U.S. patent application number 11/271021 was filed with the patent office on 2006-03-30 for beta-sheet breaker peptide analogs that inhibit beta-pleated sheet formation in amyloid beta-peptide.
This patent application is currently assigned to Axonyx, Inc.. Invention is credited to Claudio Soto-Jara.
Application Number | 20060069058 11/271021 |
Document ID | / |
Family ID | 30772493 |
Filed Date | 2006-03-30 |
United States Patent
Application |
20060069058 |
Kind Code |
A1 |
Soto-Jara; Claudio |
March 30, 2006 |
Beta-sheet breaker peptide analogs that inhibit beta-pleated sheet
formation in amyloid beta-peptide
Abstract
The present invention provides peptide analogs and peptide
mimetics that inhibit pleated sheet formation in amyloid
.beta.-peptide, pharmaceutical compositions thereof and their
therapeutic use. The inhibitory peptides possess activity as
inhibitors in the formation of amyloid-like deposits and are useful
in the treatment of Alzheimer's Disease.
Inventors: |
Soto-Jara; Claudio; (Geneva,
CH) |
Correspondence
Address: |
TRASK BRITT
P.O. BOX 2550
SALT LAKE CITY
UT
84110
US
|
Assignee: |
Axonyx, Inc.
New York
NY
|
Family ID: |
30772493 |
Appl. No.: |
11/271021 |
Filed: |
November 9, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10726366 |
Dec 3, 2003 |
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11271021 |
Nov 9, 2005 |
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09706540 |
Nov 4, 2000 |
6689753 |
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10726366 |
Dec 3, 2003 |
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60163911 |
Nov 5, 1999 |
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Current U.S.
Class: |
514/44R |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/4711 20130101; C07K 14/47 20130101; C07C 2601/08 20170501;
C07K 5/0207 20130101; C07C 229/34 20130101 |
Class at
Publication: |
514/044 |
International
Class: |
A61K 31/70 20060101
A61K031/70; A01N 43/04 20060101 A01N043/04 |
Claims
1. A compound capable of inhibiting conformational changes in a
prion PrP protein associated with amyloidosis, said compound
comprising: a .beta.-sheet breaker peptide analog produced by
chemical modification of a 13-residue prion inhibitor peptide iPrP
13 (SEQ ID NO:2), and able to inhibit said conformational changes
in prion PrP protein associated with amyloidosis.
2. The compound of claim 1, wherein alteration of the N- and
C-terminal ends of said iPrP13 is achieved by a process selected
from the group consisting of acetylation, amidation, desamination,
alcoholization, and combinations thereof.
3. A compound comprising: a .beta.-sheet breaker peptide analog
produced by chemically modifying iPrP 13 (SEQ ID NO:2), capable of
inhibiting a conformational change in a prion PrP protein
associated with amyloidosis, wherein said chemical modification is
achieved by a process selected from the group consisting of:
alteration of the N- and C-terminal ends of said prion inhibitor
peptide iPrP13; replacing at least one residue of said prion
inhibitor peptide iPrP 13 with .alpha.-aminoisobuiric acid (Aib);
methylation of the .alpha. carbon of at least one residue of said
prion inhibitor peptide iPrP13 with a D-enantiomeric residue;
forming head-to-tail cyclization of said prion inhibitor peptide
iPrP13; replacing amide bonds in said prion inhibitor peptide
iPrP13 with an amide bond surrogate; and combinations thereof.
4. The compound of claim 3, wherein alteration of the N- and
C-terminal ends of said iPrP13 is achieved by a process selected
from the group consisting of acetylation, amidation, desamination,
alcoholization, and combinations thereof.
5. The compound of claim 4, wherein said .beta.-sheet breaker
peptide analog is selected from the group consisting of: ac-Asp Ala
Pro Ala Ala Pro Ala Gly Pro Ala Val Pro Val-am, des-Asp Ala Pro Ala
Ala Pro Ala Gly Pro Ala Val Pro Val-am, ac-Asp Ala Pro Ala Ala Pro
Ala Gly Pro Ala Val Pro Val-alc, and des-Asp Ala Pro Ala Ala Pro
Ala Gly Pro Ala Val Pro Val-alc.
6. The compound of claim 4, wherein said 1-sheet breaker peptide
analog is selected from the group consisting of: Asp Ala Alb Ala
Ala Aib Ala Gly Aib Ala Val Aib Val (SEQ ID NO: 4); Asp Ala Pro Ala
Ala Pro Ala Gly Pro Ala (Me) Val Pro Val; Asp Ala Pro Ala Ala Pro
Ala Gly Pro Ala Val Pro (Me) Val; Asp Ala Pro Ala Ala Pro Ala Gly
Pro Ala (Me) Val Pro (Me) Val; asp ala pro ala ala pro ala gly pro
ala val pro val; asp Ala Pro Ala Ala Pro Ala Gly Pro Ala Val Pro
val; asp Ala Pro ala Ala Pro ala Gly Pro ala Val Pro val;
Asp.psi.[CH.sub.2CH.sub.2]Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Ala
Pro.psi.[CH.sub.2CH.sub.2]Ala Gly Pro.psi.[CH.sub.2CH.sub.2]Ala
ValPro.psi.[CH.sub.2CH.sub.2]Val; Asp.psi.[CH.sub.2S]Ala
Pro.psi.[CH.sub.2S]Ala Ala Pro.psi.[CH.sub.2S]Ala Gly
Pro.psi.[CH.sub.2S]Ala Val Pro.psi.[CH.sub.2S]Val; Ac-Asp Ala
Pro.psi.[CH.sub.2CH.sub.2]Ala Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Gly
Pro.psi.[CH.sub.2CH.sub.2]Ala Val Pro Val-Am. asp Ala
Pro.psi.[CH.sub.2CH.sub.2]Ala Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Gly
Pro.psi.[CH.sub.2CH.sub.2]Ala Val Pro val; Ac-Asp Ala
Pro.psi.[CH.sub.2S]Ala Ala Pro.psi.[CH.sub.2S]Ala Gly
Pro.psi.[CH.sub.2S]Ala Val Pro Val-Am; asp Ala
Pro.psi.[CH.sub.2S]Ala Ala Pro.psi.[CH.sub.2S]Ala Gly
Pro.psi.[CH.sub.2S]Ala Val Pro val; Ac-Asp Ala Aib Ala Ala Aib Ala
Gly Aib Ala Val Pro Val-Am (SEQ ID NO:5); Ac-Asp Ala
Pro.psi.[CH.sub.2CH.sub.2]Ala Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Gly
Pro.psi.[CH.sub.2CH.sub.2]Ala Val Pro (Me) Val; Ac-Asp Ala pro Ala
Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Gly pro Ala Val Pro Val-Am; asp
Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Ala Pro.psi.[CH.sub.2CH.sub.2]Ala
Gly Pro.psi.[CH.sub.2CH.sub.2]Ala Val Pro (Me) Val; asp Ala Aib Ala
Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Gly pro Ala Val Pro (Me)Val (SEQ
ID NO:6); asp Ala Aib Ala Ala Pro.psi.[CH.sub.2S]Ala Gly pro Ala
Val Pro (Me) Val; asp Ala Pro.psi.[CH.sub.2S]Ala Ala
Pro.psi.[CH.sub.2S]Ala Gly Pro.psi.[CH.sub.2S]Ala Val Pro (Me) Val;
Ac-Asp Ala Aib Ala Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Gly Aib Ala
Val Pro (Me) Val (SEQ ID NO:7); ##STR8## Asp Ala pro Ala Ala
Pro.psi.[CH.sub.2CH.sub.2]Ala Gly pro Ala Val Pro Val Asp Ala Aib
Ala Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Gly Aib Ala (Me) Val Pro Val
Pro Val Ac-Asp Ala Pro.psi.[CH.sub.2S]Ala ala
Pro.psi.[CH.sub.2S]Ala gly Pro.psi.[CH.sub.2S]Ala (Me) Val Pro
Val-Am; Ac-Asp Ala Aib ala Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Gly
pro Ala Val Pro (Me) Val; asp Ala Aib Ala Ala
Pro.psi.[CH.sub.2CH.sub.2]Ala Gly Aib ala Val Pro Val-Am; Ac-Asp
Ala pro Ala Ala Pro.psi.[CH.sub.2CH.sub.2]Ala gly pro Ala (Me) Val
Pro Val-Am; asp Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Ala
Pro.psi.[CH.sub.2CH.sub.2]Ala gly Pro.psi.[CH.sub.2CH.sub.2]Ala val
Pro val; Ac-Asp Ala pro Ala ala Aib Ala gly pro Ala (Me)Val Pro
Val-Am (SEQ ID NO:8); Asp Ala pro Ala Ala
Pro.psi.[CH.sub.2CH.sub.2]Ala Gly pro Ala Val Pro Val; Asp Ala Aib
Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Gly Aib Ala (Me) Val Pro Val; and
##STR9##
7. A pharmaceutical composition, comprising the .beta.-sheet
breaker peptide analog of claim 3 and a pharmaceutically acceptable
excipient or stabilizer.
8. The pharmaceutical composition of claim 7, wherein alteration of
the N- and C-terminal ends of said iPrP13 is achieved by a process
selected from the group consisting of acetylation, amidation,
desamination, alcoholization, and combinations thereof.
9. The pharmaceutical composition of claim 8, wherein said
.beta.-sheet breaker peptide analog is selected from the group
consisting of: ac-Asp Ala Pro Ala Ala Pro Ala Gly Pro Ala Val Pro
Val-am, des-Asp Ala Pro Ala Ala Pro Ala Gly Pro Ala Val Pro Val-am,
ac-Asp Ala Pro Ala Ala Pro Ala Gly Pro Ala Val Pro Val-alc, and
des-Asp Ala Pro Ala Ala Pro Ala Gly Pro Ala Val Pro Val-alc.
10. The pharmaceutical composition of claim 8, wherein said
.beta.-sheet breaker peptide analog is selected from the group
consisting of: Asp Ala Alb Ala Ala Aib Ala Gly Aib Ala Val Aib Val
(SEQ ID NO: 4); Asp Ala Pro Ala Ala Pro Ala Gly Pro Ala (Me) Val
Pro Val; Asp Ala Pro Ala Ala Pro Ala Gly Pro Ala Val Pro (Me) Val;
Asp Ala Pro Ala Ala Pro Ala Gly Pro Ala (Me) Val Pro (Me) Val; asp
ala pro ala ala pro ala gly pro ala val pro val; asp Ala Pro Ala
Ala Pro Ala Gly Pro Ala Val Pro val; asp Ala Pro ala Ala Pro ala
Gly Pro ala Val Pro val; Asp.psi.[CH.sub.2CH.sub.2]Ala
Pro.psi.[CH.sub.2CH.sub.2]Ala Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Gly
Pro.psi.[CH.sub.2CH.sub.2]Ala ValPro.psi.[CH.sub.2CH.sub.2]Val;
Asp.psi.[CH.sub.2S]Ala Pro.psi.[CH.sub.2S]Ala Ala
Pro.psi.[CH.sub.2S]Ala Gly Pro.psi.[CH.sub.2S]Ala Val
Pro.psi.[CH.sub.2S]Val; Ac-Asp Ala Pro.psi.[CH.sub.2CH.sub.2]Ala
Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Gly Pro.psi.[CH.sub.2CH.sub.2]Ala
Val Pro Val-Am. asp Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Ala
Pro.psi.[CH.sub.2CH.sub.2]Ala Gly Pro.psi.[CH.sub.2CH.sub.2]Ala Val
Pro val; Ac-Asp Ala Pro.psi.[CH.sub.2S]Ala Ala
Pro.psi.[CH.sub.2S]Ala Gly Pro.psi.[CH.sub.2S]Ala Val Pro Val-Am;
asp Ala Pro.psi.[CH.sub.2S]Ala Ala Pro.psi.[CH.sub.2S]Ala Gly
Pro.psi.[CH.sub.2S]Ala Val Pro val; Ac-Asp Ala Aib Ala Ala Aib Ala
Gly Aib Ala Val Pro Val-Am (SEQ ID NO:5); Ac-Asp Ala
Pro.psi.[CH.sub.2CH.sub.2]Ala Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Gly
Pro.psi.[CH.sub.2CH.sub.2]Ala Val Pro (Me) Val; Ac-Asp Ala pro Ala
Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Gly pro Ala Val Pro Val-Am; asp
Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Ala Pro.psi.[CH.sub.2CH.sub.2]Ala
Gly Pro.psi.[CH.sub.2CH.sub.2]Ala Val Pro (Me) Val; asp Ala Aib Ala
Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Gly pro Ala Val Pro (Me)Val (SEQ
ID NO:6); asp Ala Aib Ala Ala Pro.psi.[CH.sub.2S]Ala Gly pro Ala
Val Pro (Me) Val; asp Ala Pro.psi.[CH.sub.2S]Ala Ala
Pro.psi.[CH.sub.2S]Ala Gly Pro.psi.[CH.sub.2S]Ala Val Pro (Me) Val;
(SEQ ID NO:7); ##STR10## Ac-Asp Ala Pro.psi.[CH.sub.2S]Ala ala
Pro.psi.[CH.sub.2S]Ala gly Pro.psi.[CH.sub.2S]Ala (Me) Val Pro
Val-Am; Ac-Asp Ala Aib ala Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Gly
pro Ala Val Pro (Me) Val; asp Ala Aib Ala Ala
Pro.psi.[CH.sub.2CH.sub.2]Ala Gly Aib ala Val Pro Val-Am; Ac-Asp
Ala pro Ala Ala Pro.psi.[CH.sub.2CH.sub.2]Ala gly pro Ala (Me) Val
Pro Val-Am; asp Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Ala
Pro.psi.[CH.sub.2CH.sub.2]Ala gly Pro.psi.[CH.sub.2CH.sub.2]Ala val
Pro val; Ac-Asp Ala pro Ala ala Aib Ala gly pro Ala (Me)Val Pro
Val-Am (SEQ ID NO:8); Asp Ala pro Ala Ala
Pro.psi.[CH.sub.2CH.sub.2]Ala Gly pro Ala Val Pro Val; Asp Ala Aib
Ala Pro.psi.[CH.sub.2CH.sub.2]Ala Gly Aib Ala (Me) Val Pro Val; and
##STR11##
11. A method for reducing formation of amyloid or amyloid like
deposits involving conformational changes in a prion PrP protein,
or reducing the amount of prion PrP protein that has already formed
into amyloid or amyloid-like deposits, said method comprising:
bringing into the presence of said prion PrP protein, either prior
to or after said conformational changes thereof into amyloid
deposits, an effective amount of the compound of claim 3.
12. A method for reducing formation of amyloid or amyloid like
deposits involving conformational changes in a prion PrP protein,
or reducing the amount of said prion PrP protein which has already
formed into amyloid or amyloid-like deposits, said method
comprising: bringing into the presence of said prion PrP protein,
either prior to or after said conformational changes thereof into
amyloid deposits, the pharmaceutical composition of claim 7 in an
amount effective to reduce the formation of amyloid or amyloid like
deposits involving conformational changes in a prion PrP
protein.
13. A method of preventing or treating a disorder or disease
associated with amyloid or amyloid-like deposits or pathological
beta-sheet-rich precursors thereof, the method comprising:
administering an effective amount of a compound of claim 3 to a
subject thought to be in need thereof.
14. The method according to claim 13, wherein the subject is
thought to be suffering from a disease selected from the group
consisting of human prion diseases, prion associated human
neurodegenerative diseases and animal prion diseases.
15. The method according to claim 14, wherein the subject is
suffering from a disease selected from the group consisting of
spongiform encephalopathy, kuru, Creutzfeldt-Jakob Disease,
Gerstmann-Strausslet-Scheinker Syndrome, scrapie, transmissible
mink encephalopathy, and chronic wasting-disease.
16. The method according to claim 13, wherein the disease is
scrapie.
17. The method according to claim 13, wherein the disease is bovine
spongiform encephalopathy.
18. A method of detecting a prion PrP protein disease, the method
comprising: contacting the inhibitory peptide of claim 3 with a
prion PrP protein of a subject; binding the inhibitory peptide to a
prion PrP protein; and visualizing binding of the inhibitory
peptide to the prion PrP protein.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/726,366, filed Dec. 3, 2003, which is a continuation of
U.S. patent application Ser. No. 09/706,540, filed Nov. 4, 2000,
now U.S. Pat. No. 6,689,753, issued Feb. 10, 2004, which claims the
benefit of U.S. Provisional Application No. 60/163,911, filed Nov.
5, 1999, the entirety of each of which is incorporated by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to peptide analogs and peptide
mimetics of .beta.-sheet breaker peptides suitable for in vivo use
in treating mammals with protein conformational diseases such as
Alzheimer's and prion disease. More particularly, the present
invention is directed to novel peptide analogs and mimetics,
pharmaceutical compositions containing one or a mixture of such
peptide analogs and mimetics, and methods for preventing, treating,
or detecting disorders or diseases associated with abnormal protein
folding into amyloid or amyloid-like deposits or precursors thereof
having a pathological .beta.-sheet structure.
BACKGROUND
[0003] Extensive evidence has been accumulated indicating that
several diverse disorders have the same molecular basis, i.e., a
change in a protein conformation (Thomas et al., Trends Biochem.
Sci. 20:456-459, 1995; Soto, J. Mol. Med. 77:412-418, 1999). These
protein conformational diseases include Alzheimer's disease,
prion-related disorders, systemic amyloidosis, serpin-deficiency
disorders, Huntington's disease and Amyotrophic Lateral Sclerosis
(Soto 1999, supra). The hallmark event in protein conformational
disorders is a change in the secondary and tertiary structure of a
normal protein without alteration of the primary structure. The
conformationally modified protein may be implicated in the disease
by direct toxic activity, by the lack of biological function of
normally-folded protein, or by improper trafficking (Thomas et al.,
1995, supra). In the cases where the protein is toxic, it usually
self-associates and becomes deposited as amyloid fibrils in diverse
organs, inducing tissue damage (Thomas et al., 1995, supra; Kelly,
Curr. Opin. Struct. Biol. 6:11-17, 1996; Soto, 1999, supra).
[0004] Alzheimer's disease (AD) is a devastating neurodegenerative
problem characterized by loss of short-term memory, disorientation,
and impairment of judgment and reasoning. AD is the most common
dementia in elderly population. It is estimated that more than
twenty-five million people worldwide are affected in some degree by
AD (Teplow, Amyloid 5:121-142, 1998). A hallmark event in AD is the
deposition of insoluble protein aggregates, known as amyloid, in
brain parenchyma and cerebral vessel walls. The main component of
amyloid is a 4.3 KDa hydrophobic peptide, named amyloid
.beta.-peptide (A.beta.) that is encoded on the chromosome 21 as
part of a much longer precursor protein (APP) (Selkoe, Science
275:630-631, 1997). Genetic, biochemical, and neuropathological
evidence accumulated in the last 10 years strongly suggest that
amyloid plays an important role in early pathogenesis of AD and
perhaps triggers the disease (Soto et al., J. Neurochem.
63:1191-1198, 1994; Selkoe, 1997, supra; Teplow, 1998, supra;
Sisodia and Price, FASEB J. 9:366-370, 1995; Soto, Mol. Med. Today
5:343-350, 1999).
[0005] Amyloid is a generic term that describes fibrillar
aggregates that have a common structural motif, i.e., the
.beta.-pleated sheet conformation (Serpell et al., Cell Mol. Life
Sci. 53:887, 1997; Sipe, Ann. Rev. Biochem. 61:947-975, 1992).
These aggregates exhibit specific tinctorial properties, including
the ability to emit a green birefringent glow after staining with
Congo red, and the capacity to bind the fluorochrome, thioflavin S
(Sipe, 1992, supra; Ghiso et al., Mol. Neurobiol. 8:49-64, 1994).
There are more than a dozen human diseases of different etiology
characterized by the extracellular deposition of amyloid in diverse
tissues, which lead to cell damage, organ dysfunction, and death.
Among the diseases involving amyloidosis, it is possible to
highlight Alzheimer's disease, prion-related disorders (also known
as transmissible spongiform encephalopathy), and systemic
amyloidosis (Table 1). The amyloid fibrils are usually composed of
proteolytic fragments of normal or mutant gene products. There are
over 16 different proteins (Table 1) involved in amyloid deposition
in distinct tissues (Ghiso et al., 1994, supra).
[0006] The formation of amyloid is basically a problem of protein
folding, whereby a mainly random coil soluble peptide becomes
aggregated, adopting a .beta.-pleated sheet conformation (Kelly,
1996, supra; Soto, 1999, supra). Amyloid formation proceeds by
hydrophobic interactions among conformationally altered
amyloidogenic intermediates, which become structurally organized
into a 0-sheet conformation upon peptide interaction. The
hydrophobicity appears to be important to induce interaction of the
monomers leading to aggregation, while the .beta.-sheet
conformation might determine the ordering of the aggregates in
amyloid fibrils. In an attempt to inhibit amyloid fibril formation,
these two properties were separated by designing short synthetic
peptides bearing sequence homology and a similar degree of
hydrophobicity as the peptide domain implicated in the
conformational change, but having a very low propensity to adopt a
.beta.-sheet conformation (called .beta.-sheet breaker peptides)
(Soto et al., 1996, supra; Soto et al., 1998, supra). The aim was
to design a peptide with the ability to bind specifically to the
amyloidogenic peptide forming a complex that stabilizes the
physiological conformation and destabilizes the abnormal
conformation of the peptide (Soto, 1999, supra). TABLE-US-00001
TABLE 1 Disorders related with amyloidosis and the protein
component of the amyloid fibrils DISEASE FIBRIL COMPONENT
Alzheimer's disease Amyloid-.beta. protein Primary systemic
amyloidosis Immunoglobulin light chain or fragments thereof
Secondary systemic amyloidosis, Fragments of serum Familial
Mediterranean fever amyloid-A Spongiform encephalopathy Fragments
of prion protein Senile systemic amyloidosis, Transthyretin and
fragments thereof Familial amyloid polyneuropathy
Hemodialysis-related amyloidosis .beta.2-microglobulin Hereditary
cerebral amyloid Cystain C angiopathy, Icelandic type Familial
amyloidosis, Finnish type Gelsolin fragments Type II diabetes
Fragments of islet amyloid polypeptide Familial amyloid
polyneuropathy Fragments of apolipoprotein A-1 Medullar carcinoma
of the thyroid Fragments of calcitonin Atrial amyloidosis Atrial
natriuretic factor Hereditary non-neuropathic systemic Lysozyme or
fragments amyloidosis thereof Hereditary renal amyloidosis
Fibrinogen fragments Islet amyloid Insulin Amyloidosis in
senescence Apolipoprotein A-II
[0007] .beta.-sheet breaker peptides have so far been designed to
block the conformational changes that occur in both A.beta.and
prion protein (PrP), which are implicated in the pathogenesis of
Alzheimer's and prion disease, respectively. The prior art has
previously shown that 11- and five-residue .beta.-sheet breaker
peptides (namely, iA.beta.1 and iA.beta.5, respectively) homologous
to the central hydrophobic region of A.beta. inhibit peptide
conformational changes that result in amyloid formation and also
dissolved preformed fibrils in vitro (Soto et al., Biochem.
Biophys. Res. Commun. 226:672-680, 1996; Soto et al., Nature Med.
4:822-826, 1998). In addition, the five-residue peptide is capable
of preventing the neuronal death induced by the formation of
.beta.-sheet rich oligomeric A.beta. structures in cell culture
experiments (Soto et al., 1998, supra). Furthermore, by using a rat
model of amyloidosis induced by intracerebral injection of
A.beta.1-42, the prior art has shown that co-injections of the
five-residue .beta.-sheet breaker peptide decreased cerebral
A.beta. accumulation and completely blocked the deposition of
fibrillar amyloid-like lesions in the rat brain (Soto et al., 1998,
supra). Finally, the .beta.-sheet breaker peptide injected eight
days after the injection of A.beta. was able to disassemble
preformed A.beta. fibrils in the rat brain in vivo, that leads to a
reduction in the size of amyloid deposits (E. M. Sigurdsson, B.
Permanne, C. Soto, T. Wisniewski, B. Frangione, J. Neuropathol.
Exp. Neurol. January 2000; 59(1):11-7). Interestingly, removal of
amyloid by the .beta.-sheet breaker peptide reverts the associated
cerebral histologic damage, including neuronal shrinkage and
microglial activation.
[0008] .beta.-sheet breaker peptides have also been designed to
prevent and to revert conformational changes caused by prions
(PrP). Based on the same principles and using as a template the PrP
sequence 114-122, the prior art has shown that when a set of
.beta.-sheet breaker peptides was synthesized, a 13-residue peptide
(iPrP13) showed the greatest activity (Soto, 1999, supra). Several
in vitro cell culture and in vivo assays were used to test for
inhibitory activity and the results clearly indicated that it is
possible not only to prevent the PrP.sup.c.fwdarw.PrP.sup.sc
conversion, but more interestingly to revert the infectious
PrP.sup.sc conformer to a biochemical and structural state similar
to PrP.sup.c (Soto et al., manuscript submitted).
[0009] Short peptides have been utilized extensively as drugs in
medicine (Rao et al., C. Basava and G. M. Anantharamaiah, eds.
Boston: Birkhauser, pp. 181-198, 1994). However, the development of
peptide drugs is strongly limited by their lack of oral
bioavailability and their short duration of action resulting from
enzymatic degradation in vivo (Fauchere and Thurieau, Adv. Drug
Res. 23:127-159, 1992). Progress in recent years toward the
production of peptide analogs (such as pseudopeptides and peptide
mimetics) with lower susceptibility to proteolysis has increased
the probability to obtain useful drugs structurally related to
their parent peptides (Fauchere and Thurieau, 1992, supra).
Improving peptide stability to proteases not only increases the
half-life of the compound in the circulation but also enhances its
ability to be transported or absorbed at different levels,
including intestinal absorption and blood-brain barrier
permeability, because transport and absorption appear to be highly
dependent upon the time of exposure of membranes or barriers to the
bioactive species (Fauchere and Thurieau, 1992, supra).
SUMMARY OF THE INVENTION
[0010] The present invention is an inhibitory peptide capable of
inhibiting .beta.-pleated sheet formation in amyloid
.beta.-peptide, the inhibitory peptide being a .beta.-sheet breaker
peptide analog designed by chemical modification of a 0-sheet
breaker peptide capable of inhibiting .beta.-pleated sheet
formation in amyloid .beta.-peptide.
[0011] The peptide is altered chemically by: (1) modifications to
the N- and C-terminal ends of the peptide; (2) changes of the
side-chain, which can involve amino acid substitutions; (3)
modification in the a-carbon including methylations, alkylations
and dehydrogenations; (4) chirality changes by replacing D- for
L-residue; (5) head-to-tail cyclizations; and (6) introduction of
amide bond replacements, i.e., changing the atoms participating in
the peptide (or amide) bond.
[0012] The present invention also includes an inhibitory peptide
capable of inhibiting conformational changes in prion PrP protein
associated with amyloidosis, the inhibitory peptide being a
.beta.-sheet breaker peptide analog designed by chemical
modification of a .beta.-sheet breaker peptide capable inhibiting
the conformational changes in prior PrP protein associated with
amyloidosis.
[0013] In addition, the present invention includes a peptide
mimetic with the following structure: ##STR1##
[0014] In another embodiment, the peptide mimetic has the following
structure: ##STR2##
[0015] In yet another embodiment, the peptide mimetic has the
following structure: ##STR3##
[0016] The present invention also includes a method for preventing,
treating, or detecting disorders or diseases associated with
abnormal protein folding into amyloid or amyloid-like deposits or
precursors thereof having a pathological .beta.-sheet structure is
claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a schematic representation of the peptide bond and
the potential target sites for peptide modifications.
[0018] FIG. 2 is a graph depicting the pharmacokinetics of an
11-residue .beta.-sheet breaker peptide inhibitor of Alzheimer's
amyloidosis (Seq. RDLPFYPVPID, SEQ ID NO:9) in its natural
L-configuration and in the non-natural D-form.
[0019] FIGS. 3A and 3B are representations of the tridimensional
structure of Alzheimer's and prion .beta.-sheet breaker peptides
iA.beta.5 and iPrP13, respectively.
[0020] FIGS. 4a and 4b are graphs showing the bioavailability and
stability of iA.beta.5 and Ac-iA.beta.5-Am, respectively over
time.
[0021] FIG. 5a provides a graphical comparison of A.beta.1-40
incubated with various other peptides.
[0022] FIG. 5b is a graph of amyloid formation vs. the
Ac-iA.beta.5-Am concentration.
[0023] FIG. 5c is a graph of amyloid formation vs. the iA.beta.5
concentration.
[0024] FIG. 6 shows a model where there is an 83% dissolution of
deposits in the ventricle area and a 30% dissolution of amyloid
plaque in the amygdala.
DETAILED DESCRIPTION OF THE INVENTION
[0025] In the present invention, the bioavailability and stability
of an inhibitory peptide is improved by chemically modifying the
parent peptide to produce a derivative more suitable for in vivo
use, which is preferably administered orally. The inhibitory
peptide is capable of inhibiting .beta.-pleated sheet formation in
an amyloid .beta.-peptide. Moreover, the inhibitory peptide is a
.beta.-sheet breaker peptide analog designed by chemical
modification of a .beta.-sheet breaker peptide capable of
inhibiting .beta.-pleated sheet formation in amyloid
.beta.-peptide.
[0026] This invention also includes an inhibitory peptide capable
of inhibiting conformational changes in prion PrP protein
associated with amyloidosis, where the inhibitory peptide is a
.beta.-sheet breaker peptide analog designed by chemical
modification of a .beta.-sheet breaker peptide and is capable of
inhibiting the conformational changes in prion PrP protein
associated with amyloidosis.
[0027] In FIG. 1, a generalized peptide backbone is shown, where
possible targets for chemical modification are highlighted. The
possible targets include the following: (1) modifications to the N-
and C-terminal ends of the peptide (targets a and b); (2) changes
of the side-chain (target c) which usually involve amino acid
substitutions; (3) modification in the a-carbon (target d)
including methylations, alkylations and dehydrogenations; (4)
chirality changes by replacing D- for L-residue; (5) head-to-tail
cyclizations; and (6) introduction of amide bond replacements
(target e), i.e., changing the atoms participating in the peptide
(or amide) bond. The latter derivatives are known as pseudopeptides
or amide bond surrogates.
[0028] Natural peptides are usually degraded by the concerted
action of specialized endopeptidases and unspecific exopeptidases.
Endopeptidases are often present in tissues and cellular
compartments and convert the peptide into two or more inactive
fragments. Exopeptidases are generally present in blood and
peripheral organs and carry out the degradation of the intact
peptides or their fragments to the constituent amino acids and
hence contribute to the disappearance of the peptides from the
circulation. Exopeptidases recognize the free amino or carboxyl
groups in peptides. Therefore, modification of those groups often
diminish or abolish exopeptidase degradation. Head-to-tail peptide
cyclization results in the absence of free end-terminal groups, and
hence also minimizes cleavage by exopeptidases. On the other hand,
endopeptidases recognize the atoms participating in the amide bond.
Thus, amide bond replacements dramatically decrease degradation by
endopeptidases. The same usually happens with modifications to the
a-carbon. Since most (if not all) of the exo- and endo-proteases
are stereospecific, substitutions of the natural L-amino acids by
the D-stereoisomers result in a clear increase in peptide
stability. Finally, peptide mimetics are usually completely
resistant to proteolytic degradation and often can be administrated
orally.
.beta.-Sheet Breaker Analogues Designed by Chemical Modifications
of the Lead Peptides.
[0029] Starting from the five-residue Alzheimer's inhibitor peptide
(iA.beta.5, Seq. LPFFD, also denoted as Leu Pro Phe Phe Asp, SEQ ID
NO:1) and the 13-residue prion inhibitor peptide (iPrP13, Seq.
DAPAAPAGPAVPV, also denoted as Asp Ala Pro Ala Ala Pro Ala Gly Pro
ala Val Pro Val, SEQ ID NO:2), the modifications described below
are designed. The peptides used in the present invention are
synthesized using standard protocols as disclosed by Bergmann et
al., and incorporated herein by reference. (Bergmann and Zervas,
Berichte der Deutschen Chemischen Gesellschaft (1932)
65:1192-1201.)
[0030] a) N- and C-terminal modifications. N-terminal acetylation
or desamination confers protection against digestion by a number of
aminopeptidases while the presence of amides or alcohols replacing
the C-terminal carboxyl group prevent splitting by several
carboxypeptidases, including carboxypeptidases A and B. The altered
peptide sequences including these modifications are the following,
where ac is acetylation, am is amidation, des is desamination, and
alc is alcoholization: TABLE-US-00002 Alzheimer's Inhibitors Prion
Inhibitors ac-Leu Pro Phe Phe Asp-am ac-Asp Ala Pro Ala Ala Pro Ala
Gly Pro Ala Val Pro Val-am des-Leu Pro Phe Phe Asp-am des-Asp Ala
Pro Ala Ala Pro Ala Gly Pro Ala Val Pro Val-am ac-Leu Pro Phe Phe
Asp-alc ac-Asp Ala Pro Ala Ala Pro Ala Gly Pro Ala Val Pro Val-alc
des-Leu Pro Phe Phe Asp-alc des-Asp Ala Pro Ala Ala Pro Ala Gly Pro
Ala Val Pro Val-alc
[0031] b) Side-chain changes. The presence of non-natural amino
acids usually increases peptide stability. In addition, at least
one of these amino acids (.alpha.-aminoisobutyric acid or Aib)
imposes significant constraints to model peptides' diminishing
their conformational flexibility. In particular, the incorporation
of Aib into .beta.-sheet model peptides induces the complete
disruption of this structure. The .beta.-sheet blocking activity of
Aib is comparable or even greater than the natural residue proline
used in the peptide as a .beta.-sheet blocker. Therefore, the
introduction of Aib is expected to enhance peptide stability and
inhibitory activity at the same time. TABLE-US-00003 Alzheimer's
Prion Inhibitors Inhibitors (SEQ ID NO:3) (SEQ ID NO:4) Leu Aib Phe
Phe Asp Asp Ala Aib Ala Ala Aib Ala Gly Aib Ala Val Aib Val
[0032] c) Modifications in the .alpha.-carbon. The most commonly
used .alpha.-carbon modification to improve peptide stability is
.alpha.-methylation. In addition, replacement of the hydrogen atom
linked to the a-carbon of Phe, Val or Leu has been shown to favor
the adoption of .beta.-bend conformation and strongly disfavor the
formation of .beta.-pleated sheet structures. According to the
present invention, methylation of those residues in the inhibitor
peptides is expected to enhance stability and potency.
[0033] Alzheimer's Inhibitors [0034] (Me)Leu Pro Phe Phe Asp [0035]
Leu Pro (Me)Phe Phe Asp [0036] Leu Pro Phe (Me)Phe Asp [0037]
(Me)Leu Pro (Me)Phe (Me)Phe Asp
[0038] Prion inhibitors [0039] Asp Ala Pro Ala Ala Pro Ala Gly Pro
Ala (Me)Val Pro Val [0040] Asp Ala Pro Ala Ala Pro Ala Gly Pro Ala
Val Pro (Me)Val [0041] Asp Ala Pro Ala Ala Pro Ala Gly Pro Ala
(Me)Val Pro (Me)Val
[0042] d) Chirality changes. Replacement of the natural L-residue
by the D-enantiomers dramatically increases resistance to
proteolytic degradation. The increase in stability by introduction
of D-residue has already been demonstrated for the 11-residue
.beta.-sheet breaker peptide (iA.beta.1). In vivo studies showed
that the peptide bearing the natural sequence rapidly degraded in
rat plasma. Indeed, approximately 90% of iA.beta.1 was degraded
within minutes after intravenous injection. Conversely, a
derivative of iA.beta.1 containing all the residue in the D-form
showed virtually no degradation in the plasma after injection for
15 minutes. For detection, the peptide was radio-iodinated using
standard procedures. Peptide stability was evaluated after i.v.
bolus injection in rats by precipitation with trichloroacetic acid.
Quantitation of the intact peptide was also done by paper
chromatography. Thus, iA.beta.B5 and iPrP13 peptides (FIGS. 3A and
3B, respectively) containing all-D residue as well as peptides
containing D-residue only at the N- and C-terminal ends to prevent
exopeptidase degradation are included in the compounds of the
invention. In addition to the latter, D-residue are used after each
proline amino acid, since it has been reported that a frequent
endopeptidase cleavage site is after this residue by an enzyme
known as prolylendopeptidase. TABLE-US-00004 Alzheimer's Inhibitors
Prion Inhibitors leu pro phe phe asp asp ala pro ala ala pro ala
gly pro ala val pro val leu Pro Phe Phe asp asp Ala Pro Ala Ala Pro
Ala Gly Pro Ala Val Pro val leu Pro phe Phe asp asp Ala Pro ala Ala
Pro ala Gly Pro ala Val Pro val
Amino Acids Written with Lower Case Letters Denote D-Residue.
[0043] e) Cyclic peptides. Conformation ally constrained cyclic
peptides represent better drug candidates than linear peptides due
to their reduced conformational flexibility and improved resistance
to exopeptidase cleavage. Two alternative strategies have been used
to convert a linear sequence into a cyclic structure. One is the
introduction of cysteine residue to achieve cyclization through the
formation of a disulfide bridge and the other is the side-chain
attachment strategy involving resin-bound head-to-tail cyclization.
To avoid modifications of the peptide sequence the latter approach
is used. .beta.-sheet breaker peptides contain the ideal sequences
for facilitating macrocyclization because proline, due to its
ability to promote turns and loops, is a constituent of many
naturally occurring or artificially synthesized cyclic peptides.
##STR4##
[0044] f) Pseudopeptides. Pseudopeptides or amide bond surrogates
refers to peptides containing chemical modifications of some (or
all) of the peptide bonds. Amide bond replacements are usually
represented by retaining the amino acid designation according to
the side-chain and specifying the changes that occur between the
a-carbons, using the nomenclature known as "psi-bracket."
[0045] For example, the term Ala .psi.[CH.sub.2CH.sub.2]Gly refers
to the moiety
NH.sub.2CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.2CO.sub.2H. Several
amide bond surrogates have been described in Table 2 below.
TABLE-US-00005 TABLE 2 Some amide bond surrogates and their
properties Surrogate Properties CH.sub.2 Short, flexible
CH.sub.2CH.sub.2 Flexible, hydrophobic CH.dbd..dbd.CH Rigid,
hydrophobic C.ident..ident.C Very rigid CH.sub.2NH Flexible,
hydrophilic COCH.sub.2 Flexible, hydrophilic CH.sub.2S Flexible,
hydrophobic CH.sub.2SO.sub.2 More rigid, hydrophilic NHCO Rigid,
hydrophilic
[0046] Some of them are found in naturally occurring peptide
analogs (such as .psi.[CHOH], .psi.[CSNH], .psi.[COO]) while others
have been artificially synthesized. The introduction of amide bond
surrogates not only decreases peptide degradation but also may
significantly modify some of the biochemical properties of the
peptides, particularly the conformational flexibility and
hydrophobicity. It is likely that an increase in conformational
flexibility will be beneficial for docking the inhibitor to the
A.beta. and PrP binding sites. On the other hand, since the
interaction between the amyloidogenic proteins and the inhibitors
seems to depend to a great extent on hydrophobic interactions, it
is likely that amide bond replacement increasing hydrophobicity may
enhance affinity and hence, potency of the inhibitors. In addition,
increased hydrophobicity could also enhance transport of the
peptide across membranes and thus, improve barrier permeability
(blood-brain barrier and intestinal barrier). Therefore, to
synthesize pseudopeptides amide bond replacement is used thereby
increasing flexibility and hydrophobicity, such as
.psi.[CH.sub.2CH.sub.2] and .psi.{[CH.sub.2S]. The amide bonds to
replace are those located at the end of the peptide to prevent
exoprotease degradation and after each of the prolines, since it
has been described that a frequent endopeptidase cleavage site
occurs after this residue by an enzyme known as
prolylendopeptidase. Additional amide bonds that need to be
protected are determined by experimental studies involving the
analysis of the degradation of .beta.-sheet breaker peptides in the
plasma and tissue.
[0047] Alzheimer's Inhibitors [0048] Leu .psi.[CH.sub.2CH.sub.2]Pro
.psi.[CH.sub.2CH.sub.2]Phe Phe .psi.[CH.sub.2CH.sub.2]Asp [0049]
Leu .psi.[CH.sub.2S]Pro .psi.[CH.sub.2S]Phe Phe
.psi.[CH.sub.2S]Asp
[0050] Prion inhibitors [0051] Asp .psi.[CH.sub.2CH.sub.2]Ala Pro
.psi.[CH.sub.2CH.sub.2]Ala Ala Pro .psi.[CH.sub.2CH.sub.2]Ala Gly
Pro .psi.[CH.sub.2CH.sub.2]Ala Val Pro .psi.[CH.sub.2CH.sub.2]Val
[0052] Asp .psi.[CH.sub.2S]Ala Pro .psi.[CH.sub.2S] Ala Ala Pro
.psi.[CH.sub.2S]Ala Gly Pro .psi.[CH.sub.2S]Ala Val Pro
.psi.[CH.sub.2S]Val
[0053] g) Mixture of several modifications. By taking into the
account the features of the peptide drugs on the market or under
current development, it is clear that most of the peptides
successfully stabilized against proteolysis consist of a mixture of
several types of the above described modifications. This conclusion
makes sense in the light of the knowledge that many different
enzymes are implicated in peptide degradation. The following
structures contain combinations of several types of chemical
modifications:
[0054] Alzheimer's Inhibitors [0055] Ac-Leu Pro
.psi.[CH.sub.2CH.sub.2]Phe Phe Asp-Am [0056] Ac-Leu Pro
.psi.[CH.sub.2S]Phe Phe Asp-Am [0057] (Me)Leu Pro
.psi.[CH.sub.2CH.sub.2]Phe Phe Asp-Am [0058] leu Pro
.psi.[CH.sub.2CH.sub.2]Phe Phe asp [0059] leu Pro
.psi.[CH.sub.2S]Phe Phe asp [0060] Ac-Leu Aib Phe Phe Asp-Am [0061]
(Me)Leu Aib Phe Phe Asp-Am [0062] Leu Pro
.psi.[CH.sub.2CH.sub.2]Phe Phe asp ##STR5## [0063] Ac-Leu pro Phe
Phe Asp-Am [0064] Ac-Leu Pro .psi.[CH.sub.2CH.sub.2]Phe phe Asp-Am
[0065] Ac-Leu Pro .psi.[CH.sub.2S]Phe phe Asp-Am [0066] Ac-Leu Pro
.psi.[CH.sub.2CH.sub.2]Phe (Me)Phe Asp-Am [0067] Ac-Leu Pro
.psi.[CH.sub.2CH.sub.2]Phe (Me)Phe asp [0068] Ac-Leu Pro phe phe
Asp-Am [0069] Ac-Leu Pro (Me)Phe phe Asp-Am [0070] leu Pro
.psi.[CH.sub.2CH.sub.2]Phe phe asp [0071] leu Pro (Me)Phe phe asp
[0072] Ac-Leu Aib Phe phe Asp-Am
[0073] Prion inhibitors [0074] Ac-Asp Ala Pro
.psi.[CH.sub.2CH.sub.2]Ala Ala Pro .psi.[CH.sub.2CH.sub.2]Ala Gly
Pro .psi.[CH.sub.2CH.sub.2]Ala Val Pro Val-Am [0075] asp Ala Pro
.psi.[CH.sub.2CH.sub.2]Ala Ala Pro .psi.[CH.sub.2CH.sub.2]Ala Gly
Pro .psi.[CH.sub.2CH.sub.2]Ala Val Pro val [0076] Ac-Asp Ala Pro
.psi.[CH.sub.2S]Ala Ala Pro .psi.[CH.sub.2S]Ala Gly Pro
.psi.[CH.sub.2S]Ala Val Pro Val-Am [0077] asp Ala Pro
.psi.[CH.sub.2S]Ala Ala Pro .psi.[CH.sub.2S]Ala Gly Pro
.psi.[CH.sub.2S]Ala Val Pro val [0078] Ac-Asp Ala Aib Ala Ala Aib
Ala Gly Aib Ala Val Pro Val-Am (SEQ ID NO:5) [0079] Ac-Asp Ala Pro
.psi.[CH.sub.2CH.sub.2]Ala Ala Pro .psi.[CH.sub.2CH.sub.2]Ala Gly
Pro .psi.[CH.sub.2CH.sub.2]Ala Val Pro (Me)Val [0080] Ac-Asp Ala
pro Ala Ala Pro .psi.[CH.sub.2CH.sub.2]Ala Gly pro Ala Val Pro
Val-Am [0081] asp Ala Pro .psi.[CH.sub.2CH.sub.2]Ala Ala Pro
.psi.[CH.sub.2CH.sub.2]Ala Gly Pro .psi.[CH.sub.2CH.sub.2]Ala Val
Pro (Me)Val [0082] asp Ala Aib Ala Ala Pro
.psi.[CH.sub.2CH.sub.2]Ala Gly pro Ala Val Pro (Me)Val (SEQ ID
NO:6) [0083] asp Ala Aib Ala Ala Pro .psi.[CH.sub.2S]Ala Gly pro
Ala Val Pro (Me)Val [0084] asp Ala Pro .psi.[CH.sub.2S]Ala Ala Pro
.psi.[CH.sub.2S]Ala Gly Pro .psi.[CH.sub.2S]Ala Val Pro (Me)Val
[0085] Ac-Asp Ala Aib Ala Ala Pro .psi.[CH.sub.2CH.sub.2]Ala Gly
Aib Ala Val Pro (Me)Val (SEQ ID NO:7) [0086] Ac-Asp Ala Pro
.psi.[CH.sub.2S]Ala ala Pro .psi.[CH.sub.2S]Ala gly Pro
.psi.[CH.sub.2S]Ala (Me)Val Pro Val-Am [0087] Ac-Asp Ala Aib ala
Ala Pro .psi.[CH.sub.2CH.sub.2]Ala Gly pro Ala Val Pro (Me)Val
[0088] asp Ala Aib Ala Ala Pro .psi.[CH.sub.2CH.sub.2]Ala Gly Aib
ala Val Pro Val-Am [0089] Ac-Asp Ala pro Ala Ala Pro
.psi.[CH.sub.2CH.sub.2]Ala gly pro Ala (Me)Val Pro Val-Am [0090]
asp Ala Pro .psi.[CH.sub.2CH.sub.2]Ala Ala Pro
.psi.[CH.sub.2CH.sub.2]Ala gly Pro .psi.[CH.sub.2CH.sub.2]Ala val
Pro val [0091] Ac-Asp Ala pro Ala ala Aib Ala gly pro Ala (Me)Val
Pro Val-Am (SEQ ID NO:8) [0092] Asp Ala pro Ala Ala Pro
.psi.[CH.sub.2CH.sub.2] Ala Gly pro Ala Val Pro Val [0093] Asp Ala
Aib Ala Ala Pro .psi.[CH.sub.2CH.sub.2]Ala Gly Aib Ala (Me) Val Pro
Val
[0094] Another approach to improve stability, which also may result
in the generation of orally active compounds, is to produce a
peptide mimetic. A peptide mimetic is a molecule that mimics the
biological activity of the peptides, but is no longer a peptide in
chemical nature. The term peptide mimetic has been used sometimes
to describe molecules that are partially peptide in nature, such as
pseudopeptides, semi-peptides or peptoids, but a strict definition
and the one that is used in the present application is an organic
molecule that no longer contains any peptide bonds. Peptide
mimetics are not derivatives of a parent peptide, but rather are
chemically synthesized de novo trying to mimic the structural and
functional properties of the peptide. The rational design of
peptide mimetics requires a sufficient knowledge of the
pharmacophoric groups that are responsible for the activity and
detailed structural information of the peptide. The objective is to
reconstruct the spatial position of the pharmaco-active groups
using an organic template to mount them. Selection of the template
is important and has to take into consideration the size and
flexibility based on the conformational model of the peptide.
Peptide Mimetics Designed to Imitate .beta.-Sheet Breaker Peptide
Properties.
[0095] The rational design of peptide mimetics requires a
sufficient knowledge of the chemical groups that are responsible
for the activity and detailed structural information of the
peptide. The objective is to reconstruct the position of the
pharmaco-active groups using an organic template to mount them.
Selection of the template is important and has to take into
consideration the size and flexibility based on the conformational
model of the peptide. From the study of the activity of different
.beta.-sheet breaker sequences bearing single amino acid
substitutions, the residues that are key for inhibition have been
determined. In addition, the tridimensional structure of the lead
Alzheimer's and prion .beta.-sheet breaker peptides (FIGS. 3A and
3B) were either modeled or experimentally determined. The
five-residue inhibitor of A.beta. fibrillogenesis was modeled by
energy minimization and Monte Carlo simulations using the computer
program ICM. The structure of the 13-residue inhibitor of prion
protein conformational changes was experimentally calculated by
2D-NMR.
[0096] There are numerous approaches to the design and synthesis of
peptide mimetics as described in recent reviews by Joachim Gante
and Iwao Ojima et al. of which are incorporated herein by
reference.
[0097] The peptide mimetics shown below represent a further aspect
of this invention.
[0098] Alzheimer's Inhibitors ##STR6##
[0099] Prion Inhibitors ##STR7##
[0100] The latter (PMiPrP5) is a shorter and easier to synthesize
version that contains the chemically active groups and is analog to
a five-residue prion .beta.-sheet breaker peptide.
[0101] As a method of preventing or treating a disorder or disease
associated with amyloid or amyloid-like deposits or pathological
.beta.-sheet-rich precursors thereof, the compound of the present
invention is administered in an effective amount to a subject in
need thereof, where the subject can be human or animal. Likewise, a
method of detecting such disorders or diseases also includes
administering a sufficient amount of the designed compound to
visualize its binding to fibril deposits or precursors thereof by
well-known imaging techniques.
[0102] As used herein, the term "prevention" of a condition, such
as Alzheimer's disease or other amyloidosis disorders, in a subject
involves administering the compound according to the present
invention prior to the clinical onset of the disease. "Treatment"
involves administration of the protective compound after the
clinical onset of the disease. For example, successful
administration of the compound of the present invention, after
development of a disorder or disease comprises "treatment" of the
disease. The invention is useful in the treatment of humans as well
as for veterinary uses in animals.
[0103] The compound of the present invention may be administered by
any means that achieves its intended purpose, preferably oral. For
example, administration may be by a number of different parenteral
routes including, but not limited to, subcutaneous, intravenous,
intradermal, intramuscular, intraperitoneal, intracerebral,
intranasal, oral, transdermal, or buccal routes. Parenteral
administration can be bolus injection or by gradual perfusion over
time.
[0104] A typical regimen for preventing, suppressing, or treating a
condition associated with amyloid or amyloid-like deposits,
comprises either: (1) administration of an effective amount in one
or two doses of a high concentration of the compound in the range
of 0.5 to 10 mg, more preferably 0.5 to 5 mg, or (2) administration
of an effective amount of the compound administered in multiple
doses of lower concentrations in the range of 10 to 10,000 .mu.g,
more preferably 50 to 500 .mu.g over a period of time up to and
including several months to several years.
[0105] It is understood that the dosage administered will be
dependent upon the age, sex, health, and weight of the recipient,
kind of concurrent treatment, if any, frequency of treatment, and
the nature of the effect desired. The total dose required for each
treatment may be administered by multiple doses or in a single
dose. By "effective amount," it is meant a concentration of the
compound which is capable of slowing down or inhibiting the
formation of amyloid or amyloid-like deposits, or pathological
.beta.-sheet precursors thereof, or of dissolving preformed fibril
deposits. Such concentrations can be routinely determined by those
of skill in the art. It will also be appreciated by those of skill
in the art that the dosage may be dependent on the stability of the
administered compound. A less stable compound may required
administration in multiple doses.
[0106] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions, which
may contain auxiliary agents or excipients which are known in the
art. Pharmaceutical compositions such as tablets and capsules can
also be prepared according to routine methods.
[0107] Pharmaceutical compositions comprising the compound of the
invention include all compositions wherein the compound is
contained in an amount effective to achieve its intended purpose.
In addition, the pharmaceutical compositions may contain suitable
pharmaceutically acceptable carriers comprising excipients and
auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically. Suitable
pharmaceutically acceptable vehicles are well known in the art and
are described for example in Gennaro, Alfonso, Ed., Remington's
Pharmaceutical Sciences, 18.sup.th Edition 1990, Mack Publishing
Co., Easton, Pa., a standard reference text in this field.
Pharmaceutically acceptable vehicles can be routinely selected in
accordance with the mode of administration and the solubility and
stability of the compound. For example, formulations for
intravenous administration may include sterile aqueous solutions
which may also contain buffers, diluents and other suitable
additives.
[0108] Suitable formulations for parenteral administration include
aqueous solutions of the active compounds in water-soluble form,
for example, water-soluble salts. In addition, suspension of the
active compound as appropriate oily injections suspensions may be
administered. Suitable lipophilic solvents or vehicles include
fatty oils, for example, sesame oil, or synthetic fatty acid
esters, for example ethyl oleate or triglycerides. Aqueous
injection suspensions that may contain substances which increase
the viscosity of the suspension include, for example, sodium
carboxymethyl cellulose, sorbitol, and/or destran. Optionally, the
suspension may also contain stabilizers.
[0109] Disorders or diseases associated with abnormal protein
folding into amyloid or amyloid-like deposits or into pathological
.beta.-sheet-rich precursors of such deposits to be treated or
prevented by administering the pharmaceutical composition of the
invention includes, but is not limited to, Alzheimer's disease,
FAF, Down's syndrome, other amyloidosis disorders, human prion
diseases, such as kuru, Creutzfeldt-Jakob Disease (CJD),
Gerstmann-Strausslet-Scheinker Syndrome (GSS), prion associated
human neurodegenerative diseases as well as animal prion diseases
such as scrapie, spongiform encephalopathy, transmissible mink
encephalopathy and chronic wasting disease of mule deer and
elk.
EXAMPLES
[0110] One of the major drawbacks for the use of peptides as drugs
is their rapid proteolytic degradation in biological fluids and
tissues. In in vitro experiments, iA.beta.5, (Seq. LPFFD, also
depicted as Leu Pro Phe Phe Asp herein) degraded very quickly in
vitro after incubation with fresh human plasma. As shown in FIG.
4a, fifty percent of the peptide iA.beta.5 disappeared in
approximately five minutes in the presence of plasma. Since it was
not possible to identify any metabolic fragments as a result of the
proteolytic digestion, it seems likely that the degradation is
mainly done by unspecific exopeptidases. This conclusion is
supported by the finding that protection of amino- and
carboxy-terminus of the peptide by acetylation and amidation,
respectively, (to form Ac-iA.beta.5-Am--also depicted as Ac-Leu Pro
Phe Phe Asp-Am herein) dramatically increases the stability of the
peptide in vitro. As shown in FIG. 4b, the end-protected modified
peptide of the present invention (Ac-iA.beta.5-Am) remained stable
for a period of more than 24 hours in human plasma. (The modified
peptide was also slowly metabolized in vitro in human and rat liver
microsomes, in which after one hour of incubation at 37.degree. C.,
81.5% and 76.3% of the peptide remained intact, in human and rat
tissue homogenate, respectively.)
[0111] Additional in vitro studies showed that Ac-iA.beta.5-Am has
similar activity as iA.beta.5 in inhibiting amyloid formation (see
FIG. 5a) and the effect followed a similar dose-dependency as the
activity of the unmodified peptide as shown in FIGS. 5b and 5c.
Returning to FIG. 5a, it can be seen that modification of the
N-terminus by Boc also retains the in vitro activity exhibited by
iA.beta.5 while several unrelated peptides (CP1: VHVSEEGTEPA, CP2:
GYLTVAAVFRG, CP10: ISEVKMDAEF) or short A.beta. fragments (such as
A.beta.18-21, A.beta.1-16) at the same concentrations had no effect
on fibrillogenesis or slightly increased amyloid formation probably
by incorporation into the fibrils.
[0112] To evaluate the effect of Ac-iA.beta.5-Am in vivo, we used a
rat model in which amyloidosis was induced by intracerebral
injection of non-aggregated A.beta.1-42. After some time, the
peptide aggregates inside the rat brain resulting in the formation
of a single amyloid-like deposit in the place of injection. These
lesions have the same tinctorial (congo red birefringence and
thioflavine S binding) and translucent (fibrillar structure under
electron microscopy) properties than Alzheimer's amyloid plaques
and induce some cerebral damage similar to that observed in AD
brain, including extensive neuronal shrinkage, astrocytosis and
microglial activation. Using this model, we have shown previously
that co-injection of the unprotected iA.beta.5 with A.beta.1-42
induce a 50% inhibition of amyloid plaque formation and i.c.
injection of iA.beta.5 in animals already containing amyloid
plaques produced a 67% dissolution of preformed deposits. (E. M.
Sigurdsson, B. Permanne, C. Soto, T. Wisniewski and B. Frangione
(2000), In vivo disassembly of amyloid-.beta. deposits in rat
brain, J. Neuropath. Exp. Neurol. 59:11-17.) In the previous
experiments, the unprotected peptide was injected directly in the
brain region where the amyloid was located. In the present
experiment the amyloid-.beta.5 peptide was injected into the
amygdala of the rats. After seven days, which is the time required
to have fully formed the amyloid deposits, one-hundred .mu.L of a
solution containing 13 mg/ml of the Ac-iA.beta.5-Am were infused
for a period of three weeks using an ALZET infusion pump connected
to the lateral ventricle. The animals were sacrificed and the brain
analyzed for the presence of amyloid deposits by
immunohistochemistry. In this model, a compacted amyloid plaque was
obtained in the place where the solution containing A.beta.1-42 was
deposited (amygdala) and also several smaller amyloid deposits were
observed throughout the cannula track in regions closer to the
ventricle (FIG. 6, left panel). The results show that infusion of
the peptide induces a 30% dissolution of preformed amyloid plaque
in the amygdala and 83% dissolution of the deposits located near
the ventricle (FIG. 6).
Experimental Procedures
[0113] In vitro assays of peptide stability. Peptides were prepared
as a 1 .mu.g/.mu.l solution in water. 20 .mu.l of the peptide
solution was diluted in 80 .mu.l of fresh human plasma. The
solution was incubated at 37.degree. C. for different time periods
and the reaction was stopped by adding a complete cocktail of
protease inhibitors. The bulk of the plasma proteins (none of the
peptide) were precipitated in cold methanol (mix/MeOH, 4/5, v/v)
for one hour at -20.degree. C. The precipitated proteins were
pelleted by centrifugation (10,000 g, 10 minutes, 4.degree. C.).
The supernatant, containing the peptide, was concentrated five
times under vacuum and separated by reverse-phase HPLC. The peak
area corresponding to the intact peptide was measured and compared
with an equivalent sample incubated without plasma.
[0114] In vitro assays of activity. Amyloid formation was
quantitatively evaluated by the fluorescence emission of
thioflavine T (ThT) bound to amyloid fibrils. Aliquots of A.beta.
at a concentration of 0.5 mg/ml prepared in 0.1M Tris, pH 7.4 were
incubated for seven days at 37.degree. C. in the absence or in the
presence of different concentrations of iA.beta.5 and derivatives.
At the end of the incubation period, 50 mM glycine, pH 9.2 and 2
.mu.M ThT were added in a final volume of 2 ml. Fluorescence was
measured at: excitation 435 nm and emission 485 nm in a Perkin
Elmer, model LS50B fluorescence spectrometer.
[0115] In vivo studies using an animal model of cerebral A.beta.
deposition. Male Fischer-344 rats weighed 250-300 g and were three
to four months of age at the time of arrival. The animals were
housed two per cage, maintained on a 12 hour light-dark cycle with
access to food and water ad libitum and were habituated to their
new environment for two to three weeks prior to surgery. Surgery
was performed under sodium pentobarbital (50 mg/kg, i.p.)
anesthesia. Atropine sulfate (0.4 mg/kg) and ampicillin sodium salt
(50 mg/kg) were injected subcutaneously once the animals were
anesthetized. A.beta. 1-42 was dissolved in dimethylsulfoxide
(DMSO) and then diluted with water to a 16.7% DMS. The animal
received a bilateral injection of 5.0 nmol A.beta. 1-42 into each
amygdala by using a Kopf stereotaxic instrument with the incisor
bar set at 3.3 mm below the interaural line. Injection coordinates
measured from the bregma and the surface of the skull (A.beta.-3.0,
ML.+-.4.6 DV-8.8) were empirically determined based on the atlas of
Paxinos and Watson. A volume of 3.0 .mu.l was administered over six
minutes (flow rate 0.5 .mu.l/minute) using a CMA/100 micrasyringe
pump. The cannula was left in situ for two minutes following
injection, then it was withdrawn 0.2 mm and left for three minutes,
and after five minutes the cannula was slowly withdrawn. Following
surgery the animals were placed on a heating pad until they
regained their righting reflex. To evaluate the effect of
Ac-i.beta.5-AM the animals were subjected to a second surgery one
week after the first one, in which an ALZET infusion pump was
connected to the cerebral ventricle following the manufacturer
indications. A total of 1.3 mg of peptide in 100 .mu.l of PBS/10%
DMSO was delivered into the lateral ventricle over a period of
three weeks. After this time, the animals were sacrificed by an
overdose of sodium pentobarbital (150 mg/kg, i.p.), perfused
transaortically. For histology, serial coronal sections (40 .mu.m)
of the brain were cut, placed in ethylene glycol cryoprotectant and
stored at -20.degree. C until stained. Tissue sections were stained
with anti A.beta. 1-42 antibodies as described in C. Soto, E.
Sigursson, L. Morelli, R. A. Kumar, E. M. Castano and B. Frangione
(1998) .beta.-sheet breaker peptides inhibit fibrillogenesis in a
rat brain model of amyloidosis: Implications for Alzheimer's
therapy, Nature Med. 4:822-826. An image analysis system was used
to determine the size of the amyloid deposits. The data was
analyzed by a two-way ANOVA followed by a Newman-Keuls' multiple
range test for post hoc comparisons. Total brain deposition was
analyzed using an unpaired t-test, two tailed.
[0116] Having now fully described this invention, it will be
appreciated by those skilled in the art that the same can be
performed within a wide range of equivalent parameters,
concentrations, and conditions without departing from the spirit
and scope of the invention and without due experimentation.
[0117] While this invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications. This application is intended to
cover any variations, uses, or adaptations of the inventions
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 as follows in the scope of the appended
claims.
[0118] All references cited herein, including journal articles or
abstracts, published or corresponding U.S. or foreign patent
applications, issued U.S. or foreign patents, or any other
references, are entirely incorporated by reference herein,
including all data, tables, figures, and text presented in the
cited references. Additionally, the entire contents of the
references cited within the references cited herein are also
entirely incorporated by reference.
[0119] Reference to known method steps, conventional methods steps,
known methods or conventional methods is not in any way an
admission that any aspect, description or embodiment of the present
invention is disclosed, taught or suggested in the relevant
art.
[0120] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying knowledge within the skill of the art (including
the contents of the references cited herein), readily modify and/or
adapt for various applications such specific embodiments, without
undue experimentation, without departing from the general concept
of the present invention. Therefore, such adaptations and
modifications are intended to be within the meaning and range of
equivalents of the disclosed embodiments, based on the teaching and
guidance presented herein. It is to be understood that the
phraseology or terminology herein is for the purpose of description
and not of limitation, such that the terminology or phraseology of
the present specification is to be interpreted by the skilled
artisan in light of the teachings and guidance presented herein, in
combination with the knowledge of one of ordinary skill in the art.
Sequence CWU 1
1
11 1 5 PRT Artificial Synthetic chemical peptide 1 Leu Pro Phe Phe
Asp 1 5 2 13 PRT Artificial Synthetic chemical peptide 2 Asp Ala
Pro Ala Ala Pro Ala Gly Pro Xaa Val Pro Val 1 5 10 3 5 PRT
Artificial Synthetic chemical peptide 3 Leu Xaa Phe Phe Asp 1 5 4
13 PRT Artificial Synthetic chemical peptide 4 Asp Ala Xaa Ala Ala
Xaa Ala Gly Xaa Ala Val Xaa Val 1 5 10 5 13 PRT Artificial
Synthetic chemical peptide 5 Asp Ala Xaa Ala Ala Xaa Ala Gly Xaa
Ala Val Pro Val 1 5 10 6 13 PRT Artificial Synthetic chemical
peptide 6 Xaa Ala Xaa Ala Ala Pro Xaa Gly Xaa Ala Val Pro Val 1 5
10 7 13 PRT Artificial Synthetic chemical peptide 7 Asp Ala Xaa Ala
Ala Pro Xaa Gly Xaa Ala Val Pro Val 1 5 10 8 13 PRT Artificial
Synthetic chemical peptide 8 Asp Ala Xaa Ala Xaa Xaa Ala Xaa Xaa
Ala Val Pro Val 1 5 10 9 11 PRT Artificial CP1 9 Val His Val Ser
Glu Glu Gly Thr Glu Pro Ala 1 5 10 10 11 PRT Artificial CP2 10 Gly
Tyr Leu Thr Val Ala Ala Val Phe Arg Gly 1 5 10 11 10 PRT Artificial
CP10 11 Ile Ser Glu Val Lys Met Asp Ala Glu Phe 1 5 10
* * * * *