U.S. patent application number 09/972475 was filed with the patent office on 2002-07-25 for modulators of amyloid aggregation.
This patent application is currently assigned to Praecis Pharmaceuticals, Inc.. Invention is credited to Benjamin, Howard, Findeis, Mark A., Garnick, Marc B., Gefter, Malcolm L., Hundal, Arvind, Kasman, Laura, Musso, Gary, Reed, Michael J., Signer, Ethan R., Wakefield, James.
Application Number | 20020098173 09/972475 |
Document ID | / |
Family ID | 27410601 |
Filed Date | 2002-07-25 |
United States Patent
Application |
20020098173 |
Kind Code |
A1 |
Findeis, Mark A. ; et
al. |
July 25, 2002 |
Modulators of amyloid aggregation
Abstract
Compounds that modulate the aggregation of amyloidogenic
proteins or peptides are disclosed. The modulators of the invention
can promote amyloid aggregation or, more preferably, can inhibit
natural amyloid aggregation. In a preferred embodiment, the
compounds modulate the aggregation of natural .beta. amyloid
peptides (.beta.-AP). In a preferred embodiment, the .beta. amyloid
modulator compounds of the invention are comprised of an A.beta.
aggregation core domain and a modifying group coupled thereto such
that the compound alters the aggregation or inhibits the
neurotoxicity of natural .beta. amyloid peptides when contacted
with the peptides. Furthermore, the modulators are capable of
altering natural .beta.-AP aggregation when the natural .beta.-APs
are in a molar excess amount relative to the modulators.
Pharmaceutical compositions comprising the compounds of the
invention, and diagnostic and treatment methods for amyloidogenic
diseases using the compounds of the invention, are also
disclosed.
Inventors: |
Findeis, Mark A.;
(Cambridge, MA) ; Benjamin, Howard; (Lexington,
MA) ; Garnick, Marc B.; (Brookline, MA) ;
Gefter, Malcolm L.; (Lincoln, MA) ; Hundal,
Arvind; (Brighton, MA) ; Kasman, Laura;
(Athens, GA) ; Musso, Gary; (Hopkinton, MA)
; Signer, Ethan R.; (Cambridge, MA) ; Wakefield,
James; (Brookline, MA) ; Reed, Michael J.;
(Marietta, GA) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Praecis Pharmaceuticals,
Inc.
|
Family ID: |
27410601 |
Appl. No.: |
09/972475 |
Filed: |
October 4, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09972475 |
Oct 4, 2001 |
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08617267 |
Mar 14, 1996 |
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08617267 |
Mar 14, 1996 |
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08475579 |
Jun 7, 1995 |
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08475579 |
Jun 7, 1995 |
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08404831 |
Mar 14, 1995 |
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08475579 |
Jun 7, 1995 |
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08548998 |
Oct 27, 1995 |
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Current U.S.
Class: |
424/94.3 ;
435/226 |
Current CPC
Class: |
C07K 14/4711 20130101;
A61K 38/00 20130101; A61P 25/00 20180101; A61P 43/00 20180101 |
Class at
Publication: |
424/94.3 ;
435/226 |
International
Class: |
A61K 038/54; C12N
009/64 |
Claims
1. An amyloid modulator compound comprising an amyloidogenic
protein, or peptide fragment thereof, coupled directly or
indirectly to at least one modifying group such that the compound
modulates the aggregation of natural amyloid proteins or peptides
when contacted with the natural amyloidogenic proteins or
peptides.
2. The compound of claim 1, which inhibits aggregation of natural
amyloidogenic proteins or peptides when contacted with the natural
amyloidogenic proteins or peptides.
3. The compound of claim 2, which inhibits aggregation of natural
amyloidogenic proteins or peptides when contacted with a molar
excess amount of natural amyloidogenic proteins or peptides.
4. The compound of claim 1, which is further modified to alter a
pharmacokinetic property of the compound.
5. The compound of claim 1, which is further modified to label the
compound with a detectable substance.
6. The compound of claim 1, wherein the amyloidogenic protein, or
peptide fragment thereof, is selected from the group consisting of
transthyretin (TTR), prion protein (PrP), islet amyloid polypeptide
(IAPP), atrial natriuretic factor (ANF), kappa light chain, lambda
light chain, amyloid A, procalcitonin, cystatin C, .beta.2
microglobulin, ApoA-I, gelsolin, procalcitonin, calcitonin,
fibrinogen and lysozyme.
7. The compound of claim 1, wherein the modifying group comprises a
cyclic, heterocyclic or polycyclic group.
8. An amyloid modulator compound comprising an amyloidogenic
protein, or peptide fragment thereof, coupled directly or
indirectly to at least one modifying group comprising a cyclic,
heterocyclic or polycyclic group.
9. The compound of claim 8, wherein the modifying group contains a
cis-decalin group.
10. The compound of claim 9, wherein the modifying group contains a
cholanoyl structure.
11. The compound of claim 10, wherein the modifying group is a
cholyl group.
12. The compound of claim 8, wherein the modifying group comprises
a biotin-containing group, a diethylene-triaminepentaacetyl group,
a (-)-menthoxyacetyl group, a fluorescein-containing group or an
N-acetylneuraminyl group.
13. The compound of claim 8, wherein the amyloidogenic protein. or
peptide fragement thereof, is selected from the group consisting of
transthyretin (TTR), prion protein (PrP), islet amyloid polypeptide
(IAPP), atrial natriuretic factor (ANF), kappa light chain, lambda
light chain, amyloid A, procalcitonin, cystatin C, .beta.2
microglobulin, ApoA-I, gelsolin, procalcitonin, calcitonin,
fibrinogen and lysozyme.
14. A pharmaceutical composition comprising a therapeutically
effective amount of the compound of claim 1 and a pharmaceutically
acceptable carrier.
15. A pharmaceutical composition comprising a therapeutically
effective amount of the compound of claim 8 and a pharmaceutically
acceptable carrier.
16. A method for altering aggregation of natural amyloid proteins
or peptides, comprising contacting the natural amyloid proteins or
peptides with the compound of claim 1 such that aggregation of the
natural amyloid proteins or peptides is altered.
17. A method for detecting aggregation of natural amyloid proteins
or peptides, comprising contacting a biological sample with the
compound of claim 5 such that aggregation of the natural amyloid
proteins or peptides in the sample is detected.
18. The method of claim 17, wherein the compound is administered to
a subject to detect aggregation of the natural amyloid proteins or
peptides in the subject.
19. The method of claim 18, wherein the compound is labeled with
radioactive iodine or technetium.
20. A method for treating a subject for a disorder associated with
amyloidosis, comprising: administering to the subject a
therapeutically or prophylactically effective amount of the
compound of claim 1 such that the subject is treated for a disorder
associated with amyloidosis.
21. A method for treating a subject for a disorder associated with
amyloidosis, comprising: administering to the subject a
therapeutically or prophylactically effective amount of the
compound of claim 8 such that the subject is treated for a disorder
associated with amyloidosis.
22. The method of claim 20, wherein the disorder is selected from
the group consisting of familial amyloid polyneuropathy
(Portuguese, Japanese and Swedish types), familial amyloid
cardiomyopathy (Danish type), isolated cardiac amyloid, systemic
senile amyloidosis, scrapie, bovine spongiform encephalopathy,
Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome,
adult onset diabetes, insulinoma, isolated atrial amyloidosis,
idiopathic (primary) amyloidosis, myeloma or
macroglobulinemia-associated amyloidosis, primary localized
cutaneous nodular amyloidosis associated with Sjogren's syndrome,
reactive (secondary) amyloidosis, familial Mediterranean Fever and
familial amyloid nephropathy with urticaria and deafness
(Muckle-Wells syndrome), hereditary cerebral hemorrage with
amyloidosis of Icelandic type, amyloidosis associated with long
term hemodialysis, hereditary non-neuropathic systemic amyloidosis
(familial amyloid polyneuropathy III), familial amyloidosis of
Finnish type, amyloidosis associated with medullary carcinoma of
the thyroid, fibrinogen-associated hereditary renal amyloidosis and
lysozyme-associated hereditary systemic amyloidosis.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 08/404,831, filed Mar. 14, 1995, and U.S.
patent application Ser. No. 08/475,579, filed Jun. 7, 1995 and U.S.
patent application Ser. No. 08/548,988, filed Oct. 27, 1995, the
entire contents of each of which are hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] Alzheimer's disease (AD), first described by the Bavarian
psychiatrist Alois Alzheimer in 1907, is a progressive neurological
disorder that begins with short term memory loss and proceeds to
disorientation, impairment of judgement and reasoning and,
ultimately, dementia. The course of the disease usually leads to
death in a severely debilitated, immobile state between four and 12
years after onset. AD has been estimated to afflict 5 to 11 percent
of the population over age 65 and as much as 47 percent of the
population over age 85. The societal cost for managing AD is
upwards of 80 billion dollars annually, primarily due to the
extensive custodial care required for AD patients. Moreover, as
adults born during the population boom of the 1940's and 1950's
approach the age when AD becomes more prevalent, the control and
treatment of AD will become an even more significant health care
problem. Currently, there is no treatment that significantly
retards the progression of the disease. For reviews on AD, see
Selkoe, D. J. Sci. Amer., November 1991, pp. 68-78; and Yankner, B.
A. et al. (1991) N. Eng. J. Med. 325:1849-1857.
[0003] It has recently been reported (Games et al. (1995) Nature
373:523-527) that an Alzheimer-type neuropathology has been created
in transgenic mice. The transgenic mice express high levels of
human mutant amyloid precursor protein and progressively develop
many of the pathological conditions associated with AD.
[0004] Pathologically, AD is characterized by the presence of
distinctive lesions in the victim's brain. These brain lesions
include abnormal intracellular filaments called neurofibrillary
tangles (NTFs) and extracellular deposits of amyloidogenic proteins
in senile, or amyloid, plaques. Amyloid deposits are also present
in the walls of cerebral blood vessels of AD patients. The major
protein constituent of amyloid plaques has been identified as a 4
kilodalton peptide called .beta.-amyloid peptide
(.beta.-AP)(Glenner, G. G. and Wong, C. W. (1984) Biochem. Biophys.
Res. Commun. 120:885-890; Masters, C. et al. (1985) Proc. Natl.
Acad Sci. USA 82:4245-4249). Diffuse deposits of .beta.-AP are
frequently observed in normal adult brains, whereas AD brain tissue
is characterized by more compacted, dense-core .beta.-amyloid
plaques. (See e.g., Davies, L. et al. (1988) Neurology
38:1688-1693) These observations suggest that .beta.-AP deposition
precedes, and contributes to, the destruction of neurons that
occurs in AD. In further support of a direct pathogenic role for
.beta.-AP, .beta.-amyloid has been shown to be toxic to mature
neurons, both in culture and in vivo. Yankner, B. A. et al. (1989)
Science 245:417-420; Yankner, B. A. et al. (1990) Proc. Natl. Acad.
Sci. USA 87:9020-9023; Roher, A. E. et al. (1991) Biochem. Biophys.
Res. Commun. 174:572-579; Kowall, N. W. et al. (1991) Proc. Natl.
Acad. Sci. USA 88:7247-7251. Furthermore, patients with hereditary
cerebral hemorrhage with amyloidosis-Dutch-type (HCHWA-D), which is
characterized by diffuse .beta.-amyloid deposits within the
cerebral cortex and cerebrovasculature, have been shown to have a
point mutation that leads to an amino acid substitution within
.beta.-AP. Levy, E. et al. (1990) Science 248:1124-1126. This
observation demonstrates that a specific alteration of the
.beta.-AP sequence can cause .beta.-amyloid to be deposited.
[0005] Natural .beta.-AP is derived by proteolysis from a much
larger protein called the amyloid precursor protein (APP). Kang, J.
et al (1987) Nature 325:733; Goldgaber, D. et al. (1987) Science
235:877; Robakis, N. K. et al. (1987) Proc. Natl. Acad. Sci. USA
84:4190; Tanzi, R. E. et al. (1987) Science 235:880. The APP gene
maps to chromosome 21, thereby providing an explanation for the
.beta.-amyloid deposition seen at an early age in individuals with
Down's syndrome, which is caused by trisomy of chromosome 21. Mann,
D. M. et al (1989) Neuropathol. Appl. Neurobiol 15:317; Rumble, B.
et al. (1989) N. Eng. J. Med. 320:1446. APP contains a single
membrane spanning domain, with a long amino terminal region (about
two-thirds of the protein) extending into the extracellular
environment and a shorter carboxy-terminal region projecting into
the cytoplasm. Differential splicing of the APP messenger RNA leads
to at least five forms of APP, composed of either 563 amino acids
(APP-563), 695 amino acids (APP-695), 714 amino acids (APP-714),
751 amino acids (APP-751) or 770 amino acids (APP-770).
[0006] Within APP, naturally-occurring .beta. amyloid peptide
begins at an aspartic acid residue at amino acid position 672 of
APP-770. Naturally-occurring .beta.-AP derived from proteolysis of
APP is 39 to 43 amino acid residues in length, depending on the
carboxy-terminal end point, which exhibits heterogeneity. The
predominant circulating form of .beta.-AP in the blood and
cerebrospinal fluid of both AD patients and normal adults is (1-40
("short .beta."). Seubert, P. et al. (1992) Nature 359:325; Shoji,
M. et al. (1992) Science 258:126. However, .beta.1-42 and
.beta.1-43 ("long .beta.") also are forms in .beta.-amyloid
plaques. Masters, C. et al. (1985) Proc. Natl. Acad. Sci. USA
82:4245, Miller, D. et al. (1993) Arch. Biochem. Biophys. 301:41;
Mori, H. et al. (1992) J. Biol. Chem. 267:17082. Although the
precise molecular mechanism leading to .beta.-APP aggregation and
deposition is unknown, the process has been likened to that of
nucleation-dependent polymerizations, such as protein
crystallization, microtubule formation and actin polymerization.
See e.g., Jarrett, J. T. and Lansbury, P. T. (1993) Cell
73:1055-1058. In such processes, polymerization of monomer
components does not occur until nucleus formation. Thus, these
processes are characterized by a lag time before aggregation
occurs, followed by rapid polymerization after nucleation.
Nucleation can be accelerated by the addition of a "seed" or
preformed nucleus, which results in rapid polymerization. The long
.beta. forms of .beta.-AP have been shown to act as seeds, thereby
accelerating polymerization of both long and short .beta.-AP forms.
Jarrett, J. T. et al. (1993) Biochemistry 32:4693.
[0007] In one study, in which amino acid substitutions were made in
.beta.-AP, two mutant .beta. peptides were reported to interfere
with polymerization of non-mutated .beta.-AP when the mutant and
non-mutant forms of peptide were mixed. Hilbich, C. et al. (1992)
J. Mol. Biol. 228:460-473. However, equimolar amounts of the mutant
and non-mutant (i.e., natural) .beta. amyloid peptides were used to
see this effect and the mutant peptides were reported to be
unsuitable for use in vivo. Hilbich, C. et al. (1992), supra.
SUMMARY OF THE INVENTION
[0008] This invention pertains to compounds, and pharmaceutical
compositions thereof, that can modulate the aggregation of
amyloidogenic proteins and peptides, in particular compounds that
can modulate the aggregation of natural .beta. amyloid peptides
(.beta.-AP) and inhibit the neurotoxicity of natural .beta.-APs. In
one embodiment, the invention provides an amyloid modulator
compound comprising an amyloidogenic protein, or peptide fragment
thereof, coupled directly or indirectly to at least one modifying
group such that the compound modulates the aggregation of natural
amyloid proteins or peptides when contacted with the natural
amyloidogenic proteins or peptides. Preferably, the compound
inhibits aggregation of natural amyloidogenic proteins or peptides
when contacted with the natural amyloidogenic proteins or peptides.
The amyloidogenic protein, or peptide fragment thereof, can be, for
example, selected from the group consisting of transthyretin (TTR),
prion protein (PrP), islet amyloid polypeptide (IAPP), atrial
natriuretic factor (ANF), kappa light chain, lambda light chain,
amyloid A, procalcitonin, cystatin C, .beta.2 microglobulin,
ApoA-I, gelsolin, procalcitonin, calcitonin, fibrinogen and
lysozyme.
[0009] In the most preferred embodiment of the invention, the
compound modulates the aggregation of natural .beta.-AP. The
invention provides a .beta.-amyloid peptide compound comprising a
formula: 1
[0010] wherein Xaa is a .beta.-amyloid peptide having an
amino-terminal amino acid residue corresponding to position 668 of
.beta.-amyloid precursor protein-770 (APP-770) or to a residue
carboxy-terminal to position 668 of APP-770, A is a modifying group
attached directly or indirectly to the .beta.-amyloid peptide of
the compound such that the compound inhibits aggregation of natural
.beta.-amyloid peptides when contacted with the natural
.beta.-amyloid peptides, and n is an integer selected such that the
compound inhibits aggregation of natural .beta.-amyloid peptides
when contacted with the natural .beta.-amyloid peptides.
[0011] In one embodiment, at least one A group is attached directly
or indirectly to the amino terminus of the .beta.-amyloid peptide
of the compound. In another embodiment, at least one A group is
attached directly or indirectly to the carboxy terminus of the
.beta.-amyloid peptide of the compound. In yet another embodiment,
at least one A group is attached directly or indirectly to a side
chain of at least one amino acid residue of the .beta.-amyloid
peptide of the compound.
[0012] The invention also provides a .beta.-amyloid modulator
compound comprising an A.beta. aggregation core domain (ACD)
coupled directly or indirectly to at least one modifying group (MG)
such that the compound modulates the aggregation or inhibits the
neurotoxicity of natural .beta.-amyloid peptides when contacted
with the natural .beta.-amyloid peptides. Preferably, the A.beta.
aggregation core domain is modeled after a subregion of natural
.beta.-amyloid peptide between 3 and 10 amino acids in length.
[0013] The invention also provides .beta.-amyloid modulator
compound comprising a formula: 2
[0014] wherein Xaa.sub.1, Xaa.sub.2 and Xaa.sub.3 are each amino
acid structures and at least two of Xaa.sub.1, Xaa.sub.2 and
Xaa.sub.3 are, independently, selected from the group consisting of
a leucine structure, a phenylalanine structure and a valine
structure;
[0015] Y, which may or may not be present, is a peptidic structure
having the formula (Xaa).sub.a, wherein Xaa is any amino acid
structure and a is an integer from 1 to 15;
[0016] Z, which may or may not be present, is a peptidic structure
having the formula (Xaa).sub.b, wherein Xaa is any amino acid
structure and b is an integer from 1 to 15; and
[0017] A is a modifying group attached directly or indirectly to
the compound and n is an integer;
[0018] Xaa.sub.1, Xaa.sub.2, Xaa.sub.3, Y, Z, A and n being
selected such that the compound modulates the aggregation or
inhibits the neurotoxicity of natural .beta.-amyloid peptides when
contacted with the natural .beta.-amyloid peptides. In a preferred
embodiment, Xaa.sub.1 and Xaa.sub.2 are each phenylalanine
structures. In another preferred embodiment Xaa.sub.2 and Xaa.sub.3
are each phenylalanine structures.
[0019] The invention further provides a .beta.-amyloid modulator
compound comprising a formula: 3
[0020] wherein Xaa.sub.1 and Xaa.sub.3 are amino acid
structures;
[0021] Xaa.sub.2 is a valine structure;
[0022] Xaa.sub.4 is a phenylalanine structure;
[0023] Y, which may or may not be present, is a peptidic structure
having the formula (Xaa).sub.a, wherein Xaa is any amino acid
structure and a is an integer from 1 to 15;
[0024] Z, which may or may not be present, is a peptidic structure
having the formula (Xaa).sub.b, wherein Xaa is any amino acid
structure and b is an integer from 1 to 15; and
[0025] A is a modifying group attached directly or indirectly to
the compound and n is an integer;
[0026] Xaa.sub.1, Xaa.sub.3, Y, Z, A and n being selected such that
the compound modulates the aggregation or inhibits the
neurotoxicity of natural .beta.-amyloid peptides when contacted
with the natural .beta.-amyloid peptides. In a preferred
embodiment, Xaa.sub.1 is a leucine structure and Xaa.sub.3 is
phenylalanine structure.
[0027] The invention still further provides a compound comprising
the formula:
A-Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7-Xa-
a.sub.8-B
[0028] wherein Xaa.sub.1 is a histidine structure;
[0029] Xaa2 is a glutamine structure;
[0030] Xaa3 is a lysine structure;
[0031] Xaa4 is a leucine structure;
[0032] Xaa5 is a valine structure;
[0033] Xaa6 is a phenylalanine structure;
[0034] Xaa7 is a phenylalanine structure;
[0035] Xaa8 is an alanine structure;
[0036] A and B are modifying groups attached directly or indirectly
to the amino terminus and carboxy terminus, respectively, of the
compound;
[0037] and wherein Xaa.sub.1-Xaa.sub.2-Xaa.sub.3,
Xaa.sub.1-Xaa.sub.2 or Xaa.sub.1 may or may not be present;
[0038] Xaa.sub.8 may or may not be present; and
[0039] at least one of A and B is present.
[0040] The invention still further provides a .beta.-amyloid
modulator compound comprising a modifying group attached directly
or indirectly to a peptidic structure, wherein the peptidic
structure comprises amino acid structures having an amino acid
sequence selected from the group consisting of
His-Gln-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO: 5),
His-Gln-Lys-Leu-Val-Phe-Phe (SEQ ID NO: 6),
Gln-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO: 7), Gln-Lys-Leu-Val-Phe-Phe
(SEQ ID NO: 8), Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO: 9),
Lys-Leu-Val-Phe-Phe (SEQ ID NO: 10), Leu-Val-Phe-Phe-Ala (SEQ ID
NO: 11), Leu-Val-Phe-Phe (SEQ ID NO: 12), Leu-Ala-Phe-Phe-Ala (SEQ
ID NO: 13), Val-Phe-Phe (SEQ ID NO: 19), Phe-Phe-Ala (SEQ ID NO:
20), Phe-Phe-Val-Leu-Ala (SEQ ID NO: 21), Leu-Val-Phe-Phe-Lys (SEQ
ID NO: 22), Leu-Val-Iodotyrosine-Phe-Ala (SEQ ID NO: 23),
Val-Phe-Phe-Ala (SEQ ID NO: 24), Ala-Val-Phe-Phe-Ala (SEQ ID NO:
25), Leu-Val-Phe-Iodotyrosine-Ala (SEQ ID NO: 26),
Leu-Val-Phe-Phe-Ala-Glu (SEQ ID NO: 27), Phe-Phe-Val-Leu (SEQ ID
NO: 28), Phe-Lys-Phe-Val-Leu (SEQ ID NO: 29), Lys-Leu-Val-Ala-Phe
(SEQ ID NO: 30), Lys-Leu-Val-Phe-Phe-.beta.Ala (SEQ ID NO: 31) and
Leu-Val-Phe-Phe-DAla (SEQ ID NO: 32).
[0041] In the compounds of the invention comprising a modifying
group, preferably the modifying group comprises a cyclic,
heterocyclic or polycyclic group. Preferred modifying groups
contains a cis-decalin group, such as a cholanoyl structure.
Preferred modifying groups include a cholyl group, a
biotin-containing group, a diethylene-triaminepentaacet- yl group,
a (-)-menthoxyacetyl group, a fluorescein-containing group or an
N-acetylneuraminyl group.
[0042] The compounds of the invention can be further modified, for
example to alter a pharmacokinetic property of the compound or to
label the compound with a detectable substance. Preferred
radioactive labels are radioactive iodine or technetium.
[0043] The invention also provides a .beta.-amyloid modulator which
inhibits aggregation of natural .beta.-amyloid peptides when
contacted with a molar excess amount of natural .beta.-amyloid
peptides.
[0044] The invention also provides a .beta.-amyloid peptide
compound comprising an amino acid sequence having at least one
amino acid deletion compared to .beta.AP.sub.1-39, such that the
compound inhibits aggregation of natural .beta.-amyloid peptides
when contacted with the natural .beta.-amyloid peptides. In one
embodiment, the compound has at least one internal amino acid
deleted compared to .beta.AP.sub.1-39. In another embodiment, the
compound has at least one N-terminal amino acid deleted compared to
.beta.AP.sub.1-39 In yet another embodiment, the compound has at
least one C-terminal amino acid deleted compared to
.beta.AP.sub.1-39. Preferred compounds include .beta.AP.sub.6-20
(SEQ ID NO: 13), .beta.AP.sub.16-30 (SEQ ID NO: 14),
.beta.AP.sub.1-20, 26-40 (SEQ ID NO: 15) and
EEVVHHHHQQ-.beta.AP.sub.16-40 (SEQ ID NO: 16).
[0045] The compounds of the invention can be formulated into
pharmaceutical compositions comprising the compound and a
pharmaceutically acceptable carrier. The compounds can also be used
in the manufacture of a medicament for the diagnosis or treatment
of an amyloidogenic disease.
[0046] Another aspect of the invention pertains to diagnostic and
treatment methods using the compounds of the invention. The
invention provides a method for inhibiting aggregation of natural
.beta.-amyloid peptides, comprising contacting the natural
.beta.-amyloid peptides with a compound of the invention such that
aggregation of the natural .beta.-amyloid peptides is inhibited.
The invention also provides a method for inhibiting neurotoxicity
of natural .beta.-amyloid peptides, comprising contacting the
natural .beta.-amyloid peptides with a compound of the invention
such that neurotoxicity of the natural .beta.-amyloid peptides is
inhibited.
[0047] In another embodiment, the invention provides a method for
detecting the presence or absence of natural .beta.-amyloid
peptides in a biological sample, comprising contacting a biological
sample with a compound of the invention and detecting the compound
bound to natural .beta.-amyloid peptides to thereby detect the
presence or absence of natural .beta.-amyloid peptides in the
biological sample. In one embodiment, the .beta.-amyloid modulator
compound and the biological sample are contacted in vitro. In
another embodiment, the .beta.-amyloid modulator compound is
contacted with the biological sample by administering the
.beta.-amyloid modulator compound to a subject. For in vivo
administration, preferably the compound is labeled with radioactive
technetium or radioactive iodine.
[0048] In another embodiment, the invention provides a method for
detecting natural .beta.-amyloid peptides to facilitate diagnosis
of a .beta.-amyloidogenic disease, comprising contacting a
biological sample with a compound of the invention and detecting
the compound bound to natural .beta.-amyloid peptides to facilitate
diagnosis of a .beta.-amyloidogenic disease. In one embodiment, the
.beta.-amyloid modulator compound and the biological sample are
contacted in vitro. In another embodiment, the .beta.-amyloid
modulator compound is contacted with the biological sample by
administering the .beta.-amyloid modulator compound to a subject.
For in vivo administration, preferably the compound is labeled with
radioactive technetium or radioactive iodine. Preferably, the
method facilitates diagnosis of Alzheimer's disease.
[0049] The invention also provides a method for treating a subject
for a disorder associated with amyloidosis, comprising
administering to the subject a therapeutically or prophylactically
effective amount of a compound of the invention such that the
subject is treated for a disorder associated with amyloidosis. The
method can be used to treat disorders is selected, for example,
from the group consisting of familial amyloid polyneuropathy
(Portuguese, Japanese and Swedish types), familial amyloid
cardiomyopathy (Danish type), isolated cardiac amyloid, systemic
senile amyloidosis, scrapie, bovine spongiform encephalopathy,
Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome,
adult onset diabetes, insulinoma, isolated atrial amyloidosis,
idiopathic (primary) amyloidosis, myeloma or
macroglobulinemia-associated amyloidosis, primary localized
cutaneous nodular amyloidosis associated with Sjogren's syndrome,
reactive (secondary) amyloidosis, familial Mediterranean Fever and
familial amyloid nephropathy with urticaria and deafness
(Muckle-Wells syndrome), hereditary cerebral hemorrhage with
amyloidosis of Icelandic type, amyloidosis associated with long
term hemodialysis, hereditary non-neuropathic systemic amyloidosis
(familial amyloid polyneuropathy III), familial amyloidosis of
Finnish type, amyloidosis associated with medullary carcinoma of
the thyroid, fibrinogen-associated hereditary renal amyloidosis and
lysozyme-associated hereditary systemic amyloidosis.
[0050] In a preferred embodiment, the invention provides a method
for treating a subject for a disorder associated with
.beta.-amyloidosis, comprising administering to the subject a
therapeutically or prophylactically effective amount of a compound
of the invention such that the subject is treated for a disorder
associated with .beta.-amyloidosis. Preferably the disorder is
Alzheimer's disease.
[0051] In yet another embodiment, the invention provides a method
for treating a subject for a disorder associated with
.beta.-amyloidosis, comprising administering to the subject a
recombinant expression vector encoding a peptide compound of the
invention such that the compound is synthesized in the subject and
the subject is treated for a disorder associated with
.beta.-amyloidosis. Preferably, the disorder is Alzheimer's
disease.
BRIEF DESCRIPTION OF THE DRAWING
[0052] FIG. 1 is a graphic representation of the turbidity of a
.beta.-AP.sub.1-40 solution, as measured by optical density at 400
nm, either in the absence of a .beta.-amyloid modulator or in the
presence of the .beta.-amyloid modulator
N-biotinyl-.beta.AP.sub.1-40 (1%, or 5%).
[0053] FIG. 2 is a schematic representation of compounds which can
be used to modify a .beta.-AP or an A.beta. aggregation core domain
to form a .beta.-amyloid modulator of the invention.
[0054] FIG. 3 is a graphic representation of the toxicity of
A.beta..sub.1-40 aggregates, but not A.beta..sub.1-40 monomers, to
cultured neuronal cells.
[0055] FIG. 4 is a graphic representation of the aggregation of
A.beta..sub.1-40 in the presence of an equimolar amount of
cholyl-A.beta..sub.6-20 (panel A), a .about.2-fold molar excess of
cholyl-A.beta..sub.6-20 (panel B) or a .about.6-fold molar excess
of cholyl-A.beta.6-20 (panel C) and the corresponding toxicity of
the aggregates of panels A, B and C to cultured neuronal cells
(panels D, E and F, respectively).
DETAILED DESCRIPTION OF THE INVENTION
[0056] This invention pertains to compounds, and pharmaceutical
compositions thereof, that can modulate the aggregation of
amyloidogenic proteins and peptides, in particular compounds that
can modulate the aggregation of natural, amyloid peptides
(.beta.-AP) and inhibit the neurotoxicity of natural .beta.-APs. A
compound of the invention that modulates aggregation of natural
.beta.-AP, referred to herein interchangeably as a .beta.amyloid
modulator compound, a .beta. amyloid modulator or simply a
modulator, alters the aggregation of natural .beta.-AP when the
modulator is contacted with natural .beta.-AP. Thus, a compound of
the invention acts to alter the natural aggregation process or rate
for .beta.-AP, thereby disrupting this process. Preferably, the
compounds inhibit .beta.-AP aggregation. Furthermore, the invention
provides subregions of the .beta.amyloid peptide that are
sufficient, when appropriately modified as described herein, to
alter (and preferably inhibit) aggregation of natural .beta.
amyloid peptides when contacted with the natural .beta. amyloid
peptides. In particular, preferred modulator compounds of the
invention are comprised of a modified form of an AP.beta.
aggregation core domain, modeled after the aforementioned A.beta.
subregion (as described further below), which is sufficient to
alter (and preferably inhibit) the natural aggregation process or
rate for .beta.-AP. This A.beta. aggregation core domain can
comprises as few as three amino acid residues (or derivative,
analogues or mimetics thereof). Moreover, while the amino acid
sequence of the A.beta. aggregation core domain can directly
correspond to an amino acid sequence found in natural .beta.-AP, it
is not essential that the amino acid sequence directly correspond
to a .beta.-AP sequence. Rather, amino acid residues derived from a
preferred subregion of .beta.-AP (a hydrophobic region centered
around positions 17-20) can be rearranged in order and/or
substituted with homologous residues within a modulator compound of
the invention and yet maintain their inhibitory activity (described
further below).
[0057] The .beta. amyloid modulator compounds of the invention can
be selected based upon their ability to inhibit the aggregation of
natural .beta.-AP in vitro and/or inhibit the neurotoxicity of
natural .beta.-AP fibrils for cultured cells (using assays
described herein). Accordingly, the preferred modulator compounds
inhibit the aggregation of natural .beta.-AP and/or inhibit the
neurotoxicity of natural .beta.-AP. However, modulator compounds
selected based on one or both of these properties may have
additional properties in vivo that may be beneficial in the
treatment of amyloidosis. For example, the modulator compound may
interfere with processing of natural .beta.-AP (either by direct or
indirect protease inhibition) or by modulation of processes that
produce toxic .beta.-AP, or other APP fragments, in vivo.
Alternatively, modulator compounds may be selected based on these
latter properties, rather than inhibition of A.beta. aggregation in
vitro. Moreover, modulator compounds of the invention that are
selected based upon their interaction with natural .beta.-AP also
may interact with APP or with other APP fragments.
[0058] As used herein, a "modulator" of .beta.-amyloid aggregation
is intended to refer to an agent that, when contacted with natural
.beta. amyloid peptides, alters the aggregation of the natural
.beta. amyloid peptides. The term "aggregation of .beta. amyloid
peptides" refers to a process whereby the peptides associate with
each other to form a multimeric, largely insoluble complex. The
term "aggregation" further is intended to encompass .beta. amyloid
fibril formation and also encompasses .beta.-amyloid plaques.
[0059] The terms "natural .beta.-amyloid peptide", "natural
.beta.-AP" and "natural A.beta. peptide", used interchangeably
herein, are intended to encompass naturally occurring proteolytic
cleavage products of the .beta. amyloid precursor protein (APP)
which are involved in .beta.-AP aggregation and .beta.-amyloidosis.
These natural peptides include .beta.-amyloid peptides having 39-43
amino acids (i.e., A.beta..sub.1-39, A.beta..sub.1-40,
A.beta..sub.1-41, A.beta..sub.1-42 and A.beta..sub.1-43). The
amino-terminal amino acid residue of natural .beta.-AP corresponds
to the aspartic acid residue at position 672 of the 770 amino acid
residue form of the amyloid precursor protein ("APP-770"). The 43
amino acid long form of natural .beta.-AP has the amino acid
sequence
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAT
[0060] (also shown in SEQ ID NO: 1), whereas the shorter forms have
1-4 amino acid residues deleted from the carboxy-terminal end. The
amino acid sequence of APP-770 from position 672 (i.e., the
amino-terminus of natural .beta.-AP) to its C-terminal end (103
amino acids) is shown in SEQ ID NO: 2. The preferred form of
natural .beta.-AP for use in the aggregation assays described
herein is A.beta..sub.1-40.
[0061] In the presence of a modulator of the invention, aggregation
of natural .beta. amyloid peptides is "altered" or "modulated". The
various forms of the term "alteration" or "modulation" are intended
to encompass both inhibition of .beta.-AP aggregation and promotion
of .beta.-AP aggregation. Aggregation of natural .beta.-AP is
"inhibited" in the presence of the modulator when there is a
decrease in the amount and/or rate of .beta.-AP aggregation as
compared to the amount and/or rate of .beta.-AP aggregation in the
absence of the modulator. The various forms of the term
"inhibition" are intended to include both complete and partial
inhibition of .beta.-AP aggregation. Inhibition of aggregation can
be quantitated as the fold increase in the lag time for aggregation
or as the decrease in the overall plateau level of aggregation
(i.e., total amount of aggregation), using an aggregation assay as
described in the Examples. In various embodiments, a modulator of
the invention increases the lag time of aggregation at least
1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 2.5-fold, 3-fold, 4-fold or
5-fold. In various other embodiments, a modulator of the invention
inhibits the plateau level of aggregation at least 10%, 20%, 30%,
40%, 50%, 75% or 100%.
[0062] A modulator which inhibits .beta.-AP aggregation (an
"inhibitory modulator compound") can be used to prevent or delay
the onset of .beta.-amyloid deposition. Moreover, as demonstrated
in Example 10, inhibitory modulator compounds of the invention
inhibit the formation and/or activity of neurotoxic aggregates of
natural A.beta. peptide (i.e., the inhibitory compounds can be used
to inhibit the neurotoxicity of .beta.-AP). Still further, also as
demonstrated in Example 10, the inhibitory compounds of the
invention can be used to reduce the neurotoxicity of preformed
.beta.-AP aggregates, indicating that the inhibitory modulators can
either bind to preformed A.beta. fibrils or soluble aggregate and
modulate their inherent neurotoxicity or that the modulators can
perturb the equilibrium between monomeric and aggregated forms of
.beta.-AP in favor of the non-neurotoxic form.
[0063] Alternatively, in another embodiment, a modulator compound
of the invention promotes the aggregation of natural A.beta.
peptides. The various forms of the term "promotion" refer to an
increase in the amount and/or rate of .beta.-AP aggregation in the
presence of the modulator, as compared to the amount and/or rate of
.beta.-AP aggregation in the absence of the modulator. Such a
compound which promotes A.beta. aggregation is referred to as a
stimulatory modulator compound. Stimulatory modulator compounds may
be useful for sequestering .beta.-amyloid peptides, for example in
a biological compartment where aggregation of .beta.-AP may not be
deleterious to thereby deplete .beta.-AP from a biological
compartment where aggregation of .beta.-AP is deleterious.
Moreover, stimulatory modulator compounds can be used to promote
A.beta. aggregation in in vitro aggregation assays (e.g., assays
such as those described in the Examples), for example in screening
assays for test compounds that can then inhibit or reverse this AP
aggregation (i.e., a stimulatory modulator compound can act as a
"seed" to promote the formation of A.beta. aggregates).
[0064] In a preferred embodiment, the modulators of the invention
are capable of altering .beta.-AP aggregation when contacted with a
molar excess amount of natural .beta.-AP. A "molar excess amount of
natural .beta.-AP" refers to a concentration of natural .beta.-AP,
in moles, that is greater than the concentration, in moles, of the
modulator. For example, if the modulator and .beta.-AP are both
present at a concentration of 1 .mu.M, they are said to be
"equimolar", whereas if the modulator is present at a concentration
of 1 .mu.M and the .beta.-AP is present at a concentration of 5
.mu.M, the .beta.-AP is said to be present at a 5-fold molar excess
amount compared to the modulator. In preferred embodiments, a
modulator of the invention is effective at altering natural
.beta.-AP aggregation when the natural .beta.-AP is present at at
least a 2-fold, 3-fold or 5-fold molar excess compared to the
concentration of the modulator. In other embodiments, the modulator
is effective at altering .beta.-AP aggregation when the natural
.beta.-AP is present at at least a 10-fold, 20-fold, 33-fold,
50-fold, 100-fold, 500-fold or 1000-fold molar excess compared to
the concentration of the modulator.
[0065] Various additional aspects of the modulators of the
invention, and the uses thereof, are described in further detail in
the following subsections.
[0066] I. Modulator Compounds
[0067] In one embodiment, a modulator of the invention comprises a
.beta.-amyloid peptide compound comprising the formula: 4
[0068] wherein Xaa is a .beta.-amyloid peptide, A is a modulating
group attached directly or indirectly to the .beta.-amyloid peptide
of the compound such that the compound inhibits aggregation of
natural .beta.-amyloid peptides when contacted with the natural
.beta.-amyloid peptides, and n is an integer selected such that the
compound inhibits aggregation of natural .beta.-amyloid peptides
when contacted with the natural .beta.-amyloid peptides.
[0069] Preferably, .beta.-amyloid peptide of the compound has an
amino-terminal amino acid residue corresponding to position 668 of
.beta.-amyloid precursor protein-770 (APP-770) or to a residue
carboxy-terminal to position 668 of APP-770. The amino acid
sequence of APP-770 from position 668 to position 770 (i.e., the
carboxy terminus) is shown below and in SEQ ID NO: 2:
EVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITL
VMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQN
[0070] More preferably, the amino-terminal amino acid residue of
the .beta.-amyloid peptide corresponds to position 672 of APP-770
(position 5 of the amino acid sequence of SEQ ID NO: 2) or to a
residue carboxy-terminal to position 672 of APP-770. Although the
.beta.-amyloid peptide of the compound may encompass the 103 amino
acid residues corresponding to positions 668-770 of APP-770,
preferably the peptide is between 6 and 60 amino acids in length,
more preferably between 10 and 43 amino acids in length and even
more preferably between 10 and 25 amino acid residues in
length.
[0071] As used herein, the term ".beta. amyloid peptide", as used
in a modulator of the invention is intended to encompass peptides
having an amino acid sequence identical to that of the natural
sequence in APP, as well as peptides having acceptable amino acid
substitutions from the natural sequence. Acceptable amino acid
substitutions are those that do not affect the ability of the
peptide to alter natural .beta.-AP aggregation. Moreover,
particular amino acid substitutions may further contribute to the
ability of the peptide to alter natural .beta.-AP aggregation
and/or may confer additional beneficial properties on the peptide
(e.g., increased solubility, reduced association with other amyloid
proteins, etc.). For example, substitution of hydrophobic amino
acid residues for the two phenylalanine residues at positions 19
and 20 of natural .beta.-AP (positions 19 and 20 of the amino acid
sequence shown in SEQ ID NO: 1) may further contribute to the
ability of the peptide to alter .DELTA.-AP aggregation (see
Hilbich, C. (1992) J. Mol. Biol. 228:460-473). Thus, in one
embodiment, the .beta.-AP of the compound consists of the amino
acid sequence shown below and in SEQ ID NO: 3:
DAEFRHDSGYEVHHQKLV(Xaa.sub.19)(Xaa.sub.20)AEDVGSNKGAIIGLMVGGVVIAT
[0072] (or an amino-terminal or carboxy-terminal deletion thereof),
wherein Xaa is a hydrophobic arm amino acid. Examples of
hydrophobic amino acids are isoleucine, leucine, threonine, serine,
alanine, valine or glycine. Preferably, F.sub.19F.sub.20 is
substituted with T.sub.19T.sub.20 or G.sub.9I.sub.20.
[0073] Other suitable amino acid substitutions include replacement
of amino acids in the human peptide with the corresponding amino
acids of the rodent .beta.-AP peptide. The three amino acid
residues that differ between human and rat .beta.-AP are at
positions 5, 10 and 13 of the amino acid sequence shown in SEQ ID
NOs: 1 and 3. A human .beta.-AP having the human to rodent
substitutions Arg.sub.5 to Gly, Tyr.sub.10 to Phe and His.sub.13 to
Arg has been shown to retain the properties of the human peptide
(see Fraser, P.E. et al. (1 992) Biochemistry 31:10716-10723; and
Hilbich, C. et al. (1991) Eur. J. Biochem. 201:61-69). Accordingly,
a human .beta.-AP having rodent .beta.-AP a.a. substitutions is
suitable for use in a modulator of the invention.
[0074] Other possible .beta.-AP amino acid substitutions are
described in Hilbich, C. et al. (I991) J. Mol. Biol. 218:149-163;
and Hilbich, C. (1992) J. Mol. Biol. 228:460-473. Moreover, amino
acid substitutions that affect the ability of .beta.-AP to
associate with other proteins can be introduced. For example, one
or more amino acid substitutions that reduce the ability of
.beta.-AP to associate with the serpin enzyme complex (SEC)
receptor, .alpha.1-antichymotrypsin (ACT) and/or apolipoprotein E
(ApoE) can be introduced. A preferred substitution for reducing
binding to the SEC receptor is L.sub.34M.sub.35 to A.sub.34A.sub.35
(at positions 34 and 35 of the amino acid sequences shown in SEQ ID
NOs: 1 and 3). A preferred substitution for reducing binding to ACT
is S.sub.8 to A.sub.8 (at position 8 of the amino acid sequences
shown in SEQ ID NOs: 1 and 3).
[0075] Alternative to .beta.-AP amino acid substitutions described
herein or known in the art, a modulator composed, at least in part,
of an amino acid-substituted .beta. amyloid peptide can be prepared
by standard techniques and tested for the ability to alter
.beta.-AP aggregation using an aggregation assay described herein.
To retain the properties of the original modulator, preferably
conservative amino acid substitutions are made at one or more amino
acid residues. A "conservative amino acid substitution" is one in
which the amino acid residue is replaced with an amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art, including basic
side chains (e.g., lysine, arginine, histidine), acidic side chains
(e.g., aspartic acid, glutamic acid), uncharged polar side chains
(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan),
.beta.-branched side chains (e.g., threonine, valine, isoleucine)
and aromatic side chains (e.g., tyrosine, phenylalanine,
tryptophan, histidine). Accordingly, a modulator composed of a
.beta. amyloid peptide having an amino acid sequence that is
mutated from that of the wild-type sequence in APP-770 yet which
still retains the ability to alter natural .beta.-AP aggregation is
within the scope of the invention.
[0076] As used herein, the term ".beta. amyloid peptide" is further
intended to include peptide analogues or peptide derivatives or
peptidomimetics that retain the ability to alter natural .beta.-AP
aggregation as described herein. For example, a .beta. amyloid
peptide of a modulator of the invention may be modified to increase
its stability, bioavailability, solubility, etc. The terms "peptide
analogue", "peptide derivative" and "peptidomimetic" as used herein
are intended to include molecules which mimic the chemical
structure of a peptide and retain the functional properties of the
peptide. Approaches to designing peptide analogs are known in the
art. For example, see Farmer, P. S. in Drug Design (E. J. Ariens,
ed.) Academic Press, New York, 1980, vol. 10, pp. 119-143; Ball. J.
B. and Alewood, P. F. (1990) J. Mol. Recognition 3:55;
[0077] Morgan, B. A. and Gainor, J. A. (1989) Ann. Rep. Med. Chem.
24:243; and Freidinger, R. M. (1989) Trends Pharmacol. Sci. 10:270.
Examples of peptide analogues, derivatives and peptidomimetics
include peptides substituted with one or more benzodiazepine
molecules (see e.g., James, G. L. et al. (1993) Science
260:1937-1942), peptides with methylated amide linkages and
"retro-inverso" peptides (see U.S. Pat. No. 4,522,752 by Sisto).
Peptide analogues, peptide derivatives and peptidomimetic are
described in further detail below with regard to compounds
comprising an A.beta. aggregation core domain.
[0078] In a modulator of the invention having the formula shown
above, a modulating group ("A") is attached directly or indirectly
to the .beta.-amyloid peptide of the modulator (As used herein, the
term "modulating group" and "modifying group" are used
interchangeably to describe a chemical group directly or indirectly
attached to an AD derived peptidic structure). For example, the
modulating group can be directly attached by covalent coupling to
the .beta.-amyloid peptide or the modulating group can be attached
indirectly by a stable non-covalent association. In one embodiment
of the invention, the modulating group is attached to the
amino-terminus of the .beta.-amyloid peptide of the modulator.
Accordingly, the modulator can comprise a compound having a
formula: 5
[0079] Alternatively, in another embodiment of the invention, the
modulating group is attached to the carboxy-terminus of the
.beta.-amyloid peptide of the modulator. Accordingly, the modulator
can comprise a compound having a formula: 6
[0080] In yet another embodiment, the modulating group is attached
to the side chain of at least one amino acid residues of the
.beta.-amyloid peptide of the compound (e.g., through the epsilon
amino group of a lysyl residue(s), through the carboxyl group of an
aspartic acid residue(s) or a glutamic acid residue(s), through a
hydroxy group of a tyrosyl residue(s), a serine residue(s) or a
threonine residue(s) or other suitable reactive group on an amino
acid side chain).
[0081] The modulating group is selected such that the compound
inhibits aggregation of natural .beta.-amyloid peptides when
contacted with the natural .beta.-amyloid peptides. Accordingly,
since the .beta.-AP peptide of the compound is modified from its
natural state, the modulating group "A" as used herein is not
intended to include hydrogen. In a preferred embodiment, the
modulating group is a biotin compound of the formula: 7
[0082] wherein X.sub.1-X.sub.3 are each independently selected from
the group consisting of S, O and NR.sub.2, wherein R.sub.2 is
hydrogen, or an aryl, lower alkyl, alkenyl or alkynyl moiety; W is
.dbd.O or NR.sub.2; R.sub.1 is a lower alkylenyl moiety and Y is a
direct bond or a spacer molecule selected for its ability to react
with a target group on a .beta.-AP. At least one of X.sub.1-X.sub.3
or W is an NR.sub.2 group.
[0083] The term "aryl" is intended to include aromatic moieties
containing substituted or unsubstituted ring(s), e.g., benzyl,
napthyl, etc. Other more complex fused ring moieties also are
intended to be included.
[0084] The term "lower alkyl or alkylenyl moiety" refers to a
saturated, straight or branched chain (or combination thereof)
hydrocarbon containing 1 to about 6 carbon atoms, more preferably
from 1 to 3 carbon atoms. The terms "lower alkenyl moiety" and
"lower alkynyl moiety" refer to unsaturated hydrocarbons containing
1 to about 6 carbon atoms, more preferably 1 to 3 carbon atoms.
Preferably, R.sub.2 contains 1 to 3 carbon atoms. Preferably,
R.sub.1 contains 4 carbon atoms.
[0085] The spacer molecule (Y) can be, for example, a lower alkyl
group or a linker peptide, and is preferably selected for its
ability to link with a free amino group (e.g., the .alpha.-amino
group at the amino-terminus of a .beta.-AP). Thus, in a preferred
embodiment, the biotin compound modifies the amino-terminus of a
.beta.-amyloid peptide.
[0086] Additional suitable modulating groups may include other
cyclic and heterocyclic compounds and other compounds having
similar steric "bulk". Non-limiting examples of compounds which can
be used to modify a .beta.-AP are shown schematically in FIG. 2,
and include N-acetylneuraminic acid, cholic acid,
trans-4-cotininecarboxylic acid, 2-imino-1-imidazolidineacetic
acid, (S)-(-)-indoline-2-carboxylic acid, (-)-menthoxyacetic acid,
2-norbomaneacetic acid, .gamma.-oxo-5-acenaphthe- nebutyric acid,
(-)-2-oxo-4-thiazolidinecarboxylic acid, tetrahydro-3-furoic acid,
2-iminobiotin-N-hydroxysuccinimide ester,
diethylenetriaminepentaacetic dianhydride, 4-morpholinecarbonyl
chloride, 2-thiopheneacetyl chloride, 2-thiophenesulfonyl chloride,
5-(and 6-)-carboxyfluorescein (succinimidyl ester), fluorescein
isothiocyanate, and acetic acid (or derivatives thereof). Suitable
modulating groups are described further in subsection II below.
[0087] In a modulator of the invention, a single modulating group
may be attached to a .beta.-amyloid peptide (e.g., n=1 in the
formula shown above) or multiple modulating groups may be attached
to the peptide. The number of modulating groups is selected such
that the compound inhibits aggregation of natural .beta.-amyloid
peptides when contacted with the natural .beta.-amyloid peptides.
However, n preferably is an integer between 1 and 60, more
preferably between 1 and 30 and even more preferably between 1 and
10 or 1 and 5.
[0088] In another embodiment, a .beta.-amyloid modulator compound
of the invention comprises an A.beta. aggregation core domain
(abbreviated as ACD) coupled directly or indirectly to a modifying
group such that the compound modulates the aggregation or inhibits
the neurotoxicity of natural .beta.-amyloid peptides when contacted
with the natural .beta.-amyloid peptides. As used herein, an
"A.beta. aggregation core domain" is intended to refer to a
structure that is modeled after a subregion of a natural
.beta.-amyloid peptide which is sufficient to modulate aggregation
of natural .beta.-APs when this subregion of the natural .beta.-AP
is appropriately modified as described herein (e.g., modified at
the amino-terminus). The term "subregion of a natural
.beta.-amyloid peptide" is intended to include amino-terminal
and/or carboxy-terminal deletions of natural .beta.-AP. The term
"subregion of natural .beta.-AP" is not intended to include
full-length natural .beta.-AP (i.e., "subregion" does not include
A.beta..sub.1-39, A.beta..sub.1-40, AP.beta..sub.1-41, AP.sub.1-42
and A.beta..sub.1-43).
[0089] Although not intending to be limited by mechanism, the ACD
of the modulators of the invention is thought to confer a specific
targeting function on the compound that allows the compound to
recognize and specifically interact with natural .beta.-AP.
Preferably, the ACD is modeled after a subregion of natural
.beta.-AP that is less than 15 amino acids in length and more
preferably is between 3-10 amino acids in length. In various
embodiments, the ACD is modeled after a subregion of .beta.-AP that
is 10, 9, 8, 7, 6, 5, 4 or 3 amino acids in length. In one
embodiment, the subregion of .beta.-AP upon which the ACD is
modeled is an internal or carboxy-terminal region of .beta.-AP
(i.e., downstream of the amino-terminus at amino acid position 1).
In another embodiment, the ACD is modeled after a subregion of
.beta.-AP that is hydrophobic. In certain specific embodiments, the
term AP aggregation core domain specifically excludes .beta.-AP
subregions corresponding to amino acid positions 1-15
(A.beta..sub.1-15), 6-20 (A.beta..sub.6-20) and 16-40
(A.beta..sub.16-40)
[0090] An A.beta. aggregation core domain can be comprised of amino
acid residues linked by peptide bonds. That is, the ACD can be a
peptide corresponding to a subregion of .beta.-AP. Alternatively,
an A.beta. aggregation core domain can be modeled after the natural
A.beta. peptide region but may be comprised of a peptide analogue,
peptide derivative or peptidomimetic compound, or other similar
compounds which mimics the structure and function of the natural
peptide. Accordingly, as used herein, an "A.beta. aggregation core
domain" is intended to include peptides, peptide analogues, peptide
derivatives and peptidomimetic compounds which, when appropriately
modified, retain the aggregation modulatory activity of the
modified natural A.beta. peptide subregion. Such structures that
are designed based upon the amino acid sequence are referred to
herein as "A.beta. derived peptidic structures." Approaches to
designing peptide analogues, derivatives and mimetics are known in
the art. For example, see Farmer, P. S. in Drug Design (E. J.
Ariens, ed.) Academic Press, New York, 1980, vol. 10, pp. 119-143;
Ball. J. B. and Alewood, P. F. (1990) J. Mol. Recognition 3:55;
Morgan, B. A. and Gainor, J. A. (1989) Ann. Rep. Med. Chem. 24:243;
and Freidinger, R. M. (1989) Trends Pharmacol. Sci. 10:270. See
also Sawyer, T. K. (1995) "Peptidomimetic Design and Chemical
Approaches to Peptide Metabolism" in Taylor, M. D. and Amidon, G.
L. (eds.) Peptide-Based Drug Design: Controlling Transport and
Metabolism, Chapter 17; Smith, A. B. 3rd, et al. (1995) J. Am.
Chem. Soc. 117:11113-11123; Smith, A. B. 3rd, et al. (1994) J. Am.
Chem. Soc. 116:9947-9962; and Hirschman, R., et al. (1993) J. Am.
Chem. Soc. 115:12550-12568.
[0091] As used herein, a "derivative" of a compound X (e.g., a
peptide or amino acid) refers to a form of X in which one or more
reaction groups on the compound have been derivatized with a
substituent group. Examples of peptide derivatives include peptides
in which an amino acid side chain, the peptide backbone, or the
amino- or carboxy-terminus has been derivatized (e.g., peptidic
compounds with methylated amide linkages). As used herein an
"analogue" of a compound X refers to a compound which retains
chemical structures of X necessary for functional activity of X yet
which also contains certain chemical structures which differ from
X. An examples of an analogue of a naturally-occurring peptide is a
peptides which includes one or more non-naturally-occurring amino
acids. As used herein, a "mimetic" of a compound X refers to a
compound in which chemical structures of X necessary for functional
activity of X have been replaced with other chemical structures
which mimic the conformation of X. Examples of peptidomimetics
include peptidic compounds in which the peptide backbone is
substituted with one or more benzodiazepine molecules (see e.g.,
James, G. L. et al. (1993) Science 260:1937-1942), peptides in
which all L-amino acids are substituted with the corresponding
D-amino acids and "retro-inverso" peptides (see U.S. Pat. No.
4,522,752 by Sisto), described further below.
[0092] The term mimetic, and in particular, peptidomimetic, is
intended to include isosteres. The term "isostere" as used herein
is intended to include a chemical structure that can be substituted
for a second chemical structure because the steric conformation of
the first structure fits a binding site specific for the second
structure. The term specifically includes peptide back-bone
modifications (i.e., amide bond mimetics) well known to those
skilled in the art. Such modifications include modifications of the
amide nitrogen, the .alpha.-carbon, amide carbonyl, complete
replacement of the amide bond, extensions, deletions or backbone
crosslinks. Several peptide backbone modifications are known,
including .PSI.[CH.sub.2S], .PSI.[CH.sub.2NH], .PSI.[CSNH.sub.2],
.PSI.[NHCO], .PSI.[COCH.sub.2], and .PSI.[(E) or (Z) CH.dbd.CH]. In
the nomenclature used above, .PSI. indicates the absence of an
amide bond. The structure that replaces the amide group is
specified within the brackets. Other examples of isosteres include
peptides substituted with one or more benzodiazepine molecules (see
e.g., James, G. L. et al. (1993) Science 260:1937-1942)
[0093] Other possible modifications include an N-alkyl (or aryl)
substitution (.PSI.[CONR]), backbone crosslinking to construct
lactams and other cyclic structures, substitution of all D-amino
acids for all L-amino acids within the compound ("inverso"
compounds) or retro-inverso amino acid incorporation (.PSI.[NHCO]).
By "inverso" is meant replacing L-amino acids of a sequence with
D-amino acids, and by "retro-inverso" or "enantio-retro" is meant
reversing the sequence of the amino acids ("retro") and replacing
the L-amino acids with D-amino acids. For example, if the parent
peptide is Thr-Ala-Tyr, the retro modified form is Tyr-Ala-Thr, the
inverso form is thr-ala-tyr, and the retro-inverso form is
tyr-ala-thr (lower case letters refer to D-amino acids). Compared
to the parent peptide, a retro-inverso peptide has a reversed
backbone while retaining substantially the original spatial
conformation of the side chains, resulting in a retro-inverso
isomer with a topology that closely resembles the parent peptide.
See Goodman et al. "Perspectives in Peptide Chemistry" pp. 283-294
(1981). See also U.S. Pat. No. 4,522,752 by Sisto for further
description of "retro-inverso" peptides.
[0094] Other derivatives of the modulator compounds of the
invention include C-terminal hydroxymethyl derivatives, 0-modified
derivatives (e.g., C-terminal hydroxymethyl benzyl ether),
N-terminally modified derivatives including substituted amides such
as alkylamides and hydrazides and compounds in which a C-terminal
phenylalanine residue is replaced with a phenethylamide analogue
(e.g., Val-Phe-phenethylamide as an analogue of the tripeptide
Val-Phe-Phe).
[0095] In a preferred embodiment, the ACD of the modulator is
modeled after the subregion of .beta.-AP encompassing amino acid
positions 17-20 (i.e., Leu-Val-Phe-Phe; SEQ ID NO: 12). As
described further in Examples 7, 8 and 9, peptide subregions of
A.beta..sub.1-40 were prepared, amino-terminally modified and
evaluated for their ability to modulate aggregation of natural
.beta.-amyloid peptides. One subregion that was effective at
inhibiting aggregation was A.beta..sub.6-20 (i.e., amino acid
residues 6-20 of the natural A.beta..sub.1-40 peptide, the amino
acid sequence of which is shown in SEQ ID NO: 4). Amino acid
residues were serially deleted from the amino-terminus or carboxy
terminus of this subregion to further delineate a minimal subregion
that was sufficient for aggregation inhibitory activity. This
process defined A.beta..sub.17-20 (i.e., amino acid residues 17-20
of the natural A.beta..sub.1-40 peptide) as a minimal subregion
that, when appropriately modified, is sufficient for aggregation
inhibitory activity. Accordingly, an "A.beta. aggregation core
domain" within a modulator compound of the invention can be modeled
after A.beta..sub.17-20. In one embodiment, the Ap aggregation core
domain comprises A.beta..sub.17-20 itself (i.e., a peptide
comprising the amino acid sequence leucine-valine-phenylalanine-p-
henylalanine; SEQ ID NO: 12). In other embodiments, the structure
of A.beta.17-20 is used as a model to design an A.beta. aggregation
core domain having similar structure and function to
A.beta..sub.17-20. For example, peptidomimetics, derivatives or
analogues of A.beta..sub.17-20 (as described above) can be used as
an A.beta. aggregation core domain. In addition to
A.beta..sub.17-20, the natural A.beta. peptide is likely to contain
other minimal subregions that are sufficient for aggregation
inhibitory activity. Such additional minimal subregions can be
identified by the processes described in Examples 7, 8 and 9,
wherein a 15 mer subregion of A.beta..sub.1-40 is serially deleted
from the amino-terminus or carboxy terminus, the deleted peptides
are appropriately modified and then evaluated for aggregation
inhibitory activity.
[0096] One form of the .beta.-amyloid modulator compound comprising
an A.beta. aggregation core domain modeled after A.beta..sub.17-20
coupled directly or indirectly to at least one modifying group has
the formula: 8
[0097] wherein Xaa.sub.1 and Xaa.sub.3 are amino acid
structures;
[0098] Xaa.sub.2 is a valine structure;
[0099] Xaa.sub.4 is a phenylalanine structure;
[0100] Y, which may or may not be present, is a peptidic structure
having the formula (Xaa).sub.a, wherein Xaa is any amino acid
structure and a is an integer from 1 to 15;
[0101] Z, which may or may not be present, is a peptidic structure
having the formula (Xaa).sub.b, wherein Xaa is any amino acid
structure and b is an integer from 1 to 15; and
[0102] A is a modifying group attached directly or indirectly to
the compound and n is an integer;
[0103] Xaa.sub.1, Xaa.sub.3, Y, Z, A and n being selected such that
the compound modulates the aggregation or inhibits the
neurotoxicity of natural .beta.-amyloid peptides when contacted
with the natural .beta.-amyloid peptides.
[0104] Preferably, a modulator compound of the above formula
inhibits aggregation of natural .beta.-amyloid peptides when
contacted with the natural .beta.-amyloid peptides and/or inhibits
A.beta. neurotoxicity. Alternatively, the modulator compound can
promote aggregation of natural .beta.-amyloid peptides when
contacted with the natural .beta.-amyloid peptides. The type and
number of modifying groups ("A") coupled to the modulator are
selected such that the compound alters (and preferably inhibits)
aggregation of natural .beta.-amyloid peptides when contacted with
the natural .beta.-amyloid peptides. A single modifying group can
be coupled to the modulator (i.e., n=1 in the above formula) or,
alternatively, multiple modifying groups can be coupled to the
modulator. In various embodiments, n is an integer between 1 and
60, between 1 and 30, between 1 and 10, between I and 5 or between
1 and 3. Suitable types of modifying groups are described further
in subsection II below.
[0105] As demonstrated in Example 9, amino acid positions 18
(Val.sub.8) and 20 (Phe.sub.20) of A.beta..sub.17-20 (corresponding
to Xaa.sub.2 and Xaa.sub.4) are particularly important within the
core domain for inhibitory activity of the modulator compound.
Accordingly, these positions are conserved within the core domain
in the formula shown above. The terms "valine structure" and
"phenylalanine structure" as used in the above formula are intended
to include the natural amino acids, as well as
non-naturally-occurring analogues, derivatives and mimetics of
valine and phenylalanine, respectively, (including D-amino acids)
which maintain the functional activity of the compound. Moreover,
although Val.sub.18 and Phe.sub.20 have an important functional
role, it is possible that Xaa.sub.2 and/or Xaa.sub.4 can be
substituted with other naturally-occurring amino acids that are
structurally related to valine or phenylalanine, respectively,
while still maintaining the activity of the compound. Thus, the
terms "valine structure" is intended to include conservative amino
acid substitutions that retain the activity of valine at Xaa.sub.2,
and the term "phenylalanine structure" is intended to include
conservative amino acid substitutions that retain the activity of
phenylalanine at Xaa.sub.4. However, the term "valine structure" is
not intended to include threonine.
[0106] In contrast to positions 18 and 20 of A.beta..sub.17-20, a
Phe to Ala substitution at position 19 (corresponding to Xaa.sub.3)
did not abolish the activity of the modulator, indicating position
19 may be more amenable to amino acid substitution. In various
embodiments of the above formula, positions Xaa.sub.1 and Xaa.sub.3
are any amino acid structure. The term "amino acid structure" is
intended to include natural and non-natural amino acids as well as
analogues, derivatives and mimetics thereof, including D-amino
acids. In a preferred embodiment of the above formula, Xaa.sub.1 is
a leucine structure and Xaa.sub.3 is a phenylalanine structure
(i.e., modeled after Leu.sub.17 and Phe.sub.19, respectively, in
the natural A.beta. peptide sequence). The term "leucine structure"
is used in the same manner as valine structure and phenylalanine
structure described above. Alternatively, an another embodiment,
Xaa.sub.3 is an alanine structure.
[0107] The four amino acid structure ACD of the modulator of the
above formula can be flanked at the amino-terminal side,
carboxy-terminal side, or both, by peptidic structures derived
either from the natural A.beta. peptide sequence or from
non-A.beta. sequences. The term "peptidic structure" is intended to
include peptide analogues, derivatives and mimetics thereof, as
described above. The peptidic structure is composed of one or more
linked amino acid structures, the type and number of which in the
above formula are variable. For example, in one embodiment, no
additional amino acid structures flank the
Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-- Xaa.sub.4 core sequence (i.e., Y
and Z are absent in the above formula). In another embodiment, one
or more additional amino acid structures flank only the
amino-terminus of the core sequences (i.e., Y is present but Z is
absent in the above formula). In yet another embodiment, one or
more additional amino acid structures flank only the
carboxy-terminus of the core sequences (i.e., Z is present but Y is
absent in the above formula). The length of flanking Z or Y
sequences also is variable. For example, in one embodiment, a and b
are integers from 1 to 15. More preferably, a and b are integers
between 1 and 10. Even more preferably, a and b are integers
between 1 and 5. Most preferably, a and b are integers between 1
and 3.
[0108] One form of the .beta.-amyloid modulator compound comprising
an A.beta. aggregation core domain modeled after A.beta..sub.17-20
coupled directly or indirectly to at least one modifying group has
the formula:
A-(Y)-Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa4-(Z)-B
[0109] wherein Xaa.sub.1 and Xaa.sub.3 are amino acids or amino
acid mimetics;
[0110] Xaa.sub.2 is valine or a valine mimetic
[0111] Xaa.sub.4 is phenylalanine or a phenylalanine mimetic;
[0112] Y, which may or may not be present, is a peptide or
peptidomimetic having the formula (Xaa).sub.a, wherein Xaa is any
amino acid or amino acid mimetic and a is an integer from 1 to
15;
[0113] Z, which may or may not be present, is a peptide or
peptidomimetic having the formula (Xaa).sub.b, wherein Xaa is any
amino acid or amino acid mimetic and b is an integer from 1 to 15;
and
[0114] A and B, at least one of which is present, are modifying
groups attached directly or indirectly to the amino terminus and
carboxy terminus, respectively, of the compound;
[0115] Xaa.sub.1, Xaa.sub.3, Y, Z, A and B being selected such that
the compound modulates the aggregation or inhibits the
neurotoxicity of natural .beta.-amyloid peptides when contacted
with the natural .beta.-amyloid peptides.
[0116] In this embodiment, the modulator compound is specifically
modified at either its amino-terminus, its carboxy-terminus, or
both. The terminology used in this formula is the same as described
above. Suitable modifying groups are described in subsection II
below. In one embodiment, the compound is modified only at its
amino terminus (i.e., B is absent and the compound comprises the
formula: A-(Y)-Xaa.sub.1-Xaa.sub.2-Xaa.sub- .3-Xaa.sub.4-(Z)). In
another embodiment, the compound is modified only at its
carboxy-terminus (i.e., A is absent and the compound comprises the
formula: (Y)-Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-(Z)-B). In yet
another embodiment, the compound is modified at both its amino- and
carboxy termini (i.e., the compound comprises the formula:
A-(Y)-Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-(Z)-B and both A and
B are present). As described above, the type and number of amino
acid structures which flank the
Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4 core sequences in the above
formula is variable. For example, in one embodiment, a and b are
integers from 1 to 15. More preferably, a and b are integers
between 1 and 10. Even more preferably, a and b are integers
between 1 and 5. Most preferably, a and b are integers between 1
and 3.
[0117] As demonstrated in Examples 7, 8 and 9, preferred A.beta.
modulator compounds of the invention comprise modified forms of
A.beta..sub.14-21 (His-Gln-Lys-Leu-Val-Phe-Phe-Ala; SEQ ID NO: 5),
or amino-terminal or carboxy-terminal deletions thereof, with a
preferred "minimal core region" comprising A.beta..sub.17-20.
Accordingly, in specific embodiments, the invention provides
compounds comprising the formula:
A-Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7-Xa-
a.sub.8-B
[0118] wherein Xaa.sub.1 is a histidine structure;
[0119] Xaa2 is a glutamine structure;
[0120] Xaa3 is a lysine structure;
[0121] Xaa4 is a leucine structure;
[0122] Xaa5 is a valine structure;
[0123] Xaa6 is a phenylalanine structure;
[0124] Xaa7 is a phenylalanine structure;
[0125] Xaa8 is an alanine structure;
[0126] A and B are modifying groups attached directly or indirectly
to the amino terminus and carboxy terminus, respectively, of the
compound;
[0127] and wherein Xaa.sub.1-Xaa.sub.2-Xaa.sub.3,
Xaa.sub.1-Xaa.sub.2 or Xaa.sub.1 may or may not be present;
[0128] Xaa.sub.8 may or may not be present; and
[0129] at least one of A and B is present.
[0130] In one specific embodiment, the compound comprises the
formula: A-Xaa.sub.4-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7-B (e.g, a
modified form of A.beta..sub.17-20, comprising an amino acid
sequence Leu-Val-Phe-Phe; SEQ ID NO: 12).
[0131] In another specific embodiment, the compound comprises the
formula: A-Xaa.sub.4-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7-Xaa.sub.8-B
(e.g, a modified form of A.beta..sub.17-21, comprising an amino
acid sequence Leu-Val-Phe-Phe-Ala; SEQ ID NO: 11).
[0132] In another specific embodiment, the compound comprises the
formula: A-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7-B
(e.g., a modified form of A.beta..sub.16-20, comprising an amino
acid sequence Lys-Leu-Val-Phe-Phe; SEQ ID NO: 10).
[0133] In another specific embodiment, the compound comprises the
formula:
A-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7-Xaa.sub.8-B
(e.g., a modified form of A.beta..sub.16-21, comprising an amino
acid sequence Lys-Leu-Val-Phe-Phe-Ala; SEQ ID NO: 9).
[0134] In another specific embodiment, the compound comprises the
formula:
A-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7-B
(e.g., a modified form of A.beta..sub.15-20, comprising an amino
acid sequence Gln-Lys-Leu-Val-Phe-Phe; SEQ ID NO: 8).
[0135] In another specific embodiment, the compound comprises the
formula:
A-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7-Xaa.sub.8-B
(e.g., a modified form of A.beta..sub.15-21, comprising an amino
acid sequence Gln-Lys-Leu-Val-Phe-Phe-Ala; SEQ ID NO: 7).
[0136] In another specific embodiment, the compound comprises the
formula:
A-Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7-B
(e.g., a modified form of A.beta..sub.14-20, comprising an amino
acid sequence His-Gln-Lys-Leu-Val-Phe-Phe; SEQ ID NO: 6).
[0137] In another specific embodiment, the compound comprises the
formula:
A-Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Xaa6-Xaa.sub.7-Xaa.su-
b.8-B (e.g., a modified form of A.beta..sub.14-21, comprising an
amino acid sequence His-Gln-Lys-Leu-Val-Phe-Phe-Ala; SEQ ID NO:
5).
[0138] In preferred embodiments of the aforementioned specific
embodiments, A or B is a cholanoyl structure or a biotin-containing
structure (described further in subsection II below).
[0139] In further experiments to delineate subregions of AP upon
which an A.beta. aggregation core domain can be modeled (the
results of which are described in Example 11), it was demonstrated
that a modulator compound having inhibitory activity can comprise
as few as three A.beta. amino acids residues (e.g., Val-Phe-Phe,
which corresponds to A.beta..sub.18-20 or Phe-Phe-Ala, which
corresponds to A.beta..sub.19-21). The results also demonstrated
that a modulator compound having a modulating group at its
carboxy-terminus is effective at inhibiting A.beta. aggregation.
Still further, the results demonstrated that the cholyl group, as a
modulating group, can be manipulated while maintaining the
inhibitory activity of the compounds and that an iodotyrosyl can be
substituted for phenylalanine (e.g., at position 19 or 20 of the
A.beta. sequence) while maintaining the ability of the compound to
inhibit A.beta. aggregation.
[0140] Still further, the results demonstrated that compounds with
inhibitory activity can be created using amino acids residues that
are derived from the A.beta. sequence in the region of about
positions 17-21 but wherein the amino acid sequence is rearranged
or has a substitution with a non-A.beta.-derived amino acid.
Examples of such compounds include PPI-426, in which the sequence
of A.beta..sub.17-21 (LVFFA) has been rearranged (FFVLA), PPI-372,
in which the sequence of A.beta..sub.16-20 (KLVFF) has been
rearranged (FKFVL), and PPI-388, -389 and -390, in which the
sequence of A.beta..sub.17-21 (LVFFA) has been substituted at
position 17, 18 or 19, respectively, with an alanine residue (AVFFA
for PPI-388, LAFFA for PPI-389 and LVAFA for PPI-390). The
inhibitory activity of these compounds indicate that the presence
in the compound of an amino acid sequence directly corresponding to
a portion of A.beta. is not essential for inhibitory activity, but
rather suggests that maintenance of the hydrophobic nature of this
core region, by inclusion of amino acid residues such as
phenylalanine, valine, leucine, regardless of their precise order,
can be sufficient for inhibition of A.beta. aggregation.
Accordingly, an A.beta. aggregation core domain can be designed
based on the direct A.beta. amino acid sequence or can be designed
based on a rearranged AD sequence which maintains the
hydrophobicity of the AP subregion, e.g., the region around
positions 17-20. This region of A.beta. contains the amino acid
residues Leu, Val and Phe. Accordingly, preferred A.beta.
aggregation core domains are composed of at least three amino acid
structures (as that term is defined hereinbefore, including amino
acid derivatives, analogues and mimetics), wherein at least two of
the amino acid structures are, independently, either a leucine
structure, a valine structure or a phenylalanine structure (as
those terms are defined hereinbefore, including derivatives,
analogues and mimetics).
[0141] Thus, in another embodiment, the invention provides a
.beta.-amyloid modulator compound comprising a formula: 9
[0142] wherein Xaa.sub.1, Xaa.sub.2 and Xaa.sub.3 are each amino
acid structures and at least two of Xaa.sub.1, Xaa.sub.2 and
Xaa.sub.3 are, independently, selected from the group consisting of
a leucine structure, a phenylalanine structure and a valine
structure;
[0143] Y, which may or may not be present, is a peptidic structure
having the formula (Xaa).sub.a, wherein Xaa is any amino acid
structure and a is an integer from 1 to 15;
[0144] Z, which may or may not be present, is a peptidic structure
having the formula (Xaa).sub.b, wherein Xaa is any amino acid
structure and b is an integer from 1 to 15; and
[0145] A is a modifying group attached directly or indirectly to
the compound and n is an integer;
[0146] Xaa.sub.1, Xaa.sub.2, Xaa.sub.3, Y, Z, A and n being
selected such that the compound modulates the aggregation or
inhibits the neurotoxicity of natural .beta.-amyloid peptides when
contacted with the natural .beta.-amyloid peptides.
[0147] Preferably, the compound inhibits aggregation of natural
.beta.-amyloid peptides when contacted with the natural
.beta.-amyloid peptides. In preferred embodiments, Xaa.sub.1 and
Xaa.sub.2 are each phenylalanine structures or Xaa.sub.2 and
Xaa.sub.3 are each phenylalanine structures. "n" can be, for
example, an integer between 1 and 5, whereas "a" and "b" can be,
for example, integers between 1 and 5. The modifying group "A"
preferably comprises a cyclic, heterocyclic or polycyclic group.
More preferably, A contains a cis-decalin group, such as cholanoyl
structure or a cholyl group In other embodiments, A can comprise a
biotin-containing group, a diethylene-triaminepentaacetyl group, a
(-)-menthoxyacetyl group, a fluorescein-containing group or an
N-acetylneuraminyl group. In yet other embodiments, the compound
may promotes aggregation of natural .beta.-amyloid peptides when
contacted with the natural .beta.-amyloid peptides, may be further
modified to alter a pharmacokinetic property of the compound or may
be further modified to label the compound with a detectable
substance.
[0148] In another embodiment, the invention provides a
.beta.-amyloid modulator compound comprising a formula:
A-(Y)-Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Z)-B
[0149] wherein Xaa.sub.1, Xaa.sub.2 and Xaa.sub.3 are each amino
acid structures and at least two of Xaa.sub.1, Xaa.sub.2 and
Xaa.sub.3 are, independently, selected from the group consisting of
a leucine structure, a phenylalanine structure and a valine
structure;
[0150] Y, which may or may not be present, is a peptidic structure
having the formula (Xaa).sub.a, wherein Xaa is any amino acid
structure and a is an integer from 1 to 15;
[0151] Z, which may or may not be present, is a peptidic structure
having the formula (Xaa).sub.b, wherein Xaa is any amino acid
structure and b is an integer from 1 to 15; and
[0152] A and B, at least one of which is present, are modifying
groups attached directly or indirectly to the amino terminus and
carboxy terminus, respectively, of the compound;
[0153] Xaa.sub.1, Xaa.sub.2, Xaa.sub.3, Y, Z, A and B being
selected such that the compound modulates the aggregation or
inhibits the neurotoxicity of natural .beta.-amyloid peptides when
contacted with the natural .beta.-amyloid peptides.
[0154] Preferably, the compound inhibits aggregation of natural
.beta.-amyloid peptides when contacted with the natural
.beta.-amyloid peptides. In preferred embodiments, Xaa.sub.1 and
Xaa.sub.2 are each phenylalanine structures or Xaa.sub.2 and
Xaa.sub.3 are each phenylalanine structures. In one subembodiment,
the compound comprises the formula:
A-(Y)-Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-(Z)
[0155] In another subembodiment, the compound comprises the
formula:
(Y)-Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-(Z)-B
[0156] "n" can be, for example, an integer between 1 and 5, whereas
"a" and "b" can be, for example, integers between 1 and 5. The
modifying group "A" preferably comprises a cyclic, heterocyclic or
polycyclic group. More preferably, A contains a cis-decalin group,
such as cholanoyl structure or a cholyl group In other embodiments,
A can comprise a biotin-containing group, a
diethylene-triaminepentaacetyl group, a (-)-menthoxyacetyl group, a
fluorescein-containing group or an N-acetylneuraminyl group. In yet
other embodiments, the compound may promote aggregation of natural
.beta.-amyloid peptides when contacted with the natural
.beta.-amyloid peptides, may be further modified to alter a
pharmacokinetic property of the compound or may be further modified
to label the compound with a detectable substance.
[0157] In preferred specific embodiments, the invention provides a
.beta.-amyloid modulator compound comprising a modifying group
attached directly or indirectly to a peptidic structure, wherein
the peptidic structure comprises amino acid structures having an
amino acid sequence selected from the group consisting of
His-Gln-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO: 5),
His-Gln-Lys-Leu-Val-Phe-Phe (SEQ ID NO: 6),
Gln-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO: 7), Gln-Lys-Leu-Val-Phe-Phe
(SEQ ID NO: 8), Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO: 9),
Lys-Leu-Val-Phe-Phe (SEQ ID NO: 10), Leu-Val-Phe-Phe-Ala (SEQ ID
NO: 11), Leu-Val-Phe-Phe (SEQ ID NO: 12), Leu-Ala-Phe-Phe-Ala (SEQ
ID NO: 13), Val-Phe-Phe (SEQ ID NO: 19), Phe-Phe-Ala (SEQ ID NO:
20), Phe-Phe-Val-Leu-Ala (SEQ ID NO: 21), Leu-Val-Phe-Phe-Lys (SEQ
ID NO: 22), Leu-Val-Iodotyrosine-Phe-Ala (SEQ ID NO: 23),
Val-Phe-Phe-Ala (SEQ ID NO: 24), Ala-Val-Phe-Phe-Ala (SEQ ID NO:
25), Leu-Val-Phe-Iodotyrosine-Ala (SEQ ID NO: 26),
Leu-Val-Phe-Phe-Ala-Glu (SEQ ID NO: 27), Phe-Phe-Val-Leu (SEQ ID
NO: 28), Phe-Lys-Phe-Val-Leu (SEQ ID NO: 29), Lys-Leu-Val-Ala-Phe
(SEQ ID NO: 30), Lys-Leu-Val-Phe-Phe-.beta.Ala (SEQ ID NO: 31) and
Leu-Val-Phe-Phe-DAla (SEQ ID NO: 32).
[0158] These specific compounds can be further modified to alter a
pharmacokinetic property of the compound and/or further modified to
label the compound with a detectable substance.
[0159] The modulator compounds of the invention can be incorporated
into pharmaceutical compositions (described further in subsection V
below) and can be used in detection and treatment methods as
described further in subsection VI below.
[0160] II. Modifying Groups
[0161] Within a modulator compound of the invention, a peptidic
structure (such as an A.beta. derived peptide, or an A.beta.
aggregation core domain, or an amino acid sequence corresponding to
a rearranged A.beta. aggregation core domain) is coupled directly
or indirectly to at least one modifying group (abbreviated as MG).
In one embodiment, a modulator compounds of the invention
comprising an aggregation core domain coupled to a modifying group,
the compound can be illustrated schematically as MG-ACD. The term
"modifying group" is intended to include structures that are
directly attached to the peptidic structure (e.g., by covalent
coupling), as well as those that are indirectly attached to the
peptidic structure (e.g., by a stable non-covalent association or
by covalent coupling to additional amino acid residues, or
mimetics, analogues or derivatives thereof, which may flank the
A.beta.-derived peptidic structure). For example, the modifying
group can be coupled to the amino-terminus or carboxy-terminus of
an A.beta.-derived peptidic structure, or to a peptidic or
peptidomimetic region flanking the core domain. Alternatively, the
modifying group can be coupled to a side chain of at least one
amino acid residue of an A.beta.-derived peptidic structure, or to
a peptidic or peptidomimetic region flanking the core domain (e.g.,
through the epsilon amino group of a lysyl residue(s), through the
carboxyl group of an aspartic acid residue(s) or a glutamic acid
residue(s), through a hydroxy group of a tyrosyl residue(s), a
serine residue(s) or a threonine residue(s) or other suitable
reactive group on an amino acid side chain). Modifying groups
covalently coupled to the peptidic structure can be attached by
means and using methods well known in the art for linking chemical
structures, including, for example, amide, alkylamino, carbamate or
urea bonds.
[0162] The term "modifying group" is intended to include groups
that are not naturally coupled to natural A.beta. peptides in their
native form. Accordingly, the term "modifying group" is not
intended to include hydrogen. The modifying group(s) is selected
such that the modulator compound alters, and preferably inhibits,
aggregation of natural .beta.-amyloid peptides when contacted with
the natural .beta.-amyloid peptides or inhibits the neurotoxicity
of natural .beta.-amyloid peptides when contacted with the natural
.beta.-amyloid peptides. Although not intending to be limited by
mechanism, the modifying group(s) of the modulator compounds of the
invention is thought to function as a key pharmacophore which is
important for conferring on the modulator the ability to disrupt
A.beta. polymerization.
[0163] In a preferred embodiment, the modifying group(s) comprises
a cyclic, heterocyclic or polycyclic group. The term "cyclic
group", as used herein, is intended to include cyclic saturated or
unsaturated (i.e., aromatic) group having from about 3 to 10,
preferably about 4 to 8, and more preferably about 5 to 7, carbon
atoms. Exemplary cyclic groups include cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, and cyclooctyl. Cyclic groups may be
unsubstituted or substituted at one or more ring positions. Thus, a
cyclic group may be substituted with, e.g., halogens, alkyls,
cycloalkyls, alkenyls, alkynyls, aryls, heterocycles, hydroxyls,
aminos, nitros, thiols amines, imines, amides, phosphonates,
phosphines, carbonyls, carboxyls, silyls, ethers, thioethers,
sulfonyls, sulfonates, selenoethers, ketones, aldehydes, esters,
--CF.sub.3, --CN, or the like.
[0164] The term "heterocyclic group" is intended to include cyclic
saturated or unsaturated (i.e., aromatic) group having from about 3
to 10, preferably about 4 to 8, and more preferably about 5 to 7,
carbon atoms, wherein the ring structure includes about one to four
heteroatoms. Heterocyclic groups include pyrrolidine, oxolane,
thiolane, imidazole, oxazole, piperidine, piperazine, morpholine.
The heterocyclic ring can be substituted at one or more positions
with such substituents as, for example, halogens, alkyls,
cycloalkyls, alkenyls, alkynyls, aryls, other heterocycles,
hydroxyl, amino, nitro, thiol, amines, imines, amides,
phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers,
thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters,
--CF.sub.3, --CN, or the like. Heterocycles may also be bridged or
fused to other cyclic groups as described below.
[0165] The term "polycycdic group" as used herein is intended to
refer to two or more saturated or unsaturated (i.e., aromatic)
cyclic rings in which two or more carbons are common to two
adjoining rings, e.g., the rings are "fused rings". Rings that are
joined through non-adjacent atoms are termed "bridged" rings. Each
of the rings of the polycyclic group can be substituted with such
substituents as described above, as for example, halogens, alkyls,
cycloalkyls, alkenyls, alkynyls, hydroxyl, amino, nitro, thiol,
amines, imines, amides, phosphonates, phosphines, carbonyls,
carboxyls, silyls, ethers, thioethers, sulfonyls, selenoethers,
ketones, aldehydes, esters, --CF.sub.3, --CN, or the like.
[0166] A preferred polycyclic group is a group containing a
cis-decalin structure. Although not intending to be limited by
mechanism, it is thought that the "bent" conformation conferred on
a modifying group by the presence of a cis-decalin structure
contributes to the efficacy of the modifying group in disrupting
A.beta. polymerization. Accordingly, other structures which mimic
the "bent" configuration of the cis-decalin structure can also be
used as modifying groups. An example of a cis-decalin containing
structure that can be used as a modifying group is a cholanoyl
structure, such as a cholyl group. For example, a modulator
compound can be modified at its amino terminus with a cholyl group
by reacting the aggregation core domain with cholic acid, a bile
acid, as described in Example 4 (the structure of cholic acid is
illustrated in FIG. 2). Moreover, a modulator compound can be
modified at its carboxy terminus with a cholyl group according to
methods known in the art (see e.g., Wess, G. et al. (1993)
Tetrahedron Letters, 34:817-822; Wess, G. et al. (1992) Tetrahedron
Letters 33:195-198; and Kramer, W. et al. (1992) J. Biol. Chem.
267:18598-18604). Cholyl derivatives and analogues can also be used
as modifying groups. For example, a preferred cholyl derivative is
Aic (3-(O-aminoethyl-iso)-cholyl), which has a free amino group
that can be used to further modify the modulator compound (e.g., a
chelation group for .sup.99mTc can be introduced through the free
amino group of Aic). As used herein, the term "cholanoyl structure"
is intended to include the cholyl group and derivatives and
analogues thereof, in particular those which retain a four-ring
cis-decalin configuration. Examples of cholanoyl structures include
groups derived from other bile acids, such as deoxycholic acid,
lithocholic acid, ursodeoxycholic acid, chenodeoxycholic acid and
hyodeoxycholic acid, as well as other related structures such as
cholanic acid, bufalin and resibufogenin (although the latter two
compounds are not preferred for use as a modifying group). Another
example of a cis-decalin containing compound is
5.beta.-cholestan-3.alpha.-ol (the cis-decalin isomer of
(+)-dihydrocholesterol). For further description of bile acid and
steroid structure and nomenclature, see Nes, W. R. and McKean, M.
L. Biochemistry of Steroids and Other Isopentanoids, University
Park Press, Baltimore, Md., Chapter 2.
[0167] In addition to cis-decalin containing groups, other
polycyclic groups may be used as modifying groups. For example,
modifying groups derived from steroids or .beta.-lactams may be
suitable modifying groups. Moreover, non-limiting examples of some
additional cyclic, heterocyclic or polycyclic compounds which can
be used to modify an A.beta.-derived peptidic structure are shown
schematically in FIG. 2. In one embodiment, the modifying group is
a "biotinyl structure", which includes biotinyl groups and
analogues and derivatives thereof (such as a 2-iminobiotinyl
group). In another embodiment, the modifying group can comprise a
"fluorescein-containing group", such as a group derived from
reacting an A.beta.-derived peptidic structure with 5-(and
6-)-carboxyfluorescein, succinimidyl ester or fluorescein
isothiocyanate. In various other embodiments, the modifying
group(s) can comprise an N-acetylneuraminyl group, a
trans-4-cotininecarboxyl group, a 2-imino-1-imidazolidineacetyl
group, an (S)-(-)-indoline-2-carboxyl group, a (-)-menthoxyacetyl
group, a 2-norbornaneacetyl group, a
.gamma.-oxo-5-acenaphthenebutyryl, a
(-)-2-oxo-4-thiazolidinecarboxyl group, a tetrahydro-3-furoyl
group, a 2-iminobiotinyl group, a diethylenetriaminepentaacetyl
group, a 4-morpholinecarbonyl group, a 2-thiopheneacetyl group or a
2-thiophenesulfonyl group.
[0168] Preferred modifying groups include groups comprising cholyl
structures, biotinyl structures, fluorescein-containing groups, a
diethylene-triaminepentaacetyl group, a (-)-menthoxyacetyl group,
and a N-acetylneuraminyl group. More preferred modifying groups
those comprising a cholyl structure or an iminiobiotinyl group.
[0169] In addition to the cyclic, heterocyclic and polycyclic
groups discussed above, other types of modifying groups can be used
in a modulator of the invention. For example, small hydrophobic
groups may be suitable modifying groups. An example of a suitable
non-cyclic modifying group is an acetyl group.
[0170] Yet another type of modifying group is a compound that
contains a non-natural amino acid that acts as a beta-turn mimetic,
such as a dibenzofuran-based amino acid described in Tsang, K. Y.
et al. (1994) J. Am. Chem. Soc. 116:3988-4005; Diaz, H and Kelly,
J. W. (1991) Tetrahedron Letters 41:5725-5728; and Diaz. H et al.
(1992) J. Am. Chem. Soc. 114:8316-8318. An example of such a
modifying group is a peptide-aminoethyldibenzofuranyl-proprionic
acid (Adp) group (e.g., DDIIL-Adp). This type of modifying group
further can comprise one or more N-methyl peptide bonds to
introduce additional steric hindrance to the aggregation of natural
.beta.-AP when compounds of this type interact with natural
.beta.-AP.
[0171] III. Additional Chemical Modifications of AD Modulators
[0172] A .beta.-amyloid modulator compound of the invention can be
further modified to alter the specific properties of the compound
while retaining the ability of the compound to alter A.beta.
aggregation and inhibit A.beta. neurotoxicity. For example, in one
embodiment, the compound is further modified to alter a
pharmacokinetic property of the compound, such as in vivo stability
or half-life. In another embodiment, the compound is further
modified to label the compound with a detectable substance. In yet
another embodiment, the compound is further modified to couple the
compound to an additional therapeutic moiety. Schematically, a
modulator of the invention comprising an A.beta. aggregation core
domain coupled directly or indirectly to at least one modifying
group can be illustrated as MG-ACD, whereas this compound which has
been further modified to alter the properties of the modulator can
be illustrated as MG-ACD-CM, wherein CM represents an additional
chemical modification.
[0173] To further chemically modify the compound, such as to alter
the pharmacokinetic properties of the compound, reactive groups can
be derivatized. For example, when the modifying group is attached
to the amino-terminal end of the aggregation core domain, the
carboxy-terminal end of the compound can be further modified.
Preferred C-terminal modifications include those which reduce the
ability of the compound to act as a substrate for
carboxypeptidases. Examples of preferred C-terminal modifiers
include an amide group, an ethylamide group and various non-natural
amino acids, such as D-amino acids and .beta.-alanine.
Alternatively, when the modifying group is attached to the
carboxy-terminal end of the aggregation core domain, the
amino-terminal end of the compound can be further modified, for
example, to reduce the ability of the compound to act as a
substrate for aminopeptidases.
[0174] A modulator compound can be further modified to label the
compound by reacting the compound with a detectable substance.
Suitable detectable substances include various enzymes, prosthetic
groups, fluorescent materials, luminescent materials and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidinibiotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; and examples of suitable radioactive material include
.sup.14C, .sup.123I, .sup.124I, .sup.125I, .sup.131I, .sup.99mTc,
.sup.35S or .sup.3H. In a preferred embodiment, a modulator
compound is radioactively labeled with .sup.14C, either by
incorporation of .sup.14C into the modifying group or one or more
amino acid structures in the modulator compound. Labeled modulator
compounds can be used to assess the in vivo pharmacokinetics of the
compounds, as well as to detect A.beta. aggregation, for example
for diagnostic purposes. A.beta. aggregation can be detected using
a labeled modulator compound either in vivo or in an in vitro
sample derived from a subject.
[0175] Preferably, for use as an in vivo diagnostic agent, a
modulator compound of the invention is labeled with radioactive
technetium or iodine. Accordingly, in one embodiment, the invention
provides a modulator compound labeled with technetium, preferably
.sup.99mTc. Methods for labeling peptide compounds with technetium
are known in the art (see e.g., U.S. Pat. Nos. 5,443,815, 5,225,180
and 5,405,597, all by Dean et al.; Stepniak-Biniakiewicz, D., et
al. (1992) J. Med. Chem. 35:274-279; Fritzberg, A. R., et al.
(1988) Proc. Natl. Acad. Sci. USA 85:4025-4029; Baidoo, K. E., et
al. (1990) Cancer Res. Suppl. 50:799s-803s; and Regan, L. and
Smith, C. K. (1995) Science 270:980-982). A modifying group can be
chosen that provides a site at which a chelation group for
.sup.99mTc can be introduced, such as the Aic derivative of cholic
acid, which has a free amino group (see Example 11). In another
embodiment, the invention provides a modulator compound labeled
with radioactive iodine. For example, a phenylalanine residue
within the A.beta. sequence (such as Phe.sub.19 or Phe.sub.20) can
be substituted with radioactive iodotyrosyl (see Example 11). Any
of the various isotopes of radioactive iodine can be incorporated
to create a diagnostic agent. Preferably, .sup.123I (half-life=13.2
hours) is used for whole body scintigraphy, .sup.124I (half life=4
days) is used for positron emission tomography (PET), .sup.125I
(half life=60 days) is used for metabolic turnover studies and
.sup.131I (half life=8 days) is used for whole body counting and
delayed low resolution imaging studies.
[0176] Furthermore, an additional modification of a modulator
compound of the invention can serve to confer an additional
therapeutic property on the compound. That is, the additional
chemical modification can comprise an additional functional moiety.
For example, a functional moiety which serves to break down or
dissolve amyloid plaques can be coupled to the modulator compound.
In this form, the MG-ACD portion of the modulator serves to target
the compound to A.beta. peptides and disrupt the polymerization of
the A.beta. peptides, whereas the additional functional moiety
serves to break down or dissolve amyloid plaques after the compound
has been targeted to these sites.
[0177] In an alternative chemical modification, a .beta.-amyloid
compound of the invention is prepared in a "prodrug" form, wherein
the compound itself does not modulate A.beta. aggregation, but
rather is capable of being transformed, upon metabolism in vivo,
into a .beta.-amyloid modulator compound as defined herein. For
example, in this type of compound, the modulating group can be
present in a prodrug form that is capable of being converted upon
metabolism into the form of an active modulating group. Such a
prodrug form of a modifying group is referred to herein as a
"secondary modifying group." A variety of strategies are known in
the art for preparing peptide prodrugs that limit metabolism in
order to optimize delivery of the active form of the peptide-based
drug (see e.g., Moss, J. (1995) in Peptide-Based Drug Design:
Controlling Transport and Metabolism, Taylor, M. D. and Amidon, G.
L. (eds), Chapter 18. Additionally strategies have been
specifically tailored to achieving CNS delivery based on
"sequential metabolism" (see e.g., Bodor, N., et al (1992) Science
257:1698-1700; Prokai, L., et al (1994) J. Am. Chem. Soc.
116:2643-2644; Bodor, N. and Prokai, L. (1995) in Peptide-Based
Drug Design: Controlling Transport and Metabolism, Taylor, M. D.
and Amidon, G. L. (eds), Chapter 14. In one embodiment of a prodrug
form of a modulator of the invention, the modifying group comprises
an alkyl ester to facilitate blood-brain barrier permeability.
[0178] Modulator compounds of the invention can be prepared by
standard techniques known in the art. The peptide component of a
modulator composed, at least in part, of a peptide, can be
synthesized using standard techniques such as those described in
Bodansky, M. Principles of Peptide Synthesis, Springer Verlag,
Berlin (1993) and Grant, G. A (ed.). Synthetic Pentides: A User's
Guide, W. H. Freeman and Company, New York (1992). Automated
peptide synthesizers are commercially available (e.g., Advanced
ChemTech Model 396; Milligen/Biosearch 9600). Additionally, one or
more modulating groups can be attached to the A.beta.-derived
peptidic component (e.g., an A.beta. aggregation core domain) by
standard methods, for example using methods for reaction through an
amino group (e.g., the alpha-amino group at the amino-terminus of a
peptide), a carboxyl group (e.g., at the carboxy terminus of a
peptide), a hydroxyl group (e.g., on a tyrosine, serine or
threonine residue) or other suitable reactive group on an amino
acid side chain (see e.g., Greene, T. W. and Wuts, P. G. M.
Protective Groups in Organic Synthesis, John Wiley and Sons, Inc.,
New York (1991). Exemplary syntheses of preferred .beta. amyloid
modulators is described further in Examples 1, 4 and 11.
[0179] IV. Screening Assays
[0180] Another aspect of the invention pertains to a method for
selecting a modulator of .beta.-amyloid aggregation. In the method,
a test compound is contacted with natural .beta. amyloid peptides,
the aggregation of the natural .beta.-AP is measured and a
modulator is selected based on the ability of the test compound to
alter the aggregation of the natural .beta.-AP (e.g., inhibit or
promote aggregation). In a preferred embodiment, the test compound
is contacted with a molar excess amount of the natural .beta.-AP.
The amount and/or rate of natural .beta.-AP aggregation in the
presence of the test compound can be determined by a suitable assay
indicative of .beta.-A.beta. aggregation, as described herein (see
e.g., Examples 2, 5 and 6).
[0181] In a preferred assay, the natural .beta.-AP is dissolved in
solution in the presence of the test compound and aggregation of
the natural .beta.-AP is assessed in a nucleation assay (see
Example 6) by assessing the turbidity of the solution over time, as
measured by the apparent absorbance of the solution at 405 nm
(described further in Example 6; see also Jarrett et al. (1993)
Biochemistry 32:4693-4697). In the absence of a .beta.-amyloid
modulator, the A.sub.405nm of the solution typically stays
relatively constant during a lag time in which the .beta.-AP
remains in solution, but then the A.sub.405nm of the solution
rapidly increases as the .beta.-AP aggregates and comes out of
solution, ultimately reaching a plateau level (i.e., the
A.sub.405nm of the solution exhibits sigmoidal kinetics over time).
In contrast, in the presence of a test compound that inhibits
.beta.-AP aggregation, the A.sub.405nm of the solution is reduced
compared to when the modulator is absent. Thus, in the presence of
the inhibitory modulator, the solution may exhibit an increased lag
time, a decreased slope of aggregation and/or a lower plateau level
compared to when the modulator is absent. This method for selecting
a modulator of .beta.-amyloid polymerization can similarly be used
to select modulators that promote .beta.-AP aggregation. Thus, in
the presence of a modulator that promotes .beta.-AP aggregation,
the A.sub.405nm of the solution is increased compared to when the
modulator is absent (e.g., the solution may exhibit an decreased
lag time, increase slope of aggregation and/or a higher plateau
level compared to when the modulator is absent).
[0182] Another assay suitable for use in the screening method of
the invention, a seeded extension assay, is also described further
in Example 6. In this assay, .beta.-AP monomer and an aggregated
.beta.-AP "seed" are combined, in the presence and absence of a
test compound, and the amount of .beta.-fibril formation is assayed
based on enhanced emission of the dye Thioflavine T when contacted
with .beta.-AP fibrils. Moreover, .beta.-AP aggregation can be
assessed by electron microscopy (EM) of the .beta.-AP preparation
in the presence or absence of the modulator. For example, .beta.
amyloid fibril formation, which is detectable by EM, is reduced in
the presence of a modulator that inhibits .beta.-AP aggregation
(i.e., there is a reduced amount or number of .beta.-fibrils in the
presence of the modulator), whereas .beta. fibril formation is
increased in the presence of a modulator that promotes .beta.-AP
aggregation (i.e., there is an increased amount or number of
.beta.-fibrils in the presence of the modulator).
[0183] An even more preferred assay for use in the screening method
of the invention to select suitable modulators is the neurotoxicity
assay described in Examples 3 and 10. Compounds are selected which
inhibit the formation of neurotoxic A.beta. aggregates and/or which
inhibit the neurotoxicity of preformed A.beta. fibrils. This
neurotoxicity assay is considered to be predictive of neurotoxicity
in vivo. Accordingly, inhibitory activity of a modulator compound
in the in vitro neurotoxicity assay is predictive of similar
inhibitory activity of the compound for neurotoxicity in vivo.
[0184] V. Pharmaceutical Compositions
[0185] Another aspect of the invention pertains to pharmaceutical
compositions of the .beta.-amyloid modulator compounds of the
invention. In one embodiment, the composition includes a .beta.
amyloid modulator compound in a therapeutically or prophylactically
effective amount sufficient to alter, and preferably inhibit,
aggregation of natural .beta.-amyloid peptides, and a
pharmaceutically acceptable carrier. In another embodiment, the
composition includes a .beta. amyloid modulator compound in a
therapeutically or prophylactically effective amount sufficient to
inhibit the neurotoxicity of natural .beta.-amyloid peptides, and a
pharmaceutically acceptable carrier. A "therapeutically effective
amount" refers to an amount effective, at dosages and for periods
of time necessary, to achieve the desired therapeutic result, such
as reduction or reversal or .beta.-amyloid deposition and/or
reduction or reversal of A.beta. neurotoxicity. A therapeutically
effective amount of modulator may vary according to factors such as
the disease state, age, sex, and weight of the individual, and the
ability of the modulator to elicit a desired response in the
individual. Dosage regimens may be adjusted to provide the optimum
therapeutic response. A therapeutically effective amount is also
one in which any toxic or detrimental effects of the modulator are
outweighed by the therapeutically beneficial effects. The potential
neurotoxicity of the modulators of the invention can be assayed
using the cell-based assay described in Examples 3 and 10 and a
therapeutically effective modulator can be selected which does not
exhibit significant neurotoxicity. In a preferred embodiment, a
therapeutically effective amount of a modulator is sufficient to
alter, and preferably inhibit, aggregation of a molar excess amount
of natural .beta.-amyloid peptides. A "prophylactically effective
amount" refers to an amount effective, at dosages and for periods
of time necessary, to achieve the desired prophylactic result, such
as preventing or inhibiting the rate of .beta.-amyloid deposition
and/or A.beta. neurotoxicity in a subject predisposed to
.beta.-amyloid deposition. A prophylactically effective amount can
be determined as described above for the therapeutically effective
amount. Typically, since a prophylactic dose is used in subjects
prior to or at an earlier stage of disease, the prophylactically
effective amount will be less than the therapeutically effective
amount.
[0186] One factor that may be considered when determining a
therapeutically or prophylactically effective amount of a .beta.
amyloid modulator is the concentration of natural .beta.-AP in a
biological compartment of a subject, such as in the cerebrospinal
fluid (CSF) of the subject. The concentration of natural .beta.-AP
in the CSF has been estimated at 3 nM (Schwartzman, (1994) Proc.
Natl. Acad. Sci. USA 91:8368-8372). A non-limiting range for a
therapeutically or prophylactically effective amounts of a .beta.
amyloid modulator is 0.01 nM-10 .mu.M. It is to be noted that
dosage values may vary with the severity of the condition to be
alleviated. It is to be further understood that for any particular
subject, specific dosage regimens should be adjusted over time
according to the individual need and the professional judgment of
the person administering or supervising the administration of the
compositions, and that dosage ranges set forth herein are exemplary
only and are not intended to limit the scope or practice of the
claimed composition.
[0187] The amount of active compound in the composition may vary
according to factors such as the disease state, age, sex, and
weight of the individual, each of which may affect the amount of
natural .beta.-AP in the individual. Dosage regimens may be
adjusted to provide the optimum therapeutic response. For example,
a single bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
mammalian subjects to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on (a) the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0188] As used herein "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
In one embodiment, the carrier is suitable for parenteral
administration. Preferably, the carrier is suitable for
administration into the central nervous system (e.g., intraspinally
or intracerebrally). Alternatively, the carrier can be suitable for
intravenous, intraperitoneal or intramuscular administration. In
another embodiment, the carrier is suitable for oral
administration. Pharmaceutically acceptable carriers include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersion. The use of such media and agents for pharmaceutically
active substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
compound, use thereof in the pharmaceutical compositions of the
invention is contemplated. Supplementary active compounds can also
be incorporated into the compositions.
[0189] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyetheylene glycol, and
the like), and suitable mixtures thereof. The proper fluidity can
be maintained, for example, by the use of a coating such as
lecithin, by the maintenance of the required particle size in the
case of dispersion and by the use of surfactants. In many cases, it
will be preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, monostearate salts and gelatin.
Moreover, the modulators can be administered in a time release
formulation, for example in a composition which includes a slow
release polymer. The active compounds can be prepared with carriers
that will protect the compound against rapid release, such as a
controlled release formulation, including implants and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters,
polylactic acid and polylactic, polyglycolic copolymers (PLG). Many
methods for the preparation of such formulations are patented or
generally known to those skilled in the art.
[0190] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., .beta.-amyloid modulator)
in the required amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle which
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying which yields a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0191] A modulator compound of the invention can be formulated with
one or more additional compounds that enhance the solubility of the
modulator compound. Preferred compounds to be added to formulations
to enhance the solubility of the modulators are cyclodextrin
derivatives, preferably hydroxypropyl-.gamma.-cyclodextrin. Drug
delivery vehicles containing a cyclodextrin derivative for delivery
of peptides to the central nervous system are described in Bodor,
N., et al. (1992) Science 257:1698-1700. For the .beta.-amyloid
modulators described herein, inclusion in the formulation of
hydroxypropyl-.gamma.-cyclodextrin at a concentration 50-200 mM
increases the aqueous solubility of the compounds. In addition to
increased solubility, inclusion of a cyclodextrin derivative in the
formulation may have other beneficial effects, since p-cyclodextrin
itself has been reported to interact with the A.beta. peptide and
inhibit fibril formation in vitro (Camilleri, P., et al. (1994)
FEBS Letters 341:256-258. Accordingly, use of a modulator compound
of the invention in combination with a cyclodextrin derivative may
result in greater inhibition of A.beta. aggregation than use of the
modulator alone. Chemical modifications of cyclodextrins are known
in the art (Hanessian, S., et al. (1995) J. Org. Chem.
60:4786-4797). In addition to use as an additive in a
pharmaceutical composition containing a modulator of the invention,
cyclodextrin derivatives may also be useful as modifying groups
and, accordingly, may also be covalently coupled to an A.beta.
peptide compound to form a modulator compound of the invention.
[0192] In another embodiment, a pharmaceutical composition
comprising a modulator of the invention is formulated such that the
modulator is transported across the blood-brain barrier (BBB).
Various strategies known in the art for increasing transport across
the BBB can be adapted to the modulators of the invention to
thereby enhance transport of the modulators across the BBB (for
reviews of such strategies, see e.g., Pardridge, W. M. (1994)
Trends in Biotechnol. 12:239-245; Van Bree, J. B. et al. (1993)
Pharm. World Sci. 15:2-9, and Pardridge, W. M. et al. (1992)
Pharmacol. Toxicol. 71:3-10). In one approach, the modulator is
chemically modified to form a prodrug with enhanced transmembrane
transport. Suitable chemical modifications include covalent linking
of a fatty acid to the modulator through an amide or ester linkage
(see e.g., U.S. Pat. No. 4,933,324 and PCT Publication WO 89/07938,
both by Shashoua; U.S. Pat. No. 5,284,876 by Hesse et al.; Toth, I.
et al (1994) J. Drug Target. 2:217-239; and Shashoua, V. E. et al.
(1984) J. Med. Chem. 27:659-664) and glycating the modulator (see
e.g., U.S. Pat. No. 5,260,308 by Poduslo et al.). Also, N-acylamino
acid derivatives may be used in a modulator to form a "lipidic"
prodrug (see e.g., U.S. Pat. No. 5,112,863 by Hashimoto et
al.).
[0193] In another approach for enhancing transport across the BBB,
a peptidic or peptidomimetic modulator is conjugated to a second
peptide or protein, thereby forming a chimeric protein, wherein the
second peptide or protein undergoes absorptive-mediated or
receptor-mediated transcytosis through the BBB. Accordingly, by
coupling the modulator to this second peptide or protein, the
chimeric protein is transported across the BBB. The second peptide
or protein can be a ligand for a brain capillary endothelial cell
receptor ligand. For example, a preferred ligand is a monoclonal
antibody that specifically binds to the transferrin receptor on
brain capillary endothelial cells (see e.g., U.S. Pat. Nos.
5,182,107 and 5,154,924 and PCT Publications WO 93/10819 and WO
95/02421, all by Friden et al.). Other suitable peptides or
proteins that can mediate transport across the BBB include histones
(see e.g., U.S. Pat. No. 4,902,505 by Pardridge and Schimmel) and
ligands such as biotin, folate, niacin, pantothenic acid,
riboflavin, thiamin, pryridoxal and ascorbic acid (see e.g., U.S.
Pat. Nos. 5,416,016 and 5,108,921, both by Heinstein).
Additionally, the glucose transporter GLUT-1 has been reported to
transport glycopeptides (L-serinyl-.beta.-D-glucoside analogues of
[Met5]enkephalin) across the BBB (Polt, R. et al. (1994) Proc.
Natl. Acad. Sci. USA 91:7114-1778). Accordingly, a modulator
compound can be coupled to such a glycopeptide to target the
modulator to the GLUT-1 glucose transporter. For example, a
modulator compound which is modified at its amino terminus with the
modifying group Aic (3-(O-aminoethyl-iso)-cholyl, a derivative of
cholic acid having a free amino group) can be coupled to a
glycopeptide through the amino group of Aic by standard methods.
Chimeric proteins can be formed by recombinant DNA methods (e.g.,
by formation of a chimeric gene encoding a fusion protein) or by
chemical crosslinking of the modulator to the second peptide or
protein to form a chimeric protein. Numerous chemical crosslinking
agents are known in the (e.g., commercially available from Pierce,
Rockford Ill.). A crosslinking agent can be chosen which allows for
high yield coupling of the modulator to the second peptide or
protein and for subsequent cleavage of the linker to release
bioactive modulator. For example, a biotin-avidin-based linker
system may be used.
[0194] In yet another approach for enhancing transport across the
BBB, the modulator is encapsulated in a carrier vector which
mediates transport across the BBB. For example, the modulator can
be encapsulated in a liposome, such as a positively charged
unilamellar liposome (see e.g., PCT Publications WO 88/07851 and WO
88/07852, both by Faden) or in polymeric microspheres (see e.g.,
U.S. Pat. No. 5,413,797 by Khan et al., U.S. Pat. No. 5,271,961 by
Mathiowitz et al. and U.S. Pat. No. 5,019,400 by Gombotz et al.).
Moreover, the carrier vector can be modified to target it for
transport across the BBB. For example, the carrier vector (e.g.,
liposome) can be covalently modified with a molecule which is
actively transported across the BBB or with a ligand for brain
endothelial cell receptors, such as a monoclonal antibody that
specifically binds to transferrin receptors (see e.g., PCT
Publications WO 91/04014 by Collins et al and WO 94/02178 by Greig
et al.).
[0195] In still another approach to enhancing transport of the
modulator across the BBB, the modulator is coadministered with
another agent which functions to permeabilize the BBB. Examples of
such BBB "permeabilizers" include bradykinin and bradykinin
agonists (see e.g., U.S. Pat. No. 5,112,596 by Malfroy-Camine) and
peptidic compounds disclosed in U.S. Pat. No. 5,268,164 by Kozarich
et al.
[0196] A modulator compound of the invention can be formulated into
a pharmaceutical composition wherein the modulator is the only
active compound or, alternatively, the pharmaceutical composition
can contain additional active compounds. For example, two or more
modulator compounds may be used in combination. Moreover, a
modulator compound of the invention can be combined with one or
more other agents that have anti-amyloidogenic properties. For
example, a modulator compound can be combined with the non-specific
cholinesterase inhibitor tacrine (Cognex.RTM., Parke-Davis).
[0197] In another embodiment, a pharmaceutical composition of the
invention is provided as a packaged formulation. The packaged
formulation may include a pharmaceutical composition of the
invention in a container and printed instructions for
administration of the composition for treating a subject having a
disorder associated with .beta.-amyloidosis, e.g Alzheimer's
disease.
[0198] VI. Methods of Using AD Modulators
[0199] Another aspect of the invention pertains to methods for
altering the aggregation or inhibiting the neurotoxicity of natural
.beta.-amyloid peptides. In the methods of the invention, natural
.beta. amyloid peptides are contacted with a .beta. amyloid
modulator such that the aggregation of the natural .beta. amyloid
peptides is altered or the neurotoxicity of the natural .beta.
amyloid peptides is inhibited. In a preferred embodiment, the
modulator inhibits aggregation of the natural .beta. amyloid
peptides. In another embodiment, the modulator promotes aggregation
of the natural .beta. amyloid peptides. Preferably, aggregation of
a molar excess amount of .beta.-AP, relative to the amount of
modulator, is altered upon contact with the modulator.
[0200] In the method of the invention, natural .beta. amyloid
peptides can be contacted with a modulator either in vitro or in
vivo. Thus, the term "contacted with" is intended to encompass both
incubation of a modulator with a natural .beta.-AP preparation in
vitro and delivery of the modulator to a site in vivo where natural
.beta.-AP is present. Since the modulator compound interacts with
natural .beta.-AP, the modulator compounds can be used to detect
natural .beta.-AP, either in vitro or in vivo. Accordingly, one use
of the modulator compounds of the invention is as diagnostic agents
to detect the presence of natural .beta.-AP, either in a biological
sample or in vivo in a subject. Furthermore, detection of natural
.beta.-AP utilizing a modulator compound of the invention further
can be used to diagnose amyloidosis in a subject. Additionally,
since the modulator compounds of the invention disrupt .beta.-AP
aggregation and inhibit .beta.-AP neurotoxicity, the modulator
compounds also are useful in the treatment of disorders associated
with .beta.-amyloidosis, either prophylactically or
therapeutically. Accordingly, another use of the modulator
compounds of the invention is as therapeutic agents to alter
aggregation and/or neurotoxicity of natural .beta.-AP.
[0201] In one embodiment, a modulator compound of the invention is
used in vitro, for example to detect and quantitate natural
.beta.-AP in sample (e.g., a sample of biological fluid). To aid in
detection, the modulator compound can be modified with a detectable
substance. The source of natural .beta.-AP used in the method can
be, for example, a sample of cerebrospinal fluid (e.g., from an AD
patient, an adult susceptible to AD due to family history, or a
normal adult). The natural .beta.-AP sample is contacted with a
modulator of the invention and aggregation of the .beta.-AP is
measured, such as by as assay described in Examples 2, 5 and 6.
Preferably, the nucleation assay and/or seeded extension assay
described in Example 6 is used. The degree of aggregation of the
.beta.-AP sample can then be compared to that of a control
sample(s) of a known concentration of .beta.-AP, similarly
contacted with the modulator and the results can be used as an
indication of whether a subject is susceptible to or has a disorder
associated with .beta.-amyloidosis. Moreover, .beta.-AP can be
detected by detecting a modulating group incorporated into the
modulator. For example, modulators incorporating a biotin compound
as described herein (e.g., an amino-terminally biotinylated
.beta.-AP peptide) can be detected using a streptavidin or avidin
probe which is labeled with a detectable substance (e.g., an
enzyme, such as peroxidase). Detection of natural .beta.-AP
aggregates mixed with a modulator of the invention using a probe
that binds to the modulating group (e.g., biotin/streptavidin) is
described further in Example 2.
[0202] In another embodiment, a modulator compound of the invention
is used in vivo to detect, and, if desired, quantitate, natural
.beta.-AP deposition in a subject, for example to aid in the
diagnosis of .beta. amyloidosis in the subject. To aid in
detection, the modulator compound can be modified with a detectable
substance, preferably .sup.99mTc or radioactive iodine (described
further above), which can be detected in vivo in a subject. The
labeled .beta.-amyloid modulator compound is administered to the
subject and, after sufficient time to allow accumulation of the
modulator at sites of amyloid deposition, the labeled modulator
compound is detected by standard imaging techniques. The
radioactive signal generated by the labeled compound can be
directly detected (e.g., whole body counting), or alternatively,
the radioactive signal can be converted into an image on an
autoradiograph or on a computer screen to allow for imaging of
amyloid deposits in the subject. Methods for imaging amyloidosis
using radiolabeled proteins are known in the art. For example,
serum amyloid P component (SAP), radiolabeled with either .sup.123I
or .sup.99mTc, has been used to image systemic amyloidosis (see
e.g., Hawkins, P. N. and Pepys, M. B. (1995) Eur. J. Nucl. Med.
22:595-599). Of the various isotypes of radioactive iodine,
preferably .sup.123I (half-life=13.2 hours) is used for whole body
scintigraphy, .sup.124I (half life=4 days) is used for positron
emission tomography (PET), .sup.125I (half life=60 days) is used
for metabolic turnover studies and .sup.131I (half life=8 days) is
used for whole body counting and delayed low resolution imaging
studies. Analogous to studies using radiolabeled SAP, a labeled
modulator compound of the invention can be delivered to a subject
by an appropriate route (e.g., intravenously, intraspinally,
intracerebrally) in a single bolus, for example containing 100
.mu.g of labeled compound carrying approximately 180 MBq of
radioactivity.
[0203] The invention provides a method for detecting the presence
or absence of natural .beta.-amyloid peptides in a biological
sample, comprising contacting a biological sample with a compound
of the invention and detecting the compound bound to natural
.beta.-amyloid peptides to thereby detect the presence or absence
of natural .beta.-amyloid peptides in the biological sample. In one
embodiment, the .beta.-amyloid modulator compound and the
biological sample are contacted in vitro. In another embodiment,
the .beta.-amyloid modulator compound is contacted with the
biological sample by administering the .beta.-amyloid modulator
compound to a subject. For in vivo administration, preferably the
compound is labeled with radioactive technetium or radioactive
iodine.
[0204] The invention also provides a method for detecting natural
.beta.-amyloid peptides to facilitate diagnosis of a
.beta.-amyloidogenic disease, comprising contacting a biological
sample with the compound of the invention and detecting the
compound bound to natural .beta.-amyloid peptides to facilitate
diagnosis of a .beta.-amyloidogenic disease. In one embodiment, the
.beta.-amyloid modulator compound and the biological sample are
contacted in vitro. In another embodiment, the .beta.-amyloid
modulator compound is contacted with the biological sample by
administering the .beta.-amyloid modulator compound to a subject.
For in vivo administration, preferably the compound is labeled with
radioactive technetium or radioactive iodine. Preferably, use of
the method facilitates diagnosis of Alzheimer's disease.
[0205] In another embodiment, the invention provides a method for
altering natural .beta.-AP aggregation or inhibiting .beta.-AP
neurotoxicity, which can be used prophylactically or
therapeutically in the treatment or prevention of disorders
associated with .beta. amyloidosis, e.g., Alzheimer's Disease. As
demonstrated in Example 10, modulator compounds of the invention
reduce the toxicity of natural .beta.-AP aggregates to cultured
neuronal cells. Moreover, the modulators not only reduce the
formation of neurotoxic aggregates but also have the ability to
reduce the neurotoxicity of preformed A.beta. fibrils. Accordingly,
the modulator compounds of the invention can be used to inhibit or
prevent the formation of neurotoxic A.beta. fibrils in subjects
(e.g., prophylactically in a subject predisposed to .beta.-amyloid
deposition) and can be used to reverse .beta.-amyloidosis
therapeutically in subjects already exhibiting .beta.-amyloid
deposition.
[0206] A modulator of the invention is contacted with natural
.beta. amyloid peptides present in a subject (e.g., in the
cerebrospinal fluid or cerebrum of the subject) to thereby alter
the aggregation of the natural .beta.-AP and/or inhibit the
neurotoxicity of the natural .beta.-APs. A modulator compound alone
can be administered to the subject, or alternatively, the modulator
compound can be administered in combination with other
therapeutically active agents (e.g., as discussed above in
subsection IV). When combination therapy is employed, the
therapeutic agents can be coadministered in a single pharmaceutical
composition, coadministered in separate pharmaceutical compositions
or administered sequentially.
[0207] The modulator may be administered to a subject by any
suitable route effective for inhibiting natural .beta.-AP
aggregation in the subject, although in a particularly preferred
embodiment, the modulator is administered parenterally, most
preferably to the central nervous system of the subject. Possible
routes of CNS administration include intraspinal administration and
intracerebral administration (e.g., intracerebrovascular
administration). Alternatively, the compound can be administered,
for example, orally, intraperitoneally, intravenously or
intramuscularly. For non-CNS administration routes, the compound
can be administered in a formulation which allows for transport
across the BBB. Certain modulators may be transported across the
BBB without any additional further modification whereas others may
need further modification as described above in subsection IV.
[0208] Suitable modes and devices for delivery of therapeutic
compounds to the CNS of a subject are known in the art, including
cerebrovascular reservoirs (e.g., Ommaya or Rikker reservoirs; see
e.g., Raney, J. P. et al (1988) J. Neurosci. Nurs. 20:23-29;
Sundaresan, N. et al. (1989) Oncology 3:15-22), catheters for
intrathecal delivery (e.g., Port-a-Cath, Y-catheters and the like;
see e.g., Plummer, J. L. (1991) Pain 44:215-220; Yaksh, T. L. et
al. (1986) Pharmacol. Biochem. Behav. 25:483-485), injectable
intrathecal reservoirs (e.g., Spinalgesic; see e.g., Brazenor, G.
A. (1987) Neurosurgery 21:484-491), implantable infusion pump
systems (e.g., Infusaid; see e.g., Zierski, J. et al. (1988) Acta
Neurochem. Suppl. 43:94-99; Kanoff, R. B. (1994) J. Am. Osteopath.
Assoc. 94:487-493) and osmotic pumps (sold by Alza Corporation). A
particularly preferred mode of administration is via an
implantable, externally programmable infusion pump. Suitable
infusion pump systems and reservoir systems are also described in
U.S. Pat. No. 5,368,562 by Blomquist and U.S. Pat. No. 4,731,058 by
Doan, developed by Pharmacia Deltec Inc.
[0209] The method of the invention for altering .beta.-AP
aggregation in vivo, and in particular for inhibiting .beta.-AP
aggregation, can be used therapeutically in diseases associated
with abnormal, amyloid aggregation and deposition to thereby slow
the rate of .beta. amyloid deposition and/or lessen the degree of
.beta. amyloid deposition, thereby ameliorating the course of the
disease. In a preferred embodiment, the method is used to treat
Alzheimer's disease (e.g., sporadic or familial AD, including both
individuals exhibiting symptoms of AD and individuals susceptible
to familial AD). The method can also be used prophylactically or
therapeutically to treat other clinical occurrences of .beta.
amyloid deposition, such as in Down's syndrome individuals and in
patients with hereditary cerebral hemorrhage with
amyloidosis-Dutch-type (HCHWA-D). While inhibition of .beta.-AP
aggregation is a preferred therapeutic method, modulators that
promote .beta.-AP aggregation may also be useful therapeutically by
allowing for the sequestration of .beta.-AP at sites that do not
lead to neurological impairment.
[0210] Additionally, abnormal accumulation of .beta.-amyloid
precursor protein in muscle fibers has been implicated in the
pathology of sporadic inclusion body myositis (IBM) (Askana, V. et
al. (1996) Proc. Natl. Acad. Sci. USA 93:1314-1319; Askanas, V. et
al. (1995) Current Opinion in Rheumatology 7:486-496). Accordingly,
the modulators of the invention can be used prophylactically or
therapeutically in the treatment of disorders in which .beta.-AP,
or APP, is abnormally deposited at non-neurological locations, such
as treatment of IBM by delivery of the modulators to muscle
fibers.
[0211] VII. Unmodified A.beta. Peptides that Inhibit Aggregation of
Natural .beta.-AP
[0212] In addition to the .beta.-amyloid modulators described
hereinbefore in which an A.beta. peptide is coupled to a modifying
group, the invention also provides .beta.-amyloid modulators
comprised of an unmodified A.beta. peptide. It has now been
discovered that certain portions of natural .beta.-AP can alter
aggregation of natural .beta.-APs when contacted with the natural
.beta.-APs (see Example 12). Accordingly, these unmodified A.beta.
peptides comprise a portion of the natural .beta.-AP sequence
(i.e., a portion of .beta.AP.sub.1-39, .beta.AP.sub.1-40,
.beta.AP.sub.1-42 and .beta.AP.sub.1-43). In particular these
unmodified A.beta. peptides have at least one amino acid deletion
compared to .beta.AP.sub.1-39, the shortest natural .beta.-AP, such
that the compound alters aggregation of natural .beta.-amyloid
peptides when contacted with the natural .beta.-amyloid peptides.
In various embodiments, these unmodified peptide compounds can
promote aggregation of natural .beta.-amyloid peptides, or, more
preferably, can inhibit aggregation of natural .beta.-amyloid
peptides when contacted with the natural .beta.-amyloid peptides.
Even more preferably, the unmodified peptide compound inhibits
aggregation of natural .beta.-amyloid peptides when contacted with
a molar excess amount of natural .beta.-amyloid peptides (e.g., a
10-fold, 33-fold or 100-fold molar excess amount of natural
.beta.-AP).
[0213] As discussed above, the unmodified peptide compounds of the
invention comprise an amino acid sequence having at least one amino
acid deletion compared to the amino acid sequence of
.beta.AP.sub.1-39. Alternatively, the unmodified peptide compound
can have at least five, ten, fifteen, twenty, twenty-five, thirty
or thirty-five amino acids deleted compared to .beta.AP.sub.1-39.
Still further the unmodified peptide compound can have 1-5, 1-10,
1-15, 1-20, 1-30 or 1-35 amino acids deleted compared to
.beta.AP.sub.1-39. The amino acid deletion(s) may occur at the
amino-terminus, the carboxy-terminus, an internal site, or a
combination thereof, of the .beta.-AP sequence. Accordingly, in one
embodiment, an unmodified peptide compound of the invention
comprises an amino acid sequence which has at least one internal
amino acid deleted compared to .beta.AP.sub.1-39. Alternatively,
the unmodified peptide compound can have at least five, ten,
fifteen, twenty, twenty-five, thirty or thirty-five internal amino
acids deleted compared to .beta.AP.sub.1-39. Still further the
unmodified peptide compound can have 1-5, 1-10, 1-15, 1-20, 1-25,
1-30 or 1-35 internal amino acids deleted compared to
.beta.AP.sub.1-39. For peptides with internal deletions, preferably
the peptide has an amino terminus corresponding to amino acid
residue 1 of natural .beta.AP and a carboxy terminus corresponding
to residue 40 of natural .beta.AP and has one or more internal
.beta.-AP amino acid residues deleted (i.e., a non-contiguous
A.beta. peptide).
[0214] In another embodiment, the unmodified peptide compound
comprises an amino acid sequence which has at least one N-terminal
amino acid deleted compared to .beta.AP.sub.1-39 Alternatively, the
unmodified peptide compound can have at least five, ten, fifteen,
twenty, twenty-five, thirty or thirty-five N-terminal amino acids
deleted compared to .beta.AP.sub.1-39. Still further the unmodified
peptide compound can have 1-5, 1-10, 1-15, 1-20, 1-1-25, 1-30 or
1-35 N-terminal amino acids deleted compared to
.beta.AP.sub.1-39.
[0215] In yet another embodiment, the unmodified peptide compound
comprises an amino acid sequence which has at least one C-terminal
amino acid deleted compared to .beta.AP.sub.1-39 Alternatively, the
unmodified peptide compound can have at least five, ten, fifteen,
twenty, twenty-five, thirty or thirty-five C-terminal amino acids
deleted compared to .beta.AP.sub.1-39. Still further the unmodified
peptide compound can have 1-5, 1-10, 1-15, 1-20, 1-25, 1-30 or 1-35
C-terminal amino acids deleted compared to .beta.AP.sub.1-39.
[0216] In addition to deletion of amino acids as compared to
.beta.AP.sub.1-39, the peptide compound can have additional
non-.beta.-AP amino acid residues added to it, for example, at the
amino terminus, the carboxy-terminus or at an internal site. In one
embodiment, the peptide compound has at least one
non-.beta.-amyloid peptide-derived amino acid at its N-terminus.
Alternatively, the compound can have, for example, 1-3, 1-5, 1-7,
1-10, 1-15 or 1-20 non-.beta.-amyloid peptide-derived amino acid at
its N-terminus. In another embodiment, the peptide compound has at
least one non-.beta.-amyloid peptide-derived amino acid at its
C-terminus. Alternatively, the compound can have, for example, 1-3,
1-5, 1-7, 1-10, 1-15 or 1-20 non-.beta.-amyloid peptide-derived
amino acid at its C-terminus.
[0217] In specific preferred embodiments, an unmodified peptide
compound of the invention comprises A.beta..sub.6-20 (the amino
acid sequence of which is shown in SEQ ID NO: 4), A.beta..sub.16-30
(the amino acid sequence of which is shown in SEQ ID NO: 14),
A.beta..sub.1-20, 26-40 (the amino acid sequence of which is shown
in SEQ ID NO: 15) or EEVVHHHHQQ-.beta.AP.sub.16-40 (the amino acid
sequence of which is shown in SEQ ID NO: 16). In the nomenclature
used herein, .beta.AP.sub.1-20, 26-40 represents .beta.AP.sub.1-40
in which the internal amino acid residues 21-25 have been
deleted.
[0218] An unmodified peptide compound of the invention can be
chemically synthesized using standard techniques such as those
described in Bodansky, M. Principles of Peptide Synthesis, Springer
Verlag, Berlin (1993) and Grant, G. A (ed.). Synthetic Peptides: A
User's Guide, W. H. Freeman and Company, New York (1992). Automated
peptide synthesizers are commercially available (e.g., Advanced
ChemTech Model 396; Milligen/Biosearch 9600). Alternatively,
unmodified peptide compounds can be prepared according to standard
recombinant DNA techniques using a nucleic acid molecule encoding
the peptide. A nucleotide sequence encoding the peptide can be
determined using the genetic code and an oligonucleotide molecule
having this nucleotide sequence can be synthesized by standard DNA
synthesis methods (e.g., using an automated DNA synthesizer).
Alternatively, a DNA molecule encoding an unmodified peptide
compound can be derived from the natural .beta.-amyloid precursor
protein gene or cDNA (e.g., using the polymerase chain reaction
and/or restriction enzyme digestion) according to standard
molecular biology techniques.
[0219] Accordingly, the invention further provides an isolated
nucleic acid molecule comprising a nucleotide sequence encoding a
.beta.-amyloid peptide compound, the .beta.-amyloid peptide
compound comprising an amino acid sequence having at least one
amino acid deletion compared to .beta.AP.sub.1-39 such that the
.beta.-amyloid peptide compound alters aggregation of natural
.beta.-amyloid peptides when contacted with the natural
.beta.-amyloid peptides. As used herein, the term "nucleic acid
molecule" is intended to include DNA molecules and RNA molecules
and may be single-stranded or double-stranded, but preferably is
double-stranded DNA. The isolated nucleic acid encodes a peptide
wherein one or more amino acids are deleted from the N-terminus,
C-terminus and/or an internal site of .beta.AP.sub.1-39, as
discussed above. In yet other embodiments, the isolated nucleic
acid encodes a peptide compound having one or more amino acids
deleted compared to .beta.AP.sub.1-39 and further having at least
one non-.beta.-AP derived amino acid residue added to it, for
example, at the amino terminus, the carboxy-terminus or at an
internal site. In specific preferred embodiments, an isolated
nucleic acid molecule of the invention encodes .beta.AP.sub.6-20,
.beta.AP.sub.16-30, .beta.AP.sub.1-20, 26-40 or
EEVVHHHHQQ-.beta.AP.sub.1- 6-40.
[0220] To facilitate expression of a peptide compound in a host
cell by standard recombinant DNA techniques, the isolated nucleic
acid encoding the peptide is incorporated into a recombinant
expression vector. Accordingly, the invention also provides
recombinant expression vectors comprising the nucleic acid
molecules of the invention. As used herein, the term "vector"
refers to a nucleic acid molecule capable of transporting another
nucleic acid to which it has been linked. One type of vector is a
"plasmid", which refers to a circular double stranded DNA loop into
which additional DNA segments may be ligated. Another type of
vector is a viral vector, wherein additional DNA segments may be
ligated into the viral genome. Certain vectors are capable of
autonomous replication in a host cell into which they are
introduced (e.g., bacterial vectors having a bacterial origin of
replication and episomal mammalian vectors). Other vectors (e.g.,
non-episomal mammalian vectors) are integrated into the genome of a
host cell upon introduction into the host cell, and thereby are
replicated along with the host genome. Moreover, certain vectors
are capable of directing the expression of genes to which they are
operatively linked. Such vectors are referred to herein as
"recombinant expression vectors" or simply "expression vectors". In
general, expression vectors of utility in recombinant DNA
techniques are often in the form of plasmids. In the present
specification, "plasmid" and "vector" may be used interchangeably
as the plasmid is the most commonly used form of vector. However,
the invention is intended to include such other forms of expression
vectors, such as viral vectors, which serve equivalent
functions.
[0221] In the recombinant expression vectors of the invention, the
nucleotide sequence encoding the peptide compound are operatively
linked to one or more regulatory sequences, selected on the basis
of the host cells to be used for expression. The term "operably
linked" is intended to mean that the sequences encoding the peptide
compound are linked to the regulatory sequence(s) in a manner that
allows for expression of the peptide compound. The term "regulatory
sequence" is intended to includes promoters, enhancers and other
expression control elements (e.g., polyadenylation signals). Such
regulatory sequences are described, for example, in Goeddel; Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). Regulatory sequences include those that
direct constitutive expression of a nucleotide sequence in many
types of host cell, those that direct expression of the nucleotide
sequence only in certain host cells (e.g., tissue-specific
regulatory sequences) and those that direct expression in a
regulatable manner (e.g., only in the presence of an inducing
agent). It will be appreciated by those skilled in the art that the
design of the expression vector may depend on such factors as the
choice of the host cell to be transformed, the level of expression
of peptide compound desired, etc. The expression vectors of the
invention can be introduced into host cells thereby to produce
peptide compounds encoded by nucleic acids as described herein.
[0222] The recombinant expression vectors of the invention can be
designed for expression of peptide compounds in prokaryotic or
eukaryotic cells. For example, peptide compounds can be expressed
in bacterial cells such as E. coli, insect cells (using baculovirus
expression vectors) yeast cells or mammalian cells. Suitable host
cells are discussed further in Goeddel, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). Alternatively, the recombinant expression vector may be
transcribed and translated in vitro, for example using T7 promoter
regulatory sequences and T7 polymerase. Examples of vectors for
expression in yeast S. cerivisae include pYepSec1 (Baldari et al.,
(1987) EMBO J. 6:229-234), pMFa (Kuijan and Herskowitz, (1982) Cell
30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and
pYES2 (Invitrogen Corporation, San Diego, Calif.). Baculovirus
vectors available for expression of proteins or peptides in
cultured insect cells (e.g., Sf 9 cells) include the pAc series
(Smith et al., (1983) Mol. Cell. Biol. 3:2156-2165) and the pVL
series (Lucklow, V. A., and Summers, M. D., (1989) Virology
170:31-39). Examples of mammalian expression vectors include pCDM8
(Seed, B., (1987) Nature 329:840) and pMT2PC (Kaufman et al.
(1987), EMBO J. 6:187-195). When used in mammalian cells, the
expression vector's control functions are often provided by viral
regulatory elements. For example, commonly used promoters are
derived from polyoma, Adenovirus 2, cytomegalovirus and Simian
Virus 40.
[0223] In addition to the regulatory control sequences discussed
above, the recombinant expression vector may contain additional
nucleotide sequences. For example, the recombinant expression
vector may encode a selectable marker gene to identify host cells
that have incorporated the vector. Such selectable marker genes are
well known in the art. Moreover, the facilitate secretion of the
peptide compound from a host cell, in particular mammalian host
cells, the recombinant expression vector preferably encodes a
signal sequence operatively linked to sequences encoding the
amino-terminus of the peptide compound such that upon expression,
the peptide compound is synthesized with the signal sequence fused
to its amino terminus. This signal sequence directs the peptide
compound into the secretory pathway of the cell and is then
cleaved, allowing for release of the mature peptide compound (i.e.,
the peptide compound without the signal sequence) from the host
cell. Use of a signal sequence to facilitate secretion of proteins
or peptides from mammalian host cells is well known in the art.
[0224] A recombinant expression vector comprising a nucleic acid
encoding a peptide compound that alters aggregation of natural
.beta.-AP can be introduced into a host cell to thereby produce the
peptide compound in the host cell. Accordingly, the invention also
provides host cells containing the recombinant expression vectors
of the invention. The terms "host cell" and "recombinant host cell"
are used interchangeably herein. It is understood that such terms
refer not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein. A host cell may be any
prokaryotic or eukaryotic cell. For example, a peptide compound may
be expressed in bacterial cells such as E. coli, insect cells,
yeast or mammalian cells. Preferably, the peptide compound is
expressed in mammalian cells. In a preferred embodiment, the
peptide compound is expressed in mammalian cells in vivo in a
mammalian subject to treat amyloidosis in the subject through gene
therapy (discussed further below). Preferably, the .beta.-amyloid
peptide compound encoded by the recombinant expression vector is
secreted from the host cell upon being expressed in the host
cell.
[0225] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, electroporation,
microinjection and viral-mediated transfection. Suitable methods
for transforming or transfecting host cells can be found in
Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd
Edition, Cold Spring Harbor Laboratory press (1989)), and other
laboratory manuals. Methods for introducing DNA into mammalian
cells in vivo are also known in the art and can be used to deliver
the vector DNA to a subject for gene therapy purposes (discussed
further below).
[0226] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Preferred selectable markers
include those that confer resistance to drugs, such as G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable
marker may be introduced into a host cell on the same vector as
that encoding the peptide compound or may be introduced on a
separate vector. Cells stably transfected with the introduced
nucleic acid can be identified by drug selection (e.g., cells that
have incorporated the selectable marker gene will survive, while
the other cells die).
[0227] A nucleic acid of the invention can be delivered to cells in
vivo using methods known in the art, such as direct injection of
DNA, receptor-mediated DNA uptake or viral-mediated transfection.
Direct injection has been used to introduce naked DNA into cells in
vivo (see e.g., Acsadi et al. (1991) Nature 332: 815-818; Wolff et
al. (1990) Science 247:1465-1468). A delivery apparatus (e.g., a
"gene gun") for injecting DNA into cells in vivo can be used. Such
an apparatus is commercially available (e.g., from BioRad). Naked
DNA can also be introduced into cells by complexing the DNA to a
cation, such as polylysine, which is coupled to a ligand for a
cell-surface receptor (see for example Wu, G. and Wu, C. H. (1988)
J. Biol. Chem. 263:14621; Wilson et al. (1992) J. Biol. Chem.
267:963-967; and U.S. Pat. No. 5,166,320). Binding of the
DNA-ligand complex to the receptor facilitates uptake of the DNA by
receptor-mediated endocytosis. Additionally, a DNA-ligand complex
linked to adenovirus capsids which naturally disrupt endosomes,
thereby releasing material into the cytoplasm can be used to avoid
degradation of the complex by intracellular lysosomes (see for
example Curiel et al. (1991) Proc. Natl. Acad. Sci. USA 88:8850;
Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA
90:2122-2126).
[0228] Defective retroviruses are well characterized for use in
gene transfer for gene therapy purposes (for a review see Miller,
A. D. (1990) Blood 76:271). Protocols for producing recombinant
retroviruses and for infecting cells in vitro or in vivo with such
viruses can be found in Current Protocols in Molecular Biology,
Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989),
Sections 9.10-9.14 and other standard laboratory manuals. Examples
of suitable retroviruses include pLJ, pZIP, pWE and pEM which are
well known to those skilled in the art. Examples of suitable
packaging virus lines include .PSI.Crip, .PSI.Cre, .PSI.2 and
.PSI.Am. Retroviruses have been used to introduce a variety of
genes into many different cell types, including epithelial cells,
endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow
cells, in vitro and/or in vivo (see for example Eglitis, et al.
(1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl.
Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad.
Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad
Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci.
USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA
88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van
Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644;
Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al (1992)
Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J.
Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No.
4,980,286; PCT Application WO 89/07136; PCT Application WO
89/02468; PCT Application WO 89/05345; and PCT Application WO
92/07573).
[0229] Alternatively, the genome of an adenovirus can be
manipulated such that it encodes and expresses a peptide compound
but is inactivated in terms of its ability to replicate in a normal
lytic viral life cycle. See for example Berkner et al. (1988)
BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434;
and Rosenfeld et al (1992) Cell 68:143-155. Suitable adenoviral
vectors derived from the adenovirus strain Ad type 5 d1324 or other
strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to
those skilled in the art. Recombinant adenoviruses are advantageous
in that they do not require dividing cells to be effective gene
delivery vehicles and can be used to infect a wide variety of cell
types, including airway epithelium (Rosenfeld et al. (1992) cited
supra), endothelial cells (Lemarchand et al (1992) Proc. Natl.
Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993)
Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin
et al. (1992) Proc. Natl Acad. Sci. USA 89:2581-2584).
Additionally, introduced adenoviral DNA (and foreign DNA contained
therein) is not integrated into the genome of a host cell but
remains episomal, thereby avoiding potential problems that can
occur as a result of insertional mutagenesis in situations where
introduced DNA becomes integrated into the host genome (e.g.,
retroviral DNA).
[0230] Adeno-associated virus (AAV) can also be used for delivery
of DNA for gene therapy purposes. AAV is a naturally occurring
defective virus that requires another virus, such as an adenovirus
or a herpes virus, as a helper virus for efficient replication and
a productive life cycle. (For a review see Muzyczka et al. Curr.
Topics in Micro. and Immunol. (1992) 158:97-129). It is also one of
the few viruses that may integrate its DNA into non-dividing cells,
and exhibits a high frequency of stable integration (see for
example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol.
7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and
McLaughlin et al. (1989) J. Virol. 62:1963-1973). Vectors
containing as little as 300 base pairs of AAV can be packaged and
can integrate. An AAV vector such as that described in Tratschin et
al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce
DNA into cells. A variety of nucleic acids have been introduced
into different cell types using AAV vectors (see for example
Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;
Tratschin et al. (1985) Mol. Cell. Biol 4:2072-2081; Wondisford et
al. (1988) Mol. Endocrinol. 2:32-39; Tratschin et al (1984) J.
Virol. 51:611-619; and Flotte et al. (1993) J. Biol. Chem.
268:3781-3790).
[0231] The invention provides a method for treating a subject for a
disorder associated with .beta.-amyloidosis, comprising
administering to the subject a recombinant expression vector
encoding a .beta.-amyloid peptide compound, the compound comprising
an amino acid sequence having at least one amino acid deletion
compared to .beta.AP.sub.1-39, such that the .beta.-amyloid peptide
compound is synthesized in the subject and the subject is treated
for a disorder associated with .beta.-amyloidosis. Preferably, the
disorder is Alzheimer's disease. In one embodiment the recombinant
expression vector directs expression of the peptide compound in
neuronal cells. In another embodiment, the recombinant expression
vector directs expression of the peptide compound in glial cells.
In yet another embodiment, the recombinant expression vector
directs expression of the peptide compound in fibroblast cells.
[0232] General methods for gene therapy are known in the art. See
for example, U.S. Pat. No. 5,399,346 by Anderson et al. A
biocompatible capsule for delivering genetic material is described
in PCT Publication WO 95/05452 by Baetge et al. Methods for
grafting genetically modified cells to treat central nervous system
disorders are described in U.S. Pat. No. 5,082,670 and in PCT
Publications WO 90/06757 and WO 93/10234, all by Gage et al.
Isolation and/or genetic modification of multipotent neural stem
cells or neuro-derived fetal cells are described in PCT
Publications WO 94/02593 by Anderson et al., WO 94/16718 by Weiss
et al., and WO 94/23754 by Major et al. Fibroblasts transduced with
genetic material are described in PCT Publication WO 89/02468 by
Mulligan et al. Adenovirus vectors for transfering genetic material
into cells of the central nervous system are described in PCT
Publication WO 94/08026 by Kahn et al. Herpes simplex virus vectors
suitable for treating neural disorders are described in PCT
Publications WO 94/04695 by Kaplitt and WO 90/09441 by Geller et
al. Promoter elements of the glial fibrillary acidic protein that
can confer astrocyte specific expression on a linked gene or gene
fragment, and which thus can be used for expression of A.beta.
peptides specifically in astrocytes, is described in PCT
Publication WO 93/07280 by Brenner et al. Furthermore, alternative
to expression of an A.beta. peptide to modulate amyloidosis, an
antisense oligonucleotide that is complementary to a region of the
.beta.-amyloid precursor protein mRNA corresponding to the peptides
described herein can be expressed in a subject to modulate
amyloidosis. General methods for expressing antisense
oligonucleotides to modulate nervous system disorders are described
in PCT Publication WO 95/09236.
[0233] Alternative to delivery by gene therapy, a peptide compound
of the invention comprising an amino acid sequence having at least
one amino acid deletion compared to .beta.AP.sub.1-39 can be
delivered to a subject by directly administering the peptide
compound to the subject as described further herein for the
modified peptide compounds of the invention. The peptide compound
can be formulated into a pharmaceutical composition comprising a
therapeutically effective amount of the .beta.-amyloid peptide
compound and a pharmaceutically acceptable carrier. The peptide
compound can be contacted with natural .beta.-amyloid peptides with
a .beta.-amyloid peptide compound such that aggregation of the
natural .beta.-amyloid peptides is inhibited. Moreover, the peptide
compound can be administered to the subject in a therapeutically
effective amount such that the subject is treated for a disorder
associated with .beta.-amyloidosis, such as Alzheimer's
disease.
[0234] VIII. Other Embodiments
[0235] Although the invention has been illustrated hereinbefore
with regard to A.beta.peptide compounds, the principles described,
involving attachment of a modifying group(s) to a peptide compound,
are applicable to any amyloidogenic protein or peptide as a means
to create a modulator compound that modulates, and preferably
inhibits, amyloid aggregation. Accordingly, the invention provides
modulator compounds that can be used to treat amyloidosis in a
variety of forms and clinical settings.
[0236] Amyloidosis is a general term used to describe pathological
conditions characterized by the presence of amyloid. Amyloid is a
general term referring to a group of diverse but specific
extracellular protein deposits which are seen in a number of
different diseases. Though diverse in their occurrence, all amyloid
deposits have 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 features and common x-ray
diffraction and infrared spectra. Amyloidosis can be classified
clinically as primary, secondary, familial and/or isolated. Primary
amyloid appears de novo without any preceding disorder. Secondary
amyloid is that form which appears as a complication of a
previously existing disorder. Familial amyloid is a genetically
inherited form found in particular geographic populations. Isolated
forms of amyloid are those that tend to involve a single organ
system.
[0237] Different amyloids are characterized by the type of
protein(s) or peptide(s) present in the deposit. For example, as
described hereinbefore, amyloid deposits associated with
Alzheimer's disease comprise the .beta.-amyloid peptide and thus a
modulator compound of the invention for detecting and/or treating
Alzheimer's disease is designed based on modification of the
.beta.-amyloid peptide. The identities of the protein(s) or
peptide(s) present in amyloid deposits associated with a number of
other amyloidogenic diseases have been elucidated. Accordingly,
modulator compounds for use in the detection and/or treatment of
these other amyloidogenic diseases can be prepared in a similar
fashion to that described herein for .beta.-AP-derived modulators.
In vitro assay systems can be established using an amyloidogenic
protein or peptide which forms fibrils in vitro, analogous to the
A.beta. assays described herein. Modulators can be identified using
such assay systems, based on the ability of the modulator to
disrupt the .beta.-sheet structure of the fibrils. Initially, an
entire amyloidogenic protein can be modified or, more preferably, a
peptide fragment thereof that is known to form fibrils in vitro can
be modified (e.g., analogous to A.beta.1-40 described herein).
Amino acid deletion and substitution analyses can then be performed
on the modified protein or peptide (analogous to the studies
described in the Examples) to delineate an aggregation core domain
that is sufficient, when modified, to disrupt fibril formation.
[0238] Non-limiting examples of amyloidogenic proteins or peptides,
and their associated amyloidogenic disorders, include:
[0239] Transthyretin (TTR)--Amyloids containing transthyretin occur
in familial amyloid polyneuropathy (Portuguese, Japanese and
Swedish types), familial amyloid cardiomyopathy (Danish type),
isolated cardiac amyloid and systemic senile amyloidosis. Peptide
fragments of transthyretin have been shown to form amyloid fibrils
in vitro. For example, TTR 10-20 and TTR 105-115 form amyloid-like
fibrils in 20-30% acetonitrile/water at room temperature (Jarvis,
J. A., et al.(1994) Int. J. Pept. Protein Res. 44:388-398).
Moreover, familial cardiomyopathy (Danish type) is associated with
mutation of Leu at position 111 to Met, and an analogue of TTR
105-115 in which the wildtype Leu at position 111 has been
substituted with Met (TTR 105-115Met 111) also forms amyloid-like
fibrils in vitro (see e.g., Hermansen, L. F., et al. (1995) Eur. J.
Biochem. 227:772-779; Jarvis et al. supra). Peptide fragments of
TTR that form amyloid fibrils in vitro are also described in
Jarvis, J. A., et al. (1993) Biochem. Biophys. Res. Commun.
192:991-998 and Gustavsson, A., et al. (1991) Biochem. Biophys.
Res. Commun. 175:1159-1164. A peptide fragment of wildtype or
mutated transthyretin that forms amyloid fibrils can be modified as
described herein to create a modulator of amyloidosis that can be
used in the detection or treatment of familial amyloid
polyneuropathy (Portuguese, Japanese and Swedish types), familial
amyloid cardiomyopathy (Danish type), isolated cardiac amyloid or
systemic senile amyloidosis.
[0240] Prion Protein (PrP)--Amyloids in a number of spongiform
encephalopathies, including scrapie in sheep, bovine spongiform
encephalopathy in cows and Creutzfeldt-Jakob disease (CJ) and
Gerstmann-Straussler-Scheinker syndrome (GSS) in humans, contain
PrP. Limited proteolysis of PrPSc (the prion protein associated
with scrapie) leads to a 27-30 kDa fragment (PrP27-30) that
polymerizes into rod-shaped amyloids (see e.g., Pan, K. M., et al.
(1993) Proc. Natl. Acad. Sci. USA 90:10962-10966; Gasset, M., et
al. (1993) Proc. Natl. Acad. Sci. USA 90:1-5). Peptide fragments of
PrP from humans and other mammals have been shown to form amyloid
fibrils in vitro. For example, polypeptides corresponding to
sequences encoded by normal and mutant alleles of the PRNP gene
(encoding the precursor of the prion protein involved in CJ), in
the regions of codon 178 and codon 200, spontaneously form amyloid
fibrils in vitro (see e.g., Goldfarb, L. G., et al (1993) Proc.
Natl. Acad. Sci. USA 90:4451-4454). A peptide encompassing residues
106-126 of human PrP has been reported to form straight fibrils
similar to those extracted from GSS brains, whereas a peptide
encompassing residues 127-147 of human PrP has been reported to
form twisted fibrils resembling scrapie-associated fibrils
(Tagliavini, F., et al. (1993) Proc. Natl. Acad. Sci. USA
90:9678-9682). Peptides of Syrian hamster PrP encompassing residues
109-122, 113-127, 113-120, 178-191 or 202-218 have been reported to
form amyloid fibrils, with the most amyloidogenic peptide being
Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala (SEQ ID NO: 17), which corresponds
to residues 113-120 of Syrian hamster PrP but which is also
conserved in PrP from other species (Gasset, M., et al. (1992)
Proc. Natl. Acad. Sci. USA 89:10940-10944). A peptide fragment of
PrP that forms amyloid fibrils can be modified as described herein
to create a modulator of amyloidosis that can be used in the
detection or treatment of scrapie, bovine spongiform
encephalopathy, Creutzfeldt-Jakob disease or
Gerstmann-Straussler-Scheink- er syndrome.
[0241] Islet Amyloid Polypeptide (IAPP, also known as
amylin)--Amyloids containing IAPP occur in adult onset diabetes and
insulinoma. IAPP is a 37 amino acid polypeptide formed from an 89
amino acid precursor protein (see e.g., Betsholtz, C., et al.
(1989) Exp. Cell. Res. 183:484-493; Westermark, P., et al. (1987)
Proc. Natl. Acad. Sci. USA 84:3881-3885). A peptide corresponding
to IAPP residues 20-29 has been reported to form amyloid-like
fibrils in vitro, with residues 25-29, having the sequence
Ala-Ile-Leu-Ser-Ser (SEQ ID NO: 18), being strongly amyloidogenic
(Westermark, P., et al. (1990) Proc. Natl. Acad. Sci. USA
87:5036-5040; Glenner, G. G., et al. (1988) Biochem. Biophys. Res.
Commun. 155:608-614). A peptide fragment of IAPP that forms amyloid
fibrils can be modified as described herein to create a modulator
of amyloidosis that can be used in the detection or treatment of
adult onset diabetes or insulinoma.
[0242] Atrial Natriuretic Factor (ANF)--Amyloids containing ANF are
associated with isolated atrial amyloid (see e.g., Johansson, B.,
et al. (1987) Biochem. Biophys. Res. Commun. 148:1087-1092). ANF
corresponds to amino acid residues 99-126 (proANF99-126) of the ANF
prohormone (proANP1 -126) (Pucci, A., et al. (1991) J. Pathol.
165:235-241). ANF, or a fragment thereof, that forms amyloid
fibrils can be modified as described herein to create a modulator
of amyloidosis that can be used in the detection or treatment of
isolated atrial amyloid.
[0243] Kappa or Lambda Light Chain--Amyloids containing kappa or
lambda light chains are associated idiopathic (primary)
amyloidosis, myeloma or macroglobulinemia-associated amyloidosis,
and primary localized cutaneous nodular amyloidosis associated with
Sjogren's syndrome. The structure of amyloidogenic kappa and lambda
light chains, including amino acid sequence analysis, has been
characterized (see e.g., Buxbaum, J. N., et al. (1990) Ann. Intern.
Med. 112:455-464; Schormann, N., et al. (1995) Proc. Natl. Acad.
Sci. USA 92:9490-9494; Hurle, M. R., et al. (1994) Proc. Natl.
Acad. Sci. USA 91:5446-5450; Liepnieks, J. J., et al. (1990) Mol.
Immunol. 27:481-485; Gertz, M. A., et al. (1985) Scand. J. Immunol.
22:245-250; Inazumi, T., et al. (1994) Dermatology 189:125-128).
Kappa or lambda light chains, or a peptide fragment thereof that
forms amyloid fibrils, can be modified as described herein to
create a modulator of amyloidosis that can be used in the detection
or treatment of idiopathic (primary) amyloidosis, myeloma or
macroglobulinemia-associated amyloidosis or primary localized
cutaneous nodular amyloidosis associated with Sjogren's
syndrome.
[0244] Amyloid A--Amyloids containing the amyloid A protein (AA
protein), derived from serum amyloid A, are associated with
reactive (secondary) amyloidosis (see e.g., Liepnieks, J. J., et
al. (1995) Biochim. Biophys. Acta 1270:81-86), familial
Mediterranean Fever and familial amyloid nephropathy with urticaria
and deafness (Muckle-Wells syndrome) (see e.g., Linke, R. P., et
al. (1983) Lab. Invest. 48:698-704). Recombinant human serum
amyloid A forms amyloid-like fibrils in vitro (Yamada, T., et al.
(1994) Biochim. Biophys. Acta 1226:323-329) and circular dichroism
studies revealed a predominant .beta. sheet/turn structure
(McCubbin, W. D., et al. (1988) Biochem J. 256:775-783). Serum
amyloid A, amyloid A protein or a fragment thereof that forms
amyloid fibrils can be modified as described herein to create a
modulator of amyloidosis that can be used in the detection or
treatment of reactive (secondary) amyloidosis, familial
Mediterranean Fever and familial amyloid nephropathy with urticaria
and deafness (Muckle-Wells, syndrome).
[0245] Cystatin C--Amyloids containing a variant of cystatin C are
associated with hereditary cerebral hemorrhage with amyloidosis of
Icelandic type. The disease is associated with a leucine to glycine
mutation at position 68 and cystatin C containing this mutation
aggregates in vitro (Abrahamson, M. and Grubb, A. (1994) Proc.
Natl. Acad. Sci. USA 91:1416-1420). Cystatin C or a peptide
fragment thereof that forms amyloid fibrils can be modified as
described herein to create a modulator of amyloidosis that can be
used in the detection or treatment of hereditary cerebral
hemorrhage with amyloidosis of Icelandic type.
[0246] .beta.2 microglobulin--Amyloids containing .beta.2
microglobulin (.beta.2M) are a major complication of long term
hemodialysis (see e.g., Stein, G., et al. (1994) Nephrol. Dial.
Transplant. 9:48-50; Floege, J., et al (1992) Kidney Int. Suppl.
38:S78-S85; Maury, C. P. (1990) Rheumatol. Int. 10:1-8). The native
.beta.2M protein has been shown to form amyloid fibrils in vitro
(Connors, L. H., et al. (1985) Biochem. Biophys. Res. Commun.
131:1063-1068; Ono, K., et al (1994) Nephron 66:404-407). .beta.2M,
or a peptide fragment thereof that forms amyloid fibrils, can be
modified as described herein to create a modulator of amyloidosis
that can be used in the detection or treatment of amyloidosis
associated with long term hemodialysis.
[0247] Apolipoprotein A-I (ApoA-I)--Amyloids containing variant
forms of ApoA-I have been found in hereditary non-neuropathic
systemic amyloidosis (familial amyloid polyneuropathy III). For
example, N-terminal fragments (residues 1-86, 1-92 and 1-93) of an
ApoA-1 variant having a Trp to Arg mutation at position 50 have
been detected in amyloids (Booth, D. R., et al. (1995) QJM
88:695-702). In another family, a leucine to arginine mutation at
position 60 was found (Soutar, A. K., et al. (1992) Proc. Natl.
Acad. Sci. USA 89:7389-7393). ApoA-I or a peptide fragment thereof
that forms amyloid fibrils can be modified as described herein to
create a modulator of amyloidosis that can be used in the detection
or treatment of hereditary non-neuropathic systemic
amyloidosis.
[0248] Gelsolin--Amyloids containing variants of gelsolin are
associated with familial amyloidosis of Finnish type. Synthetic
gelsolin peptides that have sequence homology to wildtype or mutant
gelsolins and that form amyloid fibrils in vitro are reported in
Maury, C. P. et al. (1994) Lab. Invest. 70:558-564. A nine residue
segment surrounding residue 187 (which is mutated in familial
gelsolin amyloidosis) was defined as an amyloidogenic region
(Maury, et al., supra; see also Maury, C. P., et al. (1992)
Biochem. Biophys. Res. Commun. 183:227-231; Maury, C. P. (1991) J.
Clin. Invest 87:1195-1199). Gelsolin or a peptide fragment thereof
that forms amyloid fibrils can be modified as described herein to
create a modulator of amyloidosis that can be used in the detection
or treatment of familial amyloidosis of Finnish type.
[0249] Procalcitonin or calcitonin--Amyloids containing
procalcitonin, calcitonin or calcitonin-like immunoreactivity have
been detected in amyloid fibrils associated with medullary
carcinoma of the thyroid (see e.g., Butler, M. and Khan, S. (1986)
Arch. Pathol. Lab. Med. 110:647-649; Sletten, K., et al. (1976) J.
Exp. Med. 143:993-998). Calcitonin has been shown to form a
nonbranching fibrillar structure in vitro (Kedar, I., et al. (1976)
Isr. J. Med. Sci. 12:1137-1140). Procalcitonin, calcitonin or a
fragment thereof that forms amyloid fibrils can be modified as
described herein to create a modulator of amyloidosis that can be
used in the detection or treatment of amyloidosis associated with
medullary carcinoma of the thyroid.
[0250] Fibrinogen--Amyloids containing a variant form of fibrinogen
alpha-chain have been found in hereditary renal amyloidosis. An
arginine to leucine mutation at position 554 has been reported in
amyloid fibril protein isolated from postmortem kidney of an
affected individual (Benson, M. D., et al. (1993) Nature Genetics
3:252-255). Fibrinogen alpha-chain or a peptide fragment thereof
that forms amyloid fibrils can be modified as described herein to
create a modulator of amyloidosis that can be used in the detection
or treatment of fibrinogen-associated hereditary renal
amyloidosis.
[0251] Lysozyme--Amyloids containing a variant form of lysozyme
have been found in hereditary systemic amyloidosis. In one family
the disease was associated with a threonine to isoleucine mutation
at position 56, whereas in another family the disease was
associated with a histidine to aspartic acid mutation at position
67 (Pepys, M. B., et al. (1993) Nature 362:553-557). Lysozyme or a
peptide fragment thereof that forms amyloid fibrils can be modified
as described herein to create a modulator of amyloidosis that can
be used in the detection or treatment of lysozyme-associated
hereditary systemic amyloidosis.
[0252] This invention is further illustrated by the following
examples which should not be construed as limiting. A modulator's
ability to alter the aggregation of .beta.-amyloid peptide in the
assays described below are predictive of the modulator's ability to
perform the same function in vivo. The contents of all references,
patents and published patent applications cited throughout this
application are hereby incorporated by reference.
EXAMPLE 1
Construction of .beta.-Amyloid Modulators
[0253] A .beta.-amyloid modulator composed of an amino-terminally
biotinylated .beta.-amyloid peptide of the amino acid sequence:
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV
[0254] (positions 1 to 40 of SEQ ID NO: 1) was prepared by
solid-phase peptide synthesis using an
N.sup..alpha.-9-fluorenylmethyloxycarbonyl (FMOC)-based protection
strategy as follows. Starting with 2.5 mmoles of FMOC-Val-Wang
resin, sequential additions of each amino acid were performed using
a four-fold excess of protected amino acids, 1-hydroxybenzotriazole
(HOBt) and diisopropyl carbodiimide (DIC). Recouplings were
performed when necessary as determined by ninhydrin testing of the
resin after coupling. Each synthesis cycle was minimally described
by a three minute deprotection (25% piperidine/N-methyl-pyrroli-
done (NMP)), a 15 minute deprotection, five one minute NMP washes,
a 60 minute coupling cycle, five NMP washes and a ninhydrin test.
To a 700 mg portion of the fully assembled peptide-resin, biotin
(obtained commercially from Molecular Probes, Inc.) was substituted
for an FMOC-amino acid was coupled by the above protocol. The
peptide was removed from the resin by treatment with
trifluoroacetic acid (TFA) (82.5%), water (5%), thioanisole (5%),
phenol (5%), ethanedithiol (2.5%) for two hours followed by
precipitation of the peptide in cold ether. The solid was pelleted
by centrifugation (2400 rpm.times.10 min.), and the ether decanted.
It was resuspended in ether, pelleted and decanted a second time.
The solid was dissolved in 10% acetic acid and lyophilized to
dryness to yield 230 mg of crude biotinylated peptide. 60 mg of the
solid was dissolved in 25% acetonitrile (ACN)/0.1% TFA and applied
to a C18 reversed phase high performance liquid chromatography
(HPLC) column. Biotinyl .beta.A.sub.1-40 was eluted using a linear
gradient of 30-45% acetonitrile/0.1% TFA over 40 minutes. One
primary fraction (4 mg) and several side fractions were isolated.
The main fraction yielded a mass spectrum of 4556 (matrix-assisted
laser desorption ionization-time of flight) which matches the
theoretical (4555) for this peptide.
[0255] A .beta.-amyloid modulator composed of an amino-terminally
biotinylated .beta.-amyloid peptide of the amino acid sequence:
DAEFRHDSGYEVHHQ
[0256] (positions 1 to 15 of SEQ ID NO: 1) was prepared on an
Advanced ChemTech Model 396 multiple peptide synthesizer using an
automated protocol established by the manufacturer for 0.025 mmole
scale synthesis. Double couplings were performed on all cycles
using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU)/N,N-diisopropylethylamine
(DIEA)/HOBt/FMOC-AA in four-fold excess for 30 minutes followed by
DIC/HOBt/FMOC-AA in four-fold excess for 45 minutes. The peptide
was deprotected and removed from the resin by treatment with
TFA/water (95%/5%) for three hours and precipitated with ether as
described above. The pellet was resuspended in 10% acetic acid and
lyophilized. The material was purified by a preparative HPLC using
15%-40% acetonitrile over 80 minutes on a Vydac C18 column
(21.times.250 mm). The main isolate eluted as a single symmetrical
peak when analyzed by analytical HPLC and yielded the expected
molecular weight when analyzed by electrospray mass spectrometry.
Result=2052.6 (2052 theoretical).
[0257] .beta.-amyloid modulator compounds comprising other regions
of the .beta.-AP amino acid sequence (e.g., an A.beta. aggregation
core domain) were similarly prepared using the synthesis methods
described above. Moreover, modulators comprising other
amyloidogenic peptides can be similarly prepared.
EXAMPLE 2
Inhibition of .beta.-Amyloid Aggregation by Modulators
[0258] The ability of .beta.-amyloid modulators to inhibit the
aggregation of natural .beta.-AP when combined with the natural
.beta.-AP was examined in a series of aggregation assays. Natural
.beta.-AP (.beta.-AP.sub.1-40) was obtained commercially from
Bachem (Torrance, Calif.). Amino-terminally biotinylated .beta.-AP
modulators were prepared as described in Example 1.
[0259] A. Optical Density Assay
[0260] In one assay, .beta.-AP aggregation was measured by
determining the increase in turbidity of a solution of natural
.beta.-AP over time in the absence or presence of various
concentrations of the modulator. Turbidity of the solution was
quantitated by determining the optical density at 400 nm (A.sub.400
nm) of the solution over time.
[0261] The aggregation of natural .beta.-AP in the absence of
modulator was determined as follows. .beta.-AP.sub.1-40 was
dissolved in hexafluoro isopropanol (HFIP; Aldrich Chemical Co.,
Inc.) at 2 mg/ml. Aliquots of the HFIP solution (87 .mu.l) were
transferred to individual 10 mm.times.75 mm test tubes. A stream of
argon gas was passed through each tube to evaporate the HFIP. To
the resulting thin film of peptide, dimethylsulfoxide (DMSO;
Aldrich Chemical Co., Inc.) (25 .mu.l) was added to dissolve the
peptide. A 2 mm.times.7 mm teflon-coated magnetic stir bar was
added to each tube. Buffer (475 .mu.L of 100 mM NaCl, 10 mM sodium
phosphate, pH 7.4) was added to the DMSO solution with stirring.
The resulting mixture was stirred continuously and the optical
density was monitored at 400 nm to observe the formation of
insoluble peptide aggregates.
[0262] Alternatively, .beta.-AP.sub.1-40 was dissolved in DMSO as
described above at 1.6 mM (6.9 mg/ml) and aliquots (25 .mu.l) were
added to stirred buffer (475 .mu.l ), followed by monitoring of
absorbance at 400 nm.
[0263] For inhibition studies in which a .beta.-amyloid modulator
was dissolved in solution together with the natural .beta.-AP, the
modulators were dissolved in DMSO either with or without prior
dissolution in HFIP. These compounds were then added to buffer with
stirring, followed by addition of .beta.-AP.sub.1-40 in DMSO.
Alternatively, HFIP solutions of modulators were combined with
.beta.-AP.sub.1-40 in HFIP followed by evaporation and
redissolution of the mixture in DMSO. Buffer was then added to the
DMSO solution to initiate the assay. The amino-terminally
biotinylated .beta.-amyloid peptide modulators
N-biotinyl-.beta.AP.sub.1-- 40, and N-biotinyl-.beta.AP.sub.1-40
were tested at concentrations of 1% and 5% in the natural
.beta.-A.sub.1-40 solution.
[0264] A representative example of the results is shown graphically
in FIG. 1, which depicts the inhibition of aggregation of natural
.beta.AP.sub.1-40 by N-biotinyl-.beta.A.sub.1-40. In the absence of
the modulator, the optical density of the natural .beta.-AP
solution showed a characteristic sigmoidal curve, with a lag time
prior to aggregation (approximately 3 hours in FIG. 1) in which the
A.sub.400 nm was low, followed by rapid increase in the A.sub.400
nm, which quickly reached a plateau level, representing aggregation
of the natural .beta. amyloid peptides. In contrast, in the
presence of as little as 1% of the N-biotinyl-.beta.AP.sub.1-40
modulator, aggregation of the natural .beta. amyloid peptides was
markedly inhibited, indicated by an increase in the lag time, a
decrease in the slope of aggregation and a decrease in the plateau
level reached for the turbidity of the solution (see FIG. 1).
N-biotinyl-.beta.AP.sub.1-40 at a concentration of 5% similarly
inhibited aggregation of the natural .beta. amyloid peptide.
Furthermore, similar results were observed when
N-biotinyl-.beta.AP.sub.1-15 was used as the modulator. These
results demonstrate that an N-terminally biotinylated .beta.-AP
modulator can effectively inhibit the aggregation of natural .beta.
amyloid peptides, even when the natural .beta. amyloid peptides are
present at as much as a 100-fold molar excess concentration.
[0265] B. Fluorescence Assay
[0266] In a second assay, .beta.-AP aggregation was measured using
a fluorometric assay essentially as described in Levine, H. (1993)
Protein Science 2:404-410. In this assay, the dye thioflavine T
(ThT) is contacted with the .beta.-AP solution. Association of ThT
with aggregated .beta.-AP, but not monomeric or loosely associated
.beta.-AP, gives rise to a new excitation (ex) maximum at 450 nm
and an enhanced emission (em) at 482 nm, compared to the 385 nm
(ex) and 445 nm (em) for the free dye. .beta.-AP aggregation was
assayed by this method as follows. Aliquots (2.9 .mu.l) of the
solutions used in the aggregation assays as described above in
section A were removed from the samples and diluted in 200 .mu.l of
potassium phosphate buffer (50 mM, pH 7.0) containing thioflavin T
(10 .mu.M; obtained commercially from Aldrich Chemical Co., Inc.).
Excitation was set at 450 nm and emission was measured at 482 nm.
Similar to the results observed with the optical density assay
described above in section A, as little as 1% of the N-biotinylated
.beta.-AP modulators was effective at inhibiting the aggregation of
natural .beta. amyloid peptides using this fluorometric assay.
[0267] C. Static Aggregation Assay
[0268] In a third assay, .beta.-AP aggregation was measured by
visualization of the peptide aggregates using SDS-polyacrylamide
gel electrophoresis (SDS-PAGE). In this assay, .beta.-AP solutions
were allowed to aggregate over a period of time and then aliquots
of the reaction were run on a standard SDS-PAGE gel. Typical
solution conditions were 200 .mu.M of .beta.-AP.sub.1-40 in PBS at
37.degree. C. for 8 days or 200 .mu.M .beta.-AP.sub.1-40 in 0.1 M
sodium acetate at 37.degree. C. for 3 days. The peptide aggregates
were visualized by Coomassie blue staining of the gel or, for
.beta.-AP solutions that included a biotinylated .beta.-AP
modulator, by western blotting of a filter prepared from the gel
with a streptavidin-peroxidase probe, followed by a standard
peroxidase assay. The .beta.-AP aggregates are identifiable as high
molecular weight, low mobility bands on the gel, which are readily
distinguishable from the low molecular weight, high mobility
.beta.-AP monomer or dimer bands.
[0269] When natural .beta.-AP.sub.1-40 aggregation was assayed by
this method in the absence of any .beta. amyloid modulators, high
molecular weight aggregates were readily detectable on the gel. In
contrast, when N-biotinyl-.beta.-A.sub.1-40 modulator
self-aggregation was assayed (i.e., aggregation of the N-biotinyl
peptide alone, in the absence of any natural .beta.-AP), few if any
high molecular weight aggregates were observed, indicating that the
ability of the modulator to self-aggregate is significantly reduced
compared to natural .beta.-AP. Finally, when aggregation of a
mixture of natural .beta.-A.sub.1-40 and N-biotinylated
.beta.-A.sub.1-40 was assayed by this method, reduced amounts of
the peptide mixture associated into high molecular weight
aggregates, thus demonstrating that the .beta. amyloid modulator is
effective at inhibiting the aggregation of the natural .beta.
amyloid peptides.
EXAMPLE 3
Neurotoxicity Analysis of .beta.-Amyloid Modulators
[0270] The neurotoxicity of the .beta.-amyloid modulators is tested
in a cell-based assay using the neuronal precursor cell line PC-12,
or primary neuronal cells, and the viability indicator
3,(4,4-dimethylthiazol-2-yl)2- ,5-diphenyl-tetrazolium bromide
(MTT). (See Shearman, M. S. et al. (1994) Proc. Natl. Acad. Sci.
USA 91:1470-1474; Hansen, M. B. et al. (1989) J. Immun. Methods
119:203-210). PC-12 is a rat adrenal pheochromocytoma cell line and
is available from the American Type Culture Collection, Rockville,
Md. (ATCC CRL 1721). MTT (commercially available from Sigma
Chemical Co. ) is a chromogenic substrate that is converted from
yellow to blue in viable cells, which can be detected
spectrophotometrically.
[0271] To test the neurotoxicity of a .beta.-amyloid modulator
(either alone or combined with natural .beta.-AP), cells first are
plated in 96-well plates at 7,000-10,000 cells/well and allowed to
adhere by overnight culture at 37.degree. C. Serial dilutions of
freshly dissolved or "aged" modulators (either alone or combined
with natural .beta.-AP) in phosphate buffered saline (PBS) are
added to the wells in triplicate and incubation is continued for
two or more days. Aged modulators are prepared by incubating an
aqueous solution of the modulator at 37.degree. C. undisturbed for
a prolonged period (e.g., five days or more). For the final two
hours of exposure of the cells to the modulator preparation, MTT is
added to the media to a final concentration of 1 mg/ml and
incubation is continued at 37.degree. C. Following the two hour
incubation with MTT, the media is removed and the cells are lysed
in isopropanol/0.4 N HCl with agitation. An equal volume of PBS is
added to each well and the absorbance of each well at 570 nm is
measured to quantitate viable cells. Alternatively, MTT is
solubilized by addition of 50% N,N-dimethyl formamide/20% sodium
dodecyl sulfate added directly to the media in the wells and viable
cells are likewise quantitated by measuring absorbance at 570 nm.
The relative neurotoxicity of a .beta.-amyloid modulator (either
alone or in combination with natural .beta.-AP) is determined by
comparison to natural .beta.-AP alone (e.g.,
.beta.1-40,.beta.1-42), which exhibits neurotoxicity in this assay
and thus can serve as a positive control.
EXAMPLE 4
Syntheses of Additional Modified .beta.-Amyloid Peptide
Compounds
[0272] In this example, a series of modified .beta.-APs, having a
variety of N-terminal or random side chain modifications were
synthesized.
[0273] A series of N-terminally modified .beta.-amyloid peptides
was synthesized using standard methods. Fully-protected resin-bound
peptides corresponding to A.beta.(1-15) and A.beta.(1-40) were
prepared as described in Example 1 on Wang resin to eventually
afford carboxyl terminal peptide acids. Small portions of each
peptide resin (13 and 20 .mu.moles, respectively) were aliquoted
into the wells of the reaction block of an Advanced ChemTech Model
396 Multiple Peptide Synthesizer. The N-terminal FMOC protecting
group of each sample was removed in the standard manner with 25%
piperidine in NMP followed by extensive washing with NMP. The
unprotected N-terminal a-amino group of each peptide-resin sample
was modified using one of the following methods:
[0274] Method A, coupling of modifying reagents containing free
carboxylic acid groups: The modifying reagent (five equivalents)
was predissolved in NMP, DMSO or a mixture of these two solvents.
HOBT and DIC (five equivalents of each reagent) were added to the
dissolved modifier and the resulting solution was added to one
equivalent of free-amino peptide-resin. Coupling was allowed to
proceed overnight, followed by washing. If a ninhydrin test on a
small sample of peptide-resin showed that coupling was not
complete, the coupling was repeated using
1-hydroxy-7-azabenzotriazole (HOAt) in place of HOBt.
[0275] Method B, coupling of modifying reagents obtained in
preactivated forms: The modifying reagent (five equivalents) was
predissolved in NMP, DMSO or a mixture of these two solvents and
added to one equivalent of peptide-resin. Diisopropylethylamine
(DIEA; six equivalents) was added to the suspension of activated
modifier and peptide-resin. Coupling was allowed to proceed
overnight, followed by washing. If a ninhydrin test on a small
sample of peptide-resin showed that coupling was not complete, the
coupling was repeated.
[0276] After the second coupling (if required) the N-terminally
modified peptide-resins were dried at reduced pressure and cleaved
from the resin with removal of side-chain protecting groups as
described in Example 1. Analytical reversed-phase HPLC was used to
confirm that a major product was present in the resulting crude
peptides which were purified using Millipore Sep-Pak cartridges or
preparative reverse-phase HPLC. Mass spectrometry was used to
confirm the presence of the desired compound in the product.
[0277] Method A was used to couple N-acetylneuraminic acid, cholic
acid, trans-4-cotininecarboxylic acid,
2-imino-1-imidazolidineacetic acid, (S)-(-)-indoline-2-carboxylic
acid, (-)-menthoxyacetic acid, 2-norbornaneacetic acid,
.gamma.-oxo-5-acenaphthenebutyric acid,
(-)-2-oxo-4-thiazolidinecarboxylic acid, and tetrahydro-3-furoic
acid. Method B was used to couple
2-iminobiotin-N-hydroxysuccinimide ester,
diethylenetriaminepentaacetic dianhydride, 4-morpholinecarbonyl
chloride, 2-thiopheneacetyl chloride, and 2-thiophenesulfonyl
chloride.
[0278] In a manner similar to the construction of N-terminally
modified A.beta.(1-15) and A.beta.(1-40) peptides described above,
N-fluoresceinyl A.beta.(1-15) and A.beta.(1-40) were prepared in
two alternative manners using the preactivated reagents 5-(and
6)-carboxyfluorescein succinimidyl ester and
fluorescein-5-isothiocyanate (FITC Isomer I). Both reagents were
obtained from Molecular Probes Inc. Couplings were performed using
four equivalents of reagent per equivalent of peptide-resin with
DIEA added to make the reaction solution basic to wet pH paper.
Couplings of each reagent to A.beta.(1-15)-resin appeared to be
complete after a single overnight coupling. Coupling to
A.beta.(1-40)-resin was slower as indicated by a positive ninhydrin
test and both reagents were recoupled to this peptide-resin
overnight in tetrahydrofuran-NMP (1:2 v/v). The resulting
N-terminally modified peptide-resins were cleaved, deprotected and
purified as described in Example A.
[0279] In addition to the N-fluoresceinyl A.beta. peptides
described above, a .beta.-amyloid modulator comprised of random
modification of A.beta.(1-40) with fluorescein was prepared.
A.beta.(1-40) purchased from Bachem was dissolved in DMSO at
approximately 2 mg/mL. 5-(and-6)-Carboxyfluorescein purchased from
Molecular Probes was added in a 1.5 molar excess and DIEA was added
to make the solution basic to wet pH paper. The reaction was
allowed to proceed for 1 hour at room temperature and was then
quenched with triethanolamine. The product was added to assays as
this crude mixture.
[0280] .beta.-amyloid modulator compounds comprising other regions
of the .beta.-AP amino acid sequence (e.g., an A.beta. aggregation
core domain) were similarly prepared using the synthesis methods
described above. Moreover, modulators comprising other
amyloidogenic peptides can be similarly prepared.
EXAMPLE 5
Identification of Additional .beta.-Amyloid Modulators
[0281] In this Example, two assays of A.beta. aggregation were used
to identify .beta.-amyloid modulators which can inhibit this
process.
[0282] The first assay is referred to as a seeded static assay
(SSA) and was performed as follows:
[0283] To prepare a solution of A.beta. monomer, the appropriate
quantity of A.beta.(1-40) peptide (Bachem) was weighed out on a
micro-balance (the amount was corrected for the amount of water in
the preparation, which, depending on lot number, was 20-30% w/w).
The peptide was dissolved in 1/25 volume of dimethysulfoxide
(DMSO), followed by water to 1/2 volume and 1/2 volume 2.times.PBS
(10.times.PBS: NaCl 137 mM, KCl 2.7 mM
Na.sub.2HPO.sub.4.cndot.7H.sub.2O 4.3 mM, KH.sub.2PO.sub.4 1.4 mM
pH 7.2) to a final concentration of 200 .mu.M.
[0284] To prepare a stock seed, 1 ml of the above A.beta. monomer
preparation, was incubated for 8 days at 37.degree. C. and sheared
sequentially through an 18, 23, 26 and 30 gauge needle 25, 25, 50,
and 100 times respectively. 2 .mu.l samples of the sheared material
was taken for fluorescence measurements after every 50 passes
through the 30 gauge needle until the fluorescence units (FU) had
plateaued (approx. 100-150.times.).
[0285] To prepare a candidate inhibitor, the required amount of
candidate inhibitor was weighed out and the stock dissolved in
1.times.PBS to a final concentration of 1 mM (10.times.stock). If
insoluble, it was dissolved in 1/10 volume of DMSO and diluted in
1.times.PBS to 1 mM. A further 1/10 dilution was also prepared to
test each candidate at both 100 .mu.M and 10 .mu.M.
[0286] For the aggregation assay, each sample was set up in
triplicate [50 .mu.l of 200 .mu.M monomer, 125 FU sheared seed
(variable quantity dependent on the batch of seed, routinely 3-6
.mu.l), 10 .mu.l of 10.times.inhibitor solution, final volume made
up to 100 .mu.l with 1.times.PBS]. Two concentrations of each
inhibitor were tested 100 .mu.M and 10 .mu.M, equivalent to a 1:1
and a 1:10 molar ratio of monomer to inhibitor. The controls
included an unseeded reaction to confirm that the fresh monomer
contained no seed, and a seeded reaction in the absence of
inhibitor, as a reference to compare against putative inhibitors.
The assay was incubated at 37.degree. C. for 6 h, taking 2 .mu.l
samples hourly for fluorescence measurements. To measure
fluorescence, a 2 .mu.l sample of A.beta. was added to 400 .mu.l of
Thioflavin-T solution (50 mM Potassium Phosphate 10 mM Thioflavin-T
pH 7.5). The samples were vortexed and the fluorescence was read in
a 0.5 ml micro quartz cuvette at EX 450 nm and EM 482 nm (Hitachi
4500 Fluorimeter). .beta.-aggregation results in enhanced emission
of Thioflavin-T. Accordingly, samples including an effective
inhibitor compound exhibit reduced emission as compared to control
samples without the inhibitor compound.
[0287] The second assay is referred to as a shaken plate
aggregation assay and was performed as follows:
[0288] A.beta.(1-40) peptide from Bachem (Torrance, Calif.) was
dissolved in HFIP (1,1,1,3,3,3-Hexafluoro-2-propanol; Aldrich
10,522-8) at a concentration of 2 mg peptide/ml and incubated at
room temperature for 30 min. HFIP solubilized peptide was sonicated
in a waterbath sonicator for 5 min at highest setting, then
evaporated to dryness under a stream of argon. The peptide film was
resuspended in anhydrous dimethylsulfoxide (DMSO) at a
concentration of 6.9 mg/ml, sonicated for 5 min as before, then
filtered through a 0.2 micron nylon syringe filter (VWR cat. No.
28196-050). Candidate inhibitors were dissolved directly in DMSO,
generally at a molar concentration 4 times that of the
A.beta.(1-40) peptide.
[0289] Candidates were assayed in triplicate. For each candidate to
be tested, 4 parts A.beta.(1-40) peptide in DMSO were combined with
1 part candidate inhibitor in DMSO in a glass vial, and mixed to
produce a 1:1 molar ratio of A.beta. peptide to candidate. For
different molar ratios, candidates were diluted with DMSO prior to
addition to A.beta.(1-40), in order to keep the final DMSO and
A.beta.(1-40) concentrations constant. Into an ultra low binding 96
well plate (Coming Costar cat. No. 2500, Cambridge Mass.) 100 .mu.l
PTL buffer (150 mM NaCl, 10 mM NaH.sub.2PO.sub.4; pH 7.4) was
aliquotted per well. For each candidate, 10 .mu.l of peptide
mixture in DMSO was aliquotted into each of three wells containing
buffer. The covered plate was vigorously vortexed on a plate shaker
at high speed for 30 seconds. An additional 100 .mu.l of PTL buffer
was added to each well and again the plate was vortexed vigorously
for 30 sec. Absorbance at 405 nm was immediately read in a plate
reader for a baseline reading. The plate was returned to the plate
shaker and vortexed at moderate speed for 5 hours at room
temperature, with absorbance readings taken at 15-20 min intervals.
Increased absorbance indicated aggregation. Accordingly, effective
inhibitor compounds cause a decrease in absorbance in the test
sample as compared to a control sample without the inhibitor
compound.
[0290] Representative results of the static seeded assay and shaken
plate assay with preferred .beta.-amyloid modulators are shown
below in Table I.
1TABLE I Effect in Effect in Seeded Candidate A.beta. Amino
Modifying shaken plate Static Inhibitor Acids Reagent assay Assay *
174 A.beta.1-15 Cholic acid Complete ++ inhibition at 100% conc 176
A.beta.1-15 Diethylene- Decreased ++ triamine penta Plateau acetic
acid 180 A.beta.1-15 (-)-Menthoxy None ++ acetic acid 190
A.beta.1-15 Fluorescein Decreased ++ carboxylic acid Plateau (FICO)
220 A.beta.16-40 h-EVHHHHQQK- Complete ++ mutant
[A.beta.(16-40)]-OH inhibition at 100%, increased lag at 10% 224
A.beta.1-40 F.sub.19F.sub.20 -> T.sub.19T.sub.20 Increased lag
++ mutant 233 A6.beta.-20 Acetic acid accelerated ++ aggregation at
10% conc * ++ = A strong inhibitor of aggregation. The rate of
aggregation in the presence of the inhibitor was decreased compared
to the control by at least 30-50%
[0291] These results indicate that .beta.-APs modified by a wide
variety of N-terminal modifying groups are effective at modulating
.beta.-amyloid aggregation.
EXAMPLE 6
Additional .beta.-Amyloid Aggregation Assays
[0292] Most preferably, the ability of .beta.-amyloid modulator
compounds to modulate (e.g., inhibit or promote) the aggregation of
natural .beta.-AP when combined with the natural .beta.-AP is
examined in one or both of the aggregation assays described below.
Natural .beta.-AP (.beta.-AP.sub.1-40) for use in the aggregation
assays is commercially available from Bachem (Torrance,
Calif.).
[0293] A. Nucleation Assay
[0294] The nucleation assay is employed to determine the ability of
test compounds to alter (e.g. inhibit) the early events in
formation of .beta.-AP fibers from monomeric .beta.-AP.
Characteristic of a nucleated polymerization mechanism, a lag time
is observed prior to nucleation, after which the peptide rapidly
forms fibers as reflected in a linear rise in turbidity. The time
delay before polymerization of .beta.-AP monomer can be quantified
as well as the extent of formation of insoluble fiber by light
scattering (turbidity). Polymerization reaches equilibrium when the
maximum turbidity reaches a plateau. The turbidity of a solution of
natural .beta.-AP in the absence or presence of various
concentrations of a .beta.-amyloid modulator compound is determined
by measuring the apparent absorbance of the solution at 405 nm
(A.sub.405 nm) over time. The threshold of sensitivity for the
measurement of turbidity is in the range of 15-20 .mu.M .beta.-AP.
A decrease in turbidity over time in the presence of the modulator,
as compared to the turbidity in the absence of the modulator,
indicates that the modulator inhibits formation of .beta.-AP fibers
from monomeric .beta.-AP. This assay can be performed using
stirring or shaking to accelerate polymerization, thereby
increasing the speed of the assay. Moreover the assay can be
adapted to a 96-well plate format to screen multiple compounds.
[0295] To perform the nucleation assay, first A.beta..sub.1-40
peptide is dissolved in HFIP (1,1,1,3,3,3-Hexafluoro-2-propanol;
Aldrich 10,522-8) at a concentration of 2 mg peptide/ml and
incubated at room temperature for 30 min. HFIP-solubilized peptide
is sonicated in a waterbath sonicator for 5 min at highest setting,
then evaporated to dryness under a stream of argon. The peptide
film is resuspended in anhydrous dimethylsulfoxide (DMSO) at a
concentration of 6.9 mg/ml (25.times.concentration), sonicated for
5 min as before, then filtered through a 0.2 micron nylon syringe
filter (VWR cat. No. 28196-050). Test compounds are dissolved in
DMSO at a 100.times.concentration. Four volumes of
25.times.A.beta..sub.1-40 peptide in DMSO are combined with one
volume of test compound in DMSO in a glass vial, and mixed to
produce a 1:1 molar ratio of A.beta. peptide to test compound. For
different molar ratios, test compounds are diluted with DMSO prior
to addition to A.beta..sub.1-40, in order to keep the final DMSO
and A.beta..sub.1-40 concentrations constant. Control samples do
not contain the test compound. Ten microliters of the mixture is
then added to the bottom of a well of a Corning Costar ultra low
binding 96-well plate (Corning Costar, Cambridge Mass.; cat. No.
2500). Ninety microliters of water is added to the well, the plate
is shaken on a rotary shaken at a constant speed at room
temperature for 30 seconds, an additional 100 .mu.l of 2.times.PTL
buffer (20 mM NaH.sub.2PO.sub.4, 300 mM NaCl, pH 7.4) is added to
the well, the plate is reshaken for 30 seconds and a baseline (t=0)
turbidity reading is taken by measuring the apparent absorbance at
405 nm using a Bio-Rad Model 450 Microplate Reader. The plate is
then returned to the shaker and shaken continuously for 5 hours.
Turbidity readings are taken at 15 minute intervals.
[0296] .beta.-amyloid aggregation in the absence of any modulators
results in enhanced turbidity of the natural .beta.-AP solution (i.
e., an increase in the apparent absorbance at 405 nm over time).
Accordingly, a solution including an effective inhibitory modulator
compound exhibits reduced turbidity as compared to the control
sample without the modulator compound (i.e., less apparent
absorbance at 405 nm over time as compared to the control
sample).
[0297] B. Seeded Extension Assay
[0298] The seeded extension assay can be employed to measure the
rate of A.beta. fiber formed in a solution of A.beta. monomer
following addition of polymeric A.beta. fiber "seed". The ability
of test compounds to prevent further deposition of monomeric AP to
previously deposited amyloid is determined using a direct indicator
of .beta.-sheet formation using fluorescence. In contrast with the
nucleation assay, the addition of seed provides immediate
nucleation and continued growth of preformed fibrils without the
need for continuous mixing, and thus results in the absence of a
lag time before polymerization starts. Since this assay uses static
polymerization conditions, the activity of positive compounds in
the nucleation assay can be confirmed in this second assay under
different conditions and with an additional probe of amyloid
structure.
[0299] In the seeded extension assay, monomeric A.beta..sub.1-40 is
incubated in the presence of a "seed" nucleus (approximately ten
mole percent of A.beta. that has been previously allowed to
polymerize under controlled static conditions). Samples of the
solution are then diluted in thioflavin T (Th-T). The
polymer-specific association of Th-T with A.beta. produces a
fluorescent complex that allows the measurement of the extent of
fibril formation (Levine, H. (1993) Protein Science 2:404-410). In
particular, association of Th-T with aggregated .beta.-AP, but not
monomeric or loosely associated .beta.-AP, gives rise to a new
excitation (ex) maximum at 450 nm and an enhanced emission (em) at
482 nm, compared to the 385 Dm (ex) and 445 nm (em) for the free
dye. Small aliquots of the polymerization mixture contain
sufficient fibril to be mixed with Th-T to allow the monitoring of
the reaction mixture by repeated sampling. A linear growth curve is
observed in the presence of excess monomer. The formation of
thioflavin T responsive .beta.-sheet fibrils parallels the increase
in turbidity observed using the nucleation assay.
[0300] A solution of A.beta. monomer for use in the seeded
extension assay is prepared by dissolving an appropriate quantity
of AP.sub.1-40 peptide in 1/25 volume of dimethysulfoxide (DMSO),
followed by water to 1/2 volume and 1/2 volume 2.times.PBS
(10.times.PBS: NaCl 137 mM, KCl 2.7 mM
Na.sub.2HPO.sub.4.cndot.7H.sub.2O 4.3 mM, KH.sub.2PO.sub.4 1.4 mM
pH 7.2) to a final concentration of 200 .mu.M. To prepare the stock
seed, 1 ml of the A.beta. monomer preparation, is incubated for
approximately 8 days at 37.degree. C. and sheared sequentially
through an 18, 23, 26 and 30 gauge needle 25, 25, 50, and 100 times
respectively. 2 .mu.l samples of the sheared material is taken for
fluorescence measurements after every 50 passes through the 30
gauge needle until the fluorescence units (FU) plateau (approx.
100-150.times.). Test compounds are prepared by dissolving an
appropriate amount of test compound in 1.times.PBS to a final
concentration of 1 mM (10.times.stock). If insoluble, the compound
is dissolved in 1/10 volume of DMSO and diluted in 1.times.PBS to 1
mM. A further 1/10 dilution is also prepared to test each candidate
at both 100 .mu.M and 10 .mu.M.
[0301] To perform the seeded extension assay, each sample is set up
with 50 .mu.l of 200 .mu.M monomer, 125 FU sheared seed (a variable
quantity dependent on the batch of seed, routinely 3-6 .mu.l) and
10 .mu.l of 10.times.modulator solution. The sample volume is then
adjusted to a final volume of 100 .mu.l with 1.times.PBS. Two
concentrations of each modulator typically are tested: 100 .mu.M
and 10 .mu.M, equivalent to a 1:1 and a 1:10 molar ratio of monomer
to modulator. The controls include an unseeded reaction to confirm
that the fresh monomer contains no seed, and a seeded reaction in
the absence of any modulators, as a reference to compare against
candidate modulators. The assay is incubated at 37.degree. C. for 6
h, taking 2 .mu.l samples hourly for fluorescence measurements. To
measure fluorescence, a 2 .mu.l sample of A.beta.is added to 400
.mu.l of Thioflavin-T solution (50 mM Potassium Phosphate 10 mM
Thioflavin-T pH 7.5). The samples are vortexed and the fluorescence
is read in a 0.5 ml micro quartz cuvette at EX 450 nm and EM 482 nm
(Hitachi 4500 Fluorimeter).
[0302] .beta.-amyloid aggregation results in enhanced emission of
Thioflavin-T. Accordingly, samples including an effective
inhibitory modulator compound exhibit reduced emission as compared
to control samples without the modulator compound.
EXAMPLE 7
Effect of Different Amino Acid Subregions of A.beta. Peptide on the
Inhibitory Activity of .beta.-Amyloid Modulator Compounds
[0303] To determine the effect of various subregions of
A.beta..sub.1-40 on the inhibitory activity of a a .beta.-amyloid
modulator, overlapping A.beta. peptide 15 mers were constructed.
For each 15 mer, four different amino-terminal modifiers were
tested: a cholyl group, an iminobiotinyl group, an N-acetyl
neuraminyl group (NANA) and a 5-(and 6-)-carboxyfluoresceinyl group
(FICO). The modulators were evaluated in the nucleation and seeded
extension assays described in Example 6.
[0304] The results of the nucleation assays are summarized below in
Table II. The concentration of AP.sub.1-40 used in the assays was
50 .mu.M. The "mole %" value listed in Table II refers to the %
concentration of the test compound relative to A.beta..sub.1-40.
Accordingly, 100% indicates that A.beta..sub.1-40 and the test
compound were equimolar. Mole % values less than 100% indicate that
A.beta..sub.1-40 was in molar excess relative to the test compound
(e.g., 10% indicates that A.beta..sub.1-40 was in 10-fold molar
excess relative to the test compound). The results of the
nucleation assays for each test compound are presented in Table II
in two ways. The "fold increase in lag time", which is a measure of
the ability of the compound to delay the onset of aggregation,
refers to the ratio of the observed lag time in the presence of the
test compound to the observed lag time in the control without the
test compound. Accordingly a fold increase in lag time of 1.0
indicates no change in lag time, whereas numbers >1.0 indicate
an increase in lag time. The "% inhibition of plateau", which is a
measure of the ability of the compound to decrease the total amount
of aggregation, refers to the reduction of the final turbidity in
the presence of the test compound expressed as a percent of the
control without the test compound. Accordingly, an inhibitor that
abolishes aggregation during the course of the assay will have a %
inhibition of 100. N-terminally modified A.beta. subregions which
exhibited inhibitory activity are indicated in bold in Table
II.
2TABLE II Fold In- % N-terminal A.beta. crease in Inhibition
Reference # Modification Peptide Mole % Lag Time of Plateau PPI-174
cholyl A.beta..sub.1-15 100 >4.5 100 PPI-264 cholyl
A.beta..sub.6-20 100 >4.5 100 PPI-269 cholyl A.beta..sub.11-25
100 1.5 .about.0 PPI-274 cholyl A.beta..sub.16-30 100 >4.5 100
PPI-279 cholyl A.beta..sub.21-35 100 1.6 51 PPI-284 cholyl
A.beta..sub.26-40 100 >4.5 87 PPI-173 NANA A.beta..sub.1-15 100
.about.1 .about.0 PPI-266 NANA A.beta..sub.6-20 100 1.3 64 PPI-271
NANA A.beta..sub.11-25 100 1.3 77 PPI-276 NANA A.beta..sub.16-30
100 .about.1 .about.0 PPI-281 NANA A.beta..sub.21-35 100 .about.1
53 PPI-286 NANA A.beta..sub.26-40 100 1.3 .about.0 PPI-172
Iminobiotinyl A.beta..sub.1-15 100 1.2 .about.0 PPI-267
Iminobiotinyl A.beta..sub.6-20 100 1.6 44 PPI-272 Iminobiotinyl
A.beta..sub.11-25 100 1.2 40 PPI-277 Iminobiotinyl
A.beta..sub.16-30 100 1.2 55 PPI-282 Iminobiotinyl
A.beta..sub.21-35 100 .about.1 66 PPI-287 Iminobiotinyl
A.beta..sub.26-40 100 2.3 .about.0 PPI-190 FICO A.beta..sub.1-15
100 .about.1 30 PPI-268 FICO A.beta..sub.6-20 100 1.9 .about.0
PPI-273 FICO A.beta..sub.11-25 100 1.7 34 PPI-278 FICO
A.beta..sub.16-30 100 1.6 59 PPI-283 FICO A.beta..sub.21-35 100 1.2
25 PPI-288 FICO A.beta..sub.26-40 100 2 75
[0305] These results indicate that certain subregions of
A.beta..sub.1-40, when modified with an appropriate modifying
group, are effective at inhibiting the aggregation of
A.beta..sub.1-40. A cholyl group was an effective modifying group
for several subregions. Cholic acid alone was tested for inhibitory
activity but had no effect on A.beta. aggregation. The
A.beta..sub.6-20 subregion exhibited high levels of inhibitory
activity when modified with several different modifying groups
(cholyl, NANA, iminobiotinyl), with cholyl-A.beta..sub.6-20
(PPI-264) being the most active form. Accordingly, this modulator
compound was chosen for further analysis, described in Example
8.
EXAMPLE 8
Identification of a Five Amino Acid Subregion of A.beta. Peptide
Sufficient for Inhibitory Activity of a .beta.-Amyloid Modulator
Compound
[0306] To further delineate a minimal subregion of
cholyl-A.beta..sub.6-20 sufficient for inhibitory activity, a
series of amino terminal and carboxy terminal amino acid deletions
of cholyl-A.beta..sub.6-20 were constructed. The modulators all had
the same cholyl amino-terminal 10 modification. Additionally, for
the peptide series having carboxy terminal deletions, the carboxy
terminus was further modified to an amide. The modulators were
evaluated as described in Example 7 and the results are summarized
below in Table III, wherein the data is presented as described in
Example 7.
3TABLE III N- C- Fold In- Term. A.beta. Term. crease in %
Inhibition Ref. # Mod. Peptide Mod. Mole % Lag Time of Plateau
PPI-264 cholyl A.beta..sub.6-20 -- 100 >4.5 100 10 2 43 PPI-341
cholyl A.beta..sub.7-20 -- 100 >4.5 100 33 2 .about.0 PPI-342
cholyl A.beta..sub.8-20 -- 100 1.5 .about.0 33 2.1 .about.0 PPI-343
cholyl A.beta..sub.9-20 -- 33 2.0 .about.0 PPI-344 cholyl
A.beta..sub.10-20 -- 33 2.1 .about.0 PPI-345 cholyl
A.beta..sub.11-20 -- 33 1.5 .about.0 PPI-346 cholyl
A.beta..sub.12-20 -- 33 2.1 .about.0 PPI-347 cholyl
A.beta..sub.13-20 -- 33 2.6 .about.0 PPI-348 cholyl
A.beta..sub.14-20 -- 33 2.0 49 PPI-349 cholyl A.beta..sub.15-20 --
33 2.3 50 PPI-350 cholyl A.beta..sub.16-20 -- 38 3.4 23 PPI-296
cholyl A.beta..sub.6-20 amide 33 1.8 .about.0 PPI-321 cholyl
A.beta..sub.6-19 amide 33 1.4 .about.0 PPI-325 cholyl
A.beta..sub.6-17 amide 33 1.8 .about.0 PPI-331 cholyl
A.beta..sub.6-14 amide 33 1.0 29 PPI-339 cholyl A.beta..sub.6-10
amide 33 1.1 13
[0307] These results indicate that activity of the modulator is
maintained when amino acid residue 6 is removed from the amino
terminal end of the modulator (i.e., cholyl-A.beta..sub.7-20
retained activity) but activity is lost as the peptide is deleted
further at the amino-terminal end 20 by removal of amino acid
position 7 through to amino acid position 12 (i.e.,
cholyl-A.beta..sub.8-20 through cholyl-A.beta..sub.13-20 did
inhibit the plateau level of A.beta. aggregation). However, further
deletion of amino acid position 13 resulted in a compound (i.e.,
cholyl-A.beta..sub.14-20) in which inhibitory activity is restored.
Furthermore, additional deletion of amino acid position 14 (i.e.,
cholyl-A.beta..sub.15-20) or positions 14 and 15 (i.e.,
cholyl-A.beta..sub.16-20) still maintained inhibitory activity.
Thus, amino terminal deletions of A.beta..sub.6-20 identified
A.beta..sub.16-20 as a minimal subregion which is sufficient for
inhibitory activity when appropriately modified. In contrast,
carboxy terminal deletion of amino acid position 20 resulted in
loss of activity which was not fully restored as the peptide was
deleted further at the carboxy-terminal end. Thus, maintenance of
position 20 within the modulator may be important for inhibitory
activity.
EXAMPLE 9
Identification of a Four Amino Acid Subregion of A.beta. Peptide
Sufficient for Inhibitory Activity of a .beta.-Amyloid Modulator
Compound
[0308] In this example, the smallest effective modulator identified
in the studies described in Example 8, cholyl-A.beta..sub.16-20
(PPI-350), was analyzed further. Additional amino- and
carboxy-terminal deletions were made with cholyl-A.beta..sub.16-20,
as well as an amino acid substitution (Val .sub.18->Thr), to
identify the smallest region sufficient for the inhibitory activity
of the modulator. A peptide comprised of five alanine residues,
(Ala).sub.5, modified at its amino-terminus with cholic acid, was
used as a specificity control. The modulators were evaluated as
described in Example 7 and the results are summarized below in
Table IV, wherein the data is presented as described in Example
7.
4TABLE IV Fold In- N- C- crease % Term. Term. Mole in Lag
Inhibition Ref. # Mod. A.beta. Peptide Mod. % Time of Plateau
PPI-264 cholyl A.beta..sub.6-20 -- 10 2.0 43 PPI-347 cholyl
A.beta..sub.13-20 -- 10 2.2 57 PPI-349 cholyl A.beta..sub.15-20 --
100 >5.0 100 33 2.6 35 10 2.1 .about.0 PPI-350 cholyl
A.beta..sub.16-20 -- 100 >5.0 100 10 2.4 40 PPI-368 cholyl
A.beta..sub.17-21 -- 100 >5.0 100 PPI-374 imino-
A.beta..sub.16-20 -- 100 1.3 86 biotinyl PPI-366 cholyl
A.beta..sub.15-19 -- 100 3.1 .about.0 10 1.6 .about.0 PPI-369
cholyl A.beta..sub.16-20 -- 100 .about.1 .about.0 (Val.sub.18 ->
Thr) PPI-370 cholyl A.beta..sub.16-20 -- 100 2.6 73 (Phe.sub.19
-> Ala) PPI-365 cholyl (Ala).sub.5 -- 100 .about.1 .about.0
PPI-319 cholyl A.beta..sub.16-20 amide 33 5.6 .about.0 10 2.7
.about.0 PPI-321 cholyl A.beta..sub.16-19 amide 100 1.2 .about.0
PPI-377 -- A.beta..sub.16-20 - 100 .about.1 .about.0
[0309] As shown in Table IV, cholyl-A.beta..sub.16-20 (PPI-350) and
cholyl-A.beta..sub.17-21 (PPI-368) both exhibited inhibitory
activity, indicating that the four-amino acid minimal subregion of
positions 17-20 is sufficient for inhibitory activity. Loss of
position 20 (e.g., in PPI-366 and PPI-321) resulted in loss of
inhibitory activity, demonstrating the importance of position 20.
Moreover, mutation of valine at position 18 to threonine (in
PPI-369) also resulted in loss of activity, demonstrating the
importance of position 18. In contrast, mutation of phenylalanine
at position 19 to alanine (cholyl-A.beta..sub.16-20
Phe.sub.19->Ala; PPI-370) resulted in a compound which still
retained detectable inhibitory activity. Accordingly, the
phenylalanine at position 19 is more amenable to substitution,
preferably with another hydrophobic amino acid residue.
Cholyl-penta-alanine (PPI-365) showed no inhibitory activity,
demonstrating the specificity of the A.beta. peptide portion of the
modulator. Moreover, unmodified A.beta..sub.16-20 (PPI-377) was not
inhibitory, demonstrating the functional importance of the
amino-terminal modifying group. The specific functional group
influenced the activity of the modulator. For example,
iminobiotinyl-A.beta..sub.16-20 (PPI-374) exhibited inhibitory
activity similar to cholyl-A.beta..sub.16-20, whereas an N-acetyl
neuraminic acid (NANA)-modified A.beta..sub.16-20 was not an
effective inhibitory modulator (not listed in Table IV). A
C-terminal amide derivative of cholyl-A.beta..sub.16-20 (PPI-3 19)
retained high activity in delaying the lag time of aggregation,
indicating that the carboxy-terminus of the modulator can be
derivatized without loss of inhibitory activity. Although this
amide-derivatized compound did not inhibit the overall plateau
level of aggregation over time, the compound was not tested at
concentrations higher than mole 33%. Higher concentrations of the
amide-derivatized compound are predicted to inhibit the overall
plateau level of aggregation, similar to cholyl-A.beta..sub.16-20
(PPI-350).
EXAMPLE 10
Effect of .beta.-Amyloid Modulators on the Neurotoxicity of Natural
.beta.-Amyloid Peptide Aggregates
[0310] The neurotoxicity of natural .beta.-amyloid peptide
aggregates, in either the presence or absence of a .beta.-amyloid
modulator, is tested in a cell-based assay using either a rat or
human neuronally-derived cell line (PC-12 cells or NT-2 cells,
respectively) and the viability indicator
3,(4,4-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT).
(See e.g., Shearman, M.S. et al. (1994) Proc. Natl. Acad. Sci. USA
91:1470-1474; Hansen, M. B. et al. (1989) J. Immun. Methods 1
19:203-210 for a description of similar cell-based viability
assays). PC-12 is a rat adrenal pheochromocytoma cell line and is
available from the American Type Culture Collection, Rockville, Md.
(ATCC CRL 1721). MTT (commercially available from Sigma Chemical
Co.) is a chromogenic substrate that is converted from yellow to
blue in viable cells, which can be detected
spectrophotometrically.
[0311] To test the neurotoxicity of natural .beta.-amyloid
peptides, stock solutions of fresh A.beta. monomers and aged
A.beta. aggregates were first prepared. A.beta..sub.1-40 in 100%
DMSO was prepared from lyophilized powder and immediately diluted
in one half the final volume in H.sub.2O and then one half the
final volume in 2.times.PBS so that a final concentration of 200
.mu.M peptide, 4% DMSO is achieved. Peptide prepared in this way
and tested immediately on cells is referred to as "fresh" A.beta.
monomer. To prepare "aged" A.beta. aggregates, peptide solution was
placed in a 1.5 ml Eppendorf tube and incubated at 37.degree. C.
for eight days to allow fibrils to form. Such "aged" A.beta.
peptide can be tested directly on cells or frozen at -80.degree. C.
The neurotoxicity of fresh monomers and aged aggregates were tested
using PCl2 and NT2 cells. PC 12 cells were routinely cultured in
Dulbeco's modified Eagle's medium (DMEM) containing 10% horse
serum, 5% fetal calf serum, 4 mM glutamine, and 1% gentamycin. NT2
cells were routinely cultured in OPTI-MEM medium (GIBCO BRL CAT.
#31985) supplemented with 10% fetal calf serum, 2 mM glutamine and
1% gentamycin. Cells were plated at 10-15,000 cells per well in 90
.mu.l of fresh medium in a 96-well tissue culture plate 3-4 hours
prior to treatment. The fresh or aged A.beta. peptide solutions (10
.mu.L) were then diluted 1:10 directly into tissue culture medium
so that the final concentration was in the range of 1-10 .mu.M
peptide. Cells are incubated in the presence of peptide without a
change in media for 48 hours at 37.degree. C. For the final three
hours of exposure of the cells to the .beta.-AP preparation, MTT
was added to the media to a final concentration of 1 mg/ml and
incubation was continued at 37.degree. C. Following the two hour
incubation with MTT, the media was removed and the cells were lysed
in 100 .mu.L isopropanol/0.4 N HCl with agitation. An equal volume
of PBS was added to each well and the plates were agitated for an
additional 10 minutes. Absorbance of each well at 570 run was
measured using a microtiter plate reader to quantitate viable
cells.
[0312] The neurotoxicity of aged (5 day or 8 day) A.beta..sub.1-40
aggregates alone, but not fresh A.beta..sub.1-40 monomers alone,
was confirmed in an experiment the results of which are shown in
FIG. 3, which demonstrates that incubating the neuronal cells with
increasing amounts of fresh A.beta..sub.1-40 monomers was not
significantly toxic to the cells whereas incubating the cells with
increasing amounts of 5 day or 8 day A.beta..sub.1-40 aggregates
led to increasing amount of neurotoxicity. The EC50 for toxicity of
aged A.beta..sub.1-40 aggregates was 1-2 .mu.M for both the PC12
cells and the NT2 cells.
[0313] To determine the effect of a .beta.-amyloid modulator
compound on the neurotoxicity of A.beta..sub.1-40 aggregates, a
modulator compound, cholyl-A.beta..sub.6-20 (PPI-264), was
preincubated with A.beta..sub.1-40 monomers under standard
nucleation assay conditions as described in Example 6 and at
particular time intervals post-incubation, aliquots of the
.beta.-AP/modulator solution were removed and 1) the turbidity of
the solution was assessed as a measure of aggregation and 2) the
solution was applied to cultured neuronal cells for 48 hours at
which time cell viability was assessed using MTT to determine the
neurotoxicity of the solution. The results of the turbidity
analysis are shown in FIG. 4, panels A, B and C. In panel A,
A.beta..sub.1-40 and cholyl-A.beta..sub.6-20 were both present at
64 .mu.M. In panel B, A.beta..sub.1-40 was present at 30 .mu.M and
cholyl-A.beta..sub.6-20 was present at 64 .mu.M. In panel C,
A.beta..sub.1-40 was present at 10 .mu.M and
cholyl-A.beta..sub.6-20 was present at 64 .mu.M. These data show
that an equimolar amount of cholyl-A.beta..sub.6-20 is effective at
inhibiting aggregation of A.beta..sub.1-40 (see FIG. 4, panel A)
and that as the concentration of A.beta..sub.1-40 is reduced, the
amount of detectable aggregation of the A.beta..sub.1-40 monomer is
correspondingly reduced (compare FIG. 4, panels B and C with panel
A). The corresponding results of the neurotoxicity analysis are
shown in FIG. 4, panels D, E, and F. These results demonstrate that
the .beta.-amyloid modulator compound not only inhibits aggregation
of A.beta..sub.1-40 monomers but also inhibits the neurotoxicity of
the A.beta..sub.1-40 solution, illustrated by the reduced percent
toxicity of the cells when incubated with the
A.beta..sub.1-40/modulator solution as compared to A.beta..sub.1-40
alone (see e.g., FIG. 4, panel D). Moreover, even when
A.beta..sub.1-40 aggregation was not detectable as measured by
light scattering, the modulator compound inhibited the
neurotoxicity of the A.beta..sub.1-40 solution (see FIG. 4, panels
E and F). Thus, the formation of neurotoxic A.beta..sub.1-40
aggregates precedes the formation of insoluble aggregates
detectable by light scattering and the modulator compound is
effective at inhibiting the inhibiting the formation and/or
activity of these neurotoxic aggregates. Similar results were seen
with other modulator compounds, such as
iminobiotinyl-A.beta..sub.6-20 (PPI-267), cholyl-A.beta..sub.16-20
(PPI-350) and cholyl-A.beta..sub.16-20-amide (PPI-319).
[0314] Additionally, the .beta.-amyloid modulator compounds have
been demonstrated to reduce the neurotoxicity of preformed
A.beta..sub.1-40 aggregates. In these experiments, A.beta..sub.1-40
aggregates were preformed by incubation of the monomers in the
absence of any modulators. The modulator compound was then
incubated with the preformed A.beta..sub.1-40 aggregates for 24
hours at 37.degree. C., after which time the .beta.-AP/modulator
solution was collected and its neurotoxicity evaluated as described
above. Incubation of preformed A.beta..sub.1-40 aggregates with the
modulator compound prior to applying the solution to neuronal cells
resulted in a decrease in the neurotoxicity of the A.beta..sub.1-40
solution. These results suggest that the modulator can either bind
to A.beta. fibrils or soluble aggregate and modulate their inherent
neurotoxicity or that the modulator can perturb the equilibrium
between monomeric and aggregated forms of A.beta..sub.1-40 in favor
of the non-neurotoxic form.
EXAMPLE 11
Characterization of Additional .beta.-Amyloid Modulator
Compounds
[0315] In this example, additional modulator compounds designed
based upon amino acids 17-20 of A.beta., LVFF (identified in
Example 9), were prepared and analyzed to further delineate the
structural features necessary for inhibition of .beta.-amyloid
aggregation. Types of compounds analyzed included ones having only
three amino acid residues of an A.beta. aggregation core domain,
compounds in which the amino acid residues of an A.beta.
aggregation core domain were rearranged or in which amino acid
substitutions had been made, compounds modified with a
carboxy-terminal modifying group and compounds in which the
modifying group had been derivatized. Abbreviations used in this
example are: h- (free amino terminus), -oh (free carboxylic acid
terminus), -nh.sub.2 (amide terminus), CA (cholyl, the acyl portion
of cholic acid), NANA (N-acetyl neuraminyl), IB (iminobiotinyl),
.beta.A (.beta.-alanyl), DA (D-alanyl), Adp
(aminoethyldibenzofuranylpropanoic acid), Aic
(3-(O-aminoethyl-iso)-cholyl, a derivative of cholic acid), IY
(iodotyrosyl), o-methyl (carboxy-terminal methyl ester), N-me
(N-methyl peptide bond), DeoxyCA (deoxycholyl) and LithoCA
(lithocholyl).
[0316] Modulator compounds having an Aic modifying group at either
the amino- or carboxy-terminus (e.g., PPI-408 and PPI-418) were
synthesized using known methods (see e.g., Wess, G. et al. (1993)
Tetrahedron Letters, 34:817-822; Wess, G. et al (1992) Tetrahedron
Letters 33:195-198). Briefly, 3-iso-O-(2-aminoethyl)-cholic acid
(3.beta.-(2-aminoethoxy)-7.alpha.,12.alpha.-dihydroxy-5.beta.-cholanoic
acid) was converted to the FMOC-protected derivative using FMOC-OSu
(the hydroxysuccinimide ester of the FMOC group, which is
commercially available) to obtain a reagent that was used to
introduce the cholic acid derivative into the compound. For
N-terminal introduction of the cholic acid moiety, the
FMOC-protected reagent was coupled to the N-terminal amino acid of
a solid-phase peptide in the standard manner, followed by standard
FMOC-deprotection conditions and subsequent cleavage from the
resin, followed by HPLC purification. For C-terminal introduction
of the cholic acid moiety, the FMOC-protected reagent was attached
to 2-chlorotrityl chloride resin in the standard manner. This amino
acyl derivatized resin was then used in the standard manner to
synthesize the complete modified peptide.
[0317] The modulators were evaluated in the nucleation and seeded
extension assays described in Example 6 and the results are
summarized below in Table V. The change in lag time (.DELTA.Lag) is
presented as the ratio of the lag time observed in the presence of
the test compound to the lag time of the control. Data are reported
for assays in the presence of 100 mole % inhibitor relative to the
concentration of A.beta..sub.1-40, except for PPI-315, PPI-348,
PPI-380, PPI-407 and PPI-418, for which the data is reported in the
presence of 33 mole % inhibitor. Inhibition (% I.sub.nucl'n) is
listed as the percent reduction in the maximum observed turbidity
in the control at the end of the assay time period. Inhibition in
the extension assay (% I.sub.ext'n) is listed as the percent
reduction of thioflavin-T fluorescence of .beta.-structure in the
presence of 25 mole % inhibitor. Compounds with a % I.sub.nucl'n of
at least 30% are highlighted in bold.
5TABLE V N-Term. C-Term. % Ref. # Mod. Peptide Mod. .DELTA.Lag
I.sub.nucl'n % I.sub.ext'n PPI-293 CA -- -oh 1.0 0 ND* PPI-315 CA
HQKLVFF -nh.sub.2 1.1 5** ND PPI-316 NANA HQKLVFF -nh.sub.2 1.5 -15
ND PPI-319 CA KLVFF -nh.sub.2 5.4 70 52 PPI-339 CA HDSGY -nh.sub.2
1.1 -18 ND PPI-348 CA HQKLVFF -oh 2.0 70** ND PPI-349 CA QKLVFF -oh
>5 100 56 PPI-350 CA KLVFF -oh 1.8 72 11 PPI-365 CA AAAAA -oh
0.8 -7 0 PPI-366 CA QKLVF -oh 3.1 -23 ND PPI-368 CA LVFFA -oh >5
100 91 PPI-369 CA KLTFF oh 1.1 -16 44 PPI-370 CA KLVAF -oh 2.6 73
31 PPI-371 CA KLVFF(.beta.A) -oh 2.5 76 80 PPI-372 CA FKFVL -oh 0.8
45 37 PPI-373 NANA KLVFF -oh 0.9 16 8 PPI-374 IB KLVFF -oh 1.3 86 0
PPI-375 CA KTVFF -oh 1.2 18 21 PPI-377 h- KLVFF -oh 1.1 0 8 PPI-379
CA LVFFAE -oh 1.4 55 16 PPI-380 CA LVFF -oh 1.8 72** 51 PPI-381 CA
LVFF(DA) -oh 2.3 56 11 PPI-382 CA LVFFA -nh.sub.2 1.0 -200 91
PPI-383 h-DDIIL- VFF -oh 0.4 14 0 (Adp) PPI-386 h- LVFFA -oh 1.0 15
11 PPI-387 h- KLVFF -nh.sub.2 1.3 -9 39 PPI-388 CA AVFFA -oh 1.4 68
44 PPI-389 CA LAFFA -oh 1.5 47 66 PPI-390 CA LVAFA -oh 2.7 25 0
PPI-392 CA VFFA -oh 2.0 76 10 PPI-393 CA LVF -oh 1.3 1 0 PPI-394 CA
VFF -oh 1.8 55 0 PPI-395 CA FFA -oh 1.0 51 6 PPI-396 CA LV(IY)FA
-oh >5 100 71 PPI-401 CA LVFFA -o-methyl ND ND 0 PPI-405 h-
LVFFA -nh.sub.2 1.3 11 70 PPI-407 CA LVFFK -oh >5 100** 85
PPI-408 h- LVFFA (Aic)-oh 3.5 46 3 PPI-418 h-(Aic) LVFFA -oh >5
100** 87 PPI-426 CA FFVLA -oh >5 100 89 PPI-391 CA LVFAA -oh 1.6
40 ND PPI-397 CA LVF(IY)A -oh >5 95 ND PPI-400 CA AVAFA -oh 1.0
-15 ND PPI-403 *** HQKLVFF -oh 1.4 -75 0 PPI-404 **** LKLVFF -oh
1.8 -29 7 PPI-424 DeoxyCA LVFFA -oh 3.0 -114 82 PPI-425 LithoCA
LVFFA -oh 2.8 -229 0 PPI-428 CA FF -oh 1.7 -78 15 PPI-429 CA FFV
-oh 2.2 -33 7 PPI-430 CA FFVL -oh 4.1 33 75 PPI-433 CA LVFFA -oh
2.8 27 ND (all D amino acids) PPI-435 t-Boc LVFFA -oh 3.0 -5 ND
PPI-438 CA GFF -oh 1.0 0 ND *ND = not done ** = 33 mol % *** =
h-DDIII(N-Me-Val)DLL(Adp) **** = h-DDII(N-Me-Leu)VEH(Adp)
[0318] Certain compounds shown in Table V (PPI-319, PPI-349,
PPI-350, PPI-368 and PPI-426) also were tested in neurotoxicity
assays such as those described in Example 10. For each compound,
the delay of the appearance of neurotoxicity relative to control
coincided with the delay in the time at which polymerization of AP
began in the nucleation assays. This correlation between the
prevention of formation of neurotoxic AP species and the prevention
of polymerization of A.beta. was consistently observed for all
compounds tested.
[0319] The results shown in Table V demonstrate that at an
effective modulator compound can comprise as few as three A.beta.
amino acids residues (see PPI-394, comprising the amino acid
sequence VFF, which corresponds to A.beta..sub.18-20, and PPI-395,
comprising the amino acid sequence FFA, which corresponds to
A.beta..sub.19-21). The results also demonstrate that a modulator
compound having a modulating group at its carboxy-terminus is
effective at inhibiting A.beta. aggregation (see PPI-408, modified
at its C-terminus with Aic). Still further, the results demonstrate
that the cholyl group, as a modulating group, can be manipulated
while maintaining the inhibitory activity of the compounds (see
PPI-408 and PPI-4 18, both of which comprise the cholyl derivative
Aic). The free amino group of the Aic derivative of cholic acid
represents a position at which a chelation group for .sup.99mTc can
be introduced, e.g., to create a diagnostic agent. Additionally,
the ability to substitute iodotyrosyl for phenylalanine at position
19 or 20 of the A.beta. sequence (see PPI-396 and PPI-397) while
maintaining the ability of the compound to inhibit A.beta.
aggregation indicates that the compound could be labeled with
radioactive iodine, e.g., to create a diagnostic agent, without
loss of the inhibitory activity of the compound.
[0320] Finally, compounds with inhibitory activity were created
using A.beta. derived amino acids but wherein the amino acid
sequence was rearranged or had a substitution with a
non-A.beta.-derived amino acid. Examples of such compounds include
PPI-426, in which the sequence of A.beta..sub.17-21 (LVFFA) has
been rearranged (FFVLA), PPI-372, in which the sequence of
A.beta..sub.16-20 (KLVFF) has been rearranged (FKFVL), and
PPI-388,-389 and -390, in which the sequence of A.beta..sub.17-21
(LVFFA) has been substituted at position 17, 18 or 19,
respectively, with an alanine residue (AVFFA for PPI-388, LAFFA for
PPI-389 and LVAFA for PPI-390). The inhibitory activity of these
compounds indicate that the presence in the compound of an amino
acid sequence directly corresponding to a portion of A.beta. is not
essential for inhibitory activity, but rather suggests that
maintenance of the hydrophobic nature of this core region, by
inclusion of amino acid residues such as phenylalanine, valine,
leucine, regardless of their precise order, can be sufficient for
inhibition of A.beta. aggregation.
EXAMPLE 12
Characterization of .beta.-Amyloid Modulator Compounds Comprising
an Unmodified .beta.-Amyloid Peptide
[0321] To examine the ability of unmodified A.beta. peptides to
modulate aggregation of natural .beta.-AP, a series of AP peptides
having amino- and/or carboxy terminal deletions as compared to
A.beta..sub.1-40, or having internal amino acids deleted (i.e.,
noncontiguous peptides), were prepared. One peptide (PPI-220) had
additional, non-A.beta.-derived amino acid residues at its
amino-terminus. These peptides all had a free amino group at the
amino-terminus and a free carboxylic acid at the carboxy-terminus.
These unmodified peptides were evaluated in assays as described in
Example 7. The results are summarized below in Table VI, wherein
the data is presented as described in Example 7. Compounds
exhibiting at least a 1.5 fold increase in lag time are highlighted
in bold.
6TABLE VI Fold Increase % Reference in Lag Inhibition # A.beta.
Peptide Mole % Time of Plateau PPI-226 A.beta..sub.6-20 100 1.66 76
PPI-227 A.beta..sub.11-25 100 .about.1 47 PPI-228 A.beta..sub.16-30
100 >4.5 100 PPI-229 A.beta..sub.21-35 100 .about.1 .about.0
PPI-230 A.beta..sub.26-40 100 0.8 .about.0 PPI-231 A.beta..sub.1-15
100 .about.1 18 PPI-247 A.beta..sub.1-30, 36-40 (.DELTA.31-35) 100
.about.1 .about.0 PPI-248 A.beta..sub.1-25, 31-40 (.DELTA.26-30)
100 1.58 .about.0 PPI-249 A.beta..sub.1-20, 26-40 (.DELTA.21-25)
100 2.37 .about.0 PPI-250 A.beta..sub.1-15, 21-40 (.DELTA.16-20)
100 1.55 .about.0 PPI-251 A.beta..sub.1-10, 16-40 (.DELTA.11-15)
100 .about.1.2 .about.0 PPI-252 A.beta..sub.1-5, 11-40
(.DELTA.6-10) 100 1.9 33 PPI-253 A.beta..sub.6-40 100 1.9 .about.0
PPI-220 EEVVHHHHQQ-A.beta..sub.16-40 100 >4 100
[0322] The results shown in Table VI demonstrate that limited
portions of the AP sequence can have a significant inhibitory
effect on natural .beta.-AP aggregation even when the peptide is
not modified by a modifying group. Preferred unmodified peptides
are A,.sub.6-20 (PPI-226), A.beta..sub.16-30PPI-228),
A.beta..sub.1-20,26-40 (PPI-249) and EEVVHHHHQQ-A.beta..sub.16-40
(PPI-220), the amino acid sequences of which are shown in SEQ ID
NOs: 4, 14, 15, and 16, respectively.
[0323] Forming part of this disclosure is the appended Sequence
Listing, the contents of which are summarize in the Table
below.
7 SEQ ID NO: Amino Acids Peptide Sequence 1 43 amino acids
A.beta..sub.1-43 2 103 amino acids APP C-terminus 3 43 amino acids
A.beta..sub.1-43 (19, 20 mutated) 4 HDSGYEVHHQKLVFF
A.beta..sub.6-20 5 HQKLVFFA A.beta..sub.14-21 6 HQKLVFF
A.beta..sub.14-20 7 QKLVFFA A.beta..sub.15-21 8 QKLVFF
A.beta..sub.15-20 9 KLVFFA A.beta..sub.16-21 10 KLVFF
A.beta..sub.16-20 11 LVFFA A.beta..sub.17-21 12 LVFF
A.beta..sub.17-20 13 LAFFA A.beta..sub.17-21 (V.sub.18 .fwdarw. A)
14 KLVFFAEDVGSNKGA A.beta..sub.16-30 15 35 amino acids
A.beta..sub.1-20, 26-40 16 35 amino acids EEVVHHHHQQ-.beta.AP.sub.-
16-40 17 AGAAAAGA PrP peptide 18 AILSS amylin peptide 19 VFF
A.beta..sub.18-20 20 FFA A.beta..sub.19-21 21 FFVLA
A.beta..sub.17-21 (scrambled) 22 LVFFK A.beta..sub.17-21 (A.sub.21
.fwdarw. K) 23 LV(IY)FA A.beta..sub.17-21 (F.sub.19 .fwdarw. IY) 24
VFFA A.beta..sub.18-21 25 AVFFA A.beta..sub.17-21 (L.sub.17
.fwdarw. A) 26 LVF(IY)A A.beta..sub.17-21 (F.sub.20 .fwdarw. IY) 27
LVFFAE A.beta..sub.17-22 28 FFVL AA.beta..sub.17-20 (scrambled) 29
FKFVL A.beta..sub.16-20 (scrambled) 30 KLVAF A.beta..sub.16-20
(F.sub.19 .fwdarw. A) 31 KLVFF(.beta.A) A.beta..sub.16-21 (A.sub.21
.fwdarw. .beta.A) 32 LVFF(DA) A.beta..sub.17-21 (A.sub.21 .fwdarw.
DA)
[0324] Equivalents
[0325] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *