U.S. patent application number 09/984304 was filed with the patent office on 2002-03-07 for components of dense microspheres derived from mammalian brain tissue detectable in cerebrospinal fluid.
Invention is credited to Averback, Paul.
Application Number | 20020028473 09/984304 |
Document ID | / |
Family ID | 22610731 |
Filed Date | 2002-03-07 |
United States Patent
Application |
20020028473 |
Kind Code |
A1 |
Averback, Paul |
March 7, 2002 |
Components of dense microspheres derived from mammalian brain
tissue detectable in cerebrospinal fluid
Abstract
DMS present in the brain of individuals susceptible to cerebral
amyloidosis disintegrate into DMS components to form cerebral
amyloid plaques and other DMS components that are removed from the
brain via circulating bodily fluids. Detecting the presence of
these removed DMS components and/or antibodies thereto in
circulating bodily fluids provides a diagnostic mechanism to
determine the onset of cerebral amyloid plaque formation. Detecting
the presence of these removed DMS components and/or antibodies
thereto in circulating bodily fluids also provides a diagnostic
mechanism to determine the efficacy of treatment regimes for
preventing cerebral amyloid plaque formation. Antibodies also can
be raised against isolated DMS components and subsequently utilized
in a diagnostic method capable of detecting the onset of cerebral
amyloid plaque formation.
Inventors: |
Averback, Paul;
(Beaconsfield, CA) |
Correspondence
Address: |
Stephen A. Bent
FOLEY & LARDNER
Washington Harbour
3000 K Street, N.W., Suite 500
Washington
DC
20007-5109
US
|
Family ID: |
22610731 |
Appl. No.: |
09/984304 |
Filed: |
October 29, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09984304 |
Oct 29, 2001 |
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08660886 |
Jun 10, 1996 |
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6309892 |
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08660886 |
Jun 10, 1996 |
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08168250 |
Dec 17, 1993 |
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5525339 |
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08168250 |
Dec 17, 1993 |
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08077641 |
Jun 17, 1993 |
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08077641 |
Jun 17, 1993 |
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07493276 |
Mar 14, 1990 |
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5231170 |
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07493276 |
Mar 14, 1990 |
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07315796 |
Feb 27, 1989 |
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4919915 |
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07315796 |
Feb 27, 1989 |
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07021242 |
Mar 3, 1987 |
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4816416 |
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07021242 |
Mar 3, 1987 |
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06901007 |
Aug 27, 1986 |
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Current U.S.
Class: |
435/7.21 ;
530/350 |
Current CPC
Class: |
Y10S 530/839 20130101;
G01N 33/5005 20130101; G01N 33/5088 20130101; C07H 19/06 20130101;
G01N 2333/4709 20130101; G01N 2800/2821 20130101; G01N 2500/00
20130101; A61K 31/40 20130101; A61K 31/655 20130101; A61K 31/505
20130101; A61K 31/55 20130101; A61K 31/70 20130101; A61K 31/35
20130101; A61K 31/63 20130101; A61K 31/42 20130101; C07K 14/4711
20130101; G01N 33/6896 20130101; C07H 17/08 20130101 |
Class at
Publication: |
435/7.21 ;
530/350 |
International
Class: |
G01N 033/567; C07K
014/00 |
Claims
What is claimed is:
1. A composition of matter consisting essentially of at least one
dense microsphere (DMS) component having a molecular weight
selected from the group consisting of 25-27, 28-30, 45-47, 50-53,
55-57, 60-65 and 76-80 kilodaltons.
2. A composition according to claim 1, wherein said at least one
component is a membrane component.
3. A component according to claim 1, that is the product of a
process comprising the steps of (a) obtaining a sample of
cerebrospinal fluid from a mammal susceptible to or suffering from
Alzheimer's disease to provide a solution comprising DMS
components; (b) separating the DMS components in the solution on a
gel by polyacrylamide gel electrophoresis; and then (c) isolating a
band that corresponds to a component, wherein said bane is located
at a kilodalton region of said gel selected from the group
consisting of 25-27, 28-30, 45-47, 50-53, 55-57, 60-65 and 76-80
kilodaltons.
4. A composition consisting essentially of antibody which
specifically binds an isolated component of DMS according to claim
1.
5. A method for diagnosing cerebral plaque formation, Alzheimer's
disease and related conditions, comprising the steps of (a)
bringing a biological sample into contact with a first antibody
directed against at least one component of DMS having a molecular
weight selected from the group consisting of 25-27, 28-30, 45-47,
50-53, 55-57, 60-65 and 76-80; and (b) determining whether said
first antibody reacts immunologically with said biological
sample.
6. A method according to claim 5, wherein step (b) comprises the
steps of (i) reacting said first antibody with said sample to form
antibody/component complexes; (ii) washing said sample to remove
any unbound first antibody; (iii) contacting said sample of step
(ii) with a labelled second antibody which binds specifically to
said first antibody; (iv) washing said sample of step (iii) to
remove any unbound labelled second antibody; and then (v) detecting
the presence of bound labelled second antibody.
7. A composition of matter consisting essentially of at least one
dense microsphere (DMS) component having a molecular weight
selected from the group consisting of 55-60, 62-70 and 82-90
kilodaltons.
8. A composition according to claim 7, wherein said at least one
component is a membrane component.
9. A component according to claim 7, that is the product of a
process comprising the steps of (a) obtaining a sample of
cerebrospinal fluid from a mammal susceptible to or suffering from
Alzheimer's disease to provide a solution comprising DMS
components; (b) separating the DMS components in the solution on a
gel by polyacrylamide gel electrophoresis; and then (c) isolating a
band that corresponds to a component, wherein said bane is located
at a kilodalton region of said gel selected from the group
consisting of 55-60, 62-70 and 82-90 kilodaltons.
10. A composition consisting essentially of antibody which
specifically binds an isolated component of DMS according to claim
7.
11. A method for diagnosing cerebral plaque formation, Alzheimer's
disease and related conditions, comprising the steps of (a)
bringing a biological sample into contact with at least one
component of DMS having a molecular weight of 55-60, 62-70 and
82-90 kilodaltons; and then (b) determining whether said component
reacts immunologically with said biological sample.
12. A method according to claim 11, wherein step (b) comprises the
steps of (i) washing said sample to remove any unbound components;
(ii) contacting a first antibody which is present in said sample,
said first antibody being bound to said component, with a labelled
second antibody which binds specifically to said first antibody;
(iii) washing said sample to remove any unbound second antibody;
and then (iv) detecting the presence of bound second antibody.
13. A composition of matter comprising a compound and a
pharmaceutically acceptable vehicle wherein said compound is
effective in decreasing the type and concentration of at least one
DMS component or anti-DMS antibody in cerebrospinal fluid when
administered to a mammal susceptible to or suffering from
Alzheimer's disease.
14. A method of preventing the formation of cerebral amyloid
plaque, inhibiting the disruption of DMS in the brain of a mammal
or treating a mammal susceptible to or suffering from Alzheimer's
disease comprising administering to said mammal a therapeutically
effective amount of a composition as claimed in claim 13 for a
therapeutically effective period of time.
15. The method as claimed in claim 14, wherein said composition is
administered periodically.
Description
[0001] This is a continuation-in-part application based on U.S.
Ser. No. 08/168,250 (filed Dec. 17, 1993), which is a
continuation-in-part based on U.S. Ser. No. 08/077,641 (filed Jun.
17, 1993), which is a continuation of U.S. Ser. No. 07/493,276
(filed Jun. 14, 1990), now U.S. Pat. No. 5,231,170, which is a
continuation-in-part of U.S. Ser. No. 07/315,796 (filed Feb. 27,
1989), which is a continuation of U.S. Ser. No. 07/021,242 (filed
Mar. 3, 1987), now U.S. Pat. No. 4,816,416, which in turn is a
continuation-in-part of U.S. Ser. No. 06/901,007 (filed Aug. 27,
1986), now abandoned. The respective contents of the aforementioned
prior applications are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to compositions consisting
essentially of dense microsphere (DMS) components. The invention
also relates to methods for detecting components of DMS in blood,
cerebrospinal fluid and other body fluids that result from the
disruption of DMS in the brain parenchyma. More specifically, the
present invention relates to the correlation of the presence of DMS
components in such fluids and the onset or progression of the
cerebral amyloid formation associated with Alzheimer's disease and
related conditions.
[0003] The methodology of the present invention is useful for
diagnosing cerebral amyloidosis and, consequently, the onset and
progression of Alzheimer's disease and related conditions and for
monitoring the progression of such disease conditions and the
effectiveness of therapeutic regimes. In particular, the present
invention relates to methods for the detection of protein and
non-protein components of DMS and antibodies thereto which, when
detected in cerebrospinal fluid, blood or other body fluids, signal
the onset or progression of cerebral amyloid plaque formation.
[0004] Classified under the rubric "amyloidosis" are a number of
pathological conditions characterized by the deposition of abnormal
fibrils ("amyloid fibrils") in extracellular spaces. The amyloid
fibril, in turn, represents a final common pathway for a diverse
array of proteins. Regardless of their biochemical composition,
however, all types of amyloid fibrils share (a) a .beta.-pleated
sheet structure, (b) green birefringence under polarized light
after staining with Congo Red dye, and (c) a fibrillar morphology
which has a typical electron-microscopic appearance.
[0005] The deposition of amyloid fibrils can affect several organs
in the systemic forms of the disorder, exemplified by familial
Mediterranean fever, familial amyloid polyneuropathy and systemic
amyloidosis, or it can be restricted to one organ in localized
forms. Among the latter are conditions classified under the rubric
"cerebral amyloidosis," which covers the Alzheimer group of
diseases, namely, Alzheimer's disease [pre-senile dementia, senile
dementia]; Alzheimer's disease associated with Down's syndrome;
Alzheimer's disease associated with other central-nervous-system
diseases, such as Parkinson's disorder; and congophilic angiopathy
[associated or not associated with Alzheimer's disease].
[0006] Alzheimer's disease in general is an incurable brain disease
affecting middle aged and elderly people throughout the world.
According to most recent estimates, it is the fourth or fifth
leading cause of death among North Americans, and is responsible
for inestimable personal and social tragedy, loss of productivity,
and custodial burden to society. There is presently no
widely-accepted effective treatment for Alzheimer's disease.
[0007] The principle symptom (manifestation) of Alzheimer's disease
is the loss of higher mental faculties, typified by the loss of
memory and behavior referred to as "dementia." Dementia is a
symptom or syndrome that can be seen in many brain diseases other
than Alzheimer's disease, such as stroke, encephalitis and
metabolic diseases. Since memory loss and dementia are relatively
nonspecific symptoms, a certain and specific definition of
Alzheimer's disease is based on the characteristic microscopic
state of the brain, described initially by Marinesco, Alzheimer and
others. See Alzheimer, A., Allegemeine Zeitschrift fur Psychiatrie
64:146-148 (1907); Marinesco, G., Comptes Rendus des Seances de la
Societe de Biologie et ses Filiales 70:606-608 (1911).
[0008] The particular microscopic feature that is a universally
accepted indicator of Alzheimer's disease, and that separates
Alzheimer's disease from other causes of dementia, is the
accumulation of large numbers of brain lesions referred to as
senile or amyloid plaques and neurofibrillary tangles. Senile or
amyloid plaques are spherical, ranging from 10 to 200 .mu.m in
diameter, and while found only occasionally in aged adult cerebral
cortex, are found in large numbers in Alzheimer-affected cortex.
These lesions, when found in suitable quantity in a brain sample,
are the definitive criteria for the diagnosis of Alzheimer's
disease.
[0009] The clinical diagnosis of Alzheimer's disease often is a
difficult and imperfect task that generally relies initially on
ruling out other treatable or clinically definable causes of
dementia. In the appropriate clinical context, if the latter causes
cannot be proven, Alzheimer's disease is often diagnosed
antemortem, by exclusion, as the most probable diagnosis. Many
indirect methods of diagnosis at present are being proposed and
tested. See Conference Report, Khachaturian, Z., Arch. Neurol.
42:1097-1105 (1985). But the only certain and acceptable method for
diagnosing Alzheimer's disease is by tissue microscopic
histological study of a brain biopsy or necropsy sample, in which
the above-mentioned sine qua non lesions are recognized by a
certified specialist.
[0010] Amyloid plaques in large quantities are essentially found
only in the Alzheimer group of diseases, whereas neurofibrillary
tangles are nonspecific and are found in at least ten other
neurological diseases. See Corsellis, J. A. N., GREENFIELD'S
NEUROPATHOLOGY 951-1025 (4th ed. 1984) (Edward Arnold, London).
Individual amyloid plaques have roughly 1000.times. the volume of
individual neurofibrillary tangles. True measurements of total
brain amyloid plaque and neurofibrillary content are not available,
but on the above basis it is likely that the volume of abnormal
brain tissue occupied by amyloid plaques is many hundreds of times
that of neurofibrillary tangles. The essential feature of the
amyloid plaque is the presence of amiyloid fibrils, which are a
congophilic red-green birefringent microfibrillar material.
Corsellis, loc. cit.
[0011] A microscopic structure referred to as the dense microsphere
(DMS) is known to exist in normal brain and in brain affected by
Alzheimer's disease. See Averback, Acta Neuropathol. 61:148-52
(1983); results confirmed by Hara, J. Neuropath. Exp. Neurol.
45(2):169-178 (1986). Some specialists believe that DMS are linked
to cerebral amyloid plaques as the source or as a precursor to the
cerebral amyloid characteristic of Alzheimer's disease and related
conditions. Evidence for the existence of DMS comes from
microscopic histological section studies of fixed whole brain
tissue, where the dense microspheres are seen to have a
proteinaceous internal or central region ("DMS protein") surrounded
by continuous membrane ("DMS membrane"). The dense microspheres are
observed as randomly dispersed, highly infrequent structures
occupying an estimated 10.sup.-9 or less of total brain volume, at
a unit frequency roughly estimated at 10.sup.-16 or less, relative
to other definable brain structures such as mitochondria.
[0012] The extraction, purification and characterization of
isolated samples of DMS nor the use of DMS material to any
advantage has only recently been documented. Thus, DMS and its
respective components are structures of unproven function and
unknown significance or usefulness, and have been effectively
unavailable in tangible form.
[0013] It is known that DMS, when disrupted in vitro will generate
a plurality of components that can be identified using
polyacrylamide gel electrophoresis. It is believed that the
presence of these components in the cerebrospinal fluid and other
body fluids indicates the disruption of DMS in the brain, and
consequently, the onset of amyloid plaque formation. Some of the
DMS components, however, may be responsible for the formation of
amyloid plaques and hence, may not be present in cerebrospinal
fluid. Detection of the specific DMS components which are present
in cerebrospinal fluid has not been documented.
[0014] Moreover, non-surgical procedures for the antemortem
diagnosis of cerebral amyloid plaque formation and, consequently,
Alzheimer's disease and related conditions have heretofore been
limited to the exclusionary process of diagnosis. Confirmation of
such disease conditions has been possible only through analysis of
brain tissue obtained by biopsy or at autopsy. Immunological
methods universally accepted as diagnostic of Alzheimer's disease
and related conditions have not heretofore been documented.
SUMMARY OF THE INVENTION
[0015] It is therefore an object of the present invention to
provide a method for the detection of components of DMS and
antibodies thereto in biological samples for the purpose of
diagnosing cerebral plaque formation, Alzheimer's disease and
related conditions.
[0016] It also is an object of the present invention to provide a
method of monitoring the progression of cerebral plaque formation,
Alzheimer's disease and related conditions by a quantitative
analysis of the DMS component content of biological samples.
[0017] Another object of the present invention is to provide a
method of determining the therapeutic efficacy of a treatment
regime for Alzheimer's disease and related conditions by
quantitatively analyzing biological samples to determine the
content of DMS components and antibodies thereto.
[0018] Another object of the present invention is to provide a
composition of matter that includes a compound and a
pharmaceutically acceptable carrier that is capable of decreasing
the amount and/or concentration of DMS components in the
cerebrospinal fluid. It is an additional object of the present
invention to provide a method of treating Alzheimer's disease,
preventing the formation of amyloid plaque and/or preventing the
disruption of DMS in a mammal's brain by administering to a mammal
suffering from or susceptible to Alzheimer's disease, a composition
of matter that includes a compound and a pharmaceutically
acceptable carrier that is capable of decreasing the amount and/or
concentration of DMS components in the cerebrospinal fluid.
[0019] It is yet another object of the present invention to provide
components of DMS, as well as antibodies against such components,
which can be used in detecting the onset and progression of
cerebral plaque formation, Alzheimer's disease and related
conditions, even when the individual may not yet have developed
clinical symptoms associated with Alzheimer's disease and related
conditions.
[0020] In accomplishing the foregoing objects, a composition of
matter has been provided, in accordance with one aspect of the
present invention, wherein the composition consists essentially of
DMS components that are detectable in cerebrospinal fluid. In
accordance with a further aspect of the present invention, the
dense microsphere components are membrane components.
[0021] According to another aspect of the present invention, a
component of dense microspheres is provided that is the product of
a process comprising the steps of:
[0022] (a) disrupting or digesting a suspension comprising DMS;
[0023] (b) separating the components of the disrupted or digested
suspension by polyacrylamide gel electrophoresis; and
[0024] (c) isolating the components from the band located at the 5,
8, 10, 12, 16, 19, 21, 25, 28, 30, 31, 33, 40, 45, 48, 52, 60, 66,
70, 80, 100, 110 and 120 kilodalton region of the gel.
[0025] According to yet another aspect of the present invention, a
component of dense microspheres is provided that is the product of
a process comprising the steps of:
[0026] (a) obtaining a biological sample of cerebrospinal
fluid;
[0027] (b) separating the components present in the cerebrospinal
fluid by polyacrylamide gel electrophoresis; and
[0028] (c) isolating the components from the band located at the
25-27; 28-30; 45-47; 50-53; 55-57; 60-65 and 76-80 kilodalton
region of the gel.
[0029] In accordance with yet another aspect of the present
invention, a composition consisting essentially of an antibody
which specifically binds an isolated component of DMS is
provided.
[0030] According to a further aspect of the present invention, a
composition is provided that consists essentially of an antibody
which specifically binds an isolated component of DMS, where the
component is a product of a process comprising the steps of:
[0031] (a) disrupting or digesting a suspension comprising DMS;
[0032] (b) separating the components of the disrupted or digested
suspension by polyacrylamide gel electrophoresis; and
[0033] (c) isolating the components from the band located at the 5,
8, 10, 12, 16, 19, 21, 25, 28, 30, 31, 33, 40, 45, 48, 52, 60, 66,
70, 80, 100, 110 and 120 kilodalton region of the gel is
provided.
[0034] According to a further aspect of the present invention, a
composition is provided that consists essentially of an antibody
which specifically binds an isolated component of DMS, where the
component is a product of a process comprising the steps of:
[0035] (a) obtaining a biological sample of cerebrospinal
fluid;
[0036] (b) separating the components present in the cerebrospinal
fluid by polyacrylamide gel electrophoresis; and
[0037] (c) isolating the components from the band located at the
25-27; 28-30; 45-47; 50-53; 55-57; 60-65 and 76-80 kilodalton
region of the gel.
[0038] In accordance with yet another aspect of the present
invention, a composition consisting essentially of an antibody
which specifically binds an isolated component of DMS is
provided.
[0039] In accordance with yet another aspect of the present
invention, there is provided a composition consisting essentially
of an antibody that specifically binds an isolated component of
dense microspheres, the composition further comprising a label
selected from the group consisting of a radionuclide, a
calorimetric agent and a fluorescent marker.
[0040] In accordance with yet another aspect of the present
invention, a method for diagnosing cerebral plaque formation,
Alzheimer's disease and related conditions is provided, comprising
the steps of (a) bringing a biological sample into contact with a
first antibody directed against at least one component of dense
microspheres; and (b) determining whether the first antibody reacts
immunologically with the biological sample.
[0041] Pursuant to still another aspect of the present invention, a
method for diagnosing cerebral plaque formation, Alzheimer's
disease and related conditions is provided, wherein step (a)
comprises bringing a biological sample into contact with a first
antibody directed against at least one component of dense
microspheres; and step (b) comprises the steps of (i) reacting the
first antibody with the sample to form antibody/component
complexes; (ii) washing the sample to remove any unbound first
antibody; (iii) contacting the sample with a labelled second
antibody which binds specifically to the first antibody; (iv)
washing the sample to remove any unbound labelled second antibody;
and then (v) detecting the presence of bound labelled second
antibody.
[0042] In accordance with a further aspect of the present
invention, a method for diagnosing cerebral plaque formation,
Alzheimer's disease and related conditions is provided, comprising
the steps of (a) bringing a biological sample into contact with at
least one component of dense microspheres; and then (b) determining
whether the component reacts immunologically with the biological
sample.
[0043] According to yet a further aspect of the present invention,
a method for diagnosing cerebral plaque formation, Alzheimer's
disease and related conditions is provided, comprising the steps of
(a) bringing a biological sample into contact with at least one
component of dense microspheres; and then (b) comprises the steps
of (i) washing the sample to remove any unbound components; (ii)
contacting a first antibody which is present in the sample, the
first antibody being bound to the component, with a labelled second
antibody which binds specifically to the first antibody; (iii)
washing the sample to remove any unbound second antibody; and then
(iv) detecting the presence of bound second antibody.
[0044] In accordance with yet a further aspect of the present
invention, a method for diagnosing cerebral plaque formation,
Alzheimer's disease and related conditions is provided, comprising
the steps of (a) bringing a biological sample into contact with at
least one component of dense microspheres; and then (b) comprises
the steps of (i) washing the sample to remove any unbound
components; (ii) contacting a first antibody which is present in
the sample, the first antibody being bound to the component, with a
labelled second antibody which binds specifically to the first
antibody; (iii) washing the sample to remove any unbound second
antibody; (iv) detecting the presence of bound second antibody; and
then (v) determining the class of the first antibody.
[0045] In accordance with another aspect of the present invention,
a method for diagnosing cerebral plaque formation, Alzheimer's
disease and related conditions is provided, comprising the steps of
(a) bringing a biological sample into contact with at least one
component of dense microspheres; and then (b) comprises the steps
of (i) washing the sample to remove any unbound components; (ii)
contacting a first antibody which is present in the sample, the
first antibody being bound to the component, with a labelled second
antibody which binds specifically to the first antibody; (iii)
washing the sample to remove any unbound second antibody; (iv)
detecting the presence of bound second antibody; (c) repeating step
(b) periodically over time; and (d) determining the quantity and
class of antibody directed against the component.
[0046] In accordance with yet another embodiment of the present
invention, there is provided a method for diagnosing cerebral
plaque formation, Alzheimer's disease and related conditions,
comprising the steps of
[0047] (a) bringing a biological sample into contact with a first
antibody directed against at least one component of dense
microspheres;
[0048] (b) determining whether said first antibody reacts
immunologically with said biological sample;
[0049] (c) determining the quantity of component of dense
microspheres present in said biological sample; and
[0050] (d) repeating steps (a), (b) and (c) periodically and
measuring any change in the quantity of component of dense
microspheres.
[0051] In accordance with a further embodiment of the present
invention, there is provided a method for diagnosing cerebral
plaque formation, Alzheimer's disease and related conditions,
comprising the steps of
[0052] (a) bringing a biological sample into contact with at least
one component of dense microspheres;
[0053] (b) determining whether said component reacts
immunologically with said biological sample indicating the presence
of anti-DMS components in said biological sample;
[0054] (c) determining the quantity of anti-DMS components present
in said biological sample; and
[0055] (d) repeating steps (a), (b) and (c) periodically and
measuring any change in the quantity of anti-DMS component.
[0056] In accordance with another object of the invention there is
provided a method of determining the efficacy of an Alzheimer's
disease treatment regime comprising the steps of (a) obtaining a
sample of cerebrospinal fluid from a patient that is subjected to
an Alzheimer's disease treatment regime and (b) measuring the type
and quantity of DMS components present in the fluid.
[0057] In accordance with a further object of the present
invention, there is provided a composition of matter comprising a
compound that is effective in preventing the formation of amyloid
plaques as measured by a decrease in the quantity and type of DMS
components present in cerebrospinal fluid of a patient that to whom
the composition of matter had been administered.
[0058] In accordance with yet another object of the present
invention, there is provided a method of treating Alzheimer's
disease, a method of preventing the formation of amyloid plaques
and a method of preventing the disruption of DMS in a mammal's
brain comprising the step of administering to a mammal a
composition of matter comprising a compound that is effective in
preventing the formation of amyloid plaques as measured by a
decrease in the quantity and type of DMS components present in
cerebrospinal fluid of the mammal.
[0059] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description. Unless otherwise specified, the
respective contents of documents cited in the following description
are hereby incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0060] FIG. 1 illustrates a polyacrylamide gel including DMS
components obtained by disrupting DMS in vitro and separating the
components by molecular weight.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0061] It has been discovered that the disintegration of DMS in the
brain parenchyma is associated with the onset and progression of
cerebral amyloid plaque formation which is characteristic of
Alzheimer's disease and related conditions. More specifically,
disintegration of DMS releases protein and non-protein components,
or mixtures of protein and non-protein components. A portion of the
DMS components form cerebral amyloid plaques, while other portions
are removed from the brain parenchyma via circulating bodily
fluids, including cerebrospinal fluid, serum and the like.
[0062] The presence of DMS components in circulating bodily fluids
thus is correlated positively with DMS disintegration and the
associated onset of cerebral amyloid plaque formation. Moreover,
since DMS are found in the brain, the presence of DMS antigens in
circulating bodily fluids and tissue other than brain tissue is
correlated positively with DMS disintegration and the associated
onset of cerebral amyloid plaque formation. In addition, since DMS
are found in the brain, the presence of DMS antigens in circulating
bodily fluids will generate autoantibodies in these circulating
bodily fluids and tissue other than brain tissue, the generation of
which is correlated positively with DMS disintegration and the
associated onset of cerebral amyloid plaque formation.
[0063] Accordingly, the detection of DMS components and DMS
component antibodies in bodily fluids indicates active cerebral
amyloid formation. Isolated DMS components pursuant to the present
invention can be used as immunogens to produce antibodies which can
be employed in conventional immunoassays to detect DMS components
in biological samples. Alternatively, DMS components can be
utilized in such immunoassays to detect the presence of antibodies
directed against DMS components in, for example, serum and other
biological fluids. Thus, immunoassays based on DMS components and
antibodies thereto can be used in the diagnosis of plaque
formation, particularly, cerebral amyloid plaque formation,
Alzheimer's disease and related conditions. Such immunoassays can
also be employed in monitoring such disease conditions and
determining the efficacy of therapeutic regimes by measuring the
type and quantity of DMS component and/or DMS component antibody
present in circulating bodily fluids.
[0064] Throughout this description, the phrase "Alzheimer's disease
and related conditions" denotes conditions classified under the
rubric "cerebral amyloidosis."Such conditions include, but are not
limited to Alzheimer's disease [pre-senile dementia, senile
dementia]; Alzheimer's disease associated with Down's syndrome;
Alzheimer's disease associated with other central-nervous-system
diseases, such as Parkinson's disorder; and congophilic angiopathy
[associated or not associated with Alzheimer's disease]. Throughout
this description, the phrase "DMS components" denotes any component
of DMS, protein, non-protein, or mixtures of protein and
non-protein, originating from the internal or central region or
from the outer membrane portion of DMS. Throughout this
description, the phrase "disrupting" or "digesting a suspension
comprising DMS" denotes any process whereby DMS are broken down
into DMS components.
[0065] DMS present in mammalian brain tissue have an increased
tendency to disintegrate into DMS components in individuals
susceptible to cerebral amyloidosis. Some of these DMS components
are believed to be responsible for forming cerebral amyloid
plaques. In addition, when DMS are isolated from mammalian brain
tissue in vitro, they have the same tendency to disintegrate.
Hence, the disruption of DMS that naturally occurs in individuals
susceptible to cerebral amyloidosis can be replicated by isolation
of DMS and dissolution in vitro. Furthermore, when a solution
containing DMS is injected into an animal for the purpose of
raising antibodies against the DMS, the DMS will break down, or
disintegrate into DMS components. The antibodies present in the
resulting animal serum therefore will be reactive against DMS
components.
[0066] DMS components can be identified by obtaining a sample of
isolated DMS, disrupting the DMS in vitro to simulate the
disruption of the DMS in the brain, and then measuring the type and
quantity of the DMS components. These components include, for
example, components separated on a gel by polyacrylamide gel
electrophoresis and isolated, where the band corresponds to a DMS
component. The band that is isolated on the gel can be located at a
kilodalton region of the gel selected from 5, 8, 10, 12, 16, 19,
21, 25, 28, 30, 31, 33, 40, 45, 48, 52, 60, 66, 70, 80, 100, 110
and 120.
[0067] That DMS disintegration is the cause of cerebral plaque
formation is supported by the observation that cerebral amyloid
plaques appear not only when DMS disappear, but also where DMS-
disappear; that is, in about the same position in the brain as the
now-disrupted DMS. Although the present invention is not restricted
by any theory, the protein components of DMS, located in the
central or internal region thereof, are believed to be the primary
source of the cerebral amyloid plaques. In addition, the DMS
membrane components are believed to comprise protein and
non-protein matter, as noted above. Accordingly, the presence of
DMS membrane components, protein, non-protein or mixtures thereof
in bodily fluid will indicate the onset of cerebral plaque
formation.
[0068] Because some of the DMS components may be responsible for
the formation of amyloid plaques and hence, the onset of
Alzheimer's disease, it is possible that not all of the DMS
components identified by disrupting the DMS in vitro will be
present in an identifiable quantity in cerebrospinal fluid and
other circulating bodily fluids. The present inventors have found
that DMS components present in circulating bodily fluids can be
detected by either bringing a solution containing the DMS
components in contact with a laboratory made anti-DMS component
antibody, or by bringing a bodily fluid containing anti-DMS
component antibodies into contact with a solution including
laboratory derived DMS components. By carrying out polyacrylamide
gel electrophoresis as described herein, the DMS components
actually present in identifiable quantities in circulating bodily
fluids can be identified using either method. Using the former
method, the DMS components can be separated and a band that
corresponds to a DMS component can be isolated, whereby the band is
located at a kilodalton region of the gel selected from 25-27,
28-30, 45-47, 50-53, 55-57, 60-65 and 76-80 kilodaltons. Using the
latter method, the DMS components can be separated and a band that
corresponds to a DMS component can be isolated, whereby the band is
located at a kilodalton region of the gel selected from 55-60,
62-70 and 82-90 kilodaltons.
[0069] In addition to the present inventors belief that circulating
bodily fluids may not include all of the DMS components,
circulating bodily fluids also already may contain a certain
quantity of DMS components without any onset of cerebral plaque
formation. In this case, the method of the present invention
measures the quantity of DMS components in the bodily fluid
periodically. Any dramatic change in the amount of DMS components
present therein will indicate the onset of cerebral plaque
formation.
[0070] Circulating bodily fluids also serve as a convenient source
of measuring the efficacy of therapeutic regimes that purport to
reduce and/or prevent the formation of amyloid plaques. If a
treatment regime, i.e., administration of a therapeutic agent to a
mammal in need thereof, prevents the formation of amyloid plaque,
then the concentration and type of DMS components present in the
bodily fluids will be altered. Hence, the efficacy of the treatment
regime can be evaluated simply by measuring the effectiveness of
the treatment in reducing the type and concentration of DMS
components in the bodily fluids.
[0071] The present invention therefore also encompasses a method of
treating Alzheimer's disease, a method of preventing the formation
of amyloid plaque and a method of preventing the disruption of DMS
in a mammal's brain by administering to a mammal a therapeutically
effective amount and for a therapeutically effective period of
time, a composition of matter that includes a compound effective in
treating Alzheimer's disease, preventing the formation of amyloid
plaque and/or preventing the disruption of DMS in the brain. The
therapeutically effective amount of the compound and the
therapeutically effective period of treatment can be readily
ascertained by an ordinarily skilled practitioner in this art. The
effectiveness of this compound can be ascertained by quantifying
the type and concentration of DMS components present in the
cerebrospinal fluid of the mammal.
[0072] DMS are derived form mammalian brain tissue and are
characterized, in essentially homogeneous form, by a range of
diameters from about 0.1 .mu.m to about 15 .mu.m, by the
above-mentioned outer membrane/proteinaceous core structure of DMS,
and by certain stainability properties. In this regard,
"homogeneous" means that the DMS represent the only structure
discernible in the subject composition at the light-microscopic
level. For example, the microspheric bodies of the present
invention are homogeneously electron-dense when stained with osmium
and lead, and can be visualized by thin-section electron
microscopy; under optical microscopic examination, they appear
eosinophilic and phloxinophilic, and are nonbirefringent when
stained with Congo Red. When the microspheric bodies of the present
invention are disrupted or disintegrated or digested, a material is
produced that displays congophilic birefringence; that is, when
stained with Congo Red the material becomes optically anisotropic
to the extent of splitting an incident light wave into two waves
with mutually perpendicular vibrational planes.
[0073] DMS are spherical, membrane-bounded, intracellular
structures, about 0.1 .mu.m to 15 .mu.m in diameter, that are found
in human and other mammalian brains. More specifically, the normal
location for DMS is in gray-matter neuropil, where the spherical
structures are enclosed in tiny, neuronal cellular processes. DMS
are solitary, non-perikaryal and non-confluent, and are not found
in cerebellum or in white matter. With regard to inter-DMS
distances, the spatial distribution of DMS in gray matter regions
is random. Compositions of DMS in homogeneous form can be produced
by extraction to give homogeneous samples of globular bodies
according to procedures described in U.S. Pat. Nos. 4,816,416 and
5,231,170, the entire contents of which are incorporated by
reference herein.
[0074] The homogeneous composition of dense microspheres prepared
according to the above-described procedure can be disrupted by
procedures described in the aforementioned U.S. Pat. Nos. 4,816,416
and 5,231,170, and then subjected to differential gradient
centrifugation as described therein to separate DMS membrane
components from DMS internal components, if desired. Materials
isolated in distinct sedimentation layers are stained with Congo
Red. The nonbirefringent membrane components can be distinguished
from birefringent internal components by microscopic examination.
Hence, the present inventor believes that the birefringent internal
components are primarily responsible for the formation of cerebral
amyloid plaques. Alternatively, DMS membrane components can be
distinguished from DMS internal components by electron microscopic
examination. The protein and lipid components of DMS membranes can
be isolated by conventional extraction methods. Further analysis of
lipid DMS membrane components can be accomplished by extraction of
such components with organic solvents and by conventional methods
such as chromatography that are well-known to those of ordinary
skill in the art.
[0075] DMS can be treated by a variety of methods to yield DMS
components (protein internal components, protein membrane
components, non-protein membrane components, or protein and
non-protein membrane components) suitable for use according to the
present invention. Exemplary of these methods are: (a) PAGE buffer
solutions including TRIS, glycerol, .beta.-mercaptoethanol,
bromophenol blue and sodium dodecyl sulfate (SDS), (b)
ultrasonication and (c) other proteolytic treatments such as
treating with various combinations of 0.25M acetic acid, 6M
guanidine HCl, formic acid, 6M urea, pepsin and cyanogen bromide.
The resulting homogeneous composition of DMS components can be
further refined by isolating the components according to their
molecular weight by polyacrylamide gel electrophoresis (PAGE) or
according to the degree of their hydrophobicity by reverse phase
high performance liquid chromatography (rpHPLC). DMS components
isolated by PAGE can be further characterized as either discrete
migrating or non-migrating components. DMS components also can be
extracted from cerebrospinal fluid and other bodily fluids using
the extraction procedures described above.
[0076] In accordance with the present invention, DMS components
isolated by PAGE or rpHPLC can be employed in conventional
immunoassays to detect the presence of antibodies to DMS components
in serum, cerebrospinal fluid and other fluids or tissues.
Immunoassays within the present invention include but are not
limited to, ELISA-type assays [see, for example, VOLLER et al., THE
ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) (Dynatch Laboratories,
Inc. 1979)], Western Blot, and other immunological tests, such as
radioimmunoassays and conventional immunoprecipitin assays, for
detecting DMS components in biological samples. See Ausbel et al.,
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons, New
York 1990), the contents of which are incorporated by
reference.
[0077] Via conventional techniques, as described, for example, by
Kennet et al., Curr. Top. Microbiol. Immunol. 81:77-91(1978),
anti-DMS component antibodies can be produced using the isolated
DMS components of the present invention and then "labelled" with a
radionuclide, a calorimetric agent or a fluorescent marker. The
labelled antibodies can be used in diagnostic tests to screen for
the presence of DMS components in serum, cerebrospinal fluid and
other fluids and tissues. The presence of such components in these
bodily fluids is indicative of cerebral DMS disintegration and the
associated onset of cerebral amyloid plaque formation. A decrease
in type and quantity of DMS components in these bodily fluids
likewise in indicative of the effectiveness of a therapeutic
regimen. In this context, the term "antibody" encompasses
monoclonal and polyclonal antibodies. Such an antibody can belong
to any antibody class (IgG, IgM, IgA, etc.). For monoclonal
antibody (Mab) production, one generally proceeds by isolating
lymphocytes and fusing them with myeloma cells, producing
hybridomas. The cloned hybridomas are then screened for production
of "anti-DMS component" antibodies, that is, antibodies that bind
preferentially to a DMS component. "Antibody" also encompasses
fragments, like Fab and F(ab').sub.2, of anti-DMS antibodies, and
conjugates of such fragments, and so-called "antigen binding
proteins" (single-chain antibodies) which are based on anti-DMS
antibodies, in accordance, for example, with U.S. Pat. No.
4,704,692, the disclosure of which is hereby incorporated by
reference in its entirety.
[0078] Alternatively, Mabs or fragments thereof within the present
invention can be produced using isolated DNA which codes for
variable regions of such an Mab in host cells like E. coli, see
Ward et al., Nature 341:544-546 (1989), or transfected murine
myeloma cells, see Gillies et al., Biotechnol. 7:799-804 (1989),
and Nakatani et al., loc. cit., 805-810. In addition, Fab molecules
can be expressed and assembled in a genetically transformed host
like E. coli. A lambda vector system is available thus to express a
population of Fabs with a potential diversity equal to or exceeding
that of the subject generating the predecessor antibody. See Huse
et al., Science 246:1275-1281 (1989).
[0079] Antibodies against DMS components can also be employed in
the generation, via conventional methodology, of anti-idiotypic
antibodies (antibodies that bind an anti-DMS antibody), for
instance, by the use of hybridomas as described above. See, for
example, U.S. Pat. No. 4,699,880.
[0080] The above-described materials, including DMS components and
antibodies thereto that react specifically to indicate the presence
of DMS components or anti-DMS antibodies, can thus be employed in
immunoassays which can be used to detect DMS disruption, or
disintegration in the brain, and which can be used to detect the
efficacy of a treatment regimen. Such testing could be used, in
accordance with the present invention, in the context of providing
a method for the detection of such materials for the purpose of
diagnosing and monitoring cerebral plaque formation, particularly,
cerebral amyloid plaque formation, Alzheimer's disease and related
conditions. More specifically, the progress of such disease
conditions could be monitored by determining, via conventional
methods, the specific class and quantity of any antibody present in
a biological sample over an extended period of time. See, for
example, Nisonoff, A., INTRODUCTION TO MOLECULAR IMMUNOLOGY 14-15
(Sinauer Associates 1982).
[0081] Alternatively, such testing could be used in the context of
providing a method for determining the therapeutic efficacy of a
treatment regime for Alzheimer's disease and related conditions by
quantitatively analyzing biological samples for the presence of DMS
components or antibodies thereto.
[0082] Diagnostic testing of this sort can be conducted by
assaying, immunologically or otherwise, for the presence of DMS
components such as DMS membrane (protein, non-protein or both), DMS
protein or fragments thereof in biological samples not derived from
brain tissue, e.g., samples of serum, cerebrospinal fluid and other
bodily fluids or tissues. Testing can also be directed to detection
in a subject of antibodies against one or more DMS components. In
addition, prophylactic therapy according to the present invention
can be administered to the nondemented population on the basis of
other factors, suggesting a risk for dementia, which are revealed
by radiological or diagnostic imaging, genetic testing,
electroencephalography or other means.
[0083] Additionally, diagnostic testing can be used in the context
of providing a method for measuring the quantity of DMS components
in biological fluids periodically. Any dramatic increase in the
quantity of DMS components indicates the onset of cerebral plaque
formation and, consequently, the risk of Alzheimer's disease and
related conditions.
[0084] Other details of the present invention are further described
by reference to the following illustrative examples.
EXAMPLE 1
In Vitro Isolation Of Components Of DMS By Polyacrylamide Gel
Electrophoresis
[0085] DMS were isolated from human brains by density gradient
centrifugation according to the methodology described in U.S. Pat.
Nos. 4,816,416 and 5,231,170. The concentrated sediment containing
isolated DMS then was subjected to digestion or disruption and
separation by the following methods.
[0086] About 100 .mu.g of solution containing purified DMS was
suspended in 50 .mu.l of a PAGE buffer consisting of 0.5M TRIS (pH
6.8), glycerol, .beta.-mercaptoethanol, 0.4% bromophenol blue
solution and sodium dodecyl sulfate (SDS). The solubilized
preparation was electrophoresed on a 15% acrylamide gel with a
resolving buffer consisting of TRIS (pH 6.8) and an electrode
buffer consisting of TRIS base (pH 8.6), glycine and SDS at 150V
for 4 hours. See Laemmli, U.K., Nature 227:680 (1970). The gel was
stained with silver using a BioRad.RTM. Kit according to the
manufacturer's instructions. Bands containing the DMS components of
interest are presented in FIG. 1.
[0087] In FIG. 1, lane A is the molecular weight standard and lane
B is a preparation of DMS components described above. The bands
containing the protein fragments of primary interest are located in
the 5, 8, 10, 12, 16, 19, 21, 25, 28, 30, 31, 33, 40, 45, 48, 52,
60, 66, 70, 80, 100, 110 and 120 kilodalton region.
[0088] In addition to the bands containing isolated protein
components, the homogenous composition of DMS components subjected
to PAGE is also comprised of certain non-migrating components.
[0089] The individual DMS component fractions listed above can be
separated by conventional mechanisms well known to those skilled in
the art. For example, a microfiltration, ultrafiltration or reverse
osmosis apparatus can be used with various molecular weight cut-off
membranes to separate the individual DMS components. Additionally,
gels produced in accordance with Example 1 can be cut between the
distinct bands thereby separating the DMS components. The
individual DMS components then can be recovered from the gel by
conventional means such as solvent precipitation and the like.
[0090] Depending on the particular antigen present on the
aforementioned DMS components, these components can be stained by
anti-DMS by well known methods including Western Blot.
Alternatively, these DMS components can be isolated as described
above and used as pure immunogens for antibodies, which in turn are
used to identify specific protein fragments by staining and other
methods described above.
EXAMPLE 2
Preparation Of Antibodies To DMS And Evaluation Of The Presence Of
DMS Antigens In Cerebrospinal Fluid
[0091] A DMS antibody was prepared by inoculation of a rabbit with
pure DMS prepared in accordance with Example 1, mixed at a molar
ratio of 1:1 with Freund's complete adjuvant. Two hundred .mu.l of
mixture was injected subcutaneously, with 100 .mu.l booster
injections at 8 week intervals. The DMS injected into the rabbits
disintegrated into DMS components in solution either in the rabbit
or prior to injection. The rabbits were bled at intervals and serum
was separated. The resulting solution containing the rabbit
anti-DMS then was tested to determine the presence of anti-DMS by
ELISA with pure DMS components or pure DMS, and by Western Blot
techniques.
[0092] Cerebrospinal fluid (50 .mu.l) was extracted from a patient
with Alzheimer's disease and blotted onto a nitrocellulose filter.
The filter was then was blotted with 5% albumin solution and
incubated for 24 hours with rabbit anti-DMS at 1:100 molar
dilution, in 5% albumin solution. The filter then was incubated
with 1:1000 diluted horseradish peroxidase goat anti-rabbit IgG in
5% albumin for 1 hour at room temperature. The filter was washed
with phosphate buffered saline and stained. The filter was
positively stained indicating the presence of DMS antigens in the
patient's cerebrospinal fluid.
[0093] The presence of DMS antigens in the patient's cerebrospinal
fluid indicates the presence of DMS components that contain one or
more DMS antigens. The amount of DMS antigen present in the
patient's cerebrospinal fluid then can be calculatedusing known
techniques such as serial dilution followed by ELISA and the
like.
[0094] Analogously to Example 1, cerebrospinal fluid extracted from
a patient with Alzheimer's disease was applied to a 10%
polyacrylamide gel in denaturing buffer and the material was
separated into its component proteins. The gel separated
cerebrospinal proteins then were transferred to nitrocellulose
paper. A Western blot then was developed from the paper with
anti-DMS and stained as described above with horseradish peroxidase
and substrate. The banding pattern on the polyacrylamide gel
exhibited reactivity in the following molecular weight regions:
25-27; 28-30; 45-47; 50-53; 55-57; 60-65 and 76-80 kiloDaltons.
[0095] The presence of some, but not all DMS components described
in Example 1 above in the cerebrospinal fluid indicates that these
selective bands are a subgroup of DMS components that have a more
selective and hence unexpected, diagnostic utility than the other
DMS components, and that antibodies to these components include a
subgroup of anti-DMS antibodies that have a more selective
diagnostic utility than other anti-DMS antibodies. This example
illustrates the identification of a group of DMS components by
measuring the DMS components in a body fluid using a
laboratory-derived anti-DMS.
EXAMPLE 3
Evaluation Of The Presence Of Anti-DMS In Serum And Spinal
Fluid
[0096] By means of a nitrocellulose blot method similar to Example
2, cerebrospinal fluid and serum from six patients with Alzheimer's
disease were incubated on nitrocellulose filters previously blotted
with pure DMS. Positive staining in each case indicated the
presence of anti-DMS in serum and spinal fluid. The presence of
anti-DMS in the serum and spinal fluid indicates the presence of
DMS components therein. Thus, the presence of anti-DMS in serum and
spinal fluid indicates cerebral amyloid plaque formation and,
consequently, the onset or existence of Alzheimer's disease.
[0097] By means of a Western blot procedure similar to that
described above in Example 2, DMS components were separated in 10%
polyacrylamide gel and transferred to nitrocellulose paper, and
then reacted with a mixture of cerebrospinal fluid diluted 10-fold
in Tris buffered with saline with 5% Carnation dry milk. The blot
was reacted with anti-human IgG conjugated with peroxidase and
substrate. The banding pattern showed immunoreactivity at the
regions of approximately 55-60; 62-70 and 82-90 kiloDaltons.
[0098] The presence of selective anti-DMS activity in cerebrospinal
fluid as above to only some DMS components indicates that these
selective DMS components are a subgroup of DMS components that have
a more selective diagnostic utility than the other DMS components
described in Example 1 above. This example illustrates the
identification of a group of DMS components using laboratory
mixtures of DMS components and body fluid derived
auto-anti-DMS.
EXAMPLE 4
Preparation Of Antibodies To DMS Components And Evaluation Of The
Presence Of DMS Component Antigens In Cerebrospinal Fluid
[0099] DMS components are prepared in accordance with the methods
outlined in Example 1 above and purified. The purified DMS
components then are subjected to the procedures of Examples 2 and 3
to raise antibodies specific to the DMS components and to evaluate
the presence of anti-DMS components in the cerebrospinal fluid and
serum of patients with Alzheimer's disease. The formation of
plaque, particularly cerebral amyloid plaque formation, Alzheimer's
disease and related conditions can be diagnosed in accordance with
this method.
EXAMPLE 5
Preparation Of A Composition That Is Effective In Preventing
Amyloid Plaque Formation As Indicated By A Decrease In The Type And
Quantity Of DMS Component Antigens Or Anti-DMS Antibodies In
Cerebrospinal Fluid
[0100] The present inventors believe that the presence of an
increased concentration of DMS components in cerebrospinal fluid
correlates with the onset of cerebral plaque formation and hence,
the onset of Alzheimer's disease. A composition that is effective
in preventing the formation of cerebral plaque therefore would
result in a decrease in the concentration of DMS components or
antibodies thereto in the cerebrospinal fluid. This example
evaluates the efficacy of a treatment regime for treating
Alzheimer's disease in a mammal.
[0101] Subjects suspected to have DMS disintegration in the brain
and resultant DMS component formation or intracerebral amyloid, or
to be at risk of having intracerebral amyloid are tested for the
presence and quantity of DMS components and anti-DMS
auto-antibodies in their cerebrospinal fluid, blood or other bodily
fluid. The baseline DMS component and anti-DMS auto-antibody levels
or concentrations are measured by periodically extracting samples
of cerebrospinal fluid from the mammals and measuring the type and
quantity of DMS components and anti-DMS antibodies in accordance
with the procedures outlined in Examples 2-4 above. This procedure
is repeated a few times monthly to evaluate the baseline
concentration for the particular DMS components present in the
mammals bodily fluids.
[0102] A test compound is administered (orally, parenterally,
intranasally or by other means) to the mammal in a therapeutically
efficacious amount and for a therapeutically effective period of
time. At various time intervals on a monthly basis after commencing
administration of the test compound, the type and concentration of
DMS components and anti-DMS auto-antibodies are measured in
accordance with the procedures outlined above. The type and
concentrations of DMS components and antibodies thereto then are
compared to the baseline levels determined above. A decrease in the
concentration of DMS components or anti-DMS auto-antibodies or the
complete disappearance thereof correlates with the effectiveness of
the compound in entering the brain, binding to DMS or fragments
thereof in the brain and preventing and/or inhibiting the
disintegration of DMS. Hence, test compounds that are shown to be
effective in decreasing the type and concentration of DMS
components and/or antibodies thereto is useful in inhibiting
cerebral amyloid formation from disintegration of DMS.
[0103] The invention has been described with reference to the
foregoing examples which exemplify preferred embodiments. Those
skilled in the art recognize that various modifications can be made
to the present invention without departing from the spirit and
scope thereof.
* * * * *