U.S. patent application number 10/226613 was filed with the patent office on 2003-02-06 for phosphono-carboxylate compounds for treating amyloidosis.
This patent application is currently assigned to Lahive & Cockfield, LLP. Invention is credited to Gorine, Boris, Kong, Xianqi, Szarek, Walter A., Thatcher, Gregory R.J..
Application Number | 20030027796 10/226613 |
Document ID | / |
Family ID | 26765549 |
Filed Date | 2003-02-06 |
United States Patent
Application |
20030027796 |
Kind Code |
A1 |
Szarek, Walter A. ; et
al. |
February 6, 2003 |
Phosphono-carboxylate compounds for treating amyloidosis
Abstract
Therapeutic compounds and methods for modulating amyloid
deposition in a subject, whatever its clinical setting, are
described. Amyloid deposition is modulated by the administration to
a subject of an effective amount of a therapeutic compound
comprising a phosphonate group and a carboxylate group, a congener
thereof, or a pharmaceutically acceptable salt or ester thereof. In
preferred embodiments, an interaction between an amyloidogenic
protein and a basement membrane constituent is modulated.
Inventors: |
Szarek, Walter A.;
(Kingston, CA) ; Kong, Xianqi;
(Dollard-des-Ormeaux, CA) ; Thatcher, Gregory R.J.;
(Kingston, CA) ; Gorine, Boris; (Edmonton,
CA) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Lahive & Cockfield, LLP
|
Family ID: |
26765549 |
Appl. No.: |
10/226613 |
Filed: |
August 23, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10226613 |
Aug 23, 2002 |
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09860255 |
May 17, 2001 |
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6440952 |
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09860255 |
May 17, 2001 |
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09288583 |
Apr 8, 1999 |
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6329356 |
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60081402 |
Apr 10, 1998 |
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Current U.S.
Class: |
514/79 ; 514/114;
514/141 |
Current CPC
Class: |
A61K 31/675 20130101;
A61P 25/00 20180101; A61K 31/662 20130101; A61K 31/66 20130101 |
Class at
Publication: |
514/79 ; 514/141;
514/114 |
International
Class: |
A61K 031/675; A61K
031/66 |
Claims
What is claimed is:
1. A method for modulating amyloid deposition in a subject,
comprising administering to a subject an effective amount of a
therapeutic compound such that modulation of amyloid deposition
occurs, wherein the therapeutic compound has the formula: 12in
which R.sup.1 and R.sup.2 are each independently hydrogen, a
substituted or unsubstituted aliphatic group, an aryl group, a
heterocyclic group, or a salt-forming cation; R.sup.3 is hydrogen,
lower alkyl, aryl, or a salt-forming cation; R.sup.4 is hydrogen,
lower alkyl, aryl or amino; X is, independently for each
occurrence, O or S; Y.sup.1 and Y.sup.2 are each independently
hydrogen, halogen, alkyl, amino, hydroxy, alkoxy, or aryloxy; Z is
XR.sup.2 or R.sup.4; and n is an integer from 0 to 12.
2. The method of claim 1, wherein Z is XR.sup.2.
3. The method of claim 2, wherein R.sup.1 and R.sup.2 are each a
pharmaceutically acceptable salt-forming cation.
4. The method of claim 3, in which R.sup.1, R.sup.2 and R.sup.3 are
each independently a sodium, potassium or calcium cation.
5. The method of claim 4, wherein n is 0.
6. The method of claim 1, wherein at least one of R.sup.1 and
R.sup.2 is a long-chain aliphatic moiety.
7. The method of claim 6, wherein R.sup.3 is a lower alkyl
group.
8. The method of claim 1, wherein Y.sup.1 and Y.sup.2 are each
hydrogen.
9. The method of claim 1, wherein the therapeutic compound is
administered orally.
10. The method of claim 1, further comprising administering the
therapeutic compound in a pharmaceutically acceptable vehicle.
11. The method of claim 1, wherein administering the therapeutic
compound to the subject inhibits amyloid deposition in the
subject.
12. The method of claim 1, wherein X is, for each occurrence,
O.
13. The method of claim 1, wherein the compound is represented by
the formula: 13
14. The method of claim 1, wherein the compound is represented by
the formula: 14in which R.sub.a and R.sub.b are each independently
hydrogen, alkyl, aryl, or heterocyclyl, or R.sub.a and R.sub.b,
taken together with the nitrogen atom to which they are attached,
form a cyclic moiety having from 3 to 8 atoms in the ring.
15. The method of claim 14, in which R.sub.a and R.sub.b are each
hydrogen.
16. A method for treating a disease state associated with
amyloidosis, comprising: administering to a subject an effective
amount of a therapeutic compound such that a disease state
associated with amyloidosis is treated, wherein the therapeutic
compound has the formula 15in which R.sup.1 and R.sup.2 are each
independently hydrogen, an aliphatic group, an aryl group, a
heterocyclyl group, or a salt-forming cation; R.sup.3 is hydrogen,
lower alkyl, aryl or a salt-forming cation; Y.sup.1 and Y.sup.2 are
each independently hydrogen, halogen, lower alkyl, amino, hydroxy,
alkoxy, or aryloxy; and n is an integer from 0 to 12.
17. The method of claim 16, wherein said disease state is amyloid
deposition associated with Alzheimer's disease.
18. The method of claim 16, wherein R.sup.1 and R.sup.2 are each a
pharmaceutically acceptable salt-forming cation.
19. The method of claim 18, in which R.sup.1, R.sup.2 and R.sup.3
are each independently a sodium, potassium or calcium cation.
20. The method of claim 19, wherein n is 0.
21. The method of claim 16, wherein at least one of R.sup.1 and
R.sup.2 is a long-chain aliphatic moiety.
22. The method of claim 21, wherein R.sup.3 is a lower alkyl
group.
23. The method of claim 16, wherein Y.sup.1 and Y.sup.2 are each
hydrogen.
24. The method of claim 16, wherein the amino group is
--NH.sub.2.
25. The method of claim 16, wherein the therapeutic compound is
administered orally.
26. The method of claim 16, further comprising administering the
therapeutic compound in a pharmaceutically acceptable vehicle.
27. A method for modulating amyloid deposition in a subject in
which said amyloid deposition is characterized by interaction
between an amyloidogenic protein and a constituent of a basement
membrane, the method comprising administering to the subject an
effective amount of a therapeutic compound such that modulation of
amyloid deposition characterized by interaction between an
amyloidogenic protein and a constituent of a basement membrane
occurs, wherein the therapeutic compound has the formula: 16in
which R.sup.1 and R.sup.2 are each independently hydrogen, an
aliphatic group, an aryl group, a heterocyclyl group, or a
salt-forming cation; R.sup.3 is hydrogen, lower alkyl, aryl or a
salt-forming cation; Y.sup.1 and Y.sup.2 are each independently
hydrogen, halogen, lower alkyl, amino, hydroxy, alkoxy, or aryloxy;
and n is an integer from 0 to 12.
28. The method of claim 27, wherein R.sup.1 and R.sup.2 are each a
pharmaceutically acceptable salt-forming cation.
29. The method of claim 28, in which R.sup.1, R.sup.2 and R.sup.3
are each independently a sodium, potassium or calcium cation.
30. The method of claim 29, wherein n is 0.
31. The method of claim 27, wherein at least one of R.sup.1 and
R.sup.2 is a long-chain aliphatic moiety.
32. A method for preparing a compound represented by the formula:
17in which R is alkyl or aryl, and R' is hydrogen, alkyl, or aryl;
the method comprising: reacting an ester of a carbonylphosphono
diacid halide with a disiylether of a vicianl diol, under
conditions such that the compound of Formula V is prepared.
33. A compound represented by the formula (Formula IV): 18wherein G
is hydrogen or one of more substituents on the aryl ring and the L
is a substituted alkyl group.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35
U.S.C. 119(e) to copending U.S. Provisional Application No.
60/081,402, filed on Apr. 10, 1998, the entire contents 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 plaque and blood vessel amyloid is
formed by the beta protein. Other systemic or localized 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.
SUMMARY OF THE INVENTION
[0004] 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.
Without wishing to be bound by theory, it is believed that 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 can be a glycoprotein or proteoglycan,
preferably heparan sulfate proteoglycan. In certain embodiments, a
therapeutic compound used in the method of the invention preferably
can interfere with binding of a basement membrane constituent to a
target binding site on an amyloidogenic protein, thereby inhibiting
amyloid deposition.
[0005] The invention relates to phosphonocarboxylate compounds,
i.e., compounds which include a phosphonate group and a carboxylate
group, or a pharmaceutically acceptable salt or ester thereof. In
one embodiment, the method of the invention involves administering
to a subject an effective amount of a therapeutic compound having
the formula (Formula I): 1
[0006] in which Z is XR.sup.2 or R.sup.4, R.sup.1 and R.sup.2 are
each independently hydrogen, a substituted or unsubstituted
aliphatic group (preferably a branched or straight-chain aliphatic
moiety having from 1 to 24 carbon atoms in the chain; or an
unsubstituted or substituted cyclic aliphatic moiety having from 4
to 7 carbon atoms in the aliphatic ring; preferred aliphatic and
cyclic aliphatic groups are alkyl groups, more preferably lower
alkyl), an aryl group, a heterocyclic group, or a salt-forming
cation; R.sup.3 is hydrogen, lower alkyl, aryl, or a salt-forming
cation; R.sup.4 is hydrogen, lower alkyl, aryl or amino (including
alkylamino, dialkylamino (including cyclic amino moieties),
arylamino, diarylamino, and alkylarylamino); X is, independently
for each occurrence, O or S; Y.sup.1 and Y.sup.2 are each
independently hydrogen, halogen (e.g., F, Cl, Br, or I), alkyl
(preferably lower alkyl), amino, hydroxy, alkoxy, or aryloxy; and n
is an integer from 0 to 12 (more preferably 0 to 6, more preferably
0 or 1); such that amyloid deposition is modulated.
[0007] In preferred embodiments, therapeutic compounds of the
invention prevent or inhibit amyloid deposition in a subject to
which the therapeutic compound is administered. Preferred
therapeutic compounds for use in the invention include compounds in
which both R.sup.1 and R.sup.2 are pharmaceutically acceptable
salt-forming cations. It will be appreciated that the stoichiometry
of an anionic compound to a salt-forming counterion (if any) will
vary depending on the charge of the anionic portion of the compound
(if any) and the charge of the counterion. In a particularly
preferred embodiment, R.sup.1, R.sup.2 and R.sup.3 are each
independently a sodium, potassium or calcium cation. In certain
embodiments in which at least one of R.sup.1 and R.sup.2 is an
aliphatic group, the aliphatic group has between 1 and 10 carbons
atoms in the straight or branched chain, and is more preferably a
lower alkyl group. In other embodiments in which at least one of
R.sup.1 and R.sup.2 is an aliphatic group, the aliphatic group has
between 10 and 24 carbons atoms in the straight or branched chain.
In certain preferred embodiments, n is 0 or 1; more preferably, n
is 0. In certain preferred embodiments of the therapeutic
compounds, Y.sup.1 and Y.sup.2 are each hydrogen.
[0008] In certain preferred embodiments, the therapeutic compound
of the invention can be represented by the formula (Formula II):
2
[0009] in which R.sup.1, R.sup.2, R.sup.3, Y.sup.1, Y.sup.2, X and
n are as defined above. In more preferred embodiments, the
therapeutic compound of the invention can be represented by the
formula (Formula III): 3
[0010] in which R.sup.1, R.sup.2, R.sup.3, Y.sup.1, Y.sup.2, and X
are as defined above, R.sub.a and R.sub.b are each independently
hydrogen, alkyl, aryl, or heterocyclyl, or R.sub.a and R.sub.b,
taken together with the nitrogen atom to which they are attached,
form a cyclic moiety having from 3 to 8 atoms in the ring, and n is
an integer from 0 to 6. In certain preferred embodiments, R.sub.a
and R.sub.b are each hydrogen. In certain preferred embodiments, a
compound of the invention comprises an .alpha.-amino acid (or
.alpha.-amino acid ester), more preferably a L-.alpha.-amino acid
or ester.
[0011] In another embodiment, the compounds of the invention can be
represented by the formula (Formula IV): 4
[0012] in which G represents hydrogen or one or more substituents
on the aryl ring (e.g., alkyl, aryl, halogen, amino, and the like)
and L is a substituted alkyl group (in certain embodiments,
preferably a lower alkyl), more preferably a hydroxy-substituted
alkyl or an alkyl substituted with a nucleoside base.
[0013] The therapeutic compounds of the invention are administered
to a subject by a route which is effective for modulation of
amyloid deposition. Suitable routes of administration include oral,
transdermal, subcutaneous, intravenous, intramuscular and
intraperitoneal injection. A preferred route of administration is
oral administration. The therapeutic compounds can be administered
with a pharmaceutically acceptable vehicle.
[0014] The invention also provides methods for treating a disease
state associated with amyloidosis by administering to a subject an
effective amount of a therapeutic compound having the formula
described supra, such that a disease state associated with
amyloidosis is treated.
[0015] The invention provides methods for modulating amyloid
deposition characterized by interaction between an amyloidogenic
protein and a constituent of a basement membrane by administering
to the subject an effective amount of a therapeutic compound having
the formula described supra, such that modulation of amyloid
deposition characterized by interaction between an amyloidogenic
protein and a constituent of a basement membrane occurs.
[0016] The invention further provides pharmaceutical compositions
for treating amyloidosis. The pharmaceutical compositions include a
therapeutic compound of the invention in an amount effective to
modulate amyloid deposition and a pharmaceutically acceptable
vehicle.
[0017] The invention also provides packaged pharmaceutical
compositions for treating amyloidosis. The packaged pharmaceutical
compositions include a therapeutic compound of the invention and
instructions for using the pharmaceutical composition for treatment
of amyloidosis.
DETAILED DESCRIPTION OF INVENTION
[0018] 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 modulates
amyloid deposition. "Modulation 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. Modulation of amyloid deposition is determined
relative to an untreated subject or relative to the treated subject
prior to treatment. In certain embodiments, amyloid deposition can
be modulated by modulating an interaction between an amyloidogenic
protein and a constituent of basement membrane.
[0019] "Basement membrane" refers to an extracellular matrix
comprising glycoproteins and proteoglycans, including laminin,
collagen type IV, fibronectin chondroitan sulfate, and/or heparan
sulfate proteoglycan (HSPG). In one embodiment, amyloid deposition
is modulated 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 preferred embodiments of
the methods of the invention, molecules which have a similar
structure to a sulfated glycosaminoglycan are used to modulate
interaction between an amyloidogenic protein and basement membrane
constituent. In particular, the therapeutic compounds of the
invention preferably comprise at least one phosphonate group (or
phosphonic ester), or a functional equivalent thereof (including
phosphorus-containing anionic groups including, but not limited to,
phosphates, phosphate esters, phosphinates, and the like), and a
carboxylate group or carboxylic ester (or a congener such as a
thioacid, thiolester, or thionoester), provided that the compound
includes, or is capable of having after reaction in vivo, at least
one anionic group. The anionic groups(s) can optionally be
covalently bound to a carrier (e.g., an aliphatic group, peptide or
peptidomimetic, or the like). 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.
[0020] 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 phosphonate group or phosphonic
ester group. The therapeutic compound is preferably capable of
modulating interaction between an amyloidogenic protein and a
glycoprotein or proteoglycan constituent of a basement membrane to
thus modulate amyloid deposition. The therapeutic compound has the
formula (Formula I): 5
[0021] in which Z is XR.sup.2 or R.sup.4, R.sup.1 and R.sup.2 are
each independently hydrogen, a substituted or unsubstituted
aliphatic group (preferably a branched or straight-chain aliphatic
moiety having from 1 to 24 carbon atoms in the chain; or an
unsubstituted or substituted cyclic aliphatic moiety having from 4
to 7 carbon atoms in the aliphatic ring; preferred aliphatic and
cyclic aliphatic groups are alkyl groups, more preferably lower
alkyl), an aryl group, a heterocyclic group, or a salt-forming
cation; R.sup.3 is hydrogen, lower alkyl, aryl, or a salt-forming
cation; X is, independently for each occurrence, O or S; R.sup.4 is
hydrogen, lower alkyl, aryl or amino; Y.sup.1 and Y.sup.2 are each
independently hydrogen, halogen (e.g., F, Cl, Br, or I), lower
alkyl, amino (including alkylamino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), hydroxy, alkoxy, or aryloxy; and
n is an integer from 0 to 12 (more preferably 0 to 6, more
preferably 0 or 1); such that amyloid deposition is modulated.
[0022] In preferred embodiments, therapeutic compounds of the
invention prevent or inhibit amyloid deposition in a subject to
which the therapeutic compound is administered. Preferred
therapeutic compounds for use in the invention include compounds in
which both R.sup.1 and R.sup.2 are pharmaceutically acceptable
salt-forming cations. It will be appreciated that the stoichiometry
of an anionic compound to a salt-forming counterion (if any) will
vary depending on the charge of the anionic portion of the compound
(if any) and the charge of the counterion. In a particularly
preferred embodiment, R.sup.1, R.sup.2 and R.sup.3 are each
independently a sodium, potassium or calcium cation. In certain
embodiments in which at least one of R.sup.1 and R.sup.2 is an
aliphatic group, the aliphatic group has between 1 and 10 carbons
atoms in the straight or branched chain, and is more preferably a
lower alkyl group. In other embodiments in which at least one of
R.sup.1 and R.sup.2 is an aliphatic group, the aliphatic group has
between 10 and 24 carbons atoms in the straight or branched chain.
In certain preferred embodiments, n is 0 or 1; more preferably, n
is 0. In certain preferred embodiments of the therapeutic
compounds, Y.sup.1 and Y.sup.2 are each hydrogen.
[0023] In certain preferred embodiments, the therapeutic compound
of the invention can be represented by the formula (Formula II):
6
[0024] in which R.sup.1, R.sup.2, R.sup.3, Y.sup.1, Y.sup.2, X and
n are as defined above. In more preferred embodiments, the
therapeutic compound of the invention can be represented by the
formula (Formula III): 7
[0025] in which R.sup.1, R.sup.2, R.sup.3, Y.sup.1, Y.sup.2, and X
are as defined above, R.sub.a and R.sub.b are each independently
hydrogen, alkyl, aryl, or heterocyclyl, or R.sub.a and R.sub.b,
taken together with the nitrogen atom to which they are attached,
form a cyclic moiety having from 3 to 8 atoms in the ring, and n is
an integer from 0 to 6. In certain preferred embodiments, R.sub.a
and R.sub.b are each hydrogen. In certain preferred embodiments, a
compound of the invention comprises an .alpha.-amino acid (or
.alpha.-amino acid ester), more preferably a L-.alpha.-amino acid
or ester.
[0026] The Z, Q, R.sup.1, R.sup.2, R.sup.3, Y.sup.1, Y.sup.2 and X
groups are each independently selected such that the
biodistribution of the compound for an intended target site is not
prevented while maintaining activity of the compound. For example,
the number of anionic groups (and the overall charge on the
therapeutic compound) should not be 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. For
example, it has been reported that esters of phosphonoformate have
biodistribution properties different from, and in some cases
superior to, the biodistribution properties of phosphonoformate
(see, e.g., U.S. Pat. Nos. 4,386,081 and 4,591583 to Helgstrand et
al., and U.S. Pat. Nos. 5,194,654 and 5,463,092 to Hostetler et
al.). Thus, in certain embodiments, at least one of R.sup.1 and
R.sup.2 is an aliphatic group (more preferably an alkyl group), in
which the aliphatic group has between 10 and 24 carbons atoms in
the straight or branched chain. The number, length, and degree of
branching of the aliphatic chains can be selected to provide a
desired characteristic, e.g., lipophilicity. In other embodiments,
at least one of R.sup.1 and R.sup.2 is an aliphatic group (more
preferably an alkyl group), in which the aliphatic group has
between 1 and 10 carbons atoms in the straight or branched chain.
Again, the number, length, and degree of branching of the aliphatic
chains can be selected to provide a desired characteristic, e.g.,
lipophilicity or ease of ester cleavage by enzymes. In certain
embodiments, a preferred aliphatic group is an ethyl group.
[0027] It has also been reported that certain thiophosphate
compounds have in vivo activity as anti-viral agents which is equal
to or greater than the activity of the corresponding oxy-phosphate
compounds (possibly due to differences in bioavailability of the
compounds). Accordingly, in certain preferred embodiments, the
therapeutic compound includes a moiety selected from the group
consisting of --P(S)(OR.sup.1)(OR.sup.2),
--P(S)(SR.sup.1)(OR.sup.2), or --P(S)(SR.sup.1)(SR.sup.2).
[0028] In another embodiment, compounds useful in the methods of
the invention can be represented by the formula (Formula IV):
[0029] In another embodiment, the compounds of the invention can be
represented by the formula (Formula IV): 8
[0030] in which G represents hydrogen or one or more substituents
on the aryl ring (e.g., alkyl, aryl, halogen, amino, and the like)
and L is a substituted alkyl group (in certain embodiments,
preferably a lower alkyl), more preferably a hydroxy-substituted
alkyl or an alkyl substituted with a nucleoside base. In certain
embodiments, G is hydrogen or an electron-donating group. In
embodiments in which G is an electron-withdrawing group, G is
preferably an electron withdrawing group at the meta position. The
term "electron-withdrawing group" is known in the art, and, as used
herein, refers to a group which has a greater electron-withdrawing
than hydrogen. A variety of electron-withdrawing groups are known,
and include halogens (e.g., fluoro, chloro, bromo, and iodo
groups), nitro, cyano, and the like. Similarly, the term
"electron-donating group", as used herein, refers to a group which
is less electron-withdrawing than hydrogen. In embodiments in which
G is an electron donating group, G can be in the ortho, meta or
para position.
[0031] In certain preferred embodiments, L is a moiety selected
from the group consisting of (Formulas IVa-IVg): 9
[0032] Table 1 lists data pertinent to the characterization of
these compounds using art-recognized techniques.
1TABLE 1 COMPOUND .sup.31P NMR .sup.13C NMR FAB-MS(-) IVa -6.33
60.97 CH.sub.2OH (d, J = 6 Hz) 245.2 (DMSO-d.sub.6) 66.76 CHOH (d,
J = 7.8 Hz) 121.65, 121.78, 121.99, 125.71, 129.48, 129.57, 126.43
Aromatic CH 134.38 Aniline C--N 159.39 Phenyl C--O (d, J = 7 Hz)
171.57 P--C.dbd.O (d, J = 234 Hz) IVb -6.41 13.94 CH.sub.3 456
(DMSO-d.sub.6) 22.11, 24.40, 28.56, 28.72, 28.99, 29.00, 31.30,
33.43, --(CH.sub.2).sub.10-- 65.03 CH.sub.2--OC(O) 66.60
CH.sub.2--OP (d, J = 5.6 Hz) 67.71 CH2--OH (d, J = 6 Hz) 121.73,
121.10, 125.64, 126.57, 129.40, 129.95, Aromatic CH 134.04 Aniline
C--N 150.31 Phenyl C--O 171.44 P--C = O (d, J = 6.7 Hz) 172.83 O--C
= O IVc -6.46 13.94 CH.sub.3 471 22.11, 25.10, 28.68, 28.72 28.85,
29.00, 30.76, 31.31, 32.10, --(CH.sub.2).sub.10-- 43.36 CH.sub.2--S
68.43 CH.sub.2--OH 68.43 CH--OH (d, J = 6.3 Hz) 68.76
P--O--CH.sub.2--9 d, J = 5.8 Hz) 121.75, 122.03, 125.62, 126.37,
129.30, 129.53, Aromatic CH 134.23 Aniline C--N 150.37 Phenyl C--O
(d, J = 6.7 Hz) 171.47 P--C.dbd.O (d, J = 234.0 Hz) 198.47
S--C.dbd.O IVd -6.61 13.94 CH.sub.3 416 (DMSO-d.sub.6) 22.06,
25.14, 28.24, 28.35, 31.09, 32.14 --CH.sub.2).sub.6-- 43.40
CH.sub.2-- 68.50 P--O--CH.sub.2-- (d, J = 5.8 Hz) 68.77 CH--OH (d,
6.4 Hz) 121.78, 122.59, 125.69, 127.06, 129.43, 129.59 Aromatic CH
133.39 Aniline C--N 150.38 Phenyl C--O (d, J = 6.7 Hz) 171.47
P--C.dbd.O (d, J = 234.4 Hz) 198.54 S--C.dbd.O IVe -5.76 N/A N/A
(D.sub.2O) IVf -7.00 N/A N/A (DMSO-d.sub.6) IVg -6.60 70.84 CH2--OH
321 (DMSO-D6) 72.17 CH--OH 121.68, 121.79, 121.85, 125.71, 127.10,
127.92, 129.36, 129.50, 129.59 Aromatic CH 134.51 Aniline C--N
142.34 Aromatic C--CH 150.37 Phenyl C--O (d, J = 6.2 Hz) 171.59
P--C.dbd.O (d, J = 232.6 Hz)
[0033] In another aspect, the invention includes novel compounds
useful for inhibiting amyloidosis, and/or compounds having
antiviral activity. The compounds of the invention can be
represented by the structures of Formula IV, e.g., a compound of
Formula IV in which G is hydrogen (e.g., the phenyl ring is
unsubstituted) and L is any of the moieties of Formulas IVa-IVg. A
more preferred compound is the compound of Formula IVc.
[0034] In another aspect, the invention provides a method for
preparing esters of phosphonates, e.g., phosphono-carboxylate
compounds of the invention, e.g., a compound of Formula IV in which
G is hydrogen and L is a moiety of Formula IVa-IVg. Illustratively,
the method includes the step of reacting a phosphonodichloridate
(or other phosphonate diacid halide) with a disilylated diol under
conditions such that a compound of Forumla IV is formed (see
Example 2, infra).
[0035] Thus, in one embodiment, the invention provides a method for
preparing a compound represented by the Formula (Formula V): 10
[0036] in which R is alkyl or aryl, and R' is hydrogen, alkyl, or
aryl (including heteroaromatic groups such as nucleosides). The
method includes the step of reacting an ester of a
carbonylphosphono diacid halide (e.g., ROOC--P(O)(A)(A'), in which
R is as described in Formula V, and A and A' are both halogen or
other good leaving groups, e.g., chloro, iodo, bromo,
pentafluorophenyl, and the like, which can be the same or
different) with a disilylether of a vicinal diol, under conditions
such that the compound of Formula V is prepared.
[0037] An anionic group (i.e., a phosphonate or carboxylate group)
of a therapeutic compound of the invention is a negatively charged
moiety that, in certain preferred embodiments, can modulate
interaction between an amyloidogenic protein and a glycoprotein or
proteoglycan constituent of a basement membrane to thus modulate
amyloid deposition.
[0038] It will be noted that the structure of some of the compounds
of this invention includes asymmetric carbon atoms. It is to be
understood accordingly that the isomers (e.g., enantiomers and
diastereomers) arising from such asymmetry are included within the
scope of this invention. Such isomers can be obtained in
substantially pure form by classical separation techniques and by
sterically controlled synthesis. For the purposes of this
application, unless expressly noted to the contrary, a compound
shall be construed to include both the R or S stereoisomers at each
chiral center.
[0039] The ability of a therapeutic compound of the invention to
modulate 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 modulates 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).
[0040] 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 modulates
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.
[0041] In certain embodiments, a therapeutic compound of the
invention comprises a cation (i.e., in certain embodiments, at
least one of R.sup.1, R.sup.2 or R.sup.3 is a cation). If the
cationic group is hydrogen, H.sup.+, then the compound is
considered an acid, e.g., phosphonoformic acid. If hydrogen is
replaced by a metal ion 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, at
least one of R.sup.1, R.sup.2 or R.sup.3 can be a pharmaceutically
acceptable alkali metal (e.g., Li, Na, or K), ammonium cation,
alkaline earth cation (e.g., Ca.sup.2+, Ba.sup.2+, Mg.sup.2+),
higher valency cation, or polycationic counter ion (e.g., a
polyammonium cation). (See, e.g., Berge et al. (1977)
"Pharmaceutical Salts", J. Pharm. Sci. 66:1-19). It will be
appreciated that the stoichiometry of an anionic compound to a
salt-forming counterion (if any) will vary depending on the charge
of the anionic portion of the compound (if any) and the charge of
the counterion. Preferred pharmaceutically acceptable salts include
a sodium, potassium or calcium salt, but other salts are also
contemplated within their pharmaceutically acceptable range.
[0042] The term "pharmaceutically acceptable esters" refers to the
relatively non-toxic, esterified products of the compounds of the
present invention. These esters can be prepared in situ during the
final isolation and purification of the compounds or by separately
reacting the purified compound in its free acid form or hydroxyl
with a suitable esterifying agent; either of which are methods
known to those skilled in the art. Carboxylic acids and phosphonic
acids can be converted into esters according to methods well known
to one of ordinary skill in the art, e.g., via treatment with an
alcohol in the presence of a catalyst. A preferred ester group
(e.g., when R.sup.3 is lower alkyl) is an ethyl ester group.
[0043] The term "alkyl" refers to the saturated aliphatic groups,
including straight-chain alkyl groups, branched-chain alkyl groups,
cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups,
and cycloalkyl substituted alkyl groups. In preferred embodiments,
a straight chain or branched chain alkyl has 30 or fewer carbon
atoms in its backbone (e.g., C.sub.1-C.sub.30 for straight chain,
C.sub.3-C.sub.30 for branched chain), and more preferably 20 or
fewer. Likewise, preferred cycloalkyls have from 4-10 carbon atoms
in their ring structure, and more preferably have 4-7 carbon atoms
in the ring structure. The term "lower alkyl" refers to alkyl
groups having from 1 to 6 carbons in the chain, and to cycloalkyls
having from 3 to 6 carbons in the ring structure.
[0044] Moreover, the term "alkyl" (including "lower alkyl") as used
throughout the specification and claims is intended to include both
"unsubstituted alkyls" and "substituted alkyls", the latter of
which refers to alkyl moieties having substituents replacing a
hydrogen on one or more carbons of the hydrocarbon backbone. Such
substituents can include, for example, halogen, hydroxyl,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,
phosphinato, cyano, amino (including alkyl amino, dialkylamino,
arylamino, diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfate, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl,
cyano, azido, heterocyclyl, aralkyl, or an aromatic or
heteroaromatic moiety. It will be understood by those skilled in
the art that the moieties substituted on the hydrocarbon chain can
themselves be substituted, if appropriate. Cycloalkyls can be
further substituted, e.g., with the substituents described above.
An "aralkyl" moiety is an alkyl substituted with an aryl (e.g.,
phenylmethyl (benzyl)).
[0045] The term "alkoxy", as used herein, refers to a moiety having
the structure --O-alkyl, in which the alkyl moiety is described
above.
[0046] The term "aryl" as used herein includes 5- and 6-membered
single-ring aromatic groups that may include from zero to four
heteroatoms, for example, unsubstituted or substituted benzene,
pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,
pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the
like. Aryl groups also include polycyclic fused aromatic groups
such as naphthyl, quinolyl, indolyl, and the like. The aromatic
ring can be substituted at one or more ring positions with such
substituents, e.g., as described above for alkyl groups. Preferred
aryl groups include unsubstituted and substituted phenyl
groups.
[0047] The term "aryloxy", as used herein, refers to a group having
the structure --O-aryl, in which the aryl moiety is as defined
above.
[0048] The term "amino," as used herein, refers to an unsubstituted
or substituted moiety of the formula --NR.sub.aR.sub.b, in which
R.sub.a and R.sub.b are each independently hydrogen, alkyl, aryl,
or heterocyclyl, or R.sub.a and R.sub.b, taken together with the
nitrogen atom to which they are attached, form a cyclic moiety
having from 3 to 8 atoms in the ring. Thus, the term "amino" is
intended to include cyclic amino moieties such as piperidinyl or
pyrrolidinyl groups, unless otherwise stated. An "amino-substituted
amino group" refers to an amino group in which at least one of
R.sub.a and R.sub.b, is further substituted with an amino
group.
[0049] In a preferred embodiment of the compounds of Formulas
I-III, R.sup.1 or R.sup.2 can be (for at least one occurrence) a
long-chain aliphatic moiety. The term "long-chain aliphatic moiety"
as used herein, refers to a moiety having a straight or branched
chain aliphatic moiety (e.g., an alkyl or alkenyl moiety) having
from 10 to 24 carbons in the aliphatic chain, e.g., the long-chain
aliphatic moiety is an aliphatic chain of a fatty acid (preferably
a naturally-occurring fatty acid). Representative long-chain
aliphatic moieties include the aliphatic chains of stearic acid,
oleic acid, linolenic acid, and the like.
[0050] 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, excipients, 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.
[0051] In certain embodiments, the therapeutic compound of the
invention can be represented by the formula: 11
[0052] in which R.sup.1 and R.sup.2 are each independently
hydrogen, an aliphatic group (preferably a branched or
straight-chain aliphatic moiety having from 1 to 24 carbon atoms,
more preferably 10-24 carbon atoms, in the chain; or an
unsubstituted or substituted cyclic aliphatic moiety having from
4-to 7 carbon atoms in the aliphatic ring), an aryl group, a
heterocyclic group, or a salt-forming cation; R.sup.3 is hydrogen,
lower alkyl, aryl, or a salt-forming cation; Y.sup.1 and Y.sup.2
are each independently hydrogen, halogen (e.g., F, Cl, Br, or I),
lower alkyl, hydroxy, alkoxy, or aryloxy; and n is an integer from
0 to 12; such that amyloid deposition is modulated. In one
preferred embodiment, therapeutic compounds of the invention
prevent or inhibit amyloid deposition in a subject to which the
therapeutic compound is administered. Preferred therapeutic
compounds for use in the invention include compounds in which both
R.sup.1 and R.sup.2 are pharmaceutically acceptable salt-forming
cations. In a particularly preferred embodiment, R.sup.1, R.sup.2
and R.sup.3 are each independently a sodium, potassium or calcium
cation, and n is 0. In certain preferred embodiments of the
therapeutic compounds, Y.sup.1 and Y.sup.2 are each hydrogen.
Particularly preferred therapeutic compounds are salts of
phosphonoformate. Trisodium phosphonoformate (foscarnet sodium or
Foscavir.RTM.) is commercially available (e.g., from Astra), and
its clinical pharmacology has been investigated (see, e.g.,
"Physician's Desk Reference", 51st Ed., pp. 541-545 (1997)).
[0053] 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 modulate amyloidosis in a pharmaceutically acceptable
vehicle.
[0054] 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 phosphonate or
carboxylate, can be esterified, e.g., with an ethyl group or a
fatty group, to yield a phosphonic or carboxylic ester. When the
phosphonic or carboxylic ester is administered to a subject, the
ester can be cleaved, enzymatically or non-enzymatically, to reveal
the anionic group. Such an ester can be cyclic, e.g., a cyclic
phosphonate, or two or more anionic moieties may be esterified
through a linking group. In a preferred embodiment, the prodrug is
a phosphonate or carboxylate. 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. Furthermore, an anionic moiety (e.g., a
phosphonate or carboxylate) 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. In certain embodiments, as described
above, compounds of the invention can have more than one phosphonic
or carboxylic ester moiety, e.g., one phosphonic ester and one
carboxylic ester, or a phosphonic diester. In such embodiments, the
parent compound may include an anioic group and may be active;
however, cleavage of any or all ester functionalities may result in
an active compound. It will be appreciated that in a compound
having multiple esterified moieties, the ester groups can be
selected to permit selective cleavage of one or more ester
functionalities, to unveil one or more anionic groups. The relative
ease of cleavage of ester groups is well known; for example, a
tert-butyloxy ester is generally cleaved more slowly than an ethyl
ester under certain conditions. Selection of appropriate moieties
to provide a desired rate or order of ester cleavage willl be
routine to the ordinarily-skilled artisan. Thus, the number of
anionic functionalities can be controlled to provide for a
seelctive activity of a compound of the invention according to the
rate or order of ester cleavage.
[0055] Carrier or substituent moieties 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. Other carrier moieties include compounds,
such as amino acids or thyroxine, which can be passively or
actively transported in vivo. Such a carrier moiety can be
metabolically removed in vivo, or can remain intact as part of an
active compound. Structural mimics of amino acids (and other
actively transported moieties), including peptidomimetics, are also
useful in the invention. As used herein, the term "peptidomimetic"
is intended to include peptide analogs which serve as appropriate
substitutes for peptides in interactions with e.g., receptors and
enzymes. The peptidomimetic must possess not only affinity, but
also efficacy and substrate function. That is, a peptidomimetic
exhibits function(s) of a peptide, without restriction of
structure. Peptidomimetics, methods for their preparation and use
are described in Morgan et al., "Approaches to the discovery of
non-peptide ligands for peptide receptors and peptidases," In
Annual Reports in Medicinal Chemistry (Virick, F. J., ed.) pp.
243-253, Academic Press, San Diego, Calif. (1989), the contents of
which are incorporated herein by reference. 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.
[0056] The targeting and prodrug strategies described above can be
combined to produce a compound that can be transported as a prodrug
to a desired site of action and then unmasked to reveal an active
compound.
[0057] In the methods of the invention, amyloid deposition (e.g.,
deposition of .beta.-amyloid) in a subject is modulated 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 modulate 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 modulate 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., phosphonoformic acid, trisodium salt) is between 0.5 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 modulate amyloid deposition) are between 5 and
500 mM, more preferably between 10 and 100 mM, and still more
preferably between 20 and 50 mM.
[0058] The therapeutic compounds of the invention can be 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 as
subcutaneous, intravenous, intramuscular or intraperitoneal
administration, and the like (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.
[0059] 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. Nos. 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. 357:140; M. Owais
et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant
protein A receptor (Briscoe et al (1995) Am. J. Physiol. 1233: 34);
gp120 (Schreier et al (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.
[0060] Delivery and in vivo distribution can also be affected by
alteration of an anionic group of compounds of the invention. For
example, anionic groups such as phosphonate or carboxylate can be
esterified to provide compounds with desirable pharmocokinetic,
pharmacodynamic, biodistributive, or other properties. Exemplary
compounds include phosphonoformate trisodium salt (Foscarnet,
Foscavir), phosphonoacetate, trisodium salt, and pharmaceutically
acceptable salts or esters thereof.
[0061] 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).
[0062] The therapeutic compound may also be administered
parenterally (e.g., intramuscularly, intravenously,
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.
[0063] 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 some 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.
[0064] 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 filter 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.
[0065] 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.
[0066] 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.
[0067] Therapeutic compositions can be administered in time-release
or depot form, to obtain sustained release of the therapeutic
compounds over time. The therapeutic compounds of the invention can
also be administered transdermally (e.g., by providing the
therapeutic compound, with a suitable carrier, in patch form).
[0068] Active compounds are administered at a therapeutically
effective dosage sufficient to modulate amyloid deposition (or
amyloid load) in a subject. A "therapeutically effective dosage"
preferably modulates 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 modulate
amyloid deposition can be evaluated in model systems that may be
predictive of efficacy in modulating amyloid deposition in human
diseases, such as animal model systems known in the art (including,
e.g., the method described in PCT Publication WO 96/28187) or by in
vitro methods, e.g., the method of Chakrabartty, described in PCT
Publication WO 97/07402, or the assay described in Example 1,
infra. Alternatively, the ability of a compound to modulate amyloid
deposition can be evaluated by examining the ability of the
compound to modulate an interaction between an amyloidogenic
protein and a basement membrane constituent, e.g., using a binding
assay such as that described hereinabove. Furthermore, the amount
or distribution of amyloid deposits in a subject can be
non-invasively monitored in vivo, for example, by use of
radiolabelled tracers which can associate with amyloid deposits,
followed by scintigraphy to image the amyloid deposits (see, e.g.,
Aprile, C. et al., Eur. J. Nucl. Med. 22:1393 (1995); Hawkins, P.
N., Baillieres Clin. Rheumatol. 8:635 (1994); and references cited
therein). Thus, for example, the amyloid load of a subject can be
evaluated after a period of treatment according to the methods of
the invention and compared to the amyloid load of the subject prior
to beginning therapy with a therapeutic compound of the invention,
to determine the effect of the therapeutic compound on amyloid
deposition in the subject.
[0069] It will be appreciated that the ability of a compound of the
invention to modulate amyloid deposition or amyloid load can, in
certain embodiments, be evaluated by observation of one or more
symptoms or signs associated with amyloid deposition or amyloid
load in vivo. Thus, for example, the ability of a compound to
decrease amyloid deposition or amyloid load may be associated with
an observable improvement in a clinical manifestation of the
underlying amyloid-related disease state or condition, or a slowing
or delay in progression of symptoms of the condition. Thus,
monitoring of clinical manifestations of disease can be useful in
evaluating the amyloid-modulating efficacy of a compound of the
invention.
[0070] 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 modulated, 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], cerebral angiopathy); 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-Scheinker syndrome,
bovine spongiform encephalitis).
[0071] Compounds for use in the methods of the invention are
commercially available and/or can be synthesized by standard
techniques known in the art. In general, phosphonic esters can be
prepared from the corresponding phosphonic acid by standard
methods. Similarly, carboxylic esters can be prepared from the free
carboxylic acid by standard techniques (for a reference to
esterification techniques, see, e.g., R. Larock, "Comprehensive
Organic Transformations," VCH Publishers (1989)). Carboxylic esters
can be converted to thionoesters by known reactions, e.g., by
treatment with Lawesson's reagent (2,4-bis(4-methoxyphenyl)-1,3--
dithia-2,4-diphosphetane-2,4-disulfide, which is commercially
available, e.g., from Aldrich Chemical Co., Milwaukee, Wis.).
Compounds of the present invention also can be prepared as
described below. The following Examples further illustrate the
present invention and are not intended to be further limiting in
anyway.
EXAMPLE 1
[0072] It is known that amyloidogenic peptides or proteins which
have formed amyloid deposits or plaques have a significant amount
of .beta.-sheet secondary structure, while the unaggregated peptide
or protein generally has less .beta.-sheet structure. It is
believed that the ability of a candidate therapeutic compound to
prevent the formation of .beta.-sheet secondary structure in vitro
may be correlated with the ability of the compound to inhibit
amyloidogenesis in vivo. Accordingly, phosphonate compounds were
assayed for ability to prevent the formation of .beta.-sheet
secondary structure in assay systems including an in vitro circular
-dichroism (CD) assay.
[0073] A.beta. is a 40 amino acid protein associated with
Alzheimer's disease. A.beta. peptide was prepared and purified as
described in Fraser, P. E. et al., Biochemistry 31, 10716 (1992).
Briefly, the peptide was synthesized by standard solid-phase
techniques and purified by HPLC according to well known
procedures.
[0074] All CD experiments were performed on a commercially
available instrument. The cell was maintained at 25.degree. C.
using a circulating water bath. Computer-averaging of traces was
performed to improve signal-to-noise ratios. The solvent signal was
subtracted. CD experiments were performed for each test compound
according to the following procedure:
[0075] A stock solution of purified peptide was made by dissolving
the peptide in phosphate-buffered saline (PBS) to a concentration
of 2 mg/ml. A test solution was made for each potential therapeutic
agent (test compound) as shown below:
2 A.beta. stock solution 25 .mu.l Test compound (20 mg/ml) 2.5
.mu.l Distilled water 2.5 .mu.l 10 mM Tris-HCl buffer, pH 7 370
.mu.l
[0076] The control sample had no test compound, and a total of 5
.mu.l distilled water was added. The test solution was incubated
for either 0 or 24 hours at 37.degree. C. before CD measurement.
The size minimum in the CD spectrum at 218 nm is believed to be
diagnostic of the presence of .beta.-pleated sheet. Comparison of
the minimum at 218 nm of a candidate compound, compared to the
minimum of a control sample, is believed to be indicative of the
ability of the candidate compound to inhibit the formation of
.beta.-pleated sheet.
[0077] Using this assay, several candidate compounds were tested.
Phosphonoformate sodium salt (foscarnet sodium) was found to
significantly and reproducibly reduce the amount of .beta.-sheet
formation, as measured by the CD assay. Phosphonoacetate was also
found to be active in this assay. Thus, phosphonoformate and
phosphonoactetate are believed to be inhibitors of amyloid
deposition. 2-carboxyethylphosphonic acid had a lower ability to
prevent .beta.-pleated sheet formation in this model system.
[0078] In a preliminary result in a different assay system (in
which the candidate compound and amyloid peptide were incubated
together overnight, followed by centrifugation and determination of
the amount of soluble peptide), phosphonoformate trisodium salt was
found to have little effect on amyloid peptide solubility; it is
believed that the buffer composition may have interfered with the
ability of the compound to inhibit amyloid deposition.
[0079] The neurotoxicity of phosphonoformate trisodium salt was
investigated in cortical/hippocampal neuronal cultures; no
significant toxicity was noted at concentrations ranging from
10.sup.-7M to 10.sup.-4 M.
EXAMPLE 2
[0080] The procedure described below is further described in Gorin
et al., Tet. Lett. 1997, 38:2791-2794, incorporated herein by
reference. The procedure has the advantage that the reactivity of
the nucleophile (e.g., the hydroxyl groups of a diol which react
with the phosphonic acid chloride) is attenuated by use of a silyl
ether (e.g., a trimethylsilyl ether), which can improve
selectivity.
[0081] To a solution of (phenoxycarbonyl)phosphoonodichloridate (5
mmol) in 10 ml dry THF cooled in an ice water bath under argon was
added a vicinal bis-trimethylsilyl ether (5 mmol) (prepared from
the vic-diol, e.g., by treatment with trimethylsilylchloride
(TMSCl) or trimethylsilyltriflate (TMSOTf), available from Aldrich
Chemical Co., Milwaukee, Wis.) in 10 mL dry THF. After addition was
complete, the reaction mixture was stirred for one hour at room
temperature, and the solvent was evaporated under reduced pressure.
The residue was taken up in dioxane containing 90 mg water (5
mmol), neutralized by adding 5 mmol aniline in 10 mL diozane, and
the product precipitated by pouring into 200 mL 1:1 diethyl
ether:hexanes. The solid product was filtered and washed with 1:1
diethyl ether:hexanes.
[0082] Compounds IVa-IVg were prepared by the above procedure using
the corresponding diols, which are commercially available and/or
can be readily prepared by one of ordinary skill in the art using
no more than routine experimentation.
EXAMPLE 3
[0083] The compounds of Formula IVa, IVc and IVd (in salt forms,
e.g., methylpyridinium salts and/or anilinium salts) were tested in
at least one assay for their ability to inhibit amyloidosis. It was
found that these compounds showed activity in at least one assay
system indicative of their ability to be an inhibitor of
amyloidosis in vivo in both free or salt forms.
[0084] Equivalents
[0085] The contents of all references, issued patents, and
published patent applications cited throughout this application are
hereby incorporated by reference.
[0086] 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.
* * * * *