U.S. patent application number 10/421126 was filed with the patent office on 2003-11-27 for compounds and methods for diagnosing and treating amyloid-related conditions.
This patent application is currently assigned to Pharmacia & Upjohn Company. Invention is credited to Bandiera, Tiziano, Buhl, Allen Edwin, Carter, Donald Bainbridge, Fici, Gregory J., Lansen, Jacqueline, Pellerano, Cesare, Raub, Thomas J., Savini, Luisa, Sawada, Geri A., Tanis, Steven P..
Application Number | 20030219377 10/421126 |
Document ID | / |
Family ID | 22882072 |
Filed Date | 2003-11-27 |
United States Patent
Application |
20030219377 |
Kind Code |
A1 |
Raub, Thomas J. ; et
al. |
November 27, 2003 |
Compounds and methods for diagnosing and treating amyloid-related
conditions
Abstract
The invention provides methods for diagnosing and treating
amyloid-related conditions and compounds useful for the same. The
invention provides for detecting, imaging, monitoring, diagnosing,
and treating conditions characterized by the binding or aggregation
of amyloid fibrils. More particularly, the invention relates to
using quinolinehydrazone compounds for diagnosing and treating
amyloidotic conditions and also as an antioxidant.
Inventors: |
Raub, Thomas J.; (Kalamazoo,
MI) ; Sawada, Geri A.; (Portage, MI) ; Tanis,
Steven P.; (Kalamazoo, MI) ; Fici, Gregory J.;
(Kalamazoo, MI) ; Buhl, Allen Edwin; (Portage,
MI) ; Carter, Donald Bainbridge; (Kalamazoo, MI)
; Bandiera, Tiziano; (Gambolo-Pavia, IT) ; Lansen,
Jacqueline; (Milan, IT) ; Pellerano, Cesare;
(Siena, IT) ; Savini, Luisa; (Siena, IT) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
6300 SEARS TOWER
233 S. WACKER DRIVE
CHICAGO
IL
60606
US
|
Assignee: |
Pharmacia & Upjohn
Company
|
Family ID: |
22882072 |
Appl. No.: |
10/421126 |
Filed: |
April 23, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10421126 |
Apr 23, 2003 |
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|
09667357 |
Sep 22, 2000 |
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6589504 |
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60234611 |
Sep 22, 2000 |
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Current U.S.
Class: |
424/1.65 ;
534/11; 546/159 |
Current CPC
Class: |
A61K 31/47 20130101;
A61K 2300/00 20130101; A61K 31/00 20130101; A61K 45/06 20130101;
A61P 25/28 20180101; A61K 31/47 20130101; C07D 215/38 20130101;
A61K 31/47 20130101; A61K 51/0455 20130101; A61P 3/10 20180101 |
Class at
Publication: |
424/1.65 ;
534/11; 546/159 |
International
Class: |
A61K 051/00; C07F
005/00; C07D 215/38 |
Claims
What is claimed is:
1. A method for chemically tagging or inhibiting the aggregation of
amyloid fibrils comprising the steps of: (a) providing a compound
of the formula: 4or a pharmaceutically acceptable salt, ester,
solvate, or prodrug thereof, wherein: R.sup.1, R.sup.2, R.sup.3,
R.sup.4, and R.sup.5 are independently selected from the group
consisting of hydrogen, alkyl, cycloalkyl, aryl, triflouromethyl,
trifluoromethylether, halo, and a group of the formula --OR.sup.7,
wherein R.sup.7 is alkyl or aryl; and R.sup.6 is a benzopyridinyl
group optionally substituted with one to three substituents
selected from the group consisting of hydrogen, alkyl, cycloalkyl,
aryl, triflouromethyl, trifluoromethylether, halo, and a group of
the formula --OR.sup.7, wherein R.sup.7 is alkyl or aryl; wherein
said alkyl groups at each occurrence are optionally substituted
with alkoxy, aryl, or halo; said aryl groups at each occurrence are
optionally substituted with alkyl, alkoxy, or halo; and one or more
atoms in the compound of formula (I) optionally is replaced with a
radiolabeled atom; and (b) allowing the compound to associate with
the amyloid fibrils.
2. The method of claim 1 wherein the radiolabeled atom is selected
from the group consisting of .sup.3H, .sup.131I, .sup.125I,
.sup.123I, .sup.76Br, .sup.18F, .sup.19F, .sup.15O, and
.sup.11C.
3. The method of claim 1 wherein R.sup.1, R.sup.2, R.sup.3,
R.sup.4, and R.sup.5 are each independently selected from the group
consisting of hydrogen, methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, t-butyl, n-pentyl, t-pentyl, n-hexyl, methoxy,
ethoxy, isopropoxy, sec-butoxy, t-butoxy, phenyl, benzyl,
trifluoromethyl, trifluoromethylether, and halo.
4. The method of claim 1 wherein the benzopyridinyl group for
R.sup.6 is quinolyl or isoquinolyl.
5. The method of claim 1 wherein the compound of formula (I) in
step (a) is incorporated in a pharmaceutically acceptable
carrier.
6. The method of claim 1 wherein the compound of formula (I) in
step (a) is selected from the group consisting of:
4-methyl-7-methoxy-2-(4-quinoly- lmethylenehydrazino)quinoline;
4-ethyl-7-methoxy-2-(4-quinolylmethylenehyd- razino)quinoline;
4-ethyl-7-ethoxy-2-(4-quinolylmethylenehydrazino)quinoli- ne;
4-methyl-7-ethoxy-2-(4-quinolylmethylenehydrazino)quinoline;
4-ethyl-7-ethoxy-2-(3-quinolylmethylenehydrazino)quinoline;
4-ethyl-7-methoxy-2-(3-quinolylmethylenehydrazino)quinoline; and
4-methyl-7-methoxy-2-(3-quinolylmethylenehydrazino)quinoline.
7. The method of claim 1 wherein the compound of formula (I) is
4-methyl-7-methoxy-2-(4-quinolylmethylene-hydrazino)quinoline.
8. A method for detecting an aggregation of amyloid fibrils
comprising the steps of: (a) providing a compound of the formula:
5or a pharmaceutically acceptable salt, ester, solvate, or prodrug
thereof, wherein: R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5
are independently selected from the group consisting of hydrogen,
alkyl, cycloalkyl, aryl, triflouromethyl, trifluoromethylether,
halo, and a group of the formula --OR.sup.7, wherein R.sup.7 is
alkyl or aryl; and R.sup.6 is a benzopyridinyl group optionally
substituted with one to three substituents selected from the group
consisting of hydrogen, alkyl, cycloalkyl, aryl, triflouromethyl,
trifluoromethylether, halo, and a group of the formula --OR.sup.7,
wherein R.sup.7 is alkyl or aryl; wherein said alkyl groups at each
occurrence are optionally substituted with alkoxy, aryl, or halo;
said aryl groups at each occurrence are optionally substituted with
alkyl, alkoxy, or halo; and at least one atom in the compound is
replaced with a radiolabeled atom; (b) allowing the compound to
associate with the amyloid fibrils to provide a labeled deposit;
and (c) detecting the amount and location-of the labeled
deposit.
9. The method of claim 8 comprising the steps of detecting the
labeled deposit by gamma imaging, magnetic resonance imaging, or
magnetic resonance spectroscopy.
10. The method of claim 8 further comprising the step of (d)
evaluating or assessing the data obtained in step (c) in an
individual and optionally comparing the data with analogous data
obtained from a normal human or mammal to identify, assess, or
diagnose the medical condition of the individual.
11. The method of claim 10 comprising assessing the condition of an
individual undergoing treatment for a condition characterized by
the aggregation of amyloid fibrils.
12. The method of claim 11 wherein the condition is selected from
the group consisting of Alzheimer's disease, Down syndrome, Type 2
diabetes mellitus, hereditary cerebral hemorrhage amyloidosis,
amyloid A, secondary amyloidosis, familial mediterranean fever,
familial amyloid nephropathy with urticaria and deafness, amyloid
lambda L-chain or amyloid kappa L-chain, A beta 2M, ATTR, familial
amyloid cardiomyopathy, isolated cardiac amyloid, AIAPP or amylin
insulinoa, atrial naturetic factor, procalcitonin, gelsolin,
crytatin C, AApo-A-I, AApo-A-II, fibrinogen-associated amyloid; and
Asor or Pr P-27 or in cases of persons who are homozygous for the
apolipoprotein E4 allele, and the condition associated with
homozygosity for the apolipoprotein E4 allele; and the treatment
comprises administering an active agent selected from the group
consisting of doxorubicin, galantamine, tacrine (COGNEX.RTM.),
selegiline, physostigmine, revistigmin, donepizil (ARICEPT.RTM.),
metrifonate, milameline, xanomeline, saeluzole, acetyl-L-carnitine,
idebenone, ENA-713, memric, quetiapine, neurestrol and
neuromidal.
13. The method of claim 8 wherein the compound of formula (I) is a
biomarker for the aggregation of amyloid fibrils in an
individual.
14. A method for treating a condition in an individual
characterized by aggregation of amyloid fibrils comprising the
steps of: (a) providing a compound of the formula: 6or a
pharmaceutically acceptable salt, ester, solvate, or prodrug
thereof, wherein: R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5
are independently selected from the group consisting of hydrogen,
alkyl, cycloalkyl, aryl, triflouromethyl, trifluoromethylether,
halo, and a group of the formula --OR.sup.7, wherein R.sup.7 is
alkyl or aryl; and R.sup.6 is a benzopyridinyl group optionally
substituted with one to three substituents selected from the group
consisting of hydrogen, alkyl, cycloalkyl,-aryl, triflouromethyl,
trifluoromethylether, halo, and a group of the formula --OR.sup.7,
wherein R.sup.7 is alkyl or aryl; wherein said alkyl groups at each
occurrence are optionally substituted with alkoxy, aryl, or halo;
and said aryl groups at each occurrence are optionally substituted
with alkyl, alkoxy, or halo; (b) allowing the compound to associate
with the amyloid fibril; and (c) optionally repeating steps (a) and
(b), as necessary, to improve or rehabilitate the condition of the
individual.
15. The method of claim 14 wherein the compound of formula (I) is
incorporated in a pharmaceutically acceptable carrier.
16. The method of claim 14 wherein the condition is selected from
the group consisting of Alzheimer's disease, Down syndrome, Type 2
diabetes mellitus, hereditary cerebral hemorrhage amyloidosis,
amyloid A, secondary amyloidosis, familial Mediterranean fever,
familial amyloid nephropathy with urticaria and deafness, amyloid
lambda L-chain or amyloid kappa L-chain, A beta 2M, ATTR, familial
amyloid cardiomyopathy, isolated cardiac amyloid, AIAPP or amylin
insulinoa, atrial naturetic factor, procalcitonin, gelsolin,
crytatin C, AApo-A-I, AApo-A-II, fibrinogen-associated amyloid; and
Asor or Pr P-27 or in cases of persons who are homozygous for the
apolipoprotein E4 allele, and the condition associated with
homozygosity for the apolipoprotein E4 allele.
17. The method of claim 14 wherein the condition is selected from
the group consisting of Dutch hereditary cerebral hemorrhage
amyloidosis amyloid A, Muckle-wells syndrome, idiopathic-associated
amyloid lambda L-chain, myeloma-associated amyloid lambda L-chain,
macroglobulinemia-associated amyloid lambda L-chain,
idiopathic-associated amyloid kappa L-chain, myeloma-associated
amyloid kappa L-chain, macroglobulinemia-associated amyloid kappa
L-chain, Portuguese familial amyloid polyneuropathy, Japanese
familial amyloid polyneuropathy, Swedish familial amyloid
polyneuropathy, Danish familial amyloid cardiomyopathy, systemic
senile amyloidosises, isolated atrial amyloid, medullary carcinoma
of the thyroid, Finnish familial amyloidosis, Icelandic hereditary
cerebral hemorrhage with amyloidosis, scrapie, Cruetzfeld-jacob
disease, Gertsmann-Straussler-Scheinker syndrome, and bovine
spongiform encephalitis.
18. A method for delivering a treatment to an individual for a
condition characterized by an aggregation of amyloid fibrils
comprising the steps of: (a) providing a composition comprising a
compound of the formula: 7or a pharmaceutically acceptable salt,
ester, solvate, or prodrug thereof, wherein: R.sup.1, R.sup.2,
R.sup.3, R.sup.4, and R.sup.5 are independently selected from the
group consisting of hydrogen, alkyl, cycloalkyl, aryl,
triflouromethyl, trifluoromethylether, halo, and a group of the
formula --OR.sup.7, wherein R.sup.7 is alkyl or aryl; and R.sup.6
is a benzopyridinyl group optionally substituted with one to three
substituents selected from the group consisting of hydrogen, alkyl,
cycloalkyl, aryl, triflouromethyl, trifluoromethylether, halo, and
a group of the formula --OR.sup.7, wherein R.sup.7 is alkyl or
aryl; wherein said alkyl groups at each occurrence are optionally
substituted with alkoxy, aryl, or halo; said aryl groups at each
occurrence are optionally substituted with alkyl, alkoxy, or halo;
in combination with an active agent; (b) administering the
composition to the individual; and (c) optionally repeating steps
(a) and (b), as necessary, to improve or rehabilitate the condition
of the individual.
19. The method of claim 18 wherein the active agent is selected
from the group consisting of proteins, peptides, carbohydrates,
polysaccharides, glycoproteins, nucleic acids, antibodies,
peptidomimetics, organic molecules, and fragments or recombinant
forms thereof.
20. The method of claim 18 wherein the active agent is selected
from the group consisting of inhibitors or activators of a molecule
that is required for inhibiting, synthesizing, post-translation
modification of, or functioning of, some element involved in the
localization or quantification of amyloid; regulators in the
spatial or temporal control of expression of a gene product;
cytokines, growth factors, hormones, signaling components kinases,
phosphatases, homeobox proteins, transcription factors, translation
factors, post-translational factors and enzymes, cholinesterase
inhibitors, muscarinic agonists, anti-oxidants, and
anti-inflammatory agents.
21. The method of claim 18 wherein the active agent is selected
from the group consisting of doxorubicin, galantamine, tacrine
(COGNEX.RTM.), selegiline, physostigmine, revistigmin, donepizil
(ARICEPT.RTM.), metrifonate, milameline, xanomeline, saeluzole,
acetyl-L-carnitine, idebenone, ENA-713, memric, quetiapine,
neurestrol and neuromidal.
22. A method for staining amyloid fibrils comprising the steps of:
(a) providing a compound of the formula: 8or a pharmaceutically
acceptable salt, ester, solvate, or prodrug thereof, wherein:
R.sup.1, R .sup.2, R.sup.3, R.sup.4, and R.sup.5 are independently
selected from the group consisting of hydrogen, alkyl, cycloalkyl,
aryl, triflouromethyl, trifluoromethylether, halo, and a group of
the formula --OR.sup.7, wherein R.sup.7 is alkyl or aryl; and
R.sup.6 is a benzopyridinyl group optionally substituted with one
to three substituents selected from the group consisting of
hydrogen, alkyl, cycloalkyl, aryl, triflouromethyl,
trifluoromethylether, halo, and a group is of the formula
--OR.sup.7, wherein R.sup.7 is alkyl or aryl; wherein said alkyl
groups at each occurrence are optionally substituted with alkoxy,
aryl, or halo; said aryl groups at each occurrence are optionally
substituted with alkyl, alkoxy, or halo; and one or more atoms in
the compound of formula (I) is replaced with a radiolabeled atom;
(b) applying the compound to a sample containing amyloid fibrils to
form a labeled deposit; and (c) detecting the labeled deposit.
23. The method of claim 22 wherein the compound is incorporated in
a pharmaceutically acceptable carrier.
24. A method for detecting amyloid deposits in biopsy or postmortem
human or animal tissue comprising the steps of: (a) incubating
formalin-fixed biopsy or postmortem human or animal tissue with a
solution of a compound of the formula: 9or a pharmaceutically
acceptable salt, ester, solvate or prodrug thereof, wherein:
R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5 are independently
selected from the group consisting of hydrogen, alkyl, cycloalkyl,
aryl, triflouromethyl, trifluoromethylether, halo, and a group of
the formula --OR.sup.7, wherein R.sup.7 is alkyl or aryl; and
R.sup.6 is a benzopyridinyl group optionally substituted with one
to three substituents selected from the group consisting of
hydrogen, alkyl, cycloalkyl, aryl, triflouromethyl,
trifluoromethylether, halo, and a group of the formula --OR.sup.7,
wherein R.sup.7 is alkyl or aryl; wherein said alkyl groups at each
occurrence are optionally substituted with alkoxy, aryl, or halo;
said aryl groups at each occurrence are optionally substituted with
alkyl, alkoxy, or halo; and one or more atoms in the compound of
formula (I) is replaced with a radiolabeled atom; to provide a
labeled deposit; and (b) detecting the labeled deposit.
25. A method for detecting the presence of aggregated prion protein
in a mammal, comprising the steps of: (a) extracting a bodily fluid
from the mammal; (b) contacting the bodily fluid with a compound of
the formula: 10or a pharmaceutically acceptable salt, ester,
solvate, or prodrug thereof, wherein: R.sup.1, R.sup.2, R.sup.3,
R.sup.4, and R.sup.5 are independently selected from the group
consisting of hydrogen, alkyl, cycloalkyl, aryl, triflouromethyl,
trifluoromethylether, halo, and a group of the formula --OR.sup.7,
wherein R.sup.7 is alkyl or aryl; and R.sup.6 is a benzopyridinyl
group optionally substituted with one to three substituents
selected from the group consisting of hydrogen, alkyl, cycloalkyl,
aryl, triflouromethyl, trifluoromethylether, halo, and a group of
the formula --OR.sup.7, wherein R.sup.7 is alkyl or aryl; wherein
said alkyl groups at each occurrence are optionally substituted
with alkoxy, aryl, or halo; said aryl groups at each occurrence are
optionally substituted with alkyl, alkoxy, or halo; and one or more
atoms in the compound of formula (I) is replaced with a
radiolabeled atom; to provide a labeled deposit; and (c) detecting
the labeled deposit.
26. A method for providing an antioxidant o an individual,
comprising administering a quinolinehydrazone compound to said
individual.
27. The method of claim 26, comprising administering a compound of
the formula: 11or a pharmaceutically acceptable salt, ester,
solvate, or prodrug thereof, wherein: R.sup.1, R.sup.2, R.sup.3,
R.sup.4, and R.sup.5 are independently selected from the group
consisting of hydrogen, alkyl, cycloalkyl, aryl, triflouromethyl,
trifluoromethylether, halo, and a group of the formula --OR.sup.7,
wherein R.sup.7 is alkyl or aryl; and R.sup.6 is a benzopyridinyl
group optionally substituted with one to three substituents
selected from the group consisting of hydrogen, alkyl, cycloalkyl,
aryl, triflouromethyl, trifluoromethylether, halo, and a group of
the formula --OR.sup.7, wherein R.sup.7 is alkyl or aryl; wherein
said alkyl groups at each occurrence are optionally substituted
with alkoxy, aryl, or halo; and said aryl groups at each occurrence
are optionally substituted with alkyl, alkoxy, or halo.
28. A complex comprising a compound of formula (I), or a
pharmaceutically acceptable salt, ester, solvate, or prodrug
thereof, in association with or bound to an amyloid fibril, wherein
said compound has the formula: 12wherein: R.sup.1, R.sup.2,
R.sup.4, R.sup.4, and R.sup.5 are independently selected from the
group consisting of hydrogen, alkyl, cycloalkyl, aryl,
triflouromethyl, trifluoromethylether, halo, and a group of the
formula --OR.sup.7, wherein R.sup.7 is alkyl or aryl; and R.sup.6
is a benzopyridinyl group optionally substituted with one to three
substituents selected from the group consisting of hydrogen, alkyl,
cycloalkyl, aryl, triflouromethyl, trifluoromethylether, halo, and
a group of the formula --OR.sup.7, wherein R.sup.7 is alkyl or
aryl; wherein said alkyl groups at each occurrence are optionally
substituted with alkoxy, aryl, or halo; and said aryl groups at
each occurrence are optionally substituted with alkyl, alkoxy, or
halo.
29. A complex comprising a compound of formula (I), or a
pharmaceutically acceptable salt, ester, solvate, or prodrug
thereof, in association with or bound to a prion, wherein said
compound has the formula: 13wherein: R.sup.1, R.sup.2, R.sup.3,
R.sup.4, and R.sup.5 are independently selected from the group
consisting of hydrogen, alkyl, cycloalkyl, aryl, triflouromethyl,
trifluoromethylether, halo, and a group of the formula --OR.sup.7,
wherein R.sup.7 is alkyl or aryl; and R.sup.6 is a benzopyridinyl
group optionally substituted with one to three substituents
selected from the group consisting of hydrogen, alkyl, cycloalkyl,
aryl, triflouromethyl, trifluoromethylether, halo, and a group of
the formula --OR.sup.7, wherein R.sup.7 is alkyl or aryl; wherein
said alkyl groups at each occurrence are optionally substituted
with alkoxy, aryl, or halo; and said aryl groups at each occurrence
are optionally substituted with alkyl, alkoxy, or halo.
30. A compound of the formula: 14or a pharmaceutically acceptable
salt, ester, solvate, or prodrug thereof, wherein: R.sup.1,
R.sup.2, R.sup.3, R.sup.4, and R.sup.5 are independently selected
from the group consisting of hydrogen, alkyl, cycloalkyl, aryl,
triflouromethyl, trifluoromethylether, halo, and a group of the
formula --OR.sup.7, wherein R.sup.7 is alkyl or aryl; and R.sup.6
is a benzopyridinyl group optionally substituted with one to three
substituents selected from the group consisting of hydrogen, alkyl,
cycloalkyl, aryl, triflouromethyl, trifluoromethylether, halo, and
a group of the formula --OR.sup.7, wherein R.sup.7 is alkyl or
aryl; wherein said alkyl groups at each occurrence are optionally
substituted with alkoxy, aryl, or halo; said aryl groups at each
occurrence are optionally substituted with alkyl, alkoxy, or halo;
and one or more atoms in the compound of formula (I) optionally is
replaced with a radiolabeled atom.
31. The compound of claim 30 wherein R.sup.1, R.sup.2, R.sup.3,
R.sup.4, and R.sup.5 are each independently selected from the group
consisting of hydrogen, methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, t-butyl, n-pentyl, t-pentyl, n-hexyl, methoxy,
ethoxy, isopropoxy, sec-butoxy, t-butoxy, phenyl, benzyl,
trifluoromethyl, trifluoromethylether, and halo.
32. The compound of claim 30 wherein the benzopyridinyl group for
R.sup.6 is quinolyl or isoquinolyl.
33. The compound of claim 30 wherein the compound is selected from
the group consisting of:
4-methyl-7-methoxy-2-(4-quinolylmethylenehydrazino)q- uinoline;
4-ethyl-7-methoxy-2-(4-quinolylmethylenehydrazino)quinoline;
4-ethyl-7-ethoxy-2-(4-quinolylmethylenehydrazino)quinoline;
4-methyl-7-ethoxy-2-(4-quinolylmethylenehydrazino)quinoline;
4-ethyl-7-ethoxy-2-(3-quinolylmethylenehydrazino)quinoline;
4-ethyl-7-methoxy-2-(3-quinolylmethylenehydrazino)quinoline; and
4-methyl-7-methoxy-2-(3-quinolylmethylenehydrazino)quinoline.
34. The compound of claim 30 wherein the radiolabeled atom is
selected from the group consisting of .sup.3H, .sup.131I,
.sup.125I, .sup.123I, .sup.76Br, .sup.18F, .sup.19F, .sup.15O, and
.sup.11C.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The invention provides methods for diagnosing and treating
amyloid-related conditions and compounds useful for the same. The
invention allows for detecting, imaging, monitoring, diagnosing,
and treating conditions characterized by the binding or aggregation
of amyloid fibrils. More particularly, the invention relates to the
use of quinolinehydrazone compounds for diagnosing and treating
amyloidotic conditions.
[0003] The invention also encompasses a method for using
quinolinehydrazone compounds as an antioxidant.
[0004] 2. Description of the Related Technology
[0005] Disease-causing agents once were believed to be limited to
pathogens containing nucleic acids, for example viruses and
bacteria. More recently, however, considerable evidence suggests
that irregular peptides or proteinaceous infective agents can
induce or transmit infectious disease.
[0006] Currently, it is widely recognized that some proteins can
contain irregular protein sequences which cause living tissue or
organs to assemble into insoluble aggregates of partially unfolded
proteins, known as amyloid fibrils. All types of amyloid are
structurally related by containing A.beta. peptides. The A.beta.
peptides aggregate to form fibrils, which typically have a
.beta.-sheet secondary structure. The fibril deposits, or plaque,
are believed to be at the root of the pathology for a number of
neurodegenerative diseases, or amyloidosis.
[0007] In general, the term "amyloidosis" refers to diseases
characterized by the tendancy of particular proteins to aggregate
and precipitate as insoluble fibrils. The fibrils collect in the
extracellular space of the surrounding organs or tissues causing
structural and functional damage. Common attributes of amyloidosis
include, for example, cell toxicity and cell degeneration. For
instance, the clinical course of the amyloidotic condition,
Alzheimer's disease, is neurodegeneration. Neurodegeneration can be
identified, for example, by the progressive loss of mental
capacity, loss of motor control, and dementia. Portmortem deposits
of amyloid plaque have been identified in patients suffering from
Alzheimer's disease, Down syndrome, Type 2 diabetes mellitus, and
other amyloid-related conditions.
[0008] Infectious protein particles, or prions, also contain
irregular protein sequences. These particles typically can be
characterized by a single irregular sequence in the protein
peptide. The expression of the irregular sequence favors a protein
conformation that, like amyloid protein, tends to aggregate. The
aggregation of the protein into a plaque has been identified in
many patients inflicted with prion disease, such as, for example,
Crutzfeld-Jacob disease (CJD), Gerstmann-Straussler-Sche- inker
syndrome (GSS), fatal familial insomnia (FFI), kuru, Alper's
syndrome, scrapie, transmissible mink encepthalopathy (TME),
chronic wasting disease (CWD), and bovine spongiform encephalopathy
(BSE). In some cases, amyloid plaque has been detected in patients
having prion-initiated disease, for example patients diagnosed with
CJD. However, prion plaque generally has not been definitively
linked to cell impairment. Instead, considerable evidence suggests
that the prions propagate by changing the conformation of
naturally-existing, non-infectious counterpart proteins into
proteins having a harmful secondary structure.
[0009] Few methods are known for identifying, treating, or
inhibiting the aggregation of amyloid proteins or prions. The
target proteins or plaque often exhibit similar properties as
healthy, unaffected tissue. As a result, it is difficult to develop
an imaging agent selective for only harmful plaque or protein.
[0010] Moreover, the harmful plaque often resides in the brain, for
example in the case of Alzheimer's disease. To provide a method
that is useful for associating with brain plaque, the compound will
have useful properties both for binding to amyloid or prion plaque
and for crossing the blood-brain barrier. Compounds useful for
staining or imaging the plaque in vitro often can not cross the
highly discriminant blood-brain barrier to provide a useful in vivo
diagnostic or therapeutic tool. Consequently, successful compounds
and methods for detecting plaque in vitro can fail as a tool for in
vivo imaging.
[0011] One compound useful for selectively identifying plaque in
vitro is the commercially available diazo dye, Congo red, having
the scientific name
3,3'-[[1,1'-biphenyl]-4,4'-diylbis(azo)]bis[4-amino-1-naphthalenesul-
fonic acid] disodium salt. Congo red has demonstrated binding to
amyloid-like proteins with a beta-pleated sheet conformation. See,
W. E. Klunk, et al., Quantitative Evaluation of Congo Red Binding
to Amyloid-like Proteins with a Beta-Pleated Sheet Conformation, J.
Histochem. Cytochem., 37:1273-1281 (1989). However, Congo red lacks
the necessary properties to cross the blood-brain barrier as shown
by P. D. Griffiths, et al., Receptor Changes in the Neocortex of
Postmortem Tissue in Parkinson's Disease and Alzheimer's Disease,
Dementia, 3:239-246 (1992). As such, the compound provides useful
properties for postmortem staining of brain plaque in vitro, but is
unsuitable for in vivo use.
[0012] Chrysamine G (CG) compounds are a class of compounds derived
from Congo red. The predominant structural difference is
replacement of sulphonic acid groups in Congo red with carboxylic
acid groups. See, for example, International Publication Nos. WO
98/47969, published Oct. 29, 1998; WO 96/34853, published Nov. 7,
1996; and WO 99/24394, published May 20, 1999. In addition,
organometallic ligands of Congo red and Chrysamine G compounds have
been investigated for use as a diagnostic tool. See, for example,
International Publication No. WO 97/41856, published Nov. 13,
1997.
[0013] The replacement of sulphonic acid groups with carboxylic
acid groups potentially would effect better blood-brain barrier
entry of the compound. However, in vivo biodistribution of
Chrysamine G compounds has shown that technetium-99m-labeled
conjugates of 2-(acetamido)-CG with bis-S-trityl protected
monoamide-monoaminedithiol were rapidly cleared from the blood,
causing low uptake of the conjugate in the brain. See, for example,
N. A. Dezutter, European Journal of Nuclear Medicine, Vol. 26, No.
11, pp. 1392-1399 (1999); W. E. Klunk, et al., Life Sciences, Vol.
63, No. 20, pp. 1807-1814 (1998); and W. E. Klunk, et al.,
Neurobiology of Aging, Vol. 16, No. 4, pp. 541-548 (1995).
[0014] Derivatives of 9-acridinone also have been reported as
inhibiting amyloid aggregation. International Publication No. WO
97/16191, published May 9, 1997, describes that 9-acridinone
compounds inhibit amyloid aggregation in vitro. In vivo diagnostic
or therapeutic activity of the compounds is not described.
[0015] Commonly-assigned U.S. Pat. Nos. 5,731,313, and 5,998,615,
issued Mar. 24, 1998, and Dec. 7, 1999, respectively, identify
fluoroanthracyclinone compounds as useful anti-infective agents and
also as a diagnostic agent for imaging amyloid plaque. The in vitro
binding of A.beta.25-35 peptides is described in International
Publication No. WO 97/49433, published Dec. 31, 1997.
[0016] More recently, naphthylazo derivatives have been prepared
for inhibiting amyloid aggregation. The in vitro binding activity
of naphthylazo compounds is described in International Publication
No. 97/16194, published May 9, 1997. However, no in vivo data is
described in the publication.
[0017] Radiolabeled hydrazine and ethylene derivatives of
benzenethiazole compounds also have been reported for binding
insulin amyloid. See, International Publication WO 97/26919,
published Jul. 31, 1997. These compounds show inhibitory activity
in vitro, however, to the best of our knowledge, no in vivo
activity has been shown.
[0018] Of the methods known, the only conclusive test is staining
for plaque postmortem. Some compounds have been investigated for in
vivo administration and imaging, but it remains beneficial to
provide a compound for demonstrating in vitro imaging of
compounds.
[0019] Accordingly, it remains beneficial to provide a compound for
demonstrating in vitro and in vivo imaging of harmful,
proteinaceous plaque or infectious agents, more particularly
amyloid plaque, prions, or prion plaque. There remains a need to
identify compounds and methods for diagnosing and treating
conditions related to the aggregation of amyloid or other
proteinaceous fibrils. Described here are compounds that provide in
vitro imaging, diagnostic opportunities, antioxidant properties and
even treatment opportunities for patients suffering from
amyloidotic conditions.
SUMMARY OF THE INVENTION
[0020] The invention provides diagnostic and therapeutic methods
related to detecting, imaging, monitoring, diagnosing, and treating
conditions related to aggregation of amyloid or other harmful
proteinacious deposits and compounds for accomplishing the methods.
Compounds demonstrating beneficial properties for the methods of
the invention generally are quinolinehydrazone compounds having the
formula: 1
[0021] or a pharmaceutically acceptable salt, ester, solvate or
prodrug thereof, wherein:
[0022] R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5 are
independently selected from the group consisting of hydrogen,
alkyl, cycloalkyl, aryl, triflouromethyl, trifluoromethylether,
halo, and a group of the formula --OR.sup.7, wherein R.sup.7 is
alkyl or aryl; and
[0023] R.sup.6 is a benzopyridinyl group optionally substituted
with one to three substituents selected from the group consisting
of hydrogen, alkyl, cycloalkyl, aryl, triflouromethyl,
trifluoromethylether, halo, and a group of the formula --OR.sup.7,
wherein R.sup.7 is alkyl or aryl;
[0024] wherein said alkyl groups at each occurrence are optionally
substituted with alkoxy, aryl, or halo; and said aryl groups at
each occurrence are optionally substituted with alkyl, alkoxy, or
halo. One or more atoms in the compound can be optionally replaced
with a radiolabeled atom, or radioisotope (radioactive isotope).
The radioisotope can be selected from the group consisting of
.sup.3H, .sup.131I, .sup.125I, .sup.123I, .sup.76Br, .sup.75Br,
.sup.18F, .sup.19F, .sup.15O, and .sup.11C. Pharmaceutically
acceptable salts, esters, solvates, or prodrugs of compounds having
the formula (I) can also be suitable for methods of the
invention.
[0025] In one aspect, therefore, the invention relates to a method
for chemically tagging or inhibiting the aggregation of amyloid
fibrils comprising administering an effective amount of a compound
of formula (I) and allowing the compound to associate with the
amyloid fibrils. In this manner, the compound prevents amyloid
fibrils from binding to each other and, accordingly, arrests the
formation of harmful amyloid plaque.
[0026] In another aspect, the invention relates to a method for
detecting aggregation of amyloid fibrils. The method comprises (a)
administering a compound of formula (I) containing at least one
radioactive isotope; (b) allowing the compound to associate with
the amyloid fibrils to provide a labeled deposit; and (c) detecting
the labeled deposit. The detection method also can be used for
monitoring the aggregation of amyloid fibrils in an individual. If
necessary or appropriate, data obtained from detecting the labeled
amyloid deposit, such as the location and the amount of amyloid
deposit, can be recorded to allow one with skill in the art to
assess the status and/or progress of the amyloidotic condition.
[0027] The method also can be used for identifying or diagnosing a
condition characterized by aggregation of amyloid fibrils, further
comprising evaluating or assessing the amount and placement of the
radioactive isotope to determine the medical condition of the
individual, or patient. To achieve the full advantage of the
invention, the data can be evaluated or assessed in light of data
obtained from a normal, i.e. free of amyloid-plaque, individual. In
this aspect, the method comprises identifying or using a biomarker
for conditions characterized by the aggregation of amyloid fibrils.
In addition, the method can also be used in a patient undergoing
treatment for a condition characterized by the aggregation of
amyloid fibrils to evaluate the progress of the course of
treatment.
[0028] In another aspect, the invention relates to a method for
treating a condition characterized by aggregation of amyloid
fibrils comprising administering a compound of formula (I),
optionally in a pharmaceutically acceptable carrier, and allowing
the compound to associate with the amyloid plaque. The treatment
can be carried out by allowing the compound to directly bind to or
inhibit the aggregation of the amyloid plaque. The steps of
administering the compound and allowing it to associate can be
repeated as necessary or desired to effectuate treatment of the
amyloid-related condition.
[0029] Alternatively, the compound can deliver therapeutic agents
to the amyloid fibrils. The method for delivering a treatment for a
condition characterized by aggregation of amyloid fibrils,
therefore comprises (a) providing a compound of formula (I) in
combination with a therapeutic agent; (b) administering the
combination to an individual having amyloidosis; and (c) optionally
repeating steps (a) and (b), as necessary, to improve or
rehabilitate the condition of the individual.
[0030] Yet another aspect of the invention relates to a method for
staining amyloid fibrils comprising (a) applying a compound of
formula (I) to a sample containing amyloid fibrils to form a
labeled deposit and (b) detecting the labeled deposit.
[0031] In addition, the invention can relate to a method for
detecting amyloid deposits in biopsy or postmortem human or animal
tissue comprising the steps of (a) incubating formalin-fixed tissue
with a solution of a compound of formula (I) to form a labeled
deposit and (b) detecting the labeled deposit.
[0032] The compounds also can be contacted with bodily fluids
extracted from a mammal to detect aggregated prion proteins. In
this aspect, a bodily fluid is extracted from the mammal and
contacted with the bodily fluid of a compound of formula (I).
[0033] In yet another aspect, a quinolinehydrazone compound can be
used as an antioxidant by administering the compound, optionally in
a pharmaceutically acceptable carrier. The quinolinehydrazone
compound can comprise a compound of formula (I).
[0034] Yet another aspect of the invention relates to a complex
comprising a compound of formula (I) in association with or bound
to an amyloid fibril. The complex also can comprise a compound of
formula (I) in association with or bound to a proteinaceous
infectious particle, or prion.
DETAILED DESCRIPTION OF THE INVENTION
[0035] Quinolinehydrazone compounds possess beneficial properties
for providing novel diagnostic and therapeutic methods for
detecting, imaging, monitoring, diagnosing, and treating conditions
related to aggregation of amyloid and other harmful proteinacious
deposits. Preferred quinolinehydrazone compounds comprise: 2
[0036] or a pharmaceutically acceptable salt, ester, solvate, or
prodrug thereof, wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4,
R.sup.5, and R.sup.6 are as previously defined, and wherein one or
more atoms of the compound optionally can be substituted with a
radiolabeled atom.
[0037] The term "alkyl" as used herein refers to a straight or
branched hydrocarbon group, preferably containing one to six carbon
atoms. Examples of useful alkyl groups include, but are not limited
to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl
(1-methylpropyl), t-butyl (1,1-dimethylethyl), n-pentyl, t-pentyl
(1,1-dimethylpropyl), n-hexyl, and the like. Alkyl groups for the
invention also can include groups wherein the hydrocarbon contains
one or more substituents such as, for example, cycloalkyl, alkoxy,
aryl, or halo as defined hereinbelow. The substituents can bond
with the same carbon or different carbons. Typically, the
hydrocarbon contains one, two or three substituents, if any.
[0038] The term "alkoxy" as used herein refers to a straight or
branched hydrocarbon group as defined above attached to the parent
molecule through an oxygen heteroatom, typically by a carbon to
oxygen bond. The hydrocarbon of the alkoxy group preferably
contains from 1 to 6 carbon atoms. Typical alkoxy groups are
methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy
(1-methylpropoxy), t-butoxy (1,1-dimethylethoxy), n-pentoxy,
t-pentoxy (1,1-dimethylpropoxy), and the like.
[0039] The term "cycloalkyl" as used herein refers to non-aromatic
cyclic hydrocarbon group, preferably containing from three to six
carbon atoms. Examples of cycloalkyl are cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, and the like. Cycloalkyl groups may be
substituted with alkyl and alkoxy groups, as defined above, as well
as halo substituents, for example, bromo, chloro, iodo, and
fluoro.
[0040] The term "aryl" as used herein refers to an aromatic cyclic
hydrocarbon, such as phenyl and naphthyl. The aryl group optionally
can be substituted with alkyl, alkoxy or a halo group, for example,
bromo, chloro, iodo, and fluoro. Examples of aryl groups include,
but are not limited to, phenyl, bromophenyl, chlorophenyl,
iodophenyl, fluorophenyl, bromonaphthyl, and the like.
[0041] The term "triflouromethyl" as used herein refers to a methyl
group (--CH.sub.3) wherein each hydrogen atom is substituted with a
fluorine atom. Similarly, "trifluoromethylether" is a
trifluoromethyl group, as defined above, linked to the parent
molecule through an oxygen heteroatom, typically by a carbon-oxygen
bond. The trifluoromethyl and trifluoromethylether groups can be
represented by the formulae --CF.sub.3 and --OCF.sub.3,
respectively.
[0042] The term "halo" as used herein refers to a monovalent
substituent derived from a halogen. Typical halogens are, for
example, bromine, chlorine, iodine, fluorine, and the like.
[0043] The term "benzopyridinyl" as used herein refers to a group
derived from a compound comprising a fused benzene and pyridine
ring, generally called a benzopyridine. Examples of benzopyridine
compounds are quinoline and isoquinoline. The corresponding groups
derived from quinoline and isoquinoline are quinolyl and
isoquinolyl, respectively. The benzopyridinyl groups can contain
one or more substituents, typically selected from alkyl, aryl,
triflouromethyl, trifluoromethylether, halo, and a group of the
formula --OR.sup.7, wherein R.sup.7 is alkyl or aryl.
[0044] The term "radiolabeled atom", "radioisotope", or
"radioactive isotope" as used herein refers to an atom, which can
be incorporated into a compound, capable of emitting waves or
particles of radioactive energy, typically light and/or heat. More
particularly, the atoms decay from a radioactive atom to a
naturally occurring atom containing the same number of protons by
emitting electrons. Examples of radiolabeled atoms or radioisotopes
are .sup.3H, .sup.131I, .sup.125I, .sup.123I, .sup.76Br, .sup.75Br,
.sup.18F, .sup.19F, .sup.15O, and .sup.11C.
[0045] It has now been discovered that quinolinehydrazone
compounds, particularly quinoline- or isoquinoline-substituted
quinolinehydrazone compounds, exhibit antioxidant properties and
can bind to amyloid fibrils, inhibiting the aggregation thereof.
The compounds demonstrate in vitro activity for inhibiting seeded
aggregation of amyloid fibrils as well as demonstrate in vitro
antioxidant properties in oxidative neuronal models. Moreover, the
compounds can cross the blood-brain barrier in vivo to provide a
beneficial diagnostic or therapeutic tool.
[0046] Certain quinolinehydrazone compounds previously have
described as antimicrobial, antiparasitic, anti-mycoplasmal, and
anti-HIV agents. See; for example, Atti Accad. Fisiocritici Siena,
Serie XIV, Vol. 8, 82-93 (1976); Arzneim.-Forson. (Drug Res.) 23,
Nr. 6, 830-839 (1973); Il Farrmaco, 47(6), 945-962 (1992); and U.S.
Pat. No. 3,646,019. However, quinolinehydrazone compounds reported
in the literature typically are substituted with monocyclic, if
any, heteroaryl groups. There is no known description of using
quinolinehydrazine compounds for binding to amyloid fibrils or
inhibiting the aggregation of such fibrils either in vitro or in
vivo. In addition, quinoline- and isoquinoline-substituted
quinolinehydrazone compounds have not been described as a compound
per se, either for demonstrating therapeutic activities or as a
diagnostic tool.
[0047] The compounds can be prepared by any suitable process. The
compounds can be prepared from 2-hydrazinochloroquinoline starting
materials or, in the alternative, prepared by reacting the
corresponding 2-chloroquinoline with hydrazine hydrate. The
2-hydrazinoquinoline group is reacted with the carboxaldehyde of
the desired benzopyridyl substituent, for example
4-quinolinecarboxaldehyde. The reaction is preferably carried out
in an organic solvent, typically alcoholic solvents, for example,
methanol, ethanol, isopropanol, and the like. The most preferred
organic solvent is ethanol. To facilitate preparation, the
compounds can be reacted in the presence of heat, typically from
about 50-70.degree. C. Additional discussion of the methods,
reagents and conditions for preparing the quinolinehydrazone
derivatives, in general, are described in Atti Accad. Fisiocritici
Siena, Serie XIV, Vol. 8, 82-93 (1976); Arzneim.-Forson. (Drug
Res.) 23, Nr. 6, 830-839 (1973); Il Farmaco, 47(6) , 945-962
(1992).
[0048] Radiolabeled compounds can be prepared by reacting a
compound of formula (I) with a radioactive isotope or a suitable
reagent containing the isotope. A thorough discussion regarding
methods for preparing radiolabeled compounds and derivatives is
provided in Kirk-Othmer, Encyclopedia of Chemical Technology,
4.sup.th ed., vol. 20, John Wiley & Sons, Inc., New York, N.Y.
(1999), and more particularly in pp. 930-962.
[0049] A compound of formula (I) can bind to A.beta. peptide
sequences with a dissociation constant (Kp) between 0.001 and 10.0
.mu.M when measured by binding to synthetic A.beta. peptide or to
amyloid fibrils in brain tissue. In addition, the compound is
capable of traversing capillary endothelial cells with continuous
junctions and no detectable transendothelial pathways to reach the
brain, commonly called the blood-brain barrier, which is beneficial
for a diagnostic tool if used in the brain. Dose-dependent
concentrations of the compound can be detected in the brain and
plasma of C57 female mice when administered in vivo, which
indicates transport across the blood-brain barrier.
[0050] Preferred compounds for the methods of the invention can be
represented by the general formula (I), or can comprise a salt,
ester, solvate, or prodrug thereof, having at least one atom of the
original parent compound replaced with a radioactive isotope. The
radioactive isotopes emit wave frequencies, which are typically
readily detectable by a variety of detectors or imagers. The scope
of the invention contemplates detectors and imagers primarily
functioning on the detection of wave frequencies, for example
photon emissions, positron emissions, gamma emissions, and the
like. The detector and imaging apparatus suitable for use with the
invention is not limited by commercial availability of the units,
however, many practical units for detecting, imaging, possibly in
combination with recording, the presence of the radioactive
isotopes in the body of the mammal are readily available. More
particularly, non-invasive units for detecting and imaging
radioactive isotopes in vivo, generally in combination with
electronic, photographic, or textual methods of recording data, are
also contemplated. The detector or imaging equipment detect wave
frequencies emitted from the labeled compounds that can be
distinguished from other non-labeled compounds, i.e. compounds not
containing a radioactive isotope. It is well within the purview of
one skilled in the art to determine the particular radioactive
isotope for an intended detection or imaging assay, considering the
frequency range and sensitivity of the detector, the chemical and
physical properties of the imaging agent, and other factors
relevant to the precision, accuracy, and validity of the assay.
[0051] The preferred substituents for R.sup.1, R.sup.2 , R.sup.3,
R.sup.4, and R.sup.5 are each independently selected from hydrogen,
methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, t-butyl,
n-pentyl, t-pentyl, n-hexyl, methoxy, ethoxy, isopropoxy,
sec-butoxy, t-butoxy, phenyl, benzyl, trifluoromethyl,
trifluoromethylether, or halo. The preferred group for the
substituent R.sup.6 in a compound of formula (I) is quinolyl or
isoquinolyl. More preferred compounds for the methods of the
invention have the formula: 3
[0052] wherein R.sup.8 and R.sup.9 are as previously defined for
the substituents R.sup.1, R.sup.2, R.sup.3, R.sup.4, and
R.sup.5.sub.1 or a pharmaceutically acceptable salt, ester,
solvate, or prodrug thereof. The more preferred substituents for
R.sup.8 and R.sup.9 are each independently selected from hydrogen,
methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, t-butyl,
n-pentyl, t-pentyl, n-hexyl, methoxy, ethoxy, isopropoxy,
sec-butoxy, t-butoxy, phenyl, benzyl, trifluoromethyl,
trifluoromethylether, or halo. Preferred radiolabeled atoms are
.sup.3H, and .sup.125I.
[0053] More particularly, the more preferred compounds for the
invention include, but are not limited to:
[0054]
4-methyl-7-methoxy-2-(4-quinolylmethylenehydrazino)quinoline;
[0055]
4-ethyl-7-methoxy-2-(4-quinolylmethylenehydrazino)quinoline;
[0056]
4-ethyl-7-ethoxy-2-(4-quinolylmethylenehydrazino)quinoline;
[0057]
4-methyl-7-ethoxy-2-(4-quinolylmethylenehydrazino)quinoline;
[0058]
4-ethyl-7-ethoxy-2-(3-quinolylmethylenehydrazino)quinoline;
[0059] 4-ethyl-7-methoxy-2-(3-quinolylmethylenehydrazino)quinoline;
and
[0060]
4-methyl-7-methoxy-2-(3-quinolylmethylenehydrazino)quinoline.
[0061] The term "pharmaceutically acceptable salt, ester, solvate,
or prodrug thereof" refers to derivatives prepared from a parent
compound, in this case the quinolinehydrazone compound. The salts
include, but are not limited to, alkali metal salts, alkaline earth
metal salts, ammonium salts, salts with an appropriate organic
amine or amino acid, and acid addition salts prepared with organic
or inorganic acids such as, for example, hydrochloric acid,
sulfuric acid, carboxylic acid, and sulfonic acids. Esters more
particularly refer to condensation products of the parent compound
with an organic acid, for example, acetate, propionate, butyrate,
valerate, benzoate, salicylate, succinate, and the like. Solvates
refer to ionic or molecular complexes Comprising the parent
compound and one or more molecules of solute, typically an organic
solvent, and preferably water. Prodrugs are obtained in vivo when a
chemical compound is converted into a parent compound, for example
a quinoline- or isoquinoline-substituted quinolinehydrazone, by
natural metabolic processes of the body.
[0062] When administered to a mammal, the compounds have
demonstrated an ability to interact with amyloid fibrils, and
particularly with amyloid fibrils in the brain. To achieve the full
advantage of the invention, the compound is allowed to interact
with the amyloid fibrils, generally by allowing a sufficient time
for bonding, associating, conjugating, complexing, or other
reaction to take place. In the presence of amyloid fibrils, the
labeled compounds interact with the amyloid fibrils to form a
labeled amyloid fibril complex. In particular, the radiolabeled
compound can be used to bind to brain amyloid fibrils including,
for example, protofibrils, type-1 fibrils, type-2 fibrils, neuritic
plaque, diffuse amyloid, and combinations thereof. In some cases,
the compound may interact with only one type of fibril, either by
nature or by design. In a preferred case, the compound specifically
interacts with .beta.-amyloid protofibrils and, more specifically,
.beta.-amyloid protofibrils in the brain.
[0063] For the convenience and ease of administration, the compound
commonly is prepared into a pharmaceutical composition. In a
typical pharmaceutical composition, a quinolinehydrazone compound
or a derivative thereof, including a radiolabeled compound prepared
therefrom, as described above, is combined with a pharmaceutically
acceptable carrier. The carrier can comprise one or more
solubilizing agents, excipients, additives, diluents, or other
agents to improve the characteristics of the composition. In
general, the compound is formulated with the carrier in any manner
suitable for providing a pharmaceutical dosage form, typically
resulting in a sterile, non-toxic mixture or blend of the active
agent with a carrier. The composition can be administered orally,
parenterally, subcutaneously, intravenously, intramuscularly,
intracisternally, or by infusion techniques. Methods for preparing
pharmaceutical compositions are readily available in the art and
are further described, for example, in Remington: The Science and
Practice of Pharmacy, Nineteenth Edition, vol. II, Mack Publishing
Company, Easton, Pa., 1995, in particular with respect to
solutions, emulsions, suspensions, and extracts on pp. 1485-1533;
intravenous admixtures on pp. 1542-1562; oral dosage forms on pp.
1615-1649; sustained-release delivery systems on pp. 1660-1675; and
aerosols on pp. 1676-1692.
[0064] In particular, the compounds can be administered in a
liquid, solid, or semi-solid oral dosage form. Examples of common
dosage forms suitable for the invention are tablets, capsules,
troches, lozenges, emulsions, solutions, tinctures, syrups,
elixirs, and the like. Oral dosage forms can optionally contain one
or more chemically non-reactive excipients, for example, sweetening
agents, flavoring agents, coloring agents, preserving agents, and
the like.
[0065] The active agent can be admixed with excipients, such as
inert diluents, granulating and disintegrating agents, binding
agents, and lubricating agents to provide a suitable tablet.
Examples of inert diluents suitable for the tablet are, for
example, calcium carbonate, sodium carbonate, lactose, calcium
phosphate and sodium phosphate. Granulating and disintegrating
agents suitable for the invention include, but are not limited to,
maize starch, alginic acid, and the like. Binding agents, for
example maize starch, gelatin or acacia, can also be incorporated
into the tablet. Lubricating agents, such as, for example magnesium
stearate, stearic acid or talc, are also suitable for the
invention.
[0066] The tablets can be optionally coated by a wide variety of
techniques to facilitate administration, improve taste, provide a
sustained action over a longer period, or delay disintegration or
adsorption in the gastrointestinal tract. For example, a time delay
material such as glyceryl monostearate or glyceryl distearate may
be employed. Techniques for coating tablets, in general, are
further described in Coating of Pharmaceutical Dosage Forms by
Stuart C. Porter, reprinted in Remington: The Science and Practice
of Pharmacy, Nineteenth Edition, vol. II, Mack Publishing Company,
Easton, Pa., 1995, in particular in pp. 1650-1659.
[0067] Capsules are another dosage form suitable for oral
administration. The term "capsules" is intended to include hard
capsules, soft capsules such as gel capsules, microcapsules, and
the like. The active ingredient can be mixed with an inert solid
diluent, for example, calcium carbonate, calcium phosphate or
kaolin, or a soft gelatin capsule wherein the active ingredient is
mixed with water or an oil medium, for example, peanut oil, liquid
paraffin, or olive oil.
[0068] Aqueous suspensions can contain the active materials in
admixture with one or more compatible excipients. Such excipients
are suspending agents, for example, sodium carboxymethylcellulose,
methylcellulose, hydroxymethylcellulose,
hydroxypropylmethylcellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents such as naturally-occurring phosphatides, for
example lecithin, or condensation products of an alkylene oxide
with fatty acids, for example polyoxyethylene stearate, or
condensation products of ethylene oxide with long chain aliphatic
alcohols, for example, heptadecaethylene-oxycetanol, or
condensation products of ethylene oxide with partial esters derived
from fatty acids and a hexitol such as polyoxyethylene sorbitol
monooleate, or condensation products of ethylene oxide with partial
esters derived from fatty acids and a hexitol anhydrides, for
example polyoxyethylene sorbitan monooleate. The aqueous
suspensions may also contain one or more preservatives, for
example, ethyl or n-propyl p-hydroxybenzoate, one or more coloring
agents, one or more flavoring agents, or one or more sweetening
agents, such as sucrose or saccharin.
[0069] In addition to aqueous suspensions, the active agent can be
formulated as an oily suspension. The oily formulation is prepared
by suspending the active ingredient in a vegetable oil, for example
arachis oil, olive oil, sesame oil, coconut oil, or mineral oil,
such as liquid paraffin. The oily suspensions may contain a
thickening agent for example beeswax, hard paraffin or cetyl
alcohol. Sweetening agents, such as those set forth above and
flavoring agents may be added to provide a palatable oral
preparation. These compositions may be perserved by the addition of
an antioxidant, such as ascorbic acid. Dispersible powders and
granules are also suitable for the oily suspension. The powders and
granules can be incorporated into the suspension with dispersing or
wetting agents, suspending agents and one or more preservatives.
Suitable dispersing or wetting agents and suspending agents are
exemplified by those already mentioned above for the aqueous
suspension. Additional excipients, for example sweetening agents or
flavoring agents, may also be present.
[0070] The pharmaceutical composition can also be oil-in-water
emulsions. The oily phase of the emulsion can be an oil selected
from vegetable oil, for example, olive oil or arachis oil; a
mineral oil, for example, liquid paraffin; or a combination
thereof. The emulsifying agents can be naturally-occurring gums,
phosphatides, esters or partial esters derived from fatty acids and
hexitol anhydrides, and condensation products of said partial
esters. Suitable emulsifying agents are gum acacia, gum tragacanth,
soybean oil, lecithin, sorbitan monooleate, polyoxyethylene
sorbitan monooleate. The emulsion can also contain sweetening and
flavoring agents including, in addition, syrups and elixirs such as
glycerol, sorbitol, or sucrose, for example. Such formulation can
also contain soothing agents (demulcents), preservatives, flavoring
agents, and coloring agents.
[0071] Aqueous and oily suspensions described for the oral dosage
forms can be formulated into parenteral dosage, for example
subcutaneously, intravenously, intramuscularly, intracisternally,
or by infusion. The suspensions can be formulated using one or more
non-toxic parenterally acceptable diluents or excipients.
Excipients suitable for the invention include dispersing agents,
wetting agents, suspending agents, and the like. The suspension can
be incorporated into a solvent vehicle, for example 1,3-butane
diol, water, Ringer's solution, isotonic sodium chloride solution,
and the like. In addition, sterile, fixed oils are also suitably
employed as a solvent or suspending medium. Examples of oils for
the parenteral composition are monoglycerides, diglycerides, and
fatty acids, for example oleic acid.
[0072] Amyloid fibrils can be present anywhere in the body of the
mammal. Typically, amyloid fibrils aggregate in a particular organ,
or part of the organ, and form a film or mass of amyloid fibrils,
called amyloid plaque. Amyloid fibrils and amyloid plaque can be
found, for example, in the brain, pancreas, vasculature, spleen,
liver, kidneys, adrenals, lymph nodes, muscle, cardiovascular
system, skin, or any combination thereof.
[0073] A significant advantage of the invention is the assessment
of the amyloid fibrils localization and the ability to quantify the
labeled amyloid fibrils. The localization or quantification of the
labeled amyloid fibrils can be determined a variety of techniques.
Examples are radioimaging, magnetic resonance imaging (MRI), single
photon emission computed tomographic imaging (SPECT), and other
suitable detection or imaging techniques.
[0074] Overproduction of the readily aggregating amyloid protein
can cause numerous disease including, but not limited to,
Alzheimer's disease, Down syndrome, Type 2 diabetes mellitus,
amyloid A (reactive), secondary amyloidosis, familial mediterranean
fever, procalcitonin, Cruetzfeld-Jacob disease, bovine spongiform
encephalitis, and the like. Moreover, mutations in the pathogenic
genes believed to cause disease, for example, presenilin and APP
genes, have been shown to cause an increase in amyloid aggregation.
Accordingly, the detection of amyloid aggregation, or the resulting
amyloid plaque, is indicative of amyloid-related pathogenic
disease.
[0075] In accordance with the method of diagnosing a person having
a condition associated with aggregated amyloid, the data is
recorded and assessed by one with skill in the art of diagnosing or
treating amyloid-related disease. The method can be used for a wide
variety of amyloid-related disorders including, for example, but
not limited to, Alzheimer's disease, Down syndrome, Type 2 diabetes
mellitus, hereditary cerebral hemorrhage amyloidosis, amyloid A,
secondary amyloidosis, familial Mediterranean fever, familial
amyloid nephropathy with urticaria and deafness, amyloid lambda
L-chain or amyloid kappa L-chain, A beta 2M, ATTR, familial amyloid
cardiomyopathy, isolated cardiac amyloid, AIAPP or amylin
insulinoa, atrial naturetic factor, procalcitonin, gelsolin,
crytatin C, AApo-A-I, AApo-A-II, fibrinogen-associated amyloid; and
Asor or Pr P-27 or in cases of persons who are homozygous for the
apolipoprotein E4 allele, and the condition associated with
homozygosity for the apolipoprotein E4 allele. More particular
examples of such diseases include, but are not limited to, Dutch
hereditary cerebral hemorrhage amyloidosis amyloid A, Muckle-wells
syndrome, idiopathic-associated amyloid lambda L-chain,
myeloma-associated amyloid lambda L-chain,
macroglobulinemia-associated amyloid lambda L-chain,
idiopathic-associated amyloid kappa L-chain, myeloma-associated
amyloid kappa L-chain, macroglobulinemia-associated amyloid kappa
L-chain, Portuguese familial amyloid polyneuropathy, Japanese
familial amyloid polyneuropathy, Swedish familial amyloid
polyneuropathy, Danish familial amyloid cardiomyopathy, systemic
senile amyloidosises, isolated atrial amyloid, medullary carcinoma
of the thyroid, Finnish familial amyloidosis, Icelandic hereditary
cerebral hemorrhage with amyloidosis, scrapie, Cruetzfeld-Jacob
disease, Gertsmann-Straussler-Scheinker syndrome, and bovine
spongiform encephalitis.
[0076] In some cases, the data obtained regarding the localization
and quantification of the labeled amyloid fibrils can be compared
to a standard, such as, for example, the localization and
quantification patterns of a mammal having normal, or typical,
levels of amyloid plaque relative to like species. In accordance
with a method of monitoring the aggregation of amyloid fibrils, the
data obtained from the detection or imaging methods can be compared
with earlier assessments of the amyloid condition in the mammal as
well as comparing the method to a standard to determine the state
of the amyloid aggregation or the progression of the disease.
[0077] The data obtained also can be used in conjunction with a
single dose or a regimen of therapy to determine the effect of the
therapy on the localization or quantification of the amyloid
fibrils. For example, the radiolabeled compound can be administered
to a patient undergoing a course of amyloid-affecting therapy, such
as doxorubicin, an organic molecule. The radiolabeled compound
interacts with the amyloid fibrils to provide an amyloid-labeled
complex, which can be used to detect, image, and preferably record,
the condition of the affected organ at desired periods in the
therapy. The state of the amyloid aggregation and the progress of
the therapy can be suitably monitored.
[0078] In another example, the radiolabeled compound is
administered to a mammal suspected of having an amyloid-related
disorder. The radiolabeled amyloid fibril complex is detected or
imaged by the described methods to obtain localization patterns and
quantification of the labeled amyloid fibrils. The data obtained
regarding the state of the amyloid aggregation can be recorded and
compared with data obtained from a normal, i.e. disease-free, brain
to determine the state of amyloid-affectedness in the patient. In
this aspect, the radioactive compound can be used as a biomarker
for determining the relative condition of a mammal potentially
suffering from an amyloid-related disorder.
[0079] The preferred formulation for the compound is an oral dose
form. More preferably, the compound is administered as a
suspension, solution, or tincture. Dosage levels of the compound
can be determined by one with skill in the art, considering the
age, weight, and condition of the subject to be treated, as well as
the severity of the disease, the frequency of the desired dosing,
and the route of administration. In particular, the compound can be
prepared in a single dose formulation for the detection and imaging
aspects of the invention. To provide guidance to the reader in
carrying out the practice of the invention, an oral dose in the
range of from about 10 to about 1000 milligrams per kilograms of
body weight per day (mg/kg/day) are suitable for the invention. The
preferred dosage for the detection and imaging aspects of the
invention are from about 250 to about 750 milligrams per kilograms
of body weight per day.
[0080] A method of the invention can also relate to treating a
condition characterized by aggregated amyloid fibrils. In this
aspect, a therapeutically effective amount of a compound of formula
(I) is administered to the mammal, typically in a pharmaceutical
carrier. A sufficient amount of time is allowed for the compound to
interact with amyloid fibrils in the body. The interaction between
the compound and the amyloid fibrils inhibits amyloid protein
fibrils from aggregating and forming amyloid plaque. A
"therapeutically effective" amount is the amount of active agent
sufficient to inhibit or treat the condition.
[0081] The compounds also can be used to deliver treatment for an
amyloid-related condition. The compound can be bound to a
therapeutic treatment, typically a drug therapy, for example,
synthetic or naturally-derived organic compounds, proteins,
antibodies, and the like. The compound, in combination with the
therapeutic treatment, are administered to a mammal, particularly a
mammal in need of treatment for an amyloid-related condition. The
compound interacts with the amyloid fibrils, transporting the
target treatment to the site where the treatment is needed. The
compounds have demonstrated an affinity for the amyloid fibrils
and, accordingly, deliver the desired treatment efficiently and
effectively to the target area, i.e. where the amyloid fibrils
aggregate. Examples of active agents that can be monitored include,
but are not limited to, specific and non-specific drug treatments
such as proteins, peptides, carbohydrates, polysaccharides,
glycoproteins, nucleic acids, antibodies, peptidomimetics, organic
molecules (preferably, less than 1500 kDa), fragments or
recombinant forms of the above; inhibitors or activators of a
molecule that is required for inhibiting, synthesizing,
post-translation modification of, or functioning of some element
involved in the localization or quantification of amyloid;
regulators in the spatial or temporal control of expression of a
gene product; cytokines, growth factors, hormones, signaling
components, kinases, phosphatases, homeobox proteins, transcription
factors, translation factors, post-translational factors and
enzymes. Broad categories of drug therapies include, but are not
limited to, cholinesterase inhibitors, muscarinic agonists,
anti-oxidants or anti-inflammatory agents. Examples of active
agents for amyloid-related disorders are doxorubicin, galantamine,
tacrine (COGNEX.RTM.), selegiline, physostigmine, revistigmin,
donepizil (ARICEPT.RTM.), metrifonate, milameline, xanomeline,
saeluzole, acetyl-L-carnitine, idebenone, ENA-713, memric,
quetiapine, neurestrol and neuromidal.
[0082] In addition to methods for treating amyloidosis, the
compounds also have demonstrated activity as an antioxidant. To
enjoy the antioxidant properties of the invention, a
quinolinehydrazone compound, for example a compound of formula (I),
is administered to a patient, preferably in a pharmaceutically
acceptable carrier as previously described. The antioxidant
properties of the compound are especially beneficial for treating
conditions characterized by oxidative damage in the body, for
example oxidative neuronal damage. Preferably, the compound is
administered as an oral or intravenous dosage form.
[0083] Dosing for the compound as a treatment agent, either for
treating amyloidosis or for treating a condition characterized by
oxidative damage, can be administered in any suitable manner.
Examples of dosing regimens suitable for the invention are orally,
parenterally, subcutaneously, intravenously, intramuscularly,
intracisternally, or by infusion, as described for the diagnostic
aspect of the invention. The prescribed dosing can be administered
within the reasonable medicament judgment of the professional
treating the patient, however, it is suggested that a suitable dose
of the compound is in the range of from about 10 to about 1000
milligrams per kilograms of body weight per day.
[0084] The invention also relates to a method for detecting the
presence of aggregated prion protein in a mammal. The compound can
be administered orally as with the aspect of the invention related
to amyloid-related detection and imaging. Prions, however, are more
common in animals. As such, it is preferred with the aspect of the
invention related to prion detection that a body fluid or tissue
from the subject mammal is obtained. The fluid or tissue is allowed
to interact with the compound to identify the presence of any
aggregated prion. The presence of the aggregated prion can be
determined by the detection and imaging techniques previously
described. In this manner, the invention can be more efficiently
and effectively carried out for the practice of detecting and
imaging the presence of aggregated prion in animals. Examples of
prion-related disease, in particular, are generally diseases that
affect livestock including, but not limited to, scrapie, a disease
common to sheep, and bovine spongiform encephalopathy (BSE), which
typically affects cows and is more commonly known as mad cow
disease.
[0085] As previously mentioned, human prion is less common than
animal prion. Human prion has been reported in elderly patients and
is believed to be transmitted to humans via BSE-infected beef.
Accordingly, the quinolinehydrazone compounds can also be used to
identify prion aggregation in humans.
[0086] Any body fluid extracted from the subject can be used for
the assay. Typically, bodily fluid is meant to refer to fluids such
as, for example, lymph, blood, or urine. The preferred bodily fluid
is lymph. In certain preferred embodiments, the bodily fluid is
filtered in vitro such that any prion infectious agent present in
the bodily fluid does not pass through the filter. For example, a
Millipore Ultrafree-MC polysulfone membrane, 3000,0000 NMWL cutoff,
can be used. The filter is contacted with the compound. The
presence of the resulting labeled prion protein is determined using
a SPECT detector, though any other method known to those skilled in
the art to detect the labeled prion can also be used.
[0087] In another aspect, the invention relates to a complex
comprising a compound of formula (I) in association with or bound
to the amyloid fibril or prion particle. As previously described
amyloid fibril and prion particles have demonstrated properties
indicative of or causing disorders such as Alzheimer's and
Crutzfeld-Jacob syndrome. By providing a compound of formula (I)
and allowing the compound to associate with an amyloid fibril or
prion, a complex of the compound with the amyloid fibril or prion
is formed. The complex is useful for the identification of the
amyloid fibril or prion. For example, the complex can include a
radiolabeled compound, which can be identified with a suitable
detection method.
[0088] The invention can be better understood in light of the
following examples which are intended as an illustration of the
practice of the invention and are not meant to limit the scope of
the invention in any way.
EXAMPLE 1
[0089] Determination of In Vivo Brain Distribution: Intraveneous
Dosing
[0090] Transgenic mice, designated "Tg", were obtained. The
transgenic mice carry the human gene for .beta.-APP, a gene
associated with .beta.-amyloid deposition in the brain. The mice
were treated with the quinolinehydrazone compound,
4-methyl-7-methoxy-2-(4-quinolylmethylhydraz- ino)quinoline. Dosing
was administered either intravenously or orally. The intravenous
dose was administered as a single injection (50 .mu.mol/kg).
Orally-treated animals received a single dose (400 .mu.mol/kg) via
oral gavage, or were dosed twice-a-day for 10 days (400
.mu.mol/kg/dose/day).
[0091] Age-matched normal mice served as a negative control, i.e.
no plaque formation in the animals was indicated. The control mice
were treated in the same manner as the transgenic mice, but with
placebo.
[0092] In addition, a group of age-matched Tg mice were treated
with the carrier vehicle only. Age-matched Tg mice served as
negative control indicating that plaque was present, but no drug
was administered.
[0093] All animals were sacrificed beginning 1, 3 and 10 h after
the final dose. Fresh brains of the animals were removed, bisected,
and positioned in o-chlorotoluene (OCT). The brains were immersed
into 2-methylbutane, pre-cooled to 150.degree. C. with liquid
nitrogen. The brains were cut into 20 .mu.m thick sections at a
temperature of -20.degree. C. Brain sections were collected onto
poly-L-lysine-coated glass microscopic slide and treated with (1
drop) of 75%, by volume (v/v), glycerin in
poly(butene-1-sulfone)(PBS). Each section was thawed briefly,
covered with a coverslip, and examined immediately under a Zeiss
LSM510 scanning laser microscope (.lambda..sub.ex is 488 nm;
.lambda..sub.em is greater than 520 nm) to determine the extent and
distribution of compound-associated fluorescence.
[0094] The section adjacent to each compound-treated section of the
brain was stained with thioflavin S (Sigma Chemicals, Milwaukee,
Wis., U.S.A.). Sections stained with thioflavin S served as a
positive control for identifying plaque. The distribution of
compound-associated fluorescence was compared with the distribution
of thioflavin S-stained section in order to confirm whether the
compound bound plaque.
[0095] All sections were examined 1 h later to assess artifactual
drug diffusion within thawed sections over time.
[0096] Adjacent brain sections of compound-treated mice displayed
intense drug-related fluorescence (.lambda..sub.ex is 365 nm;
.lambda..sub.em is 400 nm) in areas of plaque formation. The data
confirm that the quinolinehydrazone compound is brain penetrable
and preferentially binds to human .beta.-amyloid containing
plaques.
[0097] All brain sections obtained from Tg mice receiving the
quinolinehydrazone compound demonstrated marked plaque burden when
compared with sections stained by thioflavin S.
EXAMPLE 2
[0098] Dose-Dependent Pharmacokinetics in Plasma and Brain
Concentrations
[0099] Dose-proportionality characteristics were assessed by
measuring the maxiumum concentration (C.sub.max) for plasma and
brain concentrations after oral dosing of normal mice. The mice
were treated with six, individual, oral doses increased
proportionally from 50 to 3,200 .mu.mole/kg. Plasma and brain
concentrations were measured by HPLC at 0.5 to 24 hours. The test
results showed that C.sub.max for plasma and brain increases
proportionally from 400 to 3,200 .mu.mol/kg without evidence of
absorption saturation.
EXAMPLE 3
[0100] In Vivo Determination of Plasma and Brain Exposure
[0101] Plasma and brain exposure were assessed by measuring area
under the curve (AUC) after oral dosing of normal mice. All mice
were treated with six, individual, oral doses increased
proportionally from 50 to 3,200 .mu.mole/kg. The data showed that
plasma exposure increased proportionally from 421 to 1,472
.mu.mol/kg, but showed evidence of saturation at 3,024 .mu.mol/kg.
Brain exposure increased more rapidly in the range between from 421
to 1,472 .mu.mol/kg, but showed a similar saturation phenomenon at
higher doses.
[0102] The brain to plasma ratios were calculated and reported
below.
1 B/P Dose Plasma Brain Ratio .mu.mole/kg AUC (0 to last) AUC (0 to
last) AUC 51 0.991 5.22 5.3 208 17 54.7 3.2 421 75.2 187 2.5 831
152 863 5.7 1472 262 1490 5.7 3024 235 1714 7.3
[0103] The results summarized in the above table evidence good
penetration of the compound to the brain tissue. As shown above,
brain levels average 5 times greater than plasma levels over the
dose range tested.
[0104] The terminal half-life of compound in the brain was measured
to determine the dosing frequency for 10-day tolerance studies. For
the three highest doses, brain terminal half-life ranged from 5-7
h.
EXAMPLE 4
[0105] Postmortem Staining of Amyloid Plaque
[0106] Brains from 12-14 month-old Tg mice were frozen and
sectioned at -20.degree. C. Each section was fixed in ice cold
methanol (Aldrich Chemical Co., Milwaukee, Wis.) for 10 minutes,
rinsed in buffer and incubated in
4-methyl-7-methoxy-2-(4-quinolylmethylenehydrazino)quinoline for 90
minutes at room temperature. Drug concentrations ranging from 0.01
M to 10 M effectively demonstrated amyloid plaque in brains of Tg
mice, but not in brains of transgene-free mice. Fluorescence
intensity in Tg mouse amyloid decreased with drug concentration.
Staining of the adjacent brains sections with thioflavin S
confirmed that the compound fluorescence does localize in amyloid
plaques. The transgene mouse brains were confirmed to be
plaque-free.
EXAMPLE 5
[0107] Determination of In Vitro Antioxidant Effect using Neuronal
Cytoprotection Model
[0108] The
4-methyl-7-methoxy-2-(4-quinolylmethylene-hydrazino)quinoline was
dissolved in 100 .times. stock of aqueous Locke's buffer to obtain
concentrations of 1, 3, 10, 30, and 100 .mu.M solution. Neuronal
cells were incubated in each compound solution with and without
ferrous ammonium sulfate (200 .mu.m) for 3 hours. High
concentration supernatant was obtained from each sample. The
samples were analyzed under liquid chromatography/mass spectrometry
(LC/MS). The quinoline demonstrated antioxidant properties.
EXAMPLE 6
[0109] Determination of In Vitro Antioxidant Effect using Ferrous
Ammonium Sulfate (FAS)-Induced Oxidative Toxicity Model
[0110] The
4-methyl-7-methoxy-2-(4-quinolylmethylene-hydrazino)quinoline was
tested in a primary neuronal model of FAS-induced lipid
peroxidation toxicity. The compound was treated in two manners. In
one manner, the compound was solubilized as 100.times. stock in
100% dimethyl sulfoxide (DMSO) to afford panel A. In another
manner, the compound was solubilized in aqueous Locke's buffer to
give panel B.
[0111] The data showed that the concentration of the drug
dependently inhibited 200 .mu.M FAS toxicity after 3 hours from
0.3-10 .mu.M and showed close to or complete neuronal protection at
10 .mu.M when cell viability was measured using .sup.14C-AIB uptake
or lactic dehydrogenase (LDH) release. Concentrations above 10
.mu.M (30 and 100 .mu.M) in 1% DMSO experiments revealed inherent
drug toxicity and no neuronal protection against FAS in panel A. In
the is aqueous preparation of the compound there was much less drug
toxicity at 30 .mu.M and considerable neuronal protection at 30 and
100 .mu.M, respectively. Brightfield photographs revealed the cell
morphology of the 10 .mu.M+FAS cells to be no different than cells
treated with no drug and no FAS (control cells).
[0112] The data suggested that antioxidant properties of the
compound could enable the compound for having a dual pharmacology.
Along with its putative ability to inhibit plaque formation in
Alzheimer's disease the compound also could inhibit any oxidative
neuronal damage associated with beta-amyloid.
[0113] The foregoing are intended to illustrate the invention and
are not meant to limit the scope of the invention, which is defined
in the appended claims.
* * * * *