U.S. patent application number 17/307471 was filed with the patent office on 2021-08-26 for methods for attenuating release of inflammatory mediators and peptides useful therein.
The applicant listed for this patent is BIOMARCK PHARMACEUTICALS LTD.. Invention is credited to Indu PARIKH.
Application Number | 20210260154 17/307471 |
Document ID | / |
Family ID | 1000005572052 |
Filed Date | 2021-08-26 |
United States Patent
Application |
20210260154 |
Kind Code |
A1 |
PARIKH; Indu |
August 26, 2021 |
METHODS FOR ATTENUATING RELEASE OF INFLAMMATORY MEDIATORS AND
PEPTIDES USEFUL THEREIN
Abstract
The present invention includes methods of inhibiting or
suppressing cellular secretory processes. More specifically the
present invention relates to inhibiting or reducing the release of
inflammatory mediators from inflammatory cells by inhibiting the
mechanism associated with the release of inflammatory mediators
from granules in inflammatory cells. In this regard, the present
invention discloses an intracellular signaling mechanism that
illustrates several novel intracellular targets for pharmacological
intervention in disorders involving secretion of inflammatory
mediators from vesicles in inflammatory cells. Peptide fragments
and variants thereof of MANS peptide as disclosed in the present
invention are useful in such methods.
Inventors: |
PARIKH; Indu; (Chapel Hill,
NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIOMARCK PHARMACEUTICALS LTD. |
Durham |
NC |
US |
|
|
Family ID: |
1000005572052 |
Appl. No.: |
17/307471 |
Filed: |
May 4, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16235880 |
Dec 28, 2018 |
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17307471 |
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15789442 |
Oct 20, 2017 |
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16235880 |
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14638208 |
Mar 4, 2015 |
9827287 |
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15789442 |
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12359892 |
Jan 26, 2009 |
8999915 |
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14638208 |
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PCT/US2007/074514 |
Jul 26, 2007 |
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12359892 |
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60833239 |
Jul 26, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 38/08 20130101; A61K 38/10 20130101; A61K 38/16 20130101; A61K
38/17 20130101 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61K 38/08 20060101 A61K038/08; A61K 38/10 20060101
A61K038/10; A61K 38/17 20060101 A61K038/17; A61K 45/06 20060101
A61K045/06 |
Claims
1. A method for inhibiting the production of one or more
inflammatory mediators from an inflammatory cell in a tissue and/or
fluid of a subject, the method comprising administering to the
subject an effective amount of a peptide comprising an amino acid
sequence selected from the group consisting of: SEQ ID NOs: 106, 1,
11, 37, 45, 79, 91, 93, 108, 121, 124, 141, 143, 153, 219, 234,
239, 248, 241, and 251, wherein the N-terminal amino acid of the
peptide is chemically modified by acetylation, and wherein the one
or more inflammatory mediators are IL-1beta, IL-8, TNFalpha, or a
combination thereof.
2. The method of claim 1, wherein the subject has a respiratory
disease.
3. The method of claim 2, wherein the respiratory disease is
asthma, chronic bronchitis, chronic obstructive respiratory
disease, or acute respiratory distress syndrome (ARDS).
4. The method of claim 1, wherein the peptide is administered by
inhalation, pulmonary administration, or nasal administration.
5. The method of claim 1, wherein the peptide is administered in
the form of an aerosol.
6. The method of claim 1, wherein the peptide is administered in
the form of a dry powder.
7. The method of claim 1, wherein the peptide is administered by a
dry powder inhaler, metered dose inhaler, or nebulizer.
8. The method of claim 1, wherein the subject is a human.
9. The method of claim 1, further comprising administering to the
subject a second therapeutic agent selected from the group
consisting of an antibiotic, an antiviral compound, an
antiparasitic compound, an anti-inflammatory compound, and an
immunomodulator.
10. The method of claim 1, wherein said peptide consists of
acetyl-peptide 106 (SEQ ID NO: 106).
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] The present application is a continuation of U.S. patent
application Ser. No. 16/235,880, filed Dec. 28, 2018, which is a
continuation of U.S. patent application Ser. No. 15/789,442, filed
Oct. 20, 2017, which is a continuation of U.S. patent application
Ser. No. 14/638,208, filed Mar. 4, 2015, now issued as U.S. Pat.
No. 9,827,287, which is a continuation of U.S. patent application
Ser. No. 12/359,892, filed Jan. 26, 2009, now issued as U.S. Pat.
No. 8,999,915, which is a continuation of of International Patent
Application No. PCT/US2007/074514, filed Jul. 26, 2007, which
claims priority to U.S. Ser. No. 60/833,239, filed on Jul. 26,
2006, each of which is incorporated in its entirety by
reference.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
[0002] The contents of the text file submitted electronically
herewith are incorporated herein by reference in their entirety: A
computer readable format of the Sequence Listing (filename:
BMRK_004_05US_SeqList_ST25.txt, date created: May 4, 2021, file
size: 81,722 bytes).
FIELD OF INVENTION
[0003] The present invention relates to peptides or peptide
compositions and methods of their use to attenuate (or inhibit or
reduce) the stimulated release of mediators of inflammation from
inflammatory cells during inflammation. The present invention also
relates to use of these peptides or peptide compositions to
modulate an intracellular signaling mechanism regulating the
secretion of inflammatory mediators from inflammatory cells.
BACKGROUND OF THE INVENTION
[0004] Inflammatory leukocytes synthesize a number of inflammatory
mediators that are isolated intracellularly and stored in
cytoplasmic membrane-bound granules. Examples of such mediators
include, but are not limited to, myeloperoxidase [MPO] in
neutrophils (see, for example, Borregaard N, Cowland J B. Granules
of the human neutrophilic polymorphonuclear leukocyte. Blood 1997;
89:3503-3521), eosinophil peroxidase [EPO] and major basic protein
[MBP] in eosinophils (see, for example, Gleich G J. Mechanisms of
eosinophil-associated inflammation. J Allergy Clin Immunol 2000;
105:651-663), lysozyme in monocytes/macrophages (see, for example,
Hoff T, Spencker T, Emmendoerffer A., Goppelt-Struebe M. Effects of
glucocorticoids on the TPA-induced monocytic differentiation. J
Leukoc Biol 1992; 52:173-182; and Balboa M A, Saez Y, Balsinde J.
Calcium-independent phospholipase A2 is required for lysozyme
secretion in U937 promonocytes. J Immunol 2003; 170:5276-5280), and
granzyme in natural killer (NK) cells and cytotoxic lymphocytes
(see, for example, Bochan M R, Goebel W S, Brahmi Z. Stably
transfected antisense granzyme B and perforin constructs inhibit
human granule-mediated lytic ability. Cell Immunol
1995;164:234-239; Gong J H., Maki G, Klingemann H G.
Characterization of a human cell line (NK-92) with phenotypical and
functional characteristics of activated natural killer cells.
Leukemia 1994; 8:652-658; Maki G, Klingemann H G, Martinson J A,
Tam Y K. Factors regulating the cytotoxic activity of the human
natural killer cell line, NK-92. J Hematother Stem Cell Res 2001;
10:369-383; and Takayama H, Trenn G, Sitkovsky M V. A novel
cytotoxic T lymphocyte activation assay. J Immunol Methods 1987;
104:183-190). Such mediators are released at sites of injury and
contribute to inflammation and tissue repair such as in the lung
and elsewhere. It is known that leukocytes release these granules
via an exocytotic mechanism (see, for example, Burgoyne R D, Morgan
A. Secretory granule exocytosis. Physiol Rev 2003; 83:581-632; and
Logan M R, Odemuyiwa S O, Moqbel R. Understanding exocytosis in
immune and inflammatory cells: the molecular basis of mediator
secretion. J Allergy Clin Immunol 2003; 111: 923-932), but
regulatory molecules and specific pathways involved in the
exocytotic process have not been fully described.
[0005] Several exogenous stimuli can provoke degranulation of
leukocytes via a pathway that involves activation of protein kinase
C and subsequent phosphorylation events (see, for example, Burgoyne
R D, Morgan A. Secretory granule exocytosis. Physiol Rev 2003;
83:581-632; Logan M R, Odemuyiwa S O, Moqbel R. Understanding
exocytosis in immune and inflammatory cells: the molecular basis of
mediator secretion. J Allergy Clin Immunol 2003; 111: 923-932;
Smolen J E, Sandborg R R. Ca2+-induced secretion by
electropermeabilized human neutrophils: the roles of Ca2+,
nucleotides and protein kinase C. Biochim Biophys Acta 1990;
1052:133-142; Niessen H W, Verhoeven A J. Role of protein
phosphorylation in the degranulation of electropermeabilized human
neutrophils. Biochim. Biophys. Acta 1994; 1223:267-273; and Naucler
C, Grinstein S, Sundler R., Tapper H. Signaling to localized
degranulation in neutrophils adherent to immune complexes. J Leukoc
Biol 2002; 71:701-710).
[0006] MARCKS protein (where MARCKS as used herein means
"Myristoylated Alanine-Rich C Kinase Substrate"), is a ubiquitous
phosphorylation target of protein kinase C (PKC), and is highly
expressed in leukocytes (see, for example, Aderem A A, Albert K A,
Keum M M, Wang J K, Greengard P, Cohn Z A. Stimulus-dependent
myristoylation of a major substrate for protein kinase C. Nature
1988; 332:362-364; Thelen M, Rosen A, Nairn A C, Aderem A.
Regulation by phosphorylation of reversible association of a
myristoylated protein kinase C substrate with the plasma membrane.
Nature 1991; 351:320-322; and Hartwig J H, Thelen M, Rosen A,
Janmey P A, Nairn A C, Aderem A. MARCKS is an actin filament
crosslinking protein regulated by protein kinase C and
calcium-calmodulin. Nature 1992; 356:618-622. MARCKS protein is
mechanistically involved in a process of exocytotic secretion of
mucin by goblet cells that line respiratory airways (see, for
example, Li et al., J Biol Chem 2001; 276:40982-40990; and Singer
et al., Nat Med 2004; 10:193-196). MARCKS is myristoylated via an
amide bond at the N-terminal amino acid in the MARCKS protein's
amino acid sequence at the alpha-amine position of the glycine
which resides at the N-terminus (i.e., at position 1) of amino acid
sequence. In airway epithelial cells, the myristoylated N-terminal
region of MARCKS appears to be integral to the secretory process.
By the N-terminus of the MARCKS protein is meant the MANS peptide
which contains Myristoyl-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1),
which are L-amino acids. Additionally, the peptide fragments of the
MANS peptide disclosed herein, also preferably are composed of
L-amino acids. The mechanism appears to involve binding of MARCKS,
a myristoylated protein, to membranes of intracellular
granules.
[0007] An N-terminal myristoylated peptide from the N-terminus of
MARCKS has been shown to block both mucin secretion and binding of
MARCKS to mucin granule membranes in goblet cells (see, for
example, Singer et al., Nat Med 2004; 10:193-196). This peptide
contains 24 amino acids of the MARCKS protein beginning with the
N-terminal glycine of the MARCKS protein which is myristoylated via
an amide bond and is known as myristoylated alpha-N-terminal
sequence (MANS); i.e., Myristoyl-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID
NO: 1). Also Vergeres et al., J Biochem. 1998, 330; 5-11, discloses
that the N-terminal glycine residue of MARCKS proteins is
myristoylated via a reaction catalyzed by myristoyl CoA:protein
N-myristoyl transferase (NMT).
[0008] In inflammatory diseases, such as asthma, COPD and chronic
bronchitis; in genetic diseases such as cystic fibrosis; in
allergic conditions (atopy, allergic inflammation); in
bronchiectasis; and in a number of acute, infectious respiratory
illnesses such as pneumonia, rhinitis, influenza or the common
cold, arthritis or auto-immune diseases, inflammatory cells are
usually found in or migrate to areas of injury or infection
associated with inflammatory disease states, especially in or to
respiratory passages or airways of patients suffering from such
diseases. These inflammatory cells can contribute greatly to the
pathology of diseases via tissue damage done by inflammatory
mediators released from these cells. One example of such tissue
damage or destruction via this chronic inflammation occurs in
cystic fibrosis patients where mediators released from neutrophils
(e.g., myeloperoxidase [MPO]) induce the desquamation of the airway
epithelial tissue.
[0009] MARCKS, a protein of approximately 82 kD, has three
evolutionarily-conserved regions (Aderem et al., Nature 1988;
332:362-364; Thelen et al., Nature 1991; 351:320-322; Hartwig et
al., Nature 1992; 356:618-622; Seykora et al., J Biol Chem 1996;
271:18797-18802): an N-terminus, a phosphorylation site domain (or
PSD), and a multiple homology 2 (MH2) domain. Human MARCKS cDNA and
protein is known and reported by Harlan et al., J. Biol. Chem.
1991, 266:14399 (GenBank Accession No. M68956) and also by Sakai et
al., Genomics 1992, 14: 175. These sequences are also provided in a
WO 00/50062, which is incorporated in its entirety by reference.
The N-terminus, an alpha-amino acid sequence comprising 24 amino
acid residues with a myristic acid moiety attached via an amide
bond to the N-terminal glycine residue is involved in binding of
MARCKS to membranes in cells (Seykora et al., J Biol Chem 1996;
271:18797-18802) and possibly to calmodulin (Matsubara et al., J
Biol Chem 2003; 278:48898-48902). This 24 amino acid sequence is
known as the MANS peptide.
SUMMARY OF THE INVENTION
[0010] Involvement of MARCKS protein in release of inflammatory
mediators from the granules of infiltrating leukocytes is relevant
to inflammation in diseases in all tissues and organs, including
lung diseases characterized by airway inflammation, such as asthma,
COPD and cystic fibrosis. However, inflammation and mucus secretion
in the airways are two separate and independent processes (Li et
al., J Biol Chem 2001; 276:40982-40990; Singer et al., Nat Med
2004; 10:193-196). While mucus production and secretion can be
provoked by a number of factors, including mediators released by
inflammatory cells, there is no known direct link whereby excess
mucus causes inflammation.
[0011] In one aspect of this invention, the MANS peptide can play a
role in the reducing the rate and/or amount of release of
inflammatory mediators granules or vesicles in inflammatory
leukocytes.
[0012] In another aspect, peptides derived from the MARCKS
N-terminus, especially from the 24 amino acid N-terminal sequence,
i.e., active contiguous peptide fragments derived from within the
N-terminal 1-to-24 amino acid sequence of MARCKS having a glycine
at position 1, as well as N-terminal amides of such fragments, such
as N-terminal acetic acid amides of such fragments, and/or as well
as C-terminal amides of such fragments, such as C-terminal amides
of ammonia, can inhibit or reduce the rate and/or amount of release
of inflammatory mediators from inflammatory leukocytes. Such
inhibition or reduction in release comprises inhibition of a
MARCKS-related release of inflammatory mediators from inflammatory
leukocytes.
[0013] In another aspect, peptides derived from the MARCKS
N-terminus, especially from the 1-to-24 amino acid N-terminal
sequence, i.e., active contiguous peptide fragments derived from
within the N-terminal 1 to 24 amino acid sequence of MARCKS having
a glycine at position 1, as well as N-terminal amides of such
fragments such as N-terminal acetic acid amides of such fragments,
and as well as C-terminal amides of such fragments such as
C-terminal amides of ammonia, can inhibit the rate of release
and/or amount of release of inflammatory mediators such as those
identified herein in this invention, by inhibiting the process of
degranulation in inflammatory leucocytes.
[0014] In another aspect, the MANS peptide and active fragments
thereof, and active amides of such fragments as described herein,
can compete for membrane binding in inflammatory cells with native
MARCKS protein to attenuate (lessen or reduce) MARCKS-related
release of mediators of inflammation from granules or vesicles
containing such mediators of inflammation in such inflammatory
cells.
[0015] Leukocyte cell types and model cell types that secrete
specific granule contents in response to phorbol ester induced
activation of PKC are useful for in vitro demonstration of efficacy
of peptides of this invention and of substituted peptides (e.g.,
alpha-N-amides, C-terminal amides and esters) of this
invention.
[0016] The attenuation of release of membrane-bound inflammatory
mediators by compounds and compositions of this invention can be
demonstrated using human leukocyte cell lines. For example,
neutrophils isolated from human blood can be used to demonstrate
attenuation or inhibition of release of myeloperoxidase (MPO). The
human promyelocytic cell line HL-60 clone 15 can be used to
demonstrate attenuation of release or inhibition of release or
secretion of eosinophil peroxidase (EPO) by compounds and
compositions of this invention (see, for example, Fischkoff S A.
Graded increase in probability of eosinophilic differentiation of
HL-60 promyelocytic leukemia cells induced by culture under
alkaline conditions. Leuk Res 1988; 12:679-686; Rosenberg H F,
Ackerman S J, Tenen D G. Human eosinophil cationic protein:
molecular cloning of a cytotoxin and helminthotoxin with
ribonuclease activity. J Exp Med 1989; 170:163-176; Tiffany H L, Li
F, Rosenberg H F. Hyperglycosylation of eosinophil ribonucleases in
a promyelocytic leukemia cell line and in differentiated peripheral
blood progenitor cells. J Leukoc Biol 1995; 58:49-54; and Badewa A
P, Hudson C E, Heiman A S. Regulatory effects of eotaxin,
eotaxin-2, and eotaxin-3 on eosinophil degranulation and superoxide
anion generation. Exp Biol Med 2002; 227:645-651). The monocytic
leukemia cell line U937 can be used to demonstrate attenuation of
release or inhibition of release or secretion of lysozyme by
compounds and compositions of this invention (see, for example,
Hoff T, Spencker T, Emmendoerffer A., Goppelt-Struebe M. Effects of
glucocorticoids on the TPA-induced monocytic differentiation. J
Leukoc Biol 1992; 52:173-182; Balboa M A, Saez Y, Balsinde J.
Calcium-independent phospholipase A2 is required for lysozyme
secretion in U937 promonocytes. J Immunol 2003; 170:5276-5280; and
Sundstrom C, Nilsson K. Establishment and characterization of a
human histiocytic lymphoma cell line (U-937). Int J Cancer 1976;
17:565-577). The lymphocyte natural killer cell line NK-92 can be
used to demonstrate attenuation or inhibition of release of
granzyme by compounds and compositions of this invention (see, for
example, Gong J H., Maki G, Klingemann H G. Characterization of a
human cell line (NK-92) with phenotypical and functional
characteristics of activated natural killer cells. Leukemia 1994;
8:652-658; Maki G, Klingemann H G, Martinson J A, Tam Y K. Factors
regulating the cytotoxic activity of the human natural killer cell
line, NK-92. J Hematother Stem Cell Res 2001; 10:369-383; and
Takayama H, Trenn G, Sitkovsky M V. A novel cytotoxic T lymphocyte
activation assay. J Immunol Methods 1987; 104:183-190). In an in
vitro method to inhibit or attenuate the release of a mediator of
inflammation such as those described herein, each of the cell types
is preincubated with a peptide compound or peptide composition of
this invention over a range of concentrations followed by
incubation of these cells by a stimulator of release of
inflammatory mediators, such as phorbol ester. The percent of
inhibition of release of a mediator of inflammation is determined
as compared to the release of the mediator in the absence of the
peptide compound or peptide composition, such as in a
specrophotometric readout of a concentration of the mediator
released.
[0017] To demonstrate the importance of the relative amino acid
sequence positioning in the peptides of the invention, the relative
ability to inhibit or reduce the amount of inflammatory mediator
released by a peptide which is identical to the 24 amino acid
sequence of the MARCKS protein N-terminus region (i.e., the MANS-
myristoylated alpha-N-terminal sequence peptide) was compared to
the ability to inhibit or reduce the amount of inflammatory
mediator released by a peptide containing the same 24 amino acid
residues present in MANS but which are sequenced in a random order
(i.e., an RNS peptide, otherwise referred to as a "Random
N-terminal sequence peptide") with respect to the sequence order in
MANS. In each of the cell types examined, the MANS peptide, but not
the RNS peptide, attenuated release of inflammatory mediators in a
concentration-dependent manner over a time course of 0.5-3.0 hrs.
These results suggest that the relative amino acid sequence
positioning in the peptides of the invention which are in the order
found in the MARCKS protein, specifically its N-terminal region,
and more specifically its 24 amino acid residue N-terminal region
are involved in at least one intracellular pathway dealing with the
inhibition of leukocyte degranulation.
[0018] The invention relates to a new use for the 24 amino acid
peptide sequence, and to the alpha-N-terminal acetylated peptide
sequence, the myristoylated polypeptide, also known as the MANS
peptide, and to active fragments thereof, which active fragments
can be selected from the group of peptides having from 4 to 23
contiguous amino acid residues of the MANS peptide amino acid
sequence, and which fragments may be N-terminal-myristoylated if
they do not begin with the N-terminal glycine at position 1 in SEQ
ID NO: 1, or which may be N-terminal-acylated with C2 to C12 acyl
groups, including N-terminal-acetylated, and/or C-terminal amidated
with an NH2 group.
[0019] The invention also relates to a new method for blocking
MARCKS-related cellular secretory processes, especially those that
involve the MARCKS-related release of inflammatory mediators from
inflammatory cells, whose stimulatory pathways involve the protein
kinase C (PKC) substrate MARCKS protein and release of contents
from intracellular vesicles or granules.
[0020] The present invention is directed to a method of inhibiting
the exocytotic release of at least one inflammatory mediator from
at least one inflammatory cell comprising contacting the at least
one inflammatory cell, which cell comprises at least one
inflammatory mediator contained within a vesicle inside the cell,
with at least one peptide selected from the group consisting of a
MANS peptide and an active fragment thereof as described herein in
an effective amount to reduce the release of the inflammatory
mediator from the inflammatory cell as compared to the release of
the inflammatory mediator from the same type of inflammatory cell
that would occur in the absence of the at least one peptide.
[0021] The present invention is further directed to a method of
inhibiting the release of at least one inflammatory mediator from
at least one inflammatory cell in a tissue or fluid of a subject
comprising the administration to the subject's tissue and/or fluid,
which comprises at least one inflammatory cell comprising at least
one inflammatory mediator contained within a vesicle inside the
cell, a therapeutically effective amount of a pharmaceutical
composition comprising at least one peptide selected from the group
consisting of a MANS peptide and an active fragment thereof in a
therapeutically effective amount to reduce the release of the
inflammatory mediator from at least one inflammatory cell as
compared to release of the inflammatory mediator from at least one
of the same type of inflammatory cell that would occur in the
absence of the at least one peptide. More specifically, inhibiting
the release of an inflammatory mediator comprises blocking or
reducing the release of an inflammatory mediator from the
inflammatory cell.
[0022] More particularly, the present invention includes a method
of reducing inflammation in a subject comprising the administration
of a therapeutically effective amount of a pharmaceutical
composition comprising a MANS peptide (i.e.,
N-myristoyl-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1)) or an active
fragment thereof. The active fragment is at least four and
preferably at least six amino acids in length. As used herein, an
"active fragment" of a MARCKS protein is one that affects (inhibits
or reduces) MARCKS protein-mediated release, such as MARCKS
protein-mediated release of an inflammatory mediator. An active
fragment can be selected from the group consisting of
GAQFSKTAAKGEAAAERPGEAAV (SEQ ID NO: 2); GAQFSKTAAKGEAAAERPGEAA (SEQ
ID NO: 4); GAQFSKTAAKGEAAAERPGEA (SEQ ID NO: 7);
GAQFSKTAAKGEAAAERPGE (SEQ ID NO: 11); GAQFSKTAAKGEAAAERPG (SEQ ID
NO: 16); GAQFSKTAAKGEAAAERP (SEQ ID NO: 22); GAQFSKTAAKGEAAAER (SEQ
ID NO: 29); GAQFSKTAAKGEAAAE (SEQ ID NO: 37); GAQFSKTAAKGEAAA (SEQ
ID NO: 46); GAQFSKTAAKGEAA (SEQ ID NO: 56); GAQFSKTAAKGEA (SEQ ID
NO: 67); GAQFSKTAAKGE (SEQ ID NO: 79); GAQFSKTAAKG (SEQ ID NO: 92);
GAQFSKTAAK (SEQ ID NO: 106); GAQFSKTAA (SEQ ID NO: 121); GAQFSKTA
(SEQ ID NO: 137); GAQFSKT (SEQ ID NO: 154); GAQFSK (SEQ ID NO:
172); GAQFS (SEQ ID NO: 191) and GAQF (SEQ ID NO: 211). These
peptides, instead of containing a myristoyl moiety at the
N-terminal amino acid, either contain no chemical moiety or a
non-myristoyl chemical moiety at the N-terminal amino acid and/or a
chemical moiety at the C-terminal amino acid, such as an N-terminal
acetyl group and/or a C-terminal amide group as described herein.
The presence of the hydrophobic N-terminal myristoyl moiety in the
MANS peptides and N-terminal myristoylated fragments thereof can
enhance their compatibility with and presumably their permeability
to plasma membranes, and potentially enable the peptides to be
taken up by cells. The hydrophobic insertion of a myristoyl group
into a membrane lipid bilayer can provide a partition coefficient
or apparent association constant with lipids of up to 10.sup.4
M.sup.-1 or a unitary Gibbs free binding energy of about 8 kcal/mol
(see, for example, Peitzsch, R. M., and McLaughlin, S. 1993,
Binding of acylated peptides and fatty acids to phospholipid
vesicles: pertinence to myristoylated proteins. Biochemistry. 32:
10436-10443) which is sufficient, at least in part, to permit a
partitioning of the MANS peptide and of myristoylated MANS peptide
fragments into the plasma membrane of a cell while additional
functional groups and their interactions within the MANS peptide
(which is myristoylated) and within myristoylated MANS peptide
fragments can potentiate their relative membrane permeabilities.
The fragments can each exhibit partition coefficients and membrane
affinities that are representative of their respective structure.
The fragments can be prepared by methods of peptide synthesis known
in the art, such as by solid phase peptide synthesis (see, for
example, the methods described in Chan, Weng C. and White, Peter D.
Eds., Fmoc Solid Phase Peptide Synthesis: A Practical Approach,
Oxford University Press, New York, New York (2000); and
Lloyd-Williams, P. et al. Chemical Approaches to the Synthesis of
Peptides and Proteins (1997)) and purified by methods known in the
art, such as by high pressure liquid chromatography. Molecular
weight of each peptide can be confirmed by mass spectroscopy with
each showing a peak with an appropriate molecular mass. Efficacy of
the individual peptides and of combinations of individual peptides
(for example, combinations of 2 of the peptides, combinations of 3
of the peptides, combinations of 4 of the peptides) in the methods
of this disclosure can be readily determined without undue
experimentation using the procedures described in the examples
disclosed herein. A preferred combination will comprise two of the
peptides; a preferred molar ratio of the peptides can be from 50:50
(i.e., 1:1) to 99.99 to 0.01, which ratio can be readily determined
using the procedures described in the examples disclosed
herein.
[0023] Preferably the MANS peptide or active fragment thereof is
contained in a pharmaceutical composition which is useful to block
inflammation. The present invention also includes methods for
inhibiting a cellular secretory process in a subject comprising the
administration of a therapeutically effective amount of a compound
comprising a MANS peptide or an active fragment thereof, that
inhibits an inflammatory mediator in a subject. The administration
is generally selected from the group consisting of topical
administration, parenteral administration, rectal administration,
pulmonary administration, inhalation and nasal or oral
administration, wherein pulmonary administration generally includes
either an aerosol, a dry powder inhaler, a metered dose inhaler, or
a nebulizer.
[0024] Administration of a composition comprising a
degranulation-inhibiting amount of the MANS peptide or a
degranulation-inhibiting amount of an active fragment thereof, such
as a pharmaceutical composition of the MANS peptide or an active
fragment thereof, for human or animal use provides the MANS peptide
or active fragment thereof at least to the site in or on a tissue
or to a fluid-containing layer in contact with the surface of a
tissue where an inflammatory granulocytic cell resides or into
which an inflammatory granulocytic cell will invade, thus enabling
the MANS peptide or an active fragment thereof to contact the
inflammatory granulocytic cell. In one aspect, administration of
such a composition can be made at the first onset or first
detection of inflammation or first perception of inflammation by
the human or animal or at the first perceptible change in the level
of inflammation in a human or animal to reduce the amount of
inflammation that would otherwise occur in the absence of the MANS
peptide or active fragment thereof. In another aspect,
administration can be made during an ongoing inflammation of a
tissue in the human or animal to reduce the amount of additional
inflammation that would otherwise occur in the absence of the MANS
peptide or active fragment thereof. While the amount and frequency
of dose can be determined by clinical evaluation and be a function
of the disease or source of inflammation and the extent of tissue
involved and the age and size of the patient, it is anticipated
that dosing of a pharmaceutical composition can be repeated after 3
to 8 hours, preferably after 6 to 8 hours after the first
administration of the pharmaceutical composition.
[0025] The present invention also includes methods of reducing
inflammation in a subject comprising the administration of a
therapeutically effective amount of a compound that inhibits the
MARCKS-related release of inflammatory mediators, whereby the
release of at least one inflammatory mediator in the subject is
reduced compared to that which would occur in the absence of said
treatment. As used herein "reducing" generally means a lessening of
the effects of inflammation. Preferably, release of inflammatory
mediators are inhibited or blocked by the methods disclosed.
[0026] Another embodiment of the present invention includes methods
of reducing inflammation in a subject comprising administering a
therapeutically effective amount of a compound that inhibits the
MARCKS-related release of inflammatory mediators, whereby the
inflammation in the subject is reduced compared to that which would
occur in the absence of said treatment. The present invention also
discloses methods of reducing or inhibiting inflammation in a
subject comprising the administration of a therapeutically
effective amount of a MANS peptide or an active fragment thereof
effective to inhibit an inflammatory mediator at the inflammation
site. The term "inhibiting" means a reduction in the amount of
inflammatory mediator secretion. The term "completely inhibiting"
means a reduction to zero in the amount of inflammatory mediator
secretion. Again, as stated above, the active fragment is at least
four and preferably at least six amino acids in length. The term
"exocytotic process" means exocytosis, i.e., a process of cellular
secretion or excretion in which substances contained in a vesicle,
which vesicle resides inside a cell, are discharged from the cell
by fusion of the vesicular membrane of the vesicle with the outer
cell membrane. "Degranulation" means the release of cellular
granule contents. The term "degranulation-inhibiting" means a
reduction in the release of the inflammatory mediators contained
within the granules of the inflammatory cell. Thus, a
degranulation-inhibiting amount of the MANS peptide and/or an
active fragment thereof is the amount of these peptides that is
sufficient to reduce the release of the inflammatory mediators
contained in the granules as compared to release in the absence of
the same peptide.
[0027] In the reference peptide, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID
NO: 1), at the N-terminal position of the reference peptide, G is
at position 1; adjacent to G at position 1 is A at position 2;
adjacent to A at position 2 is Q at position 3; adjacent to Q at
position 3 is F at position 4; adjacent to F at position 4 is S at
position 5; adjacent to S at position 5 is K at position 6;
adjacent to K at position 6 is T at position 7; adjacent to T at
position 7 is A at position 8; adjacent to A at position 8 is A at
position 9; adjacent to A at position 9 is K at position 10;
adjacent to K at position 10 is G at position 11; adjacent to G at
position 11 is Eat position 12; adjacent to E at position 12 is A
at position 13; adjacent to A at position 13 is A at position 14;
adjacent to A at position 14 is A at position 15; adjacent to A at
position 15 is E at position 16; adjacent to E at position 16 is R
at position 17; adjacent to R at position 17 is P at position 18;
adjacent to P at position 18 is G at position 19; adjacent to G at
position 19 is E at position 20; adjacent to E at position 20 is A
at position 21; adjacent to A at position 21 is A at position 22;
adjacent to A at position 22 is V at position 23; and adjacent to V
at position 23 is A at position 24, wherein position 24 is the
C-terminal position of the reference peptide.
[0028] A "variant" of a reference peptide or a variant of a 4 to 23
amino acid segment of a reference peptide is a peptide which has an
amino acid sequence which differs from the amino acid sequence of
the reference peptide or from the amino acid sequence of the
segment of the reference peptide, respectively, in at least one
amino acid position in the reference peptide or reference peptide
segment amino acid sequence, respectively, but which retains mucin-
or mucus-inhibiting activity, which activity is typically between
0.1 to 10 times the activity of the reference peptide or segment,
respectively, preferably between 0.2 to 6 times the activity of the
reference peptide or segment, respectively, more preferably between
0.3 to 5 times the activity of the reference peptide or segment,
respectively. A "variant" of a reference amino acid sequence or a
variant of a 4 to 23 amino acid segment of a reference amino acid
sequence is an amino acid sequence that differs by at least one
amino acid from the reference amino acid sequence or from the
segment of the reference amino acid sequence, respectively, but has
an amino acid sequence of a peptide that retains mucin- or
mucus-inhibiting activity of the peptide or segment, respectively,
encoded by the reference amino acid sequence, which activity is
typically between 0.1 to 10 times the activity of the peptide or
segment, respectively, of the reference sequence, preferably
between 0.2 to 6 times the activity of the peptide or segment of
the reference sequence, respectively, more preferably between 0.3
to 5 times the activity of the peptide or segment of the reference
sequence, respectively. A substitution variant peptide or a
substitution variant amino acid sequence may vary (i.e., differ)
from a reference peptide or reference amino acid sequence by one or
more amino acid substitutions in the reference amino acid sequence;
a deletion variant peptide or a deletion variant amino acid
sequence may vary (i.e., differ) from a reference peptide or
reference amino acid sequence by one or more amino acid deletions
in the reference amino acid sequence; and an addition variant
peptide or an addition variant amino acid sequence may vary (i.e.,
differ) from a reference peptide sequence or reference amino acid
sequence by one or more amino acid additions in the reference
sequence. A variant peptide or variant amino acid sequence can
result from a substitution of one or more amino acids (e.g.,
substitution of at least 1, 2, 3, 4, 5, 6, 7, or 8 amino acids) in
a reference sequence, or can result from a deletion of one or more
amino acids (e.g., deletion of at least 1, 2, 3, 4, 5, 6, 7, or 8
amino acids) in a reference sequence, or can result from an
addition of one or more amino acids (e.g., addition of at least 1,
2, 3, 4, 5, 6, 7, or 8 amino acids) in a reference sequence, or a
combination thereof in any order. A substitution variant 4 to 23
amino acid peptide segment or a substitution variant 4 to 23 amino
acid segment sequence may vary (i.e., differ) from a reference 4 to
23 amino acid peptide segment or reference 4 to 23 amino acid
segment sequence by one or more amino acid substitutions in the
reference amino acid segment sequence; a deletion variant 4 to 23
amino acid peptide segment or a 4 to 22 amino acid deletion variant
amino acid segment sequence may vary (i.e., differ) from a 5 to 23
reference peptide segment or a 5 to 23 amino acid reference amino
acid segment sequence by one or more amino acid deletions in the
reference amino acid segment sequence; and an 4 to 23 amino acid
addition variant peptide or a 4 to 23 amino acid addition variant
amino acid sequence may vary (i.e., differ) from a 4 to 22 amino
acid reference peptide sequence or a 4 to 22 amino acid reference
amino acid sequence by one or more amino acid additions in the
reference sequence. A 4 to 23 amino acid variant peptide or a 4 to
23 amino acid variant amino acid sequence can result from a
substitution of one or more amino acids (e.g., substitution of at
least 1, 2, 3, 4, 5, 6, 7, 8 amino acids) in a 4 to 23 amino acid
segment of a reference amino acid sequence, or can result from a
deletion of one or more amino acids (e.g., deletion of at least 1,
2, 3, 4, 5, 6, 7, or 8 amino acids) in a respectively larger
reference amino acid sequence, or can result from an addition of
one or more amino acids (e.g., addition of at least 1, 2, 3, 4, 5,
6, 7, or 8 amino acids) in a respectively smaller reference amino
acid sequence, or from a combination thereof. Preferably, a variant
peptide or amino acid sequence varies from a reference peptide or
from a segment of a reference peptide or from a reference amino
acid sequence or from a segment of a reference amino acid sequence,
respectively, by less than 10 amino acid substitutions, deletions,
and/or additions; more preferably less than 8 amino acid
substitutions, deletions, and/or additions; even more preferably
less than 6 amino acid substitutions, deletions, and/or additions;
and even more preferably less than 5 amino acid substitutions,
deletions, and/or additions; and yet even more preferably less than
4 amino acid substitutions, deletions, and/or additions. Most
preferably the variant amino acid sequence differs from a reference
peptide or segment amino acid sequence by one or two or three amino
acids.
[0029] "Sequence identity" means, with respect to amino acid
sequences of two peptides, the number of positions with identical
amino acids divided by the number of amino acids in the shorter of
the two sequences.
[0030] "Substantially identical" means, with respect to comparison
of the amino acid sequences of two peptides or comparison of the
amino acid sequences of two peptide segments (e.g. segments of a
reference peptide amino acid sequence), that the amino acid
sequence of the peptides or segments of peptides have at least 75%
sequence identity, preferably at least 80% sequence identity, more
preferably at least 90% sequence identity, and most preferably at
least 95% sequence identity.
[0031] The term "peptide" as used herein includes the peptide as
well as pharmaceutically acceptable salts of the peptide.
[0032] An "isolated" peptide, as used herein, means a
naturally-occurring peptide that has been separated or
substantially separated from the cellular components (e.g., nucleic
acids and other peptides) that naturally accompany it by
purification, recombinant synthesis, or chemical synthesis, and
also encompasses non-naturally-occurring recombinantly or
chemically synthesized peptides that have been purified or
substantially purified from cellular components, biological
materials, chemical precursors, or other chemicals.
[0033] The following three-letter and one-letter amino acid
abbreviations are used throughout the text: Alanine: (Ala) A;
Arginine: (Arg) R; Asparagine: (Asn) N; Aspartic acid: (Asp) D;
Cysteine: (Cys) C; Glutamine: (Gln) Q; Glutamic acid: (Glu) E;
Glycine: (Gly) G; Histidine: (His) H; Isoleucine: (Ile) I; Leucine:
(Leu) L; Lysine: (Lys) K; Methionine: (Met) M; Phenylalanine: (Phe)
F; Proline: (Pro) P; Serine: (Ser) S; Threonine: (Thr) T;
Tryptophan: (Trp) W; Tyrosine: (Tyr) Y; Valine: (Val) V. Additional
three letter symbols of amino acids useful herein include, in
brackets, (Hyp) for hydroxyproline, (Nle) for norleucine, (Orn) for
ornithine, (Pyr) for pyroglutamic acid and (Sar) for sarcosine. By
convention, the amino (or N-terminal) end of a peptide appears at
the left end of a written amino acid sequence of the peptide and
the carboxy (or C-terminal) end appears at the right end of a
written amino acid sequence. The amino acid sequence of a peptide
can be written in single letter symbols to represent the amino
acids which are covalently linked by peptide amide bonds in the
peptide.
[0034] Active fragments of the MANS peptide can be useful in the
prevention or reduction in amount of inflammation in a tissue in an
animal caused by inflammatory mediators. Active fragments of the
MANS peptide also can be useful in the prevention or reduction in
amount of tissue damage in an animal produced or caused by
inflammatory mediators. An active fragment of the MANS peptide is
composed of at least 4 contiguous amino acids and no more than 23
contiguous amino acids of the MANS peptide (SEQ ID NO: 1). The term
"active fragment" within the context of the present invention is
intended to encompass those fragments of the MANS peptides that are
capable of preventing or reducing the release of inflammatory
mediators from an inflammatory cell. The reduction of release of
inflammatory mediators by the active fragments can range from at
least 5% to at least 99% reduction as compared to a reference
peptide, such as MANS peptide.
[0035] Table 1 contains a list of amino acid sequences in single
letter abbreviation format together with a respectively
corresponding peptide number and SEQ ID NO. The reference peptide
amino acid sequence (MANS peptide) is listed as peptide 1. Amino
acid sequences of peptides of the invention having an amino acid
sequence of from 4 to 23 contiguous amino acids of the reference
amino acid sequence are listed as peptides 2 to 231, together with
the amino acid sequence of a random N-terminal sequence (RNS)
comprising amino acids of the MANS peptide as peptide 232. Amino
acid sequences of representative variants of amino acid sequences
of peptides of the invention as described herein and are also
listed as peptides 233 to 245 and 247 to 251. This variant peptides
listed are not intended to be a limiting group of peptides, but are
presented only to serve as representative examples of variant
peptides of the invention. Also presented is a representative
reverse amino acid sequence (peptide 246) and a representative
random amino acid sequence of peptide (peptide 232) of the
invention. The reverse and random amino acid sequences in the table
are not intended to be representative of the invention.
[0036] Table 1 contains a listing of peptides of this invention and
their respective amino acid sequences and corresponding SEQ ID
NOS.
TABLE-US-00001 TABLE 1 Peptides and Amino Acid Sequences Sequence
Peptide No. Sequence ID No. peptide 1 GAQFSKTAAKGEAAAERPGEAAVA SEQ
ID NO. 1 peptide 2 GAQFSKTAAKGEAAAERPGEAAV SEQ ID NO. 2 peptide 3
AQFSKTAAKGEAAAERPGEAAVA SEQ ID NO. 3 peptide 4
GAQFSKTAAKGEAAAERPGEAA SEQ ID NO. 4 peptide 5
AQFSKTAAKGEAAAERPGEAAV SEQ ID NO. 5 peptide 6
QFSKTAAKGEAAAERPGEAAVA SEQ ID NO. 6 peptide 7 GAQFSKTAAKGEAAAERPGEA
SEQ ID NO. 7 peptide 8 AQFSKTAAKGEAAAERPGEAA SEQ ID NO. 8 peptide 9
QFSKTAAKGEAAAERPGEAAV SEQ ID NO. 9 peptide 10 FSKTAAKGEAAAERPGEAAVA
SEQ ID NO. 10 peptide 11 GAQFSKTAAKGEAAAERPGE SEQ ID NO. 11 peptide
12 AQFSKTAAKGEAAAERPGEA SEQ ID NO. 12 peptide 13
QFSKTAAKGEAAAERPGEAA SEQ ID NO. 13 peptide 14 FSKTAAKGEAAAERPGEAAV
SEQ ID NO. 14 peptide 15 SKTAAKGEAAAERPGEAAVA SEQ ID NO. 15 peptide
16 GAQFSKTAAKGEAAAERPG SEQ ID NO. 16 peptide 17 AQFSKTAAKGEAAAERPGE
SEQ ID NO. 17 peptide 18 QFSKTAAKGEAAAERPGEA SEQ ID NO. 18 peptide
19 FSKTAAKGEAAAERPGEAA SEQ ID NO. 19 peptide 20 SKTAAKGEAAAERPGEAAV
SEQ ID NO. 20 peptide 21 KTAAKGEAAAERPGEAAVA SEQ ID NO. 21 peptide
22 GAQFSKTAAKGEAAAERP SEQ ID NO. 22 peptide 23 AQFSKTAAKGEAAAERPG
SEQ ID NO. 23 peptide 24 QFSKTAAKGEAAAERPGE SEQ ID NO. 24 peptide
25 FSKTAAKGEAAAERPGEA SEQ ID NO. 25 peptide 26 SKTAAKGEAAAERPGEAA
SEQ ID NO. 26 peptide 27 KTAAKGEAAAERPGEAAV SEQ ID NO. 27 peptide
28 TAAKGEAAAERPGEAAVA SEQ ID NO. 28 peptide 29 GAQFSKTAAKGEAAAER
SEQ ID NO. 29 peptide 30 AQFSKTAAKGEAAAERP SEQ ID NO. 30 peptide 31
QFSKTAAKGEAAAERPG SEQ ID NO. 31 peptide 32 FSKTAAKGEAAAERPGE SEQ ID
NO. 32 peptide 33 SKTAAKGEAAAERPGEA SEQ ID NO. 33 peptide 34
KTAAKGEAAAERPGEAA SEQ ID NO. 34 peptide 35 TAAKGEAAAERPGEAAV SEQ ID
NO. 35 peptide 36 AAKGEAAAERPGEAAVA SEQ ID NO. 36 peptide 37
GAQFSKTAAKGEAAAE SEQ ID NO. 37 peptide 38 AQFSKTAAKGEAAAER SEQ ID
NO. 38 peptide 39 QFSKTAAKGEAAAERP SEQ ID NO. 39 peptide 40
FSKTAAKGEAAAERPG SEQ ID NO. 40 peptide 41 SKTAAKGEAAAERPGE SEQ ID
NO. 41 peptide 42 KTAAKGEAAAERPGEA SEQ ID NO. 42 peptide 43
TAAKGEAAAERPGEAA SEQ ID NO. 43 peptide 44 AAKGEAAAERPGEAAV SEQ ID
NO. 44 peptide 45 AKGEAAAERPGEAAVA SEQ ID NO. 45 peptide 46
GAQFSKTAAKGEAAA SEQ ID NO. 46 peptide 47 AQFSKTAAKGEAAAE SEQ ID NO.
47 peptide 48 QFSKTAAKGEAAAER SEQ ID NO. 48 peptide 49
FSKTAAKGEAAAERP SEQ ID NO. 49 peptide 50 SKTAAKGEAAAERPG SEQ ID NO.
50 peptide 51 KTAAKGEAAAERPGE SEQ ID NO. 51 peptide 52
TAAKGEAAAERPGEA SEQ ID NO. 52 peptide 53 AAKGEAAAERPGEAA SEQ ID NO.
53 peptide 54 AKGEAAAERPGEAAV SEQ ID NO. 54 peptide 55
KGEAAAERPGEAAVA SEQ ID NO. 55 peptide 56 GAQFSKTAAKGEAA SEQ ID NO.
56 peptide 57 AQFSKTAAKGEAAA SEQ ID NO. 57 peptide 58
QFSKTAAKGEAAAE SEQ ID NO. 58 peptide 59 FSKTAAKGEAAAER SEQ ID NO.
59 peptide 60 SKTAAKGEAAAERP SEQ ID NO. 60 peptide 61
KTAAKGEAAAERPG SEQ ID NO. 61 peptide 62 TAAKGEAAAERPGE SEQ ID NO.
62 peptide 63 AAKGEAAAERPGEA SEQ ID NO. 63 peptide 64
AKGEAAAERPGEAA SEQ ID NO. 64 peptide 65 KGEAAAERPGEAAV SEQ ID NO.
65 peptide 66 GEAAAERPGEAAVA SEQ ID NO. 66 peptide 67 GAQFSKTAAKGEA
SEQ ID NO. 67 peptide 68 AQFSKTAAKGEAA SEQ ID NO. 68 peptide 69
QFSKTAAKGEAAA SEQ ID NO. 69 peptide 70 FSKTAAKGEAAAE SEQ ID NO. 70
peptide 71 SKTAAKGEAAAER SEQ ID NO. 71 peptide 72 KTAAKGEAAAERP SEQ
ID NO. 72 peptide 73 TAAKGEAAAERPG SEQ ID NO. 73 peptide 74
AAKGEAAAERPGE SEQ ID NO. 74 peptide 75 AKGEAAAERPGEA SEQ ID NO. 75
peptide 76 KGEAAAERPGEAA SEQ ID NO. 76 peptide 77 GEAAAERPGEAAV SEQ
ID NO. 77 peptide 78 EAAAERPGEAAVA SEQ ID NO. 78 peptide 79
GAQFSKTAAKGE SEQ ID NO. 79 peptide 80 AQFSKTAAKGEA SEQ ID NO. 80
peptide 81 QFSKTAAKGEAA SEQ ID NO. 81 peptide 82 FSKTAAKGEAAA SEQ
ID NO. 82 peptide 83 SKTAAKGEAAAE SEQ ID NO. 83 peptide 84
KTAAKGEAAAER SEQ ID NO. 84 peptide 85 TAAKGEAAAERP SEQ ID NO. 85
peptide 86 AAKGEAAAERPG SEQ ID NO. 86 peptide 87 AKGEAAAERPGE SEQ
ID NO. 87 peptide 88 KGEAAAERPGEA SEQ ID NO. 88 peptide 89
GEAAAERPGEAA SEQ ID NO. 89 peptide 90 EAAAERPGEAAV SEQ ID NO. 90
peptide 91 AAAERPGEAAVA SEQ ID NO. 91 peptide 92 GAQFSKTAAKG SEQ ID
NO. 92 peptide 93 AQFSKTAAKGE SEQ ID NO. 93 peptide 94 QFSKTAAKGEA
SEQ ID NO. 94 peptide 95 FSKTAAKGEAA SEQ ID NO. 95 peptide 96
SKTAAKGEAAA SEQ ID NO. 96 peptide 97 KTAAKGEAAAE SEQ ID NO. 97
peptide 98 TAAKGEAAAER SEQ ID NO. 98 peptide 99 AAKGEAAAERP SEQ ID
NO. 99 peptide 100 AKGEAAAERPG SEQ ID NO. 100 peptide 101
KGEAAAERPGE SEQ ID NO. 101 peptide 102 GEAAAERPGEA SEQ ID NO. 102
peptide 103 EAAAERPGEAA SEQ ID NO. 103 peptide 104 AAAERPGEAAV SEQ
ID NO. 104 peptide 105 AAERPGEAAVA SEQ ID NO. 105 peptide 106
GAQFSKTAAK SEQ ID NO. 106 peptide 107 AQFSKTAAKG SEQ ID NO. 107
peptide 108 QFSKTAAKGE SEQ ID NO. 108 peptide 109 FSKTAAKGEA SEQ ID
NO. 109 peptide 110 SKTAAKGEAA SEQ ID NO. 110 peptide 111
KTAAKGEAAA SEQ ID NO. 111 peptide 112 TAAKGEAAAE SEQ ID NO. 112
peptide 113 AAKGEAAAER SEQ ID NO. 113 peptide 114 AKGEAAAERP SEQ ID
NO. 114 peptide 115 KGEAAAERPG SEQ ID NO. 115 peptide 116
GEAAAERPGE SEQ ID NO. 116 peptide 117 EAAAERPGEA SEQ ID NO. 117
peptide 118 AAAERPGEAA SEQ ID NO. 118 peptide 119 AAERPGEAAV SEQ ID
NO. 119 peptide 120 AERPGEAAVA SEQ ID NO. 120 peptide 121 GAQFSKTAA
SEQ ID NO. 121 peptide 122 AQFSKTAAK SEQ ID NO. 122
peptide 123 QFSKTAAKG SEQ ID NO. 123 peptide 124 FSKTAAKGE SEQ ID
NO. 124 peptide 125 SKTAAKGEA SEQ ID NO. 125 peptide 126 KTAAKGEAA
SEQ ID NO. 126 peptide 127 TAAKGEAAA SEQ ID NO. 127 peptide 128
AAKGEAAAE SEQ ID NO. 128 peptide 129 AKGEAAAER SEQ ID NO. 129
peptide 130 KGEAAAERP SEQ ID NO. 130 peptide 131 GEAAAERPG SEQ ID
NO. 131 peptide 132 EAAAERPGE SEQ ID NO. 132 peptide 133 AAAERPGEA
SEQ ID NO. 133 peptide 134 AAERPGEAA SEQ ID NO. 134 peptide 135
AERPGEAAV SEQ ID NO. 135 peptide 136 ERPGEAAVA SEQ ID NO. 136
peptide 137 GAQFSKTA SEQ ID NO. 137 peptide 138 AQFSKTAA SEQ ID NO.
138 peptide 139 QFSKTAAK SEQ ID NO. 139 peptide 140 FSKTAAKG SEQ ID
NO. 140 peptide 141 SKTAAKGE SEQ ID NO. 141 peptide 142 KTAAKGEA
SEQ ID NO. 142 peptide 143 TAAKGEAA SEQ ID NO. 143 peptide 144
AAKGEAAA SEQ ID NO. 144 peptide 145 AKGEAAAE SEQ ID NO. 145 peptide
146 KGEAAAER SEQ ID NO. 146 peptide 147 GEAAAERP SEQ ID NO. 147
peptide 148 EAAAERPG SEQ ID NO. 148 peptide 149 AAAERPGE SEQ ID NO.
149 peptide 150 AAERPGEA SEQ ID NO. 150 peptide 151 AERPGEAA SEQ ID
NO. 151 peptide 152 ERPGEAAV SEQ ID NO. 152 peptide 153 RPGEAAVA
SEQ ID NO. 153 peptide 154 GAQFSKT SEQ ID NO. 154 peptide 155
AQFSKTA SEQ ID NO. 155 peptide 156 QFSKTAA SEQ ID NO. 156 peptide
157 FSKTAAK SEQ ID NO. 157 peptide 158 SKTAAKG SEQ ID NO. 158
peptide 159 KTAAKGE SEQ ID NO. 159 peptide 160 TAAKGEA SEQ ID NO.
160 peptide 161 AAKGEAA SEQ ID NO. 161 peptide 162 AKGEAAA SEQ ID
NO. 162 peptide 163 KGEAAAE SEQ ID NO. 163 peptide 164 GEAAAER SEQ
ID NO. 164 peptide 165 EAAAERP SEQ ID NO. 165 peptide 166 AAAERPG
SEQ ID NO. 166 peptide 167 AAERPGE SEQ ID NO. 167 peptide 168
AERPGEA SEQ ID NO. 168 peptide 169 ERPGEAA SEQ ID NO. 169 peptide
170 RPGEAAV SEQ ID NO. 170 peptide 171 PGEAAVA SEQ ID NO. 171
peptide 172 GAQFSK SEQ ID NO. 172 peptide 173 AQFSKT SEQ ID NO. 173
peptide 174 QFSKTA SEQ ID NO. 174 peptide 175 FSKTAA SEQ ID NO. 175
peptide 176 SKTAAK SEQ ID NO. 176 peptide 177 KTAAKG SEQ ID NO. 177
peptide 178 TAAKGE SEQ ID NO. 178 peptide 179 AAKGEA SEQ ID NO. 179
peptide 180 AKGEAA SEQ ID NO. 180 peptide 181 KGEAAA SEQ ID NO. 181
peptide 182 GEAAAE SEQ ID NO. 182 peptide 183 EAAAER SEQ ID NO. 183
peptide 184 AAAERP SEQ ID NO. 184 peptide 185 AAERPG SEQ ID NO. 185
peptide 186 AERPGE SEQ ID NO. 186 peptide 187 ERPGEA SEQ ID NO. 187
peptide 188 RPGEAA SEQ ID NO. 188 peptide 189 PGEAAV SEQ ID NO. 189
peptide 190 GEAAVA SEQ ID NO. 190 peptide 191 GAQFS SEQ ID NO. 191
peptide 192 AQFSK SEQ ID NO. 192 peptide 193 QFSKT SEQ ID NO. 193
peptide 194 FSKTA SEQ ID NO. 194 peptide 195 SKTAA SEQ ID NO. 195
peptide 196 KTAAK SEQ ID NO. 196 peptide 197 TAAKG SEQ ID NO. 197
peptide 198 AAKGE SEQ ID NO. 198 peptide 199 AKGEA SEQ ID NO. 199
peptide 200 KGEAA SEQ ID NO. 200 peptide 201 GEAAA SEQ ID NO. 201
peptide 202 EAAAE SEQ ID NO. 202 peptide 203 AAAER SEQ ID NO. 203
peptide 204 AAERP SEQ ID NO. 204 peptide 205 AERPG SEQ ID NO. 205
peptide 206 ERPGE SEQ ID NO. 206 peptide 207 RPGEA SEQ ID NO. 207
peptide 208 PGEAA SEQ ID NO. 208 peptide 209 GEAAV SEQ ID NO. 209
peptide 210 EAAVA SEQ ID NO. 210 peptide 211 GAQF SEQ ID NO. 211
peptide 212 AQFS SEQ ID NO. 212 peptide 213 QFSK SEQ ID NO. 213
peptide 214 FSKT SEQ ID NO. 214 peptide 215 SKTA SEQ ID NO. 215
peptide 216 KTAA SEQ ID NO. 216 peptide 217 TAAK SEQ ID NO. 217
peptide 218 AAKG SEQ ID NO. 218 peptide 219 AKGE SEQ ID NO. 219
peptide 220 KGEA SEQ ID NO. 220 peptide 221 GEAA SEQ ID NO. 221
peptide 222 EAAA SEQ ID NO. 222 peptide 223 AAAE SEQ ID NO. 223
peptide 224 AAER SEQ ID NO. 224 peptide 225 AERP SEQ ID NO. 225
peptide 226 ERPG SEQ ID NO. 226 peptide 227 RPGE SEQ ID NO. 227
peptide 228 PGEA SEQ ID NO. 228 peptide 229 GEAA SEQ ID NO. 229
peptide 230 EAAV SEQ ID NO. 230 peptide 231 AAVA SEQ ID NO. 231
peptide 232 GTAPAAEGAGAEVKRASAEAKQAF SEQ ID NO. 232 peptide 233
GKQFSKTAAKGE SEQ ID NO. 233 peptide 234 GAQFSKTKAKGE SEQ ID NO. 234
peptide 235 GKQFSKTKAKGE SEQ ID NO. 235 peptide 236 GAQASKTAAK SEQ
ID NO. 236 peptide 237 GAQASKTAAKGE SEQ ID NO. 237 peptide 238
GAEFSKTAAKGE SEQ ID NO. 238 peptide 239 GAQFSKTAAAGE SEQ ID NO. 239
peptide 240 GAQFSKTAAKAE SEQ ID NO. 240 peptide 241 GAQFSKTAAKGA
SEQ ID NO. 241 peptide 242 AAQFSKTAAK SEQ ID NO. 242 peptide 243
GAAFSKTAAK SEQ ID NO. 243 peptide 244 GAQFAKTAAK SEQ ID NO. 244
peptide 245 GAQFSATAAK SEQ ID NO. 245 peptide 246 KAATKSFQAG SEQ ID
NO. 246 peptide 247 GAQFSKAAAK SEQ ID NO. 247 peptide 248
GAQFSKTAAA SEQ ID NO. 248
peptide 249 GAQFSATAAA SEQ ID NO. 249 peptide 250 GAQASKTA SEQ ID
NO. 250 peptide 251 AAGE SEQ ID NO. 251 peptide 252 GKASQFAKTA SEQ
ID NO. 252
[0037] An amino acid sequence of a peptide listed in Table 1 can be
chemically modified. For example, if an amino acid sequence of a
peptide listed in Table 1 is chemically modified at the N-terminal
amine to form an amide with a carboxylic acid, the resulting
peptide is sometimes referred to herein by a combination of an
identifier for the carboxylic acid as a prefix linked by a hyphen
to the peptide number. For example, with respect to peptide 79 as
an example, an N-terminal myristoylated peptide 79 may sometimes be
referred to herein as "myristoylated-peptide 79" or "myr-peptide
79"; an N-terminal acetylated peptide 79 may sometimes be referred
to herein as "acetyl-peptide 79" or "Ac-peptide 79". A cyclic
version of peptide 79 may be referred to as "cyclic-peptide 79" or
"cyc-peptide 79". Also, for example, if an amino acid sequence of a
peptide listed in Table 1 is chemically modified at the C-terminal
carboxylic group, for example by an amine such as ammonia to form a
C-terminal amide, the resulting peptide is sometimes referred to
herein by a combination of an identifier for the amine residue as a
suffix linked by a hyphen to the peptide number. Thus, for example,
a C-terminal amide of peptide 79 can be sometimes referred to as
"peptide-NH2". When the N-terminal amine of the peptide (e.g.,
peptide 79) is chemically modified by, for example, a myristoyl
group and the C-terminal carboxylic group is chemically modified
by, for example, an ammonia group to form an amide as above, the
resulting peptide can be sometimes referred to, using both prefix
and suffix notation, as "myr-peptide 79-NH2".
[0038] The invention involves peptides having amino acid sequences
comprising less than 24 amino acids with amino acid sequences
related to the amino acid sequence of MANS peptide (i.e., the MANS
peptide is myristoyl-peptide 1 and the reference 24-amino acid
sequence of the MANS peptide is peptide 1). The peptides of the
current invention consist of amino acid sequences containing less
than 24 amino acids, and may consist of from 8 to 14, from 10 to
12, from 9 to 14, from 9 to 13, from 10 to 13, from 10 to 14, at
least 9, at least 10, or the like amino acids. The peptides are
typically straight chains, but may be cyclic peptides as well. In
addition, the peptides may be isolated peptides.
[0039] With respect to peptide 1 (SEQ ID NO: 1), the reference 24
amino acid sequence, a segment of 23 continuous amino acids of the
reference amino acid sequence is sometimes referred to herein as a
23-mer. Analogously, a segment of 22 continuous amino acids of the
reference sequence is sometimes referred to herein as a 22-mer; a
21 amino acid sequence as a 21-mer; a 20 amino acid sequence as a
20-mer; a 19 amino acid sequence as a 19-mer; an 18 amino acid
sequence as an 18-mer; a 17 amino acid sequence as a 17-mer; a 16
amino acid sequence as a 16-mer; a 15 amino acid sequence as a
15-mer; a 14 amino acid sequence as a 14-mer; a 13 amino acid
sequence as a 13-mer; a 12 amino acid sequence as a 12-mer; an 11
amino acid sequence as an 11-mer; a 10 amino acid sequence as a
10-mer; a 9 amino acid sequence as a 9-mer; an 8 amino acid
sequence as an 8-mer; a 7 amino acid sequence as a 7-mer; a 6 amino
acid sequence as a 6-mer; a 5 amino acid sequence as a 5-mer; and a
4 amino acid sequence as a 4-mer. In one aspect, any of these "4-
to 23-mer" amino acid sequences, which are themselves peptides
(sometimes herein denoted as H2N-peptide-COOH), can be
independently chemically modified, for example, by chemical
modification, which chemical modification can be selected from the
group consisting of (i) amide formation at the N-terminal amine
group (H2N-peptide-) such as with, for example, a C1 or preferably
with a C2 (acetic acid) to C22 carboxylic acid; (ii) amide
formation at the C-terminal carboxylic group (-peptide-COOH) such
as with, for example, ammonia or with a C1 to C22 primary or
secondary amine; and (iii) a combination of thereof.
[0040] The peptides have an amino acid sequence selected from the
group consisting of (a) an amino acid sequence having from 4 to 23
contiguous amino acids of the reference sequence, peptide 1; (b) a
sequence substantially similar to the amino acid sequence defined
in (a); and (c) a variant of the amino acid sequence defined in
(a), which variant is selected from the group consisting of a
substitution variant, a deletion variant, an addition variant, and
combinations thereof. In some embodiments, the peptides have an
amino acid sequence selected from the group consisting of: (a) an
amino acid sequence having from 8 to 14 contiguous amino acids of
the reference sequence, peptide 1; (b) an amino acid sequence
substantially identical to the sequence defined in (a); and (c) a
variant of the amino acid sequence defined in (a), which variant is
selected from the group consisting of a substitution variant, a
deletion variant, an addition variant, and combinations thereof. In
yet other embodiments, the peptides have an amino acid sequence
selected from the group consisting of: (a) an amino acid sequence
having from 10 to 12 contiguous amino acids of the reference
sequence, peptide 1; (b) an amino acid sequence substantially
identical to the sequence defined in (a); and (c) a variant of the
amino acid sequence defined in (a), which variant is selected from
the group consisting of a substitution variant, a deletion variant,
an addition variant, and combinations thereof. In further
embodiments, the peptides have an amino acid sequence having at
least 9, at least 10, from 9 to 14, from 9 to 13, from 10 to 13,
from 10 to 14, or the like contiguous amino acids of the reference
sequence, peptide 1; an amino acid sequence substantially identical
thereto; or a variant thereof, which variant is selected from the
group consisting of a substitution variant, a deletion variant, an
addition variant, and combinations thereof. As explained further
below, one or more of the amino acids of the peptides (e.g., the
N-terminal and/or C-terminal amino acids) may be optionally
independently chemically modified; in some embodiments, one or more
amino acids of a peptide will be chemically modified while in other
embodiments none of the amino acids of the peptide will be
chemically modified. In one aspect, preferred modification can
occur at the amine (--NH2) group of the N-terminal amino acid of
the peptide or peptide segment (which amine group would form a
peptide amide bond if present internally within a peptide sequence
rather than at the N-terminal position). In another aspect,
preferred modification can occur at the carboxy (--COOH) group of
the C-terminal amino acid of the peptide or peptide segment (which
carboxy group would form a peptide amide bond if present internally
within a peptide sequence rather than at the C-terminal position).
In another aspect, preferred modification can occur at both the
N-terminal amine (--NH2) group and at the C-terminal carboxylic
(--COOH) group.
[0041] In some embodiments, the amino acid sequence of the peptide
begins from the N-terminal amino acid of the reference sequence
peptide 1. For example, the peptides may have an amino acid
sequence selected from the group consisting of (a) an amino acid
sequence having from 4 to 23 contiguous amino acids of the
reference sequence peptide 1, wherein the amino acid sequence
begins from the N-terminal amino acid of the reference sequence
(i.e., peptide 2, peptide 4, peptide 7, peptide 11, peptide 16,
peptide 22, peptide 29, peptide 37, peptide 46, peptide 56, peptide
67, peptide 79, peptide 92, peptide 106, peptide 121, peptide 137,
peptide 154, peptide 172, peptide 191, or peptide 211); (b) a
sequence substantially similar to the amino acid sequence defined
in (a); and (c) a variant of the amino acid sequence defined in
(a). These peptides contain no chemical moiety or a chemical moiety
on the N-terminal glycine other than a myristoyl group. Preferably,
the chemical moiety is an acyl group, in the form of an amide bond,
such as an acetyl group, or alkyl group.
[0042] In other embodiments, the amino acid sequence of the peptide
ends at the C-terminal amino acid of the reference sequence peptide
1. For example, the peptides may have an amino acid sequence
selected from the group consisting of (a) an amino acid sequence
having from 4 to 23 contiguous amino acids of the reference
sequence peptide 1, wherein the amino acid sequence ends at the
C-terminal amino acid of the reference sequence (i.e., peptide 3,
peptide 6, peptide 10, peptide 15, peptide 21, peptide 28, peptide
36, peptide 45, peptide 55, peptide 66, peptide 78, peptide 91,
peptide 105, peptide 120, peptide 136, peptide 153, peptide 171,
peptide 190, peptide 210, or peptide 231); (b) a sequence
substantially similar to the amino acid sequence defined in (a);
and (c) a variant of the amino acid sequence defined in (a).
[0043] In other embodiments, the amino acid sequence of the peptide
does not begin at the N-terminal amino acid of the reference
sequence, peptide 1, (SEQ ID NO: 1) but rather begins at the amino
acid at position 2 through the amino acid at position 21 of the
reference sequence peptide 1. For example, the peptides may have an
amino acid sequence selected from the group consisting of (a) an
amino acid sequence having from 4 to 23 contiguous amino acids of
the reference sequence peptide 1, wherein the amino acid sequence
begins at any amino acid between position 2 through position 21of
the reference sequence. These peptides may be between 4 and 23
contiguous amino acids long and may represent peptides in the
middle of the reference sequence, peptide 1; (b) a sequence
substantially similar to the amino acid sequence defined in (a);
and (c) a variant of the amino acid sequence defined in (a). These
peptides are disclosed in Tables 1 or 2. These peptides may contain
no covalently bound chemical moiety or a chemical moiety on the
N-terminal amino acid which is not the N-terminal glycine from or
equivalent to the N-terminal glycine of the amino acid sequence SEQ
ID NO: 1. Preferably, the chemical moiety is an acyl group, such as
an acetyl group or a myristoyl group, in the form of an amide bond,
or an alkyl group.
[0044] Peptide amino acid sequences which are useful in the current
invention to inhibit mucin hypersecretion in a mammal, and which
are useful to reduce the amount of mucin hypersecretion in a
mammal, and which are useful in the methods of inhibition of mucin
hypersecretion and in the methods of reduction of mucin
hypersecretion include amino acid sequences of isolated peptides
and amino acid sequences of peptides which optionally contain
N-terminal- and/or C-terminal-chemically modified groups of the
current invention, which peptide amino acid sequences are selected
from the group consisting of the 23-mers (i.e., peptides having a
23 amino acid sequence): peptide 2; and peptide 3; the 22-mers
(i.e., peptides having a 22 amino acid sequence): peptide 4;
peptide 5; and peptide 6; the 21-mers (i.e., peptides having a 21
amino acid sequence): peptide 7; peptide 8; peptide 9; and peptide
10; the 20-mers (i.e., peptides having a 20 amino acid sequence):
peptide 11; peptide 12; peptide 13; peptide 14; and peptide 15; the
19-mers (i.e., peptides having a 19 amino acid sequence): peptide
16; peptide 17; peptide 18; peptide 19; peptide 20; and peptide 21;
the 18-mers (i.e., peptides having a 18 amino acid sequence):
peptide 22; peptide 23; peptide 25; peptide 26; peptide 27; and
peptide 28; the 17-mers (i.e., peptides having a 17 amino acid
sequence): peptide 29; peptide 30; peptide 31; peptide 32; peptide
33; peptide 34; peptide 35; and peptide 36; the 16-mers (i.e.,
peptides having a 16 amino acid sequence): peptide 37; peptide 38;
peptide 39; peptide 40; peptide 41; peptide 42; peptide 43; peptide
44; and peptide 45; the 15-mers (i.e., peptides having a 15 amino
acid sequence): peptide 46; peptide 47; peptide 48; peptide 49;
peptide 50; peptide 51; peptide 52; peptide 53; peptide 54; and
peptide 55; the 14-mers (i.e., peptides having a 14 amino acid
sequence): peptide 56; peptide 57; peptide 58; peptide 59; peptide
60; peptide 61; peptide 62; peptide 63; peptide 64; peptide 65; and
peptide 66; the 13-mers (i.e., peptides having a 13 amino acid
sequence): peptide 67; peptide 68; peptide 69; peptide 70; peptide
71; peptide 72; peptide 73; peptide 74; peptide 75; peptide 76;
peptide 77; and peptide 78; the 12-mers (i.e., peptides having a 12
amino acid sequence): peptide 79; peptide 80; peptide 81; peptide
82; peptide 83; peptide 84; peptide 85; peptide 86; peptide 87;
peptide 88; peptide 89; peptide 90; and peptide 91; the 11-mers
(i.e., peptides having a 11 amino acid sequence): peptide 92;
peptide 93; peptide 94; peptide 95; peptide 96; peptide 97; peptide
98; peptide 99; peptide 100; peptide 101; peptide 102; peptide 103;
peptide 104; and peptide 105; the 10-mers (i.e., peptides having a
10 amino acid sequence): peptide 106; peptide 107; peptide 108;
peptide 109; peptide 110; peptide 111; peptide 112; peptide 113;
peptide 114; peptide 115; peptide 116; peptide 117; peptide 118;
peptide 119; and peptide 120; the 9-mers (i.e., peptides having a 9
amino acid sequence): peptide 121; peptide 122; peptide 123;
peptide 124; peptide 125; peptide 126; peptide 127; peptide 128;
peptide 129; peptide 130; peptide 131; peptide 132; peptide 133;
peptide 134; peptide 135; and peptide 136; the 8-mers (i.e.,
peptides having a 8 amino acid sequence): peptide 137; peptide 138;
peptide 139; peptide 140; peptide 141; peptide 142; peptide 143;
peptide 144; peptide 145; peptide 146; peptide 147; peptide 148;
peptide 149; peptide 150; peptide 151; peptide 152; and peptide
153; the 7-mers (i.e., peptides having a 7 amino acid sequence):
peptide 154; peptide 155; peptide 156; peptide 157; peptide 158;
peptide 159; peptide 160; peptide 161; peptide 162; peptide 163;
peptide 164; peptide 165; peptide 166; peptide 167; peptide 168;
peptide 169; peptide 170; and peptide 171; the 6-mers (i.e.,
peptides having a 6 amino acid sequence): peptide 172; peptide 173;
peptide 174; peptide 175; peptide 176; peptide 177; peptide 178;
peptide 179; peptide 180; peptide 181; peptide 182; peptide 183;
peptide 184; peptide 185; peptide 186; peptide 187; peptide 188;
peptide 189; and peptide 190; the 5-mers (i.e., peptides having a 5
amino acid sequence): peptide 191; peptide 192; peptide 193;
peptide 194; peptide 195; peptide 196; peptide 197; peptide 198;
peptide 199; peptide 200; peptide 201; peptide 202; peptide 203;
peptide 204; peptide 205; peptide 206; peptide 207; peptide 208;
peptide 209; and peptide 210; and the 4-mers (i.e., peptides having
a 4 amino acid sequence): peptide 211; peptide 212; peptide 213;
peptide 214; peptide 215; peptide 216; peptide 217; peptide 218;
peptide 219; peptide 220; peptide 221; peptide 222; peptide 223;
peptide 224; peptide 225; peptide 226; peptide 227; peptide 228;
peptide 229; peptide 230; and peptide 231.
[0045] Preferred amino acid sequences of isolated peptides and of
N-terminal- and/or C-terminal-chemically modified peptides of the
current invention are selected from the group consisting of the
23-mers: peptide 2; and peptide 3; the 22-mers: peptide 4; peptide
5; and peptide 6; the 21-mers: peptide 7; peptide 8; peptide 9; and
peptide 10; the 20-mers: peptide 11; peptide 12; peptide 13;
peptide 14; and peptide 15; the 19-mers: peptide 16; peptide 17;
peptide 18; peptide 19; peptide 20; and peptide 21; the 18-mers:
peptide 22; peptide 23; peptide 24; peptide 25; peptide 26; peptide
27; and peptide 28; the 17-mers: peptide 29; peptide 30; peptide
31; peptide 32; peptide 33; peptide 34; peptide 35; and peptide 36;
the 16-mers: peptide 37; peptide 38; peptide 39; peptide 40;
peptide 41; peptide 42; peptide 43; peptide 44; and peptide 45; the
15-mers: peptide 46; peptide 47; peptide 48; peptide 49; peptide
50; peptide 51; peptide 52; peptide 53; and peptide 54; the
14-mers: peptide 56; peptide 57; peptide 58; peptide 59; peptide
60; peptide 61; peptide 62; peptide 63; and peptide 64; the
13-mers: peptide 67; peptide 68; peptide 69; peptide 70; peptide
71; peptide 72; peptide 73; peptide 74; and peptide 75; the
12-mers: peptide 79; peptide 80; peptide 81; peptide 82; peptide
83; peptide 84; peptide 85; peptide 86; and peptide 87; the
11-mers: peptide 92; peptide 93; peptide 94; peptide 95; peptide
96; peptide 97; peptide 98; peptide 99; and peptide 100; the
10-mers: peptide 106; peptide 107; peptide 108; peptide 109;
peptide 110; peptide 111; peptide 112; peptide 113; and peptide
114; the 9-mers: peptide 122; peptide 123; peptide 124; peptide
125; peptide 126; peptide 127; peptide 128; and peptide 129; the
8-mers: peptide 139; peptide 140; peptide 141; peptide 142; peptide
143; peptide 144; and peptide 145; the 7-mers: peptide 157; peptide
158; peptide 159; peptide 160; peptide 161; and peptide 162; the
6-mers: peptide 176; peptide 177; peptide 178; peptide 179; and
peptide 180; the 5-mers: peptide 196; peptide 197; peptide 198; and
peptide 199; and the 4-mers: peptide 217; and peptide 219.
[0046] More preferred amino acid sequences of isolated peptides and
of N-terminal- and/or C-terminal-chemically modified peptides of
the current invention are selected from the group consisting of the
23-mers: peptide 2; and peptide 3; the 22-mers: peptide 4; peptide
5; and peptide 6; the 21-mers: peptide 7; peptide 8; peptide 9; and
peptide 10; the 20-mers: peptide 11; peptide 12; peptide 13;
peptide 14; and peptide 15; the 19-mers: peptide 16; peptide 17;
peptide 18; peptide 19; peptide 20; and peptide 21; the 18-mers:
peptide 22; peptide 23; peptide 24; peptide 25; peptide 26; peptide
27; and peptide 28; the 17-mers: peptide 29; peptide 30; peptide
31; peptide 32; peptide 33; peptide 34; peptide 35; and peptide 36;
the 16-mers: peptide 37; peptide 38; peptide 39; peptide 40;
peptide 41; peptide 42; peptide 43; peptide 44; and peptide 45; the
15-mers: peptide 46; peptide 47; peptide 48; peptide 49; peptide
50; peptide 51; peptide 52; peptide 53; and peptide 54; the
14-mers: peptide 56; peptide 57; peptide 58; peptide 59; peptide
60; peptide 61; peptide 62; peptide 63; and peptide 64; the
13-mers: peptide 67; peptide 68; peptide 69; peptide 70; peptide
71; peptide 72; peptide 73; peptide 74; peptide 80; peptide 81;
peptide 82; peptide 83; peptide 84; peptide 85; peptide 86; and
peptide 87; the 11-mers: peptide 92; peptide 93; peptide 94;
peptide 95; peptide 96; peptide 97; peptide 98; peptide 99; and
peptide 100; the 10-mers: peptide 106; peptide 108; peptide 109;
peptide 110; peptide 111; peptide 112; peptide 113; and peptide
114; the 9-mers: peptide 124; peptide 125; peptide 126; peptide
127; peptide 128; and peptide 129; the 8-mers: peptide 141; peptide
142; peptide 143; peptide 144; and peptide 145; the 7-mers: peptide
159; peptide 160; peptide 161; and peptide 162; the 6-mers: peptide
178; peptide 179; and peptide 180; the 5-mers: peptide 198; and
peptide 199; and the 4-mer: peptide 219.
[0047] In yet other embodiments, the amino acid sequence of the
peptide includes the contiguous residues A,K,G, and E as in peptide
219 of the reference sequence peptide 1. For example, the peptides
may have an amino acid sequence selected from the group consisting
of (a) an amino acid sequence having from 4 to 23 contiguous amino
acids of the reference sequence peptide 1, wherein the amino acid
sequence of the peptide includes the contiguous residues A,K,G, and
E as in peptide 219 of the reference peptide 1 (e.g., peptide 219,
peptide 45, peptide 79, peptide 67, peptide 80, etc.); (b) a
sequence substantially similar to the amino acid sequence defined
in (a); and (c) a variant of the amino acid sequence defined in
(a).
[0048] Examples of peptide segments which contain the amino acid
sequence AKGE of the reference peptide amino acid sequence, peptide
1, include (a) the 23-mers: peptide 2; and peptide 3; the 22-mers:
peptide 4; peptide 5; and peptide 6; the 11-mers: peptide 7;
peptide 8; peptide 9; and peptide 10; the 20-mers: peptide 11;
peptide 12; peptide 13; peptide 14; and peptide 15; the 19-mers:
peptide 16; peptide 17; peptide 18; peptide 19; peptide 20; and
peptide 21; the 18-mers: peptide 22; peptide 23; peptide 24;
peptide 25; peptide 26; peptide 27; and peptide 28; the 17-mers:
peptide 29; peptide 30; peptide 31; peptide 32; peptide 33; peptide
34; peptide 35; and peptide 36; the 16-mers: peptide 37; peptide
38; peptide 39; peptide 40; peptide 41; peptide 42; peptide 43;
peptide 44; and peptide 45; the 15-mers: peptide 46; peptide 47;
peptide 48; peptide 49; peptide 50; peptide 51; peptide 52; peptide
53; and peptide 54; the 14-mers: peptide 56; peptide 57; peptide
58; peptide 59; peptide 60; peptide 61; peptide 62; peptide 63; and
peptide 64; the 13-mers: peptide 67; peptide 68; peptide 69;
peptide 70; peptide 71; peptide 72; peptide 73; peptide 74; and
peptide 75; the 12-mers: peptide 79; peptide 80; peptide 81;
peptide 82; peptide 83; peptide 84; peptide 85; peptide 86; and
peptide 87; the 11-mers: peptide 93; peptide 94; peptide 95;
peptide 96; peptide 97; peptide 98; peptide 99; and peptide 100;
the 10-mers: peptide 108; peptide 109; peptide 110; peptide 111;
peptide 112; peptide 113; and peptide 114; the 9-mers: peptide 124;
peptide 125; peptide 126; peptide 127; peptide 128; and peptide
129; the 8-mers: peptide 141; peptide 142; peptide 143; peptide
144; and peptide 145; the 7-mers: peptide 159; peptide 160; peptide
161; and peptide 162; the 6-mers: peptide 178; peptide 179; and
peptide 180; the 5-mers: peptide 198; and peptide 199; and the
4-mer: peptide 219, (b) a sequence substantially similar to the
amino acid sequence defined in (a); and (c) a variant of the amino
acid sequence defined in (a), which variant is selected from the
group consisting of a substitution variant, a deletion variant, an
addition variant, and combinations thereof, wherein the segment
comprises or consists of from 4 to 23 contiguous amino acids.
[0049] In another embodiment, preferred peptide sequences have an
amino acid sequence selected from the group consisting of (a) an
amino acid sequence having from 10 to 23 contiguous amino acids of
the reference sequence, peptide 1; (b) a sequence substantially
similar to the amino acid sequence defined in (a); and (c) a
variant of the amino acid sequence defined in (a), which variant is
selected from the group consisting of a substitution variant, a
deletion variant, an addition variant, and combinations thereof,
wherein the preferred amino acid sequences comprise the 23-mer:
peptide 2; the 22-mer: peptide 4; the 21-mer: peptide 7; the
20-mer: peptide 11; the 19-mer: peptide 16; the 18-mer: peptide 22;
the 17-mer: peptide 29; the 16-mer: peptide 37; the 15-mer: peptide
46; the 14-mer: peptide 56; the 13-mer: peptide 67; the 12-mer:
peptide 79; the 11-mer: peptide 92; and the 10-mer: peptide
106.
[0050] In further embodiments, the amino acid sequence of the
peptide begins from the N-terminal amino acid of the reference
sequence peptide 1 and includes the contiguous residues A,K,G, and
E as in peptide 219 of the reference sequence peptide 1, while in
other embodiments the amino acid sequence of the peptide ends at
the C-terminal amino acid of the reference sequence peptide 1 and
includes the contiguous residues A,K,G, and E as in peptide 219 of
the reference sequence peptide 1.
[0051] The peptides may include one or more amino acid deletions,
substitutions, and/or additions with respect to the reference amino
acid sequence. Preferably, the substitutions may be conservative
amino acid substitutions, or the substitutions may be
non-conservative amino acid substitutions. In some embodiments, the
peptides, including the peptides with amino acid sequences that are
substantially identical to or variants of the reference amino acid
sequence, will not have deletions or additions as compared to the
corresponding contiguous amino acids of the reference amino acid
sequence, but may have conservative or non-conservative
substitutions. Amino acid substitutions that may be made to the
reference amino acid sequence in the peptides of the invention
include, but are not limited to, the following: alanine (A) may be
substituted with lysine (K), valine (V), leucine (L), or isoleucine
(I); glutamic acid (E) may be substituted with aspartic acid (D);
glycine (G) may be substituted with proline (P); lysine (K) may be
substituted with arginine (R), glutamine (Q), or asparagine (N);
phenylalanine (F) may be substituted with leucine (L), valine (V),
isoleucine (I), or alanine (A); proline (P) may be substituted with
glycine (G); glutamine (Q) may be substituted with glutamic acid
(E) or asparagine (N); arginine (R) may be substituted with lysine
(K), glutamine (Q), or asparagine (N); serine (S) may be
substituted with threonine; threonine (T) may be substituted with
serine (S); and valine (V) may be substituted with leucine (L),
isoleucine (I), methionine (M), phenylalanine (F), alanine (A), or
norleucine (Nle). For example, substitutions that could be made to
the reference amino acid sequence in the peptides of the invention
include substituting alanine (A) for phenylalanine (F) (e.g., at
amino acid position 4 of the reference amino acid sequence),
glutamic acid (E) for glutamine (Q) (e.g., at amino acid position 3
of the reference amino acid sequence), lysine (K) for alanine (A)
(e.g., at amino acid positions 2 and/or 8 of the reference amino
acid sequence), and/or serine (S) for threonine (T) (e.g., at amino
acid position 7 of the reference amino acid sequence).
[0052] When substitutions are included in the amino acid sequences
of the peptides of the invention (which peptides comprise
unmodified as well as peptides which are chemically modified for
example by N-terminal and/or C-terminal modification such as by
amide formation) with respect to the reference amino acid sequence,
there is preferably at least 80% sequence identity between the
amino acid sequence of the peptide and the reference amino acid
sequence. Peptides having 5 to 23 amino acids and including one
amino acid substitution with respect to the reference amino acid
sequence will have between about 80% to about 96% (i.e.,
.about.95.7%) sequence identity to the reference amino acid
sequence. Peptides having 10 to 23 amino acids and including one
amino acid substitution with respect to the reference amino acid
sequence will have between about 90% to about 96% (i.e.,
.about.95.7%) sequence identity to the reference amino acid
sequence. Peptides having 20 to 23 amino acids and including one
amino acid substitution with respect to the reference amino acid
sequence will have between about 95% to about 96% (i.e.,
.about.95.7%) sequence identity to the reference amino acid
sequence. Peptides having 10 to 23 amino acids and including two
amino acid substitutions with respect to the reference amino acid
sequence will have between about 80% to about 92% (i.e.,
.about.91.3%) sequence identity to the reference amino acid
sequence. Peptides having 16 to 23 amino acids and including two
amino acid substitutions with respect to the reference amino acid
sequence will have between about 87.5% to about 92% (i.e.,
.about.91.3%) sequence identity to the reference amino acid
sequence. Peptides having 20 to 23 amino acids and including two
amino acid substitutions with respect to the reference amino acid
sequence will have between about 90% to about 92% (i.e.,
.about.91.3%) sequence identity to the reference amino acid
sequence. Peptides having 15 to 23 amino acids and including three
amino acid substitutions with respect to the reference amino acid
sequence will have between about 80% to about 87% sequence identity
to the reference amino acid sequence. Peptides having 20 to 23
amino acids and including three amino acid substitutions with
respect to the reference amino acid sequence will have between
about 85% to about 87% sequence identity to the reference amino
acid sequence. Peptides having 20 to 23 amino acids and including
four amino acid substitutions with respect to the reference amino
acid sequence will have between about 80% to about 83% (i.e.,
.about.82.6%) sequence identity to the reference amino acid
sequence.
[0053] In peptides of the current invention, with respect to the
contiguous amino acid sequence of the reference peptide (which is a
24-mer) substitution of one amino acid in a contiguous 23 amino
acid sequence (a 23-mer) selected from the reference 24 amino acid
sequence provides a peptide with an amino acid sequence which has a
95.65% (or .about.96%) sequence identity to the amino acid segment
in the reference peptide with which the 23-mer has identity.
Analogously, substitution of two, three, four, and five amino acids
in said 23-mer provides a peptide with an amino acid sequence which
has a 91.30% (or .about.91%), 86.96% (or .about.87%), 82.61% (or
.about.83%), and 78.27% (or .about.78%) sequence identity,
respectively, to the reference peptide amino acid sequence.
Analogously, substitution of one, two, three, four, and five amino
acids in a 22-mer provides a peptide with an amino acid sequence
which has a 95.45% (or .about.95%), 90.91% (or .about.91%), 86.36%
(or .about.86%), 81.82% (or .about.82%), and 77.27% (or .about.77%)
sequence identity, respectively, to the reference peptide amino
acid sequence. Analogously, substitution of one, two, three, four,
and five amino acids in a 21-mer provides a peptide with an amino
acid sequence which has a 95.24% (.about.95%), 90.48 (.about.91%),
85.71% (.about.86%), 80.95 (.about.81%), and 76.19% (.about.76%)
sequence identity, respectively, to the reference peptide amino
acid sequence. Analogously, substitution of one, two, three, four,
and five amino acids in a 20-mer provides a peptide with an amino
acid sequence which has a 95.00% (95%), 90.00% (90%), 85.00% (85%),
80.00% (80%), and 75.00% (75%) sequence identity, respectively, to
the reference peptide amino acid sequence. Analogously,
substitution of one, two, three, and four amino acids in a 19-mer
provides a peptide with an amino acid sequence which has a 94.74%
(.about.95%), 89.47% (.about.89%), 84.21% (.about.84%), and 78.95%
(.about.79%) sequence identity, respectively, to the reference
peptide amino acid sequence. Analogously, substitution of one, two,
three, and four amino acids in an 18-mer provides a peptide with an
amino acid sequence which has a 94.44% (.about.94%), 88.89%
(.about.89%), 83.33% (.about.83%), and 77.78% (.about.78%) sequence
identity, respectively, to the reference peptide amino acid
sequence. Analogously, substitution of one, two, three, and four
amino acids in an 17-mer provides a peptide with an amino acid
sequence which has a 94.12% (.about.94%), 88.23% (.about.88%),
82.35% (.about.82%), and 76.47% (.about.76%) sequence identity,
respectively, to the reference peptide amino acid sequence.
Analogously, substitution of one, two, three, and four amino acids
in a 16-mer provides a peptide with an amino acid sequence which
has a 93.75% (.about.94%), 87.50% (.about.88%), 81.25%
(.about.81%), and 75.00% (75%) sequence identity, respectively, to
the reference peptide amino acid sequence. Analogously,
substitution of one, two, and three amino acids in a 15-mer
provides a peptide with an amino acid sequence which has a 93.33%
(.about.93%), 86.67% (.about.87%), and 80.00% (80%) sequence
identity, respectively, to the reference peptide amino acid
sequence. Analogously, substitution of one, two, and three amino
acids in a 14-mer provides a peptide with an amino acid sequence
which has a 92.86% (.about.93%), 85.71% (.about.86%), and 78.57%
(79%) sequence identity, respectively, to the reference peptide
amino acid sequence. Analogously, substitution of one, two, and
three amino acids in a 13-mer provides a peptide with an amino acid
sequence which has a 92.31% (.about.92%), 84.62% (.about.85%), and
76.92% (.about.77%) sequence identity, respectively, to the
reference peptide amino acid sequence. Analogously, substitution of
one, two, and three amino acids in a 12-mer provides a peptide with
an amino acid sequence which has a 91.67% (.about.92%), 83.33%
(.about.83%), and 75.00% (75%) sequence identity, respectively, to
the reference peptide amino acid sequence. Analogously,
substitution of one and two amino acids in an 11-mer provides a
peptide with an amino acid sequence which has a 90.91% (.about.91%)
and 81.82% (.about.82%) sequence identity, respectively, to the
reference peptide amino acid sequence. Analogously, substitution of
one and two amino acids in a 10-mer provides a peptide with an
amino acid sequence which has a 90.00% (90%) and 80.00% (80%)
sequence identity, respectively, to the reference peptide amino
acid sequence. Analogously, substitution of one and two amino acids
in a 9-mer provides a peptide with an amino acid sequence which has
a 88.89% (.about.89%) and 77.78% (.about.78%) sequence identity,
respectively, to the reference peptide amino acid sequence.
Analogously, substitution of one and two amino acids in an 8-mer
provides a peptide with an amino acid sequence which has a 87.50%
(.about.88%) and 75.00% (75%) sequence identity, respectively, to
the reference peptide amino acid sequence. Analogously,
substitution of one amino acid in a 7-mer, 6-mer, 5-mer, and 4-mer
provides a peptide with an amino acid sequence which has a 85.71%
(.about.86%), 83.33% (.about.83.3%), 80.00% (80%), and 75.00% (75%)
sequence identity, respectively, to the reference peptide.
Preferred amino acid sequences of this invention have greater than
80% sequence identity to the amino acid sequence in the reference
sequence, more preferably between 81% and 96% sequence identity to
the amino acid sequence in the reference sequence, and more
preferably between 80% and 96% sequence identity to the amino acid
sequence in the reference sequence. The preferred amino acid
sequences can be optionally N-terminally chemically bonded at the
terminal peptide amino group to a C2 to C22 linear aliphatic
carboxylic acid moiety, more preferably to a C2 to C16 linear
aliphatic carboxylic acid moiety, most preferably to a C2 or C16
linear aliphatic carboxylic acid moiety, by an amide bond, and
optionally C-terminally chemically bonded at the terminal peptide
carboxylic group to an amine such as ammonia or a primary or
secondary amine such as a C1 to C16 linear aliphatic primary amine,
by an amide bond.
[0054] Examples of substitution variants of peptide 79, a 12-mer,
include, for example, peptide 238, where Q at position 3 in peptide
79 has been substituted by E in sequence 238; peptide 233, where A
at position 2 in peptide 79 has been substituted by K in peptide
233; peptide 234, where A at position 8 in peptide 79 has been
substituted by K in peptide 234; peptide 235, where A at positions
2 and 8 in peptide 79 have been substituted by K in peptide 235;
peptide 237, where F at position 4 in peptide 79 has been
substituted by A in peptide 237; peptide 239, where K at position
10 in peptide 79 has been substituted by A in peptide 239; peptide
240, where G at position 11 in peptide 79 has been substituted by A
in peptide 240; and peptide 241, where E at position 12 in peptide
79 has been substituted by A in peptide 241.
[0055] Examples of substitution variants of peptide 106, a 10-mer,
include, for example, peptide 236, where F at position 4 in peptide
106 has been substituted by A in peptide 236; peptide 242, where G
at position 1 in peptide 106 has been substituted by A in peptide
242; peptide 243, where Q at position 3 in peptide 106 has been
substituted by A in peptide 243; peptide 244, where S at position 5
in peptide 106 has been substituted by A in peptide 244; peptide
245, where K at position 6 in peptide 106 has been substituted by A
in peptide 245; peptide 247, where T at position 7 in peptide 106
has been substituted by A in peptide 247; peptide 248, where K at
position 10 in peptide 106 has been substituted by A in peptide
248; peptide 249, where K at positions 6 and 10 in peptide 106 have
both been substituted, each by A, in peptide 249.
[0056] Examples of a substitution variant of peptide 137, an 8-mer,
include for example, peptide 250, where F at position 4 in peptide
137 has been substituted by A in peptide 250.
[0057] Examples of a substitution variant of peptide 219, a 4-mer,
include for example, peptide 251, where K at position 2 in peptide
219 has been substituted by A in peptide 251.
[0058] A substitution variant peptide such as described herein can
be in the form of an isolated peptide or in the form of a
chemically modified peptide such as, for example, an N-terminal
amide such as a myristoyl amide, an acetyl amide, and the like as
described herein, and such as, for example, a C-terminal amide such
as an amide formed with ammonia, and such as both an N-terminal
amide and a C-terminal amide.
[0059] When deletions are included in the amino acid sequences of
the peptides of the invention with respect to the reference amino
acid sequence, there is preferably at least 80% sequence identity
between the amino acid sequence of the peptide to the reference
amino acid sequence. Peptides having 5 to 23 amino acids and
including one amino acid deletion with respect to the reference
peptide will have between 80% to about 96% (i.e., .about.95.7%)
sequence identity to the reference amino acid sequence. Peptides
having 10 to 23 amino acids and including one amino acid deletion
with respect to the reference peptide will have between about 90%
to about 96% (i.e., .about.95.7%) sequence identity to the
reference amino acid sequence. Peptides having 20 to 23 amino acids
and including one amino acid deletion with respect to the reference
peptide will have between 95% to about 96% (i.e., .about.95.7%)
sequence identity to the reference amino acid sequence. Peptides
having 10 to 23 amino acids and including two amino acid deletions
with respect to the reference peptide will have between about 80%
to about 92% (i.e., .about.91.3%) sequence identity to the
reference amino acid sequence. Peptides having 16 to 23 amino acids
and including two amino acid deletions with respect to the
reference peptide will have between about 87.5% to about 92% (i.e.,
.about.91.3%) sequence identity to the reference amino acid
sequence. Peptides having 20 to 23 amino acids and including two
amino acid deletions with respect to the reference peptide will
have between about 90% to about 92% (i.e., .about.91.3%) sequence
identity to the reference amino acid sequence. Peptides having 15
to 23 amino acids and including three amino acid deletions with
respect to the reference peptide will have between about 80% to
about 87% sequence identity to the reference amino acid sequence.
Peptides having 20 to 23 amino acids and including three amino acid
deletions with respect to the reference peptide will have between
about 85% to about 87% sequence identity to the reference amino
acid sequence. Peptides having 20 to 23 amino and including four
amino acid deletions with respect to the reference peptide will
have between about 80% to about 83% (i.e., .about.82.6%) sequence
identity to the reference amino acid sequence.
[0060] As stated above, one or more of the amino acids of the
peptides may also be chemically modified. Any amino acid
modifications known in the art may be made to the amino acids of
the peptides using any method known in the art.
[0061] In some embodiments, the N-terminal and/or C-terminal amino
acid may be modified. For example, the alpha-N-terminal amino acid
of the peptides may be alkylated, amidated, or acylated at the
alpha-N-terminal (N-terminal) amino (alpha-H2N-) group, and, for
example, the C-terminal amino acid of the peptides may be amidated
or esterified at the C-terminal carboxyl (--COOH) group. For
example, the N-terminal amino group may be modified by acylation to
include any acyl or fatty acyl group to form an amide, including an
acetyl group (i.e., CH3-C(.dbd.O)-- or a myristoyl group, both of
which are currently preferred groups). In some embodiments, the
N-terminal amino group may be modified to include an acyl group
having formula --C(O)R, wherein R is a linear or branched alkyl
group having from 1 to 15 carbon atoms, or may be modified to
include an acyl group having formula --C(O)R1, wherein R1 is a
linear alkyl group having from 1 to 15 carbon atoms. The N-amide
can also be a formamide (R.dbd.H). The C-terminal amino acid of the
peptides may also be chemically modified. For example, the
C-terminal carboxyl group of the C-terminal amino acid may be
chemically modified by conversion to a carboxamide group in place
of the carboxyl group. (i.e., amidated). In some embodiments, the
N-terminal and/or C-terminal amino acids are not chemically
modified. In some embodiments, the N-terminal group is modified and
the C-terminal group is not modified. In some embodiments, both the
N-terminal and the C-terminal groups are modified.
[0062] The peptide may be acylated at the amino group of the
N-terminal amino acid to form an N-terminal amide with an acid
selected from the group consisting of:
[0063] (i-a) a C2 (acetyl) to C13 aliphatic (saturated or
optionally unsaturated) carboxylic acid (for example, an N-terminal
amide with acetic acid (which is a preferred group), with propanoic
acid, with butanoic acid, with hexanoic acid, with octanoic acid,
with decanoic acid, with dodecanoic acid) which may be linear,
branched (greater than C3), or comprise a ring (greater than
C3);
[0064] (i-b) a saturated C14 aliphatic carboxylic acid, which may
be linear, branched or comprise a ring;
[0065] (i-c) an unsaturated C14 aliphatic carboxylic acid, which
may be linear, branched or comprise a ring;
[0066] (i-d) C15 to C24 aliphatic (saturated or optionally
unsaturated) carboxylic acid, which may be linear, branched or
comprise a ring (for example, with tetradecanoic acid (myristic
acid which is a preferred group), with hexadecanoic acid, with
9-hexadecenoic acid, with octadecanoic acid, with 9-octadecenoic
acid, with 11-octadecenoic acid, with 9,12-octadecadienoic acid,
with 9,12,15-octadecatrienoic acid, with 6,9,12-octadecatrienoic
acid, with eicosanoic acid, with 9-eicosenoic acid, with
5,8,11,14-eicosatetraenoic acid, with 5,8,11,14,17-eicosapentaenoic
acid, with docosanoic acid, with 13-docosenoic acid, with
4,7,10,13,16,19-docosahexaenoic acid, with tetracosanoic acid, and
the like);
[0067] (ii) trifluoroacetic acid;
[0068] (iii) benzoic acid; and
[0069] (iv-a) a C1 to C12 aliphatic alkyl sulfonic acid which forms
an aliphatic alkyl sulfonamide, wherein the C1 to C12 aliphatic
alkyl carbon chain structure of the sulfonic acid is analogous to
that of the aliphatic alkyl carboxylic acid chains in the aliphatic
alkyl carboxylic acids described above. For example, a peptide may
be acylated using a carboxylic acid group represented as
(C1-C11)-alkyl-C(O)OH through dehydrative coupling by way of
activation of the carboxylic acid group to form an amide
represented as (C1-C11-alkyl-C(O)--NH-peptide. Analogously, a
sulfonamide may be formed by reacting a sulfonic acid species
(represented as (C1-C12)-alkyl-S(O2)-X, e.g., where X is halogen or
OCH3 or other compatible leaving group) with an N-terminal amino
group to form a sulfonamide represented as
(C1-C12)-alkyl-S(O2)-NH-peptide.
[0070] (iv-b) a C14 to C24 aliphatic alkyl sulfonic acid which
forms an aliphatic alkyl sulfonamide, wherein the C14 to C24
aliphatic alkyl carbon chain structure of the sulfonic acid is
analogous to that of the aliphatic alkyl carboxylic acid chains in
the aliphatic alkyl carboxylic acids described above . . . For
example, a peptide may be acylated using a carboxylic acid group
represented as (C13-C23)-alkyl-C(O)OH through dehydrative coupling
by way of activation of the carboxylic acid group to form an amide
represented as (C13-C23)-alkyl-C(O)--NH-peptide. Analogously, a
sulfonamide may be formed by reacting a sulfonic acid species
(represented as (C14-C24)-alkyl-S(O2)-X, e.g., where X is halogen
or OCH3 or other compatible leaving group) with an N-terminal amino
group to form a sulfonamide represented as
(C14-C24)-alkyl-S(O2)-NH-peptide.
[0071] As another example, the N-terminal amino group of the
N-terminal amino acid may be alkylated with a C1 to C12 aliphatic
alkyl group, the structure of which aliphatic alkyl group is as
described above. Alkylation may be effected, for example, using an
aliphatic alkyl halide or an aliphatic alkyl sulfonic acid ester
(mesylate, tosylate, etc.), preferably using a primary alkyl halide
or a primary alkyl sulfonic acid ester. The N-terminal amino acid
may be also modified at the terminal amino to include any acyl or
aliphatic acyl fatty acyl group as an amide, including an acetyl
group (i.e., --C(O)CH3, which is a preferred group), a myristoyl
group (which is a preferred group), a butanoyl group, a hexanoyl
group, a octanoyl group, a decanoyl group, a dodecanoyl group, a
tetradecanoyl group, a hexadecanoyl group, a 9-hexadecenoyl group,
a octadecanoyl group, a 9-octadecenoyl group, a 11-octadecenoyl
group, a 9,12-octadecadienoyl group, a 9,12,15-octadecatrienoyl
group, a 6,9,12-octadecatrienoyl group, a eicosanoyl group, a
9-eicosenoyl group, a 5,8,11,14-eicosatetraenoyl group, a
5,8,11,14,17-eicosapentaenoyl group, a docosanoyl group, a
13-docosenoyl group, a 4,7,10,13,16,19-docosahexaenoyl group, a
tetracosanoyl group, which groups are covalently attached to the
terminal amino group of the peptide by an amide bond.
[0072] The C-terminal carboxylic acid group of the C-terminal amino
acid of the peptides of the invention may also be chemically
modified. For example, the C-terminal amino acid may be chemically
modified by reaction of the C-terminal carboxylic acid group of the
peptide with an amine to form an amide group such as an amide of
ammonia which is a preferred group; an amide of a C1 to C12
aliphatic alkyl amine, preferably a linear aliphatic alkyl amine;
an amide of a hydroxyl-substituted C2 to C12 aliphatic alkyl amine;
an amide of a linear 2-(C1 to C12 aliphatic alkyl)oxyethylamine
group; and an amide of an
omega-methoxy-poly(ethyleneoxy)n-ethylamine group (also referred to
as an omega-methoxy-PEG-alpha-amine group or an
omega-methoxy-(polyethylene glycol)amine group), where n is from 0
to 10. The C-terminal carboxylic acid group of the C-terminal amino
acid of the peptide may also be in the form of an ester selected
from the group consisting of an ester of a C1 to C12 aliphatic
alkyl alcohol and an ester of a
2-(omega-methoxy-poly(ethyleneoxy)n)-ethanol (MPEG) group, where n
is from 0 to 10. In one aspect, a polyethylene glycol component
such as in a PEG ester, an 1VIPEG ester, a PEG amide, an MPEG amide
and the like preferably has a molecular weight of from about 500 to
40,000 Daltons, more preferably from 1000 to 25,000 Daltons, and
most preferably from about 1000 to about 10,000 Daltons.
[0073] The C-terminal carboxylic acid group on the peptide, which
may be represented by the formula peptide-C(O)OH, may also be
amidated by conversion to an activated form such as a carboxylic
acid halide, carboxylic acid anhydride, N-hydroxysuccinimide ester,
pentafluorophenyl (OPfp) ester,
3-hydroxy-2,3-dihydro-4-oxo-benzo-triazone (ODhbt) ester, and the
like to facilitate reaction with ammonia or a primary or secondary
amine, preferably ammonia or a primary amine, and preferably while
any other reactive groups in the peptide are protected by synthetic
chemically compatible protecting groups well known in the art of
peptide synthesis, especially of peptide solid phase synthesis,
such as a benzyl ester, a t-butyl ester, a phenyl ester, etc. A
resulting peptide amide could be represented by the formula
peptide-C(O)--NR3R4 (the amide being at the C-terminal end of the
peptide) wherein R3 and R4 are independently selected from the
group consisting of hydrogen; C1 to C12 alkyl such as methyl,
ethyl, butyl, isobutyl, cyclopropylmethyl, hexyl, dodecyl, and
optionally higher e.g., from C14 to C24 such as tetradecyl, and the
like as described above.
[0074] The C-terminal carboxylic acid of the C-terminal amino acid
may also be converted to an amide of a hydroxyl-substituted C2 to
C12 aliphatic alkyl amine (the hydroxyl group being attached to a
carbon atom rather than a nitrogen atom of the amine) such as
2-hydroxyethylamine, 4-hydroxybutylamine, and
12-hydroxydodecylamine, and the like.
[0075] The C-terminal carboxylic acid may also be converted to an
amide of a hydroxyl-substituted C2 to C12 aliphatic alkyl amine,
wherein the hydroxyl group can be acylated to form an ester with a
C2 to C12 aliphatic carboxylic acid as described above. Preferably,
in the peptide amide at the C-terminal end of the peptide
represented by the formula peptide-C(O)NR5R6, R5 is hydrogen and R6
is selected from the group consisting of hydrogen, C1 to C12 alkyl,
and hydroxyl-substituted C2 to C12 alkyl.
[0076] The C-terminal carboxylic acid of the C-terminal amino acid
may be converted to an amide of a linear 2-(C1 to C12 aliphatic
alkyl)oxyethylamine. Such an amide may be prepared, for example, by
reaction of a linear C1 to C12 aliphatic alcohol with potassium
hydride in diglyme with 2-chloroethanol to provide a linear C1 to
C12 aliphatic alkyl ethanol, which can be converted to an amine by
oxidation to an aldehyde, followed by reductive amination to an
amine (for example using ammonia), or by conversion to an alkyl
halide (e.g. using thionyl chloride) followed by treatment with an
amine such as ammonia.
[0077] The C-terminal carboxylic acid of the C-terminal amino acid
may be converted to an amide of a linear PEG-amine (e.g.,
omega-hydroxy-PEG-alpha-amine; omega-(C1-to-C12)-PEG-alpha-amine
such as omega-methoxy- PEG-alpha-amine, i.e., MPEG-amine). In one
aspect, the polyethylene glycol or PEG component preferably has a
molecular weight of from about 500 to 40,000 Daltons, more
preferably from 1000 to 25,000 Daltons, and most preferably from
about 1000 to about 10,000 Daltons.
[0078] The C-terminal carboxylic acid of the C-terminal amino acid
may also be converted to an amide of an
omega-methoxy-poly(ethyleneoxy)n-ethylamine, where n is from 0 to
10, which can be prepared from the corresponding
omega-methoxy-poly(ethyleneoxy)n-ethanol, for example, by
conversion of the alcohol to an amine as described above.
[0079] In another embodiment, the C-terminal carboxyl may be
converted to an amide represented by the formula
peptide-C(O)-NR7R8, wherein R7 is hydrogen and R8 is a linear 2-(C1
to C12 aliphatic alkyl)oxyethyl group wherein the C1 to C12
aliphatic alkyl portion is as described above and includes groups
such as methoxyethyl (i.e., CH3O--CH2CH2-), 2-dodecyloxyethyl, and
the like; or R7 is hydrogen and R8 is an
omega-methoxy-poly(ethyleneoxy)n-ethyl group where the n of the
poly(ethyleneoxy) portion is from 0 to 10, such as 2-methoxyethyl
(i.e., CH3O--CH2CH2-), omega-methoxyethoxyethyl (i.e.,
CH3)--CH2CH2O--CH2CH2-) up to CH3O--(CH2CH2O)10-CH2CH2-.
[0080] The C-terminal carboxylic acid group of the C-terminal amino
acid of the peptide may also be in the form of an ester of a C1 to
C12 aliphatic alkyl alcohol, the aliphatic alkyl portion of the
alcohol as described above. The C-terminal carboxylic acid group of
the C-terminal amino acid of the peptide may also be in the form of
an ester of a 2-(omega-methoxy-poly(ethyleneoxy)n)-ethanol group
where n is from 0 to 10, which can be prepared from reaction of
2-methoxyethanol as a sodium 2-methoxyethanolate with
stoichiometric amounts of ethylene oxide, the stoichiometric amount
dependent on the size of n.
[0081] A side chain in an amino acid of the peptides may also be
chemically modified. For example, a phenyl group in phenylalanine
or tyrosine may be substituted with a substituent selected from the
group consisting of:
[0082] a C1 to C24 aliphatic alkyl group (i.e., linear or branched,
and/or saturated or unsaturated, and/or containing a cyclic group)
such as methyl (preferred), ethyl, propyl, isopropyl, butyl,
isobutyl, cyclopropyl, 2-methylcyclopropyl, cyclohexyl, octyl,
decyl, dodecyl, hexadecyl, octadecyl, eicosanyl, docosanyl,
tetracosanyl, 9-hexadecenyl, 9-octadecenyl, 11-octadecenyl,
9,12-octadecadienyl, 9,12,15-octadecatrienyl,
6,9,12-octadecatrienyl, 9-eicosenyl, 5,8,11,14-eicosatetraenyl,
5,8,11,14,17-eicosapentaenyl, 13-docosenyl, and
4,7,10,13,16,19-docosahexaenyl; a C 1 to C12 aliphatic alkyl group
substituted with a hydroxyl group at least one carbon atom away
from a site of unsaturation, examples of which hydroxyalkyl group
include hydroxymethyl, hydroxyethyl, hydroxydodecyl, and the
like;
[0083] a C1 to C12 alkyl group substituted with a hydroxyl group
that is esterified with a C2 to C25 aliphatic carboxyl group of an
acid such as acetic acid, butanoic acid, hexanoic acid, octanoic
acid, decanoic acid, dodecanoic acid, tetradecanoic acid,
hexadecanoic acid, 9-hexadecenoic acid, octadecanoic acid,
9-octadecenoic acid, 11-octadecenoic acid, 9,12-octadecadienoic
acid, 9,12,15-octadecatrienoic acid, 6,9,12-octadecatrienoic acid,
eicosanoic acid, 9-eicosenoic acid, 5,8,11,14-eicosatetraenoic
acid, 5,8,11,14,17-eicosapentaenoic acid, docosanoic acid,
13-docosenoic acid, 4,7,10,13,16,19-docosahexaenoic acid,
tetracosanoic acid, and the like, a dicarboxylic acid such as
succinic acid, or a hydroxyacid such as lactic acid, wherein the
total number of carbon atoms of the ester substituent is between 3
and 25;
[0084] halogen such as fluoro-, chloro-, bromo-, and iodo-;
nitro-;
[0085] amino- such as NH2, methyl amino, dimethylamino;
trifluoromethyl-;
[0086] carboxyl (--COOH);
[0087] a C1 to C24 alkoxy (such as can be formed by alkylation of
tyrosine) such as methoxy, ethoxy, propyloxy, isopropyloxy,
butyloxy, isobutyloxy, cyclopropyloxy, 2-methoxycyclopropyloxy,
cyclohexyloxy, octyloxy, decyloxy, dodecyloxy, hexadecyloxy,
octadecyloxy, eicosanyloxy, docosanyloxy, tetracosanyloxy,
9-hexadecenyloxy, 9-octadecenyloxy, 11-octadecenyloxy,
9,12-octadecadienyloxy, 9,12,15-octadecatrienyloxy,
6,9,12-octadecatrienyloxy, 9-eicosenyloxy,
5,8,11,14-eicosatetraenyloxy, 5,8,11,14,17-eicosapentaenyloxy, 13
-docosenyloxy, and 4,7,10,13,16,19-docosahexaenyloxy; and
[0088] a C2 to C12 hydroxyalkyloxy such as 2-hydroxyethyloxy and
esters thereof with carboxylic acids as described above or with
trifluoroacetic acid.
[0089] A serine hydroxyl group may be esterified with a substituent
selected from the group consisting of:
[0090] a C2 to C12 aliphatic carboxylic acid group such as
described above;
[0091] a trifluoroacetic acid group; and
[0092] a benzoic acid group.
[0093] The epsilon amino group in lysine may be chemically
modified, for example, by amide formation with: a C2 to C12
aliphatic carboxylic acid group (for example, by reaction of the
amine with a chemically activated form of a carboxylic acid such as
an acid chloride, an anhydride, an N-hydroxysuccinimide ester, a
pentafluorophenyl (OPfp) ester, a
3-hydroxy-2,3-dihydro-4-oxo-benzo-triazone (ODhbt) ester, and the
like) such as described above, or a benzoic acid group, or an amino
acid group. Additionally, the epsilon amino group in lysine may be
chemically modified by alkylation with one or two C1 to C4
aliphatic alkyl groups.
[0094] The carboxylic acid group in glutamic acid may be modified
by formation of an amide with an amine such as: ammonia; a C1 to
C12 primary aliphatic alkyl amine (the alkyl portion of which is as
described above) including with methylamine; or an amino group of
an amino acid.
[0095] The carboxylic acid group in glutamic acid may be modified
by formation of an ester with a C1 to C12 aliphatic hydroxyalkyl
group as described above, preferably an ester with a primary
alcohol of a C1 to C12 aliphatic alkyl such as methanol, ethanol,
propan-1-ol, n-dodecanol, and the like as described above.
[0096] In a preferred embodiment, the present invention comprises a
method of inhibiting the release of at least one inflammatory
mediator from a granule in at least one inflammatory cell in a
tissue and/or fluid of a subject comprising administration to said
tissue and/or fluid a therapeutically effective amount of a
pharmaceutical composition comprising at least one peptide having
an amino acid sequence selected from the group consisting of:
[0097] (a) an amino acid sequence having from 4 to 23 contiguous
amino acids of a reference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ
ID NO. 1);
[0098] (b) an amino acid sequence having the sequence,
GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1); and
[0099] (c) an amino acid sequence substantially identical to the
sequence defined in (a), wherein the C-terminal amino acid of the
peptide is optionally independently chemically modified, and the
N-terminal amino acid of the peptide is independently chemically
modified by acylation with a carboxylic acid selected from the
group consisting of a C2 to C13 saturated or unsaturated aliphatic
carboxylic acid, a C14 saturated (myristic acid) or unsaturated
aliphatic carboxylic acid, a C15 to C24 saturated or unsaturated
aliphatic carboxylic acid, and trifluoroacetic acid, or is not
chemically modified, with the proviso that said peptide can be
modified by acylation when its amino acid sequence begins with the
sequence GAQF of the reference sequence by acylation only with a
carboxylic acid selected from the group consisting of a C2 to C13
saturated or unsaturated aliphatic carboxylic acid, a C14
unsaturated aliphatic carboxylic acid, a C15 to C24 saturated or
unsaturated aliphatic carboxylic acid, and trifluoroacetic acid, or
is not chemically modified, wherein said peptide, optionally
combined with a pharmaceutically acceptable carrier, and in a
therapeutically effective inflammatory mediator release-reducing
amount to reduce the release of said inflammatory mediator from at
least one inflammatory cell as compared to release of said
inflammatory mediator from at least one of the same type of
inflammatory cell that would occur in the absence of said at least
one peptide.
[0100] The method preferably employs a peptide that can be
acetylated at the alpha N-terminal amino acid. This peptide can
consist of at least ten contiguous amino acid residues and is
preferably embodied by acetyl-peptide 106 (SEQ ID NO: 106).
[0101] The method also employs a peptide consisting of at least
four contiguous amino acid residues and more preferably at least
six contiguous amino acid residues. Further, the peptide can be
myristoylated at the alpha N-terminal amino acid when the peptide.
The method also can utilized peptide that can be amidated with
ammonia at the alpha C-terminal amino acid.
[0102] The method in a further embodiment utilizes a peptide
comprises an amino acid sequence of (a) an amino acid sequence
having from 4 to 23 contiguous amino acids of a reference sequence,
GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1), wherein the N-terminal
amino acid of the amino acid sequence of (a) is selected from amino
acid position 2 to 21 of the reference sequence,
GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1). Further, these peptides
can be myristoylated at the alpha N-terminal amino acid and also
can be amidated with ammonia at the alpha C-terminal amino
acid.
[0103] The method of administration according to the present
invention defines the reduction of the release of an inflammatory
mediator as blocking or inhibiting the mechanism that releases an
inflammatory mediator from the inflammatory cell in said
subject.
[0104] The method of aministration includes incorporating or mixing
the disclosed peptides with a pharmaceutically acceptable carrier
to form a pharmaceutical composition.
[0105] The method of administration of the present invention
release of at least one inflammatory mediator release-reducing
amount to reduce the release of said inflammatory mediator from at
least one inflammatory cell as compared to release of said
inflammatory mediator from at least one of the same type of
inflammatory cell that would occur in the absence of said at least
one peptide. The inflammatory cell in said subject can be a
leukocyte, a granulocyte, a basophil, an eosinophil, monocyte,
macrophage or a combination thereof.
[0106] The inflammatory mediator released from at least one granule
of at least one inflammatory cell is selected from the group
consisting of myeloperoxidase (MPO), eosinophil peroxidase (EPO),
major basic protein [MBP], lysozyme, granzyme, histamine,
proteoglycan, protease, a chemotactic factor, cytokine, a
metabolite of arachidonic acid, defensin, bactericidal
permeability-increasing protein (BPI), elastase, cathepsin G,
cathepsin B, cathepsin D, beta-D-glucuronidase, alpha-mannosidase,
phospholipase A2, chondroitin-4-sulphate, proteinase 3,
lactoferrin, collagenase, complement activator, complement
receptor, N-formylmethionyl-leucyl-phenylalanine (FMLP) receptor,
laminin receptor, cytochrome b558, monocyte-chemotactic factor,
histaminase, vitamin B12 binding protein, gelatinase, plasminogen
activator, beta-D-glucuronidase, and a combination thereof.
Preferably the inflammatory mediator is selected from the group
consisting of myeloperoxidase (MPO), eosinophil peroxidase (EPO),
major basic protein (MBP), lysozyme, granzyme and a combination
thereof.
[0107] The method according to claim 13, wherein said effective
inflammatory mediator release-reducing amount of said peptide
comprises a degranulation-inhibiting amount of peptide that reduces
the amount of an inflammatory mediator released from at least one
inflammatory cell from about 1% to about 99% or preferably about
5-50% to about 99%, as compared to the amount released from at
least one inflammatory cell in the absence of the peptide.
[0108] The method of the present invention is useful for the
treatment of a subject afflicted by or suffering from a respiratory
disease. This respiratory disease may be asthma, chronic
bronchitis, chronic obstructive pulmonary disease (COPD) and cystic
fibrosis. The subjects that can be treated by the present invention
are preferably mammals, such as humans, canines, equines and
felines.
[0109] The method of administration of the peptides of the present
invention are by topical administration, parenteral administration,
rectal administration, pulmonary administration, nasal
administration, and oral administration. More preferably, the
pulmonary administration comprises an aerosol, which can be
generated from a dry powder inhaler, a metered dose inhaler or
nebulizer. Additionally, the administration to the subject can
further include the administration of a second molecule selected
from the group consisting of an antibiotic, an antiviral compound,
an antiparasitic compound, an anti-inflammatory compound, and an
immunomodulator.
[0110] The method is also useful for the treatment of a subject who
is afflicted by or suffering from a disease selected from the group
consisting of a bowel disease, a skin disease, an autoimmune
disease, a pain syndrome, and combinations thereof. More
specifically, the bowel disease is selected from the group
consisting of ulcerative colitis, Crohn's disease and irritable
bowel syndrome. Skin diseases also treatable by the present method
includes rosacea, eczema, psoriasis and severe acne. Additiona a
subject suffering from arthritis may also be treated by the present
invention.
[0111] The present invention in one embodiment encompasses the
administration of peptides comprising an amino acid sequence
substantially identical to the amino acid sequence of (a) having
from 4 to 23 contiguous amino acids of a reference sequence,
GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1). These peptides preferably
are selected from the group consisting of SEQ ID NOS: 233, 234,
235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 247, 248,
249, 250, 251 and 252. These peptides can be further acetylated at
the alpha N-terminal amino acid or myristoylated at the alpha
N-terminal amino acid and optionally amidated with ammonia at the
alpha C-terminal amino acid.
[0112] The method of the present invention also is useful for
reducing mucus hypersecretion in a subject by the administration of
the peptides of the present invention as described herein for also
reducing MARCKS-related mucus hypersecretion from at least one
mucus secreting cell or tissue in the subject, whereby mucus
hypersecretion in the subject is reduced compared to that which
would occur in the absence of said administration of said
peptide.
[0113] The present invention is directed to an isolated peptide
having an amino acid sequence selected from the group consisting
of:
[0114] (a) an amino acid sequence having from 4 to 23 contiguous
amino acids of a reference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ
ID NO. 1);
[0115] (b) an amino acid sequence having the sequence,
GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1); and
[0116] (c) an amino acid sequence substantially identical to the
sequence defined in (a), wherein the C-terminal amino acid of the
peptide is optionally independently chemically modified, and the
N-terminal amino acid of the peptide is independently chemically
modified by acylation with a carboxylic acid selected from the
group consisting of a C2 to C13 saturated or unsaturated aliphatic
carboxylic acid, a C14 saturated or unsaturated aliphatic
carboxylic acid, a C15 to C24 saturated or unsaturated aliphatic
carboxylic acid, and trifluoroacetic acid, or is not chemically
modified, with the proviso that said peptide is modified by
acylation when its amino acid sequence begins with the sequence
GAQF of the reference sequence by acylation only with a carboxylic
acid selected from the group consisting of a C2 to C13 saturated or
unsaturated aliphatic carboxylic acid, a C14 unsaturated aliphatic
carboxylic acid, a C15 to C24 saturated or unsaturated aliphatic
carboxylic acid, and trifluoroacetic acid, or is not chemically
modified, wherein said peptide, optionally combined with a
pharmaceutically acceptable carrier, and in a therapeutically
effective inflammatory mediator release-reducing amount to reduce
the release of said inflammatory mediator from at least one
inflammatory cell as compared to release of said inflammatory
mediator from at least one of the same type of inflammatory cell
that would occur in the absence of said at least one peptide.
[0117] The isolated peptide can be acetylated at the alpha
N-terminal amino acid. The isolated peptide consists of at least
ten contiguous amino acid residues and preferably is an isolated
peptide consists of acetyl-peptide 106 (SEQ ID NO: 106).
[0118] In a further embodiment, the peptide consists of at least
four contiguous amino acid residues or peptide consists of at least
six contiguous amino acid residues.
[0119] The peptide can also be myristoylated at the alpha
N-terminal amino acid and/or peptide can be amidated with ammonia
at the alpha C-terminal amino acid.
[0120] The isolated peptide can further comprise an amino acid
sequence of (a) described above, (a) an amino acid sequence having
from 4 to 23 contiguous amino acids of a reference sequence,
GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1); wherein the N-terminal
amino acid of the amino acid sequence of (a) is selected from amino
acid position 2 to 21 of the reference sequence,
GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1). This peptide can be
further myristoylated or acetylated at the alpha N-terminal amino
acid or optionally amidated with ammonia at the alpha C-terminal
amino acid.
[0121] The isolated peptide in a further embodiment, wherein the
amino acid sequence is substantially identical to the amino acid
sequence of (a) having from 4 to 23 contiguous amino acids of a
reference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1). These
peptides preferably are selected from the group consisting of SEQ
ID NOS: 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,
245, 247, 248, 249, 250, 251 and 252. These peptides can be further
acetylated at the alpha N-terminal amino acid or myristoylated at
the alpha N-terminal amino acid and optionally amidated with
ammonia at the alpha C-terminal amino acid. amino acid sequence of
(c) substantially identical to the amino acid sequence of (a) is
selected from the group consisting of SEQ ID NOS: 233, 234, 235,
236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 247, 248, 249,
250, 251 and 252.
[0122] The invention also emcompasses a composition comprising an
isolated peptide as described in the paragraphs above and described
herein and an excipient. The invention also encompasses a
pharmaceutical composition comprising an isolated peptide an
isolated peptide as described in the paragraphs above and described
herein and a pharmaceutically acceptable carrier. The
pharmaceutical composition can further preferably be sterile,
sterilizable or sterilized. These peptides can be contained in a
kit with reagents useful for administration.
BRIEF DESCRIPTION OF THE DRAWINGS
[0123] FIGS. 1A-1B illustrate that PKC-dependent phosphorylation
releases MARCKS from the plasma membrane to the cytoplasm.
[0124] FIGS. 2A-2C show that PKG induces dephosphorylation of
MARCKS by activating PP2A.
[0125] FIG. 3 depicts bar graphs that demonstrate that PP2A is an
essential component of the mucin secretory pathway.
[0126] FIG. 4 is a gel that illustrates that MARCKS associates with
actin and myosin in the cytoplasm.
[0127] FIG. 5 depicts a signaling mechanism controlling MPO
secretion in neutrophils.
[0128] FIG. 6 is a bar graph depicting the ability of MANS peptide
to block secretion of myloperoxidase from isolated canine
neutrophils.
[0129] FIG. 7 is a bar graph depicting the ability of MANS peptide
to block secretion of myloperoxidase from isolated human
neutrophils.
[0130] FIG. 8 is a bar graph showing that PMA stimulates a small
increase in MPO secretion from LPS-stimulated human neutrophils
which is enhanced in a concentration-dependent manner by
co-stimulation with 8-Br-cGMP.
[0131] FIG. 9 is a bar graph showing that 8-Br-cGMP simulation has
little effect on MPO secretion from LPS-stimulated human
neutrophils until a co-stimulation with PMA occurs in a
concentration-dependent manner.
[0132] FIG. 10 is a bar graph showing that PMA stimulates a small
increase in MPO secretion from LPS-stimulated canine neutrophils
which is enhanced in a concentration-dependent manner by
co-stimulation with 8-Br-cGMP.
[0133] FIG. 11 is a bar graph showing that 8-Br-cGMP simulation has
little effect on MPO secretion from LPS-stimulated canine
neutrophils until a co-stimulation with PMA occurs in a
concentration-dependent manner.
[0134] FIG. 12 is a bar graph showing that co-stimulation with
PMA+8-Br-cGMP is required for maximal MPO secretion from
LPS-stimulated canine neutrophils.
DETAILED DESCRIPTION OF THE INVENTION
[0135] The present invention will now be described more fully
hereinafter with reference to the accompanying figures, in which
preferred embodiments of the invention are illustrated. This
invention may, however, be embodied in different forms and should
not be construed as limited to the embodiments set forth herein.
Rather, these embodiments are provided so that this disclosure will
be thorough and complete, and will fully convey the scope of the
invention to those skilled in the art.
[0136] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
The use of the words "a" or "an" herein to describe any aspect of
the present invention is to be interpreted as indicating one or
more.
[0137] The present invention is directed to a method of inhibiting
the exocytotic release of at least one inflammatory mediator from
at least one inflammatory cell comprising contacting the at least
one inflammatory cell, which cell comprises at least one
inflammatory mediator contained within a vesicle inside the cell,
with at least one peptide selected from the group consisting of a
MANS peptide and an active fragment thereof in an effective amount
to reduce the release of the inflammatory mediator from the
inflammatory cell as compared to the release of the inflammatory
mediator from the same type of inflammatory cell that would occur
in the absence of the at least one peptide.
[0138] The present invention is further directed to a method of
inhibiting the release of at least one inflammatory mediator from
at least one inflammatory cell in a tissue or fluid of a subject
comprising the administration to the subject's tissue and/or fluid,
which comprises at least one inflammatory cell comprising at least
one inflammatory mediator contained within a vesicle inside the
cell, a therapeutically effective amount of a pharmaceutical
composition comprising at least one peptide selected from the group
consisting of a MANS peptide and an active fragment thereof in a
therapeutically effective amount to reduce the release of the
inflammatory mediator from at least one inflammatory cell as
compared to release of the inflammatory mediator from at least one
of the same type of inflammatory cell that would occur in the
absence of the at least one peptide. More specifically, reducing
the release of an inflammatory mediator comprises blocking or
inhibiting the mechanism that releases an inflammatory mediator
from the inflammatory cell.
[0139] The present invention is directed to the contact and/or
administration of the peptide described above and throughout the
specification with any known inflammatory cell that may be
contained in the tissue or fluid of a subject which contains at
least one inflammatory mediator contained within a vesicle inside
the cell. The inflammatory cell is preferably a leukocyte, more
preferably a granulocyte, which can be further classified as a
neutrophil, a basophil, an eosinophil or a combination thereof. The
inflammatory cells contacted in the present method may also be a
monocyte/macrophage.
[0140] The present invention is directed to reducing the release of
inflammatory mediators contained within the vesicles of
inflammatory cells and these inflammatory mediators are selected
from the group consisting of myeloperoxidase (MPO), eosinophil
peroxidase (EPO), major basic protein (MBP), lysozyme, granzyme,
histamine, proteoglycan, protease, a chemotactic factor, cytokine,
a metabolite of arachidonic acid, defensin, bactericidal
permeability-increasing protein (BPI), elastase, cathepsin G,
cathepsin B, cathepsin D, beta-D-glucuronidase, alpha-mannosidase,
phospholipase A2, chondroitin-4-sulphate, proteinase 3,
lactoferrin, collagenase, complement activator, complement
receptor, N-formylmethionyl-leucyl-phenylalanine (FMLP) receptor,
laminin receptor, cytochrome b558, monocyte-chemotactic factor,
histaminase, vitamin B12 binding protein, gelatinase, plasminogen
activator, beta-D-glucuronidase, and a combination thereof.
Preferably, these inflammatory mediators are selected from the
group consisting of myeloperoxidase (MPO), eosinophil peroxidase
(EPO), major basic protein (MBP), lysozyme, granzyme and a
combination thereof.
[0141] The present invention contacts an effective amount of the
peptide with an inflammatory cell, wherein the effective amount is
defined as a degranulation-inhibiting amount of MANS peptide or an
active fragment thereof that reduces the amount of an inflammatory
mediator released from at least one inflammatory cell from about 1%
to about 99% as compared to the amount released from at least one
inflammatory cell in the absence of MANS peptide or an active
fragment thereof. This amount is also known as an effective
inflammatory mediator release-reducing amount. More preferably,
this effective amount of the contacted peptide comprises a
degranulation-inhibiting amount of MANS peptide or an active
fragment thereof that reduces the amount of an inflammatory
mediator released from at least one inflammatory cell from between
about 5-50% to about 99% as compared to the amount released from at
least one inflammatory cell in the absence of MANS peptide or an
active fragment thereof
[0142] The present invention in one embodiment is directed to the
administration of at least one peptide comprising a MANS peptide
and an active fragment thereof in a therapeutically effective
amount into tissue or fluid of a subject where the subject is
afflicted by a respiratory disease, which is preferably asthma,
chronic bronchitis or COPD. In a further embodiment, the subject
may be afflicted by a bowel disease, a skin disease, an autoimmune
disease, a pain syndrome, and combinations thereof. The bowel
disease may be ulcerative colitis, Crohn's disease or irritable
bowel syndrome. The subject may be afflicted with a skin disease,
such as rosacea, eczema, psoriasis or severe acne. The subject may
also be afflicted with arthritis, such as rheumatoid arthritis,
psoriatic arthritis, systemic lupus erythematosus. Subjects
afflicted by cystic fibrosis may also be treated by the present
method and peptides. The present method is preferably useful for
the treatment of subjects, such as mammals, and preferably humans,
canines, equines and felines.
[0143] The present method of treatment of subjects is by the
administration of one or more peptides including the MANS peptide
or an active fragment described herein to include topical
administration, parenteral administration, rectal administration,
pulmonary administration, nasal administration, or oral
administration. More specifically, pulmonary administration is
selected from the group of aerosol, dry powder inhaler, metered
dose inhaler, and nebulizer. Additionally, the disclosed method may
further comprise the administration to the subject of a second
molecule selected from the group consisting of an antibiotic, an
antiviral compound, an antiparasitic compound, an anti-inflammatory
compound, and an immunomodulator.
[0144] In one aspect, the invention relates to a method of
administering a pharmaceutical composition. The pharmaceutical
composition comprises a therapeutically effective amount of a known
compound and a pharmaceutically acceptable carrier. A
"therapeutically effective" amount as used herein is an amount of a
compound that is sufficient to ameliorate symptoms exhibited by a
subject. The therapeutically effective amount will vary with the
age and physical condition of the patient, the severity of the
condition of the patient being treated, the duration of the
treatment, the nature of any concurrent treatment, the
pharmaceutically acceptable carrier used and like factors within
the knowledge and expertise of those skilled in the art.
Pharmaceutically acceptable carriers are preferably solid dosage
forms such as tablets or capsules. Liquid preparations for oral
administration also may be used and may be prepared in the form of
syrups or suspensions, e.g., solutions containing an active
ingredient, sugar, and a mixture of ethanol, water, glycerol, and
propylene glycol. If desired, such liquid preparations may include
one or more of following: coloring agents, flavoring agents, and
saccharin. Additionally, thickening agents such as
carboxymethylcellulose also may be used as well as other acceptable
carriers, the selection of which are known in the art.
[0145] As stated above, the present invention relates to methods
for regulating cellular secretory processes, especially those
releasing inflammatory mediators from inflammatory cells. As used
herein, the term "regulating" means blocking, inhibiting,
decreasing, reducing, increasing, enhancing or stimulating. A
number of cellular secretory processes involve the release of
contents from membrane-bound vesicles or granules within cells A
membrane-bound vesicle or granule is defined as an intracellular
particle, which is primarily vesicular (or a vesicle inside a cell)
and which contains stored material that can be secreted. Some of
the contents of these vesicles, such as those contained in
inflammatory cells, have been found to be responsible for a variety
of pathologies in numerous mammalian tissues. Some of the effects
of these secretions appear to include damage of previously healthy
tissue during inflammation. This invention provides a means of
blocking secretion from any membrane-bound vesicle, including those
found in inflammatory cells, by targeting a specific molecule
important in the intracellular secretory pathway with a synthetic
peptide. This approach may be of therapeutic importance for the
treatment of a wide variety of hypersecretory and inflammatory
conditions in humans and animals.
[0146] More specifically, the present invention targets
inflammatory cells that contain the inflammatory mediators in one
or more granules or vesicles within the cells' cytoplasm. The cells
are contacted with one or more peptides that are selected from the
MANS peptide or an active fragment thereof, all of which are
described in detail herein. Preferably the contact of the peptide
with the inflammatory cell is via administration to a subject
afflicted by or suffering from a disease in which these
inflammatory cells are present in specific tissue or fluid within
the tissue. Upon administration or contact of the peptide with the
cell, the peptide competitively competes for and competitively
inhibits the binding of the native MARCKS protein to the membrane
of the intracellular granules or vesicles which contain the
inflammatory mediators. As a result of blocking the binding of the
MARCKS protein to the vesicles in the inflammatory cells, these
vesicles in these cells do not move to the plasma membrane of the
cells as they would normally do when stimulated to exocytotically
release their contents of inflammatory mediators out of the cells.
Thus, the method of the present invention inhibits the movement of
the vesicles to the cells' plasma membrane, which in turn, reduces
the release of the inflammatory mediators from the inflammatory
cells. The amount of inflammatory mediators released from the cells
over time is reduced because both the rate of release and the
amount of release of the mediators from the inflammatory cells is
dependent upon the concentration of the peptide administered and
contacted with the inflammatory cells.
[0147] One benefit of the present invention is that it may combine
a therapy that includes the direct blocking of mucus secretion with
a unique anti-inflammatory therapy. A benefit of the present
invention over current anti-inflammation therapies that affect a
general suppression of the immune system is that the peptide is
thought to block secretion of only intracellular components
secreted from inflammatory cells. Thus, many aspects of the immune
system should still function even with the inhibition of the
inflammatory mediators.
[0148] The compounds of the invention may regulate, i.e. block,
inflammatory mediator release from cells. This inhibition of
release of inflammatory mediators is an attractive means for
preventing and treating a variety of disorders, e.g., diseases and
pathological conditions involving inflammation. Thus, the compounds
of the invention may be useful for the treatment of such
conditions. These encompass airway diseases and chronic
inflammatory diseases including, but not limited to,
osteoarthritis, multiple sclerosis, Guillain-Barre syndrome,
Crohn's disease, ulcerative colitis, psoriasis, graft versus host
disease and systemic lupus erythematosus. The compounds of the
invention can also be used to treat other disorders associated with
the activity of elevated levels of proinflammatory mediators and
enzymes such as responses to various infectious agents and a number
of diseases of autoimmunity such as rheumatoid arthritis, toxic
shock syndrome, diabetes and inflammatory bowel diseases.
[0149] Uses of the peptide and methods of the invention include
therapies to combat inflammation along with therapies that will
combine the anti-inflammatory activity of the peptide with its
ability to block mucus secretion. Diseases that may be treated by
the peptide's ability to block both inflammation and mucus
secretion include but are not limited to inflammatory bowel
diseases, digestive disorders (i.e., inflamed gall bladder,
Menetier's disease) and inflammatory airway diseases.
[0150] Other proinflammatory mediators have been correlated with a
variety of disease states that correlate with influx of neutrophils
into sites of inflammation or injury. Blocking antibodies have been
demonstrated as useful therapies against the neutrophil-associated
tissue injury in acute inflammation (Harada et al., 1996, Molecular
Medicine Today 2, 482). Cells other than neutrophils that may
release inflammatory mediators include other leukocytes, such as
basophils, eosinophils, monocytes and lymphocytes, and therapies
may be directed against secretion from these cells. Neutrophils,
eosinophils, and basophils are each a type of granulocyte, i.e., a
leukocyte that has granules in its cytoplasm. Leukocytes synthesize
a number of inflammatory mediators that are packaged and stored in
cytoplasmic granules. Among these mediators are, for example,
myeloperoxidase [MPO] in neutrophils (Borregaard N, Cowland J B.
Granules of the human neutrophilic polymorphonuclear leukocyte.
Blood 1997; 89:3503-3521), eosinophil peroxidase [EPO] and major
basic protein [MBP] in eosinophils (Gleich G J. Mechanisms of
eosinophil-associated inflammation. J Allergy Clin Immunol 2000;
105:651-663), lysozyme in monocytes/macrophages (Hoff T, Spencker
T, Emmendoerffer A., Goppelt-Struebe M. Effects of glucocorticoids
on the TPA-induced monocytic differentiation. J Leukoc Biol 1992;
52:173-182; Balboa M A, Saez Y, Balsinde J. Calcium-independent
phospholipase A2 is required for lysozyme secretion in U937
promonocytes. J Immunol 2003; 170:5276-5280), and granzyme in
natural killer (NK) cells and cytotoxic lymphocytes (Bochan M R,
Goebel W S, Brahmi Z. Stably transfected antisense granzyme B and
perforin constructs inhibit human granule-mediated lytic ability.
Cell Immunol 1995; 164:234-239; Gong J H., Maki G, Klingemann H G.
Characterization of a human cell line (NK-92) with phenotypical and
functional characteristics of activated natural killer cells.
Leukemia 1994; 8:652-658; Maki G, Klingemann H G, Martinson J A,
Tam Y K. Factors regulating the cytotoxic activity of the human
natural killer cell line, NK-92. J Hematother Stem Cell Res 2001;
10:369-383; and Takayama H, Trenn G, Sitkovsky M V. A novel
cytotoxic T lymphocyte activation assay. J Immunol Methods 1987;
104:183-1907-10). These mediators can be released at sites of
injury and can contribute to inflammation and repair, such as in
the lung and elsewhere, as a result of the infiltration of these
cells to the tissue site of injury or disease. Leukocytes release
these granules via an exocytotic mechanism (Burgoyne R D, Morgan A.
Secretory granule exocytosis. Physiol Rev 2003; 83:581-632; Logan M
R, Odemuyiwa S O, Moqbel R. Understanding exocytosis in immune and
inflammatory cells: the molecular basis of mediator secretion. J
Allergy Clin Immunol 2003; 111: 923-932),
[0151] Mast cells, which usually do not circulate in the blood
stream, and basophils contain secretory cytoplasmic granules which
store and can release, upon cell activation, preformed inflammatory
(anaphylactic) mediators, such as histamine; proteoglycans, such as
heparin and chondroitin sulphate; proteases such as tyrptase,
chymase, carboxypeptidase, and cathepsin G-like protease;
chemotactic factors, cytokines and metabolites of arachidonic acid
that act on the vasculature, smooth muscle, connective tissue,
mucous glands and inflammatory cells.
[0152] Neutrophils, also known as polymorphonuclear leukocytes
(PMN), comprise 50 to 60% of the total circulating leukocytes.
Neutrophils act against infectious agents, such as bacteria, fungi,
protozoa, viruses, virally infected cells, as well as tumor cells,
that penetrate the body's physical barriers at sites of infection
or injury. Neutrophils mature through six morphological stages:
myeloblast, promyeloblast, myelocyte, metamyelocyte, non-segmented
(band) neutrophil, and segmented (functionally active)
neutrophil.
[0153] In neutrophils, inflammatory mediators are stored in primary
(azurophil), secondary (specific), and tertiary (gelatinase)
granules, as well as in secretory vesicles. Among numerous
mediators of inflammation, primary (azurophil) granules contain
myeloperoxidase (MPO), lysozyme, defensins, bactericidal
permeability-increasing protein (BPI), elastase, cathepsin G,
cathepsin B, cathepsin D, beta-D-glucuronidase, alpha-mannosidase,
phospholipase A.sub.2, chondroitin-4-sulphate, and proteinase 3
(see, for example, Hartwig J H, Thelen M, Rosen A, Janmey P A,
Nairn A C, Aderem A. MARCKS is an actin filament crosslinking
protein regulated by protein kinase C and calcium-calmodulin.
Nature 1992; 356:618-622); secondary (specific) granules contain
lysozyme, lactoferrin, collagenase, complement activator,
phospholipase A2, complement receptors, e.g., CR3, CR4,
N-formylmethionyl-leucyl-phenylalanine (FMLP) receptors, laminin
receptors, cytochrome b.sub.558, monocyte-chemotactic factor,
histaminase, and vitamin B12 binding protein; and small storage
granules contain gelatinase, plasminogen activator, cathepsin B,
cathepsin D, beta-D-glucuronidase, alpha-mannosidase, and
cytochrome b.sub.558.
[0154] Neutrophil granules contain antimicrobial or cytotoxic
substances, neutral proteinases, acid hydrolases and a pool of
cytoplasmic membrane receptors. Among azurophil granule
constituents myeloperoxidase (MPO) is a critical enzyme in the
conversion of hydrogen peroxide to hypochlorous acid. Together with
hydrogen peroxide and a halide cofactor it forms an effective
microbicidal and cytotoxic mechanism of leukocytes--the
myeloperoxidase system.
[0155] Defensins, which constitute 30 to 50% of azurophilic granule
protein, are small (molecule weight <4000) potent antimicrobial
peptides that are cytotoxic to a broad range of bacteria, fungi and
some viruses. Their toxicity may be due to membrane
permeabilization of the target cell which is similar to other
channel-forming proteins (perforins).
[0156] Bacterial permeability-increasing (BPI) protein is a member
of perforins. It is highly toxic to gram-negative bacteria but not
to gram-positive bacteria or fungi and can also neutralize
endotoxin, the toxic lipopolysaccharide component of gram-negative
bacterial cell envelope.
[0157] Lactoferrin sequesters free iron, thereby preventing the
growth of ingested microorganisms that survive the killing process
and increases bacterial permeability to lysozyme.
[0158] Serine proteases such as elastase and cathepsin G hydrolyze
proteins in bacterial cell envelopes. Substrates of granulocyte
elastase include collagen cross-linkages and proteoglycans, as well
as elastin components of blood vessels, ligaments, and cartilage.
Cathepsin D cleaves cartilage proteoglycans, whereas granulocyte
collagenases are active in cleaving type I and, to a lesser degree,
type III collagen from bone, cartilage, and tendon. Collagen
breakdown products have chemotactic activity for neutrophils,
monocytes, and fibroblasts.
[0159] Regulation of tissue destructive potential of lysosomal
proteases is mediated by protease inhibitors such as
alpha2-macroglobulin and alphal-antiprotease. These antiproteases
are present in serum and synovial fluids. They may function by
binding to and covering the active sites of proteases.
Protease-antiprotease imbalance can be important in the
pathogenesis of emphysema.
[0160] Azurophil granules function predominantly in the
intracellular milieu (in the phagolysosomal vacuole), where they
are involved in the killing and degradation of microorganisms.
Neutrophil specific granules are susceptible to release their
contents extracellularly and have an important role in initiating
inflammation. Specific granules represent an intracellular
reservoir of various plasma membrane components including
cytochrome b (component of NADPH oxidase, an enzyme responsible for
the production of superoxide), receptors for complement fragment
iC3b (CR3, CR4), for laminin, and formylmethionyl-peptide
chemoattractants. In addition to others, there is histaminase which
is relevant for the degradation of histamine, vitamin binding
protein, and plasminogen activator which is responsible for plasmin
formation and cleavage of C5a from C5.
[0161] The importance of neutrophil granules in inflammation is
apparent from studies of several patients with congenital
abnormalities of the granules. Patients with Chediak-Higashi
syndrome have a profound abnormality in the rate of establishment
of an inflammatory response and have abnormally large lysosomal
granules. The congenital syndrome of specific granule deficiency is
an exceedingly rare disorder characterized by diminished
inflammatory responses and severe bacterial infections of skin and
deep tissues.
[0162] Although mechanisms regulating exocytotic secretion of these
granules are only partially understood, several key molecules in
the process have been identified, including intracellular Ca2+
transients (Richter et al. Proc Natl Acad Sci USA 1990;
87:9472-9476; Blackwood et al., Biochem J 1990; 266:195-200), G
proteins, tyrosine and protein kinases (PK, especially PKC) (Smolen
et al., Biochim Biophys Acta 1990; 1052:133-142; Niessen et al.,
Biochim. Biophys. Acta 1994; 1223:267-273; Naucler et al.,
Pettersen et al., Chest 2002; 121; 142-150), Rac2 (Abdel-Latif et
al., Blood 2004; 104:832-839; Lacy et al., J Immunol 2003;
170:2670-2679) and various SNARE's, SNAP's and VAMP's (Sollner et
al., Nature 1993; 362: 318-324; Lacy, Pharmacol Ther 2005;
107:358-376).
[0163] SNARE (Soluble N-ethylmaleimide attachment protein receptor)
proteins are a family of membrane-associated proteins characterized
by an alpha-helical coiled-coil domain called the SNARE motif (Li
et al., Cell. Mol. Life Sci. 60: 942-960 (2003)). These proteins
are classified as v-SNAREs and t-SNAREs based on their localization
on vesicle or target membrane; another classification scheme
defines R-SNAREs and Q-SNAREs, as based on the conserved arginine
or glutamine residue in the centre of the SNARE motif. SNAREs are
localized to distinct membrane compartments of the secretory and
endocytic trafficking pathways, and contribute to the specificity
of intracellular membrane fusion processes. The t-SNARE domain
consists of a 4-helical bundle with a coiled-coil twist. The SNARE
motif contributes to the fusion of two membranes. SNARE motifs fall
into four classes: homologues of syntaxin la (t-SNARE), VAMP-2
(v-SNARE), and the N- and C-terminal SNARE motifs of SNAP-25. One
member from each class may interact to form a SNARE complex. The
SNARE motif is found in the N-terminal domains of certain syntaxin
family members such as syntaxin 1a, which is required for
neurotransmitter release (Lerman et al., Biochemistry 39: 8470-8479
(2000)), and syntaxin 6, which is found in endosomal transport
vesicles (Misura et al., Proc. Natl. Acad. Sci. U.S.A. 99:
[0164] SNAP-25 (synaptosome-associated protein 25 kDa) proteins are
components of SNARE complexes, which may account for the
specificity of membrane fusion and to directly execute fusion by
forming a tight complex (the SNARE or core complex) that brings the
synaptic vesicle and plasma membranes together. The SNAREs
constitute a large family of proteins that are characterized by
60-residue sequences known as SNARE motifs, which have a high
propensity to form coiled coils and often precede carboxy-terminal
transmembrane regions. The synaptic core complex is formed by four
SNARE motifs (two from SNAP-25 and one each from synaptobrevin and
syntaxin 1) that are unstructured in isolation but form a parallel
four-helix bundle on assembly. The crystal structure of the core
complex has revealed that the helix bundle is highly twisted and
contains several salt bridges on the surface, as well as layers of
interior hydrophobic residues. A polar layer in the centre of the
complex is formed by three glutamines (two from SNAP-25 and one
from syntaxin 1) and one arginine (from synaptobrevin) (Rizo et
al., Nat Rev Neurosci 3: 641-653 (2002)). Members of the SNAP-25
family contain a cluster of cysteine residues that can be
palmitoylated for membrane attachment (Risinger et al., J. Biol.
Chem. 268: 24408-24414 (1993)).
[0165] The major role of neutrophils is to phagocytose and destroy
infectious agents. They also limit the growth of some microbes,
prior to onset of adaptive (specific) immunological responses.
Although neutrophils are essential to host defense, they have also
been implicated in the pathology of many chronic inflammatory
conditions and in ischemia-reperfusion injury. Hydrolytic enzymes
of neutrophil origin and oxidatively inactivated protease
inhibitors can be detected in fluid isolated from inflammatory
sites. Under normal conditions, neutrophils can migrate to sites of
infection without damage to host tissues. However, undesirable
damage to a host tissue can sometimes occur. This damage may occur
through several independent mechanisms. These include premature
activation during migration, extracellular release of toxic
products during the killing of some microbes, removal of infected
or damage host cells and debris as a first step in tissue
remodeling, or failure to terminate acute inflammatory responses.
Ischemia-reperfusion injury is associated with an influx of
neutrophils into the affected tissue and subsequent activation.
This may be triggered by substances released from damaged host
cells or as a consequence of superoxide generation through xantine
oxidase.
[0166] Under normal conditions, blood may contain a mixture of
normal, primed, activated and spent neutrophils. In an inflammatory
site, mainly activated and spent neutrophils are present. Activated
neutrophils have enhanced production of reactive oxygen
intermediates (ROI). A subpopulation of neutrophils with the
enhanced respiratory burst has been detected in the blood of people
with an acute bacterial infection and patients with the adult
respiratory distress syndrome (ARDS). This is an example of a
neutrophil paradox. Neutrophils have been implicated in the
pathology of this condition because of the large influx of these
cells into the lung and the associated tissue damage caused by
oxidants and hydrolytic enzymes released from activated
neutrophils. The impairment of neutrophil microbicidal activity
that occurs as the ARDS worsens may be a protective response on the
part of the host, which is induced locally by inflammatory
products.
[0167] The acute phase of thermal injury is also associated with
neutrophil activation, and this is followed by a general impairment
in various neutrophil functions. Activation of neutrophils by
immune complexes in synovial fluid contributes to the pathology of
rheumatoid arthritis. Chronic activation of neutrophils may also
initiate tumor development because some ROI generated by
neutrophils damage DNA and proteases promote tumor cell migration.
In patients suffering from severe burns, a correlation has been
established between the onset of bacterial infection and reduction
in the proportion and absolute numbers of neutrophils positive for
antibody and complement receptors. Oxidants of neutrophil origin
have also been shown to oxidize low-density lipoproteins (LDL),
which are then more effectively bound to the plasma membrane of
macrophages through specific scavenger receptors. Uptake of these
oxidized LDL by macrophages may initiate atherosclerosis. In
addition, primed neutrophils have been found in people with
essential hypertension, Hodgkin's disease, inflammatory bowel
disease, psoriasis, sarcoidosis, and septicemia, where priming
correlates with high concentrations of circulating TNF-alpha
(cachectin).
[0168] Hydrolytic damage to host tissue and therefore chronic
inflammatory conditions may occur when antioxidant and antiprotease
screens are overwhelmed. Antiprotease deficiency is thought to be
responsible for the pathology of emphysema. Many antiproteases are
members of the serine protease inhibitor (SERPIN) family. Although
the circulation is rich in antiproteases, these large proteins may
be selectively excluded at sites of inflammation because
neutrophils adhere to their targets. Oxidative stress may initiate
tissue damage by reducing the concentration of extracellular
antiproteases to below the level required to inhibit released
proteases. Chlorinated oxidants and hydrogen peroxide can
inactivate antiproteases such as alphal-protease inhibitor and
alpha2-macroglobulin, which are endogenous inhibitors of elastase,
but simultaneously activate latent metalloproteases such as
collagenases and gelatinase, which contribute to the further
inactivation of antiproteases.
[0169] Cytoplasmic constituents of neutrophils may also be a cause
of formation of specific anti-neutrophil cytoplasmic antibodies
(ANCA), which are closely related to the development of systemic
vasculitis and glomerulonephritis. ANCA are antibodies directed
against enzymes that are found mainly within the azurophil or
primary granules of neutrophils. There are three types of ANCA that
can be distinguished by the patterns they produce by indirect
immunofluorescence on normal ethanol-fixed neutrophils. Diffuse
fine granular cytoplasmic fluorescence (cANCA) is typically found
in Wegener's granulomatosis, in some cases of microscopic
polyarteritis and Churg Strauss syndrome, and in some cases of
crescentic and segmental necrotizing glomerulonephritis. The target
antigen is usually proteinase 3. Perinuclear fluorescence (pANCA)
is found in many cases of microscopic polyarteritis and
glomerulonephritis. These antibodies are often directed against
myeloperoxidase but other targets include elastase, cathepsin G,
lactoferrin, lysozyme and beta-D-glucuronidase. The third group
designated "atypical" ANCA includes neutrophil nuclear fluorescence
and some unusual cytoplasmic patterns and while a few of the target
antigens are shared with pANCA, the others have not been identified
yet. pANCA are also found in a third of patients with Crohn's
disease. The reported incidence of ANCA in rheumatoid arthritis and
SLE varies considerably but the patterns are predominantly pANCA
and atypical ANCA.
[0170] The eosinophil is a terminally differentiated, end-stage
leukocyte that resides predominantly in submucosal tissue and is
recruited to sites of specific immune reactions, including allergic
diseases. The eosinophil cytoplasm contains large ellipsoid
granules with an electron-dense crystalline nucleus and partially
permeable matrix. In addition to these large primary crystalloid
granules, there is another granule type that is smaller (small
granule) and lacks the crystalline nucleus. The large specific
granules of eosinophils contain at least four distinct cationic
proteins, which exert a range of biological effects on host cells
and microbial targets: major basic protein (MBP), eosinophil
cationic protein (ECP), eosinophil derived neurotoxin (EDN), and
eosinophil peroxidase (EPO). Basophils contain about one fourth as
much major basic protein as eosinophils together with detectable
amounts of EDN, ECP and EPO. Small amounts of EDN and ECP are also
found in neutrophils (Gleich G J. Mechanisms of
eosinophil-associated inflammation. J Allergy Clin Immunol 2000;
105:651-663). MBP appears to lack enzymatic activity but is a
highly cationic polypeptide which may exert its toxic activities by
interactions with lipid membranes leading to their derangement.
Both MBP and EPO can act as selective allosteric inhibitors of
agonist binding to M2 muscarinic receptors. These proteins may
contribute to M2 receptor dysfunction and enhance vagally mediated
bronchoconstriction in asthma. EDN can specifically damage the
myelin coat of neurons. Histaminase and a variety of hydrolytic
lysosomal enzymes are also present in the large specific granules
of eosinophils. Among the enzymes in small granules of eosinophils
are aryl sulphatase, acid phosphatase, and a 92 kDa
metalloproteinase, a gelatinase. Eosinophils can elaborate
cytokines which include those with potential autocrine
growth-factor activities for eosinophils and those with potential
roles in acute and chronic inflammatory responses. Three cytokines
have growth-factor activities for eosinophils:
granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3 and
IL-5. Other cytokines produced by human eosinophils that may have
activities in acute and chronic inflammatory responses include
IL-1-alpha, IL-6, IL-8, TNF-alpha and both transforming growth
factors, TGF-alpha and TGF-beta.
[0171] Eosinophils contain crystalloid granules that contain MBP,
eosinophil cationic protein, EPO, and eosinophil-derived neurotoxin
(Gleich, J Allergy Clin Immunol 2000; 105:651-663). The human
promyelocytic cell line HL-60 clone 15 can be used to examine
secretion of EPO. This cell line was established from a clone of
HL-60 that had been grown at an elevated pH for two months
(Fischkoff, Leuk Res 1988; 12:679-686) and then treated with
butyric acid to allow the cells to differentiate so as to exhibit
many of the characteristics of peripheral blood eosinophils,
including expression of eosinophil-specific granule proteins
(Rosenberg et al., J Exp Med 1989; 170:163-176; Tiffany et al., J
Leukoc Biol 1995; 58:49-54; Badewa et al., Exp Biol Med 2002;
227:645-651).
[0172] Eosinophils can participate in hypersensitivity reactions,
especially through two lipid inflammatory mediators, leukotriene
C.sup.4 (LTC.sup.4) and platelet activating factor (PAF). Both
mediators contract airway smooth muscle, promote the secretion of
mucus, alter vascular permeability and elicit eosinophil and
neutrophil infiltration. In addition to the direct activities of
these eosinophil-derived mediators, MBP can stimulate the release
of histamine from basophils and mast cells, and MBP can stimulate
the release of EPO from mast cells. Eosinophils can serve as a
local source of specific lipid mediators as well as induce the
release of mediators from mast cells and basophils. Eosinophil
granule content is released following similar stimuli to neutrophil
granules, e.g. during phagocytosis of opsonized particles and by
chemotactic factors. Neutrophil lysosomal enzymes act primarily on
material engulfed in phagolysosomes, while the eosinophil granule
contents act mainly on extracellular target structure such as
parasites and inflammatory mediators.
[0173] Monocyte and macrophage development takes place in the bone
marrow and passes through the following steps: stem cell; committed
stem cell; monoblast; promonocyte; monocyte in bone marrow;
monocyte in peripheral blood; and macrophage in tissues. Monocyte
differentiation in the bone marrow proceeds rapidly (1.5 to 3
days). During differentiation, granules are formed in monocyte
cytoplasm and these can be divided as in neutrophils into at least
two types. However, they are fewer and smaller than their
neutrophil counterparts (azurophil and specific granules). Their
enzyme content is similar.
[0174] Granule-bound enzymes of monocytes/macrophages include
lysozyme, acid phosphatase, and beta-glucuronidase. As a model for
in vivo studies, lysozyme secretion from U937 cells was used. This
cell line is derived from a human histiocytic lymphoma and has been
used as a monocytic cell line that can be activated by a variety of
agonists, such as PMA (Hoff et al., J Leukoc Biol 1992; 52:173-182;
Balboa et al., J Immunol 2003; 170:5276-5280; Sundstrom et al., Int
J Cancer 1976; 17:565-577).
[0175] Natural killer (NK) cells and cytotoxic lymphocytes contain
potent cytotoxic granules including perforin, a pore-forming
protein, and granzymes, lymphocyte-specific serine proteases. For
example, the NK-92 cell line is an IL-2-dependent human line
established from a patient with rapidly progressive non-Hodgkin's
lymphoma (Gong JH., Maki G, Klingemann HG. Characterization of a
human cell line (NK-92) with phenotypical and functional
characteristics of activated natural killer cells. Leukemia 1994;
8:652-658). NK-92 cells express high levels of molecules involved
in the perforin-granzyme cytolytic pathway that targets a wide
range of malignant cells (Gong et al, vide infra, and Maki G,
Klingemann H G, Martinson J A, Tam Y K. Factors regulating the
cytotoxic activity of the human natural killer cell line, NK-92. J
Hematother Stem Cell Res 2001; 10:369-383).
[0176] Granzymes are exogenous serine proteases that are released
by cytoplasmic granules within cytotoxic T cells and natural killer
cells. Granzymes can induce apoptosis within virus-infected cells,
thus destroying them.
[0177] Extracellular release of a mediator of inflammation
(inflammatory mediator) from a granulocyte (or leukocyte), and
extracellular release of more than one mediator of inflammation
(inflammatory mediator) from a granulocyte (or leukocyte) is
sometimes referred to herein as degranulation. In a preferred
embodiment, the release of a mediator of inflammation comprises
release of said mediator from a granule located in the interior of
a granulocyte or leukocyte. The release of inflammatory mediator is
preferably the release of an inflammatory mediator from these
granules.
[0178] Neutrophils and macrophages, upon priming by
pro-inflammatory agents (inflammatory stimulants) such as
TNF.alpha., dramatically increase their synthesis of MARCKS
protein: as much as 90% of the new protein formed by neutrophils in
response to either TNF.alpha. or lipopolysaccharide (LPS) is MARCKS
(Thelen M, Rosen A, Nairn A C, Aderem A. Tumor necrosis factor
alpha modifies agonist-dependent responses in human neutrophils by
inducing the synthesis and myristoylation of a specific protein
kinase C substrate. Proc Natl Acad Sci USA 1990; 87:5603-5607).
MARCKS can thus have an important role in subsequent release of
inflammatory mediators when granule-containing cells, such as
neutrophils and macrophages, are stimulated by agonists, especially
those that work by activating PKC (Burgoyne et al., Physiol Rev
2003; 83:581-632; Logan et al. J Allergy Clin Immunol 2003; 111:
923-932; Smolen et al., Biochim Biophys Acta 1990; 1052:133-142;
Niessen et al., Biochim. Biophys. Acta 1994; 1223:267-273 ; Naucler
et al., J Leukoc Biol 2002; 71:701-710).
[0179] In one aspect of this invention, administration of a
degranulation-inhibiting amount of MANS peptide or an active
fragment thereof as described herein to a site of inflammation in a
subject, which site of inflammation has resulted from the onset of
entry of a disease, a condition, a trauma, a foreign body, or a
combination thereof at the site of inflammation in the subject, can
reduce the amount of a mediator of inflammation released from
infiltrating leukocytes at the site of inflammation, where the
leukocytes are preferably granulocytes. The administration of the
MANS peptide and/or at least one active fragment thereof can reduce
the amount of a mediator of inflammation released from leukocytes
such as granulocytes infiltrating into the site of inflammation.
The degranulation-inhibiting amount of MANS peptide, or the
degranulation-inhibiting amount of an active fragment thereof, is
sufficient to reduce or inhibit the exocytotic release of
inflammatory mediators from granules contained within the
inflammatory cells infiltrating into the site.
Degranulation-inhibiting efficacy is measured at a time after
administration of the MANS peptide or the active fragment thereof
by comparison of the percent of inhibition (i.e., percent of
reduction) of the release of mediators of inflammation from said
cells (leukocytes or granulocytes or other inflammatory cells)
relative to the level or amount or concentration of said mediators
of inflammation released or produced at approximately the same time
in the absence of MANS peptide and/or in the absence of the active
fragment thereof. Additionally, a skilled clinician can determine
whether inflammation at the tissue site has been reduced by
measuring symptoms and parameters of inflammation known as
indicators of the disease to determine whether a sufficient or
therapeutically effective amount MANS peptide and/or an active
fragment thereof has been administered. A sufficient
degranulation-inhibiting amount is the amount which produces a
percentage of reduction of a mediator of inflammation released from
a granulocyte, at the site of inflammation, which percentage is
from about 1% to about 99%, preferably from 5% to about 99%, more
preferably from about 10% to about 99%, even more preferably from
about 25% to 99%, and even more preferably from about 50% to about
99% of the amount of said mediator of inflammation released from
said granulocyte in the absence of MANS peptide or an active
fragment thereof tested under the same conditions.
[0180] In one aspect of this invention, administration of a
degranulation-inhibiting amount of MANS peptide to a site of
inflammatory stimulation in an animal, which site of inflammatory
stimulation has been created by administration of an
inflammation-stimulating amount of an inflammatory stimulant to
said site, can reduce the amount of a mediator of inflammation
released from a granulocyte, which granulocyte is stimulated by
said inflammatory stimulant at said site of inflammatory
stimulation, from about 1% to about 99%, preferably from 5% to
about 99%, more preferably from about 10% to about 99%, even more
preferably from about 25% to 99%, and even more preferably from
about 50% to about 99% of the amount of said mediator of
inflammation released from said granulocyte in the absence of MANS
peptide in the presence of the identical inflammation-stimulating
amount of said inflammatory stimulant.
[0181] In another aspect of this invention, administration of a
degranulation-inhibiting amount of MANS peptide to a site of
inflammatory stimulation in an animal, which site of inflammatory
stimulation has been created by administration of an
inflammation-stimulating amount of an inflammatory stimulant to
said site, can reduce the amount of a mediator of inflammation
released from a granulocyte, which granulocyte is stimulated by
said inflammatory stimulant at said site of inflammatory
stimulation, by 100% of the amount of said mediator of inflammation
released from said granulocyte in the absence of MANS peptide in
the presence of the identical inflammation-stimulating amount of
said inflammatory stimulant.
[0182] An example of an inflammatory stimulant used in in vitro
examples herein is phorbol 12-myristate 13-acetate (PMA). Monocyte
chemoattractant protein (MCP-1) is nearly as effective as C5a, and
much more potent than IL-8, in the degranulation of basophils,
resulting in histamine release. Histamine release can occur after
stimulation with chemokines (i.e., chemoattractant cytokines),
RANTES and MIP-1.
[0183] In a preferred embodiment, relative to the basal
concentration of MARCKS peptide present at the site of inflammatory
stimulation, the degranulation-inhibiting amount of MANS peptide
administered to a site of inflammatory stimulation in an animal
comprises from about 1 time to about 1,000,000 times the
concentration of the MARCKS peptide at said site of inflammatory
stimulation, preferably from about 1 time to about 100,000 times
the concentration of the MARCKS peptide at said site of
inflammatory stimulation, more preferably from about 1 time to
about 10,000 times the concentration of the MARCKS peptide at said
site of inflammatory stimulation, even more preferably from about 1
time to about 1,000 times the concentration of the MARCKS peptide
at said site of inflammatory stimulation, even more preferably from
about 1 time to about 100 times the concentration of the MARCKS
peptide at said site of inflammatory stimulation, and even more
preferably from about 1 time to about 10 times the concentration of
the MARCKS peptide at said site of inflammatory stimulation.
[0184] In a preferred embodiment, the granulocyte resides on or in
the airway of an animal, preferably a human, and the MANS peptide
is administered by inhalation, such as by inhalation of a
pharmaceutical composition comprising the MANS peptide, for example
a pharmaceutical composition comprising the MANS peptide and an
aqueous solution, which composition is administered in the form of
an aerosol, or a pharmaceutical composition comprising the MANS
peptide in the form of a dry powder, which composition is
administered using a dry powder inhaler. Other methods and devices
known in the art for administration of a solution or powder by
inhalation such as, for example, droplets, sprays, and nebulizers,
can be useful.
[0185] In some embodiments, it is possible that the peptide of the
present invention may block secretory processes that are
physiologically important, including basal secretory functions.
Although inventors do not wish to be bound to any particular theory
of the invention, it is thought that the mechanisms regulating such
basal secretion are different than those regulating stimulated
secretion. Alternatively, basal secretory mechanisms may require
less MARCKS protein than stimulated secretion. Basal secretion may
be preserved since all therapies to block MARCKS-mediated secretion
may not eliminate all MARCKS function.
[0186] As used herein, the term "MARCKS nucleotide sequence" refers
to any nucleotide sequence derived from a gene encoding a MARCKS
protein, including, for example, DNA or RNA sequence, DNA sequence
of the gene, any transcribed RNA sequence, RNA sequence of the
pre-mRNA or mRNA transcript, and DNA or RNA bound to protein.
[0187] Precise delivery of the MARCKS-blocking peptide may also
overcome any potential limitations of blocking important secretory
processes. Delivering such agents to the respiratory tract should
be readily accomplished with inhaled formulations. Since these
agents may be useful in treating inflammatory bowel disease, one
can envision delivery of the blocking agents into the
rectum/colon/intestinal tract via enema or suppositories.
Intraarticular injections or transdermal delivery into inflamed
joints may yield relief to patients with arthritic or autoimmune
diseases by limiting the secretion from localized inflammatory
cells. Injection into areas surrounding nerve endings may inhibit
secretion of some types of neurotransmitters, blocking transmission
of severe pain or uncontrolled muscle spasms. Delivery of the
peptide for the treatment of inflammatory skin diseases should be
readily accomplished using various topical formulations known in
the art.
[0188] It is believed that MARCKS interacts with actin and myosin
in the cytoplasm and thus may be able to tether the granules to the
cellular contractile apparatus, thus, mediating subsequent granule
movement and exocytosis. Secretion of the inflammatory mediatory
MPO from neutrophils may also be maximized by activation of both
PKC and PKG. It is possible that MARCKS serves as the point of
convergence for coordinating actions of these two protein kinases
that control secretion from membrane-bound compartments in
inflammatory cells (i.e. secretion of MPO from neutrophils).
[0189] The present invention demonstrates secretion of the
inflammatory mediator MPO from canine or human neutrophils was
enhanced by concurrent activation of both PKC and PKG, while
activation of either kinase alone was insufficient to induce a
maximal secretory response. An enhanced secretory response to PMA
alone has been documented in NHBE cells and in neutrophils as
demonstrated herein, although the magnitude of the response was
much less than that observed by others in a rat goblet-like cell
line. See, Abdullah et al, supra. In addition, although it was
reported previously that a cGMP analogue could induce significant
mucin secretion from cultured guinea pig tracheal epithelial cells
(Fischer et al., supra), it should be noted that this response did
not reach significant levels until 8 h of exposure. A secretory
response with such a long lag period is unlikely to be a direct
effect and probably involves de novo protein synthesis as opposed
to release of preformed and stored cytoplasmic granules.
[0190] As stated above, the present invention may be used in a
pharmaceutical formulation. In certain embodiments, the drug
product is present in a solid pharmaceutical composition that may
be suitable for oral administration. A solid composition of matter
according to the present invention may be formed and may be mixed
with and/or diluted by an excipient. The solid composition of
matter also may be enclosed within a carrier, which may be, for
example, in the form of a capsule, sachet, tablet, paper, or other
container. When the excipient serves as a diluent, it may be a
solid, semi-solid, or liquid material that acts as a vehicle,
carrier, or medium for the composition of matter.
[0191] Various suitable excipients will be understood by those
skilled in the art and may be found in the National Formulary, 19:
2404-2406 (2000), the disclosure of pages 2404 to 2406 being
incorporated herein in their entirety. Examples of suitable
excipients include, but are not limited to, starches, gum arabic,
calcium silicate, microcrystalline cellulose, methacrylates,
shellac, polyvinylpyrrolidone, cellulose, water, syrup, and
methylcellulose. The drug product formulations additionally can
include lubricating agents such as, for example, talc, magnesium
stearate and mineral oil; wetting agents; emulsifying and
suspending agents; preserving agents such as methyl- and propyl
hydroxybenzoates; sweetening agents; or flavoring agents. Polyols,
buffers, and inert fillers also may be used. Examples of polyols
include, but are not limited to, mannitol, sorbitol, xylitol,
sucrose, maltose, glucose, lactose, dextrose, and the like.
Suitable buffers include, but are not limited to, phosphate,
citrate, tartrate, succinate, and the like. Other inert fillers
that may be used include those that are known in the art and are
useful in the manufacture of various dosage forms. If desired, the
solid formulations may include other components such as bulking
agents and/or granulating agents, and the like. The drug products
of the invention may be formulated so as to provide quick,
sustained, or delayed release of the active ingredient after
administration to the patient by employing procedures well known in
the art.
[0192] To form tablets for oral administration, the composition of
matter of the present invention may be made by a direct compression
process. In this process, the active drug ingredients may be mixed
with a solid, pulverant carrier such as, for example, lactose,
saccharose, sorbitol, mannitol, starch, amylopectin, cellulose
derivatives or gelatin, and mixtures thereof, as well as with an
antifriction agent such as, for example, magnesium stearate,
calcium stearate, and polyethylene glycol waxes. The mixture may
then be pressed into tablets using a machine with the appropriate
punches and dies to obtain the desired tablet size. The operating
parameters of the machine may be selected by the skilled artisan.
Alternatively, tablets for oral administration may be formed by a
wet granulation process. Active drug ingredients may be mixed with
excipients and/or diluents. The solid substances may be ground or
sieved to a desired particle size. A binding agent may be added to
the drug. The binding agent may be suspended and homogenized in a
suitable solvent. The active ingredient and auxiliary agents also
may be mixed with the binding agent solution. The resulting dry
mixture is moistened with the solution uniformly. The moistening
typically causes the particles to aggregate slightly, and the
resulting mass is pressed through a stainless steel sieve having a
desired size. The mixture is then dried in controlled drying units
for the determined length of time necessary to achieve a desired
particle size and consistency. The granules of the dried mixture
are sieved to remove any powder. To this mixture, disintegrating,
antifriction, and/or anti-adhesive agents may be added. Finally,
the mixture is pressed into tablets using a machine with the
appropriate punches and dies to obtain the desired tablet size. The
operating parameters of the machine may be selected by the skilled
artisan.
[0193] If coated tablets are desired, the above prepared core may
be coated with a concentrated solution of sugar or cellulosic
polymers, which may contain gum arabic, gelatin, talc, titanium
dioxide, or with a lacquer dissolved in a volatile organic solvent
or a mixture of solvents. To this coating various dyes may be added
in order to distinguish among tablets with different active
compounds or with different amounts of the active compound present.
In a particular embodiment, the active ingredient may be present in
a core surrounded by one or more layers including enteric coating
layers.
[0194] Soft gelatin capsules may be prepared in which capsules
contain a mixture of the active ingredient and vegetable oil. Hard
gelatin capsules may contain granules of the active ingredient in
combination with a solid, pulverulent carrier, such as, for
example, lactose, saccharose, sorbitol, mannitol, potato starch,
corn starch, amylopectin, cellulose derivatives, and/or
gelatin.
[0195] Liquid preparations for oral administration may be prepared
in the form of syrups or suspensions, e.g., solutions containing an
active ingredient, sugar, and a mixture of ethanol, water,
glycerol, and propylene glycol. If desired, such liquid
preparations may comprise one or more of following: coloring
agents, flavoring agents, and saccharin. Thickening agents such as
carboxymethylcellulose also may be used.
[0196] In the event that the above pharmaceuticals are to be used
for parenteral administration, such a formulation may comprise
sterile aqueous injection solutions, non-aqueous injection
solutions, or both, comprising the composition of matter of the
present invention. When aqueous injection solutions are prepared,
the composition of matter may be present as a water soluble
pharmaceutically acceptable salt. Parenteral preparations may
contain anti-oxidants, buffers, bacteriostats, and solutes which
render the formulation isotonic with the blood of the intended
recipient. Aqueous and non-aqueous sterile suspensions may comprise
suspending agents and thickening agents. The formulations may be
presented in unit-dose or multi-dose containers, for example sealed
ampules and vials. Extemporaneous injection solutions and
suspensions may be prepared from sterile powders, granules and
tablets of the kind previously described.
[0197] The composition of matter also may be formulated such that
it may be suitable for topical administration (e.g., skin cream).
These formulations may contain various excipients known to those
skilled in the art. Suitable excipients may include, but are not
limited to, cetyl esters wax, cetyl alcohol, white wax, glyceryl
monostearate, propylene glycol, monostearate, methyl stearate,
benzyl alcohol, sodium lauryl sulfate, glycerin, mineral oil,
water, carbomer, ethyl alcohol, acrylate adhesives, polyisobutylene
adhesives, and silicone adhesives.
[0198] In a preferred embodiment, peptide fragments are disclosed
in Table 2 and are of a length of at least 4 to 23 amino acid
residues in length and having amino acid sequences identical to an
amino acid sequence of the MANS peptide, wherein the N-terminal
amino acid of the peptides are selected from position 2 to 21 of
the MANS peptide sequence (SEQ ID NO: 1). The more preferred
peptide fragment length is from at least 6 amino acids to 23 amino
acids. Preferably these peptides are acylated at the alpha
N-terminal amino acid, and more preferably these peptides are
myristoylated at the alpha-N-terminal amino acid position.
TABLE-US-00002 TABLE 2 Sequence Peptide No. Sequence ID NO. peptide
3 AQFSKTAAKGEAAAERPGEAAVA SEQ ID NO. 3 peptide 5
AQFSKTAAKGEAAAERPGEAAV SEQ ID NO. 5 peptide 8 AQFSKTAAKGEAAAERPGEAA
SEQ ID NO. 8 peptide 12 AQFSKTAAKGEAAAERPGEA SEQ ID NO. 12 peptide
17 AQFSKTAAKGEAAAERPGE SEQ ID NO. 17 peptide 23 AQFSKTAAKGEAAAERPG
SEQ ID NO. 23 peptide 30 AQFSKTAAKGEAAAERP SEQ ID NO. 30 peptide 38
AQFSKTAAKGEAAAER SEQ ID NO. 38 peptide 47 AQFSKTAAKGEAAAE SEQ ID
NO. 47 peptide 57 AQFSKTAAKGEAAA SEQ ID NO. 57 peptide 68
AQFSKTAAKGEAA SEQ ID NO. 68 peptide 80 AQFSKTAAKGEA SEQ ID NO. 80
peptide 93 AQFSKTAAKGE SEQ ID NO. 93 peptide 107 AQFSKTAAKG SEQ ID
NO. 107 peptide 122 AQFSKTAAK SEQ ID NO. 122 peptide 138 AQFSKTAA
SEQ ID NO. 138 peptide 155 AQFSKTA SEQ ID NO. 155 peptide 173
AQFSKT SEQ ID NO. 173 peptide 192 AQFSK SEQ ID NO. 192 peptide 212
AQFS SEQ ID NO. 212 peptide 6 QFSKTAAKGEAAAERPGEAAVA SEQ ID NO. 6
peptide 9 QFSKTAAKGEAAAERPGEAAV SEQ ID NO. 9 peptide 13
QFSKTAAKGEAAAERPGEAA SEQ ID NO. 13 peptide 18 QFSKTAAKGEAAAERPGEA
SEQ ID NO. 18 peptide 24 QFSKTAAKGEAAAERPGE SEQ ID NO. 24 peptide
31 QFSKTAAKGEAAAERPG SEQ ID NO. 31 peptide 39 QFSKTAAKGEAAAERP SEQ
ID NO. 39 peptide 48 QFSKTAAKGEAAAER SEQ ID NO. 48 peptide 58
QFSKTAAKGEAAAE SEQ ID NO. 58 peptide 69 QFSKTAAKGEAAA SEQ ID NO. 69
peptide 81 QFSKTAAKGEAA SEQ ID NO. 81 peptide 94 QFSKTAAKGEA SEQ ID
NO. 94 peptide 108 QFSKTAAKGE SEQ ID NO. 108 peptide 123 QFSKTAAKG
SEQ ID NO. 123 peptide 139 QFSKTAAK SEQ ID NO. 139 peptide 156
QFSKTAA SEQ ID NO. 156 peptide 174 QFSKTA SEQ ID NO. 174 peptide
193 QFSKT SEQ ID NO. 193 peptide 213 QFSK SEQ ID NO. 213 peptide 10
FSKTAAKGEAAAERPGEAAVA SEQ ID NO. 10 peptide 14 FSKTAAKGEAAAERPGEAAV
SEQ ID NO. 14 peptide 19 FSKTAAKGEAAAERPGEAA SEQ ID NO. 19 peptide
25 FSKTAAKGEAAAERPGEA SEQ ID NO. 25 peptide 32 FSKTAAKGEAAAERPGE
SEQ ID NO. 32 peptide 40 FSKTAAKGEAAAERPG SEQ ID NO. 40 peptide 49
FSKTAAKGEAAAERP SEQ ID NO. 49 peptide 59 FSKTAAKGEAAAER SEQ ID NO.
59 peptide 70 FSKTAAKGEAAAE SEQ ID NO. 70 peptide 82 FSKTAAKGEAAA
SEQ ID NO. 82 peptide 95 FSKTAAKGEAA SEQ ID NO. 95 peptide 109
FSKTAAKGEA SEQ ID NO. 109 peptide 124 FSKTAAKGE SEQ ID NO. 124
peptide 140 FSKTAAKG SEQ ID NO. 140 peptide 157 FSKTAAK SEQ ID NO.
157 peptide 175 FSKTAA SEQ ID NO. 175 peptide 194 FSKTA SEQ ID NO.
194 peptide 214 FSKT SEQ ID NO. 214 peptide 15 SKTAAKGEAAAERPGEAAVA
SEQ ID NO. 15 peptide 20 SKTAAKGEAAAERPGEAAV SEQ ID NO. 20 peptide
26 SKTAAKGEAAAERPGEAA SEQ ID NO. 26 peptide 33 SKTAAKGEAAAERPGEA
SEQ ID NO. 33 peptide 41 SKTAAKGEAAAERPGE SEQ ID NO. 41 peptide 50
SKTAAKGEAAAERPG SEQ ID NO. 50 peptide 60 SKTAAKGEAAAERP SEQ ID NO.
60 peptide 71 SKTAAKGEAAAER SEQ ID NO. 71 peptide 83 SKTAAKGEAAAE
SEQ ID NO. 83 peptide 96 SKTAAKGEAAA SEQ ID NO. 96 peptide 110
SKTAAKGEAA SEQ ID NO. 110 peptide 125 SKTAAKGEA SEQ ID NO. 125
peptide 141 SKTAAKGE SEQ ID NO. 141 peptide 158 SKTAAKG SEQ ID NO.
158 peptide 176 SKTAAK SEQ ID NO. 176 peptide 195 SKTAA SEQ ID NO.
195 peptide 215 SKTA SEQ ID NO. 215 peptide 21 KTAAKGEAAAERPGEAAVA
SEQ ID NO. 21 peptide 27 KTAAKGEAAAERPGEAAV SEQ ID NO. 27 peptide
34 KTAAKGEAAAERPGEAA SEQ ID NO. 34 peptide 42 KTAAKGEAAAERPGEA SEQ
ID NO. 42 peptide 51 KTAAKGEAAAERPGE SEQ ID NO. 51 peptide 61
KTAAKGEAAAERPG SEQ ID NO. 61 peptide 72 KTAAKGEAAAERP SEQ ID NO. 72
peptide 84 KTAAKGEAAAER SEQ ID NO. 84 peptide 97 KTAAKGEAAAE SEQ ID
NO. 97 peptide 111 KTAAKGEAAA SEQ ID NO. 111 peptide 126 KTAAKGEAA
SEQ ID NO. 126 peptide 142 KTAAKGEA SEQ ID NO. 142 peptide 159
KTAAKGE SEQ ID NO. 159 peptide 177 KTAAKG SEQ ID NO. 177 peptide
196 KTAAK SEQ ID NO. 196 peptide 216 KTAA SEQ ID NO. 216 peptide 28
TAAKGEAAAERPGEAAVA SEQ ID NO. 28 peptide 35 TAAKGEAAAERPGEAAV SEQ
ID NO. 35 peptide 43 TAAKGEAAAERPGEAA SEQ ID NO. 43 peptide 52
TAAKGEAAAERPGEA SEQ ID NO. 52 peptide 62 TAAKGEAAAERPGE SEQ ID NO.
62 peptide 73 TAAKGEAAAERPG SEQ ID NO. 73 peptide 85 TAAKGEAAAERP
SEQ ID NO. 85 peptide 98 TAAKGEAAAER SEQ ID NO. 98 peptide 112
TAAKGEAAAE SEQ ID NO. 112 peptide 127 TAAKGEAAA SEQ ID NO. 127
peptide 143 TAAKGEAA SEQ ID NO. 143 peptide 160 TAAKGEA SEQ ID NO.
160 peptide 178 TAAKGE SEQ ID NO. 178 peptide 197 TAAKG SEQ ID NO.
197 peptide 217 TAAK SEQ ID NO. 217 peptide 36 AAKGEAAAERPGEAAVA
SEQ ID NO. 36 peptide 44 AAKGEAAAERPGEAAV SEQ ID NO. 44 peptide 53
AAKGEAAAERPGEAA SEQ ID NO. 53 peptide 63 AAKGEAAAERPGEA SEQ ID NO.
63 peptide 74 AAKGEAAAERPGE SEQ ID NO. 74 peptide 86 AAKGEAAAERPG
SEQ ID NO. 86 peptide 99 AAKGEAAAERP SEQ ID NO. 99 peptide 113
AAKGEAAAER SEQ ID NO. 113 peptide 128 AAKGEAAAE SEQ ID NO. 128
peptide 144 AAKGEAAA SEQ ID NO. 144 peptide 161 AAKGEAA SEQ ID NO.
161 peptide 179 AAKGEA SEQ ID NO. 179 peptide 198 AAKGE SEQ ID NO.
198 peptide 218 AAKG SEQ ID NO. 218 peptide 45 AKGEAAAERPGEAAVA SEQ
ID NO. 45 peptide 54 AKGEAAAERPGEAAV SEQ ID NO. 54 peptide 64
AKGEAAAERPGEAA SEQ ID NO. 64
peptide 75 AKGEAAAERPGEA SEQ ID NO. 75 peptide 87 AKGEAAAERPGE SEQ
ID NO. 87 peptide 100 AKGEAAAERPG SEQ ID NO. 100 peptide 114
AKGEAAAERP SEQ ID NO. 114 peptide 129 AKGEAAAER SEQ ID NO. 129
peptide 145 AKGEAAAE SEQ ID NO. 145 peptide 162 AKGEAAA SEQ ID NO.
162 peptide 180 AKGEAA SEQ ID NO. 180 peptide 199 AKGEA SEQ ID NO.
199 peptide 219 AKGE SEQ ID NO. 219 peptide 55 KGEAAAERPGEAAVA SEQ
ID NO. 55 peptide 65 KGEAAAERPGEAAV SEQ ID NO. 65 peptide 76
KGEAAAERPGEAA SEQ ID NO. 76 peptide 88 KGEAAAERPGEA SEQ ID NO. 88
peptide 101 KGEAAAERPGE SEQ ID NO. 101 peptide 115 KGEAAAERPG SEQ
ID NO. 115 peptide 130 KGEAAAERP SEQ ID NO. 130 peptide 146
KGEAAAER SEQ ID NO. 146 peptide 163 KGEAAAE SEQ ID NO. 163 peptide
181 KGEAAA SEQ ID NO. 181 peptide 200 KGEAA SEQ ID NO. 200 peptide
220 KGEA SEQ ID NO. 220 peptide 66 GEAAAERPGEAAVA SEQ ID NO. 66
peptide 77 GEAAAERPGEAAV SEQ ID NO. 77 peptide 89 GEAAAERPGEAA SEQ
ID NO. 89 peptide 102 GEAAAERPGEA SEQ ID NO. 102 peptide 116
GEAAAERPGE SEQ ID NO. 116 peptide 131 GEAAAERPG SEQ ID NO. 131
peptide 147 GEAAAERP SEQ ID NO. 147 peptide 164 GEAAAER SEQ ID NO.
164 peptide 182 GEAAAE SEQ ID NO. 182 peptide 201 GEAAA SEQ ID NO.
201 peptide 221 GEAA SEQ ID NO. 221 peptide 78 EAAAERPGEAAVA SEQ ID
NO. 78 peptide 90 EAAAERPGEAAV SEQ ID NO. 90 peptide 103
EAAAERPGEAA SEQ ID NO. 103 peptide 117 EAAAERPGEA SEQ ID NO. 117
peptide 132 EAAAERPGE SEQ ID NO. 132 peptide 148 EAAAERPG SEQ ID
NO. 148 peptide 165 EAAAERP SEQ ID NO. 165 peptide 183 EAAAER SEQ
ID NO. 183 peptide 202 EAAAE SEQ ID NO. 202 peptide 222 EAAA SEQ ID
NO. 222 peptide 91 AAAERPGEAAVA SEQ ID NO. 91 peptide 104
AAAERPGEAAV SEQ ID NO. 104 peptide 118 AAAERPGEAA SEQ ID NO. 118
peptide 133 AAAERPGEA SEQ ID NO. 133 peptide 149 AAAERPGE SEQ ID
NO. 149 peptide 166 AAAERPG SEQ ID NO. 166 peptide 184 AAAERP SEQ
ID NO. 184 peptide 203 AAAER SEQ ID NO. 203 peptide 223 AAE SEQ ID
NO. 223 peptide 105 AAERPGEAAVA SEQ ID NO. 105 peptide 119
AAERPGEAAV SEQ ID NO. 119 peptide 134 AAERPGEAA SEQ ID NO. 134
peptide 150 AAERPGEA SEQ ID NO. 150 peptide 167 AAERPGE SEQ ID NO.
167 peptide 185 AAERPG SEQ ID NO. 185 peptide 204 AERP SEQ ID NO.
204 peptide 224 AER SEQ ID NO. 224 peptide 120 AERPGEAAVA SEQ ID
NO. 120 peptide 135 AERPGEAAV SEQ ID NO. 135 peptide 151 AERPGEAA
SEQ ID NO. 151 peptide 168 AERPGEA SEQ ID NO. 168 peptide 186
AERPGE SEQ ID NO. 186 peptide 205 AERPG SEQ ID NO. 205 peptide 225
AERP SEQ ID NO. 225 peptide 136 ERPGEAAVA SEQ ID NO. 136 peptide
152 ERPGEAAV SEQ ID NO. 152 peptide 169 ERPGEAA SEQ ID NO. 169
peptide 187 ERPGEA SEQ ID NO. 187 peptide 206 ERPGE SEQ ID NO. 206
peptide 226 ERPG SEQ ID NO. 226 peptide 153 RPGEAAVA SEQ ID NO. 153
peptide 170 RPGEAAV SEQ ID NO. 170 peptide 188 RPGEAA SEQ ID NO.
188 peptide 207 RPGEA SEQ ID NO. 207 peptide 227 RPGE SEQ ID NO.
227 peptide 171 PGEAAVA SEQ ID NO. 171 peptide 189 PGEAAV SEQ ID
NO. 189 peptide 208 PGEAA SEQ ID NO. 208 peptide 228 PGEA SEQ ID
NO. 228 peptide 190 GEAAVA SEQ ID NO. 190 peptide 209 GEAAV SEQ ID
NO. 209 peptide 229 GEAA SEQ ID NO. 229 peptide 210 EAAVA SEQ ID
NO. 210 peptide 230 EAAV SEQ ID NO. 230 peptide 231 AAVA SEQ ID NO.
231
[0199] As illustrated in FIG. 5, MARCKS was phosphorylated by PKC
and consequently translocated from the membrane to the cytoplasm.
Here, PKG appeared to induce dephosphorylation of MARCKS (FIG. 2A,
lane 4, and FIG. 2B). This dephosphorylation was reversed by the
PKG inhibitor R.sub.p-8-Br-PET-cGMP (FIG. 2A, lane 5), indicating
the dephosphorylation was specifically PKG-dependent. In FIG. 2,
the NHBE cells were labeled with [.sup.32P]orthophosphate and then
exposed to the indicated reagents. MARCKS phosphorylation in
response to the treatments was evaluated by immunoprecipitation
assay. In FIG. 2A, 8-Br-cGMP reversed MARCKS phosphorylation
induced by PMA, and this effect of 8-Br-cGMP could be blocked by
R.sub.p-8-Br-PET-cGMP (PKG inhibitor) or okadaic acid (PP1/2A
inhibitor). For FIG. 2B, PMA-induced phosphorylation of MARCKS was
reversed by subsequent exposure of cells to 8-Br-cGMP. Lane 1,
medium alone for 8 min; lane 2, 100 nM PMA for 3 min; lane 3, 100
nM PMA for 3 min and then with 1.mu.M 8-Br-cGMP for 5 min; lane 4,
100 nM PMA for 8 min; lane 5, medium alone for 3 min and then 100
nM PMA+1 .mu.M 8-Br-cGMP for 5 min. In FIG. 2C, 8-Br-cGMP-induced
MARCKS dephosphorylation was attenuated by fostriecin in a
concentration-dependent manner.
[0200] It is believed that PKG acts to dephosphorylate MARCKS via
activation of a protein phosphatase. As illustrated in FIG. 2A
(lane 6), okadaic acid at 500 nM, a concentration that could
inhibit both PP1 and PP2A, blocked PKG-induced dephosphorylation of
MARCKS, suggesting that PKG caused dephosphorylation by activating
PP1 and/or PP2A. Further studies with fostriecin and direct assay
of phosphatase activities indicated that only PP2A was activated by
PKG and was responsible for removal of the phosphate groups from
MARCKS (FIG. 2C). It is likely that either okadaic acid or
fostriecin, at concentrations that inhibited PKG-induced
dephosphorylation of MARCKS, attenuated mucin secretion induced by
PMA+8-Br-cGMP or UTP as exhibited in FIG. 3. FIG. 3 helps to
demonstrate that PP2A is an essential component of the mucin
secretory pathway. NHBE cells were preincubated with the indicated
concentration of fostriecin, okadaic acid (500 nM), or medium alone
for 15 min and then stimulated with PMA (100 nM)+8-Br-cGMP (1
.mu.M) for 15 min or with UTP (100 .mu.M) for 2 h. Secreted mucin
was measured by ELISA. Data are presented as mean.+-.S.E. (n=6 at
each point) wherein * stands for significantly different from
medium control (p<0.05); .dagger. stands for significantly
different from PMA+8-Br-cGMP stimulation (p<0.05); and
.dagger-dbl. stands for significantly different from UTP
stimulation p<0.05). Thus, dephosphorylation of MARCKS by a
PKG-activated PP2A appears to be an essential component of the
signaling pathway leading to mucin granule exocytosis.
[0201] To reveal molecular events by which MARCKS links kinase
activation to mucin secretion, phosphorylation of MARCKS in
response to PKC/PKG activation was investigated in depth. As
illustrated in FIG. 1A, PMA (100 nM) likely induced a significant
increase (3-4-fold) in MARCKS phosphorylation in NHBE cells, and
this phosphorylation was attenuated by the PKC inhibitor calphostin
C (500 nM). Once phosphorylated, MARCKS was translocated from the
plasma membrane to the cytoplasm (FIG. 1B). More specifically, FIG.
1A shows the activation of PKC results in MARCKS phosphorylation in
NHBE cells. Cells were labeled with [.sup.32P]orthophosphate for 2
h and then exposed to the stimulatory and/or inhibitory reagents.
MARCKS phosphorylation in response to the treatments was evaluated
by immunoprecipitation as described. Lane 1, medium control; lane 2
the vehicle, 0.1% Me.sub.2SO; lane 3, 100 nM 4a-PMA; lane 4, 100 nM
PMA; lane 5, 100 nM PMA+500 nM calphostin C; lane 6, 500 nM
calphostin C. FIG. 1B demonstrates phosphorylated MARCKS is
translocated from the plasma membrane to the cytoplasm.
.sup.32P-Labeled cells were exposed to PMA (100 nM) or medium alone
for 5 min, and then the membrane and the cytosol fractions were
isolated. Activation of PKG by 8-Br-cGMP (1 .mu.M, another kinase
activation event necessary for provoking mucin secretion, did not
lead to MARCKS phosphorylation, but, in fact, the opposite effect
was observed: MARCKS phosphorylation induced by PMA was reversed by
8-Br-cGMP (FIG. 2A). This effect of 8-Br-cGMP was not due to
suppression of PKC activity, as the PMA-induced phosphorylation
could be reversed by subsequent addition of 8-Br-cGMP to the cells
(FIG. 2B). Therefore, PKG activation likely results in
dephosphorylation of MARCKS.
[0202] Further investigation demonstrated that PKG-induced MARCKS
dephosphorylation was blocked by 500 nM okadaic acid, a protein
phosphatase (type 1 and/or 2A (PP1/2A)) inhibitor (FIG. 2A, lane
6). Thus, it appeared that the dephosphorylation was mediated by
PP1 and/or PP2A. To define the subtype of protein phosphatase
involved, a novel and more specific inhibitor of PP2A, fostriecin
(IC.sub.50=3 .2 nM), was utilized in additional phosphorylation
studies. As illustrated in FIG. 2C, fostriecin inhibited
PKG-induced MARCKS dephosphorylation in a concentration-dependent
manner (1-500 nM), suggesting that PKG induced the
dephosphorylation via activation of PP2A. To confirm further
activation of PP2A by PKG in NHBE cells, cytosolic PP1 and PP2A
activities were determined after exposure of the cells to
8-Br-cGMP. PP2A activity was increased approximately 3-fold (from
0.1 to 0.3 nmol/min/mg proteins, p<0.01) at concentrations of
8-Br-cGMP as low as 0.1 .mu.M, whereas PP1 activity remained
unchanged. This data indicates that PP2A may be activated by PKG
and is responsible for the dephosphorylation of MARCKS.
Accordingly, this PP2A activity appeared critical for mucin
secretion to occur; when PKG-induced MARCKS dephosphorylation was
blocked by okadaic acid or fostriecin, the secretory response to
PKC/PKG activation or UTP stimulation was ameliorated (FIG. 3).
MARCKS Associates with Actin and Myosin in the Cytoplasm
[0203] FIG. 4 depicts a radiolabeled immunoprecipitation assay
which reveals that MARCKS may associate with two other proteins
(about 200 and about 40 kDa) in the cytoplasm. In FIG. 4 NHBE cells
were labeled with [.sup.3H]leucine and [.sup.3H]proline overnight,
and the membrane and the cytosol fractions were prepared as
described under "Experimental Procedures." Isolated fractions were
precleared with the nonimmune control antibody (6F6). The cytosol
was then divided equally into two fractions and used for
immunoprecipitation carried out in the presence of 10 .mu.M
cytochalasin D (Biomol, Plymouth Meeting, Pa.) with the anti-MARCKS
antibody 2F12 (lane 2) and the nonimmune control antibody 6F6 (lane
3), respectively. MARCKS protein in the membrane fraction was also
assessed by immunoprecipitation using the antibody 2F12 (lane 1).
The precipitated protein complex was resolved by 8%
SDS-polyacrylamide gel electrophoresis and visualized by enhanced
autoradiography. MARCKS appeared to associate with two cytoplasmic
proteins with molecular masses of about 200 and about 40 kDa,
respectively. These two MARCKS-associated proteins were excised
from the gel and analyzed by matrix-assisted laser desorption
ionization/time of flight mass spectrometry/internal sequencing
(the Protein/DNA Technology Center of Rockefeller University,
N.Y.). The obtained peptide mass and sequence data were used to
search protein databases via Internet programs ProFound and MS-Fit.
Results indicate that they are myosin (heavy chain, non-muscle type
A) and actin, respectively. Matrix-assisted laser desorption
ionization/time of flight mass spectrometry/internal sequence
analysis indicates that these two MARCKS-associated proteins were
myosin (heavy chain, non-muscle type A) and actin,
respectively.
[0204] These studies suggest a new paradigm for the signaling
mechanism controlling exocytotic secretion of airway mucin granules
as well as providing what is believed to be the first direct
evidence demonstrating a specific biological function of MARCKS in
a physiological process. MARCKS serves as a key mediator molecule
regulating mucin granule release in human airway epithelial cells.
It is believed that elicitation of airway mucin secretion requires
dual activation and synergistic actions of PKC and PKG. Activated
PKC phosphorylates MARCKS, resulting in translocation of MARCKS
from the inner face of the plasma membrane into the cytoplasm.
Activation of PKG in turn activates PP2A, which dephosphorylates
MARCKS in the cytoplasm. Because the membrane association ability
of MARCKS is dependent on its phosphorylation state this
dephosphorylation may allow MARCKS to regain its membrane-binding
capability and may enable MARCKS to attach to membranes of
cytoplasmic mucin granules. By also interacting with actin and
myosin in the cytoplasm (FIG. 4), MARCKS may then be able to tether
granules to the cellular contractile apparatus, mediating granule
movement to the cell periphery and subsequent exocytotic release.
The wide distribution of MARCKS suggests the possibility that this
or a similar mechanism may regulate secretion of membrane-bound
granules in various cell types under normal or pathological
conditions.
[0205] As indicated in FIG. 5, MARCKS may function as a molecular
linker by interacting with granule membranes at its N-terminal
domain and binding to actin filaments at its PSD site, thereby
tethering granules to the contractile cytoskeleton for movement and
exocytosis. FIG. 5 shows a possible mechanism depicting that mucin
secretagogue interacts with airway epithelial (goblet) cells and
activates two separate protein kinases, PKC and PKG. Activated PKC
phosphorylates MARCKS, causing MARCKS translocation from the plasma
membrane to the cytoplasm, whereas PKG, activated via the nitric
oxide (NO).fwdarw.GC-S.fwdarw.cGMP.fwdarw.PKG pathway, in turn
activates a cytoplasmic PP2A, which dephosphorylates MARCKS. This
dephosphorylation stabilizes MARCKS attachment to the granule
membranes. In addition, MARCKS also interacts with actin and
myosin, thereby linking granules to the cellular contractile
machinery for subsequent movement and exocytotic release of
inflammatory mediators, such as MPO. The attachment of MARCKS to
the granules after it is released into the cytoplasm may also be
guided by specific targeting proteins or some other forms of
protein-protein interactions in which the N-terminal domain of
MARCKS is involved. In either case, the MANS peptide, or an active
fragment thereof, comprising at least 4 amino acids, would act to
inhibit competitively targeting of MARCKS to the membranes of mucin
granules, thereby blocking secretion.
[0206] The invention also relates to a new method for blocking any
cellular exocytotic secretory process, especially those releasing
inflammatory mediators from granules contained within inflammatory
cells, whose stimulatory pathways involve the protein kinase C
(PKC) substrate MARCKS protein and release of contents from
membrane-bound vesicles. Specifically, the inventors have shown
that stimulated release of the inflammatory mediator myloperoxidase
from human (FIG. 6) or canine (FIG. 7) neutrophils can be blocked
in a concentration-dependent manner by the MANS peptide.
Specifically, FIG. 6 shows isolated neutrophils that were
stimulated to secrete myloperoxidase (MPO) with 100 nM PMA and 10
.mu.M 8-Br-cGMP. 100 MANS peptide decreased secretion of MPO to
control levels (*=p<0.05). 10 .mu.M MANS causes a slight
decrease in MPO secretion. 10 or 100 .mu.M of a control peptide
(RNS) has no effect on MPO secretion. In FIG. 7, isolated
neutrophils were stimulated to secrete myloperoxidase (MPO) with
100 nM PMA and 10 .mu.M 8-Br-cGMP. 100 .mu.M MANS peptide decreased
secretion of MPO to control levels (*=p<0.05). 10 .mu.M MANS
causes a slight decrease in MPO secretion. 10 or 100 .mu.M of a
control peptide (RNS) has no effect on MPO secretion. Thus, the
peptide may be used therapeutically to block the release of
mediators of inflammation secreted from infiltrating inflammatory
cells in any tissues. Many of these released mediators are
responsible for the extensive tissue damage observed in a variety
of chronic inflammatory diseases (i.e., respiratory diseases such
as asthma, chronic bronchitis and COPD, inflammatory bowel diseases
including ulcerative colitis and Crohn's disease, autoimmune
diseases, skin diseases such as rosacea, eczema; and severe acne,
arthritic and pain syndromes such as rheumatoid arthritis and
fibromyalgia). This invention may be useful for treating diseases
such as arthritis, chronic bronchitis, COPD and cystic fibrosis.
This invention is accordingly useful for the treatment in both
human and animal diseases, especially those affecting equines,
canines, felines, and other household pets.
[0207] FIGS. 8-12 show MPO secretion for both humans and canines.
In all of these experiments, isolated neutrophils were stimulated
with LPS at a concentration of 1.times.10.sup.-6 M for 10 minutes
at 37.degree. C. prior to adding the stimuli as indicated in the
figures. The LPS primes the cells so they can respond to a
secretagogue.
[0208] In one embodiment, this invention discloses a method of
regulating an inflammation in a subject comprising administering a
therapeutically effective amount of a pharmaceutical composition
comprising a MANS peptide or an active fragment thereof. In one
aspect of this embodiment, said active fragment of the MANS protein
comprises at least four and preferably six amino acids. In another
aspect, said inflammation is caused by respiratory diseases, bowel
diseases, skin diseases, autoimmune diseases and pain syndromes. In
another aspect, said respiratory diseases are selected from the
group consisting of asthma, chronic bronchitis, and COPD. In
another aspect, said bowel diseases are selected from the group
consisting of ulcerative colitis, Crohn's disease and irritable
bowel syndrome. In another aspect, said skin diseases are selected
from the group consisting of rosacea, eczema, psoriasis and severe
acne. In another aspect, said inflammation is caused by arthritis
or cystic fibrosis. In another aspect, said subject is a mammal.
Additionally, in another aspect, said mammal is selected from the
group consisting of humans, canines, equines and felines. In
another aspect, said administering step is selected from the group
consisting of topical administration, parenteral administration,
rectal administration, pulmonary administration, nasal
administration, inhalation and oral administration. In another
aspect, said pulmonary administration is selected from the group of
aerosol, dry powder inhaler, metered dose inhaler, and
nebulizer.
[0209] In another embodiment, this invention discloses a method for
regulating a cellular secretory process in a subject comprising
administering a therapeutically effective amount of a
pharmaceutical composition comprising at least one compound
comprising a MANS peptide or an active fragment thereof, that
regulates an inflammatory mediator in a subject. In one aspect of
this embodiment, said active fragment of the MANS protein comprises
at least four, and preferably six amino acids. In another aspect,
said regulating a cellular secretory process is blocking or
reducing a cellular secretory process. In another aspect, said
inflammatory mediator is caused by respiratory diseases, bowel
diseases, skin diseases, autoimmune diseases and pain syndromes. In
another aspect, said respiratory diseases are selected from the
group consisting of asthma, chronic bronchitis, and COPD. In
another aspect, said bowel diseases are selected from the group
consisting of ulcerative colitis, Crohn's disease and irritable
bowel syndrome. In another aspect, said skin diseases are selected
from the group consisting of rosacea, eczema, psoriasis and severe
acne. In another aspect, said inflammatory mediator is caused by
arthritis or cystic fibrosis. In another aspect, said subject is a
mammal. In another aspect, said mammal is selected from the group
consisting of humans, canines, equines and felines. In another
aspect, said administering step is selected from the group
consisting of topical administration, parenteral administration,
rectal administration, pulmonary administration, nasal
administration, inhalation and oral administration. In another
aspect, said pulmonary administration is selected from the group of
aerosol, dry powder inhaler, metered dose inhaler, and
nebulizer.
[0210] In another embodiment, this invention discloses a method of
reducing inflammation in a subject comprising administering a
therapeutically effective amount of a compound that inhibits the
MARCKS-related release of inflammatory mediators, whereby the
release of inflammatory mediators in the subject is reduced
compared to that which would occur in the absence of said
treatment. In one aspect of this embodiment, said compound is at
least one active fragment of a MARCKS protein. In another aspect,
said active fragment is at least four and preferably six amino
acids in length. In another aspect, said compound is a MANS peptide
or an active fragment thereof. In another aspect, said compound is
an antisense oligonucleotide directed against the coding sequence
of a MARCKS protein or an active fragment thereof. In another
aspect, said active fragment is at least four and preferably six
amino acids in length.
[0211] In another embodiment, this invention discloses a method of
reducing inflammation in a subject comprising administering a
therapeutically effective amount of a pharmaceutically active
composition comprising a compound that inhibits the MARCKS-related
release of inflammatory mediators, whereby the inflammation in the
subject is reduced compared to that which would occur in the
absence of said treatment. In one aspect of this embodiment, said
compound is an active fragment of a MARCKS protein. In another
aspect, said active fragment is at least four and preferably six
amino acids in length. In another aspect, said compound is a MANS
peptide or an active fragment thereof. In another aspect, said
compound is an antisense oligonucleotide directed against the
coding sequence of a MARCKS protein or an active fragment thereof.
In another aspect, said active fragment is at least four and
preferably six amino acids in length. The present invention is
intended to encompass a composition that contains one or more of
the MANS peptide or its active fragments and use thereof in the
treatment of inhibiting the release of inflammatory mediators from
granules or vesicles of inflammatory cells.
[0212] In another embodiment, this invention discloses a method of
reducing or inhibiting inflammation in a subject comprising
administering a therapeutically effective amount of at least one
peptide comprising MANS peptide or an active fragment thereof
effective to inhibit or suppress release of an inflammatory
mediator at the inflammation site. In one aspect of this
embodiment, said active fragment is at least four and preferably at
least six amino acids in length. In another aspect, said
inflammatory mediators are produced by cells selected from the
group consisting of neutrophils, basophils, eosinophils, monocytes
and leukocytes. Preferably the cells are leukocytes, more
preferably granulocytes, and even more preferably neutrophils,
basophils, eosinophils or a combination thereof. In another aspect,
the agent is administered orally, parenterally, cavitarily,
rectally or through an air passage. In another aspect, said
composition further comprises a second molecule selected from the
group consisting of an antibiotic, an antiviral compound, an
antiparasitic compound, an anti-inflammatory compound, and an
immunosuppressant.
[0213] An active fragment of a MANS peptide can be selected from
the group consisting of the peptides of disclosed in Table 1. As
disclosed herein, these peptides may be contain optional chemical
moieties at the N-terminal and/or C-terminal amino acid.
[0214] In another aspect of this invention, the methods disclosed
in this invention can be accomplished by use of or administering of
combinations of the peptides disclosed in the invention in Table 1,
i.e., by use of or administering of one or more of these peptides.
Preferably a single peptide is used or administered in the methods
disclosed herein.
[0215] In response to protein kinase C (PKC) activation by an
inflammatory stimulant, degranulation in a cell selected from the
group consisting of neutrophils, eosinophils, monocytes/macrophages
and lymphocytes can be attenuated by pre-incubation and by
co-incubation with a peptide identical to the N-terminal region of
MARCKS protein, wherein the peptide is selected from the group of
MANS peptide fragments as disclosed in Table 1. Although time
courses and concentrations can vary among cell types, in all cases
the MANS peptide attenuates PKC-induced degranulation.
[0216] Having now described the invention, the same will be
illustrated with reference to certain examples, which are included
herein for illustration purposes only, and which are not intended
to be limiting of the invention.
EXAMPLES
Methods and Materials
[0217] Radiolabeled Immunoprecipitation Assay--When labeling with
[.sup.32P]phosphate, cells were preincubated for 2 h in
phosphate-free Dulbecco's modified Eagle's medium containing 0.2%
bovine serum albumin and then labeled with 0.1 mCi/ml
[.sup.32P]orthophosphate (9000 Ci/mmol, PerkinElmer Life Sciences)
for 2 h. For labeling with [.sup.3H]myristic acid or .sup.3H -amino
acids, cells were incubated overnight in medium containing 50
.mu.Ci/ml [.sup.3H]myristic acid (49 Ci/mmol, PerkinElmer Life
Sciences) or 0.2 mCi/ml [.sup.3H]leucine (159 Ci/mmol, PerkinElmer
Life Sciences) plus 0.4 mCi/ml [.sup.3H]proline (100 Ci/mmol,
PerkinElmer Life Sciences). Following labeling, cells were exposed
to stimulatory reagents for 5 min. When an inhibitor was used,
cells were preincubated with the inhibitor for 15 min prior to
stimulation. At the end of the treatments, cells were lysed in a
buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA,
10% glycerol, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride,
1 mM benzamidine, 10 .mu.g/ml pepstatin A, and 10 .mu.g/ml
leupeptin. Trichloroacetic acid precipitation and scintillation
counting may determine the radiolabeling efficiency in each
culture. Immunoprecipitation of MARCKS protein was carried out
according to the method of Spizz and Blackshear using cell lysates
containing equal counts/min. Spizz et al., J. Biol. Chem. 271,
553-562 (1996). Precipitated proteins were resolved by 8%
SDS-polyacrylamide gel electrophoresis and visualized by
autoradiography. Anti-human MARCKS antibody (2F12) and nonimmune
control antibody (6F6) were used in this assay.
[0218] To assess MARCKS or MARCKS-associated protein complexes in
different subcellular fractions, radiolabeled and treated cells
were scraped into a homogenization buffer (50 mM Tris-HCl (pH 7.5),
10 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM
benzamidine, 10 .mu.g/ml pepstatin A, 10 .mu.g/ml leupeptin) and
then disrupted by nitrogen cavitation (800 pounds/square inch for
20 min at 4.degree. C.). Cell lysates were centrifuged at
600.times.g for 10 min at 4.degree. C. to remove nuclei and
unbroken cells. Post-nuclear supernatants were separated into
membrane and cytosol fractions by ultracentrifugation at
400,000.times.g for 30 min at 4.degree. C. The membrane pellet was
solubilized in the lysis buffer by sonication. Immunoprecipitation
was then carried out as described above.
[0219] MARCKS-related Peptides--Both the myristoylated N-terminal
sequence (MANS) and the random N-terminal sequence (RNS) peptides
were synthesized at Genemed Synthesis, Inc. (San Francisco,
Calif.), then purified by high pressure liquid chromatography
(>95% pure), and confirmed by mass spectroscopy with each
showing one single peak with an appropriate molecular mass. The
MANS peptide consisted of sequence identical to the first 24 amino
acids of MARCKS, i.e. the myristoylated N-terminal region that
mediates MARCKS insertion into membranes,
MA-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1 (where MA is N-terminal
myristoyl chain). The corresponding control peptide (RNS) contained
the same amino acid composition as the MANS but arranged in random
order, MA-GTAPAAEGAGAEVKRASAEAKQAF (SEQ ID NO: 232). The presence
of the hydrophobic myristate moiety in these synthetic peptides
enhances their permeability to the plasma membranes, enabling the
peptides to be taken up readily by cells. To determine the effects
of these peptides on mucin secretion, cells were preincubated with
the peptides for 15 min prior to addition of secretagogues, and
mucin secretion was then measured by ELISA.
[0220] Antisense Oligonucleotides--MARCKS antisense oligonucleotide
and its corresponding control oligonucleotide were synthesized at
Biognostik GmbH (Gottingen, Germany). NHBE cells were treated with
5 .mu.M antisense or control oligonucleotide apically for 3 days
(in the presence of 2 .mu.g/ml lipofectin for the first 24 h).
Cells were then incubated with secretagogues, and mucin secretion
was measured by ELISA. Total RNA and protein were isolated from
treated cells. MARCKS mRNA was assessed by Northern hybridization
according to conventional procedures using human MARCKS cDNA as a
probe. MARCKS protein level was determined by Western blot using
purified anti-MARCKS IgG1 (clone 2F12) as the primary detection
antibody.
[0221] Transient Transfection--The phosphorylation site domain
(PSD) of MARCKS contains the PKC-dependent phosphorylation sites
and the actin filament-binding site. To construct a PSD-deleted
MARCKS cDNA, two fragments flanking the PSD sequence (coding for 25
amino acids) were generated by polymerase chain reaction and then
ligated through the Xhol site that was attached to the 5'-ends of
oligonucleotide primers designed for the polymerase chain reaction.
The resultant mutant cDNA and the wild-type MARCKS cDNA were each
inserted into a mammalian expression vector pcDNA4/TO (Invitrogen,
Carlsbad, Calif). Isolated recombinant constructs were confirmed by
restriction digests and DNA sequencing.
[0222] HBE1 is a papilloma virus-transformed human bronchial
epithelial cell line capable of mucin secretion when cultured in
air/liquid interface. Transfection of HBE1 cells was carried out
using the Effectene transfection reagent (Qiagen, Valencia, Calif.)
according to the manufacturer's instructions. Briefly,
differentiated HBE1 cells grown in air/liquid interface were
dissociated by trypsin/EDTA and re-seeded in 12-well culture plates
at 1.times.10.sup.5 cells/cm.sup.2. After overnight incubation,
cells were transfected with the wild-type MARCKS cDNA, the
PSD-truncated MARCKS cDNA, or vector DNA. Cells were cultured for
48 h to allow gene expression and then exposed to secretagogues and
mucin secretion measured by ELISA. All transfections were carried
out in the presence of pcDNA4/TO/lacZ plasmid (Invitrogen) (DNA
ratio 6:1, total 1 .mu.g DNA, ratio of DNA to Effectene
reagent=1:25) to monitor variations in transfection efficiency.
Results showed no significant difference in .beta.-galactosidase
activities in cell lysates isolated from the transfected cells,
indicating similar transfection efficiency among different DNA
constructs (data not shown).
[0223] Protein Phosphatase Activity Assay--PP1 and PP2A activities
were measured using a protein phosphatase assay system (Life
Technologies, Inc.) as known in the art with slight modification.
Huang et al., Adv. Exp. Med Biol. 396, 209-215 (1996). Briefly,
NHBE cells were treated with 8-Br-cGMP or medium alone for 5 min.
Cells were then scraped into a lysis buffer (50 mM Tris-HCl (pH
7.4), 0.1% .beta.-mecaptoethanol, 0.1 mM EDTA, 1 mM benaamidine, 10
.mu.g/ml pepstatin A, 10 .mu.g/ml leupeptin) and disrupted by
sonication for 20 s at 4.degree. C. Cell lysates were centrifuged
and the supernatants saved for phosphatase activity assay. The
assay was performed using .sup.32P-labeled phosphorylase A as a
substrate. Released .sup.32P, was counted by scintillation. The
protein concentration of each sample was determined by the Bradford
assay. PP2A activity was expressed as the sample total phosphatase
activity minus the activity remaining in the presence of 1 nM
okadaic acid. PP1 activity was expressed as the difference between
the activities remaining in the presence of 1 nM and 1.mu.M okadaic
acid, respectively. Protein phosphatase activities were reported as
nmol of P.sub.i released per min/mg total protein.
[0224] Cytotoxicity Assay--All reagents used in treating NHBE cells
were examined for cytotoxicity by measuring the total release of
lactate dehydrogenase from the cells. The assay was carried out
using the Promega Cytotox 96 Kit according to the manufacturer's
instructions. All experiments were performed with reagents at
non-cytotoxic concentrations.
[0225] Statistical Analysis--Data were analyzed for significance
using one-way analysis of variance with Bonferroni post-test
corrections. Differences between treatments were considered
significant at p<0.05.
[0226] Isolation of PMNs from canine blood--The steps involved in
isolating PMN include collecting 10 ml ACD anticoagulated blood.
Then layering 5 ml on 3.5 ml PMN isolation media while ensuring
that the PMN isolation media (IM) was at room temperature (RI).
Next, the blood was centrifuged at room temperature for 30',
550.times.g at 1700 RPMs. The low lower white band was transferred
into 15 ml conical centrifuge tube (CCFT). Next, 2V HESS with 10%
fetal bovine serum (PBS) was added and centrifuged at room
temperature for 10', 400.times.g at 1400 RPMs. The pellet was then
resuspended in 5 ml 1-1ESS with PBS. The cell suspension was added
to 50 ml CCFT containing 20 ml of ice cold 0.88% NH.sub.4Cl and
inverted two to three times. The resulting product was centrifuged
for 10', 800.times.g at 2000 RPMs, then aspirated and resuspended
in 5 ml HBSS with FBS. The prep was examined by counting and
cytospin and preferably for whole blood, the cell number should be
between 10.sup.9-10.sup.-11 cells and for PMNs, cell number should
be between 2-4.times.10.sup.7 cells. See generally, Wang et al., J.
Immunol., "Neutrophil-induced changes in the biomechanical
properties of endothelial cells: roles of ICAM-1 and reactive
oxygen species," 6487-94 (2000).
[0227] MPO Colorimetric Enzyme Assay--Samples were assayed for MPO
activity in 96 well round bottom microtiter plates using a sandwich
ELISA kit (R & D Systems, Minneapolis, Minn.). Briefly, 20
microliters of sample is mixed with 180 microliters of substrate
mixture containing 33 mM potassium phosphate, pH 6.0, 0.56% Triton
X-100, 0.11 mM hydrogen peroxide, and 0.36 mM O-Diannisidine
Dihydrochloride in an individual microtiter well. The final
concentrations in the assay mixture are: 30 mM potassium phosphate,
pH 6.0, 0.05% Triton X-100, 0.1 mM hydrogen peroxide, and 0.32 mM
O-Diannisidine Dihydrochloride. After mixing, the assay mixture was
incubated at room temperature for 5 minutes, and MPO enzyme
activity determined spectrophotometrically at 550 nanometers.
Samples were assayed in duplicate.
Example 1
Inflammatory Mediator Secretion Studies
[0228] Four different leukocyte types or models that secrete
specific granule contents in response to phorbol ester induced
activation of PKC were used. Neutrophils were isolated from human
blood and in vitro release of MPO by these cells was assessed.
Release of membrane-bound inflammatory mediators from
commercially-available human leukocyte cell lines was also
evaluated. The human promyelocytic cell line HL-60 clone 15 was
used to assess secretion of EPO (Fischkoff S A. Graded increase in
probability of eosinophilic differentiation of HL-60 promyelocytic
leukemia cells induced by culture under alkaline conditions. Leuk
Res 1988; 12:679-686; Rosenberg H F, Ackerman S J, Tenen D G. Human
eosinophil cationic protein: molecular cloning of a cytotoxin and
helminthotoxin with ribonuclease activity. J Exp Med 1989;
170:163-176; Tiffany H L, Li F, Rosenberg H F. Hyperglycosylation
of eosinophil ribonucleases in a promyelocytic leukemia cell line
and in differentiated peripheral blood progenitor cells. J Leukoc
Biol 1995; 58:49-54; Badewa A P, Hudson C E, Heiman A S. Regulatory
effects of eotaxin, eotaxin-2, and eotaxin-3 on eosinophil
degranulation and superoxide anion generation. Exp Biol Med 2002;
227:645-651). The monocytic leukemia cell line U937 was used to
assess secretion of lysozyme (Hoff T, Spencker T, Emmendoerffer A.,
Goppelt-Struebe M. Effects of glucocorticoids on the TPA-induced
monocytic differentiation. J Leukoc Biol 1992; 52:173-182; Balboa M
A, Saez Y, Balsinde J. Calcium-independent phospholipase A2 is
required for lysozyme secretion in U937 promonocytes. J Immunol
2003; 170:5276-5280; Sundstrom C, Nilsson K. Establishment and
characterization of a human histiocytic lymphoma cell line (U-937).
Int J Cancer 1976; 17:565-577). The lymphocyte natural killer cell
line NK-92 used to assess release of granzyme (Gong J H., Maki G,
Klingemann H G. Characterization of a human cell line (NK-92) with
phenotypical and functional characteristics of activated natural
killer cells. Leukemia 1994; 8:652-658; Maki G, Klingemann H G,
Martinson J A, Tam Y K. Factors regulating the cytotoxic activity
of the human natural killer cell line, NK-92. J Hematother Stem
Cell Res 2001; 10:369-383; Takayama H, Trenn G, Sitkovsky M V. A
novel cytotoxic T lymphocyte activation assay. J Immunol Methods
1987; 104:183-190). In all cases, the cells were preincubated with
a range of concentrations of a synthetic peptide identical to the
24 amino acid MARCKS N-terminus (MANS- myristoylated N-terminal
sequence peptide; MA-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1) wherein
MA is myristoyl attached to the N-terminal amine of the peptide by
an amide bond), or a missense control peptide (RNS: Random
N-terminal sequence peptide; MA-GTAPAAEGAGAEVKRASAEAKQAF, SEQ ID
NO: 232) which consists of the same 24 amino acids but arranged in
random order sequence which possesses less than 13% sequence
identity to the MANS peptide sequence. Alternatively, the cells
were pretreated with one of the synthetic truncated peptides listed
in Table 3 below.
[0229] In each of the cell types, MANS, but not RNS, attenuates
release of inflammatory mediators in a concentration-dependent
manner. A useful time course of observation is 0.5-3.0 hrs. The
results are consistent with the N-terminal region of the MARCKS
protein being involved in intracellular pathways leading to
leukocyte degranulation.
[0230] Human neutrophil isolation--These studies were approved by
the human studies Institutional Review Board (IRB). Human
neutrophils were isolated as previously described (see Takashi S,
OkuboY, Horie S. Contribution of CD54 to human eosinophil and
neutrophil superoxide production. J Appl Physiol 2001; 91:613-622)
with slight modifications. Briefly, heparinized venous blood was
obtained from normal healthy volunteers, diluted with RPMI-1640
(Cellgro; Mediatech, Inc., Herndon, Va.) at a ratio of 1:1, layered
onto a Histopaque (density, 1.077 g/ml; Sigma-Aldrich Co., St.
Louis, Mo.) and centrifuged at 400 g for 20 min at 4.degree. C. The
supernatant and mononuclear cells at the interface were carefully
removed, and erythrocytes in the sediment were lysed in chilled
distilled water. Isolated granulocytes were washed twice with
Hanks' balanced salts solution (HBSS) and resuspended in HBSS on
ice. The neutrophils used for the experiments were of >98%
purity with <2% contamination by eosinophils, and the viability
was >99% as determined by Trypan blue dye exclusion.
[0231] Measurement of released neutrophil MPO activity--For
measurement of MPO release, purified human neutrophils suspended in
HBSS were aliquoted at 4.times.10.sup.6 cells/ml in 15 ml tubes and
preincubated with either 50 or 100 .mu.M of MANS, RNS, or one of
the peptides of invention for 10 min at 37.degree. C. The cells
then were stimulated with 100 nM phorbol 12-myristate 13-acetate
(PMA) for up to 3 hrs. A control reference (PMA control reference)
was established using purified human neutrophils suspended in HBSS
aliquoted at 4.times.10.sup.6 cells/ml in 15 mL tubes and
stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA) in the
absence of a test peptide for the same time periods. The reaction
was terminated by placing the tubes on ice and centrifugation at
400 g for 5 min at 4.degree. C.
[0232] MPO activity in the cell supernatant was assayed using
tetramethylbenzidine (TMB) based on a previously established
technique (Abdel-Latif D, Steward M, Macdonald D L, Francis G A.,
Dinauer M C, Lacy P. Rac2 is critical for neutrophil primary
granule exocytosis. Blood 2004; 104:832-839). Briefly, 100 .mu.L of
TMB substrate solution was added to 50 .mu.L of cell supernatants
or standard human MPO (EMD Biosciences, Inc., San Diego, Calif.) in
a 96-well microplate followed by incubation at room temperature for
15 min. The reaction was terminated by addition of 50 .mu.L of 1M
H.sub.2SO.sub.4 and absorbance was read at 450 nm in a
spectrophotometric microplate reader (VERSA max, Molecular Devices,
Sunnyvale, Calif.).
Leukocyte Culture Studies
[0233] Three types of human leukocyte cell lines, specifically the
promyelocytic cell line HL-60 clone 15, the monocytic cell line
U937, and the lymphocyte natural killer cell line NK-92 were
purchased from American Type Culture Collection (ATCC; Rockville,
Md.). HL-60 clone 15 cells (ATCC CRL-1964) were maintained in
medium consisting of RPMI 1640 with L-glutamine supplemented with
10% heat-inactivated fetal bovine serum (Gibco; Invitrogen Co.,
Carlsbad, Calif.), 50 IU/ml penicillin, 50 .mu.g/mL streptomycin,
and 25 mM HEPES buffer, pH 7.8, at 37.degree. C. in an atmosphere
containing 5% CO2. Final differentiation to an eosinophil-like
phenotype was initiated by culturing cells at 5.times.10.sup.5
cells/ml in the above medium containing 0.5 mM butyric acid
(Sigma-Aldrich Co.) for 5 days as previously described (Tiffany H
L, Li F, Rosenberg H F. Hyperglycosylation of eosinophil
ribonucleases in a promyelocytic leukemia cell line and in
differentiated peripheral blood progenitor cells. J Leukoc Biol
1995; 58:49-54; Tiffany H L, Alkhatib G, Combadiere C, Berger E A,
Murphy P M. CC chemokine receptors 1 and 3 are differentially
regulated by IL-5 during maturation of eosinophilic HL-60 cells. J
Immunol 1998; 160:1385-1392). U937 cells (ATCC CRL-1593.2) were
grown at 37.degree. C. in an atmosphere of 5% CO.sub.2 in complete
medium consisting of RPMI 1640 with L-glutamine supplemented with
10% FBS, 50 IU/ml penicillin, and 50 .mu.g/mL streptomycin. NK-92
cells (ATCC CRL-2407) were maintained in alpha-MEM medium
(Sigma-Aldrich Co.) supplemented with 20% FBS, 100 U/ml of
interleukin-2 (IL-2) (Chemicon International, Inc., Temecula,
Calif.), 5.times.10.sup.-5 M of 2-mercaptoethanol, 50 IU/mL
penicillin, and 50 .mu.g/ml streptomycin at 37.degree. C. in an
atmosphere containing 5% CO.sub.2. Cell morphology was judged by
assessment of Wright-Giemsa-stained cells. Viability of cells
harvested for experiments was assessed by trypan blue exclusion and
populations of cells with viability >95% were used.
Incubation of Cells for Degranulation Assays.
[0234] HL-60 clone 15, U937, and NK-92 cells were washed and
resuspended at 2.5.times.10.sup.6 cells/ml in phenol red-free
RPMI-1640 (Cellgro; Mediatech, Inc.) for all degranulation assays.
Aliquots of cells in 15 ml tubes were preincubated with indicated
concentrations of MANS, RNS, or a test peptide for 10 min at
37.degree. C. The cells then were stimulated with PMA for up to 2
hr. A control reference (PMA control reference) was established for
each cell type using HL-60 clone 15, U937, and NK-92 cells,
respectively, which were washed and resuspended at
2.5.times.10.sup.6 cells/ml in phenol red-free RPMI-1640 and
stimulated with PMA but in the absence of MANS, RNS, or a test
peptide for the same time periods. The reaction was terminated by
placing tubes on ice and centrifuging cells at 400 g for 5 min at
4.degree. C.
[0235] For measurements of released MPO from neutrophils and
released lysozyme from U937 cells, we were able to quantify
secretion by using as standards human MPO and egg white ovalbumin,
respectively. For released EPO from HL-60 clone 15 cells and for
released granzyme from NK-92 cells, no standards were available to
use for quantification. Hence, both released and intracellular
(from lysed cells) levels of EPO and granzyme were measured, and
the released EPO and granzyme were expressed as a percentage of
total (intracellular and released) for each. To measure
intracellular EPO in HL-60 clone 15 cells and intracellular
granzyme in NK-92 cells, appropriate aliquots of 0.1% triton
X-100-lysed cells were taken for quantification of intracellular
granule proteins as described below. All treatments were expressed
as percentage of control to minimize variability between
cultures.
Measurement of HL-60 EPO Release.
[0236] EPO activity released by HL-60 clone 15 cells was assayed
using TMB according to a previously established technique (Lacy P,
Mahmudi-Azer S, Bablitz B, Hagen S C, Velazquez J R, Man S F,
Moqbel R. Rapid mobilization of intracellularly stored RANTES in
response to interferon-gamma in human eosinophils. Blood 1999;
94:23-32). Thus, 100 .mu.L of TMB substrate solution was added to
50=microliters) of sample in a 96-well microplate and incubated at
room temperature for 15 min (min=minutes). The reaction was
terminated by addition of 50 of 1.0M H.sub.2SO.sub.4 and absorbance
was read at 450 nm (nm=nanometers) in a spectrophotometric
microplate reader. The amount of secreted EPO was expressed as
percentage of total content, using the amount obtained in the same
number of triton X-100-lysed cells.
Measurement of Monocyte Lysozyme Secretion.
[0237] Lysozyme secreted by U937 cells was measured using a
spectrophotometric assay as described previously (Balboa M A, Saez
Y, Balsinde J. Calcium-independent phospholipase A2 is required for
lysozyme secretion in U937 promonocytes. J Immunol 2003;
170:5276-5280) with slight modification. Thus, 100 .mu.L of sample
was mixed with 100 .mu.L of a Micrococcus lysodeikticus
(Sigma-Aldrich Co.) suspension (0.3 mg/ml in 0.1 M sodium phosphate
buffer, pH 7.0) in a 96-well microplate. The decrease in absorbance
at 450 nm was measured at room temperature. A calibration curve was
constructed using chicken egg white lysozyme (EMD Biosciences,
Inc.) as a standard.
Measurement of NK Cell Granzyme Secretion.
[0238] Granzyme secreted from NK-92 cells was assayed by measuring
hydrolysis of Na-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT)
essentially as described previously (Takayama H, Trenn G, Sitkovsky
M V. A novel cytotoxic T lymphocyte activation assay. J Immunol
Methods 1987; 104:183-190). Briefly, 50 .mu.L of supernatant was
transferred to a 96-well plate, and 150 .mu.L of BLT solution (0.2
mM BLT; EMD Biosciences, Inc., and 0.22 mM DTNB; Sigma-Aldrich Co.)
(mM=millimolar) in phosphate-buffered saline (PBS, pH 7.2) was
added to the supernatant. Absorbance at 410 nm was read after
incubation for 30 min at room temperature. Results were expressed
as percentage of total cellular enzyme content, using the amount
obtained in the same number of triton X-100-lysed cells.
Statistical Analysis.
[0239] Statistical significance of the differences between various
treatment groups was assessed with one-way ANOVA. P values of
<0.05 were taken as significant.
[0240] Inhibition of MPO Release from Human Neutrophils
[0241] It was found that 100 nM PMA (as a stimulator of
inflammatory mediator release) increased human neutrophil MPO
release by approximately threefold versus control level at 30 min
in the PMA control reference, the release of MPO increasing to
approximately 5-6 fold after 3 hrs. At 30 minutes, relative to the
control MPO activity as 100% absent PMA and absent PMA plus MANS,
RNS,or test peptide, MPO activity of the PMA control reference was
about 275%, PMA plus 50 .mu.M MANS was about 275%, and 100 .mu.M
MANS was about 305%. Thus, the MANS peptide had no detected effect
at 30 min. However, by 1 hr the higher concentration of MANS (100
.mu.M) had a significant inhibitory effect (measured at about 260%
of control) or about 25% reduction in MPO release versus the PMA
control reference level (which was measured at about 340% of
control). The 50 .mu.M MANS sample measured about 290% of control
or about 15% reduction relative to the PMA control reference. By 2
hrs and persisting at 3 hrs, the MANS peptide significantly
attenuated MPO activity in a concentration-dependent manner. At 2
hours, the PMA control reference MPO activity was about 540% of
control, the 50 .mu.M MANS (measuring about 375% of control) caused
an approximately 30% reduction of MPO release versus the PMA
control reference; and 100 .mu.M MANS (measuring about 295% of
control) caused an approximately 45% reduction of MPO release
versus the PMA control reference. At 3 hours, the PMA control
reference MPO activity was about 560% of control, 50 .mu.M MANS
(measuring about 375% of control) caused an approximately 33%
reduction of MPO release versus the PMA control reference; 100
.mu.M MANS (measuring about 320% of control) caused an
approximately 40% reduction of MPO release versus the PMA control
reference. The RNS peptide did not affect PMA-induced MPO release
at any of the time points or concentrations tested. The data
presented in the table below represents 100 .mu.M concentration of
test peptides and a two hour incubation with 100 nM PMA.
Inhibition of EPO Release from HL-60 Cells
[0242] EPO activity in the supernatant of HL-60 clone 15 cells was
significantly enhanced at 1 and 2 hrs after PMA stimulation. At 1
hour, relative to EPO activity of the control as 100%, the PMA
control reference measured at about 110%; the sample containing 10
.mu.M MANS measured at about 95% to give about 15% reduction in EPO
activity relative to the PMA control reference; the sample
containing 50 .mu.M MANS measured at about 78% to give about 30%
reduction in EPO activity relative to the PMA control reference;
and the sample containing 100 .mu.M MANS measured at about 65% to
give about 40% reduction in EPO activity relative to the PMA
control reference. At 2 hour, relative to EPO activity of the
control as 100%, the PMA control reference measured at about 145%;
the sample containing 10 .mu.M MANS measured at about 130% to give
about 10% reduction in EPO activity relative to the PMA control
reference; the sample containing 50 .mu.M MANS measured at about
70% to give about 50% reduction in EPO activity relative to the PMA
control reference; and the sample containing 100 .mu.M MANS
measured at about 72% to give about 50% reduction in EPO activity
relative to the PMA control reference. Thus, at both 1 and 2 hrs,
MANS at 50 or 100 .mu.M significantly attenuated EPO release. The
RNS peptide did not affect PMA-enhanced EPO release at any of the
time points or concentrations tested. The data presented in the
table below represents 50 .mu.M concentration of test peptides and
a two hour incubation with 100 nM PMA.
Inhibition of Lysozyme Release from U937 Cells
[0243] Lysozyme secretion by U937 cells was increased by PMA
stimulation by 1 hr after incubation, and increased even more at 2
hrs. At 1 hour, relative to lysozyme secretion by U937 cells of the
control as 100%, the PMA control reference measured at about 210%;
the sample containing 10 .mu.M MANS measured at about 170% to give
about 20% reduction in lysozyme secretion by U937 cells relative to
the PMA control reference; the sample containing 50 .mu.M MANS
measured at about 170% to give about 20% reduction in lysozyme
secretion by U937 cells relative to the PMA control reference; and
the sample containing 100 .mu.M MANS measured at about 115% to give
about 45% reduction in lysozyme secretion by U937 cells relative to
the PMA control reference. At 2 hour, relative to lysozyme
secretion by U937 cells of the control as 100%, the PMA control
reference measured at about 240%; the sample containing 10 .mu.M
MANS measured at about 195% to give about 20% reduction in lysozyme
secretion by U937 cells relative to the PMA control reference; the
sample containing 50 .mu.M MANS measured at about 185% to give
about 25% reduction in lysozyme secretion by U937 cells relative to
the PMA control reference; and the sample containing 100 .mu.M MANS
measured at about 140% to give about 40% reduction in lysozyme
secretion by U937 cells relative to the PMA control reference.
Thus, lysozyme secretion was significantly attenuated at both 1 and
2 hours post-stimulation by 100 .mu.M of MANS but not as much by 50
or 10 .mu.M of MANS. The RNS peptide did not affect PMA-enhanced
lysozyme secretion at any of the time points or concentrations
tested. The data presented in the table below represents 50 .mu.M
concentration of test peptides and a two hour incubation with 100
nM PMA.
Inhibition of Granzyme Release from NK-92 Cells
[0244] The lymphocyte natural killer cell line NK-92 was used to
assess release of granzyme (Gong J H, Maki G, Klingemann H G.
Characterization of a human cell line (NK-92) with phenotypical and
functional characteristics of activated natural killer cells.
Leukemia 8:652-658, 1994; Maki G, Klingemann H G, Martinson J A,
Tam Y K. Factors regulating the cytotoxic activity of the human
natural killer cell line, NK-92. J. Hematother. Stem Cell Res.,
10:369-383, 2001; Takayama H, Trenn G, Sitkovsky M V. A novel
cytotoxic T lymphocyte activation assay. J. Immunol. Methods
104:183-190, 1987).
[0245] Measurement of NK cell granzyme secretion: Granzyme secreted
from NK-92 cells was assayed by measuring hydrolysis of
Na-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT, EMD
Bioscience, Inc.) essentially as described previously (Takayama H,
Trenn G, Sitkovsky M V. A novel cytotoxic T lymphocyte activation
assay. J. Immunol. Methods 104:183-190, 1987). An aliquot of 50
.mu.L of supernatant was transferred to a 96-well plate, and 150
.mu.L of 0.2 mM solution of BLT and 0.22 mM DTNB (Sigma-Aldrich
Co.) in phosphate-buffered saline (PBS, pH 7.2) was added to the
supernatant. Absorbance at 410 nm was measured after incubation for
30 min at room temperature. Results were expressed as percentage of
total cellular enzyme content, using the amount obtained in the
same number of triton X-100-lysed cells.
[0246] Because standard granzyme from NK-92 cells was not available
to use for quantification, we measured both released and
intracellular (from lysed cells) levels of granzyme, and expressed
the released granzyme as a percentage of total (intracellular and
released) for each. To measure intracellular granzyme from NK-92
cells, appropriate aliquots of 0.1% triton X-100-lysed cells were
taken for quantification of the enzyme as described above. All data
are expressed as percentage of control to minimize variability
between cultures. The data presented in the table below represents
50 .mu.M concentration of test peptides and a two hour incubation
with 100 nM PMA.
[0247] Cytotoxicity
[0248] Because standard None of the treatments generated a toxic
response in the cells, as assessed by LDH retention/release (data
not shown) (see also Park J-A, He F, Martin L D, Li Y, Adler K B.
Human neutrophil elastase induces hypersecretion of mucin from
human bronchial epithelial cells in vitro via a
PKC.delta.--mediated mechanism. Am J Pathol 2005; 167:651-661).
[0249] In preliminary experiments, the following peptides which are
presented in the table below demonstrate respective percent
inhibition of release of MPO from human neutrophils, of EPO from
HL-60 clone 15 cells, of lysozyme from U937 cells, and of granzyme
from NK-92 cells, wherein MA- signifies the presence of a myristoyl
substituent group at the alpha-N-terminal position of the peptide;
Ac- signifies the presence of an acetyl substituent group at the
alpha-N-terminal position of the peptide; H signifies no group
attached to the peptide; and NH2 signifies the presence of an amide
at the C-terminal position. Inhibition data are averaged from
multiple experiments. Qualitative solubility of the peptides in 0.5
N saline at pH 6.5 is given in mg/mL in Table 3 below. Changing the
N-terminal chemical moiety from a myristoyl group can lead to
changes in solubility of the peptides disclosed herein in aqueous
media. For example, changing the myristoyl group to an acetyl group
results in the increased aqueous solublility shown in Table 3.
TABLE-US-00003 TABLE 3 Results of Enzyme inhibition assays
solubilities for representative peptides and substituted peptides
SEQ ID NO.: N--.sup.1 Amino Acid Sequence C--.sup.2 EPO Lysozyme
MPO Granzyme Solubility.sup.3 219 Ac AKGE 87.6 7.2 >200 45 Ac
AKGEAAAERPGEAAVA 72.3 34.3 37 Ac GAQFSKTAAKGEAAAE 56.6 8.4 239 Ac
GAQFSKTAAAGE 55.8 37.2 >50 248 Ac GAQFSKTAAA 55.2 28.3 >100
91 Ac AAAERPGEAAVA 51.2 29.5 11 Ac GAQFSKTAAKGEAAAERPGE 48.8 0.0 79
Ac GAQFSKTAAKGE 46.7 43.3 >100 153 Ac RPGEAAVA 45.8 0.0 219 Ac
AKGE NH2 45.6 26.8 >200 93 Ac AQFSKTAAKGE NH2 42.8 51.8 >90
141 Ac SKTAAKGE NH2 42.2 0 >200 241 Ac GAQFSKTAAKGA 40.9 24.1
>50 143 Ac TAAKGEAA 40.4 0.5 >230 251 Ac AAGE 39.1 36.9
>200 106 Ac GAQFSKTAAK 35.7 41.2 25.3 >100 249 Ac GAQFSATAAA
35.7 3.2 <10 1 Ac GAQFSKTAAKGEAAAERPGEAAVA 33.7 39.8 >250 121
Ac GAQFSKTAA 33.3 28.9 >20 106 Ac GAQFSKTAAK (all d) 26.9 8.9
40.0 >100 124 Ac FSKTAAKGE NH2 25.3 56.7 >100 79 Ac
GAQFSKTAAKGE NH2 24.7 38.6 26.5 >60 108 Ac QFSKTAAKGE NH2 15.7
60.7 >150 179 Ac AAKGEA 10.6 9.2 >150 159 Ac KTAAKGE NH2 0
24.3 >200 137 Ac GAQFSKTA 0 0 >200 79 H GAQFSKTAAKGE 27.9
>60 1 MA GAQFSKTAAKGEAAAERPGEAAVA 46.1 40.8 31.2 76.0 <5.0
106 MA GAQFSKTAAK 37.4 56.6 >10 11 MA GAQFSKTAAKGEAAAERPGE 33.6
99 179 MA AAKGEA 31.4 28.6 <1.0 37 MA GAQFSKTAAKGEAAAE 30.3 99
>2.0 79 MA GAQFSKTAAKGE 25.2 85.2 43.2 >2.0 91 MA
AAAERPGEAAVA 21.6 98 <20 45 MA AKGEAAERPGEAAVA 18.1 98 >80
153 MA RPGEAAVA 0 99 15 MA SKTAAKGEAAAERPGEAAVA 0 99 >80 143 MA
TAAKGEAA 0 80.2 <1.0 219 MA AKGE 0 28.6 <1.0 232 MA
GTAPAAEGAGEVKRASAEAKQAF 0 0 0 29.5 >15 234 MA GAQFSKTKAKGE 65.2
>3.0 .sup.1N-- = N-terminal group .sup.2C-- = C-terminal group
.sup.30.5 N Saline, pH 6.5
Example 2
[0250] In vivo Inhibition of Lipopolysaccharide (LPS)-Induced Lung
Inflammation by MANS and Related Peptides
[0251] This example was performed essentially according to methods
described by Cox, G, Crossley, J., and Xing, Z.; Macrophage
engulfment of apoptotic neutrophils contributes to the resolution
of acute pulmonary inflammation in vivo; Am. J. Respir. Cell Mol.
Biol. 12:232-237, 1995; Hirano S., Quantitative time-course
profiles of bronchoalveolar lavage cells following intratracheal
instillation of lipopolysaccharide in mice, Ind. Health 35:353-358,
1997; and Ulich T R, Watson L R, Yin S M, Guo K Z, Wang P, Thang H,
and del Castillo, J. Am. J. Pathol. 138:1485-1496, 1991.
[0252] Thus, six to seven week old CD1 female mice weighing 15-20
grams were obtained from Charles River laboratories and housed in
groups of 5 mice per cage. The animals received standard rodent
diet and filtered water ad libitum. The animals were housed under
NIH prescribed guidelines at standard temperature (64.degree. to
79.degree. F.) and relative humidity of 30 to 70%.
[0253] Five treatment groups of mice, with 5 animals in each group,
were treated either with PBS followed by PBS, with PBS followed by
LPS, with (myristoylated) MANS peptide followed by LPS, with
acetylated peptide of SEQ ID NO: 1, followed by LPS, or with
acetylated peptide of SEQ ID NO: 106, followed by LPS.
[0254] Intranasal peptide instillation pre-treatment: A peptide of
the invention to be evaluated in vivo for its ability to inhibit or
reduce LPS-induced lung inflammation was dissolved in PBS at a
concentration of 1 mM. Animals, anesthetized with 0.8% isofluorane
by inhalation, were pretreated with 2.times.10 .mu.L intranasal
bolus of the peptide solution into one nostril 30 minutes prior to
subsequent instillation with LPS.
[0255] Intranasal LPS instillation: Lipopolysaccharide (LPS)
Endotoxin (Escherichia coli Serotype 011:B4 derived endotoxin;
Sigma, St Louis, Mo.; see Sigma product information sheet L4130
titled Lipopolysaccharides from Escherichia coli 011:B4) was
dissolved into phosphate buffered saline (PBS) at 2,500 .mu.g/mL.
To expose animals to endotoxin, a 10 .mu.L intranasal bolus of
2,500 .mu.g/ml endotoxin solution was administered to animals which
had been anesthetized with 0.8% isofluorane by inhalation. The 10
.mu.L bolus was applied into one nostril. Animals were monitored
for labored breathing, lethargy, and decreased water/food intake
following the endotoxin instillations.
[0256] Bronchoalveolar Lavage (BAL): Six hours after the last
instillation, the animals were anesthetized (90 mg/kg Nembutal) and
sacrificed by exsanguination. The lung was serially lavaged 2 times
with 1.0 mL aliquots of PBS. The collected BAL fluid was
centrifuged to remove the cells for subsequent counting and
differential analysis. Recovered lavage fluid was used for analysis
of total protein, myeloperoxidase (MPO), LDH, and hemoglobin.
[0257] Analysis: Aliquots of the BAL fluid were used immediately to
assay for the levels of LDH, total protein, or hemoglobin using the
COBAS Fara analyzer (COBAS FARA II automated analyzer; Roche
Diagnostic Systems Inc., Montclair, N.J.). An aliquot of BAL fluid
was frozen at -80.degree. C. for subsequent quantitation of
myeloperoxidase (MPO) with a mouse-specific ELISA assay (Cell
Sciences, Inc., Canton, Mass). BAL data were analyzed by standard
techniques to examine differences between the control and treatment
groups. Results demonstrating inhibition or reduction of
inflammation by Test peptide are provided in the following
tables.
TABLE-US-00004 TABLE 4 Average values of markers of inflammation in
the presense of MANS peptide, MA-GAQFSKTAAKGEAAAERPGEAAVA, SEQ ID
NO.: 1 Total Total % Total Treatment cells neutrophils Neutrophils
MPO Protein LDH Hb Regime counted counted of total cells (ng/mL)
(ug/ml) (units/L) (g/dl) PBS/PBS 157,020 29317 18.7 3.28 125.60
68.20 0.00 n = 5 PBS/LPS 264,200 110,061 41.7 28.98 272.40 60.40
0.19 n = 5 MANS/LPS 208,457 64,481 30.9 9.49 175.00 68.57 0.05 n =
7
TABLE-US-00005 TABLE 5 Average values of markers of inflammation in
the presence of an N-terminal acetylated analog of MANS peptide,
Ac-GAQFSKTAAKGEAAAERPGEAAVA, SEQ ID NO: 1 Total Total % Total
Treatment Cell Neutrophil Neutrophils MPO Protein LDH Hb Regime
Counts counts of total counts (ng/mL) (.mu.g/mL) (units/L) (g/dL)
PBS/PBS 89,440 19,770 22.1 5.45 230.6 84.0 0.00 n = 5 PBS/LPS
251,360 164,578 65.5 37.90 153.4 89.9 0.01 n= 5 Ac-SEQ ID 254,400
105,499 41.47 30.79 182.75 74.5 0.01 NO.: 1/LPS n = 5
TABLE-US-00006 TABLE 6 Average values of markers of inflammation in
the presence of acetylated peptide Ac-GAQFSKTAAK, SEQ ID NO.: 106
Total Total % MPO Total Treatment Cell Neutrophil Neutrophils MPO
Protein LDH Hb Regime Counts counts of total counts (ng/mL)
(.mu.g/mL) (units/L) (g/dL) PBS/PBS 312,620 66,521 21.3 4.88 113.8
61.80 0.00 n = 5 PBS/LPS 327,680 80,077 24.4 7.19 116.4 78.20 0.00
n = 5 Ac-SEQ ID 305,688 9,170 3.0 1.50 131.0 106.86 0.00 NO.:
106/LPS n = 5
TABLE-US-00007 TABLE 7 Inhibition of markers of inflammation by
MANS peptide (Myr-SEQ ID NO: 1), test peptides
(Ac-GAQFSKTAAKGEAAAERPGEAAVA), SEQ ID NO: 1, and Ac-GAQFSKTAAK, SEQ
ID NO: 106, relative to PBS/LPS treatment: Inhibition of Treatment
neutrophil Inhibition Regime migration of MPO MANS/LPS 41.4% 67.2%
SEQ ID NO: 1/LPS 35.9% 18.75% Ac-SEQ ID NO. 106/ 88.5% 79.1%
LPS
[0258] PBS/PBS indicates only PBS control was administered, and no
LPS endotoxin was added to stimulate chemotactic neutrophil
migration; PBS/LPS indicates LPS (endotoxin) was added to stimulate
chemotactic neutrophil migration; MANS/LPS indicates pretreatment
with MANS peptide in PBS followed by LPS stimulation to induce
neutrophil migration. The percent of neutrophils in the total cell
count in the LPS treatment groups was reduced from 41.7% to 30.9%
by treatment with MANS peptide; from 65.5% to 41.47% by treatment
with the peptide Ac-GAQFSKTAAKGEAAAERPGEAAVA, SEQ ID NO. 1; from
24.4% to 3.0% by treatment with the peptide Ac-GAQFSKTAAK, SEQ ID
NO. 106. The measured MPO levels in the LPS treatment groups was
reduced from 28.98 ng/mL to 9.49 ng/mL by treatment with MANS
peptide; from 37.9 ng/mL to 30.79 ng/mL by treatment with the
peptide with acetylated SEQ ID NO:1 and from 7.19 ng/mL to 1.50
ng/mL by treatment with the peptide with acetylated SEQ ID
NO:106.
Example 3
Mouse Model of Ozone-Induced COPD
[0259] Oxidative stress by chemical irritants such as ozone is a
widely recognized feature of chronic obstructive respiratory
disease (COPD). See: Repine J E, Bast A, Lankhorst I, and the
Oxidative Stress Study Group, Am. J. Respir. Crit. Care Med.
156:341-357, 1997; and also Harkema J R and Hotchkiss J A,
Toxicology Letters, 68:251-263, 1993.
[0260] Ten-week-old Balb/C female mice were obtained from Charles
River laboratories and housed under NIH guidelines in groups of 5
per cage. The animals received standard rodent diet and filtered
water ad libitum. Three treatment groups of mice, 5 animals in each
group, were each anesthetized by intraperitoneal injection of
Ketamine (100 mg/kg) and Xylazine (20 mg/kg) and then pretreated by
intratracheal administration with 25 .mu.L of either PBS alone, or
a solution of 1.0 mM MANS peptide in PBS, or a solution of a 1.0 mM
of an acetylated MANS-fragment-peptide Ac-GAQFSKTAAK designated as
acetylated SEQ ID NO: 106 in PBS. After 30 minutes, the animals
were then placed in the appropriate custom-made chamber for ozone
or forced air exposures. The animals were exposed to ozone for 2
hours (at ozone concentrations of 1-10 ppm by a slightly modified
method described by Haddad et al, 1995. (Haddad E-B, Salmon M, Sun
J, Liu S, Das A, Adcock I, Barnes P J, and Chung K F, FEBS Letters,
363:285-288, 1995). The ozone was generated using an ozone
generator apparatus model OL80F/B from OzoneLab, Burton, British
Columbia, Canada. Ozone concentration was continuously monitored
using a Teledyne Photometric 03 Analyzer (model 400E, Teledyne
Instruments, City of Industry, Calif.). Two additional groups of
mice, each without any pretreatment, were either exposed to ozone
under the same conditions or exposed to forced air under conditions
similar to the ozone treatment group but absent ozone. After
exposure, the animals were sacrificed by exsanguination and the
lungs were serially lavaged 2 times with 1.0 mL aliquots of PBS.
The collected bronchoalveolar lavage (BAL) fluid was centrifuged to
remove the cells for subsequent counting and differential analysis.
Recovered lavage fluid was used for protein and additional analysis
of IL-6, IFN.gamma., and KC (murine IL-8 analog) by ELISA assay
(assay kits obtained from R&D Systems, Minneapolis, Minn.).
[0261] The percent inhibition of neutrophil migration into the BAL
fluid as a function of treatment groups and relative to a control
group treated with PBS alone are provided in the table.
TABLE-US-00008 TABLE 8 Inhibition of ozone-induced neutrophil
migration by MANS peptide and by peptide acetylated SEQ ID NO: 106,
Ac-GAQFSKTAAK. % Inhibition of neutrophil migration into Treatment
Group BAL fluid MANS + Ozone 93.0 Ac-SEQ ID NO: 106 81.2 Ozone PBS
+ Ozone Not applicable Forced air alone Not applicable
[0262] Concentrations of IL-6 in pg/mL in BAL fluid, as a function
of intratracheal injection pretreatment and subsequent treatment
with ozone, were obtained as follows. IL-6 levels were found to be:
approximately 364.5 pg/mL in a group of mice pretreated with MANS
peptide and then exposed to ozone; approximately 130.4 pg/mL in a
group of mice pretreated with acetylated MANS-fragment-peptide,
Ac-GAQFSKTAAK (SEQ ID NO: 106), and then exposed to ozone;
approximately 1041.3 pg/mL in a group of mice pretreated with PBS
and exposed to ozone; approximately 43.2 pg/mL in a group of mice
exposed directly to forced air without any pretreatment.
[0263] Concentrations of KC in pg/mL in BAL fluid, as a function of
intratracheal injection pretreatment and subsequent treatment with
ozone, were obtained as follows. KC levels were found to be:
approximately 183.6 pg/mL in a group of mice pretreated with MANS
peptide and then exposed to ozone; approximately 159.7 pg/mL in a
group of mice pretreated with acetylated MANS-fragment-peptide,
Ac-GAQFSKTAAK (SEQ ID NO:106), and then exposed to ozone;
approximately 466.6 pg/mL in a group of mice pretreated with PBS
and exposed to ozone; approximately 36.3 pg/ml in a group of mice
exposed to forced air without pretreatment.
[0264] Concentrations of IFN.gamma. in pg/mL in BAL fluid as a
function of intratracheal injection pretreatment and subsequent
treatment with ozone were obtained as follows. IFN.gamma. levels
were found to be: approximately 7.4 pg/mL in a group of mice
pretreated with MANS peptide and then exposed to ozone;
approximately 3.6 pg/ml in a group of mice pretreated with
acetylated MANS-fragment-peptide, Ac-GAQFSKTAAK (SEQ ID NO:106),
and then exposed to ozone; approximately 8.6 pg/mL in a group of
mice pretreated with PBS and exposed to ozone; and approximately
5.0 pg/mL in a group of mice exposed to forced air.
[0265] Administration of ozone to mice significantly increased
infiltrated neutrophil cell numbers, as well as IL-6 and KC levels
in the BAL. In comparison to the control group in which the mice
were pretreated with PBS, the group pretreated with MANS peptide
and the group pretreated with acetylated peptide, Ac-GAQFSKTAAK,
acetylated SEQ ID NO:106. each exhibited reduced neutrophil cell
infiltration in the BAL fluid after ozone exposure (e.g.,
93%.+-.10% and 81%.+-.10%, respectively vs. PBS control). In
parallel, MANS peptide and acetylated peptide acetylated SEQ ID
NO:106 also markedly diminished KC concentrations (e.g.,
65.8%.+-.10% and 71.3%.+-.10%, respectively, vs. PBS control) and
IL-6 levels (e.g., 67.8%.+-.15%, MANS and 91.3%.+-.15% acetylated
SEQ ID NO:106 vs. PBS control) after ozone exposure but had little
effect on interferon-y levels. Collectively, these data evidence
that MANS peptide and acetylated SEQ ID NO:106 peptides markedly
diminish or inhibit ozone-induced neutrophil migration into the
airways as well as decrease selective chemokine and cytokine. The
IL-6 levels in the BAL fluids from animals pretreated with MANS
peptide or acetylated peptide SEQ ID NO:106 showed approximately
68% and 91% inhibition, respectively, compared to those pretreated
with PBS. Also the KC levels in the BAL fluids from animals
pretreated with MANS peptide or acetylated peptide SEQ ID NO:106
showed approximately 65% and 71% inhibition compared to those
pretreated with PBS.
Example 4
Chronic Bronchitis Model
[0266] The procedure is described by Voynow J A, Fischer B M,
Malarkey D E, Burch L H, Wong T, Longphre M, Ho S B, Foster W M,
Neutrophil Elastase induces mucus cell metaplasia in mouse lung,
Am. J. Physiol. Lung Cell Mol. Physiol. 287:L1293-L1302, 2004 and
is followed to develop a model of chronic bronchitis in the mouse.
Specifically, goblet cell hyperplasia, a signature pathological
feature of chronic bronchitis, is induced by chronic exposure of
mice to human Neutrophil Elastase (NE) instilled into the
airways.
[0267] Human NE are aspirated intratracheally by male Balb/c mice.
A total of 30 mice (about 25-30 g in weight) are obtained
commercially from a supplier such as Jackson Laboratories, Bar
Harbor, Me. The mice are maintained on a 12 hr diurnal cycle, with
food and water provided ad libitum. The animals receive NE by
oropharyngeal aspiration on days 1, 4, and 7. Immediately after
inhalational anesthesia with isofluorane (IsoFlo from Abbott
Laboratories and Open-Circuit Gas Anesthesia System from
Stoelting), animals are suspended by their upper incisors on a
60.degree. incline board, and a liquid volume of human NE [50 ug
(43.75 units)/40 .mu.L PBS (Elastin Products, Owensville, Mo.) is
delivered with the animal's tongue extended to the distal part of
the oropharynx. With the tongue extended, the animal is unable to
swallow, and the liquid volume is aspirated in the respiratory
tract.
[0268] At 7 days after the last NE exposure, when the goblet cell
hyperplasia modeling the airways in chronic bronchitis is at its
maximum (see Voynow et al, 2004), mice (5 animals per group) are
instilled intra-tracheally with 50 .mu.L of either PBS (as
control), or 100 uM of a solution of MANS peptide, a solution of
RNS peptide, or a solution of a peptide such as acetylated peptide
SEQ ID NO:106 dissolved in PBS. Fifteen minutes later, mucus
secretion is triggered by administration of methacholine, using a
Buxco system Nebulizer to provide a fine aerosol delivering
methacholine at approximately 60 mM for 3 min. Fifteen minutes
after methacholine administration, mice are sacrificed by
inhalational exposure to 100% CO2 gas.
[0269] Histochemistry. After exposures described above, lungs from
animals are flushed to remove blood, then are inflated with OCT
(Optimum Cutting Temperature medium (Sakura Finetck, Torrance,
Calif.), half diluted in saline. The lungs are immersed in 10%
formaldehyde in PBS overnight at 4.degree. C., and processed to
paraffin wax. Five p.m sections are treated with Periodic acid
Schiff/haematoxylin to stain mucins in the airways, for example as
described by Singer M, Vargaftig B B, Martin L D, Park J J, Gruber
A D, Li Y, Adler K B, A MARCKS-related peptide blocks mucus
hypersecretion in a murine model of asthma., Nature Medicine
10:193-196, 2004.
[0270] Histological mucus index. A histological mucus index
(Whittaker L, Niu N, Temann U-A, Stoddard A, Flavell R A, Ray A,
Homer R J, and Cohn L, Interleukin-13 mediates a fundamental
pathway for airway epithelial mucus induced by CD4 T cells and
interleukin-9, Am. J. Respir. Cell Mol. Biol. 27:593-602, 2002) is
performed on AB/PAS-stained sections that include both central and
peripheral airways. The slides are examined with a 10.times.
objective, and images captured with a digital camera. A minimum of
four representative cross- or sagittally sectioned airways is
imaged per animal. Only airways where the complete circumference of
the airway can be visualized and included in the image are
analyzed. Airways that open directly in an alveolar space are not
included. The extent of PAS-positive staining in each airway imaged
will be semi-quantitatively determined by an examiner who does not
know the treatment conditions for each section, using the following
five-tier grading system: grade 0, no PAS staining; grade 1, 25% or
less of the airway epithelium has PAS staining; grade 2, 26-50% of
the airway epithelium has PAS staining; grade 3, 51-75% of the
airway epithelium has PAS staining; and grade 4, >75% of the
airway epithelium has PAS staining. This grading system is used to
calculate a mucus index score for each group, and results are
presented as means.+-.SE.
[0271] All results are presented as means.+-.standard error (n=5
animals, 10-20 sections for each). Significance levels will be
calculated using one way ANOVA followed by Scheffe's test, using
SPSS 6.1 software (*=significance between data with a threshold of
p<0.05).
Example 5
In Vivo Assays
[0272] The objective of the following set of experiments is to
establish the effects of the peptides of this invention after in
vivo delivery, either by local instillation at the site of
inflammation or i.v. injection, on inflammation compared to the
control peptides such as RNS. Two models are useful for this
determination: (i) the murine air pouch inflammation model and (ii)
the murine thioglycollate induced peritonitis model. Both are
well-characterized models of inflammation in which neutrophils have
an essential role. The air pouch model enables determination of the
effects of the peptides on a short time course of inflammation
(approximately 4 hrs) and the peritonitis model is useful with
respect to a longer time course of inflammation (approximately 24
hrs).
Overall Experimental Design:
[0273] Four studies, two for each model, one testing i.v. delivery
of the peptides and one testing local delivery of the peptides are
useful for studying the effect of the peptides disclosed in this
application. Each study consists of 2 experimental groups, a
non-inflamed control (treated with vehicle) and an inflamed group
(i.e., treated with an inflammatory stimulus). Each group is
divided into 5 and optionally 6 treatment subgroups, n=6 for each
subgroup. Treatments subgroups are, for example: vehicle, MANS,
RNS, test peptide, optionally a peptide having a scrambled sequence
of the test peptide which scrambled sequence are dubbed
"peptide-SCR", and dexamethasone. Dexamethasone serves as a
reference anti-inflammatory agent. The selection of appropriate
doses for i.v. injection or local instillation are determined from
preliminary dose response experiments. Tentative doses based on the
inhibitory activity of MANS in human neutrophils are: 1 mg/kg for
i.v. delivery administered once or a final concentration of 50
.mu.M delivered locally (into the air pouch or i.p.). The dose for
i.v. delivery are chosen assuming a volume of distribution of 2
L/kg.
Air Pouch Inflammation Model:
[0274] Assays for neutrophil infiltration and inflammation in the
mouse air pouch are performed as described in Clish C B, O'Brien J
A, Gronert K, Stahl G L, Petasis N A, Serhan C N. Local and
systemic delivery of a stable aspirin-triggered lipoxin prevents
neutrophil recruitment in vivo. Proc Natl Acad Sci U S A. 1999 Jul.
6; 96(14):8247-52. Thus, white male BALB/c mice (6-8 wk) are
anesthetized with isoflurane, and dorsal air pouches are raised by
injecting 3 ml of sterile air subcutaneously on days 0 and 3. On
day 6 and while the mice are anesthetized with isoflurane, vehicle,
MANS, RNS, test peptide, or optionally peptide-SCR are delivered as
a bolus injection either i.v. into the tail vein in 100 .mu.L of
sterile 0.9% saline or locally into the air pouch in 900 .mu.L of
PBS-/31 (Dulbecco's Phosphate Buffered Saline without magnesium or
calcium ions, BioWhittaker). Dexamethasone (Sigma) delivered i.v.
as 0.1 mg/kg in 100 .mu.l sterile 0.9% saline or locally as 10
.mu.g in 900 .mu.L of PBS-/-, serves as a reference
anti-inflammatory agent. Inflammation in the air pouch is induced
by local injection of recombinant murine tumor necrosis factor a
(TNF-.alpha., 20 ng) (Boehringer Mannheim) dissolved in 100 .mu.L
of sterile PBS. While the mice are anesthetized with isoflurane,
the air pouches are lavaged twice with 3 mL of sterile PBS 4 hr
after the initial TNF-a injection. Aspirates are centrifuged at
2,000 rpm for 15 min at 23.degree. C. The supernatants are removed,
and the cells suspended in 500 .mu.L of PBS. Aliquots of the
supernatant are assayed for inflammatory mediator concentrations
(optionally except for TNF.alpha.), MPO activity, and lipid
peroxidation.
[0275] Total leukocytes are enumerated in the cell suspension by
light microscopy using a hemocytometer. Resuspended aspirate cells
(50 .mu.L) are added to 150 .mu.L of 30% BSA and centrifuged onto
microscope slides at 2,200 rpm for 4 min by using a cytofuge.
Differential leukocyte counts are determined in cytospins stained
with Wright Giemsa stain and used to calculate the absolute number
of each leukocyte per air pouch exudate. For microscopic analysis,
tissues are obtained with a 6-mm tissue biopsy punch (Acu-Punch,
Acuderm) and fixed in 10% buffered formaldehyde. Samples are then
embedded in paraffin, sliced and stained with hematoxylin-eosin.
Neutrophils are enumerated in histological sections by counting
number of cells/hpf. Distant dermis serve as a control for the
inflamed air pouch dermis.
[0276] Data are presented as total number of neutrophils,
monocytes, eosinophils, basophils, and lymphocytes per exudate or
the number neutrophils per tissue high power field. Values are
reported as the mean.+-.SEM (n=6). The significance of any
treatment on migration are determined by ANOVA. P<0.05 is to be
considered significant.
Example 6
Inflamed Peritoneum Model:
[0277] Male BALB/c mice (6-8 wk) are used and the
thioglycollate-induced peritonitis models performed as described in
Tedder T F, Steeber D A, Pizcueta P. L-selectin-deficient mice have
impaired leukocyte recruitment into inflammatory sites. J Exp Med.
1995 Jun. 1; 181(6):2259-64. Vehicle, MANS, RNS, test peptide, and
optionally peptide-SCR are delivered as a bolus injection either
into the tail vein in 100 .mu.L of sterile 0.9% saline or locally
into the peritoneum 900 .mu.l of PBS-/- immediately prior to i.p.
injection of thioglycollate. Dexamethasone delivered i.v. as 0.1
mg/kg in 100 .mu.L sterile 0.9% saline or locally as 10 .mu.g in
900 .mu.l of PBS-/-, serves as a reference anti-inflammatory agent.
Inflammation is induced by injection of 1 mL of thioglycollate
solution (3% wt/vol; Sigma Immunochemicals) intraperitoneally into
the mice.
[0278] Mice are humanely euthanized 24 hrs following induction of
inflammation and 5 mL of warm (37.degree. C..about.medium (RPMI
1640, 2% FCS, and 2 mM EDTA) injected into the peritoneum followed
by gentle massage of the abdomen. Aspirates of the abdominal lavage
fluid are centrifuged at 2,000 rpm for 15 min at 23.degree. C. The
supernatants are removed, and the cells suspended in 500 .mu.L of
PBS. Aliquots of the supernatant are assayed for MPO activity,
inflammatory mediator concentrations, and lipid peroxidation.
[0279] Total leukocytes are enumerated in the cell suspension by
light microscopy using a hemocytometer. Resuspended aspirate cells
(50 .mu.L) are added to 150 .mu.L of 30% BSA and centrifuged onto
microscope slides at 2,200 rpm for 4 min by using a cytofuge.
Differential leukocyte counts are determined in cytospins stained
with Wright Giemsa stain and used to calculate the absolute number
of each leukocyte per air pouch aspirate.
[0280] Data are presented as total number of neutrophils,
monocytes, eosinophils, basophils, and lymphocytes per exudate.
Values are reported as the mean.+-.SEM (n=6). The significance of
any treatment on migration is determined by ANOVA. P<0.05 is to
be considered significant.
Degranulation:
[0281] Myeloperoxidase is used as a marker of degranulation.
Myeloperoxidase activity in the cell supernatant obtained from the
air pouch or peritoneal lavage fluid is assayed and analyzed as
described above using the TMB method.
Inflammatory Mediator Concentrations:
[0282] Concentrations of the key pro-inflammatory mediators
TNF.alpha., IL-1.beta., IL-10, IL-6, KC, and PGE2 in air pouch and
peritoneal lavage fluid are determined using commercial ELISA kits
(R&D Systems) according to the manufactures instructions.
Lipid Peroxidation:
[0283] The concentration of F2-isoprostanes is a sensitive and
specific measure of oxidative injury resulting from release of
reactive oxygen intermediates from neutrophils and other cells
{Milne G L, Musiek E S, Morrow J D. F2-isoprostanes as markers of
oxidative stress in vivo: an overview. Biomarkers. 2005 November;
10 Suppl 1: S10-23}. F2-isoprostane concentration is determined in
air pouch and peritoneal exudate supernatants using a commercially
available ELISA (8-Isoprostane EIA, Cayman Chemical) according to
the manufactures instructions.
End Point:
[0284] The experiment is considered to be successful if either
local or systemic delivery of the test peptide reduces inflammation
by one or more of the above measures of inhibition of release of
inflammatory mediator.
[0285] The active fragment peptides of this invention inhibit
neutrophil influx into and degranulation in inflamed air pouch or
peritoneum, resulting in reduced MPO activity, reduced lipid
peroxidation, and reduced inflammatory mediator production.
[0286] The foregoing examples are illustrative of the present
invention and are not to be construed as limiting thereof. The
invention is defined by the following claims, with equivalents of
the claims to be included therein.
Sequence CWU 1
1
252124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 1Gly Ala Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala
Ala Glu1 5 10 15Arg Pro Gly Glu Ala Ala Val Ala 20223PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 2Gly Ala Gln Phe
Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu1 5 10 15Arg Pro Gly
Glu Ala Ala Val 20323PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 3Ala Gln Phe Ser Lys Thr Ala Ala Lys
Gly Glu Ala Ala Ala Glu Arg1 5 10 15Pro Gly Glu Ala Ala Val Ala
20422PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 4Gly Ala Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala
Ala Glu1 5 10 15Arg Pro Gly Glu Ala Ala 20522PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 5Ala Gln Phe Ser
Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg1 5 10 15Pro Gly Glu
Ala Ala Val 20622PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 6Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala
Ala Glu Arg Pro1 5 10 15Gly Glu Ala Ala Val Ala 20721PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 7Gly Ala Gln Phe
Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu1 5 10 15Arg Pro Gly
Glu Ala 20821PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 8Ala Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala
Ala Ala Glu Arg1 5 10 15Pro Gly Glu Ala Ala 20921PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 9Gln Phe Ser Lys
Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro1 5 10 15Gly Glu Ala
Ala Val 201021PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 10Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala
Glu Arg Pro Gly1 5 10 15Glu Ala Ala Val Ala 201120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 11Gly Ala Gln Phe
Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu1 5 10 15Arg Pro Gly
Glu 201220PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 12Ala Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala
Glu Arg1 5 10 15Pro Gly Glu Ala 201320PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 13Gln Phe Ser Lys
Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro1 5 10 15Gly Glu Ala
Ala 201420PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 14Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg
Pro Gly1 5 10 15Glu Ala Ala Val 201520PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 15Ser Lys Thr Ala
Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu1 5 10 15Ala Ala Val
Ala 201619PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 16Gly Ala Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala
Ala Glu1 5 10 15Arg Pro Gly1719PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 17Ala Gln Phe Ser Lys Thr Ala Ala Lys
Gly Glu Ala Ala Ala Glu Arg1 5 10 15Pro Gly Glu1819PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 18Gln Phe Ser Lys
Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro1 5 10 15Gly Glu
Ala1919PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 19Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg
Pro Gly1 5 10 15Glu Ala Ala2019PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 20Ser Lys Thr Ala Ala Lys Gly Glu Ala
Ala Ala Glu Arg Pro Gly Glu1 5 10 15Ala Ala Val2119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 21Lys Thr Ala Ala
Lys Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu Ala1 5 10 15Ala Val
Ala2218PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 22Gly Ala Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala
Ala Glu1 5 10 15Arg Pro2318PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 23Ala Gln Phe Ser Lys Thr Ala Ala Lys
Gly Glu Ala Ala Ala Glu Arg1 5 10 15Pro Gly2418PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 24Gln Phe Ser Lys
Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro1 5 10 15Gly
Glu2518PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 25Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg
Pro Gly1 5 10 15Glu Ala2618PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 26Ser Lys Thr Ala Ala Lys Gly Glu Ala
Ala Ala Glu Arg Pro Gly Glu1 5 10 15Ala Ala2718PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 27Lys Thr Ala Ala
Lys Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu Ala1 5 10 15Ala
Val2818PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 28Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu
Ala Ala1 5 10 15Val Ala2917PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 29Gly Ala Gln Phe Ser Lys Thr Ala Ala
Lys Gly Glu Ala Ala Ala Glu1 5 10 15Arg3017PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 30Ala Gln Phe Ser
Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg1 5 10
15Pro3117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 31Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu
Arg Pro1 5 10 15Gly3217PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 32Phe Ser Lys Thr Ala Ala Lys Gly Glu
Ala Ala Ala Glu Arg Pro Gly1 5 10 15Glu3317PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 33Ser Lys Thr Ala
Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu1 5 10
15Ala3417PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 34Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro Gly
Glu Ala1 5 10 15Ala3517PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 35Thr Ala Ala Lys Gly Glu Ala Ala Ala
Glu Arg Pro Gly Glu Ala Ala1 5 10 15Val3617PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 36Ala Ala Lys Gly
Glu Ala Ala Ala Glu Arg Pro Gly Glu Ala Ala Val1 5 10
15Ala3716PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 37Gly Ala Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala
Ala Glu1 5 10 153816PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 38Ala Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala
Ala Ala Glu Arg1 5 10 153916PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 39Gln Phe Ser Lys Thr Ala Ala Lys Gly
Glu Ala Ala Ala Glu Arg Pro1 5 10 154016PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 40Phe Ser Lys Thr
Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro Gly1 5 10
154116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 41Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro
Gly Glu1 5 10 154216PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 42Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg
Pro Gly Glu Ala1 5 10 154316PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 43Thr Ala Ala Lys Gly Glu Ala Ala Ala
Glu Arg Pro Gly Glu Ala Ala1 5 10 154416PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 44Ala Ala Lys Gly
Glu Ala Ala Ala Glu Arg Pro Gly Glu Ala Ala Val1 5 10
154516PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 45Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu Ala Ala
Val Ala1 5 10 154615PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 46Gly Ala Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu
Ala Ala Ala1 5 10 154715PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 47Ala Gln Phe Ser Lys Thr Ala Ala Lys
Gly Glu Ala Ala Ala Glu1 5 10 154815PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 48Gln Phe Ser Lys
Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg1 5 10
154915PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 49Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg
Pro1 5 10 155015PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 50Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu
Arg Pro Gly1 5 10 155115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 51Lys Thr Ala Ala Lys Gly Glu Ala Ala
Ala Glu Arg Pro Gly Glu1 5 10 155215PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 52Thr Ala Ala Lys
Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu Ala1 5 10
155315PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 53Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu Ala
Ala1 5 10 155415PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 54Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu
Ala Ala Val1 5 10 155515PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 55Lys Gly Glu Ala Ala Ala Glu Arg Pro
Gly Glu Ala Ala Val Ala1 5 10 155614PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 56Gly Ala Gln Phe
Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala1 5 105714PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 57Ala Gln Phe Ser
Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala1 5 105814PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 58Gln Phe Ser Lys
Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu1 5 105914PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 59Phe Ser Lys Thr
Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg1 5 106014PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 60Ser Lys Thr Ala
Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro1 5 106114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 61Lys Thr Ala Ala
Lys Gly Glu Ala Ala Ala Glu Arg Pro Gly1 5 106214PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 62Thr Ala Ala Lys
Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu1 5 106314PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 63Ala Ala Lys Gly
Glu Ala Ala Ala Glu Arg Pro Gly Glu Ala1 5 106414PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 64Ala Lys Gly Glu
Ala Ala Ala Glu Arg Pro Gly Glu Ala Ala1 5 106514PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 65Lys Gly Glu Ala
Ala Ala Glu Arg Pro Gly Glu Ala Ala Val1 5 106614PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptidePeptide may or may not be C-term and/or N-term modified
66Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu Ala Ala Val Ala1 5
106713PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 67Gly Ala Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala1 5
106813PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 68Ala Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala1 5
106913PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 69Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala1 5
107013PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 70Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu1 5
107113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 71Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg1 5
107213PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 72Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro1 5
107313PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 73Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro Gly1 5
107413PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 74Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu1 5
107513PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 75Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu Ala1 5
107613PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 76Lys Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu Ala Ala1 5
107713PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 77Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu Ala Ala Val1 5
107813PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 78Glu Ala Ala Ala Glu Arg Pro Gly Glu Ala Ala Val Ala1 5
107912PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 79Gly Ala Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu1 5
108012PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 80Ala Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala1 5
108112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 81Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala1 5
108212PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 82Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala1 5
108312PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 83Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu1 5
108412PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 84Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg1 5
108512PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 85Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro1 5
108612PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 86Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro Gly1 5
108712PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 87Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu1 5
108812PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 88Lys Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu Ala1 5
108912PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 89Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu Ala Ala1 5
109012PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 90Glu Ala Ala Ala Glu Arg Pro Gly Glu Ala Ala Val1 5
109112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 91Ala Ala Ala Glu Arg Pro Gly Glu Ala Ala Val Ala1 5
109211PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 92Gly Ala Gln Phe Ser Lys Thr Ala Ala Lys Gly1 5
109311PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 93Ala Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu1 5
109411PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 94Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala1 5
109511PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 95Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala1 5
109611PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 96Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala1 5
109711PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 97Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu1 5
109811PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 98Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg1 5
109911PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 99Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro1 5
1010011PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 100Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro Gly1 5
1010111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 101Lys Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu1 5
1010211PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 102Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu Ala1 5
1010311PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 103Glu Ala Ala Ala Glu Arg Pro Gly Glu Ala Ala1 5
1010411PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 104Ala Ala Ala Glu Arg Pro Gly Glu Ala Ala Val1 5
1010511PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 105Ala Ala Glu Arg Pro Gly Glu Ala Ala Val Ala1 5
1010610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 106Gly Ala Gln Phe Ser Lys Thr Ala Ala Lys1 5
1010710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 107Ala Gln Phe Ser Lys Thr Ala Ala Lys Gly1 5
1010810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 108Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu1 5
1010910PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 109Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala1 5
1011010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 110Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala1 5
1011110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 111Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala1 5
1011210PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 112Thr Ala Ala Lys Gly Glu Ala Ala Ala Glu1 5
1011310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 113Ala Ala Lys Gly Glu Ala Ala Ala Glu Arg1 5
1011410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 114Ala Lys Gly Glu Ala Ala Ala Glu Arg Pro1 5
1011510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 115Lys Gly Glu Ala Ala Ala Glu Arg Pro Gly1 5
1011610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 116Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu1 5
1011710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 117Glu Ala Ala Ala Glu Arg Pro Gly Glu Ala1 5
1011810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 118Ala Ala Ala Glu Arg Pro Gly Glu Ala Ala1 5
1011910PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 119Ala Ala Glu Arg Pro Gly Glu Ala Ala Val1 5
1012010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 120Ala Glu Arg Pro Gly Glu Ala Ala Val Ala1 5
101219PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 121Gly Ala Gln Phe Ser Lys Thr Ala Ala1 51229PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 122Ala Gln Phe Ser
Lys Thr Ala Ala Lys1 51239PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 123Gln Phe Ser Lys Thr Ala Ala Lys
Gly1 51249PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 124Phe Ser Lys Thr Ala Ala Lys Gly Glu1 51259PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 125Ser Lys Thr Ala
Ala Lys Gly Glu Ala1 51269PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 126Lys Thr Ala Ala Lys Gly Glu Ala
Ala1 51279PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 127Thr Ala Ala Lys Gly Glu Ala Ala Ala1 51289PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 128Ala Ala Lys Gly
Glu Ala Ala Ala Glu1 51299PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 129Ala Lys Gly Glu Ala Ala Ala Glu
Arg1 51309PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 130Lys Gly Glu Ala Ala Ala Glu Arg Pro1 51319PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 131Gly Glu Ala Ala
Ala Glu Arg Pro Gly1 51329PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 132Glu Ala Ala Ala Glu Arg Pro Gly
Glu1 51339PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 133Ala Ala Ala Glu Arg Pro Gly Glu Ala1 51349PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 134Ala Ala Glu Arg
Pro Gly Glu Ala Ala1 51359PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 135Ala Glu Arg Pro Gly Glu Ala Ala
Val1 51369PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 136Glu Arg Pro Gly Glu Ala Ala Val Ala1 51378PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 137Gly Ala Gln Phe
Ser Lys Thr Ala1 51388PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 138Ala Gln Phe Ser Lys Thr Ala Ala1
51398PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 139Gln Phe Ser Lys Thr Ala Ala Lys1 51408PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 140Phe Ser Lys Thr
Ala Ala Lys Gly1 51418PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 141Ser Lys Thr Ala Ala Lys Gly Glu1
51428PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 142Lys Thr Ala Ala Lys Gly Glu Ala1 51438PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 143Thr Ala Ala Lys
Gly Glu Ala Ala1 51448PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 144Ala Ala Lys Gly Glu Ala Ala Ala1
51458PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 145Ala Lys Gly Glu Ala Ala Ala Glu1 51468PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 146Lys Gly Glu Ala
Ala Ala Glu Arg1 51478PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 147Gly Glu Ala Ala Ala Glu Arg
Pro1
51488PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 148Glu Ala Ala Ala Glu Arg Pro Gly1 51498PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 149Ala Ala Ala Glu
Arg Pro Gly Glu1 51508PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 150Ala Ala Glu Arg Pro Gly Glu Ala1
51518PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 151Ala Glu Arg Pro Gly Glu Ala Ala1 51528PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 152Glu Arg Pro Gly
Glu Ala Ala Val1 51538PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 153Arg Pro Gly Glu Ala Ala Val Ala1
51547PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 154Gly Ala Gln Phe Ser Lys Thr1 51557PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 155Ala Gln Phe Ser
Lys Thr Ala1 51567PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 156Gln Phe Ser Lys Thr Ala Ala1 51577PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 157Phe Ser Lys Thr
Ala Ala Lys1 51587PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 158Ser Lys Thr Ala Ala Lys Gly1 51597PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 159Lys Thr Ala Ala
Lys Gly Glu1 51607PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 160Thr Ala Ala Lys Gly Glu Ala1 51617PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 161Ala Ala Lys Gly
Glu Ala Ala1 51627PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 162Ala Lys Gly Glu Ala Ala Ala1 51637PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 163Lys Gly Glu Ala
Ala Ala Glu1 51647PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 164Gly Glu Ala Ala Ala Glu Arg1 51657PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 165Glu Ala Ala Ala
Glu Arg Pro1 51667PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 166Ala Ala Ala Glu Arg Pro Gly1 51677PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 167Ala Ala Glu Arg
Pro Gly Glu1 51687PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 168Ala Glu Arg Pro Gly Glu Ala1 51697PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 169Glu Arg Pro Gly
Glu Ala Ala1 51707PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 170Arg Pro Gly Glu Ala Ala Val1 51717PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 171Pro Gly Glu Ala
Ala Val Ala1 51726PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 172Gly Ala Gln Phe Ser Lys1 51736PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 173Ala Gln Phe Ser
Lys Thr1 51746PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 174Gln Phe Ser Lys Thr Ala1 51756PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 175Phe Ser Lys Thr
Ala Ala1 51766PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 176Ser Lys Thr Ala Ala Lys1 51776PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 177Lys Thr Ala Ala
Lys Gly1 51786PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 178Thr Ala Ala Lys Gly Glu1 51796PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 179Ala Ala Lys Gly
Glu Ala1 51806PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 180Ala Lys Gly Glu Ala Ala1 51816PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 181Lys Gly Glu Ala
Ala Ala1 51826PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 182Gly Glu Ala Ala Ala Glu1 51836PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 183Glu Ala Ala Ala
Glu Arg1 51846PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 184Ala Ala Ala Glu Arg Pro1 51856PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 185Ala Ala Glu Arg
Pro Gly1 51866PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 186Ala Glu Arg Pro Gly Glu1 51876PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 187Glu Arg Pro Gly
Glu Ala1 51886PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 188Arg Pro Gly Glu Ala Ala1 51896PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 189Pro Gly Glu Ala
Ala Val1 51906PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 190Gly Glu Ala Ala Val Ala1 51915PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 191Gly Ala Gln Phe
Ser1 51925PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 192Ala Gln Phe Ser Lys1 51935PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 193Gln Phe Ser Lys
Thr1 51945PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 194Phe Ser Lys Thr Ala1 51955PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 195Ser Lys Thr Ala
Ala1 51965PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 196Lys Thr Ala Ala Lys1 51975PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 197Thr Ala Ala Lys
Gly1 51985PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 198Ala Ala Lys Gly Glu1 51995PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 199Ala Lys Gly Glu
Ala1 52005PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 200Lys Gly Glu Ala Ala1 52015PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 201Gly Glu Ala Ala
Ala1 52025PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 202Glu Ala Ala Ala Glu1 52035PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 203Ala Ala Ala Glu
Arg1 52045PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 204Ala Ala Glu Arg Pro1 52055PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 205Ala Glu Arg Pro
Gly1 52065PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 206Glu Arg Pro Gly Glu1 52075PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 207Arg Pro Gly Glu
Ala1 52085PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 208Pro Gly Glu Ala Ala1 52095PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 209Gly Glu Ala Ala
Val1 52105PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 210Glu Ala Ala Val Ala1 52114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 211Gly Ala Gln
Phe12124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 212Ala Gln Phe Ser12134PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 213Gln Phe Ser Lys12144PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 214Phe Ser Lys
Thr12154PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 215Ser Lys Thr Ala12164PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 216Lys Thr Ala Ala12174PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 217Thr Ala Ala
Lys12184PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 218Ala Ala Lys Gly12194PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 219Ala Lys Gly Glu12204PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 220Lys Gly Glu
Ala12214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 221Gly Glu Ala Ala12224PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 222Glu Ala Ala Ala12234PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 223Ala Ala Ala
Glu12244PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 224Ala Ala Glu Arg12254PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 225Ala Glu Arg Pro12264PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 226Glu Arg Pro
Gly12274PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 227Arg Pro Gly Glu12284PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 228Pro Gly Glu Ala12294PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 229Gly Glu Ala
Ala12304PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 230Glu Ala Ala Val12314PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 231Ala Ala Val Ala123224PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 232Gly Thr Ala Pro
Ala Ala Glu Gly Ala Gly Ala Glu Val Lys Arg Ala1 5 10 15Ser Ala Glu
Ala Lys Gln Ala Phe 2023312PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 233Gly Lys Gln Phe Ser Lys Thr Ala
Ala Lys Gly Glu1 5 1023412PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 234Gly Ala Gln Phe Ser Lys Thr Lys
Ala Lys Gly Glu1 5 1023512PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 235Gly Lys Gln Phe Ser Lys Thr Lys
Ala Lys Gly Glu1 5 1023610PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptidePeptide may or may not be
C-term and/or N-term modified 236Gly Ala Gln Ala Ser Lys Thr Ala
Ala Lys1 5 1023712PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptidePeptide may or may not be C-term and/or
N-term modified 237Gly Ala Gln Ala Ser Lys Thr Ala Ala Lys Gly Glu1
5 1023812PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 238Gly Ala Glu Phe Ser Lys Thr Ala Ala Lys Gly Glu1 5
1023912PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 239Gly Ala Gln Phe Ser Lys Thr Ala Ala Ala Gly Glu1 5
1024012PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 240Gly Ala Gln Phe Ser Lys Thr Ala Ala Lys Ala Glu1 5
1024112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 241Gly Ala Gln Phe Ser Lys Thr Ala Ala Lys Gly Ala1 5
1024210PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 242Ala Ala Gln Phe Ser Lys Thr Ala Ala Lys1 5
1024310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 243Gly Ala Ala Phe Ser Lys Thr Ala Ala Lys1 5
1024410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 244Gly Ala Gln Phe Ala Lys Thr Ala Ala Lys1 5
1024510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 245Gly Ala Gln Phe Ser Ala Thr Ala Ala Lys1 5
1024610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 246Lys Ala Ala Thr Lys Ser Phe Gln Ala Gly1 5
1024710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 247Gly Ala Gln Phe Ser Lys Ala Ala Ala Lys1 5
1024810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 248Gly Ala Gln Phe Ser Lys Thr Ala Ala Ala1 5
1024910PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 249Gly Ala Gln Phe Ser Ala Thr Ala Ala Ala1 5
102508PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 250Gly Ala Gln Ala Ser Lys Thr Ala1 52514PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptidePeptide
may or may not be C-term and/or N-term modified 251Ala Ala Gly
Glu125210PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptidePeptide may or may not be C-term and/or N-term
modified 252Gly Lys Ala Ser Gln Phe Ala Lys Thr Ala1 5 10
* * * * *