Note: Descriptions are shown in the official language in which they were submitted.
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WO 99/08685 PCT/IB98/00967
PHOSPHONO-CARBOXYLATE COMPOUNDS FOR TREATING
AMYLOIDOSIS
Background of Invention
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.
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
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
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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.
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):
x
11
~P-(CYlY2~C(X)XR3
RIX I
z
in which Z is XR2 or R4, Rl and R2 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; R3 is hydrogen,
lower alkyl,
aryl, or a salt-forming cation; R4 is hydrogen, lower alkyl, aryl or amino
(including
alkylamino, dialkylamino (including cyclic amino moieties), arylamino,
diarylamino,
and alkylarylamino); X is, independently for each occurrence, 0 or S; Y1 and
Y2 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.
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 R1 and R2 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
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embodiment, RI, R2 and R3 are each independently a sodium, potassium or
calcium
cation. In certain embodiments in which at least one of R1 and R2 is an
aliphatic group,
the aliphatic group has between I 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 RI and R2 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 1 and Y2 are each hydrogen.
In certain preferred embodiments, the therapeutic compound of the invention
can
be represented by the formula (Formula II):
x
P-(CY1Y2)nC(O)OR3
R1X' I
XR2
in which Rl, R2, R3, YI, Y2, 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):
x
11
P-(CY1Y2)nCH(NRaRb)C(O)OR3
R10~ 1
OR2
in which Rl, R2, R3, Y1, Y2, and X are as defmed above, Ra and Rb are each
independently hydrogen, alkyl, aryl, or heterocyclyl, or Ra and Rb, 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, Ra
and Rb are each hydrogen. In certain preferred embodiments, a compound of the
invention comprises an a-amino acid (or a-amino acid ester), more preferably a
L-a-
amino acid or ester.
In another embodiment, the compounds of the invention can be represented by
the formula (Formula IV):
- II II
O OO
O-C-P-O-L
G
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
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4
embodiments, preferably a lower alkyl), more preferably a hydroxy-substituted
alkyl or an alkyl
substituted with a nucleoside base.
In another embodiment, the compounds of the invention can be represented by
the
formula:
X Y1
I 1 I 3
' R x-~IP CXXR ()
2
z Y
n
in which Z is R4 or XR2; R1 and R2 are each independently hydrogen, an
aliphatic group, an
aryl group, a heterocyclic group, or a salt-forming cation; R3 is hydrogen,
lower alkyl, aryl, or a
salt-forming cation; R4 is hydrogen, lower alkyl, aryl or -NRaRb; X is,
independently for each
occurrence, 0 or S; Y1 and Y2 are each independently hydrogen, halogen, alkyl,
NRaRb,
hydroxy, alkoxy, or aryloxy; n is an integer from 0 to 12; wherein in each
occurrence, Ra and Rb
are each independently hydrogen, alkyl, aryl, or heteroaryl, or Ra and Rb
taken together with the
nitrogen atom to which they are attached form a cyclic moiety having from 3 to
8 atoms in the
cycle; and wherein when any of RI, R2, R3, R4, Y1, and Y2 is an aliphatic, an
alkyl or an aryl
group, then the aliphatic, alkyl or aryl group is optionally substituted with
one or more
substituents selected from halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkoxycarbonyl,
aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,
phosphinato, cyano,-NH2,
alkyl amino, dialkylamino, arylamino, diarylamino, alkylarylamino, acylamino,
amidino, imino,
sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, sulfonato,
sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, aralkyl, and an aromatic or
heteroaromatic moiety;
wherein said therapeutic compound is other than 2-amino-7-phosphono heptanoic
acid and other
than 2-amino-5-phosphonovaleric acid.
In yet another embodiment, the compounds of the invention can be represented
by the
formula:
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4a
X Y,
llCH(NRaRb)C(O)OR3
R~ O~-~ I I 2
OR Y
n
in which R1 and R2 are each independently hydrogen, an aliphatic group, an
aryl group, a
heterocyclic group, or a salt-forming cation; R3 is hydrogen, lower alkyl,
aryl, or a salt-forming
cation; X is, independently for each occurrence, 0 or S; Y1 and Y2 are each
independently
hydrogen, halogen, alkyl, NRaRb, hydroxy, alkoxy, or aryloxy; n is an integer
from 0 to 12; and
wherein in each occurrence Ra and Rb are each independently hydrogen, alkyl,
aryl, or
heterocyclyl, or Ra and Rb, taken together with the nitrogen atom to which
they are attached,
form a cyclic moiety having from 3 to 8 atoms in the cycle; wherein when any
of R1, R2, R3,
Y1, and Y2 is an aliphatic, an alkyl or an aryl group, then the aliphatic,
alkyl or aryl group is
optionally substituted with one or more substituents selected from halogen,
hydroxyl,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate,
alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl,
phosphate,
phosphonato, phosphinato, cyano, -NH2, alkyl amino, dialkylamino, arylamino,
diarylamino,
alkylarylamino, acylamino, amidino, imino, sulfhydryl, alkylthio, arylthio,
thiocarboxylate,
sulfate, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano,
azido, heterocyclyl,
aralkyl, and an aromatic or heteroaromatic moiety; wherein said therapeutic
compound is other
than 2-amino-7-phosphono heptanoic acid and other than 2-amino-5-
phosphonovaleric acid.
In a further embodiment, the compounds of the invention can be represented by
the
formula:
O Y1
I I I C O OR3
R1~~ I I ( )
OR Y2
n
in which R1 and R2 are each independently hydrogen, an aliphatic group, an
aryl group, a
heterocyclyl group, or a salt-forming cation; R3 is hydrogen, lower alkyl,
aryl or a salt-forming
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4b
cation; Y 1 and Y2 are each independently hydrogen, halogen, lower alkyl,
NRaRb, hydroxy,
alkoxy, or aryloxy, wherein Ra and Rb are each independently hydrogen, alkyl,
aryl, or
heteroaryl, or Ra and Rb taken together with the nitrogen atom to which they
are attached for a
cyclic moiety having from 3 to 8 atoms in the cycle; and n is an integer from
0 to 12; wherein
said therapeutic compound is other than 2-amino-7-phosphono heptanoic acid and
other than 2-
amino-5-phosphonovaleric acid.
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, transdennal, 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.
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.
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.
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.
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.
The invention also provides pharmaceutical compositions for modulating amyloid
deposition in a subject, the pharmaceutical compositions including a
therapeutic compound of
the invention in an amount effective to modulate amyloid deposition and a
pharmaceutically
acceptable vehicle. The invention further provides pharmaceutical compositions
for treating a
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4c
disease state associated with amyloidosis, the pharmaceutical compositions
including a
therapeutic compound of the invention in an amount effective to treat a
disease state associated
with amyloidosis and a pharmaceutically acceptable vehicle. In a further
embodiment, the
invention provides pharmaceutical compositions 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 pharmaceutical
compositions including a
therapeutic compound of the invention in an amount effective to modulate
amyloid deposition
and a pharmaceutically acceptable vehicle.
The invention further provides the use of a therapeutic compound of the
invention in the
preparation of a medicament for modulating amyloid deposition in a subject.
The invention also
provides the use of a therapeutic compound of the invention in the preparation
of a medicament
for treating a disease state associated with amyloidosis. Also provided is the
use of a therapeutic
compound of the invention in the preparation of a medicament 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
medicaments include
an effective amount of a therapeutic compound of the invention.
The invention provides the use of an effective amount of a therapeutic
compound of the
invention for modulating amyloid deposition in a subject. There is further
provided the use of an
effective amount of a therapeutic compound of the invention 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 invention
further
provides the use of a therapeutic compound of the invention for reducing
amyloid load in a
human subject. There is also provided the use of a therapeutic compound of the
invention for
treating or preventing a neurodegenerative disorder in a human subject. There
is further
provided the use of a therapeutic compound of the invention in the preparation
of a medicament
for treating or preventing a neurodegenerative disorder in a human subject.
Detailed Description of Invention
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
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4d
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.
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"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.
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):
x
11
~P-(CYI~~C~~3
RIX
I
Z
in which Z is XR2 or R4, R1 and R2 are each independently hydrogen, a
substituted or
unsubstituted aliphatic group (preferably a branched or straight-chain
aliphatic moiety
having from I to 24 carbon atoms in the chain; or an unsubstituted or
substituted cyclic
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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; R3 is hydrogen,
lower alkyl,
aryl, or a salt-forming cation; X is, independently for each occurrence, 0 or
S; R4 is
hydrogen, lower alkyl, aryl or amino; Y1 and Y2 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.
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 Ri and R2 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, R1, R2 and R3 are each independently a sodium, potassium or
calcium
cation. In certain embodiments in which at least one of Rt and R2 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 Rt and R2 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 1 and Y2 are each hydrogen.
In certain preferred embodiments, the therapeutic compound of the invention
can
be represented by the formula (Formula II):
x
~ P-(CYlY2)nC(O)OR3
RIX {
XR2
in which R1, R2, R3, Y1, Y2, 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):
X
~ P-(C Y IY2)nCH(NRaRb)C(O)OR3
RIO OR2
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in which R1, R2, R3, Y1, Y2, and X are as defined above, Ra and Rb are each
independently hydrogen, alkyl, aryl, or heterocyclyl, or Ra and Rb, 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, Ra
and Rb are each hydrogen. In certain preferred embodiments, a compound of the
invention comprises an a-amino acid (or a-amino acid ester), more preferably a
L-a-
amino acid or ester.
The Z, Q, Rl, R2, R3, YI, Y2 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. Patent Nos.
4,386,081
and 4,591583 to Helgstrand et al., and U.S. Patent Nos. 5,194,654 and
5,463,092 to
Hostetler et al.). Thus, in certain embodiments, at least one of R1 and R2 is
an aliphatic
group (more preferably an aikyl 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 RI and R2 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.
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)(OR1)(OR2), -
P(S)(SRI)(OR2), or -P(S)(SR1)(SR2).
In another embodiment, compounds useful in the methods of the invention can
be represented by the formula (Formula IV):
In another embodiment, the compounds of the invention can be represented by
the formula (Formula IV):
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O O
II II
KI7_-o_c__o- L
O
G
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.
In certain preferred embodiments, L is a moiety selected from the group
consisting of (Formulas IVa-IVg):
}OH }OH TOH
IVa OC(O)CIIH23 SC(O)C11H23 (O)C7H15
NH2 IVb NH2 IVc IVd
N~ N N
I~ OH
ic
N N N N
_.__y ____y I
OH OH
IVe IVf IVg
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Table 1 lists data pertinent to the characterization of these compounds using
art-
recognized techniques.
Table 1
COMPOUND 31 P NMR 1 jC NMR FAB-
MS(_)
IVa -6.33(DMSO-d6) 60.97 CH2OH(d, J=6Hz) 245.2
66.76 CHOH(d, J=7.8Hz)
121.65, 121.78, 121.99, 125.71,
129.48, 129.57, 126.43
Aromatic CH
134.38 Aniline C-N
150.39 Phenyl C-O(d, J=7Hz)
171.57 P-C=O(d, J=234Hz)
IVb -6.41(DMSO-d6) 13.94 CH3 456
22.11, 24.40, 28.56, 28.72, 28.99,
29.00, 31.30, 33.43, -(CH2)10
65.03 CH2-OC(O)
66.60 CH2-OP(d, J=5.6Hz)
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(DMSO-d6) 13.94 CH3 471
22.11, 25.10, 28.68, 28.72,
28.85, 29.00, 30.76, 31.31, 32.10,
-(CH2)10-
43.36 CH2-S
68.43 CH2-OH
68.43 CH-OH(d, J=6.3 Hz)
68.76 P-O-CH2-9d, J=5.8 Hz)
121.75, 122.03, 125.62, 126.37,
129.30, 129.53, Aromatic CH
134.23 Aniline C-N
150.37 Phenyl C-O(d, J=6.7 Hz)
171.47 P-C=O(d, J=234.0 Hz)
198.47 S-C=O
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COMPOUND j 1 P NMR 13C NMR FAB-
MSl-)
IVd -6.61(DMSO-d6) 13.94 CH3 416
22.06, 25.14, 28.24, 28.35,
31.09, 32.14
-CH2)6-
43.40 CH2-S
68.50 P-O-CH2-(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=O(d, J=234.4 Hz)
198.54 S-C=O
IVe -5.76(D20) N/A N/A
IVf -7.00(DMSO-d6) N/A N/A
IVg -6.60(DMSO-D6) 70.84 CH2-OH 321
72.17 CH-OH
121.68, 121.79, 121.85, 125.71
127.10,
127.92, 129.36, 129.50, 129.59
Aromatic CH
134.51 Aniline C-N
142.34 Aromatic C-CH
150.37 Phenyl C-O(d, J=6.2 Hz)
171.59 P-C=O(d, J=232.6 Hz)
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.
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
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other phosphonate diacid halide) with a disilylated diol under conditions such
that a
compound of Forumla IV is formed (see Example 2, infra).
Thus, in one embodiment, the invention provides a method for preparing a
compound represented by the Formula (Formula V):
O O OH
RO-C-P-O~
0 5
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, whicb can be the same or
different) with a
disilylether of a vicinal diol, under conditions such that the compound of
Formula V is
prepared.
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.
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.
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. Patent 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 P-amyloid precursor protein ((3-
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
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constituent (e.g., HSPG) to the amyloidogenic protein (e.g., (3-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).
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.
In certain embodiments, a therapeutic compound of the invention comprises a
cation (i.e., in certain embodiments, at least one of Rl, R2 or R3 is a
cation). If the
cationic group is hydrogen, H+, 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
RI, R2 or
R3 can be a pharmaceutically acceptable alkali metal (e.g., Li, Na, or K),
ammonium
cation, alkaline earth cation (e.g., Ca2+, Ba2+, Mg2+), 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.
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
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alcohol in the presence of a catalyst. A preferred ester group (e.g., when R3
is lower
alkyl) is an ethyl ester group.
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., C1-C30 for straight chain, C3-C30 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.
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)).
The term "alkoxy", as used herein, refers to a moiety having the structure -0-
alkyl, in which the alkyl moiety is described above.
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.
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The term "aryloxy", as used herein, refers to a group having the structure -0-
aryl,
in which the aryl moiety is as defined above.
The term "amino," as used herein, refers to an unsubstituted or substituted
moiety of the formula -NRaRb, in which Ra and Rb are each independently
hydrogen,
alkyl, aryl, or heterocyclyl, or Ra and Rb, 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 Ra and Rb, is further
substituted with
an amino group.
In a preferred embodiment of the compounds of Formulas I-III, Rl or R2 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.
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 NaC1). 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.
In certain embodiments, the therapeutic compound of the invention can be
represented by the formula:
O
11
P-(CY'Y2)nCOOR3
R1O/OR2
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in which RI and R2 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; R3 is hydrogen, lower alkyl,
aryl, or a salt-
forming cation; Y1 and Y2 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 RI and R2 are pharmaceutically
acceptable
salt-forming cations. In a particularly preferred embodiment, R1,R2 and R3 are
each
independently a sodium, potassium or calcium cation, and n is 0. In certain
preferred
embodiments of the therapeutic compounds, Y 1 and YZ are each hydrogen.
Particularly
preferred therapeutic compounds are salts of phosphonoformate. Trisodium
phosphonoformate (foscarnet sodium or Foscavir ) is commercially available
(e.g.,
from Astra), and its clinical pharmacology has been investigated (see, e.g.,
"Physician's
Desk Reference", 51 st Ed., pp. 541-545 (1997)).
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.
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
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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.
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. Patents 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-
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253, Academic Press, San Diego, CA (1989). Many targeting moieties are known,
and
include, for example, asialoglycoproteins (see, e.g. Wu, U.S. Patent
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.
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.
In the methods of the invention, amyloid deposition (e.g., deposition of (3-
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.
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.
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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. Patents 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. Patent 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) FEBSLett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother.
39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol.
1233:134);
gp120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen;
M.L.
Laukkanen (1994) FEBSLett. 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.
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 (Foscamet, Foscavir), phosphonoacetate,
trisodium
salt, and pharmaceutically acceptable salts or esters thereof.
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).
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.
Pharmaceutical compositions suitable for injectable use include sterile
aqueous
solutions (where water soluble) or dispersions and sterile powders for the
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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.
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.
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.
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
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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.
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).
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.
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
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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.
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): (3-
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 x L-chain or amyloid X L-chain
(idiopathic [primary], myeloma or macroglobulinemia-associated); A(32M
(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).
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, WI).
Compounds
of the present invention also can be prepared as described below. The
following
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Examples further illustrate the present invention and are not intended to be
further
limiting in anyway.
Example 1
It is known that amyloidogenic peptides or proteins which have formed amyloid
deposits or plaques have a significant amount of (3-sheet secondary structure,
while the
unaggregated peptide or protein generally has less P-sheet structure. It is
believed that
the ability of a candidate therapeutic compound to prevent the formation of P-
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 (3-sheet secondary structure in assay
systems
including an in vitro circular dichroism (CD) assay.
Al3 is a 40 amino acid protein associated with Alzheimer's disease. A13
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.
All CD experiments were performed on a commercially available instrument.
The cell was maintained at 25 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:
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:
A13 stock solution 25 l
Test compound (20 mg/mi) 2.5 l
Distilled water 2.5 l
10 mM Tris-HC1 buffer, pH 7 370 l
The control sample had no test compound, and a total of 5 l distilled water
was added.
The test solution was incubated for either 0 or 24 hours at 37 C before CD
measurement. The size minimum in the CD spectrum at 218 nm is believed to be
diagnostic of the presence of 0-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 (3-pleated
sheet.
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Using this assay, several candidate compounds were tested. Phosphonoformate
sodium salt (foscarnet sodium) was found to significantly and reproducibly
reduce the amount
of (3-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 (3-pleated sheet formation in this model system.
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.
The neurotoxicity of phosphonoformate trisodium salt was investigated in
cortical/hippocampal neuronal cultures; no significant toxicity was noted at
concentrations
ranging from 10-' to 10-4 M.
Example 2
The procedure described below is further described in Gorin et al., Tel. Lett.
1997,
38:2791-2794. 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.
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, WI) 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.
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.
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Example 3
The compounds of Formula IVa, lVc 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.
Eguivalents
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.
