U.S. patent application number 10/705028 was filed with the patent office on 2008-12-25 for compounds and methods for modulating cerebral amyloid angiopathy.
This patent application is currently assigned to Neurochem (International) Limited. Invention is credited to Francine Gervais, Allan M. Green.
Application Number | 20080317834 10/705028 |
Document ID | / |
Family ID | 22625484 |
Filed Date | 2008-12-25 |
United States Patent
Application |
20080317834 |
Kind Code |
A1 |
Green; Allan M. ; et
al. |
December 25, 2008 |
Compounds and methods for modulating cerebral amyloid
angiopathy
Abstract
The invention provides methods of inhibiting cerebral amyloid
angiopathy. The invention further provides methods of treating a
disease state characterized by cerebral amyloid angiopathy in a
subject.
Inventors: |
Green; Allan M.; (Cambridge,
MA) ; Gervais; Francine; (IIe Bizard, CA) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP;FLOOR 30, SUITE 3000
ONE POST OFFICE SQUARE
BOSTON
MA
02109
US
|
Assignee: |
Neurochem (International)
Limited
Walchwil
CH
|
Family ID: |
22625484 |
Appl. No.: |
10/705028 |
Filed: |
November 10, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09747408 |
Dec 22, 2000 |
6670399 |
|
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10705028 |
|
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60171877 |
Dec 23, 1999 |
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Current U.S.
Class: |
514/1.1 ;
514/109; 514/578 |
Current CPC
Class: |
A61P 25/28 20180101;
A61K 31/662 20130101; A61K 31/00 20130101; A61P 7/00 20180101; A61P
9/14 20180101; A61K 31/185 20130101; A61P 43/00 20180101; A61P 9/00
20180101; A61P 9/10 20180101 |
Class at
Publication: |
424/450 ;
514/578; 514/2; 514/109 |
International
Class: |
A61K 31/185 20060101
A61K031/185; A61K 9/127 20060101 A61K009/127; A61K 38/02 20060101
A61K038/02; A61K 31/662 20060101 A61K031/662; A61P 7/00 20060101
A61P007/00 |
Claims
1. A method of inhibiting cerebral amyloid angiopathy, comprising
contacting a blood vessel wall cell with an A.beta.40 inhibitor,
such that cerebral amyloid angiopathy is inhibited, provided said
A.beta.40 inhibitor is not 3-amino-1-propanesulfonic acid.
2. The method of claim 1, wherein the A.beta.40 inhibitor has the
following structure: Q-[--Y.sup.-X.sup.+].sub.n
3. The method of claim 1, wherein said A.beta.40 inhibitor is
selected from the group consisting of ethanesulfonic acid,
1,2-ethanedisulfonic acid, 1-propanesulfonic acid,
1,3-propanedisulfonic acid, 1,4-butanedisulfonic acid,
1,5-pentanedisulfonic acid, 2-aminoethanesulfonic acid,
4-hydroxy-1-butanesulfonic acid, and pharmaceutically acceptable
salts thereof.
4. The method of claim 1, wherein said A.beta.40 inhibitor is
selected from the group consisting of 1-butanesulfonic acid,
1-decanesulfonic acid, 2-propanesulfonic acid, 3-pentanesulfonic
acid, 4-heptanesulfonic acid, and pharmaceutically acceptable salts
thereof.
5. The method of claim 1, wherein said A.beta.40 inhibitor is
1,7-dihydroxy-4-heptanesulfonic acid, or a pharmaceutically
acceptable salt thereof.
6. The method of claim 1, wherein said blood vessel wall cell is
selected from the group consisting of blood vessel wall smooth
muscle cells, pericytes and endothelial cells.
7. The method of claim 1, wherein said blood vessel wall cell is a
blood vessel wall smooth muscle cell.
8. The method of claim 1, wherein the death of said blood vessel
wall cell is prevented.
9. The method of claim 1, wherein structural changes to said blood
vessel wall cell are prevented.
10. The method of claim 1, wherein said A.beta.40 inhibitor is a
peptide or a peptidomimetic which interacts with specific regions
of the A.beta. peptide.
11. The method of claim 1, wherein said A.beta.40 inhibitor has the
following structure: ##STR00272## wherein Z is XR.sup.2 or R.sup.4;
R.sup.1 and R.sup.2 are each independently hydrogen, a substituted
or unsubstituted aliphatic group, an aryl group, a heterocyclic
group, or a salt-forming cation; R.sup.3 is hydrogen, lower alkyl,
aryl, or a salt-forming cation; R.sup.4 is hydrogen, lower alkyl,
aryl or amino; X is, independently for each occurrence, O or S;
Y.sup.1 and Y.sup.2 are each independently hydrogen, halogen,
alkyl, amino, hydroxy, alkoxy, or aryloxy; and n is an integer from
0 to 12.
12. The method of claim 1, wherein said A.beta.40 inhibitor is
administered in a pharmaceutically acceptable formulation.
13. The method of claim 1, wherein said pharmaceutically acceptable
formulation is a dispersion system.
14. The method of claim 13, wherein said pharmaceutically
acceptable formulation comprises a lipid-based formulation.
15. The method of claim 14, wherein said pharmaceutically
acceptable formulation comprises a liposome formulation.
16. The method of claim 15, wherein said pharmaceutically
acceptable formulation comprises a multivesicular liposome
formulation.
17. The method of claim 12, wherein said pharmaceutically
acceptable formulation comprises a polymeric matrix.
18. The method of claim 17, wherein said polymeric matrix is
selected from the group consisting of naturally derived polymers,
such as albumin, alginate, cellulose derivatives, collagen, fibrin,
gelatin, and polysaccharides.
19. The method of claim 17, wherein said polymeric matrix is
selected from the group consisting of synthetic polymers such as
polyesters (PLA, PLGA), polyethylene glycol, poloxomers,
polyanhydrides, and pluronics.
20. The method of claim 17, wherein said polymeric matrix is in the
form of microspheres.
21. The method of claim 12, wherein the pharmaceutically acceptable
formulation provides sustained delivery of said A.beta.40 inhibitor
to a subject.
22. A method of treating a disease state characterized by cerebral
amyloid angiopathy in a subject, comprising administering an
A.beta.40 inhibitor to said subject, such that said disease state
characterized by cerebral amyloid angiopathy is treated, provided
said A.beta.40 inhibitor is not 3-amino-1-propanesulfonic acid.
23. The method of claim 22, wherein said A.beta.40 inhibitor has
the structure: Q-[--Y.sup.-X.sup.+].sub.n wherein Y.sup.- is an
anionic group at physiological pH; Q is a carrier group; X.sup.+ is
a cationic group; and n is an integer selected such that the
biodistribution of the A.beta.40 inhibitor for an intended target
site is not prevented while maintaining activity of the A.beta.40
inhibitor, such that cerebral amyloid angiopathy is inhibited.
24. The method of claim 22, wherein said A.beta.40 inhibitor is
selected from the group consisting of ethanesulfonic acid,
1,2-ethanedisulfonic acid, 1-propanesulfonic acid,
1,3-propanedisulfonic acid, 1,4-butanedisulfonic acid,
1,5-pentanedisulfonic acid, 2-aminoethanesulfonic acid,
4-hydroxy-1-butanesulfonic acid, and pharmaceutically acceptable
salts thereof.
25. The method of claim 22, wherein said A.beta.40 inhibitor is
selected from the group consisting of 1-butanesulfonic acid,
1-decanesulfonic acid, 2-propanesulfonic acid, 3-pentanesulfonic
acid, 4-heptanesulfonic acid, and pharmaceutically acceptable salts
thereof.
26. The method of claim 22, wherein said A.beta.40 inhibitor is
1,7-dihydroxy-4-heptane sulfonic acid, or a pharmaceutically
acceptable salt thereof.
27. The method of claim 22, wherein said blood vessel wall cell is
selected from the group consisting of blood vessel wall smooth
muscle cells, pericytes and endothelial cells.
28. The method of claim 22, wherein said blood vessel wall cell is
a blood vessel wall smooth muscle cell.
29. The method of claim 22, wherein the death of said blood vessel
wall cell is prevented.
30. The method of claim 22, wherein structural changes to said
blood vessel wall cell are prevented.
31. The method of claim 22, wherein said A.beta.40 inhibitor is a
peptide or a peptidomimetic which interacts with specific regions
of the A.beta. peptide.
32. The method of claim 22, wherein said A.beta.40 inhibitor has
the following structure: ##STR00273## wherein Z is XR.sup.2 or
R.sup.4; R.sup.1 and R.sup.2 are each independently hydrogen, a
substituted or unsubstituted aliphatic group, an aryl group, a
heterocyclic group, or a salt-forming cation; R.sup.3 is hydrogen,
lower alkyl, aryl, or a salt-forming cation; R.sup.4 is hydrogen,
lower alkyl, aryl or amino; X is, independently for each
occurrence, O or S; Y.sup.1 and Y.sup.2 are each independently
hydrogen, halogen, alkyl, amino, hydroxy, alkoxy, or aryloxy; and n
is an integer from 0 to 12.
33. The method of claim 22, wherein said A.beta.40 inhibitor is
administered in a pharmaceutically acceptable formulation.
34. The method of claim 33, wherein said pharmaceutically
acceptable formulation is a dispersion system.
35. The method of claim 34, wherein said pharmaceutically
acceptable formulation comprises a lipid-based formulation.
36. The method of claim 35, wherein said pharmaceutically
acceptable formulation comprises a liposome formulation.
37. The method of claim 36, wherein said pharmaceutically
acceptable formulation comprises a multivesicular liposome
formulation.
38. The method of claim 33, wherein said pharmaceutically
acceptable formulation comprises a polymeric matrix.
39. The method of claim 38, wherein said polymeric matrix is
selected from the group consisting of naturally derived polymers,
such as albumin, alginate, cellulose derivatives, collagen, fibrin,
gelatin, and polysaccharides.
40. The method of claim 38, wherein said polymeric matrix is
selected from the group consisting of synthetic polymers such as
polyesters (PLA, PLGA), polyethylene glycol, poloxomers,
polyanhydrides, and pluronics.
41. The method of claim 38, wherein said polymeric matrix is in the
form of microspheres.
42. The method of claim 33, wherein the pharmaceutically acceptable
formulation provides sustained delivery of said A.beta.40 inhibitor
to a subject.
43. A method of inhibiting cerebral amyloid angiopathy in a
subject, comprising administering an A.beta.40 inhibitor to said
patient in an effective amount and manner such that said A.beta.40
inhibitor contacts a blood vessel wall cell in said patient and
that cerebral amyloid angiopathy is inhibited, provided said
A.beta.40 inhibitor is not 3-amino-1-propanesulfonic acid.
44. The method of claim 43, wherein the A.beta.40 inhibitor has the
following structure: Q-[--Y.sup.-X.sup.+].sub.n wherein Y.sup.- is
an anionic group at physiological pH; Q is a carrier group; X.sup.+
is a cationic group; and n is an integer selected such that the
biodistribution of the A.beta.40 inhibitor for an intended target
site is not prevented while maintaining activity of the A.beta.40
inhibitor, such that cerebral amyloid angiopathy is inhibited.
45. The method of claim 43, wherein said A.beta.40 inhibitor is
selected from the group consisting of ethanesulfonic acid,
1,2-ethanedisulfonic acid, 1-propanesulfonic acid,
1,3-propanedisulfonic acid, 1,4-butanedisulfonic acid,
1,5-pentanedisulfonic acid, 2-aminoethanesulfonic acid,
4-hydroxy-1-butanesulfonic acid, and pharmaceutically acceptable
salts thereof.
46. The method of claim 43, wherein said A.beta.40 inhibitor is
selected from the group consisting of 1-butanesulfonic acid,
1-decanesulfonic acid, 2-propanesulfonic acid, 3-pentanesulfonic
acid, 4-heptanesulfonic acid, and pharmaceutically acceptable salts
thereof.
47. The method of claim 43, wherein said A.beta.40 inhibitor is
1,7-dihydroxy-4-heptanesulfonic acid, or a pharmaceutically
acceptable salt thereof.
48. The method of claim 43, wherein said blood vessel wall cell is
selected from the group consisting of blood vessel wall smooth
muscle cells, pericytes and endothelial cells.
49. The method of claim 43, wherein said blood vessel wall cell is
a blood vessel wall smooth muscle cell.
50. The method of claim 43, wherein the death of said blood vessel
wall cell is prevented.
51. The method of claim 43, wherein structural changes to said
blood vessel wall cell are prevented.
52. The method of claim 43, wherein said A.beta.40 inhibitor is a
peptide or a peptidomimetic which interacts with specific regions
of the A.beta. peptide.
53. The method of claim 43, wherein said A.beta.40 inhibitor has
the following structure: ##STR00274## wherein Z is XR.sup.2 or
R.sup.4; R.sup.1 and R.sup.2 are each independently hydrogen, a
substituted or unsubstituted aliphatic group, an aryl group, a
heterocyclic group, or a salt-forming cation; R.sup.3 is hydrogen,
lower alkyl, aryl, or a salt-forming cation; R.sup.4 is hydrogen,
lower alkyl, aryl or amino; X is, independently for each
occurrence, O or S; Y.sup.1 and Y.sup.2 are each independently
hydrogen, halogen, alkyl, amino, hydroxy, alkoxy, or aryloxy; and n
is an integer from 0 to 12.
54. The method of claim 43, wherein said A.beta.40 inhibitor is
administered in a pharmaceutically acceptable formulation.
55. The method of claim 54, wherein said pharmaceutically
acceptable formulation is a dispersion system.
56. The method of claim 55, wherein said pharmaceutically
acceptable formulation comprises a lipid-based formulation.
57. The method of claim 56, wherein said pharmaceutically
acceptable formulation comprises a liposome formulation.
58. The method of claim 57, wherein said pharmaceutically
acceptable formulation comprises a multivesicular liposome
formulation.
59. The method of claim 54, wherein said pharmaceutically
acceptable formulation comprises a polymeric matrix.
60. The method of claim 59, wherein said polymeric matrix is
selected from the group consisting of naturally derived polymers,
such as albumin, alginate, cellulose derivatives, collagen, fibrin,
gelatin, and polysaccharides.
61. The method of claim 59, wherein said polymeric matrix is
selected from the group consisting of synthetic polymers such as
polyesters (PLA, PLGA), polyethylene glycol, poloxomers,
polyanhydrides, and pluronics.
62. The method of claim 59, wherein said polymeric matrix is in the
form of microspheres.
63. The method of claim 54, wherein the pharmaceutically acceptable
formulation provides sustained delivery of said A.beta.40 inhibitor
to a subject.
64. A method of inhibiting cerebral amyloid angiopathy, comprising
contacting a blood vessel wall cell with a A.beta.40 inhibitor
having the structure: ##STR00275## wherein Z is XR.sup.2 or
R.sup.4; R.sup.1 and R.sup.2 are each independently hydrogen, a
substituted or unsubstituted aliphatic group, an aryl group, a
heterocyclic group, or a salt-forming cation; R.sup.3 is hydrogen,
lower alkyl, aryl, or a salt-forming cation; R.sup.4 is hydrogen,
lower alkyl, aryl or amino; X is, independently for each
occurrence, O or S; Y.sup.1 and Y.sup.2 are each independently
hydrogen, halogen, alkyl, amino, hydroxy, alkoxy, or aryloxy; and n
is an integer from 0 to 12, such that cerebral amyloid angiopathy
is inhibited.
65. A method of inhibiting cerebral amyloid angiopathy in a
subject, comprising administering an A.beta.40 inhibitor to said
patient in an effective amount and manner such that said A.beta.40
inhibitor contacts a blood vessel wall cell in said patient, said
A.beta.40 inhibitor having the structure: ##STR00276## wherein Z is
XR.sup.2 or R.sup.4; R.sup.1 and R.sup.2 are each independently
hydrogen, a substituted or unsubstituted aliphatic group, an aryl
group, a heterocyclic group, or a salt-forming cation; R.sup.3 is
hydrogen, lower alkyl, aryl, or a salt-forming cation; R.sup.4 is
hydrogen, lower alkyl, aryl or amino; X is, independently for each
occurrence, O or S; Y.sup.1 and Y.sup.2 are each independently
hydrogen, halogen, alkyl, amino, hydroxy, alkoxy, or aryloxy; and n
is an integer from 0 to 12, such that cerebral amyloid angiopathy
is inhibited.
66. The method of claim 65, wherein said A.beta.40 inhibitor has
the structure: ##STR00277##
67. The method of claim 65, wherein said A.beta.40 inhibitor has
the structure: ##STR00278## wherein R.sup.a and R.sup.b are each
independently hydrogen, alkyl, aryl, or heterocyclyl, or R.sup.a
and R.sup.b, taken together with the nitrogen atom to which they
are attached, form a cyclic moiety having from 3 to 8 atoms in the
ring, and n is an integer from 0 to 6.
68. The method of claim 67, wherein R.sup.a and R.sup.b are each
hydrogen.
69. The method of claim 65, wherein said A.beta.40 inhibitor has
the structure: ##STR00279## wherein R.sup.1 and R.sup.2 are each
independently hydrogen, an aliphatic group, an aryl group, a
heterocyclic group, or a salt-forming cation; R.sup.3 is hydrogen,
lower alkyl, aryl, or a salt-forming cation; Y.sup.1 and Y.sup.2
are each independently hydrogen, halogen, lower alkyl, hydroxy,
alkoxy, or aryloxy; and n is an integer from 0 to 12.
70. The method of claim 65, wherein R.sup.1 and R.sup.2 are an
aliphatic group selected from the group consisting of a branched or
straight-chain aliphatic moiety having from about 1 to 24 carbon
atoms or a branched or straight-chain aliphatic moiety having from
about 10 to 24 carbon atoms, in the chain; and an unsubstituted or
substituted cyclic aliphatic moiety having from 4 to 7 carbon atoms
in the aliphatic ring.
71. A method of inhibiting cerebral amyloid angiopathy in a
subject, comprising administering an A.beta.40 inhibitor to said
patient in an effective amount and manner such that said A.beta.40
inhibitor contacts a blood vessel wall cell in said patient, said
A.beta.40 inhibitor having the structure: ##STR00280## wherein G
represents hydrogen or one or more substituents on the aryl ring
and L is a substituted alkyl group, and M.sup.+ is a counter ion,
such that cerebral amyloid angiopathy is inhibited.
72. The method of claim 71, where G is hydrogen or an
electron-donating group.
73. The method of claim 71, where G is an electron-withdrawing
group at the meta position.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/747,408, filed Dec. 22, 2000, which claims the benefit of
priority under 35 U.S.C. 119(e) to copending U.S. Provisional
Application No. 60/171,877, filed Dec. 23, 1999, the entire
contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Cerebral amyloid angiopathy (CAA) remains a largely
untreatable disease often not diagnosed until autopsy. It ranges in
severity from asymptomatic amyloid deposition in otherwise normal
cerebral vessels to complete replacement and breakdown of the
cerebrovascular wall. Severe CAA can cause lobar cerebral
hemorrhage, transient neurologic symptoms, and dementia with
leukoencephalopathy. (see Greenberg, Neurology 1998,
51:690-694).
[0003] Amyloid-.beta. (A.beta.) is a toxic peptide which is
implicated in the pathogenesis of CAA. A.beta. peptide is derived
from a normal proteolytic cleavage of the precursor protein, the
Amyloid-.beta. precursor protein (.beta.APP). Advanced cases of CAA
demonstrate structural changes to the walls of the amyloid-laden
vessel such as cracking between layers, smooth muscle cell
toxicity, microaneuryism formation, and fibrinoid necrosis.
[0004] The exact mechanisms involved in the genesis of cerebral
amyloid angiopathy (CAA) have not been completely established, but
it appears that a preponderance of the form of the 39-40 amino acid
A.beta. peptide (A.beta.40) is responsible for the deposits on
blood vessel wall cells which lead to CAA, in comparison to the
42-43 amino acid A.beta. peptides (A.beta.42 and A.beta.43), which
are implicated in other amyloid-related conditions such as
Alzheimer's Disease (AD).
SUMMARY OF THE INVENTION
[0005] The present invention provides methods for modulating, e.g.,
inhibiting and/or preventing, cerebral amyloid angiopathy. The
present invention is based, at least in part, on the discovery that
compounds which interfere with the deposition of A.beta. peptide,
e.g., the A.beta.40 peptide, in blood vessel wall cells, prevent
the structural changes to cerebral blood vessels like capillaries,
that lead to CAA. It is believed, without intending to limit the
invention as claimed herein, that the compounds of the invention
interfere with the association of the A.beta.40 peptide, e.g., the
association of the A.beta.40 peptide to the sulfate GAGs present at
the smooth muscle cell surface, and thus prevent intracellular and
extracellular amyloid deposition. However, while it is believed
that inhibition of A.beta.40 is a significant factor in inhibiting
CAA, the A.beta.40 inhibitors of the invention may well work in
other ways to inhibit or prevent CAA, and these are intended to be
part of the present invention.
[0006] Accordingly, this invention pertains to a method of
modulating, e.g., inhibiting and/or preventing, cerebral amyloid
angiopathy. The method includes contacting a blood vessel wall cell
with an A.beta.40 inhibitor, such that the compound inhibits or
prevents cerebral amyloid angiopathy. The A.beta.40 inhibitor is
believed to at least interfere with the ability of the A.beta.40
peptide to form amyloid fibrils and/or with the ability of the
A.beta.40 peptide to bind to a cell (e.g., blood vessel wall smooth
muscle cells, pericytes or endothelial cells) surface molecule or
structure, forming deposits on the walls of the blood vessel and
thus prevent A.beta.-induced cell death and/or the structural
changes to cerebral blood vessels, e.g., capillaries, medium sized
arteries, or arterioles, that lead to CAA. The A.beta.40 peptide
can be either in a soluble form or in a fibril form.
[0007] In one embodiment, the A.beta.40 inhibitor may be
ethanesulfonic acid, 1,2-ethanedisulfonic acid, 1-propanesulfonic
acid, 1,3-propanedisulfonic acid, 1,4-butanedisulfonic acid,
1,5-pentanedisulfonic acid, 2-aminoethanesulfonic acid, or
4-hydroxy-1-butanesulfonic acid, and pharmaceutically acceptable
salts thereof. In other preferred embodiments, the A.beta.40
inhibitor may be 1-butanesulfonic acid, 1-decanesulfonic acid,
2-propanesulfonic acid, 3-pentanesulfonic acid, or
4-heptanesulfonic acid, and pharmaceutically acceptable salts
thereof. In yet further preferred embodiments, the A.beta.40
inhibitor may be 1,7-dihydroxy-4-heptanesulfonic acid,
3-amino-1-propanesulfonic acid, or a pharmaceutically acceptable
salt thereof. In another embodiment the A.beta.40 inhibitor is a
peptide or a peptidomimetic which interacts with specific regions
of the A.beta. peptide such as the regions responsible for cellular
adherence (aa 10-16), GAG binding site region (13-16) or the region
responsible for the .beta.-sheet formation (16-21). These peptides
are the d-stereoisomers of the A.beta. or complementary image of
the A.beta. peptide.
[0008] In one embodiment, the A.beta.40 inhibitor is administered
in a pharmaceutically acceptable formulation. The pharmaceutically
acceptable formulation can be a dispersion system like a
lipid-based formulation, a liposome formulation, or a
multivesicular liposome formulation. The pharmaceutically
acceptable formulation can also comprise a polymeric matrix, e.g.,
synthetic polymers such as polyesters (PLA, PLGA), polyethylene
glycol, poloxomers, polyanhydrides, and pluronics; or naturally
derived polymers, such as albumin, alginate, cellulose derivatives,
collagen, fibrin, gelatin, and polysaccharides. In other preferred
embodiments, the pharmaceutically acceptable formulation provides
sustained delivery of the A.beta.40 inhibitor to the target
site.
[0009] Yet another aspect of the invention pertains to a method of
treating a disease state characterized by cerebral amyloid
angiopathy in a subject. The method includes administering an
A.beta.40 inhibitor to the subject, such that the disease state
characterized by cerebral amyloid angiopathy is treated, e.g.,
inhibited or prevented.
[0010] Another aspect of the invention pertains to a method of
modulating, e.g., inhibiting and/or preventing, cerebral amyloid
angiopathy, including contacting a blood vessel wall cell with an
A.beta.40 inhibitor having the structure:
Q-[--Y.sup.-X.sup.+].sub.n
[0011] wherein Y.sup.- is an anionic group at physiological pH; Q
is a carrier group; X.sup.+ is a cationic group; and n is an
integer selected such that the biodistribution of the A.beta.40
inhibitor for an intended target site is not prevented while
maintaining activity of the A.beta.40 inhibitor, provided that the
A.beta.40 inhibitor is not chondroitin sulfate A, such that
cerebral amyloid angiopathy is inhibited or prevented.
[0012] In yet another aspect, the invention features a method of
modulating, e.g., inhibiting and/or preventing, cerebral amyloid
angiopathy, including contacting a blood vessel wall cell with an
A.beta.40 inhibitor having the structure:
##STR00001##
[0013] wherein Z is XR.sup.2 or R.sup.4, R.sup.1 and R.sup.2 are
each independently hydrogen, a substituted or unsubstituted
aliphatic group (preferably a branched or straight-chain aliphatic
moiety having from 1 to 24 carbon atoms in the chain; or an
unsubstituted or substituted cyclic aliphatic moiety having from 4
to 7 carbon atoms in the aliphatic ring; preferred aliphatic and
cyclic aliphatic groups are alkyl groups, more preferably lower
alkyl), an aryl group, a heterocyclic group, or a salt-forming
cation; R.sup.3 is hydrogen, lower alkyl, aryl, or a salt-forming
cation; R.sup.4 is hydrogen, lower alkyl, aryl or amino (including
alkylamino, dialkylamino (including cyclic amino moieties),
arylamino, diarylamino, and alkylarylamino); X is, independently
for each occurrence, O or S; Y.sup.1 and Y.sup.2 are each
independently hydrogen, halogen (e.g., F, Cl, Br, or I), alkyl
(preferably lower alkyl), amino, hydroxy, alkoxy, or aryloxy; and n
is an integer from 0 to 12 (more preferably 0 to 6, more preferably
0 or 1), such that cerebral amyloid angiopathy is inhibited or
prevented.
[0014] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
DETAILED DESCRIPTION OF THE INVENTION
[0015] The present invention is based, at least in part, on the
discovery that compounds which interfere with the ability of the
A.beta.40 peptide to form deposits in cerebral blood vessels, e.g.,
on the smooth muscle cells thereof, and thus prevent the structural
changes to cerebral blood vessels that lead to CAA.
[0016] As used herein, the language "contacting" is intended to
include both in vivo, in vitro, or ex vivo methods of bringing an
A.beta.40 inhibitor into proximity with a blood vessel wall cell,
such that the A.beta.40 inhibitor can inhibit or prevent CAA, e.g.,
via inhibiting the deposition of the A.beta.40 peptide. For
example, the blood vessel wall cell can be contacted with an
A.beta.40 inhibitor in vivo by administering the A.beta.40
inhibitor to a subject either parenterally, e.g., intravenously,
intradermally, subcutaneously, orally (e.g., via inhalation),
transdermally (topically), transmucosally, or rectally. A blood
vessel wall cell can also be contacted in vitro by, for example,
adding an A.beta.40 inhibitor into a tissue culture dish in which
blood vessel wall smooth muscle cells are grown.
[0017] As used herein, the term "subject" is intended to include
animals susceptible to states characterized by cerebral amyloid
angiopathy, preferably mammals, most preferably humans. In a
preferred embodiment, the subject is a primate. In an even more
preferred embodiment, the primate is a human. Other examples of
subjects include experimental animals such as mice, rats, dogs,
cats, goats, sheep, pigs, and cows. The experimental animal can be
an animal model for a disorder, e.g., a transgenic mouse.
[0018] The term "blood vessel wall cell" includes smooth muscle
cells, pericytes and endothelial cells. In a preferred embodiment
the blood vessel wall cell is a smooth muscle cell.
A.beta.40 Inhibitors
[0019] In one embodiment, the method of the invention includes
contacting a blood vessel wall cell in vitro or administering to a
subject in vivo, an effective amount of an A.beta.40 inhibitor,
which has at least one anionic group covalently attached to a
carrier molecule. As used herein, an "A.beta.40 inhibitor" includes
compounds which can interfere with the ability of a CAA-associated
A.beta. peptide, e.g., A.beta.40, to either form fibrils or
interact with a cell surface molecule such as a proteoglycan
constituent of a basement membrane, e.g., a glycosaminoglycan. An
A.beta.40 inhibitor can interfere with the ability of both
fibrillar or non-fibrillar CAA-associated A.beta. peptide, e.g.,
A.beta.40, to interact with a cell surface molecule.
[0020] The A.beta.40 inhibitor can have the structure:
Q-[--Y.sup.-X.sup.+].sub.n
[0021] wherein Y.sup.- is an anionic group at physiological pH; Q
is a carrier group; X.sup.+ is a cationic group; and n is an
integer. The number of anionic groups ("n") is selected such that
the biodistribution of the A.beta.40 inhibitor for an intended
target site is not prevented while maintaining activity of the
A.beta.40 inhibitor. For example, the number of anionic groups is
not so great as to prevent traversal of an anatomical barrier, such
as a cell membrane, or entry across a physiological barrier, such
as the blood-brain barrier. In one embodiment, n is an integer
between 1 and 10. In another embodiment, n is an integer between 3
and 8. These compounds are described in U.S. Pat. Nos. 5,643,562,
5,972,328, 5,728,375, 5,840,294, and U.S. Application No.
60/131,464. Such compounds also include or can be described as
glycosaminoglycan ("GAG") mimics or mimetics. Other compounds which
may be included are those described in, e.g., Pillot et al., Eur.
J. Biochem vol. 243 No. 3, 1997 (apoE2, apoE3); WO98/22441;
WO98/22430; WO96/10220; WO96/07425; and WO96/39834.
[0022] An anionic group of an A.beta.40 inhibitor of the invention
is a negatively charged moiety that, when attached to a carrier
group, can interfere with the ability of a CAA-associated A.beta.
peptide, e.g., A.beta.40, to either form fibrils or interact with a
cell surface molecule such as a proteoglycan constituent of a
basement membrane, e.g., a glycosaminoglycan ("GAG"). As such,
A.beta.40 is inhibited from forming deposits in blood vessels,
e.g., cerebral blood vessel wall smooth muscle cells, thus
preventing hardening of the vessel walls and, therefore, cerebral
amyloid angiopathy.
[0023] For purposes of this invention, the anionic group is
negatively charged at physiological pH. Preferably, the anionic
A.beta.40 inhibitor mimics the structure of a sulfated
proteoglycan, i.e., is a sulfated compound or a functional
equivalent thereof. "Functional equivalents" of sulfates are
intended to include compounds such as sulfamates as well as
bioisosteres. Bioisosteres encompass both classical bioisosteric
equivalents and non-classical bioisosteric equivalents. Classical
and non-classical bioisosteres of sulfate groups are known in the
art (see e.g., Silverman, R. B. The Organic Chemistry of Drug
Design and Drug Action, Academic Press, Inc.: San Diego, Calif.,
1992, pp. 19-23). Accordingly, an A.beta.40 inhibitor of the
invention can comprise at least one anionic group including
sulfonates, sulfates, sulfamates, phosphonates, phosphates,
carboxylates, and heterocyclic groups of the following
formulae:
##STR00002##
[0024] Depending on the carrier group, more than one anionic group
can be attached thereto. When more than one anionic group is
attached to a carrier group, the multiple anionic groups can be the
same structural group (e.g., all sulfonates) or, alternatively, a
combination of different anionic groups can be used (e.g.,
sulfonates, phosphonates, and sulfates, etc.).
[0025] The ability of an A.beta.40 inhibitor of the invention to
inhibit an interaction between A.beta.40 peptide and a glycoprotein
or proteoglycan constituent of a basement membrane can be assessed
by an in vitro binding assay, such as the one described in Leveugle
B. et al. (1998) J. of Neurochem. 70(2):736-744. Briefly, a
constituent of the basement membrane, preferably a
glycosaminoglycan (GAG) can be radiolabeled, e.g., at a specific
activity of 10,000 cpm, and then incubated with A.beta.40
peptide-Sepharose beads at, for example, a ratio of 5:1 (v/v) in
the presence or absence of the A.beta.40 inhibitor. The A.beta.40
peptide-Sepharose beads and the radiolabeled GAG can be incubated
for approximately 30 minutes at room temperature and then the beads
can be successively washed with a Tris buffer solution containing
NaCl (0.55 M and 2 M). The binding of the basement membrane
constituent (e.g., GAG) to the A.beta.40 peptide can then be
measured by collecting the fractions from the washings and
subjecting them to scintillation counting. An A.beta.40 inhibitor
which inhibits an interaction between A.beta.40 and a glycoprotein
or proteoglycan constituent of a basement membrane, e.g., GAG, will
increase the amount of radioactivity detected in the washings.
[0026] In the same manner, the invention relates to a method of
diagnosing CAA in vivo, whereas an labeled inhibitor of the
invention is administered to a subject and the disposition of the
inhibitor is determined to see whether a CAA-related condition
exists. The label may be one conventionally known in the art which
allows for detection of the compound either in vivo or in vitro,
e.g., radiolabel, fluorescent, etc. Using techniques with which
those of ordinary skill in the art will be familiar, e.g., PET
scan, bound tagged inhibitor of the invention may be visualized,
e.g., in regions where CAA would be found, such as near the
cerebellum.
[0027] Preferably, an A.beta.40 inhibitor of the invention
interacts with a binding site for a basement membrane glycoprotein
or proteoglycan in A.beta.40 and thereby inhibits the binding of
the A.beta.40 peptide to the basement membrane constituent, e.g.,
GAG. Basement membrane glycoproteins and proteoglycans include GAG,
laminin, collagen type IV, fibronectin, chondroitin sulfate,
perlecan, and heparan sulfate proteoglycan (HSPG). In a preferred
embodiment, the therapeutic compound inhibits an interaction
between an A.beta.40 peptide and a GAG. Consensus binding site
motifs for GAG in amyloidogenic proteins have been described (see,
for example, Hileman R. E. et al. (1998) BioEssays 20:156-167). For
example, a GAG consensus binding motif can be of the general
formula X--B--B--X--B--X or X--B--B--B--X--X--B--X, wherein B are
basic amino acids (e.g., lysine or arginine) and X are hydropathic
amino acids. A GAG consensus binding motif can further be of the
general formula T-X--X--B--X--X-T-B--X--X--X-T-B--B, wherein T
defines a turn of a basic amino acid, Bs are basic amino acids
(e.g., lysine, arginine, or occasionally glutamine) and X are
hydropathic amino acids. The distance between the first and the
second turn can range from approximately 12 .ANG. to 17 .ANG.. The
distance between the second and the third turn can be approximately
14 .ANG.. The distance between the first and the third turn can
range from approximately 13 .ANG. to 18 .ANG..
[0028] Accordingly, in the A.beta.40 inhibitors of the invention,
when multiple anionic groups are attached to a carrier group, the
relative spacing of the anionic groups can be chosen such that the
anionic groups (e.g., sulfonates or phosphonates) optimally
interact with the basic residues within the GAG binding site
(thereby inhibiting interaction of GAG with the site). For example,
anionic groups can be spaced approximately 15.+-.1.5 .ANG.,
14.+-.1.5 .ANG. and/or 16.+-.1.5 .ANG. apart, or appropriate
multiples thereof, such that the relative spacing of the anionic
groups allows for optimal interaction with a binding site for a
basement membrane constituent (e.g., GAG) in an A.beta.40
peptide.
[0029] A.beta.40 inhibitors of the invention typically further
comprise a counter cation (i.e., X.sup.+ in the general formula:
Q-[--Y.sup.-X.sup.+].sub.n). Cationic groups include positively
charged atoms and moieties. If the cationic group is hydrogen,
H.sup.+, then the compound is considered an acid, e.g.,
ethanesulfonic acid. If hydrogen is replaced by a metal or its
equivalent, the compound is a salt of the acid. Pharmaceutically
acceptable salts of the A.beta.40 inhibitor are within the scope of
the invention. For example, X.sup.+ can be a pharmaceutically
acceptable alkali metal, alkaline earth, higher valency cation,
polycationic counter ion or ammonium. A preferred pharmaceutically
acceptable salt is a sodium salt but other salts are also
contemplated within their pharmaceutically acceptable range.
[0030] Within the A.beta.40 inhibitor, the anionic group(s) is
covalently attached to a carrier group. Suitable carrier groups
include aliphatic groups, alicyclic groups, heterocyclic groups,
aromatic groups, and groups derived from carbohydrates, polymers,
peptides, peptide derivatives, or combinations thereof. A carrier
group can be substituted, e.g., with one or more amino, nitro,
halogen, thiol or hydroxyl groups.
[0031] As used herein, the term "carbohydrate" is intended to
include substituted and unsubstituted mono-, oligo-, and
polysaccharides. Monosaccharides are simple sugars usually of the
formula C.sub.6H.sub.12O.sub.6 that can be combined to form
oligosaccharides or polysaccharides. Monosaccharides include
enantiomers and both the D and L stereoisomers of monosaccharides.
Carbohydrates can have multiple anionic groups attached to each
monosaccharide moiety. For example, in sucrose octasulfate, four
sulfate groups are attached to each of the two monosaccharide
moieties.
[0032] As used herein, the term "polymer" is intended to include
molecules formed by the chemical union of two or more combining
subunits called monomers. Monomers are molecules or compounds which
usually contain carbon and are of relatively low molecular weight
and simple structure. A monomer can be converted to a polymer by
combination with itself or other similar molecules or compounds. A
polymer may be composed of a single identical repeating subunit or
multiple different repeating subunits (copolymers). Polymers within
the scope of this invention include substituted and unsubstituted
vinyl, acryl, styrene and carbohydrate-derived polymers and
copolymers and salts thereof. In one embodiment, the polymer has a
molecular weight of approximately 800-1000 Daltons. Examples of
polymers with suitable covalently attached anionic groups (e.g.,
sulfonates or sulfates) include
poly(2-acrylamido-2-methyl-1-propanesulfonic acid);
poly(2-acrylamido-2-methyl-1-propanesulfonic
acid-co-acrylonitrile);
poly(2-acrylamido-2-methyl-1-propanesulfonic acid-co-styrene);
poly(vinylsulfonic acid); poly(sodium 4-styrenesulfonic acid); and
sulfates and/or sulfonates derived from: poly(acrylic acid);
poly(methyl acrylate); poly(methyl methacrylate); and poly(vinyl
alcohol); and pharmaceutically acceptable salts thereof. Examples
of polymers with suitable covalently attached anionic groups
include those of the formula:
##STR00003##
[0033] wherein R is SO.sub.3H or OSO.sub.3H; and pharmaceutically
acceptable salts thereof.
[0034] Peptides and peptide derivatives can also act as carriers.
The term "peptide" includes two or more amino acids covalently
attached through a peptide bond. Amino acids which can be used in
peptide carriers include those naturally occurring amino acids
found in proteins such as glycine, alanine, valine, cysteine,
leucine, isoleucine, serine, threonine, methionine, glutamic acid,
aspartic acid, glutamine, asparagine, lysine, arginine, proline,
histidine, phenylalanine, tyrosine, and tryptophan. The term "amino
acid" further includes analogs, derivatives and congeners of
naturally occurring amino acids, one or more of which can be
present in a peptide derivative. For example, amino acid analogs
can have lengthened or shortened side chains or variant side chains
with appropriate functional groups. Also included are the D and L
stereoisomers of an amino acid when the structure of the amino acid
admits of stereoisomeric forms. The term "peptide derivative"
further includes compounds which contain molecules which mimic a
peptide backbone but are not amino acids (so-called
peptidomimetics), such as benzodiazepine molecules (see e.g.,
James, G. L. et al. (1993) Science 260:1937-1942). The anionic
groups can be attached to a peptide or peptide derivative through a
functional group on the side chain of certain amino acids or other
suitable functional group. For example, a sulfate group can be
attached through the hydroxyl side chain of a serine residue. A
peptide can be designed to interact with a binding site for a
basement membrane constituent (e.g., a GAG) in an A.beta.40 peptide
(as described above). Accordingly, in one embodiment, the peptide
comprises four amino acids and anionic groups (e.g., sulfonates)
are attached to the first, second and fourth amino acid. For
example, the peptide can be Ser-Ser-Y-Ser, wherein an anionic group
is attached to the side chain of each serine residue and Y is any
amino acid. In addition to peptides and peptide derivatives, single
amino acids can be used as carriers in the A.beta.40 inhibitor of
the invention. For example, cysteic acid, the sulfonate derivative
of cysteine, can be used. Peptides such as disclosed in
International Application No. WO 00/68263 may be used, also, e.g.,
Lys-Ile-Val-Phe-Phe-Ala (SEQ ID NO:1); Lys-Lys-Leu-Val-Phe-Phe-Ala
(SEQ ID NO:2); Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO:3);
Lys-Phe-Val-Phe-Phe-Ala (SEQ ID NO:4); Ala-Phe-Phe-Val-Leu-Lys (SEQ
ID NO:5); Lys-Leu-Val-Phe (SEQ ID NO:6); Lys-Ala-Val-Phe-Phe-Ala
(SEQ ID NO:7); Lys-Leu-Val-Phe-Phe (SEQ ID NO:8);
Lys-Val-Val-Phe-Phe-Ala (SEQ ID NO:9); Lys-Ile-Val-Phe-Phe-Ala-NH,
(SEQ ID NO:10); Lys-Leu-Val-Phe-Phe-Ala-NH, (SEQ ID NO:11);
Lys-Phe-Val-Phe-Phe-Ala-NH, (SEQ ID NO:12);
Ala-Phe-Phe-Val-Leu-Lys-NH.sub.2 (SEQ ID NO:13);
Lys-Leu-Val-Phe-NH.sub.2 (SEQ ID NO:14);
Lys-Ala-Val-Phe-Phe-Ala-NH.sub.2 (SEQ ID NO:15);
Lys-Leu-Val-Phe-Phe-NH.sub.2 (SEQ ID NO:16);
Lys-Val-Val-Phe-Phe-Ala-NH.sub.2 (SEQ ID NO:17);
Lys-Leu-Val-Phe-Phe-Ala-Gln (SEQ ID NO:18);
Lys-Leu-Val-Phe-Phe-Ala-Gln-NH.sub.2 (SEQ ID NO:19);
His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-NH.sub.2 (SEQ ID NO:20);
Asp-Asp-Asp (SEQ ID NO:21); Lys-Val-Asp-Asp-Gln-Asp (SEQ ID NO:22);
His-His-Gln-Lys (SEQ ID NO:23); and
Gln-Lys-Leu-Val-Phe-Phe-NH.sub.2 (SEQ ID NO:24).
[0035] The term "aliphatic group" is intended to include organic
compounds characterized by straight or branched chains, typically
having between 1 and 22 carbon atoms. Aliphatic groups include
alkyl groups, alkenyl groups and alkynyl groups. In complex
structures, the chains can be branched or cross-linked. Alkyl
groups include saturated hydrocarbons having one or more carbon
atoms, including straight-chain alkyl groups and branched-chain
alkyl groups. Such hydrocarbon moieties may be substituted on one
or more carbons with, for example, a halogen, a hydroxyl, a thiol,
an amino, an alkoxy, an alkylcarboxy, an alkylthio, or a nitro
group. Unless the number of carbons is otherwise specified, "lower
aliphatic" as used herein means an aliphatic group, as defined
above (e.g., lower alkyl, lower alkenyl, lower alkynyl), but having
from one to six carbon atoms. Representatives of such lower
aliphatic groups, e.g., lower alkyl groups, are methyl, ethyl,
n-propyl, isopropyl, 2-chloropropyl, n-butyl, sec-butyl,
2-aminobutyl, isobutyl, tert-butyl, 3-thiopentyl, and the like. As
used herein, the term "amino" means --NH.sub.2; the term "nitro"
means --NO.sub.2; the term "halogen" designates --F, --Cl, --Br or
--I; the term "thiol" means SH; and the term "hydroxyl" means --OH.
Thus, the term "alkylamino" as used herein means --NHR wherein R is
an alkyl group as defined above. The term "alkylthio" refers to
--SR, wherein R is an alkyl group as defined above. The term
"alkylcarboxyl" as used herein means --COOR, wherein R is an alkyl
group as defined above. The term "alkoxy" as used herein means
--OR, wherein R is an alkyl group as defined above. Representative
alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the
like. The terms "alkenyl" and "alkynyl" refer to unsaturated
aliphatic groups analogous to alkyls, but which contain at least
one double or triple bond respectively.
[0036] The term "alicyclic group" is intended to include closed
ring structures of three or more carbon atoms. Alicyclic groups
include cycloparaffins or naphthenes which are saturated cyclic
hydrocarbons, cycloolefins which are unsaturated with two or more
double bonds, and cycloacetylenes which have a triple bond. They do
not include aromatic groups. Examples of cycloparaffins include
cyclopropane, cyclohexane, and cyclopentane. Examples of
cycloolefins include cyclopentadiene and cyclooctatetraene.
Alicyclic groups also include fused ring structures and substituted
alicyclic groups such as alkyl substituted alicyclic groups. In the
instance of the alicyclics such substituents can further comprise a
lower alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a
lower alkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl,
--CF.sub.3, --CN, or the like.
[0037] The term "heterocyclic group" is intended to include closed
ring structures in which one or more of the atoms in the ring is an
element other than carbon, for example, nitrogen, or oxygen.
Heterocyclic groups can be saturated or unsaturated and
heterocyclic groups such as pyrrole and furan can have aromatic
character. They include fused ring structures such as quinoline and
isoquinoline. Other examples of heterocyclic groups include
pyridine and purine. Heterocyclic groups can also be substituted at
one or more constituent atoms with, for example, a halogen, a lower
alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a lower
alkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl, --CF.sub.3,
--CN, or the like.
[0038] The term "aromatic group" is intended to include unsaturated
cyclic hydrocarbons containing one or more rings. Aromatic groups
include 5- and 6-membered single-ring groups which may include from
zero to four heteroatoms, for example, benzene, pyrrole, furan,
thiophene, imidazole, oxazole, thiazole, triazole, pyrazole,
pyridine, pyrazine, pyridazine and pyrimidine, and the like. The
aromatic ring may be substituted at one or more ring positions
with, for example, a halogen, a lower alkyl, a lower alkenyl, a
lower alkoxy, a lower alkylthio, a lower alkylamino, a lower
alkylcarboxyl, a nitro, a hydroxyl, --CF.sub.3, --CN, or the
like.
[0039] In a preferred embodiment of the method of the invention,
the A.beta.40 inhibitor administered to the subject is comprised of
at least one sulfonate group covalently attached to a carrier
group, or a pharmaceutically acceptable salt thereof. Accordingly,
an A.beta.40 inhibitor can have the structure:
Q-[--SO.sub.3.sup.-X.sup.+].sub.n
[0040] wherein Q is a carrier group; X.sup.+ is a cationic group;
and n is an integer. Suitable carrier groups and cationic groups
are those described hereinbefore. The number of sulfonate groups
("n") is selected such that the biodistribution of the compound for
an intended target site is not prevented while maintaining activity
of the compound as discussed earlier. In one embodiment, n is an
integer between 1 and 10. In another embodiment, n is an integer
between 3 and 8. As described earlier, an A.beta.40 inhibitor with
multiple sulfonate groups can have the sulfonate groups spaced such
that the compound interacts optimally with an HSPG binding site
within the A.beta.40 peptide.
[0041] In preferred embodiments, the carrier group for a
sulfonate(s) is a lower aliphatic group (e.g., a lower alkyl, lower
alkenyl or lower alkynyl), a heterocyclic group, and group derived
from a disaccharide, a polymer or a peptide or peptide derivative.
Furthermore, the carrier can be substituted, e.g., with one or more
amino, nitro, halogeno, sulfhydryl or hydroxyl groups. In certain
embodiments, the carrier for a sulfonate(s) is an aromatic
group.
[0042] Examples of suitable sulfonated polymeric A.beta.40
inhibitors include poly(2-acrylamido-2-methyl-1-propanesulfonic
acid); poly(2-acrylamido-2-methyl-1-propanesulfonic
acid-co-acrylonitrile);
poly(2-acrylamido-2-methyl-1-propanesulfonic acid-co-styrene);
poly(vinylsulfonic acid); poly(4-styrenesulfonic acid); a sulfonic
acid derivative of poly(acrylic acid); a sulfonic acid derivative
of poly(methyl acrylate); a sulfonic acid derivative of poly(methyl
methacrylate); and pharmaceutically acceptable salts thereof.
[0043] A preferred sulfonated polymer is poly(vinylsulfonic acid)
(PVS) or a pharmaceutically acceptable salt thereof, preferably the
sodium salt thereof. In one embodiment, PVS having a molecular
weight of about 800-1000 Daltons is used. PVS may be used as a
mixture of isomers or as a single active isomer.
[0044] Preferred sulfonated saccharides include
5-deoxy-1,2-O-isopropylidene-.alpha.-D-xylofuranose-5-sulfonic acid
(XXIII, shown as the sodium salt).
[0045] Preferred lower aliphatic sulfonated A.beta.40 inhibitors
for use in the invention include ethanesulfonic acid;
2-aminoethanesulfonic acid (taurine); cysteic acid (3-sulfoalanine
or .alpha.-amino-.beta.-sulfopropionic acid); 1-propanesulfonic
acid; 1,2-ethanedisulfonic acid; 1,3-propanedisulfonic acid;
1,4-butanedisulfonic acid; 1,5-pentanedisulfonic acid; and
4-hydroxy-1-butanesulfonic acid (VIII, shown as the sodium salt);
and pharmaceutically acceptable salts thereof. Other aliphatic
sulfonated A.beta.40 inhibitors contemplated for use in the
invention include 1-butanesulfonic acid (XLVII, shown as the sodium
salt), 2-propanesulfonic acid (XLIX, shown as the sodium salt),
3-pentanesulfonic acid (L, shown as the sodium salt),
4-heptanesulfonic acid (LII, shown as the sodium salt),
1-decanesulfonic acid (XLVIII, shown as the sodium salt); and
pharmaceutically acceptable salts thereof. Sulfonated substituted
aliphatic A.beta.40 inhibitors contemplated for use in the
invention include 3-amino-1-propanesulfonic acid (XXII, shown as
the sodium salt), 3-hydroxy-1-propanesulfonic acid sulfate (XXXV,
shown as the disodium salt), 1,7-dihydroxy-4-heptanesulfonic acid
(LIII, shown as the sodium salt); and pharmaceutically acceptable
salts thereof. Yet other sulfonated compounds contemplated for use
in the invention include 2-[(4-pyridinyl)amido]ethanesulfonic acid
(LIV, depicted as the sodium salt), and pharmaceutically acceptable
salts thereof.
[0046] Preferred heterocyclic sulfonated A.beta.40 inhibitors
include 3-(N-morpholino)-1-propanesulfonic acid; and
tetrahydrothiophene-1,1-dioxide-3,4-disulfonic acid; and
pharmaceutically acceptable salts thereof.
[0047] Aromatic sulfonated A.beta.40 inhibitors include
1,3-benzenedisulfonic acid (XXXVI, shown as the disodium salt),
2,5-dimethoxy-1,4-benzenedisulfonic acid (depicted as the disodium
salt, XXXVII, or the dipotassium salt, XXXIX),
4-amino-3-hydroxy-1-naphthalenesulfonic acid (XLIII),
3,4-diamino-1-naphthalenesulfonic acid (XLIV); and pharmaceutically
acceptable salts thereof.
[0048] In another embodiment of the method of the invention, the
A.beta.40 inhibitor administered to the subject is comprised of at
least one sulfate group covalently attached to a carrier group, or
a pharmaceutically acceptable salt thereof. Accordingly, the
A.beta.40 inhibitor can have the structure:
Q-[--OSO.sub.3.sup.-X.sup.+].sub.n
[0049] wherein Q is a carrier group; X.sup.+ is a cationic group;
and n is an integer. Suitable carriers and cationic groups are
those described hereinbefore. The number of sulfate groups ("n") is
selected such that the biodistribution of the compound for an
intended target site is not prevented while maintaining activity of
the A.beta.40 inhibitor as discussed earlier. In one embodiment, n
is an integer between 1 and 10. In another embodiment, n is an
integer between 3 and 8. As described earlier, an A.beta.40
inhibitor with multiple sulfate groups can have the sulfate groups
spaced such that the compound interacts optimally with a GAG
binding site within an A.beta. peptide.
[0050] In preferred embodiments, the carrier group for a sulfate(s)
is a lower aliphatic group (e.g., a lower alkyl, lower alkenyl or
lower alkynyl), an aromatic group, a group derived from a
disaccharide, a polymer or a peptide or peptide derivative.
Furthermore, the carrier can be substituted, e.g., with one or more
amino, nitro, halogeno, sulfhydryl or hydroxyl groups.
[0051] Examples of suitable sulfated polymeric A.beta.40 inhibitors
include poly(2-acrylamido-2-methyl-1-propyl sulfuric acid);
poly(2-acrylamido-2-methyl-1-propyl sulfuric
acid-co-acrylonitrile); poly(2-acrylamido-2-methyl-propyl sulfuric
acid-co-styrene); poly(vinylsulfuric acid); poly(sodium
4-styrenesulfate); a sulfate derivative of poly(acrylic acid); a
sulfate derivative of poly(methyl acrylate); a sulfate derivative
of poly(methyl methacrylate); and a sulfate derivative of
poly(vinyl alcohol); and pharmaceutically acceptable salts
thereof.
[0052] A preferred sulfated polymer is poly(vinylsulfuric acid) or
pharmaceutically acceptable salt thereof.
[0053] A preferred sulfated disaccharide is sucrose octasulfate or
pharmaceutically acceptable salt thereof. Other sulfated
saccharides contemplated for use in the invention include the acid
form of methyl .alpha.-D-glucopyranoside 2,3-disulfate (XVI),
methyl 4,6-O-benzylidene-.alpha.-D-glucopyranoside 2,3-disulfate
(XVII), 2,3,4,3',4'-sucrose pentasulfate (XXXIII),
1,3:4,6-di-O-benzylidene-D-mannitol 2,5-disulfate (XLI), D-mannitol
2,5-disulfate (XLII), 2,5-di-O-benzyl-D-mannitol tetrasulfate
(XLV); and pharmaceutically acceptable salts thereof.
[0054] Preferred lower aliphatic sulfated A.beta.40 inhibitors for
use in the invention include ethyl sulfuric acid; 2-aminoethan-1-ol
sulfuric acid; 1-propanol sulfuric acid; 1,2-ethanediol disulfuric
acid; 1,3-propanediol disulfuric acid; 1,4-butanediol disulfuric
acid; 1,5-pentanediol disulfuric acid; and 1,4-butanediol
monosulfuric acid; and pharmaceutically acceptable salts thereof.
Other sulfated aliphatic A.beta.40 inhibitors contemplated for use
in the invention include the acid form of 1,3-cyclohexanediol
disulfate (XL), 1,3,5-heptanetriol trisulfate (XIX),
2-hydroxymethyl-1,3-propanediol trisulfate (XX),
2-hydroxymethyl-2-methyl-1,3-propanediol trisulfate (XXI),
1,3,5,7-heptanetetraol tetrasulfate (XLVI), 1,3,5,7,9-nonane
pentasulfate (LI); and pharmaceutically acceptable salts thereof.
Other sulfated A.beta.40 inhibitors contemplated for use in the
invention include the acid form of
2-amino-2-hydroxymethyl-1,3-propanediol trisulfate (XXIV),
2-benzyloxy-1,3-propanediol disulfate (XXIX),
3-hydroxypropylsulfamic acid sulfate (XXX), 2,2'-iminoethanol
disulfate (XXXI), N,N-bis(2-hydroxyethyl)sulfamic acid disulfate
(XXXII); and pharmaceutically acceptable salts thereof.
[0055] Preferred heterocyclic sulfated A.beta.40 inhibitors include
3-(N-morpholino)-1-propyl sulfuric acid; and
tetrahydrothiophene-3,4-diol-1,1-dioxide disulfuric acid; and
pharmaceutically acceptable salts thereof.
[0056] The invention further contemplates the use of prodrugs which
are converted in vivo to the A.beta.40 inhibitors used in the
methods of the invention (see, e.g., R. B. Silverman, 1992, "The
Organic Chemistry of Drug Design and Drug Action", Academic Press,
Chp. 8). Such prodrugs can be used to alter the biodistribution
(e.g., to allow compounds which would not typically cross the
blood-brain barrier to cross the blood-brain barrier) or the
pharmacokinetics of the A.beta.40 inhibitor. For example, an
anionic group, e.g., a sulfate or sulfonate, can be esterified,
e.g, with a methyl group or a phenyl group, to yield a sulfate or
sulfonate ester. When the sulfate or sulfonate ester is
administered to a subject, the ester is cleaved, enzymatically or
non-enzymatically, reductively or hydrolytically, to reveal the
anionic group. Such an ester can be cyclic, e.g., a cyclic sulfate
or sultone, or two or more anionic moieties may be esterified
through a linking group. Exemplary cyclic A.beta.40 inhibitors
include, for example, 2-sulfobenzoic acid cyclic anhydride (LV),
1,3-propane sultone (LVI), 1,4-butane sultone (LVII),
1,3-butanediol cyclic sulfate (LVIII),
.alpha.-chloro-.alpha.-hydroxy-o-toluenesulfonic acid
.gamma.-sultone (LIX), and 6-nitronaphth-[1,8-cd]-1,2,-oxathiole
2,2-dioxide (LX). In a preferred embodiment, the prodrug is a
cyclic sulfate or sultone. An anionic group can be esterified with
moieties (e.g., acyloxymethyl esters) which are cleaved to reveal
an intermediate A.beta.40 inhibitor which subsequently decomposes
to yield the active A.beta.40 inhibitor. In another embodiment, the
prodrug is a reduced form of a sulfate or sulfonate, e.g., a thiol,
which is oxidized in vivo to the A.beta.40 inhibitor. Furthermore,
an anionic moiety can be esterified to a group which is actively
transported in vivo, or which is selectively taken up by target
organs. The ester can be selected to allow specific targeting of
the A.beta.40 inhibitors to particular organs, as described below
for carrier moieties.
[0057] Carrier groups useful in the A.beta.40 inhibitors include
groups previously described, e.g., aliphatic groups, alicyclic
groups, heterocyclic groups, aromatic groups, groups derived from
carbohydrates, polymers, peptides, peptide derivatives, or
combinations thereof. Suitable polymers include substituted and
unsubstituted vinyl, acryl, styrene and carbohydrate-derived
polymers and copolymers and salts thereof. Preferred carrier groups
include a lower alkyl group, a heterocyclic group, a group derived
from a disaccharide, a polymer, a peptide, or peptide
derivative.
[0058] Carrier groups useful in the present invention may also
include moieties which allow the A.beta.40 inhibitor to be
selectively delivered to a target organ or organs. For example, for
a desirable delivery of an A.beta.40 inhibitor to the brain, the
carrier group may include a moiety capable of targeting the
A.beta.40 inhibitor to the brain, by either active or passive
transport (a "targeting moiety"). Illustratively, the carrier group
may include a redox moiety, as described in, for example, U.S. Pat.
Nos. 4,540,564 and 5,389,623, both to Bodor. These patents disclose
drugs linked to dihydropyridine moieties which can enter the brain,
where they are oxidized to a charged pyridinium species which is
trapped in the brain. Thus, drug accumulates in the brain.
Exemplary pyridine/dihydropyridine compounds of the invention
include sodium 2-(nicotinylamido)-ethanesulfonate (LXII), and
1-(3-sulfopropyl)-pyridinium betaine (LXIII). Other carrier
moieties include groups, such as those derived from amino acids or
thyroxine, which can be passively or actively transported in vivo.
An illustrative compound is phenylalanyltaurine (LXIX), in which a
taurine molecule is conjugated to a phenylalanine (a large neutral
amino acid). Such a carrier moiety can be metabolically removed in
vivo, or can remain intact as part of an active A.beta.40
inhibitor. Structural mimics of amino acids (and other actively
transported moieties) are also useful in the invention (e.g.,
1-(aminomethyl)-1-(sulfomethyl)-cyclohexane (LXX)). Other exemplary
amino acid mimetics include p-(sulfomethyl)phenylalanine (LXXII),
p-(1,3-disulfoprop-2-yl)phenylalanine (LXXIII), and
O-(1,3-disulfoprop-2-yl)tyrosine (LXXIV). Exemplary thyroxine
mimetics include compounds LXXV, LXVI, and LXXVII. Many targeting
moieties are known, and include, for example, asialoglycoproteins
(see, e.g., Wu, U.S. Pat. No. 5,166,320) and other ligands which
are transported into cells via receptor-mediated endocytosis (see
below for further examples of targeting moieties which may be
covalently or non-covalently bound to a carrier molecule).
Furthermore, the A.beta.40 inhibitors of the invention may bind to
amyloidogenic proteins, e.g., A.beta.40, in the circulation and
thus be transported to the site of action.
[0059] The targeting and prodrug strategies described above can be
combined to produce an A.beta.40 inhibitor that can be transported
as a prodrug to a desired site of action and then unmasked to
reveal an active A.beta.40 inhibitor. For example, the
dihydropyridine strategy of Bodor (see supra) can be combined with
a cyclic prodrug, as for example in the compound
2-(1-methyl-1,4-dihydronicotinoyl)amidomethyl-propanesultone
(LXXI).
[0060] In one embodiment, the A.beta.40 inhibitor in the
pharmaceutical compositions is a sulfonated polymer, for example
poly(2-acrylamido-2-methyl-1-propanesulfonic acid);
poly(2-acrylamido-2-methyl-1-propanesulfonic
acid-co-acrylonitrile);
poly(2-acrylamido-2-methyl-1-propanesulfonic acid-co-styrene);
poly(vinylsulfonic acid); poly(4-styrenesulfonic acid); a sulfonate
derivative of poly(acrylic acid); a sulfonate derivative of
poly(methyl acrylate); a sulfonate derivative of poly(methyl
methacrylate); and a sulfonate derivative of poly(vinyl alcohol);
and pharmaceutically acceptable salts thereof.
[0061] In another embodiment, the A.beta.40 inhibitor in the
pharmaceutical compositions is a sulfated polymer, for example
poly(2-acrylamido-2-methyl-1-propyl sulfuric acid);
poly(2-acrylamido-2-methyl-1-propyl sulfuric
acid-co-acrylonitrile); poly(2-acrylamido-2-methyl-1-propyl
sulfuric acid-co-styrene); poly(vinyl sulfuric acid);
poly(4-styrenesulfate); a sulfate derivative of poly(acrylic acid);
a sulfate derivative of poly(methyl acrylate); a sulfate derivative
of poly(methyl methacrylate); and pharmaceutically acceptable salts
thereof.
[0062] The A.beta.40 inhibitor can also have the structure:
##STR00004##
[0063] wherein Z is XR.sup.2 or R.sup.4, R.sup.1 and R.sup.2 are
each independently hydrogen, a substituted or unsubstituted
aliphatic group (preferably a branched or straight-chain aliphatic
moiety having from 1 to 24 carbon atoms in the chain; or an
unsubstituted or substituted cyclic aliphatic moiety having from 4
to 7 carbon atoms in the aliphatic ring; preferred aliphatic and
cyclic aliphatic groups are alkyl groups, more preferably lower
alkyl), an aryl group, a heterocyclic group, or a salt-forming
cation; R.sup.3 is hydrogen, lower alkyl, aryl, or a salt-forming
cation; X is, independently for each occurrence, O or S; R.sup.4 is
hydrogen, lower alkyl, aryl or amino; Y.sup.1 and Y.sup.2 are each
independently hydrogen, halogen (e.g., F, Cl, Br, or I), lower
alkyl, amino (including alkylamino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), hydroxy, alkoxy, or aryloxy; and
n is an integer from 0 to 12 (more preferably 0 to 6, more
preferably 0 or 1). These compounds are described in U.S. Pat. No.
5,869,469, the contents of which is incorporated herein by
reference.
[0064] Preferred A.beta.40 inhibitors for use in the invention
include compounds in which both R.sup.1 and R.sup.2 are
pharmaceutically acceptable salt-forming cations. It will be
appreciated that the stoichiometry of an anionic compound to a
salt-forming counterion (if any) will vary depending on the charge
of the anionic portion of the compound (if any) and the charge of
the counterion. In a particularly preferred embodiment, R.sup.1,
R.sup.2 and R.sup.3 are each independently a sodium, potassium or
calcium cation. In certain embodiments in which at least one of
R.sup.1 and R.sup.2 is an aliphatic group, the aliphatic group has
between 1 and 10 carbons atoms in the straight or branched chain,
and is more preferably a lower alkyl group. In other embodiments in
which at least one of R.sup.1 and R.sup.2 is an aliphatic group,
the aliphatic group has between 10 and 24 carbons atoms in the
straight or branched chain. In certain preferred embodiments, n is
0 or 1; more preferably, n is 0. In certain preferred embodiments
of the therapeutic compounds, Y.sup.1 and Y.sup.2 are each
hydrogen.
[0065] In certain preferred embodiments, the A.beta.40 inhibitor of
the invention can have the structure:
##STR00005##
[0066] in which R.sup.1, R.sup.2, R.sup.3, Y.sup.1, Y.sup.2, X and
n are as defined above. In more preferred embodiments, the
A.beta.40 inhibitor of the invention can have the structure:
##STR00006##
[0067] wherein R.sup.1, R.sup.2, R.sup.3, Y.sup.1, Y.sup.2, and X
are as defined above, R.sub.a and R.sub.b are each independently
hydrogen, alkyl, aryl, or heterocyclyl, or R.sub.a and R.sub.b,
taken together with the nitrogen atom to which they are attached,
form a cyclic moiety having from 3 to 8 atoms in the ring, and n is
an integer from 0 to 6. In certain preferred embodiments, R.sub.a
and R.sub.b are each hydrogen. In certain preferred embodiments, a
compound of the invention comprises an .alpha.-amino acid (or
.alpha.-amino acid ester), more preferably a L-.alpha.-amino acid
or ester.
[0068] The Z, R.sup.1, R.sup.2, R.sup.3, Y.sup.1, Y.sup.2 and X
groups are each independently selected such that the
biodistribution of the A.beta.40 inhibitor for an intended target
site is not prevented while maintaining activity of the A.beta.40
inhibitor. For example, the number of anionic groups (and the
overall charge on the therapeutic compound) should not be so great
as to prevent traversal of an anatomical barrier, such as a cell
membrane, or entry across a physiological barrier, such as the
blood-brain barrier, in situations where such properties are
desired. For example, it has been reported that esters of
phosphonoformate have biodistribution properties different from,
and in some cases superior to, the biodistribution properties of
phosphonoformate (see, e.g., U.S. Pat. Nos. 4,386,081 and 4,591,583
to Helgstrand et al., and U.S. Pat. Nos. 5,194,654 and 5,463,092 to
Hostetler et al.). Thus, in certain embodiments, at least one of
R.sup.1 and R.sup.2 is an aliphatic group (more preferably an alkyl
group), in which the aliphatic group has between 10 and 24 carbons
atoms in the straight or branched chain. The number, length, and
degree of branching of the aliphatic chains can be selected to
provide a desired characteristic, e.g., lipophilicity. In other
embodiments, at least one of R.sup.1 and R.sup.2 is an aliphatic
group (more preferably an alkyl group), in which the aliphatic
group has between 1 and 10 carbons atoms in the straight or
branched chain. Again, the number, length, and degree of branching
of the aliphatic chains can be selected to provide a desired
characteristic, e.g., lipophilicity or ease of ester cleavage by
enzymes. In certain embodiments, a preferred aliphatic group is an
ethyl group.
[0069] In another embodiment, the A.beta.40 inhibitor of the
invention can have the structure:
##STR00007##
[0070] wherein G represents hydrogen or one or more substituents on
the aryl ring (e.g., alkyl, aryl, halogen, amino, and the like) and
L is a substituted alkyl group (in certain embodiments, preferably
a lower alkyl), more preferably a hydroxy-substituted alkyl or an
alkyl substituted with a nucleoside base, and M.sup.+ is a counter
ion. In certain embodiments, G is hydrogen or an electron-donating
group. In embodiments in which G is an electron-withdrawing group,
G is preferably an electron withdrawing group at the meta position.
The term "electron-withdrawing group" is known in the art, and, as
used herein, refers to a group which has a greater
electron-withdrawing than hydrogen. A variety of
electron-withdrawing groups are known, and include halogens (e.g.,
fluoro, chloro, bromo, and iodo groups), nitro, cyano, and the
like. Similarly, the term "electron-donating group", as used
herein, refers to a group which is less electron-withdrawing than
hydrogen. In embodiments in which G is an electron donating group,
G can be in the ortho, meta or para position. In certain
embodiments, M.sup.+ is a cationic species selected from, e.g.,
H.sup.+ and pharmaceutically acceptable organic or inorganic ions,
including, without limitation, Na.sup.+, K.sup.+, NH.sub.4.sup.+,
Ca.sup.+2, RNH.sub.3.sup.+, RR'NH.sub.2.sup.+. In one preferred
embodiment, M+ is an anilinium ion.
[0071] In certain preferred embodiments, L may be one of the
following moieties:
##STR00008##
[0072] Table 1 lists data pertinent to the characterization of
these compounds using art-recognized techniques. The compounds
IVa-IVg in Table 1 correspond to the following structure, wherein L
is a group selected from the above-listed (Groups IVa-IVg) having
the same number.
##STR00009##
TABLE-US-00001 TABLE 1 COMPOUND .sup.31P NMR .sup.13C NMR FAB-MS(-)
IVa -6.33(DMSO-d.sub.6) 60.97 CH.sub.2OH(d, J = 6 Hz) 245.2 66.76
CHOH(d, J = 7.8 Hz) 121.65, 121.78, 121.99, 125.71, 129.48, 129.57,
126.43 Aromatic CH 134.38 Aniline C--N 150.39 Phenyl C--O(d, J = 7
Hz) 171.57 P--C.dbd.O(d, J = 234 Hz) IVb -6.41(DMSO-d.sub.6) 13.94
CH.sub.3 456 22.11, 24.40, 28.56, 28.72, 28.99, 29.00, 31.30,
33.43, --(CH.sub.2).sub.10.sub.- 65.03 CH.sub.2--OC(O) 66.60
CH.sub.2--OP(d, J = 5.6 Hz) 67.71 CH2--OH(d, J = 6 Hz) 121.73,
121.10, 125.64, 126.57, 129.40, 129.95, Aromatic CH 134.04 Aniline
C--N 150.31 Phenyl C--O 171.44 P--C.dbd.O(d, J = 6.7 Hz) 172.83
O--C.dbd.O IVc -6.46(DMSO-d.sub.6) 13.94 CH.sub.3 471 22.11, 25.10,
28.68, 28.72, 28.85, 29.00, 30.76, 31.31, 32.10,
--(CH.sub.2).sub.10.sub.- 43.36 CH.sub.2--S 68.43 CH.sub.2--OH
68.43 CH--OH(d, J = 6.3 Hz) 68.76 P--O--CH.sub.2-9d, J = 5.8 Hz)
121.75, 122.03, 125.62, 126.37, 129.30, 129.53, Aromatic CH 134.23
Aniline C--N 150.37 Phenyl C--O(d, J = 6.7 Hz) 171.47 P--C.dbd.O(d,
J = 234.0 Hz) 198.47 S--C.dbd.O IVd -6.61(DMSO-d6) 13.94 CH.sub.3
416 22.06, 25.14, 28.24, 28.35, 31.09, 32.14 --CH.sub.2).sub.6-
43.40 CH.sub.2--S 68.50 P--O--CH.sub.2-(d, J = 5.8 Hz) 68.77
CH--OH(d, 6.4 Hz) 121.78, 122.59, 125.69, 127.06, 129.43, 129.59
Aromatic CH 133.39 Aniline C--N 150.38 Phenyl C--O(d, J = 6.7 Hz)
171.47 P--C.dbd.O(d, J = 234.4 Hz) 198.54 S--C.dbd.O IVe
-5.76(D.sub.2O) N/A N/A IVf -7.00(DMSO-d.sub.6) N/A N/A IVg
-6.60(DMSO-D6) 70.84 CH2--OH 321 72.17 CH--OH 121.68, 121.79,
121.85, 125.71 127.10, 127.92, 129.36, 129.50, 129.59 Aromatic CH
134.51 Aniline C--N 142.34 Aromatic C--CH 150.37 Phenyl C--O(d, J =
6.2 Hz) 171.59 P--C.dbd.O(d, J = 232.6 Hz)
[0073] Note that the structure of some of the A.beta.40 inhibitors
of this invention includes asymmetric carbon atoms. It is to be
understood accordingly that the isomers (e.g., enantiomers and
diastereomers) arising from such asymmetry are included within the
scope of this invention. Such isomers can be obtained in
substantially pure form by classical separation techniques and by
sterically controlled synthesis. For the purposes of this
application, unless expressly noted to the contrary, an A.beta.40
inhibitor shall be construed to include both the R or S
stereoisomers at each chiral center.
[0074] In certain embodiments, an A.beta.40 inhibitor of the
invention comprises a cation (i.e., in certain embodiments, at
least one of R.sup.1, R.sup.2 or R.sup.3 is a cation). If the
cationic group is hydrogen, H.sup.+, then the A.beta.40 inhibitor
is considered an acid, e.g., phosphonoformic acid. If hydrogen is
replaced by a metal ion or its equivalent, the A.beta.40 inhibitor
is a salt of the acid. Pharmaceutically acceptable salts of the
A.beta.40 inhibitor are within the scope of the invention. For
example, at least one of R.sup.1, R.sup.2 or R.sup.3 can be a
pharmaceutically acceptable alkali metal (e.g., Li, Na, or K),
ammonium cation, alkaline earth cation (e.g., Ca.sup.2+, Ba.sup.2+,
Mg.sup.2+), higher valency cation, or polycationic counter ion
(e.g., a polyammonium cation). (See, e.g., Berge et al. (1977)
"Pharmaceutical Salts", J. Pharm. Sci. 66:1-19). It will be
appreciated that the stoichiometry of an anionic compound to a
salt-forming counterion (if any) will vary depending on the charge
of the anionic portion of the compound (if any) and the charge of
the counterion. Preferred pharmaceutically acceptable salts include
a sodium, potassium or calcium salt, but other salts are also
contemplated within their pharmaceutically acceptable range.
[0075] The term "pharmaceutically acceptable esters" refers to the
relatively non-toxic, esterified products of the A.beta.40
inhibitors of the present invention. These esters can be prepared
in situ during the final isolation and purification of the
A.beta.40 inhibitors or by separately reacting the purified
A.beta.40 inhibitor in its free acid form or hydroxyl with a
suitable esterifying agent; either of which are methods known to
those skilled in the art. Carboxylic acids and phosphonic acids can
be converted into esters according to methods well known to one of
ordinary skill in the art, e.g., via treatment with an alcohol in
the presence of a catalyst. A preferred ester group (e.g., when
R.sup.3 is lower alkyl) is an ethyl ester group.
[0076] The term "alkyl" refers to the saturated aliphatic groups,
including straight-chain alkyl groups, branched-chain alkyl groups,
cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups,
and cycloalkyl substituted alkyl groups. In preferred embodiments,
a straight chain or branched chain alkyl has 30 or fewer carbon
atoms in its backbone (e.g., C.sub.1-C.sub.30 for straight chain,
C.sub.3-C.sub.30 for branched chain), and more preferably 20 or
fewer. Likewise, preferred cycloalkyls have from 4-10 carbon atoms
in their ring structure, and more preferably have 4-7 carbon atoms
in the ring structure. The term "lower alkyl" refers to alkyl
groups having from 1 to 6 carbons in the chain, and to cycloalkyls
having from 3 to 6 carbons in the ring structure.
[0077] Moreover, the term "alkyl" (including "lower alkyl") as used
throughout the specification and claims is intended to include both
"unsubstituted alkyls" and "substituted alkyls", the latter of
which refers to alkyl moieties having substituents replacing a
hydrogen on one or more carbons of the hydrocarbon backbone. Such
substituents can include, for example, halogen, hydroxyl,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,
phosphinato, cyano, amino (including alkyl amino, dialkylamino,
arylamino, diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfate, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl,
cyano, azido, heterocyclyl, aralkyl, or an aromatic or
heteroaromatic moiety. It will be understood by those skilled in
the art that the moieties substituted on the hydrocarbon chain can
themselves be substituted, if appropriate. Cycloalkyls can be
further substituted, e.g., with the substituents described above.
An "aralkyl" moiety is an alkyl substituted with an aryl (e.g.,
phenylmethyl (benzyl)).
[0078] The term "alkoxy", as used herein, refers to a moiety having
the structure --O-alkyl, in which the alkyl moiety is described
above.
[0079] The term "aryl" as used herein includes 5- and 6-membered
single-ring aromatic groups that may include from zero to four
heteroatoms, for example, unsubstituted or substituted benzene,
pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,
pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the
like. Aryl groups also include polycyclic fused aromatic groups
such as naphthyl, quinolyl, indolyl, and the like. The aromatic
ring can be substituted at one or more ring positions with such
substituents, e.g., as described above for alkyl groups. Preferred
aryl groups include unsubstituted and substituted phenyl
groups.
[0080] The term "aryloxy", as used herein, refers to a group having
the structure --O-aryl, in which the aryl moiety is as defined
above.
[0081] The term "amino," as used herein, refers to an unsubstituted
or substituted moiety of the formula --NR.sub.aR.sub.b, wherein
R.sub.a and R.sub.b are each independently hydrogen, alkyl, aryl,
or heterocyclyl, or R.sub.a and R.sub.b, taken together with the
nitrogen atom to which they are attached, form a cyclic moiety
having from 3 to 8 atoms in the ring. Thus, the term "amino" is
intended to include cyclic amino moieties such as piperidinyl or
pyrrolidinyl groups, unless otherwise stated. An "amino-substituted
amino group" refers to an amino group in which at least one of
R.sub.a and R.sub.b, is further substituted with an amino
group.
[0082] In a preferred embodiment, R.sup.1 or R.sup.2 can be (for at
least one occurrence) a long-chain aliphatic moiety. The term
"long-chain aliphatic moiety" as used herein, refers to a moiety
having a straight or branched chain aliphatic moiety (e.g., an
alkyl or alkenyl moiety) having from 10 to 24 carbons in the
aliphatic chain, e.g., the long-chain aliphatic moiety is an
aliphatic chain of a fatty acid (preferably a naturally-occurring
fatty acid). Representative long-chain aliphatic moieties include
the aliphatic chains of stearic acid, oleic acid, linolenic acid,
and the like.
[0083] In certain embodiments, the A.beta.40 inhibitor of the
invention can have the structure:
##STR00010##
[0084] wherein R.sup.1 and R.sup.2 are each independently hydrogen,
an aliphatic group (preferably a branched or straight-chain
aliphatic moiety having from 1 to 24 carbon atoms, more preferably
10-24 carbon atoms, in the chain; or an unsubstituted or
substituted cyclic aliphatic moiety having from 4 to 7 carbon atoms
in the aliphatic ring), an aryl group, a heterocyclic group, or a
salt-forming cation; R.sup.3 is hydrogen, lower alkyl, aryl, or a
salt-forming cation; Y.sup.1 and Y.sup.2 are each independently
hydrogen, halogen (e.g., F, Cl, Br, or I), lower alkyl, hydroxy,
alkoxy, or aryloxy; and n is an integer from 0 to 12. Preferred
A.beta.40 inhibitors for use in the invention include compounds
wherein both R.sup.1 and R.sup.2 are pharmaceutically acceptable
salt-forming cations. In a particularly preferred embodiment,
R.sup.1, R.sup.2 and R.sup.3 are each independently a sodium,
potassium or calcium cation, and n is 0. In certain preferred
embodiments of the therapeutic compounds, Y.sup.1 and Y.sup.2 are
each hydrogen. Particularly preferred A.beta.40 inhibitors are
salts of phosphonoformate. Trisodium phosphonoformate (foscarnet
sodium or Foscavir.RTM.) is commercially available (e.g., from
Astra), and its clinical pharmacology has been investigated (see,
e.g., "Physician's Desk Reference", 51st Ed., pp. 541-545
(1997)).
[0085] In another embodiment, the A.beta.40 inhibitor used in the
invention can be an aminophosphonate, a bisphosphonate, a
phosphonocarboxylate derivative, a phosphonate derivative, or a
phosphono carbohydrate. For example, the A.beta.40 inhibitor can be
one of the compounds described in--Tables III and IV.
[0086] Pharmaceutically Acceptable Formulations
[0087] In the methods of the invention, the A.beta.40 inhibitor can
be administered in a pharmaceutically acceptable formulation. The
present invention pertains to any pharmaceutically acceptable
formulations, such as synthetic or natural polymers in the form of
macromolecular complexes, nanocapsules, microspheres, or beads, and
lipid-based formulations including oil-in-water emulsions,
micelles, mixed micelles, synthetic membrane vesicles, and resealed
erythrocytes.
[0088] In one embodiment, the pharmaceutically acceptable
formulations comprise a polymeric matrix.
[0089] The terms "polymer" or "polymeric" are art-recognized and
include a structural framework comprised of repeating monomer units
which is capable of delivering an A.beta.40 inhibitor, such that
treatment of a targeted condition occurs. The terms also include
co-polymers and homopolymers e.g., synthetic or naturally
occurring. Linear polymers, branched polymers, and cross-linked
polymers are also meant to be included.
[0090] For example, polymeric materials suitable for forming the
pharmaceutically acceptable formulation employed in the present
invention, include naturally derived polymers such as albumin,
alginate, cellulose derivatives, collagen, fibrin, gelatin, and
polysaccharides, as well as synthetic polymers such as polyesters
(PLA, PLGA), polyethylene glycol, poloxomers, polyanhydrides, and
pluronics. These polymers are biocompatible and biodegradable
without producing any toxic byproducts of degradation, and they
possess the ability to modify the manner and duration of A.beta.40
inhibitor release by manipulating the polymer's kinetic
characteristics. As used herein, the term "biodegradable" means
that the polymer will degrade over time by the action of enzymes,
by hydrolytic action and/or by other similar mechanisms in the body
of the subject. As used herein, the term "biocompatible" means that
the polymer is compatible with a living tissue or a living organism
by not being toxic or injurious and by not causing an immunological
rejection.
[0091] Polymers can be prepared using methods known in the art
(Sandier, S. R.; Karo, W. Polymer Syntheses; Harcourt Brace:
Boston, 1994; Shalaby, W.; Ikada, Y.; Langer, R.; Williams, J.
Polymers of Biological and Biomedical Significance (ACS Symposium
Series 540; American Chemical Society: Washington, D.C., 1994).
Polymers can be designed to be flexible; the distance between the
bioactive side-chains and the length of a linker between the
polymer backbone and the group can be controlled. Other suitable
polymers and methods for their preparation are described in U.S.
Pat. Nos. 5,455,044 and 5,576,018.
[0092] The polymeric formulations are preferably formed by
dispersion of the A.beta.40 inhibitor within liquefied polymer, as
described in U.S. Pat. No. 4,883,666, the teachings of which are
incorporated herein by reference, or by such methods as bulk
polymerization, interfacial polymerization, solution polymerization
and ring polymerization as described in Odian G., Principles Of
Polymerization And Ring Opening Polymerization, 2nd ed., John Wiley
& Sons, New York, 1981. The properties and characteristics of
the formulations are controlled by varying such parameters as the
reaction temperature, concentrations of polymer and A.beta.40
inhibitor, types of solvent used, and reaction times.
[0093] In addition to the A.beta.40 inhibitor and the
pharmaceutically acceptable polymer, the pharmaceutically
acceptable formulation used in the method of the invention can
comprise additional pharmaceutically acceptable carriers and/or
excipients. 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.
For example, the carrier can be suitable for injection into the
cerebrospinal fluid. Excipients include pharmaceutically acceptable
stabilizers and disintegrants.
[0094] The A.beta.40 inhibitor can be encapsulated in one or more
pharmaceutically acceptable polymers, to form a microcapsule,
microsphere, or microparticle, terms used herein interchangeably.
Microcapsules, microspheres, and microparticles are conventionally
free-flowing powders consisting of spherical particles of 2 mm or
less in diameter, usually 500 .mu.m or less in diameter. Particles
less than 1 .mu.m are conventionally referred to as nanocapsules,
nanoparticles or nanospheres. For the most part, the difference
between a microcapsule and a nanocapsule, a microsphere and a
nanosphere, or microparticle and nanoparticle is size; generally
there is little, if any, difference between the internal structure
of the two. In one aspect of the present invention, the mean
average diameter is less than about 45 .mu.m, preferably less than
20 .mu.m, and more preferably between about 0.1 and 10 .mu.m.
[0095] In another embodiment, the pharmaceutically acceptable
formulations comprise lipid-based formulations. Any of the known
lipid-based drug delivery systems can be used in the practice of
the invention. For instance, multivesicular liposomes (MVL),
multilamellar liposomes (also known as multilamellar vesicles or
"MLV"), unilamellar liposomes, including small unilamellar
liposomes (also known as unilamellar vesicles or "SUV") and large
unilamellar liposomes (also known as large unilamellar vesicles or
"LUV"), can all be used so long as a sustained release rate of the
encapsulated A.beta.40 inhibitor can be established. In one
embodiment, the lipid-based formulation can be a multivesicular
liposome system. Methods of making controlled release
multivesicular liposome drug delivery systems is described in PCT
Application Nos. US96/11642, US94/12957 and US94/04490.
[0096] The composition of the synthetic membrane vesicle is usually
a combination of phospholipids, usually in combination with
steroids, especially cholesterol. Other phospholipids or other
lipids may also be used.
[0097] Examples of lipids useful in synthetic membrane vesicle
production include phosphatidylglycerols, phosphatidylcholines,
phosphatidylserines, phosphatidylethanolamines, sphingolipids,
cerebrosides, and gangliosides. Preferably phospholipids including
egg phosphatidylcholine, dipalmitoylphosphatidylcholine,
distearoylphosphatidylcholine, dioleoylphosphatidylcholine,
dipalmitoylphosphatidylglycerol, and dioleoylphosphatidylglycerol
are used.
[0098] In preparing lipid-based vesicles containing an A.beta.40
inhibitor, such variables as the efficiency of A.beta.40 inhibitor
encapsulation, lability of the A.beta.40 inhibitor, homogeneity and
size of the resulting population of vesicles, A.beta.40
inhibitor-to-lipid ratio, permeability, instability of the
preparation, and pharmaceutical acceptability of the formulation
should be considered (see Szoka, et al., Annual Reviews of
Biophysics and Bioengineering, 9:467, 1980; Deamer, et al., in
Liposomes, Marcel Dekker, New York, 1983, 27; and Hope, et al.,
Chem. Phys. Lipids, 40:89, 1986, the contents of which are
incorporated herein by reference).
Administration of the Pharmaceutically Acceptable Formulation
[0099] The A.beta.40 inhibitor may be administered to a subject,
e.g., parenterally, e.g., intravenously, intradermally,
subcutaneously, orally (e.g., via inhalation), transdermally
(topically), transmucosally, or rectally. In one embodiment, the
A.beta.40 inhibitor is administered by introduction into the
central nervous system of the subject, e.g., into the cerebrospinal
fluid of the subject. In certain aspects of the invention, the
A.beta.40 inhibitor is introduced intrathecally, e.g., into a
cerebral ventricle, the lumbar area, or the cisterna magna.
[0100] The pharmaceutically acceptable formulations can easily be
suspended in aqueous vehicles and introduced through conventional
hypodermic needles or using infusion pumps. Prior to introduction,
the formulations can be sterilized with, preferably, gamma
radiation or electron beam sterilization.
[0101] In another embodiment of the invention, the A.beta.40
inhibitor formulation is administered into a subject intrathecally.
As used herein, the term "intrathecal administration" is intended
to include delivering an A.beta.40 inhibitor formulation directly
into the cerebrospinal fluid of a subject, by techniques including
lateral cerebroventricular injection through a burrhole or
cisternal or lumbar puncture or the like (described in Lazorthes et
al. Advances in Drug Delivery Systems and Applications in
Neurosurgery, 143-192 and Omaya et al., Cancer Drug Delivery, 1:
169-179, the contents of which are incorporated herein by
reference). The term "lumbar region" is intended to include the
area between the third and fourth lumbar (lower back) vertebrae.
The term "cisterna magna" is intended to include the area where the
skull ends and the spinal cord begins at the back of the head. The
term "cerebral ventricle" is intended to include the cavities in
the brain that are continuous with the central canal of the spinal
cord. Administration of an A.beta.40 inhibitor to any of the above
mentioned sites can be achieved by direct injection of the
A.beta.40 inhibitor formulation or by the use of infusion pumps.
For injection, the A.beta.40 inhibitor formulation of the invention
can be formulated in liquid solutions, preferably in
physiologically compatible buffers such as Hank's solution or
Ringer's solution. In addition, the A.beta.40 inhibitor formulation
may be formulated in solid form and re-dissolved or suspended
immediately prior to use. Lyophilized forms are also included. The
injection can be, for example, in the form of a bolus injection or
continuous infusion (e.g., using infusion pumps) of the
formulation.
Duration and Levels of Administration
[0102] In another embodiment of the method of the invention, the
pharmaceutically acceptable formulation provides sustained
delivery, e.g., "slow release" of the A.beta.40 inhibitor to a
subject for at least one, two, three, or four weeks after the
pharmaceutically acceptable formulation is administered to the
subject.
[0103] As used herein, the term "sustained delivery" is intended to
include continual delivery of an A.beta.40 inhibitor in vivo over a
period of time following administration, preferably at least
several days, a week or several weeks. Sustained delivery of the
A.beta.40 inhibitor can be demonstrated by, for example, the
continued therapeutic effect of the A.beta.40 inhibitor over time
(e.g., sustained delivery of the A.beta.40 inhibitor can be
demonstrated by continued inhibition of cerebral amyloid angiopathy
over time). Alternatively, sustained delivery of the A.beta.40
inhibitor may be demonstrated by detecting the presence of the
A.beta.40 inhibitor in vivo over time.
[0104] In one embodiment, the pharmaceutically acceptable
formulation provides sustained delivery of the A.beta.40 inhibitor
to a subject for less than 30 days after the A.beta.40 inhibitor is
administered to the subject. For example, the pharmaceutically
acceptable formulation, e.g., "slow release" formulation, can
provide sustained delivery of the A.beta.40 inhibitor to a subject
for one, two, three or four weeks after the A.beta.40 inhibitor is
administered to the subject. Alternatively, the pharmaceutically
acceptable formulation may provide sustained delivery of the
A.beta.40 inhibitor to a subject for more than 30 days after the
A.beta.40 inhibitor is administered to the subject.
[0105] The pharmaceutical formulation, used in the method of the
invention, contains a therapeutically effective amount of the
A.beta.40 inhibitor. A "therapeutically effective amount" refers to
an amount effective, at dosages and for periods of time necessary,
to achieve the desired result. A therapeutically effective amount
of the A.beta.40 inhibitor may vary according to factors such as
the disease state, age, and weight of the subject, and the ability
of the A.beta.40 inhibitor (alone or in combination with one or
more other agents) to elicit a desired response in the subject.
Dosage regimens may be adjusted to provide the optimum therapeutic
response. A therapeutically effective amount is also one in which
any toxic or detrimental effects of the A.beta.40 inhibitor are
outweighed by the therapeutically beneficial effects. A
non-limiting range for a therapeutically effective concentration of
an A.beta.40 inhibitor is 100 .mu.M to 1 mM. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the A.beta.40 inhibitor and that
dosage ranges set forth herein are exemplary only and are not
intended to limit the scope or practice of the claimed
invention.
In Vitro Treatment of Blood Vessel Wall Cells
[0106] Blood vessel wall cells, or isolated blood vessel wall
cells, can further be contacted with a therapeutically effective
amount of a A.beta.40 inhibitor, in vitro. Accordingly, such cells
can be isolated from a subject and grown in vitro, using techniques
well known in the art. Briefly, a smooth muscle cell culture can be
obtained by allowing smooth muscle cells to migrate out of
fragments of tissue adhering to a suitable substrate such as a
culture dish, or by disaggregating the tissue, e.g., mechanically
or enzymatically, to produce a suspension of cells. For example,
the enzymes trypsin, collagenase, elastase, hyaluronidase, DNAse,
pronase, dispase, or various combinations thereof can be used.
Trypsin and pronase give the most complete disaggregation but may
damage the cells. Collagenase and dispase give a less complete
disaggregation but are less harmful. Methods for isolating tissue
such as neuronal tissue, and the disaggregation of tissue to obtain
cells such as neuronal cells are described in Freshney R. I.,
Culture of Animal Cells, A Manual of Basic Technique, Third
Edition, 1994, the contents of which are incorporated herein by
reference.
[0107] Such cells can be subsequently contacted with an A.beta.40
inhibitor at levels and for a duration of time as described above.
Once inhibition of cerebral amyloid angiopathy has been achieved,
these neuronal cells can be re-administered to the subject, e.g.,
by implantation.
States Characterized by CAA
[0108] The present invention further pertains to a method of
treating a disease state characterized by cerebral amyloid
angiopathy in a subject. As used herein, the term "state" is art
recognized and includes a disorder, disease or condition
characterized by cerebral amyloid angiopathy. Examples of such
disorders include Alzheimer's Disease, HCHWA-D, and hemorrhagic
stroke.
[0109] The invention is further illustrated by the following
examples, which should not be construed as further limiting. The
contents of all references, patents and published patent
applications cited throughout this application are hereby
incorporated by reference.
EXAMPLE 1
[0110] A compound of the invention is administered in a therapeutic
amount to a subject having a clinical diagnosis of `probable CAA`,
defined for present purposes as: multiple hemorrhages confined to
the lobar brain regions diagnosed by CT or MRI scan and no other
cause of hemorrhage. The ability of the compound of the invention
to prevent recurrence of CAA-related hemorrhages is determined by
clinical exams (new neurologic symptoms or death with acute
hemorrhage confirmed by CT scan or autopsy) or by gradient-echo MRI
scans which mark the progression of CAA by the appearance of new
hemorrhages. The ability of the compound to inhibit the progression
of CAA can also be assessed through cognitive decline (MMSE) or
functional decline (NIHSS, FIM). The APOE-2 and APOE-4 are
associated with increasing risk and earlier age of first
hemorrhage, but are neither specific nor sensitive for CAA.
EXAMPLE 2
[0111] The ability of compounds of the invention to inhibit CAA was
measured in the following example. Nine week old hAPP transgenic
mice were treated for a period of 8 weeks with two different
concentrations of a compound of the present invention,
3-amino-1-propanesulfonic acid, sodium salt, 100 and 30 mg/kg. Mice
were administered the compound for 8 weeks, after which they were
sacrificed and their brains were perfused and processed for
histological staining with Thioflavin S. This method may also be
used as a screening method for determining activity of a candidate
compound for inhibiting CAA.
[0112] The extent of CAA in brain sections obtained from these
animals was qualitatively determined following staining. The extent
of CAA, if any, was graded as follows: [0113] + Slight deposition
[0114] ++ Moderate deposition [0115] +++ Severe deposition
[0116] The results shown in Table II, below, indicate that the test
compound was effective in 1) reducing the number of mice showing
CAA, and 2) showing an effect on the severity of the deposition
seen in the brain vasculature of these animals.
TABLE-US-00002 TABLE II # of CAA animals/ # animals animals total
CAA Severity Treatment in study with CAA animals + ++ +++ Vehicle
16 15 15/16 5/15 9/15 1/15 30 mg/kg 11 10 10/11 6/10 4/10 -- 100
mg.kg 15 10 10/15 9/10 -- 1/10
TABLE-US-00003 TABLE III Phosphonoacetic acid ##STR00011##
Phosphonoformic acid, trisodium salt hexahydrate ##STR00012##
Diethylphosphonoacetic acid ##STR00013##
3-[2-(1,2,3,4-Tetrahydroisoquinolinyl)]-1-propanephosphonic acid
##STR00014## 3-Aminopropylphosphonic acid
NH.sub.2CH.sub.2CH.sub.2CH.sub.2PO.sub.3H.sub.2 Propylphosphonic
acid CH.sub.3CH.sub.2CH.sub.2PO.sub.3H.sub.2 Ethylphosphonic acid
CH.sub.3CH.sub.2PO.sub.3H.sub.2 Methylphosphonic acid
CH.sub.3PO.sub.3H.sub.2 tert-Butylphosphonic acid
(CH.sub.3).sub.3CPO.sub.3H.sub.2 Phenylphosphonic acid ##STR00015##
(dl)-2-Amino-3-phosphonopropanoic acid ##STR00016##
(1-Aminopropyl)phosphonic acid ##STR00017##
(dl)-2-Amino-5-phosphonopentanoic acid ##STR00018## Diethyl
phosphoramidate ##STR00019##
(S)-2-Amino-2-methyl-4-phosphonobutanoic acid ##STR00020##
D-(-)-2-Amino-4-phosphonobutanoic acid ##STR00021##
L-(+)-2-Amino-4-phosphonobutanoic acid ##STR00022##
D-(-)-2-Amino-7-phosphonoheptanoic acid ##STR00023##
L-(+)-2-Amino-7-phosphonoheptanoic acid ##STR00024##
D-(-)-2-Amino-6-phosphonohexanoic acid ##STR00025##
L-(+)-2-Amino-6-phosphonohexanoic acid ##STR00026##
D-(-)-2-Amino-4-phosphonopentanoic acid ##STR00027##
L-(+)-2-Amino-4-phosphonopentanoic acid ##STR00028##
D-(-)-2-Amino-3-phosphonopropanoic acid ##STR00029##
L-(+)-2-Amino-3-phosphonopropanoic acid ##STR00030##
3-Aminopropyl(methyl)phosphinic acid, hydrochloride ##STR00031##
(R)-(-)-3-(2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid
(D-CPP) ##STR00032## L-4-[Difluoro(phosphono)methyl)]-phenylalanine
##STR00033##
(R,E)-4-(3-Phosphonoprop-2-enyl)piperazine-2-carboxylic acid
##STR00034## trans-L-4-Phosphonomethylproline, trisodium salt
##STR00035## cis-L-4-Phosphonomethylproline, trisodium salt
##STR00036## Thiophosphonoformic acid, trisodium salt ##STR00037##
Thiophosphonoacetic acid ##STR00038## Thiophosphonoacetic acid,
trisodium salt ##STR00039## Thiophosphonoacetic acid, triethyl
ester ##STR00040## Chloro(thiophosphono)acetic acid, trisodium salt
##STR00041## Dichloro(thiophosphono)acetic acid,trisodium salt
##STR00042## Thiophosphonomethylthiophosphonic acid,
tetrasodiumsalt ##STR00043##
Phenylthiophosphinomethylthio-phosphonic acid,trisodium salt
##STR00044## Propylthiophosphonic acid ##STR00045##
Ethylthiophosphonic acid ##STR00046## Methylthiophosphonic acid
##STR00047## tert-Butylthiophosphonic acid ##STR00048##
3-Thiophosphonopropanoic acid ##STR00049## Phenylthiophosphonic
acid ##STR00050## 3-Aminopropylthiophosphonic acid ##STR00051##
(dl)-2-Amino-3-thiophosphonopropanoic acid ##STR00052##
(1-Aminopropyl)thiophosphonic acid ##STR00053##
(dl)-2-Amino-5-thiophosphonopentanoic acid ##STR00054##
(S)-2-Amino-2-methyl-4-thiophosphonobutanoic acid ##STR00055##
D-2-Amino-4-thiophosphonobutanoic acid ##STR00056##
L-2-Amino-4-thiophosphonobutanoic acid ##STR00057##
D-2-Amino-7-thiophosphonoheptanoic acid ##STR00058##
L-2-Amino-7-thiophosphonoheptanoic acid ##STR00059##
D-2-Amino-6-thiophosphonohexanoic acid ##STR00060##
L-2-Amino-6-thiophosphonohexanoic acid ##STR00061##
D-2-Amino-5-thiophosphonopentanoic acid ##STR00062##
L-2-Amino-5-thiophosphonopentanoic acid ##STR00063##
D-2-Amino-3-thiophosphonopropanoic acid ##STR00064##
L-2-Amino-3-thiophosphonopropanoic acid ##STR00065##
3-Aminopropyl(methyl)thiophosphinic acid,hydrochloride ##STR00066##
(R)-3-(3-Carboxy-1-piperazinyl)-1-propyl-thiophosphonic acid
##STR00067## L-4-[Difluoro(thiophosphono)methyl)]-phenylalanine
##STR00068##
(R,E)-4-(3-Thiophosphonoprop-2-enyl)piperazine-2-carboxylic acid
##STR00069## 4-Amino-1-butylphosphonic acid, disodium salt
##STR00070## 4-Amino-1-butylthiophosphonic acid, disodium salt
##STR00071## 1-(3-Phosphonopropyl)-benzimidazole, disodium salt
##STR00072## 1-(3-Thiophosphonopropyl)-benzimidazole, disodiumsalt
##STR00073## 3-Dimethylamino-1-propylphosphonic acid, disodiumsalt
##STR00074## N,N-Diethylphosphonoacetamide, disodium salt
##STR00075## N,N-Diethylthiophosphonoacetamide, disodium salt
##STR00076## Diphenylamine-4-phosphonic acid, disodium salt
##STR00077## Diphenylamine-4-thiophosphonic acid, disodium salt
##STR00078## Selenophosphonoformic acid, trisodium salt
##STR00079## Selenophosphonoacetic acid, trisodium salt
##STR00080## D-2-Amino-3-selenophosphonopropanoic acid ##STR00081##
L-2-Amino-3-selenophosphonopropanoic acid ##STR00082##
D-2-Amino-4-selenophosphonobutanoic acid ##STR00083##
L-2-Amino-4-selenophosphonobutanoic acid ##STR00084##
N-Cyclohexylphosphonoacetamide, disodium salt ##STR00085##
N-Cyclohexylthiophosphonoacetamide, disodium salt ##STR00086##
N-Cyclohexylselenophosphonoacetamide, disodium salt ##STR00087##
Phosphonoacetic hydrazide, disodium salt ##STR00088##
N-Hydroxyphosphonoacetamide, disodium salt ##STR00089##
N-Hydroxythiophosphonoacetamide, disodium salt ##STR00090##
Thiophosphonoacetic hydrazide, disodium salt ##STR00091##
N-Phosphonoacetyl-L-alanine, trisodium salt ##STR00092##
N-Thiophosphonoacetyl-L-alanine, trisodium salt ##STR00093##
N-Phosphonoacetyl-Glycine, trisodium salt ##STR00094##
N-Thiophosphonoacetyl-Glycine, trisodium salt ##STR00095##
N-(Phosphonoactyl)-L-asparagine-Glycine, tetrasodiumsalt
##STR00096##
N-(Thiophosphonoactyl)-L-asparagine-Glycine,tetrasodium salt
##STR00097## (S)-2-Pyrrolidinemethylthiophosphonic acid,
disodiumsalt ##STR00098## (dl)-3-Amino-butylphosphonic acid,
disodium salt ##STR00099## (dl)-3-Amino-pentylphosphonic acid,
disodium salt ##STR00100## (dl)-3-Amino-hexylphosphonic acid,
disodium salt ##STR00101## (dl)-3-Amino-heptylphosphonic acid,
disodium salt ##STR00102## (dl)-3-Amino-octylphophonic acid,
disodium salt ##STR00103## (dl)-3-Amino-4-methyl-pentylphosphonic
acid, disodiumsalt ##STR00104## 3-Amino-3-methyl-butylphosphonic
acid, disodium salt ##STR00105##
(dl)-3-Amino-3-phenyl-propylphosphonic acid,disodium salt
##STR00106## (dl)-3-Amino-4-phenyl-butylphosphonic acid,
disodiumsalt ##STR00107## (dl)-3-Amino-4-phenyl-pentylphosphonic
acid, disodiumsalt ##STR00108##
(dl)-3-Amino-3-phenyl-butylphosphonic acid, disodiumsalt
##STR00109##
(dl)-2-Amino-2-(2-phosphonoethyl)-1,2,3,4-tetrahydronaphthalene,
disodium salt ##STR00110##
1-Amino-1-(2-phosphonoethyl)-cyclohexane, disodiumsalt ##STR00111##
(dl)-2-(2-Amino-4-phosphonobutoxy)tetrahydropyran,disodium salt
##STR00112## (dl)-3-Amino-4-hydroxybutylphosphonic acid,
disodiumsalt ##STR00113## 3-Phosphonopropanesulfonic acid,
trisodium salt ##STR00114## Pamidronic acid
(3-Amino-1-hydroxypropane-1,1-bisphosphonic acid) ##STR00115##
3-Amino-1-hydroxypropane-1,1-bisphosphonic acid,tetrasodium salt
##STR00116## Diethyl 2-pyrrolidinylphosphonate ##STR00117##
2-Pyrrolidinylphosphonic acid, disodium salt ##STR00118##
1,1-Dioxo-2-(3-phosphonopropyl)-isothiazoline,disodium salt
##STR00119##
1,1-Dioxo-2-(3-thiophosphonopropyl)-isothiazolidine,disodium salt
##STR00120## 2-Deoxy-2-phosphonoacetylamino-D-glucose ##STR00121##
2-Deoxy-2-thiophosphonoacetylamino-D-glucose ##STR00122##
1-Amino-3-sulfopropane-1,1-bisphosphonic acid ##STR00123##
1-Amino-3-sulfopropane-1,1-bisphosphonic acid,pentasodium salt
##STR00124##
3-Hydroxy-3-(2-pyridyl)propenyl-2-phosphonic acid,disodium salt
##STR00125## 3-Hydroxy-3-(3-pyridyl)propenyl-2-phosphonic
acid,disodium salt ##STR00126##
3-Hydroxy-3-(4-pyridyl)propenyl-2-phosphonic acid,disodium salt
##STR00127## 3-Amino-3-(2-pyridyl)propenyl-2-phosphonic
acid,disodium salt ##STR00128##
3-Amino-3-(3-pyridyl)propenyl-2-phosphonic acid,disodium salt
##STR00129## 3-Amino-3-(4-pyridyl)propenyl-2-phosphonic
acid,disodium salt ##STR00130##
1,4-Diamino-1-(3-pyridyl)butyl-2-phosphonic acid,disodium salt
##STR00131##
1,4-Diamino-4-methyl-1-(3-pyridyl)pentyl-2-phosphonicacid, disodium
salt ##STR00132##
1,4-Diamino-4-methyl-1-(2-pyridyl)pentyl-2-phosphonicacid, disodium
salt ##STR00133##
1,4-Diamino-4-methyl-1-(4-pyridyl)pentyl-2-phosphonicacid, disodium
salt ##STR00134## 1,3-Diaminopropane-1,1-bisphosphonic
acid,tetrasodium salt ##STR00135##
1-Amino-3-dimethylaminopropane-1,1-bisphosphonicacid, tetrasodium
salt ##STR00136##
3-Dimethylamino-1-hydroxypropane-1,1-bisphosphonicacid, tetrasodium
salt ##STR00137##
1-Hydroxy-3-(methylphenylamino)-propane-1,1-bisphosphonic acid,
tetrasodium salt ##STR00138##
1-Amino-3-(methylphenylamino)propane-1,1-bisphosphonic acid,
tetrasodium salt ##STR00139##
3-(2-Amino-4,5,7,8-tetrahydro-6H-thiazolo[4,5-d]azepin-6-yl)propyl-phospho-
nic acid, disodium salt ##STR00140##
3-(2-Amino-4,5,7,8-tetrahydro-6H-thiazolo[4,5-d]azepin-6-yl)propyl-thiopho-
sphonic acid, disodium salt ##STR00141## Ibandronic acid,
tetrasodium salt
(1-Hydroxy-3-(methylpentylamino)-propane-1,1-bisphosphonic
acid,tetrasodium salt) ##STR00142##
1-Amino-3-(methylpentylamino)propane-1,1-bisphosphonic acid,
tetrasodium salt ##STR00143##
1-Amino-3-(1-benzimidazolyl)propane-1,1-bisphosphonic acid
##STR00144## 1-Amino-3-(1-benzimidazolyl)propane-1,1-bisphosphonic
acid, tetrasodium salt ##STR00145##
3-Aminopropane-1,1-bisphosphonic acid, tetrasodiumsalt ##STR00146##
(dl)-3-Aminobutane-1,1-bisphosphonic acid,tetrasodium salt
##STR00147## (dl)-3-Aminopentane-1,1-bisphosphonic acid,tetrasodium
salt ##STR00148## (dl)-3-Aminohexane-1,1-bisphosphonic
acid,tetrasodium salt ##STR00149##
(dl)-3-Aminoheptane-1,1-bisphosphonic acid,tetrasodium salt
##STR00150## (dl)-3-Aminooctane-1,1-bisphosphonic acid,
tetrasodiumsalt ##STR00151##
(dl)-3-Amino-4-methylpentane-1,1-bisphosphonic acid,tetrasodium
salt ##STR00152## 3-Amino-3-methylbutane-1,1-bisphosphonic
acid,tetrasodium salt ##STR00153##
(dl)-3-Amino-3-phenylpropane-1,1-bisphosphonic acid,tetrasodium
salt ##STR00154## (dl)-3-Amino-4-phenylbutane-1,1-bisphosphonic
acid,tetrasodium salt ##STR00155##
3-Amino-4-phenylpentane-1,1-bisphosphonic acid,tetrasodium salt
##STR00156## (dl)-3-Amino-3-phenylbutane-1,1-bisphosphonic
acid,tetrasodium salt ##STR00157##
(dl)-2-(2-Amino-1,2,3,4-tetrahydronaphthalenyl)ethane-1,1-bisphosphonic
acid, tetrasodium salt ##STR00158##
2-(1-Aminocyclohexyl)ethane-1,1-bisphosphonic acid,tetrasodium salt
##STR00159##
2-(2-Amino-4,4-bisphosphonobutoxy)-tetrahydropyran,tetrasodium salt
##STR00160## (dl)-3-Amino-4-hydroxybutane-1,1-bisphosphonic
acid,tetrasodium salt ##STR00161##
(S)-Hydroxy(2-pyrrolidinyl)methane-bisphosphonicacid, sodium salt
##STR00162## Hydroxy[(2S,
4R)-4-hydroxy-2-pyrrolidinyl]methanebisphosphonic acid
tetrasodiumsalt ##STR00163##
2-Amino-1-hydroxyethane-1,1-bisphosphonic acid,tetrasodium salt
##STR00164## 1,2-Diaminoethane-1,1-bisphonphonic acid,
tetrasodiumsalt ##STR00165##
4-Amino-1-hydroxybutane-1,1-bisphosphonic acid,sodium salt
##STR00166## 1,4-Diaminobutane-1,1-bisphosphonic acid,
tetrasodiumsalt ##STR00167##
5-Amino-1-hydroxypentane-1,1-bisphosphonic acid,tetrasodium salt
##STR00168## 1,5-Diaminopentane-1,1-bisphosphonic acid,tetrasodium
salt ##STR00169## (S)-2-Amino-1-hydroxypropane-1,1-bisphosphonic
acid,tetrasodium salt ##STR00170##
(S)-2-Amino-1-hydroxybutane-1,1-bisphosphonic acid,tetrasodium salt
##STR00171## (S)-2-Amino-1-hydroxy-3-methylbutane-1,1-bisphosphonic
acid, tetrasodium salt ##STR00172##
(S)-2-Amino-1-hydroxy-3-phenylpropane-1,1-bisphosphonic acid,
tetrasodium salt ##STR00173##
(S)-2-Amino-1,3-dihydroxypropane-1,1-bisphosphonicacid, tetrasodium
salt ##STR00174##
(S)-2,3-Diamino-1-hydroxypropane-1,1-bisphosphonicacid, tetrasodium
salt ##STR00175##
(dl)-3-Amino-1-hydroxy-3-phenylpropane-1,1-bisphosphonic acid,
tetrasodium salt ##STR00176##
(S)-3-Amino-2-(4-chlorophenyl)-1-hydroxypropane-1,1-bisphosphonic
acid, tetrasodium salt ##STR00177##
(S)-2-Amino-3-(4-aminophenyl)-1-hydroxypropane-1,1-bisphosphonic
acid, tetrasodium salt ##STR00178## N-Phosphonomethylglycine
##STR00179## N-Phosphonomethylglycine, trisodium salt ##STR00180##
2-Phosphonomethylglutaric acid, tetrasodium salt ##STR00181##
2-Phosphonomethylsuccinic acid, tetrasodium salt ##STR00182##
(2R,4S)-4-Phosphonomethylpipecolinic acid, trisodiumsalt
##STR00183## (2R,4S)-4-Phosphonomethylpipecolinamide, disodiumsalt
##STR00184## N-Phosphonomethylglycine ##STR00185##
N-Phosphonomethylglycine, trisodium salt ##STR00186##
3-[6-Methoxy-2-(1,2,3,4-tetrahydro-isoquinolinyl)]propylphosphonic
acid, disodium salt ##STR00187##
3-[8-Methoxy-2-(1,2,3,4-tetrahydro-isoquinolinyl)]propylphosphonic
acid, disodium salt ##STR00188##
3-[2-(3-Methoxycarbonyl-1,2,3,4-tetrahydroisoquinolinyl)]-propylphosphonic
aciddisodium salt ##STR00189##
2-(3-Phosphonopropyl)-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole,
disodium salt ##STR00190## .beta.-D-Glucopyranosylmethylphosphonic
acid, disodiumsalt ##STR00191##
.alpha.-D-Glucopyranosylmethylphosphonic acid, disodiumsalt
##STR00192##
6-Deoxy-6-C-phosphonomethyl-D-glucono-.delta.-lactone,disodium salt
##STR00193## 6-Deoxy-6-C-phosphonomethyl-D-glucose, disodiumsalt
##STR00194## 4-Deoxy-4-C-phosphonomethyl-D-glucose, disodiumsalt
##STR00195## 3-Deoxy-3-C-phosphonomethyl-D-glucose, disodiumsalt
##STR00196## 1-Deoxy-N-phosphonoacetylnojirimycin, disodium salt
##STR00197##
(1,5-Dideoxy-1,5-imino-.alpha.-D-glucopyranosyl)methylphosphonic
acid, disodium salt ##STR00198##
1,6-Dideoxy-6-C-phosphonomethyl-nojirimycin,disodium salt
##STR00199##
TABLE-US-00004 TABLE IV
Na.sup.+-O.sub.3SO(CH.sub.2).sub.3OSO.sub.3.sup.-Na.sup.+ I II
##STR00200## ##STR00201## III ##STR00202## IV ##STR00203## V
##STR00204## VI ##STR00205## VII
HOCH.sub.2CH.sub.2CH.sub.2CH.sub.2SO.sub.3.sup.-Na.sup.+ VIII
(Na.sup.+-O.sub.3SCH.sub.2CH.sub.2CH.sub.2CH.sub.2).sub.2O IX
##STR00206## X ##STR00207## XI ##STR00208## XII XIII ##STR00209##
##STR00210## XIV ##STR00211## XV ##STR00212## XVI ##STR00213## XVII
XVIII ##STR00214## XIX ##STR00215## ##STR00216## XX ##STR00217##
XXI ##STR00218## XXII XXIII ##STR00219## ##STR00220## XXIV XXV
##STR00221## XXVI ##STR00222## ##STR00223## XXVII ##STR00224##
XXVIII ##STR00225## XXIX XXX ##STR00226## XXXI ##STR00227##
##STR00228## XXXII ##STR00229## XXXIII ##STR00230## XXXIV
##STR00231## XXXV ##STR00232## XXXVI XXXVII ##STR00233##
##STR00234## XXXVIII XXXIX ##STR00235## XL ##STR00236## XLI
##STR00237## ##STR00238## XLII ##STR00239## XLIII ##STR00240## XLIV
##STR00241## XLV ##STR00242## XLVI
CH.sub.3CH.sub.2CH.sub.2CH.sub.2SO.sub.3Na XLVII
CH.sub.3(CH.sub.2).sub.8CH.sub.2SO.sub.3Na XLVIII XLIX ##STR00243##
L ##STR00244## ##STR00245## LI ##STR00246## LII ##STR00247## LIII
##STR00248## LIV LV ##STR00249## LVI ##STR00250## ##STR00251## LVII
##STR00252## LVIII ##STR00253## LIX LX ##STR00254## LXI
##STR00255## ##STR00256## LXII ##STR00257## LXIII ##STR00258## LXIV
##STR00259## LXV ##STR00260## LXVI LXVII ##STR00261## ##STR00262##
LXVIII LXIX ##STR00263## ##STR00264## LXX ##STR00265## LXXI LXXII
##STR00266## ##STR00267## LXXIII ##STR00268## LXXIV ##STR00269##
LXXV ##STR00270## LXXVI LXXVII ##STR00271##
EQUIVALENTS
[0117] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures described herein. Such
equivalents are considered to be within the scope of the present
invention and are covered by the following claims. The contents of
all references, issued patents, and published patent applications
cited throughout this application are hereby incorporated by
reference. The appropriate components, processes, and methods of
those patents, applications and other documents may be selected for
the present invention and embodiments thereof.
Sequence CWU 1
1
2416PRTHomo sapiens 1Lys Ile Val Phe Phe Ala 1 527PRTHomo sapiens
2Lys Lys Leu Val Phe Phe Ala 1 536PRTHomo sapiens 3Lys Leu Val Phe
Phe Ala 1 546PRTHomo sapiens 4Lys Phe Val Phe Phe Ala 1 556PRTHomo
sapiens 5Ala Phe Phe Val Leu Lys 1 564PRTHomo sapiens 6Lys Leu Val
Phe 176PRTHomo sapiens 7Lys Ala Val Phe Phe Ala 1 585PRTHomo
sapiens 8Lys Leu Val Phe Phe 1 596PRTHomo sapiens 9Lys Val Val Phe
Phe Ala 1 5106PRTHomo sapiens 10Lys Ile Val Phe Phe Ala 1
5116PRTHomo sapiens 11Lys Leu Val Phe Phe Ala 1 5126PRTHomo sapiens
12Lys Phe Val Phe Phe Ala 1 5136PRTHomo sapiens 13Ala Phe Phe Val
Leu Lys 1 5144PRTHomo sapiens 14Lys Leu Val Phe 1156PRTHomo sapiens
15Lys Ala Val Phe Phe Ala 1 5165PRTHomo sapiens 16Lys Leu Val Phe
Phe 1 5176PRTHomo sapiens 17Lys Val Val Phe Phe Ala 1 5187PRTHomo
sapiens 18Lys Leu Val Phe Phe Ala Gln 1 5197PRTHomo sapiens 19Lys
Leu Val Phe Phe Ala Gln 1 5209PRTHomo sapiens 20His His Gln Lys Leu
Val Phe Phe Ala 1 5213PRTHomo sapiens 21Asp Asp Asp 1226PRTHomo
sapiens 22Lys Val Asp Asp Gln Asp 1 5234PRTHomo sapiens 23His His
Gln Lys 1246PRTHomo sapiens 24Gln Lys Leu Val Phe Phe 1 5
* * * * *