U.S. patent application number 11/434997 was filed with the patent office on 2006-09-07 for stereoselective antifibrillogenic peptides and peptidomimetics thereof.
This patent application is currently assigned to Neurochem (International) Limited. Invention is credited to Robert Chalifour, Francine Gervais, Ajay Gupta.
Application Number | 20060199771 11/434997 |
Document ID | / |
Family ID | 36576427 |
Filed Date | 2006-09-07 |
United States Patent
Application |
20060199771 |
Kind Code |
A1 |
Chalifour; Robert ; et
al. |
September 7, 2006 |
Stereoselective antifibrillogenic peptides and peptidomimetics
thereof
Abstract
The present invention relates to antifibrillogenic agents for
inhibiting amyloidosis and/or for cytoprotection for the treatment
of amyloidosis disorders. These agents include peptides, isomers
thereof and peptidomimetic compounds thereof. These agents comprise
a peptide having a sequence identified from the glycosaminoglycan
(GAG) binding region and the prot-prot interaction region of the
amyloid protein. The peptide has at least one [D] amino acid isomer
substitution. The invention also relates to the peptide bound to a
label for in vivo imaging of amyloid deposits.
Inventors: |
Chalifour; Robert; (Ile
Bizard, CA) ; Gervais; Francine; (Ile Bizard, CA)
; Gupta; Ajay; (Pointe Claire, CA) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Assignee: |
Neurochem (International)
Limited
Lausanne
CH
|
Family ID: |
36576427 |
Appl. No.: |
11/434997 |
Filed: |
May 16, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10009122 |
Jun 18, 2002 |
7060670 |
|
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PCT/CA00/00515 |
May 4, 2000 |
|
|
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11434997 |
May 16, 2006 |
|
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60132592 |
May 5, 1999 |
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Current U.S.
Class: |
514/17.8 ;
514/21.8; 530/329; 530/330 |
Current CPC
Class: |
A61K 38/08 20130101 |
Class at
Publication: |
514/017 ;
530/329; 530/330 |
International
Class: |
A61K 38/08 20060101
A61K038/08; C07K 7/06 20060101 C07K007/06 |
Claims
1. A peptide for the prevention or treatment of an amyloidosis
disorder in a human, said peptide comprising a sequence of
[D]-amino acids selected from the group consisting of: (i)
Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4 and (ii) retro-isomer
sequences of Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4, wherein:
Xaa.sub.1 is Lys; Xaa.sub.2 is Ile, Lys-Leu, Phe, Ala, Val, or Leu;
Xaa.sub.3 is Val; and Xaa.sub.4 is Phe-Phe-Ala,
Phe-Phe-Ala-NH.sub.2, Phe-Phe-Ala-Gln, or Phe-Phe-Ala-Gln-NH.sub.2;
with the proviso that Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4 is
not Lys-Leu-Val-Phe-Phe-Ala.
2. The peptide of claim 1, wherein said sequence of [D]-amino acids
is selected from the group consisting of: TABLE-US-00003
Lys-Ile-Val-Phe-Phe-Ala; (SEQ ID NO:1) Lys-Lys-Leu-Val-Phe-Phe-Ala;
(SEQ ID NO:2) Lys-Phe-Val-Phe-Phe-Ala; (SEQ ID NO:4)
Lys-Ala-Val-Phe-Phe-Ala; (SEQ ID NO:7) Lys-Val-Val-Phe-Phe-Ala;
(SEQ ID NO:9) Lys-Ile-Val-Phe-Phe-Ala-NH.sub.2; (SEQ ID NO:10)
Lys-Phe-Val-Phe-Phe-Ala-NH.sub.2; (SEQ ID NO:12)
Lys-Ala-Val-Phe-Phe-Ala-NH.sub.2; (SEQ ID NO:15)
Lys-Val-Val-Phe-Phe-Ala-NH.sub.2; (SEQ ID NO:17)
Lys-Leu-Val-Phe-Phe-Ala-Gln; (SEQ ID NO:18)
Lys-Leu-Val-Phe-Phe-Ala-Gln-NH.sub.2; (SEQ ID NO:19)
and retro-isomer sequences thereof.
3. The peptide of claim 1, wherein said peptide is associated to a
pharmaceutically acceptable carrier.
4. The peptide of claim 1, wherein said amyloidosis disorder is
Alzheimer's disease.
5. The peptide of claim 1, wherein said peptide is for preventing
the development of Alzheimer's disease in a human.
6. The peptide of claim 1, wherein said peptide is for arresting
the development of Alzheimer's disease in a human.
7. A peptide comprising a sequence of [D]-amino acids, wherein said
sequence [D]-amino acids is Lys-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID
NO:2) or the retro-isomer sequence thereof.
8. A medicament for the prevention or treatment of an amyloidosis
disorder in a human, said medicament comprising a peptide as
defined in claim 1.
9. The medicament of claim 8, wherein said medicament is for
preventing or for arresting the development of Alzheimer's disease
in a human.
10. A method for blocking or preventing an amyloidosis disorder in
a patient, said method comprising administering to said patient a
peptide comprising a sequence of [D]-amino acids selected from the
group consisting of: (i) Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4
and (ii) retro-isomer sequences of
Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4, wherein: Xaa.sub.1 is Lys;
Xaa.sub.2 is Ile, Lys-Leu, Phe, Ala, Val, or Leu; Xaa.sub.3 is Val;
and Xaa.sub.4 is Phe-Phe-Ala, Phe-Phe-Ala-NH.sub.2,
Phe-Phe-Ala-Gln, or Phe-Phe-Ala-Gln-NH.sub.2; with the proviso that
Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4 is not
Lys-Leu-Val-Phe-Phe-Ala.
11. The method of claim 10, wherein said sequence of [D]-amino
acids is selected from the group consisting of: TABLE-US-00004
Lys-Ile-Val-Phe-Phe-Ala; (SEQ ID NO:1) Lys-Lys-Leu-Val-Phe-Phe-Ala;
(SEQ ID NO:2) Lys-Phe-Val-Phe-Phe-Ala; (SEQ ID NO:4)
Lys-Ala-Val-Phe-Phe-Ala; (SEQ ID NO:7) Lys-Val-Val-Phe-Phe-Ala;
(SEQ ID NO:9) Lys-Ile-Val-Phe-Phe-Ala-NH.sub.2; (SEQ ID NO:10)
Lys-Phe-Val-Phe-Phe-Ala-NH.sub.2; (SEQ ID NO:12)
Lys-Ala-Val-Phe-Phe-Ala-NH.sub.2; (SEQ ID NO:15)
Lys-Val-Val-Phe-Phe-Ala-NH.sub.2; (SEQ ID NO:17)
Lys-Leu-Val-Phe-Phe-Ala-Gln; (SEQ ID NO:18)
Lys-Leu-Val-Phe-Phe-Ala-Gln-NH.sub.2; (SEQ ID NO:19)
and retro-isomer sequences thereof.
12. The method of claim 10, wherein said peptide is associated to a
pharmaceutically acceptable carrier.
13. The method of claim 10, wherein said patient is in need of a
treatment against an amyloidosis disorder.
14. The method of claim 10, wherein said amyloidosis disorder is
Alzheimer's disease.
15. The method of claim 10, wherein said method is for preventing
the development of Alzheimer's disease.
16. The method of claim 10, wherein said method is for arresting
the development of Alzheimer's disease.
17. A method for preventing or for arresting the development of
Alzheimer's disease in a human, said method comprising
administering to a patient a peptide comprising a sequence of
[D]-amino acids, wherein said sequence is
Lys-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO:2) or the retro-isomer
thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
10/009,122, filed Jun. 18, 2002 (pending), which claims priority
under 35 U.S.C. .sctn. 371 from PCT/CA00/00515, filed May 4, 2000,
which claims priority from U.S. Ser. No. 60/132,592, filed May 5,
1999, the contents of each of which are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] (a) Field of the Invention
[0003] The invention relates to agents having potent
antifibrillogenic activity for the treatment of amyloidosis
disorders and for imaging of amyloid deposits. These agents include
peptides and peptidomimetic compounds thereof.
[0004] (b) Description of Prior Art
[0005] Amyloidosis refers to a pathological condition characterized
by the presence of amyloid fibers. Amyloid is a generic term
referring to a group of diverse but specific extracellular protein
deposits that are seen in a number of different diseases. Though
diverse in their occurrence, all amyloid deposits share common
morphologic properties, stain with specific dyes (e.g. Congo red),
and have a characteristic red-green birefringent appearance in
polarized light after staining. They also share common
ultrastructural, x-ray diffraction and infrared spectra
features.
[0006] Some amyloidotic diseases can be idiopathic but most of
these diseases appear as a complication of a previously existing
disorder. For example, primary amyloidosis can appear without any
other pathology or can follow plasma cell dyscrasia or multiple
myeloma. Secondary amyloidosis is usually seen associated with
chronic infection (such as tuberculosis) or chronic inflammation
(such as rheumatoid arthritis). A familial form of secondary
amyloidosis is also seen in Familial Mediterranean Fever (FMF).
This familial type of amyloidosis, as one of the other types of
familial amyloidosis, is genetically inherited and is found in
specific population groups. Isolated forms of amyloidosis are those
that tend to involve a single organ system. Different amyloids are
also characterized by the type of protein present in the deposit.
For example, neurodegenerative diseases such as scrapie, bovine
spongiform encephalitis, Creutzfeldt-Jakob disease and the like are
characterized by the appearance and accumulation of a
protease-resistant form of a prion protein (referred to as AScr or
PrP-27) in the central nervous system. Similarly, Alzheimer's
disease, another neurodegenerative disorder, is characterized by
congophilic cerebral angiopathy, neuritic plaques and
neurofibrillary tangles. In this case, the plaque and blood vessel
amyloid is formed by the deposition of fibrillar A.beta. amyloid
protein. In adult-onset diabetes, amyloids containing the IAPP
amyloid protein accumulate in the pancreas. Other systemic
diseases, complications of long-term hemodialysis and sequelae of
long-standing inflammation or plasma cell dyscrasias are
characterized by the accumulation of amyloids systemically. In each
of these cases, a different amyloidogenic protein is involved in
amyloid deposition.
[0007] Once these amyloids have formed, there is no known, widely
accepted therapy or treatment that significantly dissolves the
deposits in situ.
[0008] Each amyloidogenic protein has the ability to organize into
.beta.-sheet and to form insoluble fibrils that get deposited
extracellularly. Each amyloidogenic protein, although different in
amino acid sequence has the same property of forming fibrils and
binding to other elements such as proteoglycan (glycosaminoglycan),
amyloid P and complement component. Moreover, each amyloidogenic
protein has amino acid sequences, which, although different, will
show similarities such as regions with the ability to bind to GAG's
(referred to as the GAG binding site) as well as other regions that
will promote .beta.-sheet formation referred to as .beta.-sheet
region.
[0009] In specific cases, amyloidotic fibrils once deposited can
become toxic to the surrounding cells. As per example, the A.beta.
fibrils organized as senile plaques have been shown to be
associated with dead neuronal cells and microgliosis in patients
with Alzheimer's disease. When tested in vitro, A.beta. peptide was
shown to be capable of triggering an activation process of the
microglia (brain macrophages), which would explain the presence of
microgliosis and brain inflammation found in the brain of patients
with Alzheimer's disease.
[0010] In another type of amyloidosis seen in patients with Type II
diabetes, the amyloidogenic protein IAPP, has been shown to induce
.beta.-islet cell toxicity in vitro. Hence, appearance of IAPP
fibrils in the pancreas of Type II diabetic patients could
contribute to the loss of the .beta. islet cells (Langerhans) and
organ dysfunction.
[0011] Particularly, in patients with Alzheimer's Disease, an agent
capable 1) of preventing amyloid fibril formation and deposition
and 2) of directly or indirectly inhibiting A.beta.-induced
neurotoxicity and inflammation (microgliosis), could be a treatment
of choice to prevent and arrest the development of Alzheimer's
disease.
[0012] It would be highly desirable to be provided with agents
having potent antifibrillogenic activity for the treatment of
amyloidosis disorders.
SUMMARY OF THE INVENTION
[0013] One aim of the present invention is to provide agents having
potent antifibrillogenic activity for the treatment of amyloidosis
disorders.
[0014] Another aim of the present invention is to provide a method
for the treatment of amyloidosis disorders, such as Alzheimer's'
disease.
[0015] A number of strategies for possible therapeutic intervention
in amyloid development have been proposed. These strategies include
reduction of the pool of precursor proteins, prevention of the
interaction of precursor proteins and disruption of preformed
amyloid. The present invention deals mainly with the second
approach, prevention of precursor protein interactions. The ideal
molecule to fulfill this function, would interact specifically with
the amyloid protein and would in so doing prevent the protein from
interacting with itself. When dealing with molecules that are
chiral, it is standard practice to identify which of the
stereoisomers possesses the activity, since in general, activity
can be attributed to one or the other of the isomers. By using a
stereochemically pure isomer, side reactions can be avoided or
reduced.
[0016] In accordance with one embodiment of the present invention
there is provided an antifibrillogenic agent for inhibiting
amyloidosis and/or for cytoprotection, which comprises a peptide of
Formula I, an isomer thereof, a retro or a retro-inverso isomer
thereof or a peptidomimetic thereof:
Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4 I wherein, Xaa.sub.1 is
absent or selected from the group consisting of Lys, Lys-Lys,
Xaa.sub.5-Lys-, and Ala; Xaa.sub.5 is absent or selected from the
group consisting of His-Gln-, His-His-Gln-, Val-His-His-Gln-,
Glu-Val-His-His-Gln-, Asp-Asp-Asp-, Lys-Val-Asp-Asp-Gln-Asp-, Gln-;
Xaa.sub.2 is absent or any amino acid; Xaa.sub.3 is absent, Val or
Phe; Xaa.sub.4 is absent or selected from the group consisting of
Phe, Phe-NH.sub.2, Phe-Phe, Phe-Phe-Ala, Phe-Phe-Ala-NH.sub.2,
Phe-Phe-Ala-Gln, Phe-Phe-Ala-Gln-NH.sub.2, Val-Leu-Lys,
Val-Leu-Lys-NH.sub.2; wherein the peptide of formula I contains at
least one Lys or Asp; and wherein the peptide has at least one [D]
amino acid residue, with the proviso that
Lys-Lys-Leu-Val-Phe-Phe-Ala is an all-[D] peptide; and with the
proviso that when Xaa.sub.5 is Lys-Val-Asp-Asp-Gln-Asp- all of
Xaa.sub.2, Xaa.sub.3, and Xaa.sub.4 are absent.
[0017] Preferably, Xaa.sub.2 is a hydrophobic amino acid residue
such as a leucine residue.
[0018] In one embodiment of the invention, the peptide of formula I
has at least two [D] amino acid residues, and more preferably at
least three [D] amino acid residues. Optionally, the peptide of
formula I has one [L] amino acid residue, or more preferably the
peptide is an all-[D] isomer peptide.
[0019] In another embodiment of the invention, the peptide of
Formula I is selected from the group consisting of: TABLE-US-00001
Lys-Ile-Val-Phe-Phe-Ala; (SEQ ID NO:1) Lys-Lys-Leu-Val-Phe-Phe-Ala;
(SEQ ID NO:2) Lys-Leu-Val-Phe-Phe-Ala; (SEQ ID NO:3)
Lys-Phe-Val-Phe-Phe-Ala; (SEQ ID NO:4) Ala-Phe-Phe-Val-Leu-Lys;
(SEQ ID NO:5) Lys-Leu-Val-Phe; (SEQ ID NO:6)
Lys-Ala-Val-Phe-Phe-Ala; (SEQ ID NO:7) Lys-Leu-Val-Phe-Phe; (SEQ ID
NO:8) Lys-Val-Val-Phe-Phe-Ala; (SEQ ID NO:9)
Lys-Ile-Val-Phe-Phe-Ala-NH.sub.2; (SEQ ID NO:10)
Lys-Leu-Val-Phe-Phe-Ala-NH.sub.2; (SEQ ID NO:11)
Lys-Phe-Val-Phe-Phe-Ala-NH.sub.2; (SEQ ID NO:12)
Ala-Phe-Phe-Val-Leu-Lys-NH.sub.2; (SEQ ID NO:13)
Lys-Leu-Val-Phe-NH.sub.2; (SEQ ID NO:14)
Lys-Ala-Val-Phe-Phe-Ala-NH.sub.2; (SEQ ID NO:15)
Lys-Leu-Val-Phe-Phe-NH.sub.2; (SEQ ID NO:16)
Lys-Val-Val-Phe-Phe-Ala-NH.sub.2; (SEQ ID NO:17)
Lys-Leu-Val-Phe-Phe-Ala-Gln; (SEQ ID NO:18)
Lys-Leu-Val-Phe-Phe-Ala-Gln-NH.sub.2; (SEQ ID NO:19)
His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala- (SEQ ID NO:20) NH.sub.2;
Asp-Asp-Asp; (SEQ ID NO:21) Lys-Val-Asp-Asp-Gln-Asp; (SEQ ID NO:22)
His-His-Gln-Lys; (SEQ ID NO:23) and
Gln-Lys-Leu-Val-Phe-Phe-NH.sub.2. (SEQ ID NO:24)
[0020] More preferably, the peptide of formula I is a peptide as
set forth in SEQ ID NO:2 or SEQ ID NO:3.
[0021] In accordance with one embodiment of the present invention
there is provided a labeled conjugate for in vivo imaging of
amyloid plaque, which comprises a conjugate of formula II: A-B-C II
wherein A is an amyloid plaque-targeting moiety selected from the
group consisting of a peptide of Formula I as defined above, an
isomer thereof, a retro or a retro-inverso isomer thereof and a
peptidomimetic thereof, wherein B is a linker portion allowing
attachment of the amyloid plaque-targeting moiety to C; and wherein
C is a label that allows for in vivo imaging. Preferably, the
linker portion B is selected from the group consisting of Glucose
and Phe. Preferably, the label C is .sup.99mTc.
[0022] Still in accordance with the present invention, there is
provided a method for the treatment of amyloidosis disorders in a
patient, which comprises administering to the patient a
therapeutically effective amount of a peptide of Formula I, or the
antifibrillogenic agent, as defined above.
[0023] Further in accordance with the present invention, there is
provided a composition for the treatment of amyloidosis disorders
in a patient, which comprises a therapeutically effective amount of
a peptide of Formula I, or of an antifibrillogenic agent, as
defined above in association with a pharmaceutically acceptable
carrier.
[0024] In accordance with the present invention, there is also
provided a composition for in vivo imaging of amyloid plaques,
which comprises a therapeutically effective amount of a labeled
conjugate as defined above in association with a pharmaceutically
acceptable carrier.
[0025] The peptide of Formula I or the antifibrillogenic agent may
be used for inhibiting amyloidosis and/or for cytoprotection.
[0026] The labeled conjugate may be used for in vivo imaging of
amyloid plaques.
[0027] The peptide of Formula I or the antifibrillogenic agent may
alternatively be used for the manufacture of a medicament for
inhibiting amyloidosis and/or for cytoprotection.
[0028] Similarly, the labeled conjugate may also be used for the
manufacture of a medicament for in vivo imaging of amyloid
plaques.
[0029] Other embodiments of these peptides include racemic mixtures
and peptides having mixed chirality, i.e., different chirality at
different chiral centers.
[0030] In accordance with the peptides Lys-Lys-Leu-Val-Phe-Phe-Ala
(SEQ ID NO:2) and Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO:3), one
stereoisomer, the D form, is found to be more active than the L
form, and the D isomer is the preferred form for use of this
peptide as a drug.
[0031] The present invention further provides similar peptides
designed for the other amyloidogenic peptides such as AA, AL, and
IAPP. In fact, the present invention also provides a peptide for
inhibiting amyloidosis and/or for cytoprotection. The peptide has a
sequence taken from the .beta.-sheet region of an amyloid protein.
Such peptide or a composition containing such peptide can be used
for inhibiting amyloidosis and/or for cytoprotection.
Alternatively, such peptide or a composition containing such
peptide can be used for the manufacture of a medicament for
inhibiting amyloidosis and/or for cytoprotection.
[0032] Accordingly, the present invention also provides a
composition for inhibiting amyloidosis and/or for cytoprotection,
which comprises a therapeutically effective amount of a peptide as
defined previously in association with a pharmaceutically
acceptable carrier.
[0033] In accordance with the present invention, the amyloidosis
disorder includes, without limitation, prion protein related
disorders, type II diabetes and Alzheimer's disease.
[0034] With regard to another aspect of the invention, diseases
caused by the death or malfunctioning of a particular type or types
of cells can be treated by transplanting into the patient healthy
cells of the relevant type of cell. Often these cells are cultured
in vitro prior to transplantation to increase their numbers, to
allow them to recover after the isolation procedure or to reduce
their immunogenicity. However, in many instances the transplants
are unsuccessful, due to the death of the transplanted cells. The
inventors have now also found that culturing of cells can lead to
the formation of fibrils from endogenous proteins. Such fibrils are
likely to continue to grow after the cells are transplanted and
cause death or dysfunction of the cells. The inventors have also
found that the peptide of the present invention or the
antifibrillogenic compound of the present invention can be used to
reduce the formation of fibrils.
[0035] Thus the invention also provides a process for the
preparation of cells suitable for transplantation into a mammal,
which cells are capable of forming fibrils. The process comprises
contacting the cells with the peptide of the present invention or
the antifibrillogenic compound of the present invention.
[0036] The peptide of Formula I or the antifibrillogenic compound
causes breakdown of amyloid deposits which have been formed by the
cells prior to the contact. Preferably, the cells are cultured in
the presence of the peptide of Formula I or the antifibrillogenic
compound.
[0037] For the purpose of the present invention the following
expressions and terms are defined below.
[0038] The term "agents having stereoselective antifibrillogenic
activity" is intended to mean any peptides, peptide analogues,
peptide derivatives, or peptidomimetics that retain the
stereoselective antifibrillogenic activity, the cytoprotective and
anti-inflammatory activity and/or the ability to alter a natural
amyloidotic protein aggregation as described herein. Peptide
analogues, peptide derivatives, or peptidomimetics include any
molecules that mimic the chemical structure of a peptide and retain
the functional properties of the peptide (Williams, W. V. and
Weiner, D. B., eds., Biologically Active Peptides: Design,
Synthesis, and Utilization, vol. 1, Technomic Publishing Company
Inc., Lancaster, Pa., 1993, pages 35-3 . . . ). Examples of peptide
analogues, peptide derivatives, or peptidomimetics include
compounds with sulfonamide, phosphoramide or non-amide
linkages.
[0039] The expression "antifibrillogenic activity" is intended to
mean the ability to block or prevent an amyloidogenic protein from
forming fibrils, preferably by preventing it from adopting its
.beta.-pleated conformation.
[0040] The term "cytoprotection" or "cytoprotective activity" is
intended to mean the ability to protect cells from amyloid-induced
toxicity.
[0041] The expression "anti-inflammatory" is intended to mean the
ability to block or reduce the A.beta.-induced microglial
activation process or to block the chemokine-induced inflammatory
reaction.
[0042] The expression "retro isomer" is intended to mean a reversal
of the direction of the peptide backbone.
[0043] The expression "inverso isomer" is intended to mean an
inversion of the amino acid chirality used to make the peptide.
[0044] The expression "retro-inverso isomer" is intended to mean a
reversal of both the peptide backbone direction and the amino acid
chirality.
[0045] Except as otherwise expressly defined herein, the
abbreviations used herein for designating the amino acids and the
protective groups are based on recommendations of the IUPAC-IUB
Commission on Biochemical Nomenclature (Biochemistry, 1972,
11:1726-1732).
[0046] Also, unless specified otherwise, the A.beta.(1-40) is the
naturally occurring A.beta.(1-40), that is the all [L]-isomer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] FIG. 1 illustrates the targeted sites of the protein-protein
interactions required for self-assembly into .beta.-sheet
fibrils;
[0048] FIG. 2 illustrates a thioflavin T fluorescence assay for
fibril formation by [L]-A.beta. (1-40) in the absence and presence
of a peptide in accordance with one embodiment of the
invention;
[0049] FIG. 3 shows the same assay as in FIG. 2 for fibril
formation by [D]-A.beta. (1-40);
[0050] FIG. 4 is a bar graph illustrating the percentage of
thioflavin T fluorescence in the presence of the [D]-peptide used
in FIG. 2, with or without single substitutions of corresponding
[L]-amino acids;
[0051] FIG. 5 is a bar graph illustrating a thioflavin T
fluorescence assay for fibril formation by [L]-A.beta. (1-40) in
the presence of the [D]-peptide used in FIG. 2, with or without
substitution of the Leu residue by other hydrophobic amino
acids;
[0052] FIG. 6 illustrates the toxicity of [L]-A.beta. (1-40) in the
absence and presence of peptides in accordance with one embodiment
of the invention; and
[0053] FIG. 7 is a bar graph illustrating the toxicity of
[L]-A.beta. (1-40) in the presence of another peptide of the
present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0054] As illustrated in FIG. 1, internal regions of the A.beta.
sequence have been shown to confer characteristics of the amyloid
protein. Indeed, the region between amino acid 13-16
(His-His-Gln-Lys, SEQ ID NO:23) of the amyloid protein is
responsible for the interaction between the A.beta. protein and the
glycosaminoglycan moiety of the proteoglycans (Kisilevsky, R., et
al., Proteoglycans and amyloid fibrillogenesis: The nature and
origin of amyloid fibrils, Wiley, Chichester (CIBA Foundation
Symposium 1997), pp. 58-72). Proteoglycans are known to promote
amyloid fibril formation as well as protect these fibrils from
proteolysis (Gupta-Bansal, R., et al., 1995, The Journal of
Biological Chemistry, 270:18666-18671). More recently, the same
region has been determined to play a role in the activation process
of microglial cells by A.beta. (Giulian, D., et al., 1998, The
Journal of Biological Chemistry, 273(45):29719-29726). This 13-16
region of A.beta., often referred to as the GAG binding site, is
also part of a larger domain, the 10-16 region of the protein which
has been suggested as the region responsible for the adherence of
A.beta. to the cell surface (Giulian, D., et al., 1996, The Journal
of Neuroscience, 16(19):6021-6037). Such adherence of A.beta. to
the cell surface will allow the interaction of A.beta. with the
specific cells leading to either microglia activation or toxicity
of neuronal cells.
[0055] These two overlapping regions of the A.beta. protein, i.e.
amino acids 13-16 and 10-16 are adjacent to the 16-21 region of
A.beta., a short hydrophobic stretch critical for the formation of
fibrillar structures (Hilbrich, C., et al., 1992, J. Mol. Biol.,
228:460-473). By having peptides capable of interacting with these
overlapping regions of A.beta., one can aim at preventing both
A.beta. fibril formation and A.beta. cellular interaction (i.e.
microglia activation, neurotoxicity).
[0056] A preferred embodiment of the present invention is novel and
arises from the unexpected finding that the all-[D] stereoisomer
peptides, Lys-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO:2) and
Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO:3), are much more potent
inhibitors of A.beta.(1-40) fibrillogenesis then the corresponding
all-[L] peptides. The all-[D] stereoisomer peptides,
Lys-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO:2) and
Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO:3) are also potent
cytoprotective agents.
[0057] This finding was unforeseen particularly because the
researchers who originally reported peptides containing the
sequence Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO:3) as an inhibitor of
fibrillogenesis, state in a second article which they published: "A
peptide entirely composed of amino acids in D configuration with
the sequence klvff (lowercase marks amino acids in D configuration)
was synthesized using the SPOT technique and assayed for
.sup.125I-LBMP1620 binding. This peptide failed to bind
.sup.125I-LBMP1620 indicating that KLVFF-KLVFF interaction is
sterospecific." Tjernberg, L. O. et al. (1997) Controlling Amyloid
.beta.-Peptide Fibril Formation with Protease-stable Ligands, J.
Biol. Chem., 272:12602.
Inhibition of Amyloidosis
[0058] The experimental work performed leading to this invention
included comparing the ability of the [D] and [L] stereoisomers of
peptide Lys-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO:2) to inhibit the
fibrillogenesis process observed with the amyloidogenic peptide
A.beta.(1-40) in a thioflavin T fluorescence assay.
[0059] The thioflavin T fluorescence assay for fibrillogenesis is
based on the principle that the fluorescent dye, thioflavin T,
binds specifically to fibrillar, but not to unaggregated A.beta.
peptide (LeVine III, H., 1993, Protein Science 2:404-410). Upon
binding, thioflavin T develops a characteristic fluorescence
(Naiki, H., et al., 1996, Lab. Invest. 74: 374-383), which can be
easily detected. The dye is believed to interact with the stacked
cross-.beta. pleated sheets, the common structural motif of all
amyloids (LeVine III, H., 1995, Amyloid: Int. J. Exp. Clin Invest.
2:1.6). Thioflavin T is widely used to assay the effect of
compounds on A.beta. peptide fibrillogenesis (Bronfman, P. C., et
al., 1995, Neuroscience Lett. 218:201-203).
[0060] In this assay test compounds are incubated with a solution
of A.beta.(1-40) (20 .mu.M) containing 10 .mu.M thioflavin T, in
0.02M Tris/0.02M acetate/0.15M NaCl/0.005% azide/pH 7.40 at
37.degree. C. in sealed 384 well microplates. Readings (ex 430
nm/em 485 nm) are taken at various time intervals with a microplate
fluorescence reader. An increase in fluorescence signifies the
appearance of amyloid or intermediates in the production of
amyloid. Inhibitors of fibrillogenesis will lead to less
fluorescence production.
[0061] The results illustrated in Table 1 below, are based on the
fluorescence production in the presence of test peptides at either
20 .mu.M or 80 .mu.M concentration, at the time intervals of 5, 19,
45, 67, 77 and 90 hours, compared to a control, buffer alone,
without added inhibitory peptide. TABLE-US-00002 TABLE 1 Order Of
Potency of Peptide Inhibitors Tested at Tested at 20 .mu.M 80 .mu.M
(strongest activity) 1 (D) KIVFFA 1 (D) AFFVLK 2 (D) KKLVFFA 1 (D)
KKLVFFA 3 (D) KLVFFA 1 (D) KLVFFA 4 (D) KFVFFA 1 (D) KFVFFA 5 (D)
AFFVLK 5 (D) KIVFFA 6 (D) KLVF 6 (D) KAVFFA 7 (D) KAVFFA 7 (L)
KKLVFFA 8 (L) KLVFFA 8 (L) KLVFFA 9 (D) KLVFF 9 (D) KLVF 10 (L)
KKLVFFA 10 (D) KLVFF (weakest activity) 11 (L) AFFVLK 11 (L)
AFFVLK
Protocol A.beta. peptide: A.beta.(1-40) 95% purity (American
Peptide Company, Inc, Sunnyvale, Cal. USA, cat. 62-0-78) is
disaggregated in trifluoroacetic acid and filtered through a 0.02
.mu.M filter, (Whatman Anotop 25 plus, 0.02 .mu.m, Catalogue no.
6809 4102) in hexafluoroisopropanol (HFIP). Solutions of
A.beta.(1-40) at 600 .mu.M in HFIP are stored at -80.degree. C.
Assay mixture: The mixture is prepared as two solutions that are
combined upon addition to the 384 well microplate (Corning Costar
cat. 3705). [0062] i) Solution A consists of test peptides in 0.02M
Tris/0.02M acetate/0.15M NaCl/0.01% azide at pH 7.40 or buffer
alone (control), [0063] ii) Solution B consists of A.beta.(1-40) 40
.mu.M, thioflavin T 20 .mu.M in 0.02M Tris/0.02M acetate/0.15M NaCl
at pH 7.40. This solution is prepared by drying the A.beta. peptide
under nitrogen and then resuspending this in 0.04M Tris base with
15 minutes sonication. An equal volume of 0.04M acetic acid
containing 0.3 M NaCl is added and the solution is adjusted to pH
7.40.+-.0.02. A small volume of 5 mM thioflavin T is added to the
solution to give a final 20 .mu.M concentration of thioflavin T.
[0064] iii) The microplate is loaded with 40 .mu.L of solution A
followed by 40 .mu.L of solution B which gives a final 20 .mu.M
A.beta.(1-40), 10 .mu.M thioflavin T, and either 20 .mu.M, 80 .mu.M
or 100 .mu.M test compound in 0.02M Tris/0.02M acetate/0.15M
NaCl/0.005% azide, pH 7.40. The plate is sealed and loaded into the
microplate fluorescence reader. Fluorescence measurement data
analysis: The HTS-7000 Bio Assay Reader, Perkin Elmer, is used to
perform kinetic runs of about 5 days. Readings were taken at
various time intervals, 5, 19, 45, 67, 77 and 90 hours, with one
minute shaking before each reading. Bandpass filters used were:
excitation 430 nm, emission 485 mm. Calculations
[0065] The rank order of efficacy of the peptides is determined by
observing which peptides allow the appearance of fluorescence,
above the background level, first. For example in the presence of
buffer control alone, fluorescence appears earlier than when any of
the peptides is present. The most active peptides prevent the
appearance of fluorescence even after 90 hours of incubation.
[0066] The results achieved in the thioflavin T fibrillogenesis
assays show that all-[D] stereoisomer peptide was about 60 times
more potent then the all-[L] stereoisomer peptide. This is based on
the observation that 400 .mu.M all-[L] stereoisomer was required to
give an equivalent inhibition to that produced with 6.1 .mu.M
all-[D] stereoisomer peptide.
[0067] The results achieved in the A.beta.-NBD environmental probe
fibrillogenesis assay showed that the all-[D] stereoisomer peptide
was at least 30 times more potent than the all-[L] stereoisomer
peptide. This estimate is based on the observation that the lowest
concentration of all-[D] peptide tested (25 .mu.M) was more potent
than the highest concentration of the all-[L] peptide (800
.mu.M).
.beta.-Sheet and GAG Binding Domains Peptides
[0068] Novel peptides and peptidomimetics that include
complementary sequences to certain portions of amyloidogenic
peptides such as A.beta., AA, AL, IAPP, and prion proteins are
designed to be capable of inhibition of Protein-Protein
interactions or self assembly. The targeted portions in the various
disease-causing proteins aforementioned, preferably contain one or
more charged residues such as aspartate, glutamate, lysine,
histidine and arginine. Such peptides and their peptidomimetics
will inhibit fibrillogenesis of the amyloidogenic peptides and
prion proteins and interfere with chemokines binding to the cell
surface proteoglycans leading to dimerization or tetramerization by
interacting with their GAG binding domains. In the case of A.beta.,
these interactions lead to cytoprotection as well as inhibition of
inflammatory response and serve as potent therapeutics for the
treatment of Alzheimer's disease. In the case of chemokine-related
disorders these interactions may lead to a decrease in the
uncontrolled inflammatory response associated with some
diseases.
[0069] Other amyloidogenic peptides such as IAPP, have also been
tested. For example, 2 peptides from the .beta.-sheet region of
IAPP have been shown to inhibit IAPP fibril formation using the
thioflavin T fluorescence assay, circular dichroism (measures
secondary structure) and the electron microscope (to look at
fibrils directly).
[0070] The full-length IAPP is 37 amino acids and the .beta.-sheet
region is the 20-29 sequence. The 20-29 sequence is critical for
forming .beta.-sheet and has been previously shown to be a key
region in modulating IAPP aggregation and folding. Hexapeptides
from this .beta.-sheet region were examined and 2 were found to be
active.
[0071] Hexapeptides spanning the 20-29 region
(Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser) of the IAPP protein were
synthesized and tested for their ability to prevent fibril
formation as determined by circular dichroism and the thioflavin T
assay. Hexapeptides were designed and were found to be capable of
blocking the formation of IAPP fibrils. These peptides
(Ser-Asn-Asn-Phe-Gly-Ala- and Asn-Asn-Phe-Gly-Ala-Ile) were
directed towards the central core of the 20-29 region.
[0072] Novel peptides containing 3-6 residues that are
complementary (in terms of their charges) to the 10-16 segment of
A.beta. peptide have been shown for the first time to strongly
interact with A.beta. peptide. They provide a starting point for
the design of BBB (blood brain barrier) permeable peptidomimetics.
In principle, the present invention provides similar peptides can
be designed for the other amyloidogenic peptides such as AA, AL,
and IAPP.
[0073] Asp-Asp-Asp (SEQ ID NO:21), a tripeptide, when incubated
with A.beta.40 under physiological conditions shows a slight
decrease at time t=0 in the amount of .beta.-sheet content as is
evident by the CD spectrum. Incubation of this tripeptide with
A.beta.40 for 24 hours shows no trace of .beta.-sheet conformation
of the A.beta.40 and clearly indicates the ability of this
tripeptide to strongly interact with A.beta.40 peptide and keep
A.beta.40 in a randomized and non-fibrillary conformation. The
anti-fibrillogenic property of this tripeptide is also supported by
the A.beta.42 solubilization assay.
[0074] Lys-Val-Asp-Asp-Gln-Asp (SEQ ID NO:22), a hexapeptide, when
incubated with A.beta.40 under physiological conditions shows an
increase at time t=0 in the amount of .beta.-sheet content as is
evident by the CD spectrum. Incubation of this hexapeptide with
A.beta.40 for 24 hours shows a dramatic increase in .beta.-sheet
content of the A.beta.40 and clearly indicates the ability of this
hexapeptide to strongly interact with A.beta.40 peptide and
organize it into a .beta.-sheet conformation. Electron microscopy
of the mixture failed to show any fibrils indicating that this
particular compound is in fact an anti-fibrillogenic compound with
regard to A.beta.. In vitro results with NBD and thioflavin-T based
fluorescence assays confirm this finding. It is the understanding
of the inventors that this interesting observation will lead to a
greater understanding of fibrillogenesis of A.beta.40 and A.beta.42
peptides and as a result, will provide important information for
the design of potent anti-fibrillogenic compounds for A.beta.,
other amyloidotic peptides such as AA, AL and IAPP for the
treatment of diseases such as Alzheimer's, Type II Diabetes and
amyloidosis related disorders. The same principle can also be
applied to the design of peptide type compounds for the inhibition
of binding of various chemokines to the cell surface as well as
inhibition of self-assembly and cellular adherence of prion
proteins.
[0075] The results illustrated in FIG. 2 show that all
[D]-Lys-Leu-Val-Phe-Phe-Ala (SEQ. ID NO: 3) is a more potent
inhibitor of A.beta. (1-40) assembly in the thioflavin T
fluorescence assay than is all [L]-Lys-Leu-Val-Phe-Phe-Ala. Since
the naturally occurring A.beta. (1-40) used in these experiments
was the all-[L] amino acid version, these results indicate that an
inhibitor peptide works best when containing amino acids of the
opposite chirality.
[0076] FIG. 3 demonstrates that the same rule of opposite chirality
illustrated in FIG. 2 applies for the assembly of A.beta. (1-40)
synthesized using amino acids of the [D] type. In this experiment
all-[L]-Lys-Leu-Val-Phe-Phe-Ala (SEQ. ID NO:3) is a more potent
inhibitor in the all-[D]-A.beta. (1-40) assembly reaction than
all-[D]-Lys-Leu-Val-Phe-Phe-Ala. This result confirms that peptides
of opposite chirality are better inhibitors.
[0077] FIG. 4 illustrates the inhibition of A.beta. (1-40) fibril
formation by all-[D]-Lys-Leu-Val-Phe-Phe-Ala (20 .mu.M) with or
without single substitutions of [L]-amino acids in the thioflavin T
fluorescence assay. In this experiment the ability of the
all-[D]-Lys-Leu-Val-Phe-Phe-Ala peptide to inhibit A.beta. (1-40)
fibril formation, measured as percentage of thioflavin T
fluorescence in the absence of peptide (control), was compared to
[D]-Lys-Leu-Val-Phe-Phe-Ala peptides with single [L]-amino acid
replacements. None of the mixed chirality Lys-Leu-Val-Phe-Phe-Ala
peptides were more potent than the original all-[D] peptide. This
result demonstrates that [D]-amino acids are more potent inhibitors
of A.beta. (1-40) fibrillogenesis than [L]-amino acids.
[0078] However as seen in FIG. 4 some peptides with single [L]
substitutions do retain inhibitory activity. In particular peptides
in which the [D] isomer of the Lys, the second Phe and the Ala are
substituted with the [L]-isomers retain inhibitory activity. The
substitutions, which reduce inhibitory activity the most, are the
Leu, the Val and the first Phe, indicating that these residues
contribute the most to the potency of the [D]-peptide. From FIG. 4,
it is apparent that peptides with mixed chirality or with at least
one [D]-substituted amino acid are also inhibitors, although not as
potent as the all-[D] peptide. These mixed-chirality peptides are
thus meant to be included in the present invention.
[0079] FIG. 5 illustrates the inhibition of A.beta. (1-40) fibril
formation in the thioflavin T fluorescence assay by
all-[D]-Lys-Leu-Val-Phe-Phe-Ala (20 .mu.M), with or without
replacement of the leucine by other hydrophobic amino acids. In
this experiment all the peptides tested retained some inhibitory
activity. This result demonstrates that the leucine residue is not
critical for inhibition of A.beta. fibril formation in the all-[D]
peptide. These results illustrated in FIG. 5 were non-obvious and
unexpected in light of a prior publication which identified the
Leucine residue as critical in an all-[L] version of the peptide
(Tjernberg L O et al., J. Biol. Chem. 271:8545, 1996).
Cytoprotection
[0080] The experimental work performed leading to this invention
also included comparing the ability of [D] and [L] stereoisomers of
the peptides of the present invention to show cytoprotective
activity, i.e. to protect cells from A.beta. toxicity.
[0081] FIG. 6 uses the MTT assay on SH-SY5Y cells.
Protocol
[0082] A SH-SY5Y human neuroblast cell line (American Type Culture
Collection, cat. CRL-2266) is cultured according to technical
specifications. Monomerized A.beta. (1-40) is prepared using
trifluoroacetic acid and hexafluoroisopropanol, in the same way
already described for the thioflavin T fluorescence assay.
Monomerized A.beta. at various concentrations in TANA buffer (0.02
M TRIS base pH 7.4, 0.02M acetate, 0.15 M NaCl) is added to 100
.mu.M test peptide and the mixture is incubated for 24 hours at
37.degree. C. with agitation, in order to allow polymerization to
occur. Cells are adhered to a 96-well microplate for 2 hours at
37.degree. C. and 5% CO.sub.2 before the A.beta.-peptide mixture,
or buffer alone (control), is added. The microplate is gently
agitated and incubated for 20-24 hours at 37.degree. C. and 5%
CO.sub.2. Cell viability is determined by a MTT-based colorimetric
assay. The MTT assay (Boehringer Mannheim, Cell Proliferation Kit
1) is based on the principle that the yellow tetrazolium salt MTT
is cleaved in metabolically active cells to produce purple formazan
crystals. The formazan crystals are solubilized and the resulting
colored solution is quantified using a scanning multiwell
spectrophotometer (ELISA reader, Absorbance A.sub.560 nm). Cellular
toxicity was calculated as follows: Toxicity .times. .times. ( % )
= 100 - ( O . D .times. . .times. sample - O . D . .times. Blank )
( O . D . .times. Control - O . D . .times. Blank ) . ##EQU1##
[0083] FIG. 6 shows the neurotoxicity of A.beta. (1-40) in the
absence or presence of various peptides of the present invention.
In this experiment the all-[D]-Lys-Lys-Leu-Val-Phe-Phe-Ala (SEQ. ID
NO: 2) peptide is a more potent inhibitor of A.beta. neurotoxicity
than the all-[L]-Lys-Lys-Leu-Val-Phe-Phe-Ala peptide in the
cytoprotection assay.
[0084] FIG. 7 uses the propidium iodide assay on primary cortical
neurons. Briefly, fetal rat primary cortical neurons are isolated
and cultured according to Durkin, J. P. et al., J. Neurochem.,
66:951-962, 1996. Neurons are plated in a 48 well microplate. 7
days after plating the neuronal culture media is supplemented with
B27 (Life Technologies, Data sheet form No. 3755). A mixture of
A.beta. and test peptide is added to the cortical neurons for 3
days at 37.degree. C. and 5% CO2.
[0085] Cell viability is then visually assessed as the proportion
of phase-bright cells that exclude propidium iodide, since only
dead cells take up propidium iodide.
[0086] FIG. 7 shows the potent cytoprotective activity of
all-[D]-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO:3). This experiment
shows the potent cytoprotective activity of
all-[D]-Lys-Leu-Val-Phe-Phe-Ala compared to Congo red, which is a
known cytoprotective agent and compared to the absence of any
cytoprotective agent (A.beta. alone).
[0087] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth, and as follows in the scope of the appended
claims.
Sequence CWU 1
1
24 1 6 PRT Artificial Sequence peptide having antifibrillogenic
activity and/or neuroprotection 1 Lys Ile Val Phe Phe Ala 1 5 2 7
PRT Artificial Sequence peptide having antifibrillogenic activity
and/or neuroprotection 2 Lys Lys Leu Val Phe Phe Ala 1 5 3 6 PRT
Artificial Sequence peptide having antifibrillogenic activity
and/or neuroprotection 3 Lys Leu Val Phe Phe Ala 1 5 4 6 PRT
Artificial Sequence peptide having antifibrillogenic activity
and/or neuroprotection 4 Lys Phe Val Phe Phe Ala 1 5 5 6 PRT
Artificial Sequence peptide having antifibrillogenic activity
and/or neuroprotection 5 Ala Phe Phe Val Leu Lys 1 5 6 4 PRT
Artificial Sequence peptide having antifibrillogenic activity
and/or neuroprotection 6 Lys Leu Val Phe 1 7 6 PRT Artificial
Sequence peptide having antifibrillogenic activity and/or
neuroprotection 7 Lys Ala Val Phe Phe Ala 1 5 8 5 PRT Artificial
Sequence peptide having antifibrillogenic activity and/or
neuroprotection 8 Lys Leu Val Phe Phe 1 5 9 6 PRT Artificial
Sequence peptide having antifibrillogenic activity and/or
neuroprotection 9 Lys Val Val Phe Phe Ala 1 5 10 6 PRT Artificial
Sequence peptide having antifibrillogenic activity and/or
neuroprotection 10 Lys Ile Val Phe Phe Ala 1 5 11 6 PRT Artificial
Sequence peptide having antifibrillogenic activity and/or
neuroprotection 11 Lys Leu Val Phe Phe Ala 1 5 12 6 PRT Artificial
Sequence peptide having antifibrillogenic activity and/or
neuroprotection 12 Lys Phe Val Phe Phe Ala 1 5 13 6 PRT Artificial
Sequence peptide having antifibrillogenic activity and/or
neuroprotection 13 Ala Phe Phe Val Leu Lys 1 5 14 4 PRT Artificial
Sequence peptide having antifibrillogenic activity and/or
neuroprotection 14 Lys Leu Val Phe 1 15 6 PRT Artificial Sequence
peptide having antifibrillogenic activity and/or neuroprotection 15
Lys Ala Val Phe Phe Ala 1 5 16 5 PRT Artificial Sequence peptide
having antifibrillogenic activity and/or neuroprotection 16 Lys Leu
Val Phe Phe 1 5 17 6 PRT Artificial Sequence peptide having
antifibrillogenic activity and/or neuroprotection 17 Lys Val Val
Phe Phe Ala 1 5 18 7 PRT Artificial Sequence peptide having
antifibrillogenic activity and/or neuroprotection 18 Lys Leu Val
Phe Phe Ala Gln 1 5 19 7 PRT Artificial Sequence peptide having
antifibrillogenic activity and/or neuroprotection 19 Lys Leu Val
Phe Phe Ala Gln 1 5 20 9 PRT Artificial Sequence peptide having
antifibrillogenic activity and/or neuroprotection 20 His His Gln
Lys Leu Val Phe Phe Ala 1 5 21 3 PRT Artificial Sequence peptide
having antifibrillogenic activity and/or neuroprotection 21 Asp Asp
Asp 1 22 6 PRT Artificial Sequence peptide having antifibrillogenic
activity and/or neuroprotection 22 Lys Val Asp Asp Gln Asp 1 5 23 4
PRT Artificial Sequence peptide having antifibrillogenic activity
and/or neuroprotection 23 His His Gln Lys 1 24 6 PRT Artificial
Sequence peptide having antifibrillogenic activity and/or
neuroprotection 24 Gln Lys Leu Val Phe Phe 1 5
* * * * *