U.S. patent application number 09/780233 was filed with the patent office on 2001-12-06 for method for treating amyloidosis.
This patent application is currently assigned to Queen's University of Kingston. Invention is credited to Kisilevsky, Robert, Szarek, Walter, Weaver, Donald.
Application Number | 20010048941 09/780233 |
Document ID | / |
Family ID | 46252868 |
Filed Date | 2001-12-06 |
United States Patent
Application |
20010048941 |
Kind Code |
A1 |
Kisilevsky, Robert ; et
al. |
December 6, 2001 |
Method for treating amyloidosis
Abstract
Therapeutic compounds and methods for inhibiting amyloid
deposition in a subject, whatever its clinical setting, are
described. Amyloid deposition is inhibited by the administration to
a subject of an effective amount of a therapeutic compound
comprising an anionic group and a carrier molecule, or a
pharmaceutically acceptable salt thereof, such that an interaction
between an amnyloidogenic protein and a basement membrane
constituent is inhibited. Preferred anionic groups are sulfonates
and sulfates. Preferred carrier molecules include carbohydrates,
polymers, peptides, peptide derivatives, aliphatic groups,
alicyclic groups, heterocyclic groups, aromatic groups and
combinations thereof
Inventors: |
Kisilevsky, Robert;
(Kingston, CA) ; Szarek, Walter; (Kingston,
CA) ; Weaver, Donald; (Kingston, CA) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Queen's University of
Kingston
|
Family ID: |
46252868 |
Appl. No.: |
09/780233 |
Filed: |
February 9, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09780233 |
Feb 9, 2001 |
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09322577 |
May 27, 1999 |
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09322577 |
May 27, 1999 |
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08463548 |
Jun 5, 1995 |
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5972328 |
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08463548 |
Jun 5, 1995 |
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08403230 |
Mar 15, 1995 |
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5643562 |
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08403230 |
Mar 15, 1995 |
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08315391 |
Sep 29, 1994 |
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08315391 |
Sep 29, 1994 |
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08219798 |
Mar 29, 1994 |
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08219798 |
Mar 29, 1994 |
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08037844 |
Mar 29, 1993 |
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Current U.S.
Class: |
424/450 ;
514/17.8; 514/378; 514/381; 514/460; 514/54 |
Current CPC
Class: |
C07H 15/04 20130101;
A61K 31/255 20130101; A61K 31/41 20130101; A61K 47/549 20170801;
A61K 47/58 20170801; A61K 31/737 20130101; A61L 33/08 20130101;
A61K 31/185 20130101; A61K 31/795 20130101; C07H 11/00 20130101;
A61K 47/555 20170801; A61K 31/70 20130101; A61K 47/54 20170801;
A61K 47/55 20170801; A61K 47/51 20170801 |
Class at
Publication: |
424/450 ; 514/2;
514/54; 514/381; 514/378; 514/460 |
International
Class: |
A61K 009/127; A61K
038/00; A61K 031/715 |
Claims
1. A method for inhibiting amyloid deposition in a subject
comprising administering to the subject an effective amount of a
therapeutic compound, the therapeutic compound comprising at least
one anionic group attached to a carrier molecule, or a
pharmaceutically acceptable salt thereof, such that the therapeutic
compound inhibits an interaction between an amyloidogenic protein
and a glycoprotein or proteoglycan constituent of a basement
membrane to inhibit amyloid deposition.
2. The method of claim 1 further comprising administering the
therapeutic compound in a pharmaceutically acceptable vehicle.
3. The method of claim 1, wherein the carrier molecule is selected
from the group consisting of a carbohydrate, a polymer, a peptide,
a peptide derivative, an aliphatic group, an alicyclic group, a
heterocyclic group, an aromatic group and combinations thereof.
4. The method of claim 1, wherein the anionic group is selected
from the group consisting of a sulfonate group, a sulfate group, a
carboxylate group, a phosphate group, a phosphonate group, and a
heterocyclic group selected from the group consisting of 3
5. The method of claim 1, wherein the carrier molecule is selected
for delivery of the therapeutic compound to the brain.
6. The method of claim 1, wherein the therapeutic compound is
delivered liposomally.
7. A method for inhibiting amyloid deposition in a subject
comprising administering to the subject an effective amount of a
therapeutic compound, the therapeutic compound comprising at least
one sulfonate group covalently attached to a carrier molecule, or a
pharmaceutically acceptable salt thereof, such that the therapeutic
compound inhibits an interaction between an amyloidogenic protein
and a glycoprotein or proteoglycan constituent of a basement
membrane to inhibit amyloid deposition.
8. A method for inhibiting amyloid deposition in a subject
comprising administering to the subject an effective amount of a
therapeutic compound, the therapeutic compound having the following
formula:Q--[--SO.sub.3.sup.-X.sup.+].sub.nwherein Q is a carrier
molecule; X.sup.+ is a cationic group; and n is an integer selected
such that the biodistribution of the therapeutic compound for an
intended target site is not prevented while maintaining activity of
the therapeutic compound, wherein the therapeutic compound inhibits
an interaction between an amyloidogenic protein and a glycoprotein
or proteoglycan constituent of a basement membrane to inhibit
amyloid deposition.
9. The method of claim 7, wherein the therapeutic compound is
administered orally.
10. The method of claim 7, further comprising administering the
therapeutic compound in a pharmaceutically acceptable vehicle.
11. The method of claim 7, wherein the carrier molecule is selected
for delivery of the therapeutic compound to the brain.
12. The method of claim 7, wherein the therapeutic compound is
delivered liposomally.
13. The method of claim 7, wherein the carrier molecule is selected
from the group consisting of a carbohydrate, a polymer, a peptide,
a peptide derivative, an aliphatic group, an alicyclic group, a
heterocyclic group, an aromatic group and combinations thereof.
14. The method of claim 13, wherein the carrier molecule is a
carbohydrate.
15. The method of claim 13, wherein the carrier molecule is a lower
aliphatic group.
16. The method of claim 13, wherein the carrier molecule is a
polymer.
17. The method of claim 16, wherein the polymer is selected from
the group consisting of substituted and unsubstituted vinyl, acryl,
styrene and carbohydrate-derived polymers and copolymers and
pharmaceutically acceptable salts thereof.
18. The method of claim 13, wherein the carrier molecule includes a
heterocylic group.
19. A method for inhibiting amyloid deposition in a subject
comprising orally administering to the subject an effective amount
of a therapeutic compound, the therapeutic compound comprising at
least one sulfonate group covalently attached to a carrier
molecule, or a pharmaceutically acceptable salt thereof.
20. The method of claim 19 further comprising administering the
therapeutic compound in a pharmaceutically acceptable vehicle.
21. A method for inhibiting amyloid deposition in a subject
comprising administering to the subject an effective amount of a
therapeutic compound, the therapeutic compound comprising at least
one sulfate group covalently attached to a carrier molecule, or a
pharmaceutically acceptable salt thereof, such that the therapeutic
compound inhibits an interaction between an amyloidogenic protein
and a glycoprotein or proteoglycan constituent of a basement
membrane to inhibit amyloid deposition.
22. A method for inhibiting amyloid deposition in a subject
comprising administering to the subject an effective amount of a
therapeutic compound, the therapeutic compound having the following
formula:Q--[--OSO.sub.3.sup.-X.sup.+].sub.nwherein Q is a carrier
molecule; X.sup.+ is a cationic group; and n is an integer selected
such that the biodistribution of the therapeutic compound for an
intended target site is not prevented while maintaining activity of
the therapeutic compound, wherein the therapeutic compound inhibits
an interaction between an amyloidogenic protein and a glycoprotein
or proteoglycan constituent of a basement membrane to inhibit
amyloid deposition.
23. The method of claim 21, wherein the therapeutic compound is
administered orally.
24. The method of claim 21, further comprising administering the
therapeutic compound in a pharmaceutically acceptable vehicle.
25. The method of claim 21, wherein the carrier molecule is
selected from the group consisting of a carbohydrate, a polymer, a
peptide, a peptide derivative, an aliphatic group, an alicyclic
group, a heterocyclic group, an aromatic group and combinations
thereof.
26. The method of claim 25, wherein the carrier molecule is a
carbohydrate.
27. The method of claim 25, wherein the carrier molecule is a lower
aliphatic group.
28. The method of claim 25, wherein the carrier molecule is a
polymer.
29. The method of claim 28, wherein the polymer is selected from
the group consisting of substituted and unsubstituted vinyl, acryl,
styrene and carbohydrate-derived polymers and copolymers and
pharmaceutically acceptable salts thereof.
30. The method of claim 25, wherein the carrier molecule includes a
heterocyclic group.
31. A method of inhibiting amyloid deposition in a subject
comprising administering to the subject an effective amount of a
therapeutic compound comprising at least one sulfonate group
covalently attached to a lower aliphatic group, or a
pharmaceutically acceptable salt thereof, optionally in a
pharmaceutically acceptable vehicle.
32. The method of claim 31, wherein the therapeutic compound 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-hydroxybutane-1-sulfonic acid, and pharmaceutically acceptable
salts thereof.
33. The method of claim 31, wherein the therapeutic compound is
administered orally.
34. A method of inhibiting amyloid deposition in a subject
comprising administering to the subject an effective amount of a
therapeutic compound comprising at least one sulfonate group
covalently attached to a disaccharide, or a pharmaceutically
acceptable salt thereof, optionally in a pharmaceutically
acceptable vehicle.
35. The method of claim 34, wherein the disaccharide is
sucrose.
36. A method of inhibiting amyloid deposition in a subject
comprising administering to the subject an effective amount of a
therapeutic compound comprising at least one sulfonate group
covalently attached to a polymer, or a pharmaceutically acceptable
salt thereof, optionally in a pharmaceutically acceptable
vehicle.
37. The method of claim 36, wherein the therapeutic compound is
selected from the group consisting of
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); a
sulfonate derivative of poly(acrylic acid); a sulfonate derivative
of poly(methyl acrylate); a sulfonate derivative of poly(methyl
methacrylate); and pharmaceutically acceptable salts thereof.
38. The method of claim 37, wherein the therapeutic compound is
poly(vinylsulfonic acid) or a pharmaceutically acceptable salt
thereof.
39. A method of inhibiting amyloid deposition in a subject
comprising administering to the subject an effective amount of a
therapeutic compound comprising at least one sulfonate group
covalently attached to carrier molecule that includes a
heterocyclic group, or a pharmaceutically acceptable salt thereof,
optionally in a pharmaceutically acceptable vehicle.
40. The method of claim 39, wherein the compound is selected from
the group consisting of 3-(N-morpholino)propanesulfonic acid,
tetrahydrothiophene-1,1-dioxide-3,4-disulfonic acid, and
pharmaceutically acceptable salts thereof.
41. A method of inhibiting amyloid deposition in a subject
comprising administering to the subject an effective amount of a
therapeutic compound comprising at least one sulfonate group
covalently attached to a peptide and a peptide derivative, or a
pharmaceutically acceptable salt thereof, optionally in a
pharmaceutically acceptable vehicle.
42. A method of inhibiting amyloid deposition in a subject
comprising administering to the subject an effective amount of a
therapeutic compound comprising at least one sulfate group
covalently attached to a lower aliphatic group, or a
pharmaceutically acceptable salt thereof, optionally in a
pharmaceutically acceptable vehicle.
43. The method of claim 42, wherein the therapeutic compound is
selected from the group consisting of ethyl sulfuric acid,
1,2-ethanediol disulfuric acid, 1-propyl sulfuric acid,
1,3-propanediol disulfuric acid, 1,4-butanediol disulfuric acid,
1,5-pentanediol disulfuric acid, 2-amino-ethanesulfuric acid,
1,4-butanediol monosulfuric acid, and pharmaceutically acceptable
salts thereof.
44. A method of inhibiting amyloid deposition in a subject
comprising administering to the subject an effective amount of a
therapeutic compound comprising at least one sulfate group
covalently attached to a disaccharide, or a pharmaceutically
acceptable salt thereof, optionally in a pharmaceutically
acceptable vehicle.
45. The method of claim 44, wherein the therapeutic compound is
sucrose octasulfate or a pharmaceutically acceptable salt
thereof.
46. A method of inhibiting amyloid deposition in a subject
comprising administering to the subject an effective amount of a
therapeutic compound comprising at least one sulfate group
covalently attached to a polymer, or a pharmaceutically acceptable
salt thereof, optionally in a pharmaceutically acceptable
vehicle.
47. The method of claim 46, wherein the therapeutic compound is
selected from the group consisting of
poly(2-acrylamido-2-methyl-1-propanesulfuric acid);
poly(2-acrylamido-2-methyl-1-propanesulfuric
acid-co-acrylonitrile);
poly(2-acrylamido-2-methyl-1-propanesulfuric acid-co-styrene);
poly(vinylsulfuric acid); poly(sodium 4-styrenesulfuric acid); 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.
48. A method of inhibiting amyloid deposition in a subject
comprising administering to the subject an effective amount of a
therapeutic compound comprising at least one sulfate group
covalently attached to a carrier molecule that includes a
heterocyclic group, or a pharmaceutically acceptable salt thereof,
optionally in a pharmaceutically acceptable vehicle.
49. The method of claim 48, wherein the compound is selected from
the group consisting of 3-(N-morpholino)propanesulfuric acid,
tetrahydrothiophene-1,1-dioxide-3,4-diol disulfuric acid, and
pharmaceutically acceptable salts thereof.
50. A method of inhibiting amyloid deposition in a subject
comprising administering to the subject an effective amount of a
therapeutic compound comprising at least one sulfate group
covalently attached to a peptide and a peptide derivative, or a
pharmaceutically acceptable salt thereof, optionally in a
pharmaceutically acceptable vehicle.
51. A pharmaceutical composition for treating amyloidosis
comprising a therapeutic compound comprising at least one sulfonate
group covalently attached to a carrier molecule, or a
pharmaceutically acceptable salt thereof, in an amount sufficient
to inhibit amyloid deposition in a subject, and a pharmaceutically
acceptable vehicle.
52. The pharmaceutical composition of claim 51, wherein the carrier
molecule is selected from the group consisting of a carbohydrate, a
polymer, a peptide, a peptide derivative, an aliphatic group, an
alicyclic group, a heterocyclic group, an aromatic group and
combinations thereof.
53. The pharmaceutical composition of claim 51, wherein the
therapeutic compound is poly(vinylsulfonic acid) or a
pharmaceutically acceptable salt thereof.
54. The pharmaceutical composition of claim 51, wherein the
therapeutic compound 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-amino-ethanesulfonic acid,
4-hydroxybutane-1-sulfonic acid, and pharmaceutically acceptable
salts thereof.
55. The pharmaceutical composition of claim 51, wherein the
therapeutic compound is selected from the group consisting of
3-(N-morpholino)propane- sulfonic acid,
tetrahydrothiophene-1,1-dioxide-3,4-disulfonic acid, and
pharmaceutically acceptable salts thereof.
56. A pharmaceutical composition for treating amyloidosis
comprising a therapeutic compound comprising at least one sulfate
group covalently attached to a carrier molecule, or a
pharmaceutically acceptable salt thereof, in an amount sufficient
to inhibit amyloid deposition in a subject, and a pharmaceutically
acceptable vehicle.
57. The pharmaceutical composition of claim 56, wherein the carrier
molecule is selected from the group consisting of a carbohydrate, a
polymer, a peptide, a peptide derivative, an aliphatic group, an
alicyclic group, a heterocyclic group, an aromatic group and
combinations thereof.
58. The pharmaceutical composition of claim 56, wherein the
therapeutic compound is sucrose octasulfate or a pharmaceutically
acceptable salt thereof.
59. The pharmaceutical composition of claim 56, wherein the
therapeutic compound is selected from the group consisting of ethyl
sulfuric acid, 1,2-ethanediol disulfuric acid, 1-propanesulfuric
acid, 1,3-propanediol disulfuric acid, 1,4-butanediol disulfuric
acid, 1,5-pentanediol disulfuric acid, 2-amino-ethanesulfuric acid,
1,4-butanediol monosulfuric acid, and pharmaceutically acceptable
salts thereof.
60. The pharmaceutical composition of claim 51, wherein the
therapeutic compound is encapsulated in a liposome.
61. The pharmaceutical composition of claim 56, wherein the
therapeutic compound is encapsulated in a liposome.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application
Ser. No. 08/403,230, filed Mar. 15, 1995, which is a
continuation-in-part of application Ser. No. 08/315,391, filed Sep.
29, 1994, which is a continuation-in-part of application Ser. No.
08/219,798, filed Mar. 29, 1994, which is a continuation-in-part of
application Ser. No. 08/037,844 filed Mar. 29, 1993, now abandoned,
the contents of all of which are incorporated herein by
reference.
BACKGROUND OF INVENTION
[0002] Amyloidosis refers to a pathological condition characterized
by the presence of amyloid. Amyloid is a generic term referring to
a group of diverse but specific extracellular protein deposits
which are seen in a number of different diseases. Though diverse in
their occurrence, all amyloid deposits have common morphologic
properties, stain with specific dyes (e.g., Congo red), and have a
characteristic red-green birefringent appearance in polarized light
after staining. They also share common ultrastructural features and
common x-ray diffraction and infrared spectra.
[0003] Amyloidosis can be classified clinically as primary,
secondary, familial and/or isolated. Primary amyloidosis appears de
novo without any preceding disorder. Secondary amyloidosis is that
form which appears as a complication of a previously existing
disorder. Familial amyloidosis is a genetically inherited form
found in particular geographic populations. Isolated forms of
amyloidosis are those that tend to involve a single organ system.
Different amyloids are also characterized by the type of protein
present in the deposit. For example, neurodegenerative diseases
such as scrapie, bovine spongiform encephalitis, Creutzfeldt-Jakob
disease and the like are characterized by the appearance and
accumulation of a protease-resistant form of a prion protein
(referred to as AScr or PrP-27) in the central nervous system.
Similarly, Alzheimer's disease, another neurodegenerative disorder,
is characterized by congophilic angiopathy, neuritic plaques and
neurofibrillary tangles, all of which have the characteristics of
amyloids. In this case, the plaques and blood vessel amyloid is
formed by the beta protein. Other systemic diseases such as
adult-onset diabetes, complications of long-term hemodialysis and
sequelae of long-standing inflammation or plasma cell dyscrasias
are characterized by the accumulation of amyloids systemically. In
each of these cases, a different amyloidogenic protein is involved
in amyloid deposition.
[0004] Once these amyloids have formed, there is no known therapy
or treatment which significantly dissolves the deposits in situ
which is widely accepted.
SUMMARY OF THE INVENTION
[0005] This invention provides methods and compositions which are
useful in the treatment of amyloidosis. The methods of the
invention involve administering to a subject a therapeutic compound
which inhibits amyloid deposition. Accordingly, the compositions
and methods of the invention are useful for inhibiting amyloidosis
in disorders in which amyloid deposition occurs. The methods of the
invention can be used therapeutically to treat amyloidosis or can
be used prophylactically in a subject susceptible to amyloidosis.
The methods of the invention are based, at least in part, on
inhibiting an interaction between an amyloidogenic protein and a
constituent of basement membrane to inhibit amyloid deposition. The
constituent of basement membrane is a glycoprotein or proteoglyean,
preferably heparan sulfate proteoglycan. A therapeutic compound
used in the method of the invention can interfere with binding of a
basement membrane constituent to a target binding site on an
amyloidogenic protein, thereby inhibiting amyloid deposition.
[0006] In one embodiment, the method of the invention involves
administering to a subject a therapeutic compound having at least
one anionic group covalently attached to a carrier molecule which
is capable of inhibiting an interaction between an amyloidogenic
protein and a glycoprotein or proteoglycan constituent of a
basement membrane to inhibit amyloid deposition. In one embodiment,
the anionic group covalently attached to the carrier molecule is a
sulfonate group. Accordingly, the therapeutic compound can have the
formula:
Q--[--SO.sub.3.sup.-X.sup.+].sub.n
[0007] wherein Q is a carrier molecule; X.sup.+is a cationic group;
and n is an integer. In another embodiment, the anionic group is a
sulfate group. Accordingly, the therapeutic compound can have the
formula:
Q--[--OSO.sub.3.sup.-X.sup.+].sub.n
[0008] wherein Q is a carrier molecule; X.sup.+is a cationic group;
and n is an integer. Carrier molecules which can be used include
carbohydrates, polymers, peptides, peptide derivatives, aliphatic
groups, alicyclic groups, heterocyclic groups, aromatic groups and
combinations thereof. Preferred therapeutic compounds for use in
the invention include poly(vinylsulfonic acid), ethanesulfonic
acid, sucrose octasulfate, 1,2-ethanediol disulfuric acid,
1,2-ethanedisulfonic acid, 1,3-propanediol disulfuric acid,
1,3-propanedisulfonic acid, 1,4-butanediol disulfuric acid,
1,4-butanedisulfonic acid, 1,5-pentanedisulfonic acid, taurine,
3-(N-morpholino)propanesulfonic acid,
tetrahydrothiophene-1,1-dioxide-3,4-disulfonic acid,
4-hydroxybutane-1-sulfonic acid, or pharmaceutically acceptable
salts thereof.
[0009] The therapeutic compounds of the invention are administered
to a subject by a route which is effective for inhibition of
amyloid deposition. Suitable routes of administration include
subcutaneous, intravenous and intraperitoneal injection. The
therapeutic compounds of the invention have been found to be
effective when administered orally. Accordingly, a preferred route
of administration is oral administration. The therapeutic compounds
can be administered with a pharmaceutically acceptable vehicle.
[0010] The invention further provides pharmaceutical compositions
for treating amyloidosis. The pharmaceutical compositions include a
therapeutic compound of the invention in an amount effective to
inhibit amyloid deposition and a pharmaceutically acceptable
vehicle.
BRIEF DESCRIPTION OF DRAWINGS
[0011] FIG. 1 is a bar graph illustrating the effect of poly
(vinylsulfonate sodium salt) administered intraperitoneally on in
vivo AA amyloid deposition in mouse spleen.
[0012] FIG. 2 is a graph illustrating the effect of poly
(vinylsulfonate sodium salt) on heparan sulfate proteoglycan
binding to .beta.-APP in tris-buffered saline (TBS).
[0013] FIG. 3 is a graph illustrating the effect of poly
(vinylsulfonate sodium salt) on heparan sulfate proteoglycan
binding to .beta.-APP in phosphate buffered saline (PBS).
[0014] FIG. 4 is a bar graph illustrating the effect of poly
(vinylsulfonate sodium salt) administered orally on in vivo AA
amyloid deposition in mouse spleen.
[0015] FIG. 5 is a graph illustrating the blood level of the
amyloid precursor, SAA, over time for animals receiving
poly(vinylsulfonate sodium salt) (open circles) and control animals
(triangles).
[0016] FIG. 6 is a graph illustrating the effect of orally
administered poly(vinylsulfonate sodium salt) on the course of AA
amyloid deposition in mouse spleen when amyloid deposits were
already present prior to treatment of the animals. The triangles
represent the control animals and the open circles represent the
treated animals.
[0017] FIG. 7 is a graph illustrating the effect of orally
administered poly(vinylsulfonate sodium salt) on splenic amyloid
deposition when the inflammatory stimulus is maintained during the
course of the experiment. The triangles represent the control
animals and the open circles represent the treated animals.
[0018] FIG. 8 is a graph illustrating the effect of orally
administered ethane monosulfonate, sodium salt (EMS) on in vivo AA
splenic amyloid deposition. The triangles represent the control
animals, the open circles represent animals receiving 2.5 mg/ml of
EMS in their drinking water, and the open squares represent animals
receiving 6 mg/ml of EMS in their drinking water.
[0019] FIGS. 9 and 10 depict the chemical structures of the WAS
compounds described in Example 9.
DETAILED DESCRIPTION OF INVENTION
[0020] This invention pertains to methods and compositions useful
for treating amyloidosis. The methods of the invention involve
administering to a subject a therapeutic compound which inhibits
amyloid deposition. "Inhibition of amyloid deposition" is intended
to encompass prevention of amyloid formation, inhibition of further
amyloid deposition in a subject with ongoing amyloidosis and
reduction of amyloid deposits in a subject with ongoing
amyloidosis. Inhibition of amyloid deposition is determined
relative to an untreated subject or relative to the treated subject
prior to treatment. Amyloid deposition is inhibited by inhibiting
an interaction between an amyloidogenic protein and a constituent
of basement membrane. "Basement membrane" refers to an
extracellular matrix comprising glycoproteins and proteoglycans,
including laminin, collagen type IV, fibronectin and heparan
sulfate proteoglycan (HSPG). In one embodiment, amyloid deposition
is inhibited by interfering with an interaction between an
amyloidogenic protein and a sulfated glycosaminoglycan such as
HSPG. Sulfated glycosaminoglycans are known to be present in all
types of amyloids (see Snow, A. D. et al. (1987) Lab. Invest.
56:120-123) and amyloid deposition and HSPG deposition occur
coincidentally in animal models of amyloidosis (see Snow, A. D. et
al. (1987) Lab. Invest. 56:665-675). In the methods of the
invention, molecules which have a similar structure to a sulfated
glycosaminoglycan are used to inhibit an interaction between an
amyloidogenic protein and basement membrane constituent. In
particular, the therapeutic compounds of the invention comprise at
least one sulfate group or a functional equivalent thereof, for
example a sulfonic acid group or other functionally equivalent
anionic group, linked to a carrier molecule. In addition to
functioning as a carrier for the anionic functionality, the carrier
molecule can enable the compound to traverse biological membranes
and to be biodistributed without excessive or premature metabolism.
Moreover, when multiple anionic functionalities are present on a
carrier molecule, the carrier molecule serves to space the anionic
groups in a correct geometric separation.
[0021] In one embodiment, the method of the invention includes
administering to the subject an effective amount of a therapeutic
compound which has at least one anionic group covalently attached
to a carrier molecule. The therapeutic compound is capable of
inhibiting an interaction between an amyloidogenic protein and a
glycoprotein or proteoglycan constituent of a basement membrane to
thus inhibit amyloid deposition. The therapeutic compound can have
the formula:
Q--[--Y.sup.-X.sup.+].sub.n
[0022] wherein Y.sup.-is an anionic group at physiological pH; Q is
a carrier molecule; 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 compound for an intended target site is
not prevented while maintaining activity of the compound. For
example, the number of anionic groups is not so great as to inhibit
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. In one
embodiment, n is an integer between 1 and 10. In another
embodiment, n is an integer between 3 and 8.
[0023] An anionic group of a therapeutic compound of the invention
is a negatively charged moiety that, when attached to a carrier
molecule, can inhibit an interaction between an amyloidogenic
protein and a glycoprotein or proteoglycan constituent of a
basement membrane to thus inhibit amyloid deposition. For purposes
of this invention, the anionic group is negatively charged at
physiological pH. Preferably, the anionic therapeutic compound
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 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, a therapeutic compound of the invention can comprise
at least one anionic group including sulfonates, sulfates,
phosphonates, phosphates, carboxylates, and heterocyclic groups of
the following formulas: 1
[0024] Depending on the carrier molecule, more than one anionic
group can be attached thereto. When more than one anionic group is
attached to a carrier molecule, the multiple anionic groups can be
the sane structural group (e.g., all sulfonates) or, alternatively,
a combination of different anionic groups can be used (e.g.,
sulfonates and sulfates, etc.).
[0025] The ability of a therapeutic compound of the invention to
inhibit an interaction between an amyloidogenic protein and a
glycoprotein or proteoglycan constituent of a basement membrane can
be assessed by an in vitro binding assay, such as that described in
the Exemplification or in U.S. Pat. No. 5,164,295 by Kisilevsky et
al. Briefly, a solid support such as a polystyrene microtiter plate
is coated with an amyloidogenic protein (e.g., serum amyloid A
protein or .beta.-amyloid precursor protein (.beta.-APP)) and any
residual hydrophobic surfaces are blocked. The coated solid support
is incubated with various concentrations of a constituent of
basement membrane, preferably HSPG, either in the presence or
absence of a compound to be tested. The solid support is washed
extensively to remove unbound material. The binding of the basement
membrane constituent (e.g., HSPG) to the amyloidogenic protein
(e.g., .beta.-APP) is then measured using an antibody directed
against the basement membrane constituent which is conjugated to a
detectable substance (e.g., an enzyme, such as alkaline
phosphatase) by detecting the detectable substance. A compound
which inhibits an interaction between an amyloidogenic protein and
a glycoprotein or proteoglycan constituent of a basement membrane
will reduce the amount of substance detected (e.g., will inhibit
the amount of enzyme activity detected).
[0026] Preferably, a therapeutic compound of the invention
interacts with a binding site for a basement membrane glycoprotein
or proteoglycan in an amyloidogenic protein and thereby inhibits
the binding of the amyloidogenic protein to the basement membrane
constituent. Basement membrane glycoproteins and proteoglycans
include laminin, collagen type IV, fibronectin and heparan sulfate
proteoglycan (HSPG). In a preferred embodiment, the therapeutic
compound inhibits an interaction between an amyloidogenic protein
and HSPG. Consensus binding site motifs for HSPG in amyloidogenic
proteins have been described (see e.g. Cardin and Weintraub (1989)
Arteriosclerosis 9:21-32). For example, an HSPG consensus binding
motif can be of the general formula X1-X2-Y-X3, wherein X1, X2 and
X3 are basic amino acids (e.g., lysine or arginine) and Y is any
amino acid. Modeling of the geometry of this site led to
determination of the following spacing between basic amino acid
residues (carboxylate to carboxylate, in Angstroms):
X1-X2 5.3.+-.1.5 .ANG.
X1-X3 7.1.+-.1.5 .ANG.
X2-X3 7.6.+-.1.5 .ANG.
[0027] These values were determined using a combination of
molecular mechanics and semi-empirical quantum mechanics
calculations. Molecular mechanics calculations were performed using
the MM2 force field equation. Semi-empirical molecular orbital
calculations were performed using the AM1Hamiltonian equation. The
conformational space of the site was sampled using a combination of
molecular dynamics (both high and low temperature) and Monte Carlo
simulations.
[0028] Accordingly, in the therapeutic compounds of the invention,
when multiple anionic groups are attached to a carrier molecule,
the relative spacing of the anionic groups can be chosen such that
the anionic groups (e.g., sulfonates) optimally interact with the
basic residues within the HSPG binding site (thereby inhibiting
interaction of HSPG with the site). For example, anionic groups can
be spaced approximately 5.3.+-.1.5 .ANG., 7.1.+-.1.5 .ANG. and/or
7.6.+-.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., HSPG) in an amyloidogenic protein.
[0029] A therapeutic compound of the invention typically further
comprises 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 therapeutic compound are within the scope
of the invention. For example, X.sup.+can be a pharmaceutically
acceptable alkali metal, alkaline earth, higher valency cation
(e.g., aluminum salt), 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 therapeutic compound, the anionic group(s) is
covalently attached to a carrier molecule. Suitable carrier
molecules include carbohydrates, polymers, peptides, peptide
derivatives, aliphatic groups, alicyclic groups, heterocyclic
groups, aromatic groups or combinations thereof. A carrier molecule
can be substituted, e.g. with one or more amino, nitro, halogen,
thiol or hydroxy 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 sulfonates derived from: poly(acrylic acid);
poly(methyl acrylate); poly(methyl methacrylate); and poly(vinyl
alcohol); and pharmaceutically acceptable salts thereof. Examples
of carbohydrate-derived polymers with suitable covalently attached
anionic groups include those of the formula: 2
[0033] wherein R is SO.sub.3-or OSO.sub.3-; and pharmaceutically
acceptable salts thereof.
[0034] Peptides and peptide derivatives can also act as carrier
molecules. The term "peptide" includes two or more amino acids
covalently attached through a peptide bond. Amino acids which can
be used in peptide carrier molecules 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 or sulfonate
group can be attached through the hydroxy side chain of a serine
residue. A peptide can be designed to interact with a binding site
for a basement membrane constituent (e.g., HSPG) in an
amyloidogenic protein (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 therapeutic compounds of the invention. For example, cysteic
acid, the sulfonate derivative of cysteine, can be used.
[0035] The term "aliphatic group" is intended to include organic
compounds characterized by straight or branched chains, typically
having between I 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. Representative 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 an alkyl group, as
defined above, having an amino group attached thereto. The term
"alkylthio" refers to an alkyl group, as defined above, having a
sulfhydryl group attached thereto. The term "alkylcarboxyl" as used
herein means an alkyl group, as defined above, having a carboxyl
group attached thereto. The term "alkoxy" as used herein means an
alkyl group, as defined above, having an oxygen atom, attached
thereto. 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] The therapeutic compound of the invention can be
administered in a pharmaceutically acceptable vehicle. As used
herein "pharmaceutically acceptable vehicle" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like which
are compatible with the activity of the compound and are
physiologically acceptable to the subject. An example of a
pharmaceutically acceptable vehicle is buffered normal saline (0.15
molar NaCl). The use of such media and agents for pharmaceutically
active substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the therapeutic
compound, use thereof in the compositions suitable for
pharmaceutical administration is contemplated. Supplementary active
compounds can also be incorporated into the compositions.
[0040] In a preferred embodiment of the method of the invention,
the therapeutic compound administered to the subject is comprised
of at least one sulfonate group covalently attached to a carrier
molecule, or a pharmaceutically acceptable salt thereof.
Accordingly, the therapeutic compound can have the formula:
Q--[--SO.sub.3.sup.-X.sup.+].sub.n
[0041] wherein Q is a carrier molecule; X.sup.+is a cationic group;
and n is an integer. Suitable carrier molecules 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, therapeutic compounds with
multiple sulfonate groups can have the sulfonate groups spaced such
that the compound interacts optimally with an HSPG binding site
within an amyloidogenic protein.
[0042] In preferred embodiments, the carrier molecule for a
sulfonate(s) is a lower aliphatic group (e.g., a lower alkyl, lower
alkenyl or lower alkynyl), a heterocyclic group, a disaccharide, a
polymer or a peptide or peptide derivative. Furthermore, the
carrier can be substituted, e.g. with one or more amino, nitro,
halogen, thiol or hydroxy groups.
[0043] Examples of suitable sulfonated polymeric therapeutic
compounds 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); 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 a sulfonate derivative of poly(vinyl
alcohol); and pharmaceutically acceptable salts thereof.
[0044] 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 stereoisomers or as a single active isomer.
[0045] A preferred sulfonated disaccharide is a fully or partially
sulfonated sucrose, or pharmaceutically acceptable salt thereof,
such as sucrose octasulfonate.
[0046] Preferred lower aliphatic sulfonated compounds for use in
the invention include ethanesulfonic acid; 2-aminoethanesulfonic
acid (taurine); cysteic acid (3-sulfoalanine or
.alpha.-amino-.beta.-sulfoprop- ionic acid); 1-propanesulfonic
acid; 1,2-ethanedisulfonic acid; 1,3-propanedisulfonic acid;
1,4-butanedisulfonic acid; 1,5-pentanedisulfonic acid; and
4-hydroxybutane-1-sulfonic acid; and pharmaceutically acceptable
salts thereof.
[0047] Preferred heterocyclic sulfonated compounds include
3-(N-morpholino)propanesulfonic acid; and
tetrahydrothiophene-1,1-dioxide- -3,4-disulfonic acid; and
pharmaceutically acceptable salts thereof.
[0048] In another embodiment of the method of the invention, the
therapeutic compound administered to the subject is comprised of at
least one sulfate group covalently attached to a carrier molecule,
or a pharmaceutically acceptable salt thereof. Accordingly, the
therapeutic compound can have the formula:
Q--[--OSO.sub.3.sup.-X.sup.+].sub.n
[0049] wherein Q is a carrier molecule; X.sup.+is a cationic group;
and n is an integer. Suitable carrier molecules 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 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, therapeutic compounds with
multiple sulfate groups can have the sulfate groups spaced such
that the compound interacts optimally with an HSPG binding site
within an amyloidogenic protein.
[0050] In preferred embodiments, the carrier molecule for a
sulfate(s) is a lower aliphatic group (e.g., a lower alkyl, lower
alkenyl or lower alkynyl), a disaccharide, a polymer or a peptide
or peptide derivative. Furthermore, the carrier can be substituted,
e.g. with one or more amino, nitro, halogen, thiol or hydroxy
groups.
[0051] Examples of suitable sulfated polymeric therapeutic
compounds include poly(2-acrylamido-2-methyl-propyl sulfuric acid);
poly(2-acrylamido-2-methyl-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.
[0054] Preferred lower aliphatic sulfated compounds 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.
[0055] Preferred heterocyclic sulfated compounds include
3-(N-morpholino)propanesulfuric acid; and
tetrahydrothiophene-1,1-dioxide- -3,4-diol disulfuric acid; and
pharmaceutically acceptable salts thereof.
[0056] A further aspect of the invention includes pharmaceutical
compositions for treating amyloidosis. The therapeutic compounds in
the methods of the invention, as described hereinbefore, can be
incorporated into a pharmaceutical composition in an amount
effective to inhibit amyloidosis in a pharmaceutically acceptable
vehicle.
[0057] In one embodiment, the pharmaceutical compositions of the
invention include a therapeutic compound that has at least one
sulfonate group covalently attached to a carrier molecule, or a
pharmaceutically acceptable salt thereof, in an amount sufficient
to inhibit amyloid deposition, and a pharmaceutically acceptable
vehicle. The therapeutic composition can have the formula:
Q--[--SO.sub.3.sup.-X.sup.+].sub.n
[0058] wherein Q is a carrier molecule; X.sup.+is a cationic group;
and n is an integer selected such that the biodistribution of the
compound for an intended target site is not prevented while
maintaining activity of the compound.
[0059] In another embodiment, the pharmaceutical compositions of
the invention include a therapeutic compound that has at least one
sulfate group covalently attached to a carrier molecule, or a
pharmaceutically acceptable salt thereof, in an amount sufficient
to inhibit amyloid deposition, and a pharmaceutically acceptable
vehicle. The therapeutic compound can have the following
formula:
Q--[--OSO.sub.3.sup.-X.sup.+].sub.n
[0060] wherein Q is a carrier molecule; X.sup.+is a cationic group;
and n is an integer selected such that the biodistribution of the
compound for an intended target site is not prevented while
maintaining activity of the compound.
[0061] The invention further contemplates the use of prodrugs which
are converted in vivo to the therapeutic compounds 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 therapeutic
compound. 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, 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. 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
compound which subsequently decomposes to yield the active
compound. 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 therapeutic compound. 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 therapeutic moieties to
particular organs, as described below for carrier moieties.
[0062] Carrier molecules useful in the therapeutic compounds
include carrier molecules previously described, e.g. carbohydrates,
polymers, peptides, peptide derivatives, aliphatic groups,
alicyclic groups, heterocyclic groups, aromatic groups or
combinations thereof. Suitable polymers include substituted and
unsubstituted vinyl, acryl, styrene and carbohydrate-derived
polymers and copolymers and salts thereof. Preferred carrier
molecules include a lower alkyl group, a heterocyclic group, a
disaccharide, a polymer or a peptide or peptide derivative.
[0063] Carrier molecules useful in the present invention may also
include moieties which allow the therapeutic compound to be
selectively delivered to a target organ or organs. For example, if
delivery of a therapeutic compound to the brain is desired, the
carrier molecule may include a moiety capable of targeting the
therapeutic compound to the brain, by either active or passive
transport (a "targeting moiety"). Illustratively, the carrier
molecule 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. 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 therapeutic compounds of
the invention may bind to amyloidogenic proteins in the circulation
and thus be transported to the site of action.
[0064] In one embodiment, the therapeutic compound in the
pharmaceutical compositions is a sulfonated polymer, for example
poly(2-acrylamido-2-met- hyl-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); 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.
[0065] In another embodiment, the therapeutic compound in the
pharmaceutical compositions is a sulfated polymer, for example
poly(2-acrylamido-2-methyl-1-propanesulfuric acid);
poly(2-acrylamido-2-methyl-1-propanesulfuric
acid-co-acrylonitrile);
poly(2-acrylamido-2-methyl-1-propanesulfuric 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.
[0066] Preferred therapeutic compounds for inclusion in a
pharmaceutical composition for treating amyloidosis of the
invention include poly(vinylsulfuric acid); poly(vinylsulfonic
acid); sucrose octasulfate; a partially or fully sulfonated
sucrose; ethyl sulfuric acid; ethanesulfonic acid;
2-aminoethanesulfonic acid (taurine); 2-(aminoethyl)sulfuric acid;
cysteic acid (3-sulfoalanine or a-amino-.beta.-sulfopropionic
acid); 1-propanesulfonic acid; propyl sulfuric acid;
1,2-ethanedisulfonic acid; 1 ,2-ethanediol disulfuric acid;
1,3-propanedisulfonic acid; 1,3-propanediol disulfuric acid;
1,4-butanedisulfonic acid; 1,4-butanediol disulfuric acid;
1,5-pentanedisulfonic acid; 1,5-pentanediol disulfuric acid;
4-hydroxybutane-1-sulfonic acid;
tetrahydrothiophene-1,1-dioxide-3,4-disu- lfonic acid;
3-(N-morpholino)propanesulfonic acid; and pharmaceutically
acceptable salts thereof.
[0067] In the methods of the invention, amyloid deposition in a
subject is inhibited by administering a therapeutic compound of the
invention to the subject. The term subject is intended to include
living organisms in which amyloidosis can occur. Examples of
subjects include humans, monkeys, cows, sheep, goats, dogs, cats,
mice, rats, and transgenic species thereof. Administration of the
compositions of the present invention to a subject to be treated
can be carried out using known procedures, at dosages and for
periods of time effective to inhibit amyloid deposition in the
subject. An effective amount of the therapeutic compound necessary
to achieve a therapeutic effect may vary according to factors such
as the amount of amyloid already deposited at the clinical site in
the subject, the age, sex, and weight of the subject, and the
ability of the therapeutic compound to inhibit amyloid deposition
in the subject. Dosage regimens can be adjusted to provide the
optimum therapeutic response. For example, several divided doses
may be administered daily or the dose may be proportionally reduced
as indicated by the exigencies of the therapeutic situation. A
non-limiting example of an effective dose range for a therapeutic
compound of the invention (e.g., poly(vinylsulfonate sodium salt))
is between 5 and 500 mg/kg of body weight/per day. In an aqueous
composition, preferred concentrations for the active compound
(i.e., the therapeutic compound that can inhibit amyloid
deposition) are between 5 and 500 mM, more preferably between 10
and 100 mM, and still more preferably between 20 and 50 mM. For
taurine, particularly preferred aqueous concentrations are between
10 and 20 mM.
[0068] As demonstrated in the Exemplification, the therapeutic
compounds of the invention are effective when administered orally.
Accordingly, a preferred route of administration is oral
administration. Alternatively, the active compound may be
administered by other suitable routes such subcutaneous,
intravenous, intraperitoneal, etc. administration (e.g. by
injection). Depending on the route of administration, the active
compound may be coated in a material to protect the compound from
the action of acids and other natural conditions which may
inactivate the compound.
[0069] The compounds of the invention can be formulated to ensure
proper distribution in vivo. For example, the blood-brain barrier
(BBB) excludes many highly hydrophilic compounds. To ensure that
the therapeutic compounds of the invention cross the BBB, they can
be formulated, for example, in liposomes. For methods of
manufacturing liposomes, see, e.g., U.S. Pat. No. 4,522,811;
5,374,548; and 5,399,331. The liposomes may comprise one or more
moieties which are selectively transported into specific cells or
organs ("targeting moieties"), thus providing targeted drug
delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol.
29:685). Exemplary targeting moieties include folate or biotin
(see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides
(Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038);
antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 3:140; M. Owais
et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant
protein A receptor (Briscoe et al. (1995) Am. J. Physiol.
1233:134); gp120 (Schreieretal. (1994) J. Biol. Chem. 269:9090);
see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J.
J. Killion; I. J. Fidler (1994) Immunomethods 4:273. In a preferred
embodiment, the therapeutic compounds of the invention are
formulated in liposomes; in a more preferred embodiment, the
liposomes include a targeting moiety.
[0070] To administer the therapeutic compound by other than
parenteral administration, it may be necessary to coat the compound
with, or co-administer the compound with, a material to prevent its
inactivation. For example, the therapeutic compound may be
administered to a subject in an appropriate carrier, for example,
liposomes, or a diluent. Pharmaceutically acceptable diluents
include saline and aqueous buffer solutions. Liposomes include
water-in-oil-in-water CGF emulsions as well as conventional
liposomes (Strejan et al., (1984) J. Neuroimmunol. 7:27).
[0071] The therapeutic compound may also be administered
parenterally, intraperitoneally, intraspinally, or intracerebrally.
Dispersions can be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations may contain a
preservative to prevent the growth of microorganisms.
[0072] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. In all cases, the
composition must be sterile and must be fluid to the extent that
easy syringability exists. It must be stable under the conditions
of manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The vehicle can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), suitable
mixtures thereof, and vegetable oils. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. Prevention of the action
of microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars, sodium
chloride, or polyalcohols such as mannitol and sorbitol, in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate or
gelatin.
[0073] Sterile injectable solutions can be prepared by
incorporating the therapeutic compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the
therapeutic compound into a sterile vehicle which contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying which yields a
powder of the active ingredient (i.e., the therapeutic compound)
plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0074] The therapeutic compound can be orally administered, for
example, with an inert diluent or an assimilable edible carrier.
The therapeutic compound and other ingredients may also be enclosed
in a hard or soft shell gelatin capsule, compressed into tablets,
or incorporated directly into the subject's diet. For oral
therapeutic administration, the therapeutic compound may be
incorporated with excipients and used in the form of ingestible
tablets, buccal tablets, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like. The percentage of the therapeutic
compound in the compositions and preparations may, of course, be
varied. The amount of the therapeutic compound in such
therapeutically useful compositions is such that a suitable dosage
will be obtained.
[0075] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit containing a predetermined
quantity of therapeutic compound calculated to produce the desired
therapeutic effect in association with the required pharmaceutical
vehicle. The specification for the dosage unit forms of the
invention are dictated by and directly dependent on (a) the unique
characteristics of the therapeutic compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such a therapeutic compound for the
treatment of amyloid deposition in subjects.
[0076] Active compounds are administered at a therapeutically
effective dosage sufficient to inhibit amyloid deposition in a
subject. A "therapeutically effective dosage" preferably inhibits
amyloid deposition by at least about 20%, more preferably by at
least about 40%, even more preferably by at least about 60%, and
still more preferably by at least about 80% relative to untreated
subjects. The ability of a compound to inhibit amyloid deposition
can be evaluated in an animal model system that may be predictive
of efficacy in inhibiting amyloid deposition in human diseases,
such as the model system used in the Examples. Alternatively, the
ability of a compound to inhibit amyloid deposition can be
evaluated by examining the ability of the compound to inhibit an
interaction between an amyloidogenic protein and a basement
membrane constituent, e.g., using a binding assay such as that
described hereinbefore.
[0077] The method of the invention is useful for treating
amyloidosis associated with any disease in which amyloid deposition
occurs. Clinically, amyloidosis can be primary, secondary, familial
or isolated. Amyloids have been categorized by the type of
amyloidogenic protein contained within the amyloid. Non-limiting
examples of amyloids which can be inhibited, as identified by their
amyloidogenic protein, are as follows (with the associated disease
in parentheses after the amyloidogenic protein): .beta.-amyloid
(Alzheimer's disease, Down's syndrome, hereditary cerebral
hemorrhage amyloidosis [Dutch]); amyloid A (reactive [secondary]
amyloidosis, familial Mediterranean Fever, familial amyloid
nephropathy with urticaria and deafness [Muckle-Wells syndrome]);
amyloid .kappa. L-chain or amyloid .lambda. L-chain (idiopathic
[primary], myeloma or macroglobulinemia-associated); A.beta.2M
(chronic hemodialysis); ATTR (familial amyloid polyneuropathy
[Portuguese, Japanese, Swedish], familial amyloid cardiomyopathy
[Danish], isolated cardiac amyloid, systemic senile amyloidosis);
AIAPP or amylin (adult onset diabetes, insulinoma); atrial
naturetic factor (isolated atrial amyloid); procalcitonin
(medullary carcinoma of the thyroid); gelsolin (familial
amyloidosis [Finnish]); cystatin C (hereditary cerebral hemorrhage
with amyloidosis [Icelandic]); AApoA-I (familial amyloidotic
polyneuropathy [Iowa]); AApoA-II (accelerated senescence in mice);
fibrinogen-associated amyloid; lysozyme-associated amyloid; and
AScr or PrP-27 (Scrapie, Creutzfeldt-Jacob disease,
Gerstmann-Straussler-Scheinke- r syndrome, bovine spongiform
encephalitis).
[0078] The sulfated and sulfonated compounds used in the methods
described herein are commercially available (e.g. Sigma Chemical
Co., St. Louis, Mo., or Aldrich Chemical Co., Milwaukee, Wis.)
and/or can be synthesized by standard techniques known in the art
(see, e.g., Stone, G. C. H. (1936) J. Am. Chem. Soc., 58:488). In
general, sulfated compounds were synthesized from the corresponding
alcohols. The alcohols corresponding to WAS-28 and WAS-29 were
obtained by reduction of 1,3-acetonedicarboxylic acid and triethyl
methanetricarboxylate, respectively, which are commercially
available. Representative syntheses of active compounds used herein
are described in further detail in Example 10.
[0079] In certain embodiments of the invention, Congo red is
excluded from sulfonated compounds used in the method of the
invention.
[0080] In certain embodiments of the invention, the following
sulfated compounds are excluded from use in the method of the
invention: dextran sulfate 500, .iota.-carrageenan,
.lambda.-carrageenan, dextran sulfate 8, .kappa.-carrageenan,
pentosan polysulfate, and/or heparan.
[0081] In certain embodiments of the invention, the compositions
and methods of the invention are used to inhibit amyloid deposition
in amyloidosis wherein the amyloidogenic protein is not the
protease-resistant form of a prion protein, AScr (also known as
PrP-27).
[0082] The invention is further illustrated by the following
examples which should not be construed as further limiting the
subject invention. The contents of all references, issued patents,
and published patent applications cited throughout this application
are hereby incorporated by reference. A demonstration of efficacy
of the therapeutic compounds of the present invention in the mouse
model described in the examples is predictive of efficacy in
humans.
Exemplification
[0083] In the following examples, a well-characterized mouse model
of amyloidosis was used. In this in vivo system, animals receive an
inflammatory stimulus and amyloid enhancing factor. For acute
amyloidosis (i.e., short term amyloid deposition), the inflammatory
stimulus is AgNO.sub.3. For chronic amyloidosis (ongoing amyloid
deposition), the inflammatory stimulus is lipopolysaccharide (LPS).
Amyloid deposition (AA amyloid) in the spleens of mice was measured
with and without therapeutic treatment.
EXAMPLE 1
[0084] The following methodologies were used:
Animals
[0085] All mice were of the CD strain (Charles Rivers, Montreal,
Quebec) and weighing 25-30 g.
Animal Treatment
[0086] All animals received AgNO.sub.3 (0.5 ml, 2% solution)
subcutaneously in the back, and amyloid enhancing factor (AEF) 100
.mu.g intravenously. The preparation of amyloid enhancing factor
has been described previously in Axelrad, M. A. et al. ("Further
Characterization of Amyloid Enhancing Factor" Lab. Invest.
47:139-146 (1982)). The animals were divided into several groups
one of which was an untreated control group which was sacrificed
six days later. The remaining animals were divided into those which
received poly(vinylsulfonate sodium salt) (PVS) at 50 mg, 40 mg, 20
mg, or 10 mg by intraperitoneal injection every 12 hours or sucrose
octasulfate ammonium salt (SOA) at 73 mg or 36.5 mg every 8 hours
by IP injection. The PVS used in this and all subsequent Examples
was a mixture of stereoisomers. Surviving animals were sacrificed
on the 5th day of treatment. In all cases the PVS or SOA was
dissolved in a sterile aqueous carrier.
Tissue Preparation
[0087] At the termination of the experiments, the animals were
sacrificed by cervical dislocation and the spleens, livers, and
kidneys were fixed in 96% ethanol, 1% glacial acetic acid and 3%
water as described in Lyon, A. W. et al. ("Co-deposition of
Basement Membrane Components During the Induction of Murine Splenic
AA Amyloid" Lab. Invest. 64:785-790 (1991)). Following fixation,
the tissues were embedded in paraffin, 8-10 micron sections were
cut and stained with Congo Red without counterstain as described in
Puchtler, H. et al. ("Application of Thiazole Dyes to Amyloid Under
Conditions of Direct Cotton Dyeing: Correlation of Histochemical
and Chemical Data" Histochemistry 77:431-445 (1983)). The
histologic sections viewed under polarized light were assessed by
image analysis for the percent of spleen occupied by amyloid. In
the case of the experiments with sucrose octasulfate, the tissues
were immunostained with an antibody to the SAA protein (described
in Lyon A. W. et al. Lab Invest. 64:785-790 (1991)) and the
immunostained sections assessed by image analysis for the percent
of tissue section occupied by amyloid.
Viabiity of Animals
[0088] All control animals survived the experiment without
incident. In the case of the animals undergoing therapy, all
animals given sucrose octasulfate at 73 mg/injection succumbed
prior to the termination of the experiment. Animals receiving 36.5
mg of sucrose octasulfate/injection all survived. Of those animals
receiving PVS (molecular weight 900-1 000) in each dosage group,
approximately half to one-third of the animals succumbed prior to
the termination of the experiment. In all cases of animal deaths
prior to the end of the experiments, the cause of death was
uncontrolled intraperitoneal hemorrhage.
Effects of Agents on Amyloid Deposition
[0089] The effect of sucrose octasulfate at 36.5 mg/injection is
shown below in Table 1. The mean area of spleen occupied by amyloid
in control animals was 7.8%.+-.1.5% S.E.M. In animals receiving the
therapeutic agent the mean area was 3.2%.+-.0.5% S.E.M. The
difference is significant at a p.ltoreq.0.02.
1TABLE 1 Effect of Sucrose Octasulfate Ammonium Salt on AA Amyloid
Deposition In vivo in Mouse Spleen % Area Occupied by Amyloid
Untreated 7.8 + 1.5 n = 5 Sucrose OctaSO.sub.4 3.2 + 0.5 n = 5 p
.ltoreq. 0.02
[0090] In the case of PVS, the data are shown in FIG. 1. There was
a profound inhibition of amyloid deposition at all doses with the
suggestion of a dose-dependent effect. An effective dose range is
between 5 and 500 mg/kg of body weight/per day.
[0091] Preliminary assessment of the plasma level of the precursor
of inflammation-associated amyloidosis, SAA, has shown that there
is no difference between the animals being treated with PVS and
those untreated.
[0092] The method of administering the agents of the present
invention is believed to have had an effect upon the mortality rate
of the animals. Intraperitoneal injection was selected as providing
a large membrane surface for ease of access to the circulating
system. However, like heparan, the compounds of the present
invention exhibit anti-coagulant properties. Repeated injections
through the peritoneal wall induced severe hemorrhaging and
ultimately resulted in filling the peritoneal cavity, with loss of
blood causing death. While subcutaneous injection would result in
slower absorption of the active compound, it is less likely that
this route would cause hemorrhaging to such an extent as to cause
death. Oral administration of the compounds was performed in
subsequent experiments (see below).
EXAMPLE 2
[0093] Swiss white mice weighing 25-30 g were given Amyloid
Enhancing Factor (AEF) and AgNO.sub.3 as described previously
(Kisilevsky, R. and Boudreau, L. (1983) "The kinetics of amyloid
deposition: I. The effect of amyloid enhancing factor and
splenectomy" Lab. Invest., 48, 53-59), to induce amyloidosis.
Twenty four (24) hours later they were divided into three groups.
One group served as a control and was maintained on standard
laboratory mouse chow and tap water ad lib. A second group received
the standard chow but its water contained 20 mg/ml of
poly(vinylsulfonate sodium salt) (PVS). The third group had 50
mg/ml of PVS in its drinking water. Fluid intake in both groups was
the same. All animals were sacrificed on day six (6) of the
experiment, their spleens collected, prepared for sectioning,
spleen sections stained with Congo red (Puchtler, H., et al. (1983)
"Application of Thiazole Dyes to Amyloid under Conditions of Direct
Cotton Dyeing: Correlation of Histochemical and Chemical Data"
Histochemistry, 11, 431-445), and the percent area occupied by
amyloid assessed by an image analysis apparatus and program (MCID
M2, Imaging Research Inc., Brock University, St. Catherines,
Ontario, Canada). As shown in FIG. 4, oral administration of PVS
interferes with amyloid deposition in a dose dependent manner.
EXAMPLE 3
[0094] Since it was possible that PVS was inhibiting the hepatic
synthesis of the amyloid precursor, and thus failure to deposit
amyloid was due to the absence of the precursor pool, the effect of
PVS on the blood level of the amyloid precursor (SAA) during the
course of the experiment was determined. Animals received AEF+AgNO3
as described above and were divided into two groups. Group 1
received no further treatment. Twenty four hours later, Group 2
received 50 mg of PVS by intraperitoneal injection every 12 hours
for a period of 5 days. To plot the level of SAA during this
process, each animal (controls and experimentals) was bled from the
tail (.noteq.25 .mu.l) each day. The SAA levels in these samples
were determined by a solid phase ELISA procedure (described in
Brissette, L., et al. (1989) J. Biol. Chem., 264, 19327-19332). The
results are shown in FIG. 5. The open circles represent the data
from the PVS-treated mice, while the triangles show the data from
the non-treated animals. SAA levels were equivalent in treated and
untreated animals, demonstrating that PVS does not mediate its
effect by preventing the synthesis of SAA.
EXAMPLE 4
[0095] In the above described experiments, therapy with PVS was
begun 24 hours into the amyloid induction protocol. This does not
mimic a clinical situation where the patient usually has well
established amyloid. To approximate a more realistic clinical
situation, a separate set of experiments were performed in which
PVS treatment was begun after amyloid deposition had already begun.
Animals received AEF+AgNO3, as described above, remained on tap
water for 7 days, after which they were separated into two groups.
Group 1 remained on standard food and tap water. Group 2 remained
on standard food but had 50 mg/ml of PVS added to their drinking
water. To assess the effect of PVS on the course of amyloid
deposition after amyloid was already present, five animals in each
group were sacrificed on days 7, 10, 14, and 17. The spleens were
processed and evaluated as described above. The data are shown in
FIG. 6. Control animals (triangles) continued to deposit amyloid
for 14 days, following which the quantity of amyloid began to
decrease. This latter decrease is most likely due to the fact that
only one injection of AgNO.sub.3, the inflammatory stimulus, was
given and, after 14 days, the SAA levels are known to decrease
(Kisilevsky, R., Boudreau, L. and Foster, D. (1983) "Kinetics of
amyloid deposition. II. The effects of dimethylsulfoxide and
colchicine therapy" Lab. Invest., 48, 60-67). In the absence of
precursor, further amyloid cannot be deposited and existing
deposits are mobilized (Kisilevsky, R. and Boudreau, L. (1983) "The
kinetics of amyloid deposition: I. The effect of amyloid enhancing
factor and splenectomy" Lab. Invest., 4, 53-59). In contrast, the
treated group of animals (open circles) stopped deposition of
amyloid within 3 days of being placed on PVS. This demonstrates
that PVS is effective at inhibiting ongoing deposition of
amyloid.
EXAMPLE 5
[0096] To maintain the inflammation and the blood SAA levels, and
allow amyloid to be continuously deposited for the duration of a
longer term experiment, the nature of the inflammatory stimulus was
changed. So as to maintain the inflammation, animals received
lipopolysaccharide (LPS, 20 .mu.g)+AEF on day 0 and LPS was given
by intraperitoneal injection every 2nd day. On day seven (7), the
animals were separated into two groups as described in Example 4.
Assessment of amyloid over the course of the experiment proceeded
as described in Example 4. The data are shown in FIG. 7. The
control group (triangles) continued to deposit amyloid for the
entire 17 day period. Those receiving PVS apparently stopped
depositing amyloid by day 14 (open circles and dashed line). The
data on day 17 represent 4 animals per group as one animal was
omitted from this time period. The quantity of amyloid in this
particular individual was so far removed from all other data points
(treated or not, it was 21%) that it is believed that this was a
statistically valid procedure. If this individual is included, the
curve is represented by the dotted line and the remaining open
circle. It should be pointed out that animals receiving PVS began
to develop a significant diarrhea as the experiment proceeded.
EXAMPLE 6
[0097] In this experiment, another sulfonated compound, ethane
monosulfonic acid was used to inhibit amyloidosis. Ethane
monosulfonic acid, sodium salt, (EMS) is structurally the monomeric
unit of PVS. Animals were given LPS+AEF as in Experiment 5, but on
day seven EMS was used in the drinking water as the therapeutic
agent. On day seven, the animals were divided into three groups.
Group 1 was the untreated group. Group 2 received 2.5 mg/ml EMS in
their drinking water. Group 3 received 6 mg/ml in their drinking
water. Animals were sacrificed on days 7, 10, 14, and 17. These
animals did not develop gastro-intestinal problems. These data are
shown in FIG. 8. Animals receiving 6 mg/ml EMS in their drinking
water (open squares) stopped depositing amyloid after day 14. Those
receiving 2.5 mg/ml EMS (open circles) seemed to have an abortive
therapeutic effect, with a slight diminution in the rate of amyloid
deposition at day 14 which was not maintained by day 17.
EXAMPLE 7
[0098] The Influence of PVS on HSPG Binding to the Alzheimer's
Amyloid Precursor Protein (Beta APP)
[0099] The binding of heparan sulfate proteoglycan to beta APP was
assessed using an enzyme-linked immunosorbent assay technique as
described in Narindrasorasak, S. et al. ("High Affinity
Interactions Between the Alzheimer's Beta-Amyloid Precursor
Proteins and the Basement Membrane Form of Heparan Sulfate
Proteoglycan" J. Biol. Chem. 266:12878-12883 (1991)). Polystyrene
microtiter plates (Linbro, Flow Laboratories) were coated with a
100 .mu.l solution, 1 .mu.g/ml of .beta.-APP, in 20 mM NaHCO.sub.3
buffer, pH 9.6. After overnight incubation at 4.degree. C., the
plates were rinsed with 0.15 M NaCl, 20 mM Tris-Cl, pH 7.5 (TBS).
The plates were then incubated with 150 .mu.l of 1% bovine serum
albumin (BSA) in TBS for 2 hours at 37.degree. C. to block the
residual hydrophobic surface on the wells. After rinsing with TBS
containing 0.05 % (w/v) Tween 20 (TBS-Tween), 100 .mu.l of various
concentrations of HSPG in TBS-Tween were added alone or 500
.mu.g/ml of PVS, either in Tris-buffered saline (TBS) or
phosphate-buffered saline (PBS), was included in the binding assay
to assess the effect of PVS on HSPG binding to P-APP. The plates
were left overnight at 4.degree. C. to permit maximum binding of
HSPG to .beta.-APP. The plates were then washed extensively and
incubated 2 hours at 37.degree. C. with 100 .mu.l of anti-HSPG
diluted in TBS-Tween containing 0.1 % BSA. The plates were washed
again and incubated for another 2 hours with 100 .mu.l of goat
anti-rabbit IgG conjugated with alkaline phosphatase (1:2000
dilution) in TBS-Tween containing BSA as above. Finally, after
further washing, the bound antibodies were detected by adding an
alkaline phosphatase substrate solution (100 .mu.l) containing 2
mg/ml p-nitrophenyl phosphate, 0.1 mM ZnCl.sub.2, 1 mM MgCl.sub.2,
and 100 mM glycine, pH 10. The plates were left at room temperature
for 15-120 minutes. The enzyme reaction was stopped by addition of
50 .mu.l of 2 M NaOH. The absorbence of the released p-nitrophenol
was measured at 405 nm with a Titertek Multiscan/MCC 340 (Flow
Laboratories). The amounts of HSPG bound were determined by the net
A.sub.405 after subtracting the A from blank wells in which the
HSPG incubation step was omitted. The effect of PVS on HSPG:
beta-APP binding is illustrated in FIG. 2 (in TBS) and FIG. 3 (in
PBS). Approximately 30-50% inhibition of binding is demonstrated
with this compound.
EXAMPLE 8
[0100] Acute amyloidosis was elicited in mice with AgNO3 and
amyloid enhancing factor as described in Examples 1 and 2.
Twenty-four hours later, the animals were divided into a control
group and six test groups. The control group was maintained on
standard laboratory mouse chow and tap water ad lib. The test
groups received standard chow but their water contained 50 mM of
one of the following six compounds: sodium ethanesulfonate, sodium
2-aminoethanesulfonate (taurine), sodium 1 -propanesulfonate,
sodium 1,2-ethanedisulfonate, sodium 1,3-propanedisulfonate, or
sodium 1,4-butanedisulfonate. Water intake was approximately
equivalent for all groups. After six days, the animals were
sacrificed and their spleens were processed as described in Example
2. For preliminary analysis, the spleen sections were examined
visually under a microscope for differences in amyloid deposition
in the treated animals versus the control animals.
[0101] The results indicated that animals treated with sodium
1-propanesulfonate, sodium 1,2-ethanedisulfonate, or sodium
1,3-propanedisulfonate had less amyloid deposition than control
animals. Under the conditions used in this experiment, animals
treated with sodium ethanesulfonate, taurine sodium salt, or sodium
1,4-butanedisulfonate were not observed to have less amyloid
deposition than control animals. However, these compounds may
exhibit effectiveness under other conditions, for example sodium
ethanesulfonate has been observed to inhibit chronic amyloid
deposition (see Example 6) and taurine inhibits acute amyloid
deposition at other concentrations (see Example 9).
[0102] This experiment suggests that oral administration of
sulfonated lower aliphatics such as sodium 1-propanesulfonate,
sodium 1,2-ethanedisulfonate and sodium 1,3-propanedisulfonate can
inhibit amyloid deposition in an acute amyloidogenic system.
EXAMPLE 9
[0103] In view of the preliminary results described in Example 8,
further experiments were conducted to determine the effect of a
panel of sulfated or sulfonated compounds on acute amyloid
deposition. Acute amyloidosis was induced in mice as described in
Examples 1 and 2. Twenty-four hours later, the animals were divided
into a control group and test groups. The control group was
maintained on standard laboratory mouse chow and tap water ad lib.
The test groups received standard chow but their water contained 20
or 50 mM of one of the compounds listed in Table 2, below (the
chemical structures of the WAS compounds listed in Table 2 are
depicted in FIGS. 9 and 10). One compound, taurine, was tested at
concentrations of 5 mM, 10 mM, 20 mM, and 50 mM. All compounds were
dissolved in water containing 1.0% sucrose. Water intake was
approximately equivalent for all groups. After six days, the
animals were sacrificed and their spleens were processed as
described in Example 2.
[0104] The results are summarized in Table 2, below.
2TABLE 2 Effect of Sulfated and Sulfonated Compounds on AA Amyloid
Deposition In vivo in Mouse Spleen Concentration Amyloid Standard
Compound (mM) Deposition.sup.* Error 1,5-Pentanedisulfonate.dagger.
50 76 11 20 60 20 1,6-Hexanedisulfonate.dagger. 50 117 17 20 98 26
1,2-Ethanediol disulfate.dagger. 50 8 2 20 36 10 1,3-Propanediol
disulfate.dagger. 50 11 4 20 32 11 1,4-Butanediol disulfate.dagger.
50 54 22 20 44 11 Taurine 50 68 15 20 45 23 10 34 16 5 95 33 WAS-10
50 79 22 20 80 23 WAS-11 50 114 20 114 WAS-12 50 55 20 74 WAS-13 50
81 20 63 WAS-14 50 135 27 20 83 28 WAS-15 50 56 13 20 102 24 WAS-16
50 48 12 20 98 30 WAS-17 50 60 21 20 54 31 WAS-18 50 110 35 20 97
50 WAS-19 50 61 13 20 117 28 WAS-20 50 192 37 20 119 19 WAS-21 50
158 19 20 130 28 WAS-22 50 83 19 20 155 28 WAS-23 50 66 12 20 94 11
WAS-24 50 103 19 20 110 15 WAS-27 50 100 18 20 86 30 WAS-28 50 56
20 53 WAS-34 50 53 20 59 WAS-35 50 51 WAS-36 50 71 WAS-37 50 100 20
102 WAS-38 50 81 .dagger.As the sodium salt. .sup.*Amyloid
deposition is given as a percentage of untreated control. All
measurements are the average of # 3-5 animals.
[0105] The results indicate that animals treated with sodium
1,2-ethanediol disulfate or sodium 1,3-propanediol disulfate had at
least about a 65% decrease in amyloid deposition at 20 mM and at
least about a 90% decrease in amyloid deposition at 50 mM. Animals
treated with sodium 1,4-butanediol disulfate (50 mM), sodium
1,5-pentanedisulfonate ((50 mM), taurine (sodium
2-amino-ethanesulfonate) (10-20 mM), 3-(cyclohexylamino)-1-propane
sulfonate (WAS-12) (50 mM),
4-(2-hydroxyethyl)-1-piperazine-ethanesulfonate (WAS-13) (20 mM),
3-(N-morpholino)propanesulfonic acid (MOPS) (WAS-15) or its sodium
salt (WAS-16) (50 mM), sodium
tetrahydrothiophene-1,1-dioxide-3,4-disulfate trihydrate (WAS-19),
sodium 4-hydroxybutane-1-sulfonate (WAS-17) (50 mM), sodium
1,3,5-pentanetriol trisulfate (WAS-28) (20 and 50 mM), 2-aminoethyl
hydrogen sulfate (WAS-34) (20 and 50 mM), indigo carmine (WAS-35)
(50 mM) had at least approximately a 40% decrease in amyloid
deposition compared to untreated control animals. Taurine was
effective at concentrations of 10-20 mM, as seen in this example,
but less effective at 5 mM or 50 mM (see also Example 8).
[0106] Certain sulfated or sulfonated compounds were not effective
in reducing the amount of amyloid deposition under the conditions
employed, but may be effective in other embodiments. Earlier in
vitro work demonstrated that dermatan sulfate and chondroitin
6-sulfate do not interfere with the binding of beta amyoid
presursor protein to HSPG. Sodium (.+-.)-10-camphorsulfonate
(WAS-22), 4,5-dihydroxy- 1,3-benzenedisulfonic acid, disodium salt
(WAS-21) and 2,5-dihydroxy-1,4-benzenedisulfonic acid, dipotassium
salt (WAS-20) were tested in the above-described mouse model and,
as shown in Table 2, were found not to reduce amyloid
deposition.
EXAMPLE 10
[0107] In this example representative syntheses of two compounds
used in the methods of the invention are described.
Sodium ethane-1,2-disulfonate
[0108] A mixture of 1,2-dibromoethane (37.6 g, 0.20 mol) and sodium
sulfite (63.0 g, 0.5 mol) in water (225 mL) was heated at reflux
temperature for 20 h. After the mixture was cooled in the
refrigerator, crystals were collected. The crude product was
repeatedly recrystallized from water-ethanol. The trace amount of
inorganic salts was removed by treating the aqueous solution with a
small amount of silver(I) oxide and barium hydroxide. The basic
solution was neutralized with Amberlite-120 ion-exchange resin and
treated three times with Amberlite-120 (sodium form) ion-exchange
resin. After removal of the water, the product was recrystallized
from water-ethanol to afford the title compound (30.5 g).
Sodium 1,3-propanedisulfonate
[0109] This compound was prepared by a modification of the method
described in Stone, G. C. H. (1936) J. Am. Chem. Soc., 58:488.
1,3-Dibromopropane (40.4 g, 0.20 mol) was treated with sodium
sulfite (60.3 g, 0.50 mol) in water at reflux temperature for 48 h.
Inorganic salts (sodium bromide and sodium sulfite) were removed by
successive treatment of the resultant reaction mixture with barium
hydroxide and silver(I)oxide. The solution was then neutralized
with Amberlite-120 (acid form) and decolorized with Norit-A. Barium
ions were removed by treatment of the aqueous solution with
Amberlite-120 (sodium form) ion-exchange resin. The solvent was
removed on a rotary evaporator, and the crude product was
recrystallized from water-ethanol several times to give the title
compound (42.5 g). The small amount of trapped ethanol was removed
by dissolving the crystals in a minimum amount of water and then
concentrating the solution to dryness. The pure product was further
dried under high vacuum at 56.degree. C. for 24 h:mp>300.degree.
C.;.sup.1H NMR (D.sub.2O).delta.:3.06-3.13 (m, 4H, H-1 and
H-3),2.13-2.29 (m, 2H, H-2);.sup.13C NMR (D.sub.2O).delta.:52.3(C-1
and C-3), 23.8(C2).
EQUIVALENTS
[0110] 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 this invention
and are covered by the following claims.
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