CA2214247C - Modulators of amyloid aggregation - Google Patents

Abstract

Compounds that modulate the aggregation of amyloidogenic proteins or peptide s 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 modifyin g 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.-AP s 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.

Description

'.IODULATORS OF Ai"VIYLOID AGGREGATIOi'~
Background of the Invention Alzheimer's disease (AD), first described by the Bavarian psychiatrist Alois Alzheimer n. 1907, is a progressive neurologica: .-lisorder 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 bean estimated to afflict 5 to 1 I percent of the population over age 55 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 far 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 significan.tlv retards the progression ofthe disease. For reviews en AD, see ~eikoe. I>.1. Sc-i. .Amen . November 1991, pp.
68-78; and Yankner. B.A. et al. (1991) N Eng. J :l~eci. 32.5:1849-1857.
It has recently been reported ('Games et al. (1990 Nature 3 13:523-527) that an Alzheimer-type neuropathology has been created in transgenic mice. The transgenic mice express high levels of human mutant amyloid precursor protein and prog:essively develop many of the pathological conditions associated with AD.
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 amylaidoaenic proteins in senile.
or amyloid. plaques. A:nyloid deposits are also present in the walls of cerebral blood vessels of AD patients. The major protein constituent. or amylc>id plaques has been identified as a 4 kilodalton peptide called ~3-amyloid peptide ((3-AP)(Glenner. G.G. and Wona, C.W. (1984) Biocj.~em. Biophvs. Res. Common. 1?0:885-890; Masters, C. et al. (1985) Proc.
Natl. Acad.
Sci. USA 02:4245-4249). Diffuse deposits of ~3-AP are frequently observed in normal adult brains, whereas AD brain tissue is characterized by morn compacted, dense-core ~i-amyloid plaques. (See e.g.. Davies, L. et al. (1988) Neurofo~ 38:1688-1693) These observations suggest that ~i-AP deposition precedes, and contributes tc>, the destruction of neurons that occurs in AD. In further support of a direct pathogenic rule far (3-AP, ~3-amyloid has been shown to 17e toxic to mature neurons. both in culture.and in vivo. Yankner, B.A. et al. (1989) Science 245:417--120: Yankner, B.A. et al. (1990) Proc. aVatl. Acad. Sci. USA
87:9020-9023;
Roher, A.E. et al. (1991) 8iochem. Biophys. Res. Common. 174:572-579; Kowall, N.W. et al.
(1991) Proc. Natl. Acad Sei. LISA 88:724?-7251. Furthermore, patients with hereditary cerebral hemorrhage with amyloidosis-Dutch-type (HCHWA-D), which is characterized by diffuse ~i-amyloid deposits within the cerebral cortex and cerebrovasculature, have been shown to have a point mutation that leads to an amino acid substitution within ~3-AP. Levy, E. et al. (1990) Science 248:1 124-I 126. T his observation demonstrates that a specific alteration of the (3-AP sequence can cause ~3-amyloid to be deposited.
Natural ~i-AP is derived by proteolysis fry ~z a much larger protein called the amy Ioid precursor protein (APP). Kung, J. et al. (I 987) Nature 3?5:733: Goldgaber, D.
et al. (1987) S Science 2J55:877; Robakis, N.K, et al. (1987) Proc. Natl. Acad Sci. USA
84:4190; Tanzi, R.E. er al (198?) Science 235:880. The APP gene maps to chromosome 21, thereby providing an explanation for the ~3-amyloid deposition seen at an early age in individuals with Down's syndrome. which is caused by trisomv of chromosome 21. Mann, D.M. et al. ( 1989) Neuropathol. Appl. Neurnbiol. 15:317; Rumble. B. et a1. ('1989) rV 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 extracellulan 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 :~63 amino acids (APF-S63), 69S amino acids (APP-69S). 714 amino acids (APP-71=~l. 7S1 amino acids (APP-7S1 l or 770 1 S amino acids (APP-770).
Within APP. naturally -occurring ~3 amyIoid peptide begins at an aspartic acid residue at amino acid position 672 of APP-770, Naturally-occurring ~i-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 ~i-.~1P in the blood and cerebrospinal fluid of both AD patients and normal adults is (31-40 ("short ~i"). Seubert, P. et al. (1992) Nature 359:325: Shoji, M. ~~t al. 1;1992) Science 258:126.
However. (31-42 and (31-43 ("long ~i") also are forms in ~-amyloid plaques. Masters. C. et al.
(1985) Proc.
Natl. Acad Sci. L'SA 8?:4245: Miller. D. er al. (19931 Arch. Biochem. Biophys.
301:41: Mori.
H. et al. (1992) J. Biol. Chem. 267:17082. Although the precise molecular mechanism leading to ~3-APP agareaation and deposition is unknown. the process has been likened to that of nucleation-dependent polvmerizations. such as protein crystallization, microtubule formation and actin polymerization. See e.g.. Jarrett. J.'T. and Lansbury, P.T. (1993) Cell 73:1 OSS-lOSB. In such processes, polymerization of monomer components does not occur until nucleus formation. Thus. these processes are characterized by a lag time before aggregation occt;rs. followed by rapid polymerization after nucleation.
r~ucleat:on can be accelerated by the addition of a "seed" or preformed nucleus, which results in rapid polymerization. The long ~3 forms of (3-AP have been shown to act as seeds.
thereby accelerating polymerization of both long and short ~3-AP forms. Jarrett, J.T.
et al. ( 1993) Biochemistry 32:4693.
3S In one study. in which amino acid substitutions were made in ~i-AP, two mutant ~3 peptides were reported to interfere with polymerization of non-mutated (3-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) (3 l amyloid peptides were used to see this effect and the :xtutant peptides were reported t~ be unsuitable for use in vivo. Hilbich, C. et a~. ( 1992), supra.
Summary of the Invention This invention pertains to compounds, and pharmaceutical compositions thereof, that can modulate the aggregation of amyloidagenic proteins and peptides. in parncular compounds that can modulate the aggregation of natural p3 amyloid peptides (j3-AP) and inhibit the neurotoxicity of natural (3-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 arnyloid 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 w~ah the natural amvloidogenic proteins or peptides. The amyloicaogenic protein. or peptide f'zagment thereof. :an be. for example. selected from the group c;onsistina of transthvr~,tin ('ITR), prion protein (PrP). islet amyIoid polypeptide (IAPP), atrial natriuretic factor (A~F). kappa light chain. lambda Iight chain, amyloid A. procalcitonin, cystatin C', ~i2 microglobulin. ApoA-I.
gelsolin, procalcitonin, c~lcitonin, fibrinogen and lysozyme.
In the most preferred embodiment of tlne invention.. the compound modulates the aggregation of natural ~3-AP. The invention provides a ~3-amyloid peptide compound comprising a formula:
.,iAn Xaa' wherein Xaa is a (3-amyloid peptide having an amino-terminal amino acid residue corresponding to position 668 of (3-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 ~i-amyloid peptide of the compound such that the compound inhibits aggregation of natural ~i-amyloid peptides 4vhen contacted with the natural (3-amyloid peptides. and n is an integer selected such that the compound inhibits aggregation of natural ~i-amyloid peptides when contacted with the natural (3-amyloid peptides.
In one embodiment, at least one A group is attached directly or indirectly to the amino terminus of the ~i-amyloid peptide of the Compound. In another embodiment. at least one A
group is attached directly or indirectly to the carboxy terminus of the (3-amyloid peptide of the compound. In vet another embodiment. at least one A group is attached directly or indirectly to a side chain of at least one amino acid residuf: of the ~i-amyloid peptide of the compound.

The~nvention also provides a ø-arnyloid modulator compound comprising an Aø
aggregation core domain (ACD) coupled directly or indirectly to at least one modifying group (MG) such that the compound modulates the ag ;regation or inhibits the neurotoxicity of nattual ø-amyloid peptides when contacted w:.:1 the natural ø-amvIoid peptides.
Preferably, the Aø aggregation core domain is modeled after a subregion of natural ø-amyloid peptide between 3 and I O amino acids in length..
The invention also provides ø-amyloid modulator compound comprising a formula:
~n ( Y-Xaai-~'~aa~-Xaa;-Z
IO
wherein Xaa~, Xaa~and Xaa~ are each amino acid structures and at least two of Xaa~, Xaa~ and Xaaz are. independently. selected frorn the group consisting of a ieucine structure. a phenvlalanine structu:e and a valine structure;
Y, which may or may not be present. is a peptidic structure having the 1 S formula (Xaa)a. wherein Xaa is any amino acid structure and :t is an integer from I to I S:
Z. which may or may not be present. is a peptidic structure having the formula (Xaa)b, wherein Xaa is any amino acid structure and b is an integer from 1 to 1 S; and A is a ;nodifvin~ group attached directly c:~r indirectly to the compound and n is an integer;
20 Xaal, Xaa,. Xaa;. Y. Z, A and n being selected such that the comvound modulates the aggregation or inhibits the neurotoxicity of natural ø-amv(oid peptides when contacted with the natural ø-amyloid peptides. In a preferred embodiment. Xaa, and Xaa, are each phenylalanine structures. In another preferred r~mbc~dime;nt .'~aa~ and Xaa;
are each phenvlalanine structures.
25 The invention further provides a ø-amyloid modulator compound comprising a formula:
~n ( Y-Xaai-Xaa~-Xaa;-Xaa4-Z'~~
30 wherein Xaal and Xaa3 are amino acid structures;
Xaa~ is a valine structure;
Xaa4 is a phenylalanine structure;
Y. wrich may or may not be present, is a peptidic structure having th4 formula (Xaa)a, wherein Xaa is any amino acid structure a.nd a is an integer from I to 1 ~;
3S Z. which may or may not be present. is a peptidic structure having the formula (Xaa)b, wherein Xaa is any amino acid structure and b is an integer from I to 1 ~: and A ss a modifying group attached directly or indirectly to the compound and n is an integer;
Xaa~, Xaa3, Y, Z, A and n being selected such that the compound modulates the aggregatl~n or inhibits the neurotoxicitv of na;°:ral ~3-amyloid peptides when contacted with the natural ~i-amyloid peptides. In a preferred embodiment, Xaaa is a leucine structure and Xaa3 is phenylalanine structure.
The invention still ftuuthher provicie~ a compound comprising the formula:
A-Xaa l -Xaa~-Xaa;-Xaa~-Xaa~-Xaa6-Xaa7-;Xaag-B
wherein Xaa 1 is a histidine structure;
Xaa2 is a glutamine structure;
Xaa~ is a lysine structure;
Xaa=1 is a leucine struc;ure:
f ~ Xaa~ is a vaIine structure;
Xaa6 is a phenvlalanine structure;
Xaa f is a phenylalanine .structure;
Xaa8 is an alanine structtue;
A and Ei are modifying groups attached direetls~ or indirectly to the amino terminus and carbory terminus, respectively, of the compound;
and wherein Xaal-Xaa~-Xaa;. Xaa1-Xaa~ or X<3a2 may or may not be present:
Xaag may or may not be present: and at least one of A and B is present The invention still further provides a ~3-amyloid modulator compound comprising a 2~ modifying group attachea directly or indirectly to a peptidic structure.
wherein the peptidic structure comprises amine acid structures having an amino acid sequence selected from the group consisting of His-Gln-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO: ~). His-Gln-Lys-Leu-Val-Phe-Phe (SEQ ID NO: 6). Gln-Lys-Leu-Val-Phe-1'he-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-Ph:,-Phe-Ala (SEQ ID NO: 11). Leu-Val-Phe-Phe (SEQ ID NO: 12). Leu-AIa-Phe-Phe-Ala (SEQ ID NO: 13), Val-Phe-Phe (SEQ ID NO:
? 9), Phe-Phe-Ala (SEQ ID NO: 20), Phe-Phe-Val-Leu-Ala e~SEQ ID NO: 21 ), Leu-Val-Phe-Phe-Lys (SEQ ID NO: 2?), Leu-Val-Iodotvrosine-Phe-Ala (SEQ ID NO: 23). Val-Phe-Phe-Ala (SEQ ID NO: 24), Ala-Val-Phe-Phe-Ala (SEQ ID NO: '.:.'S), Leu-Val-Phe-Iodotyrosine-Ala (SEQ ID NO: 26), Leu-Val-Phe-Phe-Ala-Glu (SEQ iD NO: ''7), Phe-Phe-Val-Leu (SEQ ID
NO: 28), Phe-Lys-Phe-Va-Leu (SEQ ID NO: 29), Lys-Leu-Val-Ala-Phe (SEQ ID NO:
3u), Lys-Leu-Val-Phe-Phe-~3A13 (SEQ ID NO: 31 ) and Leu-Val-Phe-Phe-~Ala (SEQ ID
NO: 32).
In the compounds of the invention comprisine a modifying group. preferably the modifying group comprises a cyclic. heterocyclic or polycyclic group.
Preferred modifying fi groups contains a cis-decalin group. such as a cholanoyl structure. Preferred modifying groups include a cholyl group, a biotin-containing group. a diethylene-triaminepentaacetyl group, a (-)-menthoxyacetyl group, a fluorescein-containing group or an N-acetylneuraminyl group.
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.
The invention also provides a ø-amyloid modulator which inhibits aggregation of natural ø-amyloid peptides when contacted with a molar excess amount of natural ø-amyloid peptides.
The invention also provides a ~i-amyloid peptide compound comprising an amino acid sequence having at least one amino acid deletion compared to øAP~-;9, such that the compound inhibits aggregation of natural ø-amyloid peptides when contacted with t:le natural ~i-amyloid peptides. In one embodiment. the compound has at least one internal amino acid 1 ~ deleted compared to øAP ~ _;9. In another embodiment. the compound has at least one N-terminal amino acid deleted compared to øAP a _;a. In yet another embodiment, the compound has at least one C-terminal amino acid deleted compared to øAP~-;9.
Preferred compounds include øAPo--,p (SEQ ID NO: 1~), øAPIS_;,a (SEQ ID NO: 14).
øAP1_?0. 26-.~0 (SEQ ID NO: 1 ~) and EEV VHHHHQQ-øAP 16..,~~ (SEQ ID NO: 16).
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 'or the diagnosis or treatment of an amvloidosenic disease.
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 ø-amvloid peptides. comprising contacting the natural ø-amyloid peptides with a compound of the invention sucr. that aggrega~c.ion oaf the natural ø-amyloid peptides is inhibited. The invention also provides a method for inhibiting neurotoxicity of natural ø-amyloid peptides. comprising contacting the natural ø-amyloid peptides with a compound of the invention such that neurotoxicity of the natural ø-amyloid peptides is inhibited.
In another embodiment. the invention provides a method for detecting the presence or absence of natural ø-amyloid peptides in a biological sample. comprising contacting a biological sample with a compound of the invention and detecting the compound bound to natural ø-amvloid peptides to thereby detect the presence or absence of natural ø-amyloid ~5 peptides in the biological sample. In one embodiment. the ø-amyloid modulator compound and the biological sample are contacted in vitro. In another embodiment, the ø-amyloid modulator compound is contacted with the biological sample by administering the ø-amyloid modulator compound to a subject. For in vivo administration, preferably the compound is labeled with radioactive technetium or radioactive iodine.

In another embodiment, the invention provides a method for detecting natural ~i-amyloid peptides to facilitate diagnosis of a (:i-amyloidogenic disease.
comprising contacting a biological sample with a compound of the invention and detecting the compound bound to natural ~3-amyloid peptides to facilitate diagnosis of a (3-amyloidogenic disease. In one embodiment, the (i-amyloid modulator compounel and the biological sample are contacted in ultra. In another embodiment, the ~3-amyloid modulator compound is contacted with the biological sample by administering the ~i-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.
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 amvloidosis. The method can be used to ~reat disorders is selected. for example, from the Group Consisting car farniiial amyloid poiynetiropathv I ~ (Portuguese. .lapanese and Swedish types). familial amvloid cardiomvopathy (Danish type).
isolated cardiac amyloid, systemic senile amyloidosis. scrapie. bovine spongiform encephalopathy. Creutzfeldt-Jakob disease. Gerstmann-~traussler-Scheinker syndrome, adult onset diabetes. insulinoma, isolated atrial amy loidosis. idiopathic (primary) amvloidosis, myeloma or macroglobulinemia-associated amyloidosis, primary localized cutaneous nodular amyloidosis associated with Sjogren's syndrome. reactive (secondary) arnyloidosis, familial Mediterranean Fever and familial amyloid nephropathy with urticaria and deafness (MuckIe-Wells syndrome), hereditan~ cerebral hemorrhalte with amyloidosis of Icelandic type, amyloidosis associated with long term hemodialysis, hereditan~ non-neuropathic systemic amyloidosis (familial arnvloid poivneurapathv III). familial amyIoidosis of Finnish 23 ry~pe, amvloidosis associated with medullary carcinoma c~f the thyroid.
fibrinogen-associated hereditary renal amyloidesis and lysozyme-associated hereditan~ systemic amyIoidosis.
In a preferred embodiment, the invention provides a method for treating a subject for a disorder associated with j3-amyioidosis. 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 j3-amyloidosis.
Preferably the disorder is Alzheimer's disease.
In yet another embodiment. the invention provides a method for treating a subject for a disorder associated with ~i-amyioidosis. comprising administering to the subject a recombinant expression vector en;,oding 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 ~3-amyloidosis. Preferably, the disorder is Alzheimer's disease.

Brief Description of the Drawin,-g Figure 1 is a graphic representation of the turbidity of a ~3-AP j~p solution, as measured by optical density at 400 nm, either in the absence of a ~3-amyloid modulator or in the presence ofthe ~i-amyloid modulator N-biotinyl-~3AP]~p ( 1 %, or ~'%).
Figure 2 is a schematic representation of compounds which can be used to modify a (3-AP or an A~i aggregation core domain to form a ~3-amyloid modulator of the invention.
Figure 3 is a graphic representation of the toxicity of A~i ~ .,;tp aggregates. but not A~3~~p monomers, to cultured neuronal cells.
Figure ~ is a graphic representation o.f the aggregation of A~il~~ in the presence of an equimolar amount of cholvl-A~6_,p (panel A}, a --?-fold molar excess of cholyl-A~3~-,p (panel B) or a ~6-fold molar excess of cholvl-A~36_~~ (panel C) and the corresponding toxicity of the aggregates of panels A, B and G to cultured neuronal cells (panels D. E
and F, respectively).
l~
Detailed Description of the Invention This invention pertains to compounds, and pharmaceutical compositions thereof, that can modulate the aggregation of amyoidogenic proteins and peptides. in particular compounds that can modulate the aggregation of natural ~3 amyloid peptides ((3-AP) and inhibit the neurotoxicity of natural ~-APs. A compound of the invention that modulates aggregation of natural (3-AP, referred to herein interchangeably as a ~i amyloid modulator compound. a ~ amyloid modulator or simply a modulatoc~. alters the aggregation of natural ~3-.AP when the modulator is contacted with natural ~3-AP. 'thus. a compound of the invention acts to alter the natural aggregation process or rate far ~~-AP. thereby disrupting this process.
2~ Preferably. the compounds inhibit ~3-AP aggregation. Furthermore. the invention provides subregions of the ~i amyloid peptide that are sufficient. ~.vhen appropriately modified as described herein. to alter (and preferably inhibit) aggregation of natural (3 amyloid peptides when contacted with the natural ø amyloid peptides. In particular. preferred modulator compounds of the invention are comprised of a modified form of an A~i aggregation core domain. modeled after the aforementioned A~i subregion (as described further below). which is sufficient to alter (and preferably inhibit) the natural aggregation process or rate for ~i-AP.
This A~i aggregation core domain can comprises as few as three amino acid residues (or dernative, analogues or mimetics thereof) Moreover. s~~hile the amino acid sequence of the A~3 aggregation core domain can directly correspond to an amino acid sequence found in natural (3-AP, it is not essential that the amino acid sequence directly correspond to a ~i-AP
sequence. Rather, amino acid residues derived from a preferred subregian of /3-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}.

C) The ~ amyloid modulator compounds of the invention can be selected based upon their ability to inhibit the aggregation of natural ~-AP ~~ra virra andJor inhibit the neurotoxicit5~
of natural ~i-AP fibrils for cultured cells (using assays described herein).
Accordingly, the preferred rriLdulator compounds inhibit the aggro:~ation of natural (3 :AP
and/or inhibit the neurotoxicity of natural ~i-AP. However. modulator compounds selected based on one or both of these properties may have additional properties a viv~o that may be beneficial in the treatment of amyloidosis. For example, the modulator compound may interfere with processing of natural ~3-AP (either by direct or indirect protease inhibition) or by modulation of processes that produce toxic (3-AP, or other APP fragments, in vivo.
Alternatively, modulator compounds may be selected based on these latter properties. rather than inhibition of A~i aggregation in vitro. Moreover. modulator compounds of the invention that are selected based upon their interaction with natural ~3-AP also may interact with APP or with other APP fragments.
As used herein. a "modulator" of ~3-amyloid aggregation is intended to refer to an 1 ~ agent that. when contacted with natural ~i amyloid peptides. alters the aggregation of the natural ø amyioid peptides. The term "aggregation of ~3 amyloid peptides"
refers to a process whereby the peptides associate with each other to form ~ multimeric. largely insoluble complex. The term "aggregation" further is intended to encompass ~i amyloid fibril formation and also encompasses (3-amyloid plaques.
The terms "natural p-amyloid peptide", "natural (3-AP"' and "natural Ap peptide", used interchangeably herein. are intended to encompass naturally occurring proteolytic cleavage products of the ~i amvloid precursor protein (APP) which are involved in (3-AP
aggregation and ~i-amyloidosis. These natural peptides include ~i-arz~vloid peptides having 39-43 amino acids (l. e.. A(3 ~-;g. A~3 ~ ~o, A~31 _:~ ~ . A~3 a _a~ and A.~i 1 _4;.7.
T'he amino-terminal amino acid 2~ residue of natural ~i-AP corresponds to the aspartic acid residue at position 672 of the 770 amino acid residue form of the amvloid precursor protein ("AfP-77C"). 'The 4~
amino acid long form of natural ~3-AP has the amina acid sequence DAEFRHDSGYEVHHQKLV FFAEDVGSNKGAIIGLMVGGVVIAT
(also shown in SEQ ID NO: 1 j, whereas the shorter forms have 1-4 amino acid residues deleted from the carboxy-tetmtinal end. The amino acid sequence of APP-770 from position 672 (l. e., the amino-terminus of natural (3-AP) to its C-terminal end ( 103 amino acids) is shown in SEQ ID NO: 2. The preferred fowl of natural ~?~-AP for use in the aggregation assays described herein is A~ y..4o.
In the presence of a modulator of the invention. aggregation of natural ~i amyloid 3~ peptides is "altered" or "modulated". 'The various forms of the term "alteration" or "modulation" are intended to encompass both inhibition of" ~i-AP aggregation and promotion of (3-AP aggregation. Aggregation of natural ~3-AP is "inhibited" in the presence of the modulator when there is a decrease in the amount and.%or rate of ~3-AP
aggregation as compared to the amount andlor rate of ~-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 ~i-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 aggregatio:~ (i.e., total amount of aggregation;), i:sing an aggregation assay as described in the Examples. In various embodiments, a madulator of the invention increases the lag time of aggregation at least 1.2-fold, I.5-fold, I.g-fold. ?-fold, *'_.5-fold, 3-fold.

4-fold or ~-fold. In va~~ious other embodiments, a modulator of the invention inhibits the plateau level of aggregation at least 10%, 20%, 30%, 40 ° o, fiU %, 7~ ~% ar 1 UO %.
A modulator which inhibits ~3-AP aggregation i;an "inhibitory modulator compound") IO can be used to prevent or delay the anset caf ~3-amyloid deposition.
Moreover, as demonstrated in Example I U, inhibitory rnodulatar compounds of the invention inhibit the formation and/or activity of neurotoxic aggregates of natural A(3 peptide (i.e., the inhibitory compounds can be used to inhibit the neurotoxicity of (~-AP) Still further, also as demonstrated in Example I0. the inhibitory c.ompaunds of the invention can be used to 1 ~ reduce the neurotoxicity of preformed ~3-AI' ag<gregates. indicating that the inhibitory modulators can either bind to preformed A~3 fibrils ar soluble aggregate and modulate their inherent neurotoxicity or that the madulators can perturb the equilibrium between monome:ic and aggregated forms of ~3-AP in favor of the man-neurclcoxic form.
Alternatively. in another embodiment. a modulator campound of the invention 20 promotes the aggregation of natural .~(~ peptides. The v ariaus forms of the term "promotion"
refer to an increase in the amount and/or rate of ~'~-AP aggregation in the presence of the modulator. as compared to the amount ancl/ar rate of ~i-.-'~ P a<~.:regation in the absence of the modulator. Such a compound which promotes .4~_s aggre<__>,.ati~>n is referred to as a stimulatory modulator compound. Stimulatorw rnadulator compourn~s may be useful for sequestering (3-2~ amyloid peptides. for example in a biological oc>mpartm~~nt where aggregation of (3-AP may not be deleterious to thereby deplete ~3-AP from a biola~:ical ~:c~mpartment where aggregation of (3-AP is deleterious. Moreover, stimulatory modulator compounds can be used to promote A~3 aggregation in in vitro aggregation assays (e.g.. assays such as those described in the Examples), for example in screening assays far test compounds that can then inhibit or 30 reverse this A~i aggregation (i.e., a stimulatory madulatc~r compoun;~ can act as a "seed'° to promote the formation of A(3 aggregates).
In a preferred embodiment, the modulators of the invention are capable of altering (3-AP aggregation when contacted with a molar excess amount of natural ~3-AP. A
"molar excess amount of natural ~i-AP" refers to a concentration of natural ~3-AP, in moles, that is 35 greater than the concentration. in moles, of the modulator. For example, if the modulator and ~3-AP are both present at a concentration of 1 ~lyz. they are said to be "equimolar", whereas if the modulator is present at a concentration of 1 ~M and the ~i-AP is present at a concentration of ~ uM. the (3-AP is said to be present at a ~-fold molar excess amount compared to the modulator. in preferred embodiments. a modulator of tl"m invention is effective at altering natural ~i-AP aggregation when the natural (3-AP is present at at least a 2-fold. 3-fold or ~-fold molar excess compared to the concentration of the modulator. In other embodiments, the modulator is effective at altering ~3-_AP aggregation when the natural ~i-AP
is present at at least a I O-fold, 20-fold, 33-fold, 50-fold, 1 ~'(~-fold, 600-fold or I 000-fold molar excess compared to the concentration of the modulator.
Various additional aspects of the modulators of the invennon, and the uses thereof, are described in further detail in the following subsections.
I. Modulator Compounds In one embodiment. a modulator of the invention comprises a (3-amyloid peptide compound comprising the formula:
~n ( Xaa~' wherein Xaa is a ~3-amvloid peptide. A is a modulating group attached directly or 16 indirectly to the ~3-amyloid peptide of the compound such that the compound inhibits aggregation of natural p-amyloid peptides when contacted wish the natural ~-amyloid peptides. and n is an integer selected such that the compound inhibits aggregation of natural ~3-amyloid peptides when contacted with the natural ~3-amvloid peptides.
Preferably. ~i-amyloid peptide of tl~ee compound has an amino-terminal amino acid residue corresponding to position 668 of ~-anrylaid precursor protein-770 (APP-770) or to a residue carboxy-terminal to position 668 of APP-77(l. T'he amino acid sequence of APP-770 from position 668 to position 770 (i.c:~.. the carboxv terminus) is show below and in SEQ ID
NO: ~:
EVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKCiAIIGLMVGGVVIATVIVITL
VMLKKKQYTSIHHGV VEVDAA~°TPEEFtHLSI~:MQQNGYENPTYKFFEQMQN
More preferably, the amino-terminal amino acid residue of the ~3-amyloid peptide corr~;sponds to position 672 of APP-7 7 0 (position 6 of tlne amino acid sequence of SEQ ID
NO: 2) or to a residue carboxy-terminal to position 672 caf AF'P-770. Although the ~i-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 4~~ amino acids in length and even more preferably between 10 and 26 amino acid residues in length.
As used herein, the term "~S amyloid pepti,ie", 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 T
ability of the peptide to alter natural ~3-AP aggregatior:. i'~Ioreover.
paricular ~.mino acid substitutions may further contribute to the ability of the peptide to alter natural (3-AP
aggregation and/or may confer additional beneficial properties on the peptide (e.g., increased solubility, reduced association with other amyla:d proteins, etc.). For example. substitution of hydrophobic amino acid residues for the two phenylalanine residues at positions 19 and 20 of natural ~i-AP (positions 19 and 20 of the amino acid sequence shower in SEQ
ID NO: 1 ) may furt:'~er contribute to the ability of the peptide to alter ~3-.AP
aggregation (see Hilbich, C.
(1992) J. .~lol. Biol. 228:460-473). Thus, in one embodiment. the ~3-AP of the compound consists of the amino acid sequence shown below and iza SEQ ID N0: 3:
DAEFRHDSGYEVHHQI~L~'(Xaai9)(xaa~o):~1~:DVGSNitGAIIGL~vGGVVIAT
1 or an amino-terminal or carboxv-terminal deletion ther~of'~, wherein Xaa is a hydrophobic amino acid. Examples of hydrophobic amino acids arc ~:soleucire. leueine.
threonine. serine.
alanine. valine or glycine. Preferablr-, FigF'~p is su°nsti4uted with T~9T~p or G~9I~p.
Other suitable amino acid substitutions include replacement of amino acids in the human peptide with the corresponding amino acids of the rodent ~-AP peptide.
The three amino acid residues that differ between human and rat ~'b-.AP are at positions 5. 10 and 13 of the amino acid sequence shown in SEQ ID NOs: 1 and >. A human p-AP having the human to rodent substitutions Args to Gly, Tyrlp Fo Phe and Hisl~ tc:~ Arg has been shown to retain the properties of the human peptide (see -Fraser. P.E. er ul. (1992) Biochemisrry 31:10716-10723; and Hilbich. _C. et al. ( 1991 ) Eur. J Biochcam. 2:(t 1:61-69).
Accordingly. a human ~i-.4P having rodent ~3-AP a.a. substitutions is suitablL for use in a modulator of the invention.
Other possible ~i-AP amino acid substitutions are: described in Hiibich. C. et of.
(1991) J ~4Tol. Biol. ?18:149-163: arid Hilbicla. C. (19~~'1 J. aloC. Bio!.
??8:460-473.
Moreover, amino acid substitutions that affect the ability of pJ-AP t~:
associate with other proteins can be introduced. For example. one or more a:mincr acid substitutions that reduce the ability of ~i-AP to associate with the serpin enzyme complex (SEC) receptor, al-antichymotrypsin (ACT) and/or apolipoprotein E (.Apal".~) can be introduced. A
preferred substitution. for reducing binding to the SEC receptor is L,;~I~'I;5 to A;;tA,;s (at positions 34 and 35 ofthe amino acid sequences shawra in SEQ ID "COs: ! and 3). A preferred substitution for reducing binding to ACT is S~ to Ag (at position 8 of the amino acid sequences shown in SEQ ID NOs: 1 and 3).
Alternative to ~i-AP amino acid substitutions described herein or known in the art. a modulator composed. at least in part. or an amino acid-substituted ~i amyloid peptide can be prepared by standard techniques and tested for the ability to alter ~i-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.~,>., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, g,lutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ~3-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phor,~ylalanine, trvptophan, histidine).
Accordingly, a modulator composed of a ~ amyloid peptide having an amino acid sequence that is mutated from that of the wild-type sequence in f~,.l?P-7'70 yet which still retains the ability to alter natural ~3 :aP aggregation is within the scope of the invention.
As used herein, the term "(~ amyloid peptide" is further intended to include peptide analogues or peptide derivatives or peptidomimetics that retain the abiliy to alter natural (3-AP aggregation as described herein. For example, a ~i amyloid peptide of a modulator of the invention may oe modified to increase its stability. bioa~;~ailai~ility, solubility, etc. The terms 1 ~ "peptide analogue"', "peptide derivative" and "peptidomimeti~:" as used herein are intended to include molecules which mimic the chemical structure or a peptide and re:ain the functional properties of the peptide. Approaches to designing= peptide analogs are I:now~n in the art. For example, see Farmer, P.S. in Drug Design t:E.J. Ariens. ed.) Academic Press.
New ~C'ork, 1980, vol. 10, pp. 119-143; Ball. J.B. and Alewood, P.I~ . ( 190) J. Mol.
.Recogr~irion 3:>j;
Morgan. 8.A. and Gainor, J. A. (1989) tlnn. Rep. ~l~Ied :hem. 24:243; and Freidinger, R.M.
(1989) Trends Pharmacol. Sci. 10:2 i0. 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 260:19 7-192). peptides with methylated amide linkages and "retro-inverso" peptides C,see I.S. Patent ~;o. 4.~?'?,?~? by Sisto). Peptide analogues, peptide derivatives and pepudomimetic are described in further detail below with regard to compounds comprising an A~ aggregation core domain.
In a modulator of the invention having the i~ormula shown above, a modulating group ("A") is attached directly or indirectly to the ~3-amvloid 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 or A~i derived peptidic structure).
For example, the modulating group can be directly attached by covalent coupling to the (3-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 ~i-amyloid peptide of the modulator. accordingly, the modulator can 3~ comprise a compound having a formula:
A -~ N --( Xaa ) 1-1.
Alternatively, in another embodiment of the invention. the modulating group is attached to the carboxy-terminus of the ~i-amylaid peptide of the modulator. Accordingly, the modulator can comprise a compound having a formula:
O
n ( Xaa ) C-A
In yet another embodiment, the modulating group is attached to the side chain of at least one amino acid residues of the ~i-amyloid peptide of the compound (e.g., through the epsilon amino group or" a lysyl residue(s), through the carboxyl lzroup of an aspaTtic acid residues) or a glutamic acid residue(s), through. a hydraxy group of a tyrosvl residue(s), a serine residues) or a threonine residues) or other suitable reactive group an an amino acid side chair..l.
The modulating group is selected such that the ~,ampaund inhibits aggregation of na:-ural Q-amyloid pc;ptides when contacted with the natural ~i-amyloid peptides.
Accordinolv. since the ~i-.~P peptide of the compound is rnoditied from its natural state, the I ~ modulating group "A" as used herein is not intended to include hydrogen.
In ~. preferred embodiment. the modulating Group is a biotin compound of the formula:
W, y X
O
I
?l ;
wherein X1-X; are each independently selected from the Group consisting of S.
O and NR~.
wherein R, is hydrogen, or an aryl. lower alkyl, alkenyl or alkynyl moiety; ~' is =O or NR~; Ri is a lower alkylenyl moiety and Y is a direct band or a spacer molecule selected for its abilin~ to react with a target group on a ~3-AP. At leas: one of X i-X; or W is an NR~
group.
The term "aryl" is intended to include aromatic moieties containing substituted or unsubstituted ring(s), e.g., benzyl, napthyl, etc. Other mare complex fused ring moieties also are intended to be included.
The term "lower alkyl or alkylenyl moiety" refers to a saturated. straight or branched chain (or combination thereof) hydrocarbon containing I 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 I to 3 carbon atoms. Preferably, R~ contains 1 to 3 carbon atoms.
Preferably, R~
contains 4 carbon atoms.

The-spacer molecule (I~ 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 a-amino group at the amino-terminus of a (3-AP). Thus, in a preferred embodiment, the biotin compound modifies the amino-terminus of a ~'~-amyloid peptide.
Additional suitable modulating groups may include other cyclic and heterocyclic compounds and other compounds having similar steric "b uk", Non-limiting examples of compounds which car. be used to modify a ~3-AP are shaven schematically in Figure ?. and include N-aeetylneuraminie acid, cholic acid. trans-4-cotininecarboxylic acid, ?-imino-1-imidazolidineacetic acid, (,5~-(-)-indoline-2-carboxylic acid, (-;f-menthoxyacetic acid. 2-norbomaneacetic acid, Y-oxo-5-acenaphthenebutyric acid, w;-)-';~y-oxo-4-thiazolidinecartioxylic acid. tetrahydro-3-furoic acid. 2-iminobiotin-~'~-hydroxvsuccinimide ester, diethylenetriaminepentaacetic dianhydride, 4-morpholinecarbonyl chloride. 2-thiopheneacetyl chloride, 2-thiophenesulfonyl chloride. ~-(and 6-)-carboxyfluorescein (succinimidyl ester), fluoreseein isothiocyanate. and acetic acid (or derivative: thereotl.
l~ Suitable modulating groups are described further in substcrion II below.
In a modulator of the invention. a single modulating group may be attached to a ~3-amyloid peptide (e.g., n=1 in the formula shown above) or multiple modulating 'roups may be attached to the peptide. The number of modulating groups is selected such that the compound inhibits aggregation of natural ~i-amyloid peptides when contacted with the natural (3-amyloid peptides. However, n preferably is an integer between 1 and 60.
more pref~rablv between 1 and 30 and even more preferably between l and 10 or l and ~..
In another embodiment. a ~-amvloid modulator compound of the invention comprises an A(3 aggregation core domain (abbreviated as ACD) coupled directly or indirectly to a modifying group such that the compound modulates the aGxaregation or inhibits the neurotoxicity of natural ~i-amyloid peptides when contacted with the natural (3-arryloid peptides. As used herein, an "A~3 aggregation core domain" is intended to refer to a structure that is modeled after a subregion of a natural ~i-amvloid peptide which is sufficient to modulate aggregation of natural (3-APs when this subregion ofthe natural ~i-AP
is appropriately modified as described herein (e.~., modified at the amino-terminus). The term "subregion of a natural ~i-amyloid peptide" is intended to include air_ino-terminal and/or carboxy-terminal deletions of natural ~i-AP. The term "subregion of natural ~i-AP" is not intended to include full-length natural (3 :aP (i.e.. "subregion" does not include A~il_;9=
A~~-ao~ A~1-~n APt-~2 ~d A~31-a~).
Although not intending to be limited by mechanism, the ACV of the modulators of the invention is thought to confer a specific targeting function on the compound that allaws the compound to recognize and specifically interact with natural (3-AP.
Preferably, the ACD
is modeled after a subregion of natural ~3-AP that is Iess than 15 amino acids in length and more preferably is between 3-10 amino acids in length. In various embodiments.
the ACD is 1 f~
modeled afee~r a subregion of ~i-AP that is 1 (), 9, 8, '~, 6, ~ , _~ or ~
amino acids in length. In one embodiment, the subregion of (J-AP upon which the ACD is modeled is an internal or carboxy-terminal region of ~i-AP (l.c., downstream of the amino-terminus at amino acid position 1 ). L: another embodiment, the ACD is r~ udeled after a subregion of ~i-AP that is hydrophobic. In certain specific embodiments, the term A~i aggregation core domain specifically excludes (3-AP subregians corresponding to amino acid positions 1-15 (A~i~_r5), 6-20 (A~36.20) and 16-40 (Al 1 ~oO
An A(3 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 (3-AP.
Alternatively. an A~i aggregation core domain can be modeled after the natural A(3 peptide region but may be comprised of a peptide analogue, peptide derivative or peptidomimetic compound. or other similar compounds which mimics the stz-ucture and function of the natural peptide. Accordingly, a5 used herein. arl "A~3 as<~regation core domain" is intended to include peptides, peptide analogues, peptide derivatives and peptidomimetic compounds 1 ~ which. when appropriately modified. retain the aggregation modulatorv activity of the modified natural A~3 peptide subregian. Such structures that are designed based upon the amino acid sequence are referred to herein as "A~i 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 'r'ork. 1980, vol. 10, pp. 119-143; Ball. J.B. and Alewood. P.F. (1990) J ,~t'nl. Recognition ~:~d:
Morgan, B.A.
and Gainor. J.A. (1989)Ann. Rep. Nled C:hem. '?4:243; and Freidinger, R.M.
(1989) Trends Pharmacol. Sci. 10:270. See also Sawyer. T.K. (1995) "Peptidomimetic Design and Chemical Approaches to Peptide lvietabolisrn" in 'havlor, :"rr.D. and Amidon.
G.L. (eds.) Peptide-Based Drug Design: Controlling Transport and 'vtetaboiism. Chapter ~?:
Smith. A.B.
2~ 3rd. et al. (1990 J. .gym. Chem. Soc. 1?:l I 11.>-111'_'3: Smith. :~.B.
3rd. et al. (1994) J. .Am.
Chem. Soc. 116:9947-9962; and Hirschman. R.. ct al. {I'>'~:_>) J .-lm. Chem.
Soc. 113:I2»0-12568.
As used herein, a "derivative" of a compound X (c.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 pep:ide backbone. or the amino- or carboxy-terminus has been derivatized (e.g., peptidic compounds with methylated amide linkages). ~s used herein an "analogue" of a compound X refers to a compound which retains chemical structures of X
necessary for functional activity of X vet 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-accurrirtg 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 1 '' compounds in which the peptide backbone is substituted with one or more benzodiazepine molecules (see e.g., James, G.L. er al. (1993) Science 260:1937-1942), peptides in which aII
L-amino acids are substituted with the corresponding D-amino acids and "retie-inverse"
peptides (sPe U.S. Patent No. 4,5?2,?52 by Sistc'. described further below.
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 static 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 modif-tcations of the amide nitrogen, the cc-carbon. amide carbonyl. complete replacement of the amide bond. extensions. deletions or backbone crosslinks. Several peptide backbone modifications are known.. including y[CH,S], ~r [CH~NH), ~r[CSNH,), W[I'iHCOj, ~r[COCH~j, and ~r[(E) or l Z) CH=CH]. In the nomenclature used above, yr 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 mare benzodiazfi.pine molecules (see E.g.. James, G.L. et al. (1993) Science 2?0:193'"-194?) Other possible modifications include an N-alkyl (or aryl) substitution (y~[CONR]), backbone crosslinkinQ to construct lactams and other cyclic structures.
substitution of all D-amino acids for all L-amino acids within the compound ("'inverse" compounds) or retro-inverso amino acid incorporation (~[NHCO]). By "inverse" is meant replacing L-amino acids of a sequence with D-2_mino acids, and by "ratio-inverse" or "enantio-ratio" is meant reversing the sequence of the amino acids (°'retro") and replacing the L-amino acids with D-omino acids. For example, if the parent peptide is ~~~hr-.~la-Tyr, the ratio modified form is Tyr-Ala-Thr, the inverse fbrm is tl~-ala-tvr. and the ratio-inverse form is tyr-ala-thr (lower case letters refer to D-amino acidsl. Compared to the parent peptide. a ratio-inverse peptide has a reversed backbone while retaining substantially the original spatial conformation of the side chains, resulting in a ratio-inverse isomer with a topology that closely resembles the parent peptide. See Goodman et al. "Perspectives fn Peptide Chemistry" pp. 283-(1981 ). See also L~.S. Patent No. 4.5''.752 by Sisto for further description of "ratio-inverse"
peptides.
Other derivatives of the modulator compounds of the invention include C-terminal hydroxymethyl derivatives. O-modined derivatives (e.g., C-terminal hydroxvmethyl 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 phenethylamid~ analogue (e.g., V<tI-Phe-phenethylamide as an analogue of the tripeptide Val-Phe-Phe).
In a preferred embodiment, the ACD of the modulator is modeled after the subregion of [i-AP encompassing amino acid positions I'7-~0 c'i.e.. Leu-Val-Phe-Phe; SEQ
ID NO: I2).

.As described further in Examples l. ~ and 9 ~ peptide subregions of A(3 ~ ~p were prepared, amino-terminally modified and evaluated for their ability to modulate aggregation of natural ~3-amyloid peptides. One subregion that was effective at inhibiting aggregation was A~36_?p (i.e., amino scid residues 6-20 of the natural A(3I..,« 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 su~cient for aggregation inhibitory acaivity. This process defined A~3 ~ ~_2p (l. e. , amino acid residues I 7-20 of the natural A~:i ~ ~p peptide) as a minimal subregion that, when appropriately modified. is sufficient for aggregation inhibitory activity.
Accordingly, an "A~i aggregation core domain" within a modulator compound of the invention can be modeled after A~3~~_~p. In one embodin ent. the A(3 aggregation core domain comprises A~il7_2p itself (i.e., a peptide comprising the amino acid sequence ieucine-valirre-phenylalanine-phenylalanine; SEQ IO N0: 12 ). Irc oth~:r embodiments, the structure of A~ij ;_~p is used as a model to design an .~~3 aggregation core domain having similar I5 structure and function to A(3l ~_?p. For example, peptidornimetics, derivatives or analogues of A~17-Zp las described above; can be used as an A(3 aggre;ation core domain. In addition to A~1~_~p. the natural A~i peptide is l:ilcelv to contain other wminimal subregions that are sufficient for aggregation inhibitorr~ activit;;. Such additional minimal subregions can be identified by the processes described in Examples %. 8 and 9, wherein a l ~mer subregion of A~il~p is serially deleted fiom the amino-terminus or carboxy terminus. the deleted peptides are appropriately modified and then evaluated for aagre~~ation inhibitory activity.
One form of the ~i-amyloid modulator compound comprising an A~i aggregation core domain modeled after A(31~_;p coupled ~iirectl~ er indirecuv tt3 3t least one modifying group has the formula:
%,.an ( Y-Xaal-Xaa~-Xaa;-Xaa,~-Z'S
wherein Xaa~ and Xaa3 are amino acid structures;
Xaa~ is a valine structure;
Xaa4 is a phenylalanine structure;
Y. which may or may not be present. is a peptidic structure having ta'~e formula (Xaa)a; whe:ein Xaa is any amino acid structure and a is an integer from 1 to I ~;
Z. which may or may not be present, is a peptidic structure having the formula (Xaa)b, wherein Xaa is any amino acid structure and b is an integer from I to 1 ~; and A is a modifying graup attached directly cyr indirectly to tl~.e compound and n is an integer;
Xaa~, Xaa;. Y, Z. A and n being selected such that the compound modulates the aggregation or inhibits the neurotoxicitv of natural ~i-arnyloid peptides when contacted with the natural ~i-amyloid peptides.

Preferably, a modulator compound of the above formula inhibits aggregation of natural ~i-amyloid peptides when contacted with the natural ~i-amyloid peptides and/or inhibits A~3 neurotoxicity. Alternatively, the modulator compound can promote aggregation of natural ~i-amyloid peptides when contacted with the natural ~-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 ~i-amyloid peptides when contacted with the natural ~-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 l and 10. between 1 and ~ or between 1 and 3.
Suitable types of modifying groups are described further in subsection II below.
As demonstrated in Example 9, amino acid positions 18 (Vallg) and 20 fPhe~p) of A~1 ~-20 (con'esponding to Xaa~ and Xaa~) are particularly important w within the core domain for inhibitory activity of the modulator compound. Accardingl~,~. these positions are 1 ~ conserved within the core domain in the fornmla 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-occumng analogues., derivatives and mimetics of valine and phenylalanine, respectively. (including Ia-amino acidsl which maintain the functional activity of the compound. Moreover. although Val t g and I'he,,~ have an important functional role, it is possible that Xaa~ andi'or Xaa4 can be substituted with other naturally-occurring amino acids that are structurally related to v<3line or phens~lalanine.
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 vaIine at Xaa-,. and the term "phenylalanine structure" is intended to include conservative amino acid substitutions that retain the activity of phenvlalanine at Xa::y. -Iowever.
the term "valine structure" is not intended to include threanine.
In contrast to positions 18 and 20 of A~i ~ ~_-,p, a Phe to AIa substitution at position 19 (,corresponding to Xaa,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~ and Xaa3 are any amino acid structure. The term "amino acid structure" is intended to include natural and non-nataral amino acids as well as analogues.
derivatives and mimerics thereof, including D-amino acids. In a preferred embodiment of the above formula. Xaai is a leucine structure and Xaa; is a phenylalanine structure (i.e., modeled after Leul~ and Phez9, respectively, in the natural A~3 peptide seauence). The term "leucine structure" is used in the same manner as valine structure and phenylalanine structure described above. Alternatively, an another embodiment, Xaa; is an alanine structure.
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~i peptide sequence or from non-A(3 sequences. The term "peptidic structure" is intended to include peptide analogues, derivatives and mimetics thereof as described above. The peptidie 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 o.ae embodiment. no additional amine :acid structures flank the Xaa1-Xaa-~-Xaa3-Xaa4 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 ir: the above formula). In vet another embodiment, one or more additional amino acid structures flank only the carboxy-terminus of the core sequences (i.e.. Z is present but ~' 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 1 ~. More preferably, a and b are integers between l and 10. Even more preferably, a and b are integers between i and ~. Most preferably. a and b are integers between i and 3.
One form of the ~i-amvioid modulator compound comprising an A~3 agare<~ation core 1 ~ domain modeled after A(3 j ~.~p coupled directly or indirectiv to at least one modifying Group has the formula:
A-(Y)-Xaa ~ -Xaa,-Xaa;-Xaat-(Z)-B
wherein Xaai and Xaa; are amino acids ur amino acid mimetics;
Xaa-, is valine or a valine mimetic Xaa4 is phenylalanine or a phenylalanine mimetic;
Y. which may or may not be present. is a peptide or peptidomimetic having the formula (Xaa)a, wherein Xaa is any amino acid or amir~m acid mimetic and a is an inte2er frornltol~:
Z, which may or may not be present. is a peptide or peptidomimetic having the formula (Xaa)b, wherein Xaa is any amino acid or amine acid mimetic and b is an integer from I to 1~; and A and B. at least one of which is present, are modifying groups attached directly or indirectly to the amino terminus and carboxy temninus, respectively. of the compound;
Xaa~. Xaa;, Y, Z, A and B being selected such that the compound :modulates the aggregation or inhibits the npurotoxicity of natural ~i-amyloid peptides when contacted with the natural (3-amyloid peptides.
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~-Xaa~-~:aa3-Xaa4-(Z)). In another embodiment. the compound is modified only at its carboxy-terminus (i.e., A is absent and the compound comprises the formula: (~-Xaat-Xaa-,-Xaa~-Xaa~-(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)-Xaat-Xaa~-Xaa3-Xaa4-(Z)-B and both A and B are present). A s described above, the type and numl~t~:r of amino acid structures which flank the Xaal-Xaa~-Xaa3-Xaa4 core sequences in the above formula is variable. For example, in one embodiment, a and b are integers from 1 to 1 ~. ivlore preferably, a and b are integers between 1 and 10. Even more preferably, a and b are intejers between 1 and ~. I~rlost preferably, a and b are intesers between 1 and 3.
As demonstrated in Examples 7. 8 and 9, preferred A~i modulator compounds of the invention comprise modified forms of A~3t,~_~ r (I-his-Gln-Lys-l.eu-VaI-Phe-Phe-Ala; SEQ ID
NO: 5), or amino-terminal or carboxy-terminal deletions thereof with a preferred "minimal core region" comprising A~31~_,p. Accordingl~,~, in specific embodiments. the invention provides compounds comprising the formula:
1 ~ A-Xaa~-.Xaa~-Xaa,-Xaa~-Xaag-Xaat;-Xaa~~~Xaag-B
wherein Xaal is a histidine structure;
Xaa2 is a glutamine structure;
Xaa3 is a lysine structure;
Xaa4 is a leucine structure;
XaaJ is a online structure;
Xaa6 is a pherxylalanine structure;
Xaa7 is a phenvlalanine structure;
Xaa8 is an alanine structure;
2~ A and B are modifying groups attached directly or indirectly to the amino terminus and carboxy terminus. respectively, of the compound;
and wherein Xaal-Xaa,-Xaa;. Xaa;-X3a, or Xaa~ may or rnay not be present:
Xaag may or may not be present: and at least one of A and B is present.
In one specific embodiment, the compound comprises the formula: A-Xaa4-Xaas-Xaa6-Xaa~-B (e.g, a modified farm of A~31~.2~, comprising an amino acid sequence Leu-Val-Phe-Phe; SEQ ID NO: 12).
In another specific embodiment. the compound comprises the formula: A-Xaa4-XaaS-Xaas-Xaa~-Xaag-B (e.g. a modified form of A(3 t ;. ~ ~ . comprising an amino acid sequence Leu-Val-Phe-Phe-AIa; SEQ ID NO: 11).
In another specific embodiment. the compound comprises the formula: A-Xaa;-Xaa4-XaaS-Xaa6-Xaa~-B (e.g., a rnoditied form of A~il~_~,p. comprising an amino acid sequence Lys-Leu-Val-Phe-Phe: SEQ ID NO: 10 j.

~'?
In ataother specific embodiment, the compound comprises the formula: A-Xaa;-Xaa4-Xaas-Xaas-Xaa~-Xaag-B (e.o., a modified form of A~3 ~ 6_T ~ , comprising an amino acid sequence Lys-Leu-Val-Phe-Phe-Ala; SEQ ID N0: 91.
In another specific embodiment, the comp~.;und comprises the formula: A-Xaa~-Xaa;-Xaa4-XaaS-Xaa6-Xaa~-B (e.g., a modified form of A~3 t 5-gyp, comprising an amino acid sequence Gln-Lys-Leu-Va1-Phe-Phe: SEQ ID N0: 8).
In another specific embodiment, the compound omprises the formula: A-Xaa~-Xaa;-Xaat-XaaS-Xaa6-Xaa~-Xaag-B (e.g., a modified form of ,~~i~5-?1, comprising an amino acid sequence GIn-Lys-Leu-Val-Phe-Phe-Ala; SEQ ID N4: 7).
In another specific embodiment, the compound comprises the formula: A-Xaai-Xaa~-Xaa;-Xaa4-Xaas-Xaa6-Xaa~-B (e.g., a modified form of A(314-20~ comprising an amino acid sequence His-Gln-Lys-Leu-Val-Phe-Phe; SEQ ID NO: 6).
In another specific embodiment, the compound comprises the formula: A-Xaa~-Xaa,-Xaa;-Xaa~-Xaas-Xaa6-Xaa~-Xaag-B (e.g., a modined form of A~3la-? ~.
comprising an I ~ amino acid sequence His-Gln-Lys-Leu-Val-fhe-Phe-Ala: SEQ 1D NO: ~).
In preferred embodiments of thv aforementioned specific embodiments, A or B is a cholanovl structure or a biotin-containing structure (described further in subsection II below).
In further experiments to delineate subregions of :~~i upon which an A~i aggregation core domain can be modeled (the results of which are described in Example 1 I
), it was demonstrated that a modulator compound having inhibitory. activity can comprise as few as three A~i amino acids residues (e.g., Val-Phe-Phe. which corresponds to A.(31g_2p or Phe-Phe-Ala. which corresponds to A~3 ~ 9_~ ~ ). The results also demonstrated that a modulator compound having a modulating group at its carboxy-terminus is effective at inhibiting A~i 2~ a~~resation. Still further. the results demonstrated that the chofy~l group. as a modulating group, can be manipulated while maintaining the inhibitor's activity of the compounds and that an iodotyrosyl can be substituted for phenylalanine (e,y., at position 19 or 20 of the A~i sequence) while maintaining the ability of the compound to inhibit A(3 aggregation.
Still further. the results demonstrated that compounds with inhibitory activity can be i0 created using amino acids residues that are derived from t;~e A~i sequence in the region of about positions 17-21 but wherein the tunino acid sequence is rearranged or has a substitution with a non-A~3-derived amino acid. E~~amples of such compounds include PPI-426. in which the sequence of A~i 1~-~ t (L.VFFA) has been rearranged (FFVL A), PPI-372, in which the sequence of A~ilb-20 (I:LVFF) has been rearranged (FKFVL), and PPI-388, -389 and -390, in 35 which the sequence of A~31~_~1 (LVFFA) has been substituted at position 17, 18 or 19, respectively, with an alanine residue (HVFFA for PPI-38~~, LAFFA for PPI-389 and LVAFA
for PPI-390). The inhibitory activity oI'these compounds indicate that the presence in the compound of an amino acid sequence directly corresponding to a portion of A~3 is not essential for inhibitory activity, but rather suggests that maintenance of the hydrophobic 7~
nature of this core region, by inclusion of amino acid residues such as phenylalanine, valine, leucine, regardless of their precise order, can be surf cient: for inhibition of A~3 aggregation.
Accordingly, an A~i aggregation core domain can be designed based on the direct A~3 amino acid se~aence or can be designed based on ci °~arranged :~.~i sequence which maintains the hydrophobicity of the A~3 subregion, e.g., the region around positions 17-20.
This region of A~i contains the amino acid residues Leu, Vai and Phe. accordingly, preferred A~3 aggregation core domains are composed of at least three amino acid structures (as that term is defined hereinbefore, including amino acid derivatives, an.alagues 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 t:~c~se terms are defined hereinbefore, including derivatives, analogues and mimetics ).
Thus. in another embodiment. the invention provides a ~3-amyioid modulator compound comprising a formula:
~rt 1 ~ ( Y-Xaa i -Xaa~-Xaa;-Z,~' wherein Xaal, Xaa,and Xaa; are each amino acid structures and at least two of Xaa~, Xaa~ and Xaa3 are. independently. selected fiom the group consisting of a Ieucine structure, a phenvlalanine structure and a vaiine structure Y, which may ar may not be present. is a peptidic structure having the formula (Xaa)a, wherein Xaa is any amino acid structure 4nd a is an integer from 1 to 1 >:
Z, which may or may not be present. is a peptic~lic structure having the formula (Xaa)b. wherein Xaa is any amino acid staucture and b is an integer from I to ? ~: and A is a modifying group attached directly or indirectly to the compound and n 2~ is an integer:
Xaal, Xaa~, Xaa3, Y, Z. A and r. being selected such that the compound modulates the aggregation or inhibits the neurotoxicity of natural (3-arnyloid peptides when contacted with the natural p-amyloid peptides.
Preferably, the compound inhibits aggregation of natural ~i-amyloid peptides when 3~ contacted with the natural (3-amyioid peptides. In preferred embodiments.
Xaa~ and Xaa~ are each phenylalanine structures or Xaa~ aid Xaa~ are each phenvlalanine stntctures. "n" can be, for example. an integer between I and ~, whereas ''a" and "b" can be. for example, integers between 1 and ~. The modifying group ".~" preferably comprises a cyclic, heterocyclic or polycyclic group. More preferably. A contains a cis-decalin group, such as 35 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 l~-acPtvlneurarninyl group. In yet other embodiments, the compound may promotes aggregation of natural ~i-amvloici peptides when contacted with the natural ~i-amyloid peptides, may be further modif ed to alter a pharmacokinetic property of the compound or may be further modified to label the compound with a detectable substance.
In another embodiment, the invention provides a ~i-amyloid modulator compound comprising ~. formula:
.r~-(Y)-Xaa ~ -Xaa~-Xaa;-(:Z;)-B
wherein Xaa2, Xaa~and Xaa~ are each amino acid structures and at least two of Xaal, Xaa~ and Xaa3 are, independently, selected from the group consisting of a leucine I O structure, a phenylalanine structure and a valine structure;
Y. which may or may not be present, is a peptidic structure having the formula (Xaa)a, wherein Xaa is any amino acid structure and a is an integer from 1 to 1 ~;
Z, which may or may not be present. is peptidic stn:cture having the formula (Xaa)b, wherein Xaa is any amino acid structure and ~ is an integer from 1 to 1 ~: and 1 ~ A and B. at least one of which is present. are modin~ir.~ Groups attached directly or indirectly to the amino terminus :~nd carboxv t~::minus, respectively. of the compound;
Xaai, Xaa-,, Xaa;, Y, Z, .~ and I3 being selected such that the compound modulates the aggregation or inhibits the neurotoxicity <v' natural ~3-amvloid peptides when 20 contacted with the natural ~3-amyloid peptides.
Preferably. the compound inhibits aggregation of natural ~i-amvIoid peptides when contacted with the natural (3-amyloid peptides. In preferred embodiments. Xaa~
and Xaa~ are each phenylalanine structures or Xaa-, and Xaa3 are each phenylalanine structures. In one subembodiment. the compound comprises the formula:
25 A-(Y)-Xaa ~ -Xaary-Xaa:~-(Z f In another subembodiment, the compound comprises the rormula:
(Y)-Xaai-X.aa~-Xaa:-(.2)-B
"n" can be, for example. an integer between l and ~, whereas "a" and "b" can be. for example.
integers between 1 and ~. The modifying group "A" preferably comprises a cyclic, 30 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-triaminepenta.acetyl group, a (-)-menthoxyacetyl group. a fluorescein-containing group or an N-acetylneuraminyl group. In yet other embodiments, the compound may promote aggregation of natural ~i-amyloid peptides when contacted with the 35 natural ~3-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.
In preferred specific embodiments. the invention provides a ~-amyloid modulator compound comprising a modifying group attached directly or indirectly to a peptidic .--.~::.crure. wherein the peptidic structure comprises amino acid structures having an amino ?S
acid sequence selected from the group consisting of His-Gln-Lys-Leu-Val-Phe-Phe-Ala (SEQ
ID NO: ~), His-Gln-Lys-Leu-Val-Phe-Phe f~SEQ ID 1~T0: e), Gln-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO: 7), Gln-Lys-Leu-Val-Phe-Phe (SEQ ID NC.~: 8), Lys-Leu-Val-Phe-Phe-Ala (SEQ ID N0: 9), Lys-Leu-Val-Phe-Phe (SEQ ID ~'~O: 10), Leu-Val-Phe-Phe-Ala (SEQ ID
NO: 11), Leu-Val-Phe-Phe (SEQ ID N0: 12), Leu-Ala-Phe-Phe-Ala (SEQ ID NO: 13), Val-Phe-Phe (SEQ ID NO: 19), Phe-Phe-Ala (SEQ ID NO: 2C~), 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-~°al-Phe-Phe-Ala-Glu (;SEQ ID NO: 27), Phe-Phe-Val-Leu (SEQ ID NO: 28), Phe-Lys-F'he-Val-Leu (SIm:Q ID NO: 29), Lys-Leu-Val-Ala-Phe (SEQ ID NO: 30), Lys-Leu-Val-Phe-Phe-~3Ala (SFQ 1D NO: 31 ) and Leu-Val-Phe-Phe-DAIa (SEQ ID NO: 32).
These specific compounds can be further modified to alter a pharmacokinetic propem°
of the compound andlor further modified to label the compou~~d with a detectable substance.
1 ~ The modulator compounds of the invention can be incorporated into pharmaceutical compositions (described further in subsection y' beloyj and can be used in detection and treatment methods as described further in subsection ~'I below.
II. Modifvina Groups Within a modulator compound of the invention, a peptidic structure (such as an A~3 derived peptide. or an A~3 aggregation core domain. or an amino acid sequence corresponding to a rearranged A(3 aggregation core domain) is coupled di.rectlv or indirectly to at least one modifying group (abbreviated as MIG). 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-AC:D. The term "modifying group" is intended to include structures that are directxv 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~i-derived peptidic structure). For example, the modifying group can be coupled to the amino-terminus or carboxy-terminus of an A~3-derived peptidic structure, or to a peptidic or peptidomimetic region flanking the core damain. Alternatively. the modifying group can be coupled to a side chain of at least one amino acid residue of an A~3-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 residues) or a glutamic acid residue(s), through a hydroxv group of a tyrosy°l residue(;s;l, a serine residues) or a threonine residues) 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 2 f) known in the art for linking chemical structures. including. for example.
amide. alkylamino, carbamate or urea bonds.
The term "modifying group" is intended to include groups that are not naturally coupled to natural A~i peptides in their native form. Accordingly, the term "modifying group" is not intended to include hydrogen. The modifying groups) is selected such that the modulator compound alters, and preferably inhibits, aggregation of natural ~i-amyloid peptides when contacted with the natural ~i-amyloid peptides or inhibits the neurotoxicity of natural ~i-amyloid peptides when contacted with the natural (3-amyloid peptides. Although not intending to be limited by mechanism, the modifying groups) 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~i polymerization.
In a preferred embodiment. the modifying groups) comprises a cyclic, heterocyclic or polycvclic 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 1 ~ to 8. and more preferably about ~ 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;, -CN, or the like.
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 ~ to 7. carbon atoms. wherein the ring structure includes about one to four heteroatoms.
Heterocvclic groups include pyrrolidine. oxolane. thiolarze. 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. thioh amines. imines, amides, phosphonates, phosphines, carbonyls, carboxyls. silyls, ethers, thioethers, sulfonyls, selenoethers, ketones. aldehydes, esters. -CF;, -CN, or the like. Heterocycles may also be bridged or fused to other cyclic groups as described below.
The term "polycyclic 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 rind's". 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. -CFA. -CN. or the like.

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-deca.lin structure contributes to the efficacy of the modifying group in disrupting Aø 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-decaiin containing structure that can be used as a modifying group is a cholanoyl structure, such as a chalyl 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 Figure 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, 34:817-82?; Wess, G. et al. (1992) Tetrahedron Letters 33:195-198: and Kramer, W. et al. (:I99'_') J. Biol. Chem.
2u7:18598-18604). Cholyl derivatives and analogues can also be used as modifying groups.
For 1 ~ 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.4., a chelation group for 99mTc can be introduced through the free amino group of Aic). As used herein, the term "cholanovl 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 S~i-cholestan-3a-of (the 2~ cis-decalin isomer of (+)-dihydrocholesterol). for further description of bile acid and steroid structure and nomenclature, see Nes, W.R. and McKean. M.Ir. Biochemistry of Steroids and Other Isopentanoids, University Park Press, Baltimore, ivlD, Chapter 2.
In addition to cis-decalin containing groups, other polycyclic groups may be used as modifying groups. For example, modifying groups derived from steroids or ~3-lactams may be suitable modifying groups. Moreover. non-limiting examples of some additional cyclic, heterocyclic or polycyciic compounds which can be used to modify an A~-derived peptidic structure are shown schematically in Figure 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~3-derived peptidic structure with 5- .(and 6-)-carboxyfluorescein, succinimidyl ester or fluorescein isothiocyanate. In various other embodiments, the modifying groups) can comprise an N-acetylneuraminyl .group. a truns-4-cotininecarboxyl group, a 2-imino-1-imidazolidineaceryl .group. an (S~-(-)-indoline-2-carboxyl group, a (-)-menthoxyacetyl group.

a 2-norbornaneacetyl group, a y-oxo-~-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.
Preferred modifying groups include groups comprising cholyl structures, biotinyl structures, fluorescein-containing groups. a diethylene-triaminepentaacetvl group, a (-)-menthoxyacetyl group, and a N-acetylneurarninyl group. More preferred modifying groups those comprising a cholyl structure or an iminiobiotinyl group.
In addition to the cyclic. heterocycIic 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.
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 dibenzoiwan-based amino acid described in 1 ~ Tsang. K.Y. et al. ( 1994) J. Am. Chem. Soc. 116:3988-400: Diaz. H and Kelly. 1.W. ( 1991 ) Tetrahedron Letters 41:572-X728: and Diaz. H et al. (1992) J. Am. Chem. Soc.
114:8316-8318. An example of such a modifying group is a peptide-aminoethvldibenzofuranyl-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 ~i-AP when compounds of this type interact with natural (3-AP.
III. Additional Chemical Modifications of :A(3 Modulators A ~i-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 23 A~3 aggregation and inhibit A~i neurotoxicitv. 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~3 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 C.'M represents an additional chemical modification.
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 J~-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 th.e compound to act as a substrate for aminopeptidases.
A modulator compound can be further modified to label the compound by reacting the compound with a detectable substancf:. Suitable detectable substances include various enzymes, prosthetic groups. fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, ~3-gaiactosidase, or aeet~rlchoIinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidinlbiotin; examples of suitable fluorescent materials include umbelliferone, fluorc~scein. fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluoreseein, darrsyl chloride or phycoerythrin; an example of a luminescent material includes luminol: and examples of suitable radioactive material include 1~ 14C, 1231, 1241. I?~I, 13l I, 99mTc,'~:S or'H. In a preferred embodiment. a modulator compound is radioactively labeled with I4C, either by incorporation of 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~i aggregation. for example for diagnostic purposes. A(3 aggregation can be detected using a labeled modulator compound either in vivv or in an in vitro sample derived from a subject.
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 99mTc.
2~ Methods for labeling peptide compounds with technetium are i:nown in the art (see e.g., U.S.
Patent Nos. ~.443,81~. .?25.180 and 5,405.97, all by Dean er 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 9gmTc can be introduced.
such as the Aic derivative of cho1ie acid, which has a free amino group twee Example 11 ). In another embodiment. the invention provides a modulator compound labeled with radioactive iodine.
For example. a phenylalanine residue within the A~i sequence t uch as Phel9 or Phe2p) can be substituted with radioactive iodotyrosyl (see Example 1 I ;~. Any of the various isotopes of 3~ radioactive iodine can be incorporated to create a dia2nostic agent.
Preferably, 1231 (half life = I3.2 hours) is used for whole body scintigraphy, ~'-'~I (half life = 4 days) is used for positron emission tomography (PET), 125I (half life = 60 days) is used for metabolic turnover studies and 1' ~ I (half life = 8 days) is used for whole body counting and delayed low resolution imagine studies.

Furthermore. an additional modification of tt modulator compound of the invention can serve to confer an additional therapeutic propeny on the compound. That is, the additional chemical modification can comprise an a~3ditional functional moiety. For example, a functional moiety which serves to break down or ci.issolve amyloid plaques can be coupled 5 to the modulator compound. in this form the MG-ACD portion of the modulator serves to target the compound to A(3 peptides and disrupt the polymerization of the A(3 peptides, whereas the additional functional moiety serves to break down or dissolve amyloid plaques after the compound has been targeted to th~:°se sites.
In an alternative chemical modification, a ø-amyloid compound of the invention is 10 prepared in a "prodrug" form, wherein the compound itself does not modulate A~3 aggregation, but rather is capable of being transformed, upon metabolism in vivo, into a ~i-amyloid modulator compound as defined herein. For example, in this type of compound, the modulating group can be present in a procirug form that is capable of being converted upon metabolism into the form of an active mo~dulatina group. Such a prodrue form of a 1 ~ modifying group is referred to herein as a "secondan~ modifying group.'" A
variety of strategies are known in the art for preparir~l; peptide prodrugs that Iimit metabolism in order to optimize delivery of the active form of the peptide-ba..sed 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 20 achieving CNS delivery based on "sequential metabolism" (see e.g., Bodor, N., er al. (1992) Science 237:1698-1700: Prokai, L., et al. (I'994),~. Am. C'hem. Soc. I 16:2643-2644; Bodor, N. and Prokai. L. (i995) in Peptide-Based I)ru~ 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 2~ to facilitate blood-brain barrier permeability.
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, 30 M. Principles ofPeptide 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). Additionally, one or more modulating groups can be attached to the A~i-derived peptidic component (e.g.. an A~i 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 ~i amyioid rncduiatars is described further in Examples 1, 4 and 11.
IV. Screening Assays Another aspect of the invention pertains to a method for selecting a modulator of (3-amyloid aggregation. In the method, a test compound is contacted with natural ~3 amyloid peptides. the aggregation of the natural ~i-AP is measured and a modulator is selected based on the ability of the,test compound to alter the aggregation of the natural (3-AP (e.g., inhibit or promote aggregation). In a preferred embodiment. the test compound is contacted with a molar excess amount of the natural ~i-AP. The amount andior rate of natural ~3-AP
aggregation in the presence of the test compound can be determined by a suitable assay indicative of ~i-AP aggregation. as described herein (see e.g., Examples ?, ~
and 6).
In a preferred assay, the natural ~i-AP is dissolved in solution in the presence of the test compound and aggregation of the natural ~-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 4t~t6 nm (described further in Example 6; see also Jarrett et al.
(1993) Biochemisrry 3?:4693-4697). In the absence of a ~3-amyloid modulator.
the A4o~~, of the solution typically stays relatively constant during a laa time in which the ~i-AP remains in solution. but then the A4p~m" of flue solutian rapidly increases as the ~i-AP
aggregates and comes out of solution, ultimately reaching a plateau level (i.e., the A405nm of the solution exhibits siemoidal kinetics over time). In contrast. in the presence of a test compound that inhibits ~i-AP aggregation. the A4o5nm 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 2~ compared to when the modulator is absent. This method for selectinE a modulator of ~i-amyloid polymerization can similarly be used to select rnoduiators that promote (3-AP
aggregation. Thus, in the presence of a modulator that promotes ~-AP
aggregation, the A4o5nm of ~e solution is increased compared to when the modulator is absent (e.g., the solution may exhibit an decreased lag time, increase slope of aggregation andlor a higher plateau level compared to when the modulator is absent).
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, ~3-AP
monomer and an aggregated (3-AP "seed" are combined. in the presence and absence of a test compound. and the amount of Q-fibril formation is assayed based on enhanced emission of the dye Thioflavine T when contacted with ~3-AP fibrils. Moreover, ~3-AP aggregation can be , assessed by electron microscopy (EM) of the ~3-AP preparation in the presence or absence of the modulator. For example, (3 amyloid fibril formation, which is detectable by EM, is reduced in the presence of a modulator that inhibits (3-AP aggregation (i.e., there is a reduced amount or number of ~3-fibrils in the presence of the modulator), whereas ~
fibril formation is ~) increased in-the presence of a modulator that promotes ~3-AP aggregation (i.e., there is an increased amount or number of (3-fibrils in the presence of the modulator).
An even more preferred assay for use in rlte 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 nettrotoxic A~i aggregates and/or which inhibit the neurotoxicity of ,preformed A~3 fibrils. This neurotoxicity assay is considered to be predictive of neurotoxicity in vivo. Accordingly, inhibitory activity of a modulator compound in the in vitro neurotc~xicity assay is predictive of similar inhibitory activity of the compound for neurotoxiciri~ in vivo.
V. Pharmaceutical Compos itions~
Another aspect of thv: invention pertains to pharmaceutical compositions of the ~i-amyloid modulator compounds of the invention. In one embodiment, the composition includes a ~3 amyloid modulator compound in a therapeutically or prophylactically effective 1 ~ amount sufficient to alter. and preferably inhibit. aggregation of natural (3-amyloid peptides.
and a pharmaceutically acceptable carrier. In another embodiment. the composition includes a ~ amyloid modulator compe~und in a therapeutically or prophylactically effective amount sufficient to inhibit the neurotoxicity of natural ~3-amyioid peptides. and a pharmaceutically acceptable carrier. A "therape:utically effective amount" refers to an amount effective, at dosages and far periods of time necessary, to achieve the desired therapeutic result. such as reduction or reversal or (3-am~~Ioid depasition and/or reduction or reversal of A(3 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 2~ provide the optimum therapeutic response. A therapeutically effective amount is also one in which anv 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 ~i-amyloid peptides.
A "prophylacticallv 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 (3-amyloid deposition and/or A~i neurotoxicity in a subject predisposed to ~i-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.

One-factor that may be considered when determining a therapeutically or prophylactically effective amount of a ~3 amyloid modulator is the concentration of natural ~i-AP in a biological compartment of a subject, such as in 'the cerebrospinal fluid (CSF) of the subject. The concentration of natural (3-AP in the CSF has been estimated at 3 nM
(Schwarizman, (1994) Proc. Natl. Acad. Sci. TISA 91:$36$-$372). A non-limiting range for a therapeutically or prophylactically effective amounts of a [3 amyioid modulator is 0.01 nM-10 ~M. It is to be noted that dosage values may vary with the severity of the condition to be alleviated. It is to 1?e 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.
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 1 ~ amount of natural (3-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.
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.
3~ 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.
Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemuIsion, 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.
Sterile injectable solutions can be prepared by incorporating the active compound (e.g., ~i-arnyloid modulator) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above. as required. followed by filtered sterilization.
2~ 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.
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-y-cvclodextrin. 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 '''57:1698-1700. For the (3-amyloid modulators described herein, inclusion in the formulation of hydroxypropyl-y-cyclodextrin at a concentration ~0-200 mM increases the aqueous solubility of the compounds. In addition to increased solubility. inclusion of a eyclodextrin derivative in the formulation may have other beneficial effects. since p-cyclodextri:c~ itself has been reported to interact with the A(3 peptide and inhibit fibril formation in vitro (C:amilleri, P., et al. (1994) FEBSLetters 341:26-2~8, Accordingly, use of a modulator compound ofthe invention in combination with a cyclodextrin derivative may result in greater inhibition of A[i aggregation than use of the modulator alone. Chemical modifications of cyclodexzrins 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[i peptide compound to form a moduhitor compound of the invention.
10 In another embodiment. a pharmaceutical compraition 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 r_iodulators across the BBB (for reviews of such strat~:eies. see e.g., Pardridge. W.M.
(1994) Trends in 1 ~ Biotechnol. I 2:239-24~: Van Bree, J.B. ~t al. ( 1993) Pharm. ~I~orld Sci.
13:?-9: and Pardridge. W,M. et al. (1992) Pharmacol. Tozicol. 71:3,-10). In one approach.
the modulator is chemically modified to form a prodru o 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. Patient 4.933.324 and PCT Publication 8, 20 both by Shashoua; U.S. Patent ~,284,8T6 by Hesse et ul.; Toth, I. et al.
(1994) J. Drug Target. 2:217-239: and Shashoua, V.E. et al. (1984) J. ~~Ted C'hem. 27:69-664) and glycating the modulator (see e.g., U.S. Patent x.260.308 by Poduslo et al.).
Also, N-acvlamino acid derivatives may be used in a modulator t:o form a "lipidic"
prodrug (see e.g., U.S. Patent No. 5,112,863 by Hashimoto et al. issued on May 12''', 1992).
2~ In anqther 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 30 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.
Patents x.182.107 and 5.14.924 and PCT Publications WO 93/i0819 and WO 9/02421, all by Friden et al.).
Other suitable peptides or proteins that can mediate transport across the BBB
include histones 35 (see e.g., U.S. Patent 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. Patents x,416,016 and 5,108,921, both by Heinstein). Additionally, the glucose transporter GLUT-1 has been reported to transport glycopeptides (L-serinyl-[3-D-glucoside analogues of [Met~Jenkephalin) across the BBB (Polt, P.. et al. (1994) Proc.
Natl. Acad. Sci.

USA 91:71 I~-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 chc~lic 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 D1~1A methods t;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 IL). A crosslinking agent can be chosen which allows for high yield coupling of the rnadulatar to the second peptide or protein and for subsequent cleavage c7f the linker to release bioactive modulator. For example, a biotin-avidin-based linker system may be used.
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 I ~ modulator can be encapsulated in a liposome, such as a positively charged unilamellar liposome (see e.g., PCT Publicatitans WO 88107831 and 'WO 8$/U78~2. both by Faden) or in polymeric microspheres (see e.g., LJ.S. Patent x,413,797 by Khan et al... U.S.
Patent x.271,961 by Mathiowitz et al. and ~s,019,400 by Gombotz et al. ). Moreover, the carrier vector can be modified to target it for transport across tlve BBB. For example, the carrier vector (e.g., liposome) can be covalenrly 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 er al. and WO 94/02178 by Greig et al.).
In still another approach to enhancing transport of the modulator across the BBB, the 2~ 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. Patent ~,112.~96 by Malfroy-Camine) and peptidic compounds disclosed in U.S.
Patent 5.268.164 by Kozarich et al.
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 RO, Parke-Davis;l.
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 compositiorrfor treating a subject having a disorder associated with (3-amyloidosis, e.g.
Alzheimer's disease.
VI. Methods of Usin AQ Modulators Another aspect of the invention pertains to methods for altering the aggregation or inhibiting the neurotoxicity of natural (i-amyloid peptides. In the methods of the invention, natural ~3 amyloid peptides are contacted with a ~ amyloid modulator such that the aggregation of the natural ~i amyloid peptides is altered or the neurotoxicity of the natural (3 amyloid peptides is inhibited. In a preferred embodiment, the modulator inhibits aggregation of the natural ø amyloid peptides. In another embodiment. the modulator promotes aggregation of the natural (3 amyloid peptides. Preferably, aggregation of a molar excess amount of ~3-AP, relative to the amount of modulator, is altered upon contact with the modulator.
In the method of the invention. natural ~ 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 ~i-AP preparation in vitro and delivery of the modulator to a site in vivo where natural ~3-AP is present. Since the modulator compound interacts with natural ~i-AP, the modulatar compounds can be used to detect natural ~i-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 (3-AI', either in a biological sample or in vivo in a subject. Furthermore, detection of natural ~-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 ~3-Al' aggregation and inhibit ~-AP
neurotoxicitv. the modulator compounds alsa are useful in the treatment of disorders 2~ associated with p-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 (3-AP.
In one embodiment. a modulator compound of the invention is used in vitro, for example to detect and quantitate natural ~3-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 ~i-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 ~3-AP sample is contacted with a modulator of the invention and aggregation of the ~3-AP is measured. such as by as assay described in 3~ Examples 2. ~ and 6. Preferably. the nucleation assay and/or seeded extension assay described in Example 6 is used. The degree of aggregation of the ~i-AP sample can then be compared to that of a control samples) of a lrnown concentration of ~3 :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 (3-amvloidosis. Moreover, (3-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 ~-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 ~-AP aggregates mixed with a modulator of the invention using a probe that binds to the modulating group {e.g., biotin/strept.avidin) is described further in Example 2.
In another embodiment, a modulator compound of the invention is used in vivo to detect, and, if desired, quantitate, natural ~3-AF deposition in a subject, for example to aid in the diagnosis of ~3 amyloidosis in the sul:~ject. To aid in detection, the modulator compound can be modified with a detectable substance, preferably 99mTc or radioactive iodine (described further above). which can be detected in vivo in a subject. The labeled ~3-amyloid modulator compound is administered to the subject and, after sufficient time to allow accumulation of the modulator at sites ot~ amyloid deposition, the labeled modulator compound is detected by standard imaging techniques. The radioactive signal generated by 1 ~ the labeled compound can be directly detected (e.g.. whale body counting), or alternatively, the radioactive signal can be converted into an image an an autoradiograph or on a computer screen to allow for imaging of amyloid deposits in the subject. Methods for imaging amyIoidosis using radiolabeled proteins are known in the art. For example.
serum amyloid P
component (SAP). radiolabeled with either A'~ I or 99m'Tc, has been used to image systemic amyloidosis (see e.g., Hawkins, P.N. and Pepys, M.B. (I99~) Eur. J. Nucl. Med.
22:59-599).
Of the various isotypes of radioactive iodine. preferably 1'-'I (half life =
13.2 hours) is used for whole body scintigraphy, l2al (half life = 4 days) is used for positron emission tomography (PET). 1-'SI (half life = 60 days) is used far metabolic turnover studies and 1311 (half life = 8 days) is used for whole body counting and delayed low resolution imaging 2~ studies. AnaloQOUS 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 wg of labeled compound carving approximately 180 MBq of radioactivity.
The invention provides a method for detecting the presence or absence of natural (3-amyloid peptides in a biological sample, comprising contacting a biological sample with a compound of the invention and detecting the compound bound to natural ~i-amyloid peptides to thereby detect the presence or absence of natural ~3-amyloid peptides in the biological sample. In one embodiment. the ~3-amylaid modulator compound and the biological sample are contacted in vitro. In another embodiment, the ~i-amvloid modulator compound is 3~ contacted with the biological sample by administering the p-amyloid modulator compound to a subject. For in vivo administration, preferably the compotmd is labeled with radioactive technetium or radioactive iodine.
The invention also provides a method for detecting natural ~i-amyloid peptides to facilitate diagnosis of a (3-amyloidogenic disease. comprising contacting a biological sample '~ r; ;:
with the compound of the invention and deter sting the compound bound to natural (3-amyloid peptides to facilitate diagnosis of a ~i-amyloi~ Iagenic disease. In one embodiment, the (3-amyloid modulator compound and the biological sample are contacted in vitro.
In another embodiment, the (3-amyloid modulator coml.:round is contacted with the biological sample by administering the ~i-amyloid modulator con ~~ pound to a subject. For in viva administration, preferably the compound is labeled with radioactive technetium or radioactive iodine.
Preferably, use of the method facilitates dial.;nosis of Alzheimer's disease.
In another embodiment, the inventio:~~ provides a method for altering natural ~i-AP
aggregation or inhibiting ~3-AP neurotoxioir.~r, which can be used prophylactically or therapeutically in the treatment or preventio n of disorders associated with ~i amyloidosis, e.g., Alzheimer's Disease. As demonstrated in E;~;ample I 0, modulator compounds of the invention reduce the toxicity of natural ~i-AI' aggregates to cultured neuronal cells.
Moreover. the modulators not only reduce floe formation of neurotoxic aggregates but also have the ability to reduce the neurotoxicity c~f preformed A~3 fibrils.
Accordingly, the modulator compounds of the invention can be used to inhibit or prevent the formation of neurotoxic A(3 fibrils in subjects (e. g., prophylactically in a subject predisposed to ~3-amyloid deposition) and can be used to reverse ~3-amylaidosis therapeutically in subjects already exhibiting ~i-amyloid deposition.
A modulator of the invention is contacted with natural ~i 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 ~3-AP and/or inhibit the neurotoxicity of the natural (3-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.
The modulator may be administered to a subject by any suitable route effective for inhibiting natural (3-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.
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 4C>
al. (1989) Oncology 3:15-22), catheters for intra~hecal delivery (e.g., Port-a-Cath, Y-catheters and the like; see e.g., Plummer, J.L. ( 1991 ) Pain 44:2 i :~-22U; 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 ~ I :484-491), implantable infusion pump S 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. 9r~ :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. Patent No. 'i~ 368,56'? by Blomquist and U.S. Patent No.
4,731,058 by Doan, developed by Pharmacia I~eltec Inc.
The method of the invention for alterir.~g ø-AP aggregation in vivo . and in particular for inhibiting ø-AP aggregation. can be used therapeutically in diseases associated with abnormal ø amyloid aggregation and depositi«n to thereby slow the rate of ø
amyloid deposition and/or lessen the degree of ø amyl oid deposition. thereby a.Tneliorating the course 1 ~ of the disease. In a preferred embodiment. tl~le method is used to treat Alzheimer's disease (e.g., sporadic or familial AD, including botri 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 (3 arnyloid deposition.
such as in Down's syndrome individuals and in patients with hereditary cerebral hemorrhage with amyloidosis-Dutch-type (HCHWA-D). While inhibition of ~3-AP aggregation is a preferred therapeutic method. modulators that promote ø :AP aggregation may also be useful therapeutically by allowing for the sequestration of ø-AP at sites that do not lead to neurological impairment.
Additionally. abnormal accumulation of ø-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 9~ :I3I4-1319; .A.skanas. V. et al.
(1995) Current Opinion in Rheumatologv .7:486-496). Accordingly. the modulators of the invention can be used prophylactically or therapeutically in the treatment of disorders in which ø-AP. or APP.
is abnormally deposited at non-neurological locations, such as treatment of IBM by delivery of the modulators to muscle fibers.
VII Unmodified Aø Peptides that inhibit Assreeation c,~fNatural I3-AP
In addition to the ø-amyloid modulators described hereinbefore in which an Aø
peptide is coupled to a modifying group, the invention also provides ø-amyloid modulators comprised of an unmodified Aø peptide. it has now been discovered that certain portions of natural ø-AP can alter aggregation of natural ø-APs when contacted with the natural ø-APs (see Example 12). Accordingly, these unmodified Aø peptides comprise a portion of the natural ø-AP sequence (i. e., a portion of øAP 1 _; g, øAP t _4a, øAP t-4, and øAP 1 ~3)- In particular these unmodified Aø peptides have at least one amino acid deletion compared to øAP1_39~ ~e shortest natural ø-AP, such that the compound alters aggregation of natural ø-amyloid peptides when contacted with the natural (3-amyloid peptides. In various embodiments, these unmodified peptide compounds can promote aggregation of natural (3-amyloid peptides, or, more preferably, can inhibit aggregation of natural ~3-amyl'oid peptides when contacted with the natural (3-amyloid peptides. Even more preferably, the unmodified peptide compound inhibits aggregation of natural p-amyloid peptides when contacted with a molar excess amount of natural ~i-amyloid peptides (e.g., a I O-fold, 33-fold or 100-fold molar excess amount of natural ~i-AP).
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 ~iAPI_;g. Alternatively, the unmodified peptide compound can have at least five, ten, fifteen. twenty. twenty-five. thirty or thirty-five amino acids deleted compared to ~3APt-fig. Still further the unmodified peptide compound can have 1-~. 1-10. 1-1~. 1-20, 1-2~, 1-30 or 1-3~ amino acids deleted compared to ~iAP~_3g. The amino acid deletionl;s) may occur at the amino-terminus. the carboxv-terminus. an internal site, or a combination thereof.
1 ~ of the ~-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 (3AP~-;g. Alternatively. the unmodified peptide compound can have at least five. ten, fifreen, twenty, twenty-five, thirty or thirty-five internal amino acids deleted compared to ~3AP1_~g. Still further the unmodified peptide compound can have 1-5, 1-10. I-I5. 1-20, 1-25. 1-30 or I-3~ internal amino acids deleted compared to ~3AP1_39. For peptides with internal deletions, preferably the peptide has an amino terminus corresponding to amino acid residue 1 of natural ~3AP and a carboxy terminus corresponding to residue 40 of natural (3AP and has one or more internal ~3-AP amino acid residues deleted (i.e.. a non-contiguous A~i peptide).
2~ In another embodiment. the unmodified peptide compound comprises an amino acid sequence which has at least one N-terminal amino acid deleted compared to j3AP~_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 ~iAPt-;g. Still further the unmodified peptide compound can have I-5, I-10, 1-l~, 1-20. I-2~, 1-30 or 1-35 N-terminal amino acids deleted compared to ~3APa_39~
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 (3AP1-;g.
Alternatively. the unmodified peptide compound can have at least five, ten, fifteen. twenty, twenty-five. thim or thirty-five C-terminal amino acids deleted compared to ~iAP~_;g. Still further the unmodified peptide compound can have 1-5, 1-1 (), 1-1 ~. I -20, 1-?~, 1-30 or I -3 ~
C-terminal amino acids deleted compared to ~iAP~-;g.
In addition to deletion of amino acids as compared to ~3AP1-;g, the peptide compound can have additional non-(3-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-(3-amyloid peptide-derived amino acid at its N-terminus.
Alternatively, the compound can have, for example, 1-3, 1-a, 1-7, 1-10, I-15 or I-20 non-~3-amyloid peptide-derived amino acid at its N-terminus. In another embodiment, the peptide compound has at least one non-p-amyloid peptide-derived amino acid at its C-terminus.
Alternatively, the compound can have, for example, 1-:i, I-'~, 1-7, I-lU, 1-1~
or 1-20 non-(3-amyloid peptide-derived amino acid at its C-terminus.
In specific preferred embodiments, an unmodified peptide compound of the invention comprises A~36_~0 (the amino acid sequence of which is shown in SEQ ID NO: 4), A~i~6_30 (the amino acid sequence of which is shown in SEQ ID NO: 14), A~ij_~p, -,6..40 {the amino acid sequence of which is shown in SEQ ID NO: 1 ~) or EEVVHHHHQQ-~3AP16-4p {the amino acid sequence of which is shown in SEQ ID NO: 16). In the nomenclature used herein. ~iAP 1 _~o, ?6-4o represents ~3AP ~ ~p in which the internal amino acid residues ~ 1-25 have been deleted.
An unmodified peptide compound of the invention can be chemically synthesized 1 ~ 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 ~r'ork (199?). Automated peptide synthesizers are commercially available (e.g., Advanced Chem'Tech 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., uszng an automated DN.~~ synthesizer).
Alternatively. a DNA
molecule encoding an unmodified peptide compound can be derived from the natural ~3-amyloid precursor protein gene or cDNA (e.~.. using tl~e polymerase chain reaction and/or restriction enzyme digestion) according to standard molecular biology techniques.
Accordingly. the invention further provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a ~-amyloid peptide compound. the ~3-amyloid peptide compound comprising an amino acid sequence having at least one amino acid deletion compared to ~iAPI_;9 such that the (3-amyloid peptide compound alters aggregation of natural ~3-amyloid peptides when contacted with the natural (3-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 (3AP1_39~ ~
discussed above. In yet other embodiments, the isolated nucleic acid encodes a peptide compound having one or more amino acids deleted compared to (3AP1_~9 and further having at least one non-(3-AP derived amino acid residue added to it, for example, at the amino terminus, the carboxy-terminus or at an internal site. In specific prei~erred embodiments, an isolated nucleic acid Fnolecule of the invention en odes (3AP~_2p, ~iAPl6-3o~ ~~'~-20, 26-40 or EE~TVHHHHQQ-aAP t 6-ao~
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~nueleic 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 lieated into the viral ~enome. 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 hast 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 commonlv 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.
In the recombinant expression vectors of the invention. the nucleotide sequence encoding the peptide compound are operatively linked to one or more regulatory sequences.
2~ selected on the basis of the host cells to be used far expression. The term "operably linked"
is intended to mean that the sequences encoding the peptide compound are linked to the regulatory sequences) 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 En..wmologv 185.
Academic Press. San Diego, CA (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. ~.~tc. The expression vectors of the invention can be introduced into host cells thereby to produce peptide compounds encoded by nucleic acids as described herein.

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. cola, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in En~,~mology 18S, Academic Press, San Diego, CA
( 1990). Alternatively, the recombinant expression vector may be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerise. Examples of vectors for expression in yeast S. cerivisae include pYepSecl (Baldari et al., (1987) EMBOJ. 6:229-234), pMFa (Kurjan and Herskowitz, 4;1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:i I3-123), and pYES2 (Invitrogen Corporation.
San Diego, CA). 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) 1'l~l0l. Cell. Biol. 3:21~6-216~) and the pVL series (Lucklow, V.A., and Summers. M.D., (1989) Virology 170:31-39).
Examples of mammalian expression vectors include pCDMB (Seed. B.. (1987) Nature 3?9:840) and pMT2PC (Kaufman et al. (1987;1. EMBC) J. 6:187-19~). 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. cvtomegalovirus and Simian Virus 40.
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 2~ 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. 'Ibis 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.
A recombinant expression vector comprising a nucleic acid encoding a peptide compound that alters aggregation of natural ~i-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 (3-amyioid peptide compound encoded by the recombinant expression vector is,secreted from the host cell upon being expressed in the host cell.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional 10 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, DEAF-dextrin-mediated transfection, lipofection, electroporation, microinjection and viral-mediated transfection. Suitable methods for transforming or 1 ~ transfecting host cells can be found in Sambrook et al. (Molecular Cloning: .4 Laboratory Manual. 2nd Edition, Cold Spring Harbor Laboratory press (19$9)), 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).
20 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 2~ confer resistance to drugs. such as 6418, hygromvcin and methotrexate.
Nucleic acid encoding a selectable marker may be introduced into a host cell on the same vector as that encocing the peptide compound or may be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selectian (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
30 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: 81S-818; Wolff e1 al. (;1990) Science 247:146-1468).
A delivery apparatus (e.g., a "gene gun") for injecting DNA into cells in vivo can be used.
35 Such an apparatus is commercially available (e.g., from. BioRad). Naked DNA
can also be introduced into cells by complexing the DNA to a canon, 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. Patent 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 Curie! et al. ( 1991 ) Proc. Nat!. Acad. Sci. US'A 88:8850;
Cristiano et al.
(1993) Proc. Nat!. Acad. Sci. USA 90:2122-2126).
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 Biolouy, Ausubel, F.M. et al. (eds.) Green Publishing Associates, (1989), Sections 9.1U-9.14 and other standard laboratory manuals.
Examples of suitable retroviruses include pLJ, pZI3', pWl? and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines include yrCrip, yrCre, y~2 and yrArn. 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. Nat!. Acad. Sci.
USA
85:6460-6464; Wilson et u1. (1988) Proc. Nut!. Acad. Sci. USA 85:3014-3018;
Armentano et al. (1990) Proc. Nat!. Acad. Sci. USA 87:6141-b145; Huber et al. (1991) Proc. Nat!.
Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Nat!. Acad. Sci. USA
88:8377-8381; Chowdhury et al. (1991) Science '254:1802-18t)5; van Beusechem et al.
(1992) Froc. Nat!. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Nat!. Acad. Sci. USA 89:10892-10895; Hwu et al.
(1993) J.
Immunol. 150:4104-4115; U.S. Patent No. 4,868,116 by Morgan et al., issued on September 29'", 1989; U.S. Patent No. 4,980,286 by Nlorgan et al., issued on December 25'", 1990; PCT Application WO 89/07136 (PGT/US89/00422); PCT
Application WO 89/02468 (PCT/US88/03089); PCT Application WO 89105345 (PCTlUS88/04383); and PCT Application WO 92/07573 (PCT/LTS91/08127)).
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) Bio Techniques 6:616;
Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.

46a 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. Z>;SA 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 I3NA becomes integrated into the host genome (e.g., retroviral DNA).
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 ~ 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. Ytrol.
63:3822-3828; and McLaughlin et al. (1989) J. Yirol. 62:1963-1973). Vectars 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) Ntol. Cell. Biol. 5:325/-3260 can be used to introduce DrIA into cells.
A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) 'roc. ,~'utl. Acact Sci. USA 81:6466-6470; Tratschin 1S et al. (I985) Mol. Cell. Biol. 4:2072-2081: Wondisford et al. (1988) Mol.
Endocrinol. 2:32 39; Tratschin et al. (1984) J Y'irol. S 1:611-619; and Flotte et al. (1993) J.
Biol. Chem.
268:3781-3790).
The invention provides a method for treating a subject for a disorder associated with ~i-amyloidosis. comprising administering to the subject a recombinant expression vector encoding a p-amyloid peptide compound, the carnpound comprising an amino acid sequence having at least one amino acid deletion compared to ~iAP1-;g, such that the ~i-amyloid peptide compound is synthesized in the subject and the subject is treated for a disorder associated with ~i-amyloidosis. Preferably, the disorder is Alzheimer's disease. In one embodiment the recombinant expression vector directs expression of the peptide compound 2S in neuronal cells. In anather embodiment, the recombinant expression vector directs expression of the peptide compound in filial cells. In yet another embodiment, the recombinant expression vector directs expression of the peptide compound in fibroblast cells.
General methods for gene therapy are known in the art. See for example. U.5.
Patent 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.
Patent No. 5,082,670 and in PCT Publications WO 90/0677 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., by Weiss et al., and WO 94/23754 by Major et al. Fibroblasts transduced with genetic material are described in PCT Publication VVO 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 94i04695 by Kapiitt and WO

90/09441 b~Gelier 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~i peptides specifically in astrocytes, is described in PCT Publication WO 93/07280 by Brenner et al. Furthermore, alternative to expression of an A~i peptide to modulate amyloidosis, an antisense oligonucieotide that is complementary to a region of the ~i-amyloid precursor protein mRNA corresponding to the peptides described herein can be expressed in a subject to modulate amyloidosis. General methods for expressing antisense oligonu~leotides to modulate nervous system disorders are described in PCT
Publication WO
95/09236.
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 (3AP1-39 c~ 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 1 ~ therapeutically effective amount of the p-amyloid peptide compound and a pharmaceutically acceptable carrier. The peptide compound can be contacted with natural p-amyloid peptides with a ~i-amyloid peptide compound such that aggregation of the natural ~3-amyloid peptides is inhibited. Moreover, the peptide comps>und can be administered to the subject in a therapeutically effective amount such that the subject is treated for a disorder associated with ~i-amyloidosis, such as Alzheimer's disease.
VIII. Other Embodiments Although the invention has been illustrated hereinbefore with regard to A~3 peptide compounds, the principles described. invorving attachment of a modiying groups) to a peptide compound. are applicable to any amyloidagenic prorein 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.
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., C'ongo red), and have a characteristic red-green birefringent appearance in polarized Light after staining. They also share common uitrastructurai 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 forfn found in particular geographic populations. Isolated forms of amyloid are those that tend to involve a single organ system.
Different amyloids are characterized by the type of proteins) or peptides) present in the deposit. For example, as described hereinbefore, amyloid deposits associated with Alzheimer's disease comprise the ~i-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 ~i-amyloid peptide. The identities of the protein(;s) or peptides) present in amyloid deposits associatedwith 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 ~-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(3 assays described herein.
Modulators can be identified using such assay systems. based on the ability of the modulator to disrupt the ~-sheet structure of the fibrils. Initially, an entire amvloidogenic protein can be 1 ~ modified or. more preferably. a peptide fragment thereof that is known to form fibrils in vitro can be modified (e.g., analogous to Aril-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.
Non-limiting examples of amyloidogenic proteins or peptides. and their associated amyloidogenic disorders. include:
Transthvretin (TTR) - Amyloids containing transthyretin occur in familial amyloid polyneuropathy (Portuguese. Japanese and Swedish types). familial amyloid cardiomyopathy (Danish type). isolated cardiac amvloid and systemic s~.nile amyloidosis.
Peptide fragments 2~ of transthyretin have been shown to form amyloid fibrils in vitro. For example. TTR 10-20 and TTR 10~-1 I ~ form amvloid-like fibrils in 20-30°~o acetonitrileiwater at room temperature (Jarvi:~, J.A., et a1.(1994) Int. J. Pept. Prcatein Res. 44:3$$-39$).
Moreover, familial cardiomyopathv (Danish type) is associated with mutation of Leu at position 111 to Met. and an analogue of TTR 105-11 ~ in which the wildtype Leu at position 111 has been substituted with Met (TTR 10~-l l~Met111) also forms amyloid-like fibrils in vitro (see e.g., Hermansen. L.F.. et al. ( 1995) Eur. J. Bi<~chem. 227:7'2-779; 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. 17w: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 amvloidosis that can be used in flag detection or treatment of familial amyloid polyneuropathv (Portuguese. Japanese and Swedish types). familial amyloid cardiomyopathy (Danish type). isolated cardiac amyloid or systemic senile amyloidosis.

Priors 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 PrPSe (the priors protein associated with scrapie) leads to a 27-30 kDa 5 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; Gasser, 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 10 of the priors protein involved in CJ), in the regions of codon 17$ and codon 200, spontaneously form amyloid fibrils in vitro (see e.g., Coldfarb, 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-I47 of human PrP has been reported to form twisted 15 fibrils resembling scrapie-associated fibrils (Tagliavini. F.. er 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 20'?-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 20 PrP from other species (Gasset, M., et al. (1992) Proc. Natl. Acad Sci. USA
$9:10940-10944). A peptide fragment of PrP that forms amyloid fibrils can be modified as described herein to create a modulator of amyloidosis that can be used in the detection or treatment of scrapie. bovine spongifonm encephalopathy, Creutzfeldt-Jakob disease or Gerstmann-Straussler-Scheinker syndrome.
25 Islet Amvloid Polvpeptide (IAPP. also known as amylin) - AmyIoids containing IAPP
occur in adult onset diabetes and insulinoma. IAPP is a ~7 amino acid polypeptide formed from an 89 amino acid precursor protein (see e.g., Betsholtz. C., et al.
(1989) Exp. Cell. Res.
183:484-493; Westennark, P., et al. (1987) Proc. Natl. ,Acad. Sci. USA 84:3881-3885). A
peptide corresponding to IAPP residues 20-29 has been reported to form amyloid-like fibrils 30 in vitro, with residues 25-29, having the sequence Ala-Ile-Leu-Ser-Ser (SEQ
ID NO: 18), being strongly amyloidogenic (Westermark, P., et al. ( 1990) Proc. Natl. Acad Sci. USA
87:5036-5040: Glenner, G.G., et al. (1988) Biochem. Biophvs. Res. Commun.
155:608-614).
A peptide fragment of IAPP that forms amyloid fibrils can be modified as described herein to create a modulator of amyloidosis that can be used in the detection or treatment of adult onset 35 diabetes or insulinoma.
Atrial Natriuretic Factor (ANF) - Amyloids containing ANF are associated with isolated atrial arnvloid (see e.g., Johansson, B., et al. (1987) Biochem.
Biophys. Res.
Commun. 14$:1087-1092). ANF corresponds to amino acid residues 99-126 (proANF99-I26) ofthe ANF prohonnone (proANPl-126) (Pucci. ~~... et al. (1991) J. Pathol.
165:235-241 ). ANF, er a fragment thereof; that forms amyloid fibrils c4m be modified as described herein to create a modulator of amyloidosis that can be used in the detection or treatment of isolated atriaI amyIoid.
Kappa .5r Lambda Lisht Chain - Amyloids ~=~,~n~~nQ kappa or lambda light chains S are associated idiopathic (primary) atnyloidosis, myeloma or macroglobulinemia-associated amyloidosis, and primary localized cutaneous nodular amyloidosis associated with Sjogren's syndrome. The structure of amyloidogenic kappa and lambda ligh~ chains, including amino acid sequence analygis, has been characterized (see e.g., ~uxbaum. J.N., et al. (1990) Ann.
Intern. Med. 112:45-464; Schermann. N., et al. (1995) Proc. avarl. Acad Sci.
USA 9x:9490-9494; Hurle, M.R., et al. (1994) Proc. .~atl. .,4cad. ScT. ~~~;4 91:5446-5450:
Liepnieks, J.J., et al. (1990) ~l~lol. Immunol. 27:481-485; Gertz, M.A.. er al. ~' 1985) Scand J.
Immunol. ~~:245-250; Inazumi, T., et al. (1994) Dermatology 1$9:I~S-12$L. Kappa or lambda light chains, or a peptide fragment thereof that forms amy loid fibrils, can be modified as descrioed herein to create a modulator of amvloidosis that can be used in the detection or treatmew of idiopathic 1 S (primary) amvloidosis. mveloma or macroglobulinemia-associated amyloidosis or primary localized cutaneous nodular amyloidosis associated with Sjogren's :.yndrome.
Amvloid A - Amyloids containing the amvIoid A protein (AA protein), derived from serum amvloid A, are associated with reactive (secondarv~~ j amvloidosis (see e.g., Liepnieks, J.J., et al. (1995) Biochim. Biop~ys. .4cta 12?0:81-86), familial Mediterranean Fever and familial amyloid .nephropathy with urtiearia and deafness (iViuckle-Wells syndrome) (see e.g., Linke. R.P., et al. (1983) Lab. Invest. 4$:698-7()4;E. Recombinant human serum amyloid A
forms amyloid-like fibrils in vitro (Yamada, T., er aa', ! 1994) B. ochim.
Biophys. Acta 1226:323-391 and circular dichroism studies revealed a predominant ~i sheet/turn structure (McCubbin. W.D., et al. (198$1 Biochem J. ~~6:7~S-i83'). Sertam amyioid A.
amyloid A
2S protein or a fragment thereof that forms amyloid fibrils can be modified as described herein to create a modulator of amvloidosis that can be used in the detcetion or treatment of reactive (secondary) amyloidosis, familial :'Mediterranean Fever arid familial amyloid nephropathy with urticaria and deafness (Muckle-Wells syndrome).
Cvstatin C - Amyloids containing a variant of cystatin C are associated with hereditary cerebral hemorrhage with amyloidosis of Icelandic type. The disease is associated with 2 leucine to glycine mutation at position 68 a.~d cystatin (..::.
containing this mutation aggregates in vitro (Abrahamson, M. and Grubb, A. ( 1994) Prvc. 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 amvloidosis that can be used in the 3S detection or treatment of hereditary cerebral hemorrhage r~zth amyloidosis of Icelandic type.
~i2 microslobulin - Amyloids containing X3'2 mi~croglobuiin ( j32M) are a major complication of long term hemodialysis (see e.g., Stein, G., et al. (1994) Nephrol. Dial.
Transplant. 9:48-S0; Floege, J.. et al. ( 19921 Kidney Int. Suppl. 38:S'78-SBS; Maury, C.P.
(1990) Rheumatol. Int. 10:1-8). The native ~i2M protein has been shown to form amyloid y~
fibrils in vitro (Connors, L.H.. et al. (1985) Biochem. rliophys. Res.
Corrunun. 131:1063-1068; Ono, K., et al. (1994) Nephron ti6:40:~-407j. Vii:-'M. or a peptide fragment thereof that forms amyloid fibrils, can be modified as described herein to create a modulator of amyloidosis t.a~t can be used in the detection or rtes tment of amyloidosis associated with long tezzn hemodiaIysis.
Apolipoprotein A-I (ApoA-I) - Amyloids containing variant forms of ApoA-I have been found in hereditary non-neuropathic systemic amyIoidosis (famili~~I
amyloid polyneu~opathy III). For example. N-terminal fragments ;residues 1-86. 1-92 and 1-93) of an ApoA-I variant having a Trp to Arg mutation at position ~f~ have been detected in amyloids (Booth, D.R., et al. (1995) OJl'1~188:695-702). In another family. a leucine to arginine mutation at position 60 was found (Soutar, :3.K.. er al. ( 1 u92) I'roc. Natl.
.=lcad Sci. L~'SA
89:?389-7393). ApoA-I or a peptide fragment thereof that forms amyloid fibrils can be modified as described herein to create a modulator of amvloidosis that can be used in the detection or treatment of hereditary non-neuropathic systemic ;;~myloidosi5.
I ~ Gelsolin - Amyloids containing variants of ~elsoliz~ are associated with familial am~; loidosis of Finnish type. Synthetic ~elsolin peptides t~h.at have sequence homology to wildtype or mutant gelsolins and that form amyloid fiQrils ~n vitro are reported in Maury, C.P. er al. (I994) Lab. Invest. 70:»8-j64. .A nine residue segment surrounding residue 187 (which is mutated in familial gelsolin amyloidosis) was defined as an amvloidogenic region (Maury, et al., supra: see also Maury. C.P.. et al. ( 3 99?) Bioc~aem.
Biophvs. Res. Commun.
183:227-='31; Maury. C.P. (1991) J. Cllr. Invest. 8?:I 19~-1199). Gelsolin or a peptide fragment thereof that forms amyloid fibrils can be modified as described herein to create a modulator of amvloidosis that can be used in the detection or treatment of familial amyioidosis of Finnish type.
Procalcitonin or calcitonin - Amvloids containing procalcitonin. calcitonin or calcitonin-like immunoreactivity have been detected in arnyloid fibrils associated with meduilary carcinoma of the thyroid (see e.o., butler. fit. :~n.d Khan. S.
(1986) Arch. Pathol.
Lab. Med. 110:647-649; Sletten, K., et al. (1976) J. E;xp. Med. 143:993-998).
Calcitonin has been shown to form a nonbranching fibrillar structure in vuro (Kedar, L, er al. ( 1976) Isr. J.
Med. Sci. 12:1137-1140). Procalcitonin. calcitonin or a fiagrnent thereof chat forms amyloid fibrils can be modified as described herein to create a rl~odulator of amyloidosis that can be used in the detection or treatment of amyloidosis associated writh medullary carcinoma of the thyroid.
Fibrinoeen - Amyloids containing a variant form of fibrinogen alpha-chain have been 3~ found in hereditary renal amyloidosis. An arginine to Ieucine mutation at position ~~4 has been reported in amyloid fibril protein isolated from posttrortem kidney of an affected individual (Benson. M.D., et al. (1993) t~'a~ure Generic.°. x:25'2-25~). Fibrinogen alpha-chain or a peptide fragment thereof that forms amvloid fibrils can be modified as described herein 1~
to create a modulator of amyloidosis that c;u ~e used n she detection or treatment of fibrinogen-associated hereditary renal arnvloidosis.
Lvsozvme - Amyloids containing a variant Porn of lysozyzne have been found in hereditary systemic amyloidosis. In one family the: dise;~e was associated with a threonine to isoleucine mutation at position ~6, whezeas in another fa~-nily the disease was associated with a histidine to aspartic acid mutation at position 6'7 (Pepys, M.B.. er al. ( 1993) ~~'ature 362:>j3-»7) Lysoryme or a peptide fragment thereof that forms amyloid fbrils can be modified as described herein to create a modulator of am loidosis that can be used in the detection or treatment of lysozyme-associated hereditarsystemic amyloidosis.
This invention is further illustrated by the following examples which should not be construed as limiting. A modulator's ability to alter the a~~~,~regation of ~-amyloid peptide in the assays described below are predictive of the modulator's abilin.~ to perform the same function in vivo.
1~
EX.4MP'_,E 1: Construction of ~3-.~.myloid ylodulators A (i-amvloid modulator composed of an amino-terminally biotinvlated p-amyloid peptide of the amino acid sequence:
DAEFRHDSGYEVHHQKLVFF:~EDVGSNKG:~",IIGL~IVGGVV
(positions 1 to 40 of SEQ ID NO: 1 ) was prepared by solid-phase peptide synthesis using an Na-9-fluorenvlinethyloxvcarbanyl (FivIOC)-based protection strategy as follows. Starting with''. mmoles of FMOC-Val-Vvang resin. seauential additions of each amino acid were 23 performed using a four-fold excess of protected amino acids. 1-hvdraxybenzotriazole (HOBt) and diisoptopyl carbodiimide (DIC;1. Recouplings were performed when :.ecessary as detemuined by ninhydrin testing of the resin after coupling. Each synthesis cycle was minimally described by a three minute deprotection (?~ ° o piperidine/N-methyl-pyrrolidone (NMP)), a 1 ~ minute deprotection, f ve one minute NI~tP washes, a 60 minute coupling cycle.
five NMP washes and a ninhydrin test. To a 704 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) (8?.~ °~'o), water (5 %), thioanisole (5 %), phenol (5 %). ethanedithiol (2.5 %) for two hours followed by precipitation of tine peptide in cold ether. The solid was pelleted .by centrifugation (2400 rpm x 10 min.).
and the ether decanted. It was resuspended in ether, pelleted and decaaited 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 2~ °/'°
acetonitrile (ACN) /0.I % TFA and applied to a C I8 reversed phase high performance liquid chromatography (HPLC) column.

1 ~.
Biotinyl (3A-I'l~p was eluted using a linear ~.Tradient of 30-q~ c~o acetonitril~.r0.1 % TFA over 40 minutes. One primary fraction (~ mg) and several side fractions were isolated. Tne main fraction yielded a mass spectrum of45~6 (matrix-assisted laser desorption ionization -time of flight) which matches the theoretical (4~55~ for this peptide.
A ~3-amyloid modulator composed of an amino-terminally biotinylated ~i-amyloid peptide of 21-.e amino acid sequence:
DAEFRIVDSOYEVHHQ
(positiolrs 1 to 1 ~ of SEQ ID NO: 1 ) was prepared on an Advanced them l-ech Modet 3y6 multiple peptide synthesizer using an automated protocol established by the manufacturer for IO 0.02 mmole scale synthesis. Double couplings wore pezdormed on all cycles using ?-(1H-benzotriazol-1-yl)-1,I.3,3-tetramethyluronium hexafluorophosphate (HBTTT)/N.N-diisopropylethylamine (DIEA')IHOBt/FMOi~-AA in four-fold excess for 30 minutes Followed by DIC/HOBv'FMOC-AA in four-fold excess for =I~ mirut~a. The peptide was deprotected and removed from the resin by treatment with ~l"F~~water 1 '~~ °'oi~
°~o) for three hours and I6 precipitated with ether as described above. The pellet w~~is resuspended in I 0 °'° acetic acid and lyophilizEd. The material was purified 1y a prepara.iv~~: HI'LC using I ~
°,'0-40 acetonit_rile over 80 minutes on a Vvdac C 18 column (? 1 :~: X50 mm). The main isolate eluted as a single symmetrical peak when analyzed bs~ anaiyical HPI_C and yielded the expected molecular weight when analyzed by electrosprav mass spectrometry. Result --?02.6 (2052 2G theoretical).
~i-amyloid modulator compounds comprisin<~ other regions of the ~3-AP amino acid sequence (e.g.. an A~3 aggregation core domain;9 were sir,~ilarly prepared using ~:he synthesis methods described above. Moreover. modulators comprising c>ther amyloidogenic peptides can be similarly prepared.
EXAMPLE 2: Inhibition of ~i-Amvloid Aggregation by Modulators The ability of ~3-amyloid modulators to inhibit the aggregation of natural ~i-AP when combined with the natural ~i-AP was examined in a series of aggregation assays. Natural ~i-30 AP (~i-API~p) was obtained commercially Pram Bachem ~~Corrance, CA). Amino-terminally biotinylated ~-AP modulators were prepared as described in Example I.
A. Optical Density Assay In one assay, ~3-AP aggregation was measured by determining the increase in turbidity 35 of a solution of natural (3-AP over time in the absence or ,c~rcaence of various concentrations of the modulator. Turbidity of the solution was quantitate:d by determining the optical density at 400 nm (A4oo nm) of the ~>olution over time.
The aggregation of natural ~3-AP in the absence of modulator was determined as follows. ~i-AP 1 ~p was dissolved in hexafluoro isopropanol (HFIP; Aldrich Chemical Co., '~ 5 inc.) at '_' mglml. Aliquots of the HFIP solution ($7 ~1) were transferred to individual 1 U mm x 7~ mm test tubes. A stream of argan gas was passed through each tube to evaporate the HFIP. To the resulting thin f lm of peptide, dimethylsulfoxide (DMSO; Aldrich Chemical TM
Co., Inc.) (~~ p,l) was added to dissolve the pep:ide. A ,." mrn x 7 mmTetlon-coated magnetic stir bar was added to each tube. Buffer {4?~ p.L of 1 UO m~mivl i~;faCl. 1 U
mM sodium phosphate, pH 7.4) was added to the DMSO solution with stirring. The resulting mixture was stirred continuously ancthe optical density was monitored at 4UU nm to observe the formation of insoluble peptide aggregates.
Alternatively, ~3-AP~~o was dissolved in DMSO as described above at 1.6 mM (6.9 mg/ml) and aliquots (25 ul) were added to stirred buffer {4'7~
~l), followed by monitoring of absorbance at 400 nm.
For inhibition studies in which a ~i-amyloid modulator was dissolved in solution together with the natural ~i-AP. the modulators were dissolved in DMSC) either with or without prior dissolution in HFIP. These c;ompaunds w~c~re then added to buffer with stirring.
1 ~ followed by addition of ~3-AP ~ _;~p in DI~iSO. Alternatively. HFIP
solutions of modulators were combined with (3 .AP ~ ~o in HFIP followed by evaporatior: and redissolutio:~ of the mixture in DivISO. Buffer was then added to the DMSO salution to initiate the assay. The amino-terminally biotinylated ~i-amyloid peptide modulators N-biotinyl-~3APi~o, and N-biotinyl-(3AP~_~5 were tested at concentrations of 1 °~o and ~
°ro in the natural ~i-AP~~o 2U solution.
A representative example of the results is showrn graphically in Figure 1, which ~:jepicts the inhibition of aggregation of natural ~i-AP ~ _<«y by 'v-hiotinyl-~3AP; _,~p. In the absence of the modulator. the optical density of the natural ~i~AP solution showed a characteristic sigmoidal cuwe. with a lag time prior to aggre~,~sation (approximately hours in Figure 1 ) in which the A,~oO nm w'as low , ioilowed by rapid increase in the A40o nm9 Which quickly reached a plateau level. representing aggregation of the natural ~i amvloid peptides.
In contrast. in the presence of as little as l % of the N-biotinyl-~3AP~~p modulator, aggregation of the natural ~i amyloid peptides was markedly inhibited.
indicated by an increase in the lag time, a decrease in the slope of aggzegation and a decrease in the plateau 30 level reached for the turbidity of the solution (see Figure I ). N-biotinyl-~3AP 1 ~p zt a concentration of S °% similarly inhibited aggregation of the natural ~3 amyloid peptide.
Furthermore, similar results were observed when N-biotinyl-~iAP 1 _ 15 "vas used as the modulator. These results demonstrate that art N-terminally biotinylated ~ .AP
modulator can effectively inhibit the aggregation of natural ~ amylaid peptides, even when the natural ~i 35 amyloid peptides are present at as much as a 1 UO-fold molar excess concentration.
B. Fluorescence Assay In a second assay. ~i :4P aggregation was measured using a fluorometric assay essentially as described in Levine, H. (1993) Protein SGZ~n~~ 2:404-41U. In this assay, the ~7 dye thioflavirte T (ThT) is contacted with tile ~3-AP solution. Association of ThT with aggregated ~i-AP, but not monomeric or loosely associated ~i-AP, gives rise to a new excitation (ex) maximum at 450 tun and an enha:~ced emission (em) at 482 nm, compared to the 385 ntn (ex) and 445 nm (em) for the free dye. ~i-AP aggregation was assayed by this method as follows. Aliquots (2.9 ~tl) of the solutions used in the aggregation assays as described above in section A were removed from the samples and diluted in 200 p.1 of potassium phosphate buffer (SO mM, pH 7.0) containing thiollavin T (10 ~tM:
obtained commerFially 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 g '~o of the N-biotinylated (3-AP
modulators was effective at inhibiting the aggregation of natural (3 a.myloicl F;eptides using this fluorometric assay.
C. Static :Agare~ation Assay 1 ~ In a third assay, (3-AP aggregation was measured by visualization of the peptide aggregates using SDS-polyacrylamide gel electrophoresis (SI)S-PAGE). In this assay. (3-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. T~.~pic:al solution conditions were 200 ulvl of ~3-AP ~ _ 4p in PBS at 37 °C for 8 days or 200 pMI ~3-~AP~_.~~ in 0.1 M sodium acetate at 37 °C for 3 days. The peptide aggregates were visualized by ~: oomassie blue staining of the gel or, for ~3-AP solutions that included a biotinvlated ~i-Ah modulator, by western blotting of a filter prepared from the gel with a streptavidin-peroxidase probe. followed by a standard perc;xvdase assay. The (3-AP aggregates are identifiable °us high molecular weight. low mobility bands on the gel. which ai-e readil.~~ distinguishable i:~om the low molecular weight.
high mobility ~3-AP monomer or dimer bands.
When natural ~i-APl_4o ag~re'Tatior: was assayed by tlTis method in the absence of any ~i amyioid modulators, high molecular weight aggregate<~ were readily detectable on the gel.
In contrast, when N-biotinyl-~3-AP l ~~ modulator self aggregation was assayed (l. e..
aggregation of the N-biotinyl peptide alone, in the absence of any natural ~3-AP), few if any high molecular weight aggregates were obsen°ed. indicating that the ability of the modulator to self aggregate is significantly reduced compared to natural ~3-AP. Finally, when aggregation of a mixture of natural ~i-AP 1 _,~p and iii -'bioti.nylated (3-AP
l _4o was assayed by this m°_thod, reduced amounts of the peptide mixture associated into high molecular weight aggregates. thus demonstrating that the ~i amyloid modulatar is effective at inhibiting the aggregation of the natural ~i amyloid peptides.

EXAMPLE-3: Neui otoxicity Analysis of ~i-Am;yloid Modulators The neurotoxicity of the ~3-amyloid modulators is tested in a cell-based assay using the neuronal precursor cell line PC-12, or primary neuronal cells, and the viability indscator 3,(4.4-dimethvlthiazol-2-;yl)2,~-diphenyl-tetrazalium brc~mido (MTT). (See Sheatman. M.S.
et al. (1994) Proc. Natl. Aced. Sci. USA 91:1470-I474~ Hansen, M.B. er al.
(1989) J. Immun.
Methods 119:203-210). PC-12 is a rat adrenal pheochramacwnoma cell line and is available from the American,Type Culture Collection, F~ockville, MD (ATCC CRL 1721 ).
MTT
(commercially available fiom Sigma Chemical C:o. ) is a chramogenic substrate that is converted from yellow to blue in viable cells. which can be detected spectrophotometrically.
To test the neurotoxicity of a (3-amvloid modulator (either alone or combined with natural (3-AP). cells first are plated in 96-well plates at i.000-10,000 cells/well and allowed to adhere by overnight culture at 37 °~. Serial dilutions of freshly dissolved or "aged"
modulators (either alone or combined with natural ~-~4P' in phosphate buffered saline (PBS) 1 ~ 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 °C
undisturbed for a prolonged period (e.g., five days car morej. For the final two hours of exposure of the cells to the modulator preparation, ivITT is. added to the media to a final concentration of I mg/ml and incubation is continued at :>7 °C'.
Following the two hour incubation with MTT. the media is remcwed and the cells are lysed in isopropano1/0.4N HCl with agitation. An equal volume oa PBS is added to eac't~ well and the absorbance of each well at X70 nm is measured to quantitaze viable cells. .~,iternazively. MTT is soIubilized by addition of ~0 % N.N-dimethvl fornamide:'20 % sc3diurr~ dadecvl sulfate added directly to the media in the wells and viable cells are likewise quantita.zed by measuring absorbance at S70 2~ nm. The relative neurotoxicir, of a ~3-amyloid modulazcar~ !;eiti~er alone or in combination with natural ~3-AP;) is determined by comparison to natural ~-AP alone (e.g., X31-40, ail--:2), whic:a exhibits neurotoxicity in this assay and thus can serve as a positive control.
EXAMPLE 4: Syntheses of Additional i~Iodified ~i-r~mvloid Peptide Compounds In this example. a series of modified ~i-APs. having a variety of N-terminal or random side chain modifications were synthesized.
A series of N terminally modified ~-amyloid peptides was synthesized using standard methods. Fully-protected resin-bound peptides corresp~rnding to Ap(1-15) and Ap(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 pmoles, respectively) were aliquoted into the wells of the reaction black 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 PIMP followed by extensive washing j~
with NMP.-The unprotected.~V-terminal ~-amino group of each peptide-resin sample was modified using one of the following methods:
Method A, coupling of modifying reagents containing free carboxylic acid groups:
The r-codifying reagent (five equivalents) ~,, :-~s predissoived in NMP, DMSU
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 wag allowed to proceed overnight, followed by washing.
If a ninhyd~in 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.
Method B, coupling of modifying reagents obtained in preactivated forms: The modifying reagent (five equivalents) w°as predissalv~d in N~MP. DMSG or a mixture of these two solvents and added to one equivalent of peptide-resin.
I)iisopropylethylamine (JIEA;
six equivalents) was added to the suspension of activated modifier and peptide-resin.
Coupling was allowed to proceed overnight. followed by washing. If a ninhvdrin test nn a 1 ~ small sample or peptide-resin showed tha: couplinn w;;~s not complete. the coupling was repeated.
After the second coupling (if required) the .~'-terminally modified Feptide-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 con: rm the presence of the desired compound in the product.
Method A was used to couple ~'~~-acetylneuraminic acid, cholic acid_ traps-4-cotininecarboxvlic acid. ?-imino-l -imida.~olidineacetic:. acid, (.S~-{-)-indoline-2-carboxylic acid. (-)-menthexvacetic acid, ?-norbomaneacetic acid, w~-oxc}-~-acenaphthene~butyric acid.
(-}-2-oxo-:~-thiazolidinecarboxylic acid, and tetrahydra-~-furoic acid. Method B was used to couple 2-iminobiotin-:V-hydroxysuccinimide ester. diethylenetriaminepennacetic dianhydride, 4-morpholinecarbonyl chloride. 2-thiopheneacetyl chloride. and 2-thiophenesulfonyl chloride.
In a manner similar to the construction of 'V-terminal )y modified All( 1-15 ) and Ap( 1-~0) peptides described above, N fluoresceinyl A(i( I-17 f and Ap(1-40) were prepared in two alternative manners using the preactivated reagents ~-(and 6)-carboxyfluortscein succinimidyl ester and fluorescein-~-isothiocyanate (FITC Isomer I}. Botl-~
reagents were obtained from Molecular Probes Lnc. 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 Ap(I-1 ~)-resin appeared to be complete after a single overnight coupling. Coupling to Ali(1-40)-resin was slower as indicated by a positive ninhydrin test and both reagents were recaupled to this peptide-resin overnight in >9 tetrahydrofuran-NMP (l :? v/v). 'Ihe resulting ,'V-terminally modified peptide-resins were cleaved, deprotected and purified as described in Example A.
In addition to the N fluoresceinyl A~3 peptides described above, a [i-amyloid modulator comprised of random modificatiorx o ~' ~ Vii( 1-:~0) with fluorescein was prepared.
S Ap(1-40) purchased from Bachem was dissolved in DMSO at approximately 2 mg/mL. S-(and-6)-Carboxyfluorescein purchased from Molecular Probes was added in a 1.S
molar excess and DIEA was added to make the solution basic to wet pH paper. The reaction was allowed to proceedfor 1 hour at room temperature and was then quenched with triethanolamine. The product was added to assays as this crude mixture.
[i-amyloid modulator compounds comprising ot~uer regions of the [i-AP amino acid sequence (e.g , an A[3 aggregation core domain) were similarly prepared using the synthesis methods described above. Moreover, modulators comprising other amvloidogenic peptides can be similarly prepared.
1S EXAMPLE 5: Identification of Additional (3-..:~myioid iYlodulatars In this Example, two assays of A~i aggregation were used to identiy [i-amyloid modulators which can inhibit this process.
The first assay is referred to as a seeded static assay (SSA) and was performed as follows:
To prepare a solution of A(3 monomer. the appropriate quantity of A~( 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 n.uml-~er, vvas ?0-30% wiw).
The peptide was dissolved in 1I?S volume of dimethysulfoxide (DMSO). followed by water to 1!?
2S volume and 1!2 volume ?x PBS (10x PBS. NaCI i ~ i m:'~1. Kiel ?.? mM
Na~HPG.~ ~ 7H?O
4.3 mM. KH~PO~ 1.4 mM pH x.23 to a final concentration o3~200 wM.
To prepare a stock seed, 1 ml of the above A[3 monomer preparation, was incubated for 8 days at 37 °C and sheared sequentially through an 1.8, ?:~, 26 and 30 gauge needle 2S, ~S, S0, and 100 times respectively. ~ pI samples of the sheared material was taken for fluorescence measurements after every SO passes through the 30 gauge needle until the fluorescence units (FLT) had plateaued (approx. 100-150:x;).
To prepare a candidate inhibitor, the required amount of candidate inhibitor was weished out and the stock dissolved in lx PBS to a final concentration of 1 mM
(10x stock).
If insoluble. it was dissolved in 1/10 volume of DMSO and diluted in lx PBS to 1 mM. A
further 1/10 dilution was also prepared to test each candidate at both 100 pM
and 10 uM.
For the aggregation assay, each sample was set up in triplicate [SO u1 of 2G0 ~M
monomer, 12S FU sheared seed (variable quantity dependent on the batch of seed. routinely 3-b p1), 10 p1 of lOx inhibitor solution, final volume made up to 100 p1 with lx PBS]. Two concentrations of each inhibitor were tested 100 ~lvi and 10 ~clvl, equivalent to a l : l and a fin 1:10 molar ratio of monomer to inhibitor. The controls included an unneeded 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. T'ne assay wan incubated at 37 °C for 6 :. taking 2 u1 samples hourly far flucacescence measurements. To measure TM
fluorescence, a 2 ~xl sample of A~i was added to 400 ~1 of Thiaflavin-T
solution (.50 mM
TM
Potassium Phosphate 10 m:~f Thiofiavin-T pH 7.~). The samples were vortexed and the fluorescence was read in a O.a ml micro ;luartz cuverte at EX 450 not and Eli 482 nm (Hitachi 400 ~luorimeter). ~-aggregation results in enhanced emission of Thioflavin-TTM
Accordingly. samples including an effective inhibitor catnpound exhibit reduced emission as compared to control samples without the inhibitor compound.
The second assay is referred to as a shaken plate aggregation assay and was performed as follows:
A~3(1-40) peptide from Bachem (Torrance. C:~)'was dissolved in HFIP
(1,1,1,3,3,3-Hexafluoro-?-propanol: Aldrich 10.5''?-8) at a concentration of? m; peptideiml and incubated at room temperature for 30 min. HFIP soiubilized peptide was sonicated in a waterbatin sonicator for 5 min at highest settin?. Then evaporated to dryness under a stream of argon. The peptide film was resuspended in anhydrous dimethylsulf~.~xide (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. i~o. 28196-050). ~:andidate inhibitors were dissolved directly in DMSO, generally at a molar concentration 4 times that of the A~3(1-40) peptide.
Candidates were assayed in triplicate. Far eacn candidate to be tested, 4 parts A(3(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 .~~i peptide to candidate. For different molar ratios.
candidates were diluted with DMSO prior tc~ addition to A~i(1-40). in order to keep the final DMSO .and A~i(1-40) concentrations constant. Into an ultra low binding 96 well plate (Corning Costar cat. No. 200. Cambridge 11A) 100 y~l PTL buffer ( 1 ~0 mM NaCI, 10 mM
NaH~P04;
pH 7.4) was aliquotted per well. For each candidate. 10 p1 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 far 30 seconds. An additional 100 ~1 of PTL buffer was added to each well and again the plate .vas vortexed vigorously for 30 sec. Absorbance at 405 nm was immediately read in a plate reader for a baseline reading. The plate was re~_urrted to the plate shaker and vortexed .at moderate speed for ~ 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.
Representative results of the static seeded assay and shaken plate assay with preferred (3-amyloid modulators are shown below in Table I.

TABLE I
(> 1 Candidate A Amino Modifying Effect in Effect in Inhibitor l Acids ~ Reagent shaken plate~ Seeded Static ;

I ' assay ~ Assay Chafic acid Complete l "~",~, ~

l 174 A~1-15 l ; inhibition ;
at 100% cone l ~ Diethyiene- Decreased , ;

176 ~ A(31-15 triamine yentaPlateau acetic acid l (-)-Menthaxy None 180 A~31-15 acetic acid , ~
l j ' Fluorescein Decreased 190 A~i1-15 carboxylic Plateau acid l ' (!~"ICC~) -~~

h-EVHHHfiQ~K-Complete 220 A(316-40 ~~~ f ~'~''tG)a-CHinhibition at t a()%, ;ncreased mutant ~~ag at ~o l ~-~ Increased ,~,~, fag ~

224 A~i 1-4O F~9F2o''T~sTzoI
~

mutant '~ ! _~
~l ~~

accelerated +,~, , Acetic acid a re ation 233 A6~i-20 at ' gg g a 10r~ cant j * A strong inhibitor ~"~' of aggregation _ The rate of aggregation in the presence of decreased the inhibitor compared srras to the control by at least 30- 50~0 T"nese results indicate that ~i-APs modified by a wide variety of N-terminal modifying grour~s are effective at modulating ~i-amyloid aggregation.
EXAMPLE 6: Additional ~i-Amyloid Aggregation Assays Most preferably, the ability of ~3-amyloid modulator compounds to modulate (e.g., inhibit or promote) the aggregation of natural ~3-:'~P when combined with the natural ~i-AP is examined in one or both of the aggregation assays descri°rred below.
Natural ~i-AP ((3-AP 1 ~G) for use in the aggregation assays is commercially available from Bachem (Torrance, CA).
A. Nucleation Assav The nucleation assay is employed to determine the ability of test compounds to alter (e.g. inhibit) the early events ir. formation of ~3-AP fibers from monomeric (3-AP.
Characteristic of a nucleated polymerization mechanism. a lag time is observed prior to f>
nucleation, alter which the peptide rapidly forms fiber: its reflected in a linear rise in turbidity. The time delay before polymerization of ~-AP mo.namer can be quantified as well as the extent of formation of insoluble fiber by light scattering (turbidit~~). Polymerization reaches equilibrium when the maximum tur''..~ idity reaches a plateau. The turbidity of a solution of natural ~i-AP in the absence or presence of various concentrations of a ~i-amyloid modulator compound is determined by measuring the apparent absorbance of the solution at 405nm (A405 nm) over time. The threshold of sensitivit~r for the measurement of turbidity is in the rage of 15-20 ~M (3-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 ~3-.~P fibers from monomeric (3-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 tc~ a 9t~-well plate format to screen multiple compounds.
To perform the nucleation assay. first .-'~(3 ~ _~p peptide is dissolved in I-IFIP
(I.1,I,3,3.3-Hexafluoro-2-propanol: Aldrich 1t1.52~'-8) pct a concentration of 2 mg peptide/ml and incubated at room temperature for .~0 min. HFIP-solubilized peptide is sonicated in a waterbath sonicator for 5 min at hi;~hest setting, then evaporated to dryness under a stream cf argon. The peptide film is resuspended in ;anhydrous dirnethy°lsulfoxide (DMSO) at a concentration of 6.9 mgiml (25x coneentrariorx), sonicated for 5 min as before. then filtered through a 0.2 micron nylon syringe filter (VWR cat. Nc>. 28196-050). Test compounds are dissolved in DMSO at a 100x concentrations. Four volurrtes of 25x A~3l..~p peptide in DMSO
are combined with one volurr~e of test compound in Di~'i;~0 in a glass vial.
and mixed to produce a I :l molar ratio of A~3 peptide to nest compound. For different molar ratios, test compounds are diluted with DMSC) prior to addition to .~,~3~~0, in order to keep the final DMSO and A~3l~o concentrations constant. C:°ontrol sarrtples do not contain the test compound. Ten microliters of the mixture is then added to the bottom of a well of a Coming Costar ultra low binding 96-well plate (Coming Costar, ~~ambridge MA; 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 ~t1 of 2x PTL buffer (20 mM NaH2P04, 300 mM NaCI, pH 7.4) is added to the ~,~~ell, 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. Tl°~e plate is then returned to the shaker and shaken continuously for ~ hours. Turbidity readings are taken at 15 minute intervals.
~i-amvloid aggregation in the absence of any modulators results in enhanced turbidity of the natural ~i-AP solution (i.e., an increase in the apparent absorbance at 405 nm over time). Accordingly. a solution including an effectiv°e inhibitory modulator compound exhibits reduced turbidity as compared to the control sample without the modulator f) 3 compound (~.e.. less apparent absorbance at ~0~ r.m over time as compared to the control sample).
B. Seeded E ~aension Assay The seeded extension assay can be employed to measure the rate of A~i fiber fotTned in a solution of A~i monomer following addition of polymeric; A~ fiber "seed".
The ability of test compounds to prevent further deposition of moaomeric A~i to previously deposited amyIoid is determined using a direct indicator of ~i-sheet formation using tZuorescence. In contrast with the nucleation assay, the addition of seed provides immediate nucleation and continued ~row~th of preformed fibrils withcaut the need for continuous mixing, and thus results in the absence of a lag time before polvmezization stares. Since this assay uses static polymerization conditions, the activity of positive compounds in the nucleation assay can be confirmed in this second assay under differE:nt conditions and with an additionzi probe of amvloid structure.
I ~ In the seeded extension assay. monomeric A~i ~ _4~ is incubated in the presence of a "seed" nucleus (approximately ten mole percent of A~i 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-st~ecific association of"'fh- I' with A~i produces a fluorescent complex that allows the measurement ofthe extent of fibril formation (Levine, H.
(1993) Protein Science 2:4()4-4I0). In particular. association of Th-'t with aggregated ~i-AP, but not monomeric or loosely associated ~3-~P, gives rise to a new' excitation (ex) maximum at 4~0 nm and an enhanced emissiorx (emj at 48 2 nrn, compared to the 38~ nm (ex) and 44~
nm (em) for the free dye. Small aliduots of the p:alymeria:ation mixture contain sufficient fibril to be mixed with Th-'C to allow the monitoring of tine reaction mixture by repeated 2~ sampling. A linear erowth curve is observed in the presence of excess monomer. The formation of thioflavin T responsive ~3-sheet fibrils parallels tl:~e increase in turbidity observed using the nucleation assay.
A solution of A~i monomer fs~r use in the seeded extension assay is prepared by dissolving an appropriate quantity ofA~ii~Q peptide in 1,"25 volume of dimethysulfoxide (DMSO), followed by water to I/2 volume and 1/2 volurrae 2x PBS (10x PBS: NaCI
137 mM, KCl 2.7 mM Na2HP04 ~ 7H20 4.3 mM, KH2PO4 1.4 mM pH 7.2) to a final concentration of 200 ~M. To prepare the stock se~;d, 1 ml of the A~i monomer preparation. is incubated for approximately 8 days at 37 °C and sheared sequentially through an 18, 2:3, 26 and 30 gauge needle 25, 2~, ~0, and I00 times respectively. 2 u1 samples ofthe sheared material is taken for fluorescence measurements after every ~0 passes through the 30 gauge needle until the fluorescence units (FU) plateau (approx. 100-1 ?0x). Test compounds are prepared by dissolving or :.ppropriate amount of test compound in 1 x fBS to a final concentration of 1 mM (I Ox stock). If insoluble. the compound is dissolved in 1,10 volume of DMSO and c;a diluted in lx PBS to 1 m.~~f. A further I~'1 G dilution is also prepared to test each candidate at both 100 pM and 10 uM.
To perform the seeded extension assay. each sample is set up with ~0 u1 of 200 p,M
monomer, 1 ~' ~ FU sheared seed (a variable quan:itZ~ dependent on the batch of seed. routinely 3-6 u1;1 and 10 p1 of lOx modulator solution. The sample volume is then adjusted to a final volume of 100 ~tl with lx PBS. Two concentrations of each modulator typicalIv are tested:
100 uM and 10 pi~~i, equivalent to a I :1 and a I : I0 :polar 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 ~T °(w for 6 h.
taking '? u1 samples hourly for fluorescence measurements. To measure tZuorescence, a 2 u1 sample of A~i is added to 400 p1 of Thioflavin-T solution (,~0 mNI Potassium Phosphate 10 mM
Thioflavin-T~
pH 7.~). The samples are vortexed and the fluorescence is read in a 0.~ mI
micro quartz TM
cuvette at EX 4~0 nm and Elf 482 nm (Hitachi 400 Fluorimeter).
TM
1~ (3-amyloid aggregation results in enhanced emission of Thiofiavin-T.
Accordingly, samples including an effective inhibitory modulator compound exhibit reduced emission as compared to control samples without the modulator .compound.
EX..4I~iPLE 7: Effect of Different :amino Acid Subr~egions of A~i Peptide on the Tnhibitory Acti~~ity of ~3-Amyioid ll~Iodulatar Compounds To determine the effect of various subregions of A~i~_,~p on the inhibitory activity of a a ~3-amyloid modulator. overlapping A(3 peptide 1 ~mers were constructed. For each 1 ~mer.
four different amino-terminal modifiers were tested: a chclyl group, an iminobiotinyl Group.
2~ an N-acetyl neuraminyl group ('NAN,~~.) and a ~-'and 6-:i-~c:arboxyfluoresceinyl group (FICO).
The modulators were evaluated in the nucleation and seeded extension assays described in Example 6.
The results of the nucleation assays are summarised below in Table IL. The concentration of A~il.~p used in the assays was ~0 ~M. The "mole %" value listed in Table II
refers to the % concentration of the test compound relative to A~i~.4p.
Accordingly, 100%
indicates that A~3~.~p and the test compound were equimolar. Mole % values less than 100%
indicate that A(31~p was in molar excess relative to the test compound (e.g., 10% indicates that A~3l~p 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 Ias 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 f) "?
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 w ggregation during the course of the assay wtIl have a % inhibition of 100. N-ter~rvinall~~ modified A~i subregions which exhibited inhibitory activity are indicated in bo(d in Tabie II.
Table II
N-terminal ~ Fold Increase% Inhibition Reference Modification' AQ PeptideI Mole ~n Lay ( of Plateau # ro E Time PPI-174 cholyl A~it_i' ! 100 >4.5 ~ 100 ~

PPI-264 ~ cholyt j A~36..2p ~ 100 >4.5 ~ 100 I

PPI-269 ~ ch I A(31 ~-.,~I 10(:) 1.~ j ~0 olyl ;

PPI-274 _ A(3i~.3o ; 10C! >4.5 100 cholylr ~

PPI-279 cholyl ' A~i~t _~~ ; 100 1.6 ( 51 ~

PPI-28-i cholyl ' A~i,~o . 10t1 >4.5 ~ 87 ~

i I

PPI-173 NANA I A~i~_~~ j t0(:;~ --1 J ~0 ~ !

PPI-266 NANA A~i~.2o ; 100 1.3 ~ 64 ~

PPI-271 NANA ; A~3t t _2~ t 100 1.3 I, 77 ~ !

PPI-276 NANA I A~ii~T;n 1()C) ~.-1 ~ ; ' ~

PPI-281 NANA I A~3y l ..; 1 (>'~~ ~ 1 ~ ~3 ~ . ~

PPI-286 N ANA A~3~~..4Q 1 C)C) 1.3 ~ --0 ( I

i PPI-172 IminobiotinylA~i~_~s 1C)0 I 1.2 j ~0 ~ I

PPI-267 Iminobiotinyt2o' 1t)t) 1.6 44 ~ A~i~

PPI-272 Iminabi~otinyt_ 100 1.2 ( 40 ~ ' A~3tt_ZS ~ ~

PPI-277 IminobiotinytA~3i6..30 lt)t'i 1.2 ~ 55 ~ ' ?PI-28~ IminobiatinylA~3-~~ .;~ 1()(~~_I --1 ( 66 i PPI-28 7 lminobiotinylA~3~6..~~ 1 ()(;~ 2.3 ; --0 J ' r PPI-190 FICO ' Alit-t~ 1C)() .~1 ~ 30 ~ ' , PPI-268 FICO I A(36_,0 if)() 1.9 ~ ~0 ( ' PPI-Z?3 FICO A~itl.-25 100 1.7 ~ 34 ~ ' F'PI-278 FICO ~ A~1~.30 x00 1.6 59 ~ ' PPI-283 FICO A~21-3s ltltr 1.2 25 ~ I

PPI-288 FICO i A~iz6..ao 1t>0 ~ 75 ~ i These results indicate that certain subregions of e~.~3)_:~p, when modified with an appropriate modifying group, are effective at inhibiting she aggregation of A~3~~p. A cllolyl group was an effective modifying group for s~weral subregions. Cholic acid alone was tested for inhibitory activity but had no effect on A~3 aggregation. 'hhe A(36_-,p subregion exhibited Nigh levels of inhibitory activity when modified with serreral different modifying groups (cholyl. NANA. iminobiotinyl), with cholvl-A~36_~p (,PPaI-264) being the most active form.

(ids Accordingly, this modulator compound was chosen for ii.uwthhet- analysis.
described in Example 8.
EXAMPLE 8: Identification of a fiwie Anxino Acid Subregion of A~3 Peptide Sufficient for Inhibitory Activity of a ~-Amyloid Modulator Compound To further delineate a minimal subregian of choIyl-Aj3~_2o sufficient for inhibitory activity, a series of amino terminal and carboxy terminal amina acid deletions of cholyl-A~6-20 were constructed. The modulators all had the same choIyl amino-terminal 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 i'.
1 ~ Table III
N-Term. ~ C-Term. ~ Fold Increase~o Inhibition Ref. Mod. ~ A(3 PeptideMad. I~iole in Lay of Plateau # ' , ~o Time PPI-264 ~ cholylA(36..2o - ~ 100 X4.5 ~ 100 t 10 2 ~ 43 PPI-341 cholyl A~-Zp - 100 ~ >4.5 100 ~ ~

33 ~ 2 ~0 PPI-342 cholyl A~ig-?o - I C?C)~ 1.3 --0 ~

~ _ 2.I _ --0 PPI-343 cholvl A~i9_~o ~ ~ 3:~ 2.U ( -0 ~ I ! - ~ 3 :~ 2.1 ~ ~-0 PPI-344 cholyl A~3 j o_-,o ~
I ! ? 3. ~

PPI-343 cholyl A(3n_~o ~- ! ~.~ 1.3 ~ ~0 I ' ~ ~ ~ .

PPI-346 cholvl A~31~_~o m~ 3:~ 2.1 ~ --U
~ ~ E ~ 3 ~ 2.6 ~ ~0 PPI-347 cholyl Aril;-~o - ~
~ I ~

PPI-348 choiyl A~it2o ' -~ 33 2.0 ~ 49 ~ ~ ~

PPi-349 cholyl A~tS-2o - 3-3 ' 2.3 ~ 50 PPI-350 cholyl ! - 38 3.4 23 ~ i A~t6-2o PPI-296 cholyl A(~6_20 ~.mide 33 1.8 --0 I I

PPI-321 cholyl A(36-19 amide 33 1.4 ~0 ~ j ( PPI-32~ cholyl A(36_r~ amide 33 I 1.8 ~ ~0 PFI-331 cholyl A(3,~_ 14 amide 3 3 j 1.0 ~ 29 ! ' PFI-339 cholyl A~6-l o amide 3 3 ; 1.1 ~ 13 r ~

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~i~_2p retained activity) but activity is .lost as the peptide is deleted further at the amino-terminal end by removal of amino acid position 7 through to amino acid position 12 (i.e..
cholyl-A~ig_2o f)~
through cholyl-A~31~_~p did inhibit the plateau level of ~,~3 aggregation).
However, further deletion of amino acid position 13 resulted in a compound (i.e., cholvl-A~il4_2o) in which inhibitory activity is restored. Funther~rnore, additional deletion of amino acid position 14 (l. e., cholyl-A(31 ~_~o) or positions 14 and 15 {z. e. . cholyl-A~i I x_20) still maintained inhibitory activity. Thus, amino terminal deletions of A~i6_~o identified A~il6_?o as a minimal subregion which is sufficient for inhibitory activity when appropriately modified. In contrast, carboxy terminal deletion of amino acid position 2(:) 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~i Peptide Sufficient for Inhibitory Activity of a ~i-Amyloid ylodulator Compound In this example, the smallest effective modulator identified in the studies described in 1 ~ Example 8. cholyl-A~316_-,0 (PPI-3 ~U;), was analyzed further. Additional amino- and carboxv terminal deletions were made with cholvl-A~il6_-,0, as well as an ami:~o acid substitution ~ Val ~ g->Thr), to identify the smallest region sufficient for the irLhibitory activity of the modulator. A peptide comprised of live alanine residues, {Ala)~, modifzed at its amino-terminus with cholic acid. was used as a specificity control. °~'he 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 E:~ample "1.
Table IV
N-Term. ~ C-Term. ~ ' Fold % Inhibition Ref. Mod. A Peptide ~ ~ylod. Mole Increase of Plateau ~ I ~ ,~o , 'n t-as Time ~

PPI-264 cholyl A~3~ZO I, - ! 10 2.0 ~ 43 j ( PPI-347 choiyl A~13-2o I - ! I0 ' 2.2 57 ~ ~

PfI-3Q9 cholyl A~31~20 . - ' 100 >~.0 100 ~

J ~ 33 ; 2.6 35 10 2.1 --0 PPI-350 ~ cholylA(31 ~2p - , 10_0 >5.0 j 100 10 2.4 ' 40 PPI-368 cholyl A~il~_Zt - 100 ~ >5.0 100 PPI-374 s imino-A(3l~zo ", - 100 1.3 86 i biotinyl PPI-366 cholyl A~i I 5_ I a> ' 1 UU 3. l ' ~U
- l 1 (.) 1.6 ~U

PPI-369 cholyl A~316_~0 - l 1 U0 ~1 ~0 (Vall8->Thr) !

PPI-370 ~ cholylA(31~20 - 100 2.6 ~ 73 (Phel9-~AIa) C !

PPI-365 j cholyl(Ala)~ ! - 100 ' ~1 ~ --U

()~S
PPI-319 _ cholyi At6",~ mide ' 33 ~ S.6 ~ --0 ~
'''' ~ IO 2.7 ~ --4 ~

PPI-321 choiyl Aa t 6-t amide 10_0 1.2 ~0 9 ~ p ~

PPI-377 - A~t6-2o I 1t)() ~1 -0 ~ ~

As shown in Table IV, cholyl-A~3t~-2p (PPI-350) and choIyl-A~3t ~_,t (PPI-368) both exhibited inhibitory activity, indicating that the four-amino acid minimal subregion of positions 17-20 is suffcient for inhibitory activity. Loss of position 20 I;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~l6.,p Phet~-:~Ala; PPI-3?()) resulted in a compound which still retained detectable inhibitory activity. :accordingly, the phenylalanine at position I9 is more amenable to substitution. preferably with anorsher hydrophobic amino acid residue.
Cholyl-pen.a-alanine (PPI-36J ) showed no inhibitory activity. demonstrating the specificity of the A~i peptide portion of the modulator. Moreover, u:zmodified A~3 t f;_~p (PPI-3?7) was not inhibitory, demonstrating the functionai importance of the amino-terminal modifying group. The specific functional group influenced the activity of the modulator.
For example, i6 iminobiotinyl-A(3t5-2o -(PPI-374) exhibited inhibiton~ activity similar to cholyl-A~it6_?0~
whereas an i~-acetyl neuraminic acid (NAhA)-modified A~it6-20 ~'as not an effective inhibitory modulator (not listed in Table IV ). A C-terminal amide derivative of cholyl-A~it6-~p (PPI-319) 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 inhi"bit the overall plateau level of aggregation over time. the compound eras not tested at ~c:~centrations 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~3t6_-,p (PPI-3~0).
EXAMPLE I0: Effect of (3-Amyloid Modulators on the Neurotoxicity of Natural (3-Amyioid Peptide Aggregates The neurotoxicity of natural ~i-amyloid peptide aggregates, in either the presence or.
absence of a (3-amyloid modulator. a tested in a cell-based assay using either a rat or human neuronally-deuved cell line (PG-12 cells or NT-2 cells, respectively) and the viability indicator 3,(4,4-dimethylthiazol-2-yl)2,~-diphenyl-tetrazo~ium bromide (MTT).
(See e.g., Sheatman, M.S. et al. (1994) Proc. :~latl. ~lcad. Sci. USrI X1:1470-1474;
Hansen, M.B. et al.
(1989) J. Immun. ~~lethods 119:203-? 10 far a description of similar cell-based viability assays). PC-12 is a rat adrenal pheochromocvtoma cell line and is available from the American Type Culture Collection. Rockville, Mr) (ATCC CRL 1721). MTT
(commercially (~~1 available from Sigma Chemical Co.) is a c:hromogenic substrate that is converted from yellow to blue in viable cells, which can be detected spectrophotometrically.
To test the neurotoxiciry of natural (3-amyloid peptides, stock solutions of fresh A(3 monomers end aged A~3 aggregates were first pT spared. A~l,~p in 100°io DMSO was prepared from lyophilized powder and immediately diluted in one half the final volume in H20 and then one half the final volume in 2X PBS so that a final concentration of 200 ~M
peptide, 4% D\rS0 is achieved. Peptide prepared in this way and tested immediately on cells is referred to ~s "fresh" Ap monomer T'o prepare ''aged" A(3 aggregates, peptide TM
solution was placed in a 1.~ ml Eppendorf tube and incubated at 37 °C
for eight days to allow fibrils to form. Such "aged" A~ peptide can be tested directly on cells or frozen at -80°C. The neurotoxicity of fresh monomers and aged aggregates were tested using PC 12 and NT2 cells. PC 12 cells ~~ere routinely ouitured in Dulbeco's modified Eagle's medium (DMEM) containing 10% horse serum, 5°ia fetal colt semen, ~mM
glutamine, and 1%
TM
gentamycin. NT2 cells were routinely cultured in OPTI-MENI medium (GIBC~BRL
CAT.
1 ~ ~3198~) supplemented with 10% fetal calf serum, '' m~1 ~tlutamine and 1 %
gentamycin.
Cells were plated at 10-1 x,000 cells per well in 90 u1 of fresh medium in a 96 -well tissue culture plate 3-4 hours prior to treatment. I'he fresh or aged A~i peptide solutions ( 10 uL) were then diluted 1:10 directly into tissue culture mediurn so that the final concentration was in the range of 1-10 ~M peptide. Cells are incubated in the presence of peptide without a chance in media for 48 hours at 37°C. for the final three hours of exposure of the cells to the ~i-AP preparation. MTT was added to the media to a final concentration of 1 mg/ml and incubation was continued at 3? .°C. Following the nvo hour incubation with MTT, the media was removed and the cells were lysed in 100 ~L isopropano1/0.4111 HCl with agitation. An equal volume of PBS was added to each well and the plates were agitated for an additional 10 2~ minutes. Absorbance of each well at 570 nm was measured using a microtiter plate reader to quantitate viable cells.
The neurotoxicitv of aged (~ day or 8 day) A~i)...~p aggregates alone, but not fresh A~3 ~~p monomers alone. was confirmed in an experiment the results of which are shown in figure 3, which demonstrates that incubating the neuronal cells with increasing amounts of fresh A~i ~.~p monomers was not significantly toxic to the cells whereas incubating the cells with increasing amounts of 5 day or 8 day A(31_~p aggregates 1:.d to increasing amount of neurotoxiciry. The EC50 for toxicity of aged A~ ~..~~ agg~'egates was 1-2 ~M
for both the PC 12 cells and the NT2 cells.
To determine the effect of a ~3-amyloid modulator compound on the neurotoxicity of A~ii.~p aggregates. a modulator compound, cholyl-A~i~-~fp (PPI-264), was preincubated with A~il~p monomers under standard nucleation assay conditions as described in Example 6 and at particular time intervals post-incubation, aliquots of the ~i-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 ~fa was assessed-using MTT to determine the neurotoxiciry of the solution. The results of the turbidity analysis are shown in Figure 4, panels A, B and C. In panel A, A~i a ~o and cholyl-Aa6-2o were both present at 64 ~M, In panel B, A~i~.~o was present at 30 ~M
and cholyl-A~3~2~ was present at 64 ~M. In panel C, :a~3 ~ ~o was present at 10 pM and cholyl-A~i~2o was present at 64 pM. These data show that an equimolar amount of cholyl-A136-2o is effective at inhibiting aggregation of A~3I.~p (see Figure 4, panel A) and that a:: the concentration of A~ii~o is reduced, the amount of detectable aggregation of the A~il~o monomer is correspondingly reduced (compare Figure 4. panels B and C with panel A). The corresponding results of the neurotoxicity° analysis are shown in Figure 4, panels D, E, and F.
These results demonstrate that the ~3-amyloid modulator compound not only inluibits aggregation of A(3 x~o monomers but also inhibits the neurotoxiciry of the A(3 ~..,tp solution, illustrated by the reduced percent toxicity of the cells when incubated with the A~3 ~ _4pimodulator solution as compared to A.~3 ~ .~o alone ~ see ~.~.g., Figure 4, panel D).
Moreover. even when A~i~~o aggregation was not detectable as measured by light scattering, 1 ~ the modulator compound inhibited the neurotoxicity of the A~ 1 ~o solution (see Figure 4, panels E and F). Thus, the formation of neurotoxic A(3a-.tn aggregates precedes the formation of insoluble aggregates detectable by light scattering and the modulator compound is effective at inhibiting the inhibiting the formation andr'or activity of these neurotoxic aggregates. Similar results were seen with other modulator compounds. such as iminobiotinyl-A~6_2o (PPI-267), cholyl-A~ii6_~o (PPI-350) and cholyl-A(3~6-?o-~.mide (PPI-319).
Additionally. the ~i-amyloid modulator compouncs have been demonstrated to reduce the neurotoxiciry of preformed A(3~~o aggregates. In these experiments. A~it~o aggregates were preformed by incubation of the monomers in the ab:~enc~: of any modulators. The modulator compound was then incubated with the preformed ,=~~ ~ ~o aggregates for ?4 hours at 37 °C, aftez which time the ~i-AP/modulator solution was ccollected and its neurotoxicity evaluated as described above. Incubation of prefarmed :~.~3~,~o aggregates with the modulator compound prior to applying the solution to neuronal cells resulted in a decrease in the neurotoxiciry of the A~3l~o solution. These results suggest that the modulator can either bind to A~i fibrils or sol~,:ble aggregate and modulate their inherent neurotoxicity or that the modulator can perturb the equilibrium between monameric and aggregated forms of A~j,.4o in favor of the non-neurotoxic form.
EXAMPLE 11: Characterization of Additional ~-Amyloid Modulator Compounds In this example, additional modulator compounds designed based upon amino acids 17-20 of A~i, LVFF (identified in Example 9), were prepared and analyzed to further delineate the structural features necessary for inhibition of (3-amyloid aggregation. Types of compounds analyzed included ones having only three amino acid residues of an A~

aggregation-core domain. compounds in which the amino acid residues of an A(3 aggregation core domaii: were rearranged or in which amino :cid 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- (rree S amino terminus), -oh (free carboxylic acid terminus), -nlm (amide terminus), CA (chc~Iyl, the acyl portion of cho1ie acid). NANA (~V acetyl neuraminyl ), IB
(iminobiotinyl), (3A (~i-alanyl), DA (D-alanyl), Adp (aminoetlhyldibenzofuranylpropanoic acid), Aic (3-(O-aminoethyl-iso)-cholyl, a derivative,of cho1ie acid), IY (iodotyrosyl;l, o-methyl (carboxy-terminal methyl ester), N me (N methyl peptide bond), DeoxyCA (deoxycholyl) and LithoCA
(lithocholyl).
Modulator compounds having an Aic rnodif~~ing 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, ~~:817-822; ~~%ess. G. et al. (1992) Tetrahedron Letters 33:19:x-198). Briefly, ~~-isa-i?-t2-aminoethyl)-cho1ie acid s;3(3-(2-aminoethoxy)-7a..12a-dihvdroxy-5~3-choIanoic acid;) was converted to the FMOC-provected derivative using FMOC-OSu (the hvdroxysuccinimide ester of'the FMOC group, which is commercially available) to obtain a reagent that was used to introduce the cho1ie acid derivative into the compound. For N-terminal introduction of the cho1ie 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 cleav~.ge from the oesin, followed by HPLC purification. For C~:-terminal introduction of the cho1ie acid moiety, the FMOC-protected reagent was attached to 2-chlorotrityl chloride resin in the standard manner. This amino acvl de~ivatized resin was then used in the standard manner to synthesize the complete modified peptide.
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 (~lLag) is presented as the ratio of the lag time observed irn 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~il~p, except for PPI-315, PPI-348, PPI-3d0, PPI-407 and PPI-418, for which the data is reported in the presence of 33 mole inhibitor. Inhibition (% Inucln) is listed as the percent reduction in the maximum observed turbidity in the control at the end of the assay time period. ~-nhibition in the extension assay f !41 (% Iexnn) is listed as the percent reduction of thioflavin-Z ~Iuorescence of ~i-structure in the presence of 25 mole % inhibitor. Compounds ~rith a % Inuclv of at least 30%
are highlighted in bold.

Table V
N-Term. C-Term.
Ref. ~ Mod. n ~ Mod. ALae ~ fo Inu~~.~fo I xt'n Pegtid~

PPI-293 CA - -oh 1.0 0 ND

PPI-3 CA HQKLVFF j -nh-, 1.1 Sx ~ ND
i 5 ~

PPI-316 NANA HQKI,VFF -nh~ _ I.S -15 j ND
~ ~
~~

PPI-319 CA KLVFF -nh~ a.4 70 ~ 52 PPI-339 CA HDSGY ! -nh2 I.1 -18 j ND

PPI-348 CA ) IrIQKLVFF -oh 2.0 70~ ND
~

PPI-349 CA QKLVFF -oh >5 100 ~ S6 PPI-350 CA KLVFF -oh 1,8 j 72 j 11 PPI-36S CA ~ ' -oh ~:p.8 -7 I 0 i PPI-366 CA QKLVF ~ -oh ~5.1 -23 ! ND

PPI-368 CA LYFFA ' :>5 100 f 91 PPI-369 CA -oh j ~ . I -16 ' 44 I~~LTFF
I oh PPl-370 C A j KLVAF ~ 2.6 ~ 73 ! 31 ( -oh !, PPI-371 CA [ KLVFF(~3A) 2.5 ~ 76 ;
~ i -oh ~ 80 PPI-372 CA ~ FKFVL . C~.B 45 37 j -oh i PPI-373 NANA IkLVFF -oh ~).9 16 ! 8 j ~

PPI-374 IB ~ kI,VFF 1..3 PPI-375 CA . -oh j ! 86 ItTVFF ~ '; 0 -oh j i .2 ! 18 f 2 i PPI-377 h- ItLVFF i i .1 -oh ~ 0 j 8 PPI-379 CA j LVFFA.E ~..4 -oh 55 i PPI-380 CA f LVFF -oh 1.8 72*~
~ ~ j 5i PPI-381 CA ! LVFF(nA) ~ -oh ~"..3 S6 j 11 _~_ PPI-382 CA ~ ~..0 ~ -200 ~ 91 LVFFA -nh-, PPI-383 h-DDIIL-(Adp)VFF -oh.
j ~).4 ~

PPI-386 h- ~ LVFFA !
-oo. ~
i~.0 ~
1 ~ _ !

PPI-387 h- KLVFF -nh-,~
I -9 i 39 PPI-388 CA ~ ,~VFFA -oh~
j 71.4 j PPI-389 CA ! LAFFA -oh 66 ~l.S 47 PPI-390 CA LVAF.~ -oh 0 ?.7 2S

PPI-392 CA ~ VFFA ' -oh 10 i ;~.0 PPI-393 CA LVF ~ -oh 0 j I l.3 a 1 j PPI-394 CA VFF -oh 55 j 0 1.8 PPI CA FF A -oh ~ ~ ~ 51 T 6 PPI-396 Ca j LV(I1~FA >5 k, 100 j 71 -oh PPI-401 CA LVFFA -~~~thy~ Nap j ND ~ 0 - .

PPI-405 h- LVFFA j 1.3 11 70 1 j -nh-~

PPI-407 CA j LVFFK j >_S 100** 85 -oh PPI-408 h- LVFF.~ j ~3.5 46 j 3 (:tic)-oh PPI-418 h-(Aic) LVFFA ~ >5 j 100*- ~ 87 -oh PPI-426 C A FFVLA-. ~,,5 100 89 ~ -oh ~

PPI-391 CA LVFAA -ah 1.6 40 ND

PPI-397 CA ( -oh j 95 j ND
LVF(I7c~_A

PPI-400 CA j '_ 1.0 ~ ND
AVAFA i -I S
-<s~

7~
PPI-403 *** HQKLVFF ~ _ah ~ i-4_ 1 _-7;

PPI-404 **** -_LKLVFF -_ _'~h f I.8 -29 ~ 7 PPI-424 DeoxyCA LVFFA -oh ~ 3.0 ~ -114 82 PPI-425 LithoCA LVFFA -oh ~?..8 -229 0 !

PPI-428 CA FF -oh L .7 -78 15 PPI-429 CA FFV ' -oh ~ :''.2 -33 7 ~

PPI-430 CA FFVL ~ ~.oh 33 75 ' ~L1 ;

PPI-433 CA LVFFA -oh :'.8 27 ND
(all D
amino acids) PPI-435 t-Boc oh a_.0 -5 ND
i a LVFFA -r PPI-438 CA _ GFF ' -oh~I.O
. 0 ND

_ND = not done _ _ -**=33mo1%
*** = h-DDIII(~"~'-Me-Val)DLL(Adp) ****= h-DDII(~'~'-Me-Leu)VEH(Adp) Certain compounds shown in Table V (PPI-3I9, PPI-349. PPI-3~0, PPI-368 and PPI-426) also were tested in neurotox:cin~ assails such as thuse 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 A~3 began in tl:e nucleation assays.
This correlation between the prevention of formation of neuratoxic A(3 species and the prevention of pulymerization of A~f was consistently obsen~ed for all compounds tested.
The results shown in T able V demonstrate that at an effective modulator compound can comprise as few as three A~i amino acids residues lsee PPI-394. comprising the amino acid sequence VFF, which corresponds to A~3lg-gyp. and PPI-39~, comprising the amino acid sequence FFA. which corresponds to A1319_~1~. The results also demonstrate that a modulator compound having a modulating group at its carboxv-terniinus is effective at in~~ibiting A~3 aggregation (;see PPI-408. mcdified at its C-terminus with Aic j. 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-4I
8, 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 g9mTc 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~i sequence (see PPI-396 and PPI-397) while maintaining the ability of the compound to inhibit A~i 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.
Finally. compounds with inhibitory activity were created using A~ deri~jed amino acids but wherein the amino acid sequence was rearranged or had a substitution with a non-A~-derived amino acid. Examples of such compounds include PPI-426, in which the sequence of A~i I ~-2I (LVFFA) has been rearranged (FFV LA), PPI-372, in which the sequence of-A~il6-2o (1~-VFF) has been rearranged yFI~~FVL;3> and PPI-388, -389 Gnu -39G, in which the sequence of A~i t 7-21 (L~JFFA) has been substituted at position 17, 18 or 19, respectively, with an alanine residue (AVFFA for PPI- i88, LAF'FA for PPI-389 and LVAFA
for PPI-390). The inhibitory activity ofthes~ compounds indicate that the presence in the compound of an amino acid sequence directly corresponding to a portion of A j3 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~3 aggregation.
EXAMPLE I2: Characterization of (3-Amyloid Madulatar Compounds Comprising an Unmodified (3-Arnylaid Peptide To examine the ability of unmodified A~3 peptides to modulate aggregation of natural ~i-AP, a series of A~i peptides having aminc:~- andlor carbcaxy terminal deletions as compared to A~31-40, or having internal amino acids deleted (r. e.. noncontiguous peptides). were prepared. One peptide (PPI-2'?0) had additional, non-A~~-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 t.~plow 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.
Table VT
Fold Increase% Inhibition Reference A(3 Peptide Mole ~o ?n Las of Plateau # Time PPI-226 100 i.66 ~ 76 j A~~2o PPI-227 100 ~l ~ 47 Aft t-=5 PPI-228 A~m3o I00 >4.5 j 100 j PPI-229 A~3zl-~5 100 - j ~1 I .-0 PPI-230 A~2~~o - 100 ~ 0.8 ~0 PPI-231 A~i~_15 ~~ X00 ~1 18 PPI-247 I AEI-3o. 36-ao 100 ~_--.l (d31-3~~

PPI-248 j Aft-ZS 3t-~o I00 j 1.58 ~0 06-30) PPI-249 j AEI-2o. 26-ao j 100 0 (~l-25) 2.37 PPI-250 ~0 I Ap-ts.
2t-ao (O1~-z0) I00 _1.5_5 PPI-251 ~ ~0 ( Ail-lo.
t6-ao (O11-15j 100 _~l,2 PPI-252 ~ A~1-$ tt-ao(06-IO) 33 I00 Im9 J

PPI-253 100 t ,~ I.9 ~0 ~ A~3,~o .

j EEW~IHHHQQ-A~it,~p ~ I00 a =~4 The results shown in Table VI demonstrate that limited portions ofthe A~i sequence can have a significant inhibitory effect on natural ~i-AP aggregation even when the peptide is not modified by a modifying group. Preferred unmodified peptides are A~3~2o (PPI-226), A~16-30 ~PPI-228), A~31_20, 2~to ~PPI-2'19) and E:~wHHHHQQ-A~il~o (PPI-220), the amino acid sequences of which are shown in SEQ ID NOs: 4, 14, 15, and 16, respectively.
Forming part of this disclosure is the appended Sequence Listing, the contents of which are summarize i n T a b 1 a V ~ I b a 1 caw .
Table VII
SEO ID N Amina Acids j Peptide Sequence O ~~
: I

_ 43 amino acids ~ A(~t-.~;
_ 1 . I

2 I 103 amino acids ~ APP ~-terminus ~~

3 43 amino acids ; A(3t~; (19. 20 mu:ated) 4 ~ HDSGYEVHHQKLVFF ~ A~6-zo ~

HQKLVFFA ~ A~ t a-2 t 6 HQKLVFF ~ A~ t 4-?0 ~

7 (?KLVFFA ' A~tS-~t A~tS-2O
- -9 K.LuFFA ~ A~16-21 KLVFF I A~ t 6-zo 11 LVFFA ~ A~t'7-zt 12 LVFF ~~ A~ t 7-20 _ ___ _ _ ' 13 LAFFA _I' Aft?-'~t CVig-~A) ~~

14 ~ hLVFFAEDVGSNKG.A ! A~16.;0 1 ~ ~ 3 5 amino acids ' A~ t -zo. 26-ao 16 ~ 35 amino acids ! EEWHHHHQQ-~iAP ! 6-40 17 I AGAAAAGA _ PrP peptide 18 AILSS ( amylin peptide _ ~

19 ~ VFF ~ A~ t s-2o - _ FFA ~ A~t9-zt ~

21 ~ FFVL j A(3 t ;~~z ! (scrambled) A .

22 ; LVFFK I A~t7_2t I,A2t-jK) 23 LV(IY)FA -______ ~ I A~t?_~t I;Ft9-DIY) 24 i VFFA _ ~ A~tB-2!
~

~ .AVFFA ~ AI~t~-2t ~Lt ~~'A) 26 LVF(IY)A ~_ ~ A~t~-2t ~F2o~IY) ~

27 ~ LVFFAE i A~t7-zz 28 ~ FFVF =~ ~ A I3 ! 7-20 scrambled) 29 - FKFVL A(3 ~ 6_~p (scrambled) 3U KLVAF A~ t 6-20 ~ 19--~A) 31 KLVFF(~A) A~ ~ 6-21 (A~ m~~A) 32 LVFF(DA) A(3~~_2~ (A~~-FDA) E~( UIVALENTS
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.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
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ZO (A) ADDRESSEE: LAHIVE & CQCKFIELD
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{A) APPLICATION NUMBER: 000000 3S (B) FILING DATE: Herewith (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
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{A) NAME: DeConti, Giulio A.
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SS (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 227-7400 {B) TELEFAX: (617)227-5941.

(2) INF9RMATION
FOR
SEQ
ID N0:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 amino acids S (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal r (xi) SEQUENCE DESCRIPTION: NO:1:
SEQ ID

Asp Ala Glu Phe Arg His Asp Gly TyrGlu Val His GlnLys 5er His IS 1 5 1t) 15 Leu Val Phe Phe Ala Glu Asp Gly SerAsn Lys Gly IleIle Val Ala Gly Leu Met Val Gly Gly Val Ile AlaThr Val {2) INFORMATION
FOR
SEQ
ID N0:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 103 amino acids {B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal 3S (xi) SEQUENCE DESCRIPTION:
SEQ ID N0:3:

Glu Val Lys Met Asp Ala G1u Arg HisAsp Ser G1y GluVal Phe Tyr 1 5 1~1 15 4U His His Gln Lys Leu Val Phe Ala GluAsp Val Gly AsnLys Phe Ser Gly Ala Ile Ile Gly Leu Met Gly G~.yVal Val Ile ThrVal Val Ala Ile Val Ile Thr Leu Val Met Lys LysLys Gln Tyr SerIle Leu Thr His His Gly Val Val Glu Val Ala AlaVal Thr Pro GluArg Asp Glu His Leu Ser Lys Met Gln Gln Gly TyrG7.u Asn Pro TyrLys Asn Thr 85 ~af7 95 SS Phe Phe Glu Gln Met Gln Asn (2) INFORMATION FOR SEQ TD N0:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 amino acids (B) TYPE: amino acid S (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (ix) FEATURE:

(A) NAME/KEY: Modifiedsite (B) LOCATION: 19 (D) OTHER INFORMATION:/note= Xaa hydrophobic amino is a 1S acid (ix) FEATURE:

(A) NAME/KEY: Modifiedsite (B) LOCATION: 20 (D) OTHER INFORMATION:/note= Xaa hydrophobic amino is a acid (xi) SEQUENCE DESCRIPTION:
SEQ ID N0:3:

2S Asp Ala Glu Phe Arg His Ser Gly Tyr Val His His Gln Asp Glu Lys Leu Val Xaa Xaa Ala Glu Val Gly Ser Lys Gly Ala Ile Asp Asn Ile Gly Leu Met Val Gly Gly Val Ile Ala Val Thr 3S (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe SO (2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid S (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:

His Gln Lys Leu val Phe Phe Ala S
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids 10 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide IS (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:°
His Gln Lys Leu Val Phe Phe (2) INFORMATION FOR SEQ ID NO:'7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids 2S (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 3O (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Gln Lys Leu Val Phe Phe Ala (2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 4S (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Gln Lys Leu Val Phe Phe SO
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids SS (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ~5 ~
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Lys Leu Val Phe Phe Ala (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 1$
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Lys Leu Val Phe Phe (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHAR.ACTERISTIC:~:
2$ (A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Leu Val Phe Phe Ala 3$
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 4$
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1.2:
Leu Val Phe Phe $0 (2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
$$ (A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ~?
(v) FRAGMENT TYPE: internal S (xi) SEQUENCE DESCRIPTION: SEQ ID N0;13:
Leu Ala Phe Phe Ala (2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids IS (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
ZS Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala (2) INFORMATION FOR SEQ ID N0:15:
( i ) SEQUENCE CFiAR.ACTERISTICS
(A) LENGTH: 35 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 ~ 5 1.0 15 Leu Val Phe Phe Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val (2) INFORMATION FOR SEQ ID N0:16:
SS (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ~3 (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
Glu Glu Val Val His His His His Gln Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val (2) INFORMATION FOR SEQ ID N0:17:
ZO (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE : internal 3O (xi) SEQUENCE DESCRIPTION; SEQ ID N0:17;
Ala Gly Ala Ala AIa Ala Gly Ala (2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Ala Ile Leu Ser Ser (2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3 amino~acids (B) TYPE: amino acid (D) TOPOLOGY: linear {ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
S
Val Phe Phe IO {2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS;
(A) LENGTH: 3 amino acids (B) TYPE: amino acid IS (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIFTION: SEQ ID N0:20:
Phe Phe Ala 2S (2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:1:
Phe Phe Val Leu Ala (2) INFORMATION FOR SEQ ID N0:22;
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid 4S (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:22;
Leu Val Phe Phe Lys SS (2) INFORMATION FOR SEQ ID N0:23:
ti) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide S (ix) FEATURE:
(A) NAME/KEY: Modified site (B) LOCATION: 3 (D) OTHER INFORMATION: /note= Xaa is iodotyrosyl 1O (xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
Leu Val Xaa Phe Ala {2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids 20 (B) TYPE: amino acid (D) TOPOLOGY: linear {ii) MOLECULE TYPE: peptide ZS (xi) SEQUENCE DESCRIPTION. SEQ ID N0:24;
Val Phe Phe Ala (2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: S amino acids 3S (B) TYPE: amino acid {D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
Ala Val Phe Phe Ala (2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHAkACTERISTICS:
(A) LENGTH: 5 amino acids SO (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide SS (ix) FEATURE:
(A) NAME/KEY: Modified site (B) LOCATION: 9 (D) OTHER INFORMATION: /note= Xaa is iodotyrosyl (x~,) SEQUENCE DESCRIPTION: SEQ TD N0:26:
Leu Val Phe Xaa Ala (2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
1~ (A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
Leu Val Phe Phe Ala Glu (2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS;
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2~:
Phe Phe Val Leu (2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:"w9:
Phe Lys Phe Val Leu SO
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide g7 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
Lys Leu Val Ala Phe 1 s (2) INFORMATION FOR SEQ ID N0:31:
IO (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 1S (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/KEY: Modified site (B) LOCATION: 6 2O (D) OTHER INFORMATION: /note= Xaa is beta-alariyl (xi) SEQUENCE DESCRIPTION; SEQ ID N0:31:
Lys Leu Val Phe Phe Xaa (2) INFORMATION FOR SEQ ID N0:32:
3O (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: S amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 3J (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/KEY: Modified site (B) LOCATION: 5 4O (D) OTHER INFORMATION: /note= Xaa is D-alanyl (xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
Leu Val Phe Phe Xaa

Claims (116)

CLAIMS:

1. A .beta.-amyloid peptide compound comprising a formula:

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, and A is a modifying group attached to the amino-terminus of the .beta.-amyloid peptide such that the compound inhibits aggregation of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides, wherein A comprises a cyclic or heterocyclic moiety.

2. A .beta.-amyloid peptide compound comprising a formula:

wherein Xaa is a .beta.-amyloid peptide and A is a modifying group attached to the carboxy-terminus of the .beta.-amyloid peptide such that the compound inhibits aggregation of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides, wherein A comprises a cyclic or heterocyclic moiety or other moiety having similar steric bulk.

3. The compound of claim 1 or 2, wherein the A group comprises a biotin compound of the formula:

wherein X1-X3 are each independently selected from the group consisting of S, O and NR', wherein R' is selected from the group consisting of hydrogen, an aryl moiety, a lower alkyl moiety, an alkenyl moiety and an alkynyl moiety;
W is ~O or N(R')2;
R1 is a lower alkylenyl moiety; and Y is a direct bond or a spacer molecule selected for its ability to react with an amino group, whereby at least one of X1-X3 is an NR' group or W is an N(R')2 group.

4. The compound of claim 1 or 2, wherein the .beta.-amyloid peptide of the compound has an amino-terminal amino acid residue corresponding to position 672 of APP-770, or to a residue carboxy-terminal to position 672 of APP-770.

5. The compound of claim 1 or 2, wherein the .beta.-amyloid peptide of the compound consists of an amino acid sequence:

DAEFRHDSGYEVHHQKLV(Xaa19)(Xaa20)AEDVGSNKGAIIGLMVGGVVIAT

(SEQ ID NO: 3), wherein Xaa19 and Xaa20 are each independently selected from the group consisting of phenylalanine, isoleucine, leucine, threonine, serine, alanine, valine and glycine, or an amino-terminal or carboxy-terminal deletion thereof having at least 6 amino acid residues.

6. The compound of claim 1 or 2, wherein the .beta.-amyloid peptide of the compound consists of an amino acid sequence: DAEFRHDSGYEVHHQ (positions 1 to 15 of SEQ ID NO: 3).

7. A .beta.-amyloid modulator compound comprising a retro-inverso isomer of a .beta.-amyloid peptide, wherein the compound inhibits the aggregation of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides.

8. The compound of claim 7, wherein the retro-inverso isomer of the .beta.-amyloid peptide further comprises a modifying group attached to the amino-terminal end of the retro-inverso isomer.

9. The compound of claim 7, wherein the retro-inverso isomer of the .beta.-amyloid peptide further comprises a modifying group attached to the carboxy-terminal end of the retro-inverso isomer.

10. The compound of claim 7, wherein the retro-inverso isomer of the .beta.-amyloid peptide further comprises a modifying group attached to the amino- and carboxy-terminal ends of the retro-inverso isomer.

11. The compound of claim 7, which is modified to label the compound with a detectable substance.

12. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 7 and a pharmaceutically acceptable carrier.

13. A packaged formulation for treating a subject for a disorder associated with .beta.-amyloidosis, comprising a therapeutically effective amount of the compound of claim 7 packaged with instructions for using the compound for treating a subject having a disorder associated with .beta.-amyloidosis.

14. A pharmaceutical composition comprising:

a therapeutically effective amount of a .beta.-amyloid peptide compound sufficient to inhibit aggregation of natural .beta.-amyloid peptides, the .beta.-amyloid peptide compound comprising a formula:

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, and A is a modifying group attached to the amino-terminus of the .beta.-amyloid peptide such that the compound inhibits aggregation of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides; and a pharmaceutically acceptable carrier.

15. A pharmaceutical composition comprising:
a therapeutically effective amount of a .beta.-amyloid peptide compound sufficient to inhibit aggregation of natural .beta.-amyloid peptides, the .beta.-amyloid peptide compound comprising a formula:

wherein Xaa is a .beta.-amyloid peptide and A is a modifying group attached to the carboxy-terminus of the .beta.-amyloid peptide such that the compound inhibits aggregation of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides; and a pharmaceutically acceptable carrier.

16. The pharmaceutical composition of claim 14 or 15, wherein the pharmaceutically acceptable carrier is suitable for parenteral administration.

17. The pharmaceutical composition of claim 14 or 15, wherein the pharmaceutically acceptable carrier is suitable for administration to the central nervous system of a subject.

18. The pharmaceutical composition of claim 14 or 15, wherein the compound is in an amount sufficient to inhibit aggregation of at least a molar excess amount of natural .beta.-amyloid peptides.

19. A packaged formulation for treating a subject for a disorder associated with .beta.-amyloidosis, comprising the compound of claim 1 or 2 packaged with instructions for using the composition for treating a subject having a disorder associated with .beta.-amyloidosis.

20. The packaged formulation of claim 19, wherein the instructions are for using the compound for treating a subject having Alzheimer's disease.

21. The packaged formulation of claim 13, wherein the instructions are for using the compound for treating a subject having Alzheimer's disease.

22. Use of a pharmaceutically effective amount of a compound having a formula:

wherein Xaa is a .beta-amyloid peptide, A is a cyclic or heterocyclic modifying group attached 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.-AP peptides.

23. The use of claim 22, wherein at least one A group is attached to the amino terminus of the .beta.-amyloid peptide of the compound.

24. The use of claim 23, wherein the at least one A group comprises a biotin compound of the formula:

wherein X1-X3 are each independently selected from the group consisting of S, O
and NR', wherein R' is selected from the group consisting of hydrogen, an aryl moiety, a lower alkyl moiety, an alkenyl moiety and an alkynyl moiety;

W is ~O or N(R')2;
R1 is a lower alkylenyl moiety; and Y is a direct bond or a spacer molecule selected for its ability to react with an amino group, whereby at least one of X1-X3 is an NR' group or W is an N(R')2 group.

25. The use of claim 23, wherein the at least one A group is attached to the .beta.-amyloid peptide by modifying the .beta.-amyloid peptide with a compound selected from the group consisting of diethylenetriaminepentaacetic dianhydride, cholic acid, (-)-menthoxyacetic acid, 5-(and 6-)-carboxyfluorescein, fluorescein isothiocyanate and acetic acid.

26. The use of claim 23, wherein the compound is in a form administrable to the central nervous system of a subject.

27. Use of a therapeutically effective amount of a compound having a formula:

for treating a disorder associated with .beta.-amyloidosis in a mammalian subject, wherein Xaa is a .beta.-amyloid peptide, A is a modifying group attached 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.

28. The use of claim 27, wherein the disorder is Alzheimer's disease.

29. Use of a therapeutically effective amount of a compound comprising a retro-inverso isomer of a .beta.-amyloid peptide, wherein said compound inhibits aggregation of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides, in treatment of a disorder associated with .beta.-amyloidosis.

30. The use of claim 29, wherein the disorder is Alzheimer's disease.

31. The use of claim 29, wherein the retro-inverso isomer of the .beta.-amyloid peptide further comprises a modifying group attached to the amino-terminal end of the retro-inverso isomer.

32. The use of claim 31, wherein the modifying group comprises a cyclic or heterocyclic compound.

33. The use of claim 29, wherein the retro-inverso isomer of the .beta.-amyloid peptide further comprises a modifying group attached to the carboxy-terminal end of the retro-inverso isomer.

34. The use of claim 33, wherein the modifying group comprises a cyclic or heterocyclic compound.

35. The use of claim 29, wherein the retro-inverso isomer of the .beta.-amyloid peptide further comprises modifying groups attached to the amino- and carboxy-terminal ends of the retro-inverso isomer.

36. Use of a compound comprising a retro-inverso isomer of a .beta.-amyloid peptide as an inhibitor of aggregation of natural .beta.-amyloid peptides.

37. The use of claim 36, wherein the retro-inverso isomer of the .beta.-amyloid peptide further comprises a modifying group attached to the amino- terminal end of the retro-inverso isomer.

38. The use of claim 37, wherein the modifying group comprises a cyclic or heterocyclic compound.

39. The use of claim 36, wherein the retro-inverso isomer of the .beta.-amyloid peptide further comprises a modifying group attached to the carboxy-terminal end of the retro-inverso isomer.

40. The use of claim 39, wherein the modifying group comprises a cyclic or heterocyclic compound.

41. The use of claim 36, wherein the retro-inverso isomer of the .beta.-amyloid peptide further comprises modifying groups attached to the amino- and carboxy-terminal ends of the retro-inverso isomer.

42. The use of claim 36, wherein the compound is in a form administrable to the central nervous system of a subject.

43. Use of a compound having a formula:

wherein Xaa is a .beta.-amyloid peptide and A is a modifying group attached to the amino-terminus of the .beta.-amyloid peptide in inhibiting aggregation of natural .beta.-amyloid peptides when contacted with natural .beta.-amyloid peptides.

44. The use of claim 43, wherein the modifying group comprises a biotin compound of the formula:

wherein X1-X3 are each independently selected from the group consisting of S, O and NR', wherein R' is selected from the group consisting of hydrogen, an aryl moiety, a lower alkyl moiety, an alkenyl moiety and an alkynyl moiety;
W is ===O or N(R')2;
R1 is a lower alkylenyl moiety; and Y is a direct bond or a spacer molecule selected for its ability to react with an amino group, whereby at least one of X1-X3 is an NR' group or W is an N(R')2 group.

45. The use of claim 43, wherein the modifying group is attached to the .beta.-amyloid peptide by modifying the .beta.-amyloid peptide with a compound selected from the group consisting of diethylenetriaminepentaacetic dianhydride, cholic acid, (-)-menthoxyacetic acid, 5-(and 6-)-carboxyfluorescein, fluorescein isothiocyanate and acetic acid.

46. The use of claim 43, wherein the modifying group comprises a cyclic or heterocyclic compound.

47. The use of claim 43, wherein the compound is in a form administrable to the central nervous system of a subject.

48. Use of a compound having a formula:

wherein Xaa is a .beta.-amyloid peptide and A is a modifying group attached to the amino-terminus of the .beta.-amyloid peptide;
in inhibiting aggregation of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides in a mammalian subject having a disorder associated with .beta.-amyloidosis.

49. The use of claim 48, wherein the modifying group comprises a cyclic or heterocyclic compound.

50. The use of claim 48, in treatment of Alzheimer's disease.

51. Use of a compound having a formula:

wherein Xaa is a .beta.-amyloid peptide and A is a modifying group attached to the carboxy-terminus of the .beta.-amyloid peptide in inhibiting aggregation of natural .beta.-amyloid peptides when contacted With natural .beta.-amyloid peptides.

52. The use of claim 51, wherein the modifying group comprises a biotin compound of the formula:

wherein X1-X3 are each independently selected from the group consisting of S, O and NR', wherein R' is selected from the group consisting of hydrogen, an aryl moiety, a lower alkyl moiety, an alkenyl moiety and an alkynyl moiety;
W is ===O or N(R')2;
R1 is a lower alkylenyl moiety; and Y is a direct bond or a spacer molecule selected for its ability to react with an amino group, whereby at least tine of X1-X3 is an NR' group or W is an N(R')2 group.

53. The use of claim 51, wherein the modifying group is attached to the .beta.-amyloid peptide by modifying the .beta.-amyloid peptide with a compound selected from the group consisting of diethylenetriaminepentaacetic dianhydride, cholic acid, (-)-menthoxyacetic acid, 5-(and 6-)-carboxyfluorescein, fluorescein isothiocyanate and acetic acid.

54. The use of claim 51, wherein the modifying group comprises a cyclic or heterocyclic compound.

55. The use of claim 51, wherein the compound is in a form admnistrable to the central nervous system of a subject.

56. Use of a compound having a formula:

wherein Xaa is a .beta.-amyloid peptide and A is a modifying group attached to the carboxy-terminus of the .beta.-amyloid peptide;
in inhibiting aggregation of natural .beta.-amyloid peptides when contacted with the natural .beta.-amyloid peptides in a mammalian subject for treatment of a disorder associated with .beta.-amyloidosis.

57. The use of claim 56, wherein the modifying group comprises a cyclic or heterocyclic compound.

58. The use of claim 56, wherein the disorder is Alzheimer's disease.

59. A .beta.-amyloid peptide compound having a structure: .beta.AP6-20(SEQ ID
NO: 4).

60. A .beta.-amyloid peptide compound having a structure: .beta.AP16-30 (SEQ
ID
NO: 14).

61. A .beta.-amyloid peptide compound having a structure: .beta.AP1-20, 26-40 (SEQ ID
NO: 15).

62. A .beta.-amyloid peptide compound having a structure: EEVVHHHHQQ-.beta.AP16-40 (SEQ ID NO: 16).

63. A .beta.-amyloid peptide compound having a structure: A.beta.6-40.

64. A .beta.-amyloid peptide compound having a structure: A.beta.11-25.

65. A .beta.-amyloid peptide compound having a structure: A.beta.1-25-, 31-40(26-30).

66. A .beta.-amyloid peptide compound having a structure: A.beta.1-15, 21-40(16-20).

67. A .beta.-amyloid peptide compound having a structure: A.beta.1-5, 11-40(6-10).

68. A composition comprising the compound of any one of claims 59-67 and a pharmaceutically acceptable carrier.

69. A compound having the structure:

wherein Xaa is an amyloidogenic protein, or peptide fragment thereof, and A is a modifying group comprising a cis-decalin group, a cholanoyl structure, a cholyl group, a diethylene-triaminepentaacetyl group, a (-)-menthoxyacetyl group, a fluorescein-containing group, or an N-acetylneuraminyl group, covalently attached to the .alpha.-amino group at the amino-terminus of the amyloidogenic protein, or peptide fragment thereof, such that the compound modulates the aggregation of natural amyloid proteins or peptides when contacted with the natural amyloidogenic proteins or peptides.

-l01-

70. A compound having the structure:

wherein Xaa is an amyloidogenic protein, or peptide fragment thereof, and A is a modifying group comprising a cis-decalin group, a cholanoyl structure, a cholyl group, a diethylene-triaminepentaacetyl group, a (-)-menthoxyacetyl group, a fluorescein-containing group, or an N-acetylneuraminyl group, covalently attached to the carboxy-terminus of the amyloidogenic protein, or peptide fragment thereof, such that the compound modulates the aggregation of natural amyloid proteins or peptides when contacted with the natural amyloidogenic proteins or peptides.

71. The compound of claim 69 or 70, which inhibits aggregation of natural amyloidogenic proteins or peptides when contacted with the natural amyloidogenic proteins or peptides.

72. The compound of claim 71, which inhibits aggregation of natural amyloidogenic proteins or peptides when contacted with a molar excess amount of natural amyloidogenic proteins or peptides.

73. The compound of claim 69 or 70, which is further modified to alter a pharmacokinetic property of the compound.

74. The compound of claim 69 or 70, which is further modified to label the compound with a detectable substance.

75. The compound of claim 69 or 70, 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, procalcitanin, cystatin C, .beta.2 microglobulin, ApoA-I, gelsolin, calcitonin, fibrinogen and lysozyme.

76. The compound of claim 69 or 70, wherein the modifying group contains a cis-decalin group.

77. The compound of claim 76, wherein the modifying group contains a cholanoyl structure.

78. The compound of claim 77, wherein the modifying group is a cholyl group.

79. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 69 or 70 and a pharmaceutically acceptable carrier.

80. A compound comprising an amyloidogenic peptide comprising at least one D-amino acid and having a modifying group attached to the peptide, wherein said modifying group comprises a cyclic, heterocyclic, or polycyclic group having 4 to 10 carbon atoms, or other moiety having similar steric bulk, and wherein the compound inhibits aggregation of natural amyloidogenic proteins or peptides when contacted with the natural amyloidogenic proteins or peptides.

81. A compound comprising an amyloidogenic peptide comprising at least one non-natural amino acid and having a modifying group attached to the peptide, wherein said modifying group comprises a cyclic, heterocyclic, or polycyclic group having 4 to 10 carbon atoms, or other moiety having similar steric bulk. and wherein the compound inhibits aggregation of natural amyloidogenic proteins or peptides when contacted with the natural amyloidogenic proteins or peptides.

82. A compound comprising a retro-inverso isomer of an amyloidogenic peptide, and having a modifying group attached to said isomer, wherein said modifying group comprises a cyclic, heterocyclic, or polycyclic group having 4 to 10 carbon atoms, or other moiety having similar steric bulk, and wherein the compound inhibits aggregation of natural amyloidogenic proteins or peptides when contacted with the natural amyloidogenic proteins or peptides.

83. The compound of claim 80, 81 or 82, wherein the modifying group is attached to the amino-terminal end of the amyloidgenic peptide.

84. The compound of claim 80, 81 or 82, wherein the modifying group is attached to the carboxy-terminal end of the amyloidogenic peptide.

85. The compound of claim 83, wherein the modifying group comprises a cyclic, heterocyclic or polycyclic group.

86. The compound of claim 84, wherein the modifying group comprises a cyclic, heterocyclic or polycyclic group.

87. The compound of claim 83, wherein the amyloidogenic peptide is comprised entirely of D-amino acids.

88. The compound of claim 84, wherein the amyloidogenic peptide is comprised entirely of D-amino acids.

89. The compound of claim 80, 81 or 82, which is further modified to label the compound with a detectable substance.

90. The compound of claim 80, 81 or 82, which is further modified to alter a pharmacokinetic property of the compound.

91. The compound of claim 80, 81 or 82, wherein the amyloidogenic peptide is from a protein or polypeptide 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, calcitonin, cystatin C, .beta.2 microglobulin, ApoA-I, gelsolin, fibrinogen and lysozyme.

92. A pharmaceutical composition comprising a compound and a pharmaceutically acceptable carrier, wherein the compound comprises an amyloidogenic peptide comprising at least one D-amino acid and having a modifying group attached to the peptide, and wherein the compound inhibits aggregation of natural amyloidogenic proteins or peptides when contacted with the natural amyloidogenic proteins or peptides.

93. A pharmaceutical composition comprising a compound and a pharmaceutically acceptable carrier, wherein the compound comprises an amyloidogenic peptide comprising at least one non-natural amino acid and having a modifying group attached to the peptide, and wherein the compound inhibits aggregation of natural amyloidogenic proteins or peptides when contacted with the natural amyloidogenic proteins or peptides.

94. A pharmaceutical composition comprising a compound and a pharmaceutically acceptable carrier, wherein the compound comprises a retro-inverso isomer of an amyloidogenic peptide and has a modifying group attached to said isomer, and wherein the compound inhibits aggregation of natural amyloidogenic proteins or peptides when contacted with the natural amyloidogenic proteins or peptides.

95. The pharmaceutical composition of claim 92, 93 or 94, wherein the modifying group is attached to the amino-terminal end of the amyloidgenic peptide.

96. The pharmaceutical composition of claim 92, 93 or 94, wherein the modifying group is attached to the carboxy-terminal end of the amyloidogenic peptide.

97. The pharmaceutical composition of claim 95, wherein the modifying group comprises a cyclic, heterocyclic or polycyclic group.

98. The pharmaceutical composition of claim 96, wherein the modifying group comprises a cyclic, heterocyclic or polycyclic group.

99. The pharmaceutical composition of claim 95, wherein the amyloidogenic peptide is comprised entirely of D-amino acids.

100. The pharmaceutical composition of claim 96, wherein the amyloidogenic peptide is comprised entirely of D-amino acids.

101. The pharmaceutical composition of claim 92, 93 or 94, wherein the compound is further modified to label the compound with a detectable substance.

102. The pharmaceutical composition of claim 92, 93 or 94, wherein the compound is further modified to alter a pharmacokinetic property of the compound.

103. The pharmaceutical composition of claim 92, 93 or 94, wherein the amyloidogenic peptide is from a protein or polypeptide selected from the group consisting of transthyretin (TTR), priori protein (PrP), islet amyloid polypeptide (IAPP), atrial natriuretic factor (ANF), kappa light chain, lambda light chain, amyloid A, procalcitonin, calcitonin, cystatin C, .beta.2 microglobulin, ApoA-I, gelsolin, fibrinogen and lysozyme.

104. Use of a therapeutically effective amount of a compound selected from the group consisting of: an amyloidogenic peptide comprising at least one D-amino acid, an amyloidogenic peptide comprising at least one non-natural amino acid, and a retro-inverso isomer of an amyloidogenic peptide, such that the compound inhibits aggregation of natural amyloidogenic proteins or peptides when contacted with the natural amyloidogenic proteins or peptides, in treatment of a disorder associated with amyloidosis.

105. The use of claim 104, wherein the compound comprises an amyloidogenic peptide comprising at least one D-amino acid.

106. The use of claim 104, wherein the compound comprises an amyloidogenic peptide comprising at least one non-natural amino acid.

107. The use of claim 104, wherein the compound comprises a retro-inverso isomer of an amyloidogenic peptide.

108. The use of claim 105, 106 or 107, wherein the compound further comprises a modifying group attached to the amyloidogenic peptide.

109. The use of claim 108, wherein the modifying group is attached to the amino-terminal end of the amyloidgenic peptide.

110. The use of claim 108, wherein themodifying group is attached to the carboxy-terminal end of the amyloidogenic peptide.

111. The use of claim 109, wherein the modifying group comprises a cyclic, heterocyclic or polycyclic group.

112. The use of claim 110, wherein the modifying group comprises a cyclic, heterocyclic or polycyclic group.

113. The use of claim 105, wherein the amyloidogenic peptide is comprised entirely of D-amino acids.

114. The use of claim 105, 106 or 107, wherein the compound is modified to label the compound with a detectable substance.

115. The use of claim 105, 106 or 107, wherein the compound is modified to alter a pharmacokinetic property of the compound.

116. The use of claim 105, 106 or 107, wherein the amyloidogenic peptide is from a protein or polypeptide selected from the group consisting of transthyretin (TTR), prion protein (PrP), islet amyloid polypeptide (LAPP), atrial natriuretic factor (ANF), kappa light chain, lambda light chain, amyloid A, procalcitonin, calcitonin, cystatin C, .beta.2 microglobulin, ApoA-I, gelsolin, fibrinogen and lysozyme.