning disorders, behavioral changes, impairment in
orientation in time and space, loss of autonomic functions, finally
results in complete dementia. The death comes on average 9 years
after diagnosis. Histopathologically, AD is characterized by two
hallmarks: intracelullar neurofibrilary tangles formed by
hyperphosphorylated Tau protein and extracellular senile plaques of
beta peptide.
[0003] Prolactin releasing peptide (PrRP) was discovered at the end
of 20.sup.th century. Naturally two isoforms of PrRP can be found
in organism: peptide containing 31 amino acids (PrRP/1-31/; PrRP31)
or 20 amino acids (PrRP/12-311; PrRP20), its amino acid composition
also exhibits small differences in various species (human, rat,
bovine) (Hinuma et al., 1998). PrRP is produced in neurons of many
brain regions, mainly in medulla oblongata (in nucleus tractus
solitarius and ventrolateral reticular nucleus), and hypothalamus
(in paraventricular and dorsomedial nuclei), less in pituitary
gland, and amygdala. In the periphery PrRP can be found in adrenal
medulla, testis, pancreas, and small and large intestines.
[0004] PrRP receptor, GPR10, is extensively expressed in the whole
brain; it can be found in anterior pituitary, amygdala,
hypothalamus, brainstem, and medulla oblongata. In the periphery
GPR10 can be found in adrenal medulla, and significantly increased
expression was observed in human and rat pancreas.
[0005] Subsequently, new modified analogs of neuropeptides PrRP31
and PrRP20 were synthesized, with changes in amino acid chain,
lipidated with fatty acid (e.g. myristoylated or palmitoylated) at
the N-terminus, however, for use in regulating food intake
(WO2014/009808) and regulating blood glucose levels (U.S. Pat. No.
61/927944).
[0006] Nowadays, drugs slowing the AD progression and improving
cognitive functions are used. These are inhibitors of
acetylcholinesterase, which increase acetylcholine concentration in
the brain and inhibitors of N-methyl-D-aspartate receptors (e.g.
memantin). Because of the high incidence of insulin resistance in
AD patients, it is not possible to use insulin as an AD treatment.
It is hypothesized that agents increasing insulin sensitivity, such
as metformin, insulin secretagogues such as glucagon-like peptide-1
(GLP-1 gastric-inhibitory peptide (GIP and their analogs could act
as AD treatment.
[0007] There is a need to provide further substances with
neuroprotective effect which could be useful in the treatment of
neurodegeneravie diseases.
DISCLOSURE OF THE INVENTION
[0008] The present invention provides lipidated neuropeptides based
on prolactin-releasing peptide (PrRP-based neuropeptides) selected
from prolactin-releasing peptide 20 (PrRP20), prolactin-releasing
peptide 31 (PrRP31) and their analogs, wherein in the C-terminal
sequence IRPVGRF-NH.sub.2 (SEQ ID NO. 1), one or more of
isoleucine, valine and phenylalanine can be replaced by another
amino acid; said PrRP-based neuropeptide containing C14 and/or C16
fatty acid chain, said fatty acid is bound in position 1 or 11 for
PrRP31 or its analogs and in position 1 or 7 for PrRP20 or its
analogs; said fatty acid being bound by a bond between an amino
acid having at least one free NH.sub.2, OH or SH group and the
carboxylic group of the fatty acid or through a hydrophilic linker
X.sup.2 selected from the group comprising polyoxyethylene moiety,
arylalkyl moiety, or a saturated or unsaturated, linear or branched
C.sub.3-C.sub.8 hydrocarbon chain, wherein some carbon atoms may be
replaced by heteroatoms selected from a group comprising N, S, and
O; said chain carrying at least one and preferably two amino groups
or carboxylic acid groups, one of which may be substituted to form
a group selected from: CONH.sub.2; NH-polyoxyethylene; COOM.sup.1
wherein M.sup.1 is alkali metal, preferably Na or K; CN;
COOR.sup.1, COR.sup.1, or CONHR.sup.1 wherein R.sup.1 is selected
from a group comprising lower alkyl, arylalkyl, polyoxyethylene,
methylpolyoxyethylene, and aminoethylpolyoxyethylene;
(CHOH).sub.nR.sup.2 where R.sup.2 is H or COOH and n is an integer
from 2 to 10; or (CH).sub.nN.sup.+R.sub.3, wherein R.sub.3 is the
same or different, selected from H and C.sub.1-C.sub.4 alkyl;
and the PrRP31 or its analogs may optionally have the amino acid in
position 11 replaced by an amino acid having a free NH.sub.2, OH or
SH group, particularly when the fatty acid is bound in position 11
for PrRP31 or its analogs; and the PrRP20 or its analogs may have
the amino acid in position 7 replaced by an amino acid having a
free NH.sub.2, OH or SH group in position 7, particularly when the
fatty acid is bound in position 7 for PrRP20 or its analogs; for
use in a method of treatment and prevention of neurodegenerative
diseases, such as Alzheimer's disease (AD), Parkinson's disease
(PD), cognitive impairment no dementia (CIND), brain trauma, and
neurodegenerative changes and disorders.
[0009] The PrRP31 and/or PrRP20 include variants found in various
animal species. Preferred are the human and rat variants.
[0010] Preferably, in the sequence of the C-terminal heptapeptide
as mentioned above, isoleucine can be replaced by phenylglycine or
alanine, valine can be replaced by phenylglycine and/or terminal
phenylalanine can be replaced by dichlorophenylalanine,
pentafluorophenylalanine, nitrophenylalanine, histidine,
benzylhistidine, naphthylalanine, tryptofane, pyroglutamic acid,
benzylcysteine, benzyl-O-glutamate, tetrachlorophenylalanine,
methyl-O-phenylalanine or methyl-NH-phenylalanine.
[0011] The binding of the fatty acid thus includes either a direct
bond between an amino acid of the PrRP chain having at least one
free amino, SH or OH group and the carboxylic group of the fatty
acid, or a bond through X.sup.2, wherein X.sup.2 is a hydrophilic
linker selected from a group comprising polyoxyethylene moiety,
arylalkyl moiety, or a saturated or unsaturated, linear or branched
C.sub.3-C.sub.8 hydrocarbon chain, wherein some carbon atoms may be
replaced by heteroatoms selected from a group comprising N, S, and
O; said chain carrying at least one and preferably two amino groups
or carboxylic acid groups, one of which may be substituted to form
a group selected from: CONH.sub.2; NH-polyoxyethylene; COOM.sup.1
wherein M.sup.1 is alkali metal, preferably Na or K; CN;
COOR.sup.1, COR.sup.1, or CONHR.sup.1 wherein R.sup.1 is selected
from a group comprising lower alkyl, arylalkyl, polyoxyethylene,
methylpolyoxyethylene, and aminoethylpolyoxyethylene;
(CHOH).sub.nR.sup.2 wherein R.sup.2 is H or COOH and n is an
integer from 2 to 10; or (CH).sub.nN.sup.+R.sub.3, where R.sub.3 is
the same or different, selected from H and C.sub.1-C.sub.4
alkyl.
[0012] Preferably, X.sup.2 is a hydrophilic linker selected from
the group comprising .beta.-alanine, .gamma.-aminobutyric acid and
.gamma.-glutamic acid.
[0013] When the fatty acid is bound in position 11 for PrRP31 or
its analogs, the PrRP31 or its analogs have an amino acid having a
free NH.sub.2, OH or SH group in position 11, and when the fatty
acid is bound in position 7 for PrRP20 or its analogs, the PrRP20
or its analogs have an amino acid having a free NH.sub.2, OH or SH
group in position 7. Amino acids having a free NH.sub.2, OH or SH
group include, for example, lysine, arginine, serine, cysteine,
tyrosine.
[0014] The present invention provides, more particularly, the
lipidated analogs of PrRP20 or PrRP31 (rat and human) according to
the formulae:
TABLE-US-00001 (1) (SEQ ID NO. 2)
(X)SRTHRHSMEIRTPDINPAWYASRGIRPVGRF-NH.sub.2, (2) (SEQ ID NO. 3)
(X)SRAHQHSMETRTPDINPAWYTGRGIRPVGRF-NH.sub.2, or (3) (SEQ ID NO. 4)
(X)TPDINPAWYASRGIRPVGRF-NH.sub.2, (4) (SEQ ID NO. 5)
(X)TPD1NPAWYTGRGIRPVGRF-NH.sub.2,
wherein X.dbd.X.sup.1 or X.sup.1X.sup.2; X.sup.1 being
tetradecanoic or hexadecanoic acid, which is bound in a position 1
to an amino acid of the above mentioned peptide chain either
directly or through X.sup.2, X.sup.2 being a hydrophilic linker as
defined above, preferably selected from the group consisting of
.beta.-alanine, .gamma.-amino butyric acid and .gamma.-glutamic
acid, and wherein in the C-terminal sequence IRPVGRF-NH.sub.2, one
or more of isoleucine, valine and phenylalanine can be replaced by
another amino acid; for use in the treatment and prevention,
preferably by peripheral administration, of neurodegenerative
diseases, which are Alzheimer's disease (AD), Parkinson's disease
(PD), cognitive impairment no dementia (CIND), brain trauma, and
neurodegenerative changes and disorders.
[0015] In a preferred embodiment, the lipidated analogs of PrRP20
or PrRP31 according to the formulae:
TABLE-US-00002 (5) (SEQ ID NO. 6)
(N-palm)SRTHRHSMEIRTPDINPAWYASRGIRPVGRF-NH.sub.2 and (6) (SEQ ID
NO. 7) (palm)TPDINPKWYASRGIRPVGRF-NH.sub.2;
wherein palm is hexadecanoic acid, and wherein in the C-terminal
sequence IRPVGRF-NH.sub.2, one or more of isoleucine, valine and
phenylalanine can be replaced by another amino acid; are provided
for use in the treatment and prevention, preferably by peripheral
administration, of neurodegenerative diseases, which are
Alzheimer's disease (AD), Parkinson's disease (PD), cognitive
impairment no dementia (CIND), brain trauma, and neurodegenerative
changes and disorders.
[0016] In another embodiment, the lipidated analogs of PrRP20 or
PrRP31 according to the formulae:
TABLE-US-00003 (7) (SEQ ID NO. 8)
SRTHRHSMEIK(palm)TPDINPAWYASRGIRPVGRF-NH.sub.2, (8) (SEQ ID NO. 9)
TPDINPK(palm)WYASRGIRPVGRF-NH.sub.2, (9) (SEQ ID NO. 10)
SRTHRHSMEIKTPDINPAWYASRGIRPVGRF-NH.sub.2, and | X.sup.2(palm) (10)
(SEQ ID NO. 11) TPDINPKWYASRGIRPVGRF-NH.sub.2; | X.sup.2(palm)
wherein palm is hexadecanoic acid and X.sup.2 is .gamma.-glutamic
acid, and wherein in the C-terminal sequence IRPVGRF-NH.sub.2, one
or more of isoleucine, valine and phenylalanine can be replaced by
another amino acid; are provided for use in the treatment and
prevention, preferably by peripheral administration, of
neurodegenerative diseases, which are Alzheimer's disease (AD),
Parkinson's disease (PD), cognitive impairment no dementia (CIND),
brain trauma, and neurodegenerative changes and disorders.
[0017] A further embodiment of the invention relates to the use of
lipidated neuropeptides based on prolactin-releasing peptide
(PrRP-based neuropeptides) selected from prolactin-releasing
peptide 20 (PrRP20), prolactin-releasing peptide 31 (PrRP31) and
their analogs, wherein in the C-terminal sequence IRPVGRF-NH.sub.2,
one or more of isoleucine, valine and phenylalanine can be replaced
by another amino acid; said PrRP-based neuropeptide containing C14
and/or C16 fatty acid chain, said fatty acid is bound in position 1
or 11 for PrRP31 or its analogs and in position 1 or 7 for PrRP20
or its analogs; said fatty acid being bound by a bond between an
amino acid having at least one free NH.sub.2, OH or SH group and
the carboxylic group of the fatty acid or through a hydrophilic
linker X.sup.2 selected from the group comprising polyoxyethylene
moiety, arylalkyl moiety, or a saturated or unsaturated, linear or
branched C.sub.3-C.sub.8 hydrocarbon chain, wherein some carbon
atoms may be replaced by heteroatoms selected from a group
comprising N, S, and 0; said chain carrying at least one and
preferably two amino groups or carboxylic acid groups, one of which
may be substituted to form a group selected from: CONH.sub.2;
NH-polyoxyethylene; COOM.sup.1 wherein M.sup.1 is alkali metal,
preferably Na or K; CN; COOR.sup.1, COR.sup.1, or CONHR.sup.1
wherein R.sup.1 is selected from a group comprising lower alkyl,
arylalkyl, polyoxyethylene, methylpolyoxyethylene, and
aminoethylpolyoxyethylene; (CHOH).sub.PR.sup.2 where R.sup.2 is H
or COOH and n is an integer from 2 to 10; or
(CH).sub.nN.sup.+R.sub.3, where R.sub.3 is the same or different,
selected from H and C.sub.1-C.sub.4 alkyl;
and the PrRP31 or its analogs may have the amino acid in position
11 replaced by an amino acid having a free NH.sub.2, OH or SH
group, particularly when the fatty acid is bound in position 11 for
PrRP31 or its analogs; and the PrRP20 or its analogs may have the
amino acid in position 7 replaced by an amino acid having a free
NH.sub.2, OH or SH group in position 7, particularly when the fatty
acid is bound in position 7 for PrRP20 or its analogs; for the
manufacture of a medicament for treatment and prevention of
neurodegenerative diseases, such as Alzheimer's disease (AD),
Parkinson's disease (PD), cognitive impairment no dementia (CIND),
brain trauma, and neurodegenerative changes and disorders.
[0018] Another embodiment of the invention provides a method of
treatment and prevention of neurodegenerative diseases, such as
Alzheimer's disease (AD), Parkinson's disease (PD), cognitive
impairment no dementia (CIND), brain trauma, and neurodegenerative
changes and disorders, comprising a step of administering to a
subject in need of such treatment or prevention, preferably by
peripheral administration, lipidated neuropeptides based on
prolactin-releasing peptide (PrRP-based neuropeptides) selected
from prolactin-releasing peptide 20 (PrRP20), prolactin-releasing
peptide 31 (PrRP31) and their analogs, wherein in the C-terminal
sequence IRPVGRF-NH.sub.2, one or more of isoleucine, valine and
phenylalanine can be replaced by another amino acid; said
PrRP-based neuropeptide containing C14 and/or C16 fatty acid chain,
said fatty acid is bound in position 1 or 11 for PrRP31 or its
analogs and in position 1 or 7 for PrRP20 or its analogs; said
fatty acid being bound by a bond between an amino acid having at
least one free NH.sub.2, OH or SH group and the carboxylic group of
the fatty acid or through a hydrophilic linker X.sup.2 selected
from the group comprising polyoxyethylene moiety, arylalkyl moiety,
or a saturated or unsaturated, linear or branched C.sub.3-C.sub.8
hydrocarbon chain, wherein some carbon atoms may be replaced by
heteroatoms selected from a group comprising N, S, and O; said
chain carrying at least one and preferably two amino groups or
carboxylic acid groups, one of which may be substituted to form a
group selected from: CONH.sub.2; NH-polyoxyethylene; COOM.sup.1
wherein M.sup.1 is alkali metal, preferably Na or K; CN;
COOR.sup.1, COR.sup.1, or CONHR.sup.1 wherein R.sup.1 is selected
from a group comprising lower alkyl, arylalkyl, polyoxyethylene,
methylpolyoxyethylene, and aminoethylpolyoxyethylene;
(CHOH).sub.nR.sup.2 wherein R.sup.2 is H or COOH and n is an
integer from 2 to 10; or (CH).sub.nN.sup.+R.sub.3, where R.sub.3 is
the same or different, selected from H and C.sub.1-C.sub.4
alkyl;
and the PrRP31 or its analogs may have the amino acid in position
11 replaced by an amino acid having a free NH.sub.2, OH or SH
group, particularly when the fatty acid is bound in position 11 for
PrRP31 or its analogs; and the PrRP20 or its analogs may have the
amino acid in position 7 replaced by an amino acid having a free
NH.sub.2, OH or SH group in position 7, particularly when the fatty
acid is bound in position 7 for PrRP20 or its analogs.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 shows the escape latency in Morris water maze test
(MWM). The experiment was performed for 5 days, with 4 sessions per
day, each session from different starting point, using MSG obese
mice and their controls. Data are mean.+-.SEM, n=10 mice per
group.
[0020] Statistical analysis is 2-way ANOVA with Bonferroni post hoc
test. Significance is *P <0.05 and **P<0.01.
[0021] FIG. 2 shows phosphorylation of GSK-3.beta. at Ser9 and Tau
phosphorylation at Ser396 and Thr231 in the hippocampi of 2 and
6-month-old MSG mice and their age-matched controls.
Phosphorylation was determined using Western blot (WB). Data are
mean.+-.SEM, n=7-10 mice per group. Statistical analysis is 1-way
ANOVA with Bonferroni post hoc test. Significance is *P<0.05 and
***P<0.001.
[0022] FIG. 3 shows insulin signaling cascade in hippocampi of
6-month-old MSG obese mice after 14-day treatment with liraglutide
and palmitoylated human PrRP31. Liraglutide (0.2 mg/kg) and
palmitoylated human PrRP31 (5 mg/kg) were subcutaneously
administered twice a day, in the morning and in the evening. Saline
treated mice served as a control. Phosphorylation was determined
using WB. Data are mean.+-.SEM, n=7-10 mice per group. Statistical
analysis is 1-way ANOVA with Bonferroni post hoc test. Significance
is *P<0.05 and ***P<0.001.
[0023] FIG. 4 shows phosphorylation at different epitopes of Tau
protein in hippocampi of 6-month-old MSG mice after 14-day
treatment with liraglutide and palmitoylated human PrRP31.
Liraglutide (0.2 mg/kg) and palmitoylated human PrRP31 (5 mg/kg)
were subcutaneously administered twice a day, in the morning and in
the evening. Saline treated mice served as a control.
Phosphorylation was determined using WB. Data are mean SEM, n=7-10
mice per group. Statistical analysis is 1-way ANOVA with Bonferroni
post hoc test. Significance is *P<0.05 and ***P<0.001.
[0024] FIG. 5 shows phosphorylation of Tau protein at different Tau
epitopes in hippocampi of 6-month-old MSG mice after 14-day
treatment with palmitoylated PrRP31 with dichlorophenylalanin in
position 31. Palmitoylated PrRP31 with dichlorophenylalanin in
position 31 (5 mg/kg) was subcutaneously administered twice a day,
in the morning and in the evening. Saline treated mice served as a
control. Phosphorylation was determined using WB. Data are
mean.+-.SEM, n=7-10 mice per group. Statistical analysis is 1-way
ANOVA with Bonferroni post hoc test. Significance is *P<0.05 and
***P<0.001.
[0025] FIG. 6 shows immunohistochemical analysis of Tau
hyperphosphorylation in CA1 region of the hippocampus of
6-month-old MSG mice and their age-matched controls, and Tau
hyperphosphorylation after 14-day treatment with liraglutide and
palmitoylated PrRP31. Liraglutide (0.2 mg/kg) and palmitoylated
human PrRP31 (5 mg/kg) were subcutaneously administered twice a
day, in the morning and in the evening. Saline treated mice served
as a control. Tau phosphorylation was determined using double
immunohistochemical fluorescent staining.
[0026] FIG. 7 shows spatial memory testing of Thy-Tau 22 mice and
their WT controls in Y-maze. The memory was tested A/ before the
beginning of the experiment and B/ after 2-month-long treatment
with LiPR31 dissolved in PBS/5% Tween 80 using SC Alzet.RTM.
osmotic pumps, the concentration was 5 mg/kg/day; PBS/5% Tween 80
was administered to the control group. Data are mean.+-.SEM,
n=10-12 animals per group. Statistical analysis is Student t-test.
Significance is *P<0.05.
[0027] FIG. 8 shows Tau phosphorylation after 2-month-long
treatment of Thy-Tau22 mice with LiPR31 dissolved in PBS/5% Tween
80 using SC Alzet.RTM. osmotic pumps, the concentration was 5
mg/kg/day; PBS/5% Tween 80 was administered to the control group.
A/ phosphorylation on Tau epitope Thr231, B/ phosphorylation on Tau
epitopes Ser396&Ser404 using AD2 antibody. Data are
mean.+-.SEM, n=7 animals per group. Statistical analysis is Student
t-test. Significance is *P<0.05.
EXAMPLES
Abbreviations
[0028] ANOVA--analysis of variance [0029] ARC--nucleus arcuatus
[0030] GSK-3.beta.--glycogen synthase kinase--3.beta. [0031]
MWM--Morris water maze [0032] PDK-1--phosphoinositide-dependent
kinase--1 [0033] SC--subcutaneous [0034] SDS--sodium dodecyl
sulfate [0035] SEM--standard error of the mean [0036] WB--Western
blot [0037] LiPR31--an analog of PrRP31 palmitoylated at Lys.sup.11
through gamma glutamic acid linker
Tested Compounds
[0038] According to the structure-activity studies, analogs of
PrRP31 and PrRP20, rat (identical to mouse) or human, lipidated at
N-terminal or amino acid containing amino group, using C14 or C16
fatty acid. Methionine in position 8 in PrRP31 was replaced by more
stable norleucine. In Thy Tau22 mice study, an analog of PrRP31
palmitoylated at Lys.sup.11 (Lys instead of Arg) through gamma
glutamic acid linker (hereinafter referred to as LiPR31) was
used.
[0039] Lipidated analogs of PrRP31 and PrRP20 were synthesized by
solid-phase synthesis at the IOCB AS CR, Prague, on the peptide
synthesis department, as described by Maixnerova et al. (Maletinska
et al., 2007).
[0040] Liraglutide was purchased from Novo Nordisk A/S (Bagsvaerd,
Denmark).
Animals
[0041] To examine neuroprotective properties, insulin resistant
animal model was used, e.g. mouse model with obesity induced by
MSG. These mice are characterized by growth hormone insufficiency,
pituitary and optic nerves atrophy, and infertility (Olney, 1969).
In their brains the reduced nucleus arcuatus, enlarged third brain
ventricle, and narrowed eminentia mediana are observed. Total
number of neurons in ARC is reduced about 75% in MSG mice compared
to their controls; however, the number of neurons does not differ
significantly in other brain regions (Elefteriou et al., 2003).
[0042] The imbalance between food intake and energy expenditure at
MSG obese mice leads to hypophagia and an increased adipose tissue;
compared to their control, MSG obese mice have even 8 times higher
weight of white adipose tissue (Maletinska et al., 2006). They have
also increased leptin and glucose blood concentration, and insulin
resistance (Maletinska et al., 2006).
[0043] For evaluation of the the neuroprotecitve effect of
palmitoylated PrRP LiPR31, the model of AD like pathology,
Thy-Tau22 mice, was used. Thy-Tau22 mice overexpress human 4R-Tau
protein with mutations G272V and P3015. These mice develop memory
deficits, Tau hyper-phosphorylation at different epitopes, such as
Ser202, Thr205, Thr212, Ser214, Thr231, Ser396, in CA1 region of
hippocampus, and neurofibrillary tangles formation (Schindowski et
al., 2006; Van der Jeugd et al., 2011).
MSG Mice
[0044] Mice of strain NMRI (Harlan, Italy) were housed at the
certified animal facility of IOCB AS CR, Prague, in the campus of
Academy of Science in Kr{hacek over (c)} at 22.+-.2.degree. C.,
they had free access to water and food. They were fed standard chow
diet St-1 (Ml n Kocanda, Jesenice, Czech Republic), which contained
66% calories as carbohydrates, 25% as protein, and 9% as fat; its
energy content was 3.4 kcal/g. Daily cycle was 12/12 hours, lights
on at 6:00 a.m. All animal experiments followed the ethical
guidelines for animal experiments and the Czech Republic Act No.
246/1992.
[0045] For obesity induction, the newborn NMRI mice were SC
administered with sodium glutamic acid (Sigma, St. Louis, USA) at
dose 4 mg/g of body weight at postnatal days 2-5. These MSG-obese
mice were fed the same standard diet as the control group. The food
and body weight was monitored once per week. For the study, MSG and
control male mice at the age of 2 and 6 months were used.
6-Month-Old MSG Mice Treatment with Peptides Increasing Insulin
Sensitivity
[0046] Groups of MSG mice (n=10 animals per group) were for 14 days
SC administered with liraglutide at a dose 0.2 mg/kg, or
palmitoylated analog of PrRP31 at a dose 5 mg/kg, or palmitoylated
analog with PrRP31 with dichlorophenylalanin in position 31 at a
dose 5 mg/kg dissolved in saline, twice a day, at 8 a.m. and 6:00
p.m. Control mice (n=10 animals per group), NMRI and MSG, were
injected with saline (the volume was always 0.2 ml/mouse).
Spatial Memory Testing at 6 Months Old MSG Mice
[0047] The spatial memory was tested using Morris water maze (MWM)
following the protocol described in article of Vorheese and
Williamse (Vorhees and Williams, 2006) in 6-month-old MSG mice and
their age-matched controls.
Thy-Tau22 Mice
[0048] Thy-Tau22 female mice and their age-matched WT controls
(C57B1/6 origin) were a kind gift from INSERM laboratory, Lille,
France, the research group "Alzheimer & Tauopathies". Mice were
obtained at the age of 7 months, and were housed 3-4 per cage in
the certified animal facility of the Institute of Physiology AS CR,
Prague, Czech Republic, with free access to water and Altromin diet
(Altromin, Eastern-Westphalia, Germany). Daily cycle was 12/12
hours, lights on at 6:00 a.m. All animal experiments followed the
ethical guidelines for animal experiments and the Czech Republic
Act No. 246/1992.
Thy-Tau22 Mice Treatment with LiPR31
[0049] Thy-Tau22 mice were infused for 2 months with LiPR31, with
doses 5 mg/kg/day dissolved in PBS/5% Tween 80 pH 6, using SC
Alzet.RTM. osmotic pumps. Control mice were infused with PBS/5%
Tween 80. Alzet.RTM. osmotic pumps were subcutaneously (SC)
implanted in short-term ether anesthesia, and were changed after
one months of experiment.
Spatial Memory Testing of Thy-Tau22 Mice
[0050] The spatial memory was tested before the beginning of the
treatment with LiPR31, and after 2 months of the treatment, using
the Y-maze. Experiment was performed following the protocol
described by Belarbi et al. (Belarbi et al., 2011)
Tissue Dissection
[0051] Overnight fasted mice with ad libitum access to water were
weighed, and their plasma glucose concentration was measured using
Glucocard glucometer. After decapitation, the brains were dissected
on ice, and cut between hemispheres. For immunohistochemical
staining the half of the brain was fixed for 24 hours in 4%
paraformaldehyde and dehydrated in 70% ethanol, afterward. For the
western blot (WB) analysis, the hippocampus was dissected, and
lysed in cold lysis buffer (62.5 mmol.l.sup.-1 Tris-HCl, pH 6.8
with 1% sodium deoxycholate, 1% Triton X-100, Complete, 50
mmol.l.sup.-1 NaF, 1 mmol.l.sup.-1 Na.sub.3VO.sub.4), homogenized,
sonicated 10 minutes and stored at -20.degree. C. The blood plasma
was prepared, and stored at -20.degree. C.
[0052] Western Blot Analysis of Proteins Implicated in Insulin
Signaling Cascade and Detection of Hyperphosphorylation of Tau
Protein
[0053] In homogenized hippocampi the protein level was measured
using BCA kit (Pierce, Thermo Fisher Scientific, Rockfor, Ill.,
USA), then the samples were diluted in sample buffer (62.5
mmol.l.sup.-1 Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 0.01%
bromfenol blue, 5% merkaptoethanol, 50 mmol/l NaF and 1 mmol/l
Na.sub.3VO.sub.4) to final concentration 1 ug/.mu.l. WB method and
analysis of the results were performed according to Nagelova et al.
(Nagelova et al., 2014). The list of the used antibodies and their
dilution is shown in table 1.
TABLE-US-00004 TABLE 1 Antibody Company Dilution Rabbit monoclonal
antibody Cell Signaling Technology, 1:1000 5% BSA TBS/tween-20
against Phospho-Akt (Ser473) Beverly, MA, USA Rabbit monoclonal
antibody Cell Signaling Technology, 1:1000 5% BSA TBS/tween-20
against Phospho-Akt (Thr308) Beverly, MA, USA Rabbit monoclonal
antibody Cell Signaling Technology, 1:1000 5% BSA TBS/tween-20
against total Akt Beverly, MA, USA Rabbit monoclonal antibody Cell
Signaling Technology, 1:1000 5% BSA TBS/tween-20 against
Phospho-GSK-3.beta. (Ser9) Beverly, MA, USA Rabbit monoclonal
antibody Cell Signaling Technology, 1:1000 5% BSA TBS/tween-20
against total GSK-3.beta. Beverly, MA, USA Rabbit monoclonal
antibody Cell Signaling Technology, 1:1000 5% BSA TBS/tween-20
against Phospho-PDK1 (ser241) Beverly, MA, USA Rabbit monoclonal
antibody Cell Signaling Technology, 1:1000 5% BSA TBS/tween-20
against total PDK1 Beverly, MA, USA AD2 rabbit monoclonal Tau Gift
from Dr. M.-C.Galas, 1:10 000 5% milk TBS/tween-20 antibody [pS396
& pS404] Inserm, Lille, Francie Rabbit polyclonal antibody
Invitrogen Grand Island, NY, 1:10 000 5% BSA TBS/tween-20 against
Tau [pS396] USA Rabbit polyclonal antibody Invitrogen Grand Island,
NY, 1:1000 5% BSA TBS/tween-20 against Tau [pT231] USA Rabbit
polyclonal antibody Invitrogen Grand Island, NY, 1:1000 5% BSA
TBS/tween-20 against Tau [pT212] USA Anti-total Tau CTer Gift from
Dr. M.-C.Galas, 1:10 000 5% milk TBS/tween-20 Inserm, Lille,
Francie Anti-total Tau NTer (M19G) Gift from Dr. M.-C.Galas, 1:10
000 5% milk TBS/tween-20 Inserm, Lille, Francie Mouse monoclonal
antibody Millipore, Billerica, MA, USA 1:10 000 5% milk
TBS/tween-20 against Tau1 (Ser195, 198, 199, 202) Mouse monoclonal
antibody Sigma, St. Louis, MO, USA 1:10 000 5% milk TBS/tween-20
against .beta.-actin
Immunohistochemical Staining of Hyperphosphorylated Tau Protein
[0054] To verify the results obtained from WB analysis the
immunohistochemical staining was performed. 10 .mu.m thick
paraffin-embedded brain slices were prepared at INSERM, Lille,
France. Immunohistochemical staining was performed according the
method from Violet et al. (Violet et al., 2014).
Statistical Analysis
[0055] Statistical analysis was calculated by 1-way ANOVA, with
Dunnett post-hoc test, or by Student t-test, using GrapPad software
(San Diego, Calif., USA). Data are presented as mean .+-.SEM.
Results:
[0056] MWM with 6-Month-Old MSG Obese Mice
[0057] The escape latency was measured in 6-month-old MSG mice and
their age-matched controls. Experiment was performed 5 days with 4
sessions per day. As shown in FIG. 1, MSG mice had significantly
increased escape latency compared to the control group.
[0058] Insulin Signaling Activation and Tau Phosphorylation in
Hippocampi of 2- and 6-Month-Old MSG Mice and Their Controls
[0059] Activation of insulin signaling cascade and Tau protein
phosphorylation was measured by WB analysis in hippocampi of MSG
obese mice and their controls aged 2 and 6 months. The
phosphorylation of GSK-3.beta. at Ser9 was detected. As shown in
FIG. 2A, the phosphorylation was decreased in MSG mice at the age
of 2 months, and furthermore significantly decreased at the age of
6 months, compared to the control mice. A decreased phosphorylation
of Ser9 at GSK-3.beta. probably caused increased phosphorylation of
Tau protein at epitopes Ser396 and Thr231, as shown in FIG. 2B and
2C. The 6-month old MSG mice were proven as suitable model for
testing the effect of insulin-sensitizing compounds.
Insulin Signaling Cascade in Hippocampi of 6-Month-Old MSG Mice
after 14-Day Treatment with Palmitoylated Analog of PrRP31 and
Liraglutide
[0060] Enhanced activation of kinases implicated in insulin
signaling cascade was observed in hippocampi of 6-month-old MSG
mice after 14-day intervention, either with palmitoylated analog of
PrRP31, or with liraglutide, as shown in FIG. 3. After liraglutide
treatment, significantly increased phosphorylation was observed in
PDK-1, Akt (Thr308), and GSK-3.beta. (Ser9); more pronounced
phosphorylation was observed in-Akt (Thr308), and GSK-3.beta.
(Ser9) after treatment with palmitoylated analog of PrRP31.
[0061] Tau Phosphorylation in Hippocampi of 6-Month-Old MSG Mice
after 14-Day Treatment with Palmitoylated Analog or PrRP31 and
Liraglutide
[0062] Tau phosphorylation in hippocampi of 6 month-old-MSG mice
after 14-day treatment was measured using WB analysis. In
accordance with previous results, the increased phosphorylation of
GSK-3.beta. at Ser9 caused decreased phosphorylation of Tau protein
at epitopes Ser396, Thr212 and Trh231 after 14-day-long treatment
either with palmitoylated analog of PrRP31, or with liraglutide, as
shown in FIG. 4A, B and C. Antibody Tau1, which recognizes
not-phosphorylated Tau, did not show any significant differences
among groups (FIG. 4D).
Phosphorylation of GSK-3.beta. and Tau Protein at Epitope Thr231 in
Hippocampi of 6-Month-Old MSG Mice after 14-Day-Long Treatment with
palmitoylated analog of PrRP31 with Dichlorophenylalanin in
Position 31
[0063] Phosphorylation was detected using the method of WB. As
shown in FIG. 5, 14-day-long treatment with palmitoylated analog of
PrRP31 with dichlorophenylalanin in position 31 increased
phosphorylation of GSK-313 at Ser9 and subsequently led to a
decreased phosphorylation of Tau protein at the epitope Thr231.
[0064] Immunohistochemical Fluorescent Double Staining of Tau
Phosphorylation in CA1 Region of Hippocampi of 6-Month-Old MSG Mice
after 14-Day-Long Intervention with Palmitoylated Analog of Prrp31
and Liraglutide
[0065] To evaluate the WB analysis the double immunohistochemical
staining was used. As shown in FIG. 6, the phosphorylation of Tau
protein at epitopes Thr212 and Ser202/Thr205 was increased in MSG
mice at the age of 6 months compared to their age-matched control,
both treated with saline. Increased phosphorylation is manifested
by a stronger fluorescent signal using the laser of the same
intensity.
[0066] After 14-day treatment with palmitoylated analog of PrRP31
and liraglutide, the Tau phosphorylation is decreased in
hippocampal region CA1, which is manifested by a weaker fluorescent
signal, using the laser of the same intensity.
Spatial Memory Testing in Thy-Tau 22 Mice before and after the
Treatment with LiPR31
[0067] The spatial memory was tested before and after the treatment
with LiPR31 in Thy-Tau22 mice and their age-matched WT control
using the Y-maze; the WT and Thy-Tau22 control group was treated
with PBS/5% Tween 80. As shown in FIG. 7A before the experiment the
Thy-Tau22 mice spent significantly less time in the newly open arm,
compared to WT animals. After the 2-month-long treatment with
LiPR31 the Thy-Tau22 mice spent significantly more time in the new
arm compared to the PBS/Tween 80 treated group, as shown in FIG.
7B.
Tau Phosphorylation in Hippocampi of 9-month-old Thy-Tau22 Mice
after 2-Month-Long Treatment with LiPR31
[0068] Tau phosphorylation was determined in the hippocampi of
Thy-Tau22 mice treated with LiPR31 and their Thy-Tau22 control
using the method of WB. Compared to the control group, the
attenuation of Tau phosphorylation at epitopes Thr231, Ser396 and
Ser404 was observed in hippocampi of Thy-Tau22 mice treated for 2
months with LiPR31, as shown in FIG. 8A and 8B.
Conclusions
[0069] AD is characterized by two pathological changes in neurons:
formation of non-soluble extracellular A.beta. plaques and
hyperphosphorylation of intracellular cytoskeletal Tau protein.
[0070] Within the framework of the present invention, the potential
neuroprotective effect of tested compound was examined in the mouse
model of obesity and insulin resistance, where obesity is caused by
the application of monosodium glutamate (MSG) to newborn animals.
Thy-Tau 22 mice, a model of AD like pathology, were also used to
verify neuroprotective effect of tested compound.
[0071] Compounds increasing insulin sensitivity were tested
regarding their effect on insulin signaling cascade and tau
hyperphosphorylation in the brain (in hippocampus), in MSG obese
mice before and after peptides application.
[0072] Compared to age-matched controls, the insulin resistance was
observed in the brain of MSG obese mice. Decreased activation of
insulin signaling cascade led to a decreased phosphorylation of
GSK-313 at Ser9, which increased its kinase activity. GSK-3.beta.
is one of the most important kinases implicated in Tau
phosphorylation. Consequently, hyperphosphorylation of Tau protein
was observed at epitopes Ser396 and Thr231. After 14-day treatment
with compounds increasing insulin sensitivity, which were
palmitoylated analog of PrRP31, palmitoylated analog of PrRP31 with
dichlorophenylalanin in position 31, and analog of GLP-1
liraglutide which served as a positive control, an enhanced
activation of insulin signaling cascade, including increased
phosphorylation of GSK-3.beta. at Ser9, and decreased
phosphorylation of Tau protein at epitopes Ser396, Thr212 and
Thr231 was observed.
[0073] Tested analogs of palmitoylated PrRP enhanced insulin
signaling cascade in the hippocampi of 6-month-old insulin
resistant MSG mice after 14-day SC treatment. Attenuated Tau
phosphorylation was also observed; Tau hyperphosphorylation is the
pathological change found in brains of AD patients.
INDUSTRIAL APPLICABILITY
[0074] New analogs of prolactin releasing peptide (PrRP) represent
neuroprotective agents for peripheral treatment and prevention of
diseases, which are Alzheimer's disease (AD), Parkinson's disease
(PD), cognitive impairment no dementia (CIND), brain trauma, and
neurodegenerative changes and disorders.
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Sequence CWU 1
1
1117PRTHomo sapiens 1Ile Arg Pro Val Gly Arg Phe 1 5 231PRTHomo
sapiens 2Ser Arg Thr His Arg His Ser Met Glu Ile Arg Thr Pro Asp
Ile Asn 1 5 10 15 Pro Ala Trp Tyr Ala Ser Arg Gly Ile Arg Pro Val
Gly Arg Phe 20 25 30 331PRTRattus sp. 3Ser Arg Ala His Gln His Ser
Met Glu Thr Arg Thr Pro Asp Ile Asn 1 5 10 15 Pro Ala Trp Tyr Thr
Gly Arg Gly Ile Arg Pro Val Gly Arg Phe 20 25 30 420PRTHomo sapiens
4Thr Pro Asp Ile Asn Pro Ala Trp Tyr Ala Ser Arg Gly Ile Arg Pro 1
5 10 15 Val Gly Arg Phe 20 520PRTRattus sp. 5Thr Pro Asp Ile Asn
Pro Ala Trp Tyr Thr Gly Arg Gly Ile Arg Pro 1 5 10 15 Val Gly Arg
Phe 20 631PRTArtificialPrRP31 analog 6Ser Arg Thr His Arg His Ser
Met Glu Ile Arg Thr Pro Asp Ile Asn 1 5 10 15 Pro Ala Trp Tyr Ala
Ser Arg Gly Ile Arg Pro Val Gly Arg Phe 20 25 30
720PRTArtificialPrRP20 analog 7Thr Pro Asp Ile Asn Pro Lys Trp Tyr
Ala Ser Arg Gly Ile Arg Pro 1 5 10 15 Val Gly Arg Phe 20
831PRTArtificialPrRP31 analog 8Ser Arg Thr His Arg His Ser Met Glu
Ile Lys Thr Pro Asp Ile Asn 1 5 10 15 Pro Ala Trp Tyr Ala Ser Arg
Gly Ile Arg Pro Val Gly Arg Phe 20 25 30 920PRTArtificialPrRP20
analog 9Thr Pro Asp Ile Asn Pro Lys Trp Tyr Ala Ser Arg Gly Ile Arg
Pro 1 5 10 15 Val Gly Arg Phe 20 1031PRTArtificialPrRP31 analog
10Ser Arg Thr His Arg His Ser Met Glu Ile Lys Thr Pro Asp Ile Asn 1
5 10 15 Pro Ala Trp Tyr Ala Ser Arg Gly Ile Arg Pro Val Gly Arg Phe
20 25 30 1120PRTArtificialPrRP20 analog 11Thr Pro Asp Ile Asn Pro
Lys Trp Tyr Ala Ser Arg Gly Ile Arg Pro 1 5 10 15 Val Gly Arg Phe
20
* * * * *