U.S. patent application number 11/583986 was filed with the patent office on 2007-04-19 for apoptotic entities for use in treatment of neurodegenerative and other neurological disorders.
This patent application is currently assigned to Vasogen Ireland Limited. Invention is credited to Anthony E. Bolton, Arkady Mandel, Daniel Sauder.
Application Number | 20070087010 11/583986 |
Document ID | / |
Family ID | 4166243 |
Filed Date | 2007-04-19 |
United States Patent
Application |
20070087010 |
Kind Code |
A1 |
Bolton; Anthony E. ; et
al. |
April 19, 2007 |
Apoptotic entities for use in treatment of neurodegenerative and
other neurological disorders
Abstract
Treatment and/or prophylaxis, in mammalian patients, of
neurodegenerative and other neurological medical disorders is
effected by administering to the patient effective amounts of
apoptotic bodies and/or apoptotic cells, preferably those derived
from the patient's own white blood cells, e.g. by extracorporeal
treatment of the patient's blood cells to induce apoptosis and
administration of the apoptotic bodies and/or cells so formed to
the patient.
Inventors: |
Bolton; Anthony E.; (Dublin,
IE) ; Mandel; Arkady; (Mississauga, CA) ;
Sauder; Daniel; (Mississauga, CA) |
Correspondence
Address: |
FOLEY & LARDNER LLP
1530 PAGE MILL ROAD
PALO ALTO
CA
94304
US
|
Assignee: |
Vasogen Ireland Limited
|
Family ID: |
4166243 |
Appl. No.: |
11/583986 |
Filed: |
October 17, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09871146 |
May 25, 2001 |
7132285 |
|
|
11583986 |
Oct 17, 2006 |
|
|
|
Current U.S.
Class: |
424/184.1 ;
435/372 |
Current CPC
Class: |
A61P 21/04 20180101;
A61P 25/02 20180101; A61P 25/24 20180101; A61P 37/00 20180101; A61P
25/28 20180101; A61P 25/00 20180101; A61K 35/17 20130101; A61P
25/16 20180101; A61P 25/14 20180101 |
Class at
Publication: |
424/184.1 ;
435/372 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C12N 5/08 20060101 C12N005/08 |
Foreign Application Data
Date |
Code |
Application Number |
May 25, 2000 |
CA |
2,309,424 |
Claims
1. The use of apoptotic bodies and/or apoptotic cells in treatment
and/or prophylaxis in mammalian patients of neurodegenerative and
other neurological medical disorders.
2. The use of apoptotic bodies and/or apoptotic cells in the
preparation of a medicament for the treatment and/or prophylaxis of
neurodegenerative and other neurological medical disorders in
mammalian patients.
3. The use of claim 2 wherein apoptotic bodies and/or apoptotic
cells are in a liquid suspension along with viable cells.
4. The use of claim 3 wherein the apoptotic bodies and/or apoptotic
cells comprise from 10% to 90% of the cellular portion of the
suspension.
5. The use of claim 4 wherein the apoptotic bodies and/or apoptotic
cells comprise from 30% to 70% of the cellular portion of the
suspension.
6. The use of claim 1 or 2 wherein the apoptotic bodies and/or
cells are derived from extracorporeal treatment of blood cells
compatible with those of the mammalian patient.
7. The use of claim 1 or 2 wherein the apoptotic bodies and/or
cells are derived from established cultured cell lines.
8. The use of claim 6 wherein the blood cells are white blood cells
of blood compatible with that of the mammalian patient.
9. The use of claim 8 wherein the blood cells are the patient's own
white blood cells.
10. The use of claim 9 wherein the blood cells are the patient's
own T lymphocytes.
11. The use of claim 1 or 2 wherein the disorder is selected from
the group consisting of Alzheimer's disease, senile dementia,
multiple sclerosis, depression, Down's syndrome, Huntington's
disease, peripheral neuropathies, spinal cord diseases, neuropathic
joint diseases, chronic inflammatory demyelinating disease (CIPD),
neuropathies including mononeuropathy, polyneuropathy, symmetrical
distal sensory neuropathy, cystic fibrosis, neuromuscular junction
disorders, myasthenias and Parkinson's disease.
12. The use of claim 1 or 2 further comprising administering to a
human patient a dosage of apoptotic bodies and/or apoptotic cells
comprising from 10,000 to 10,000,000 apoptotic bodies and/or
apoptotic cells per kilogram body weight of the patient.
13. The use of claim 12 wherein the dosage contains from 500,000 to
5,000,000 apoptotic bodies and/or apoptotic cells per kilogram body
weight of the patient.
14. The use of claim 12 wherein the dosage contains from 1,500,000
to 4,000,000 apoptotic bodies and/or apoptotic cells per kilogram
body weight of the patient.
15. A method for treatment of or prophylaxis against
T-cell-mediated and inflammatory disorders in a mammalian patient,
which comprises administering to the patient an effective amount of
apoptotic bodies and/or apoptotic cells.
16. A pharmaceutical composition comprising a pharmaceutically
acceptable excipient and an effective amount of apoptotic bodies
and/or apoptotic cells.
17. The pharmaceutical composition of claim 16 which is suitable
for administration to a mammalian patient to treat or to effect
prophylaxis against neurodegenerative and other neurological
medical disorders.
18. The composition of claim 16 or 17 comprising a liquid
suspension of cellular material, from 10% to 90% of the cellular
material being apoptotic bodies and/or apoptotic cells.
19. A unit dosage composition for administration to a human patient
for alleviation or prophylaxis of a nuerological or
neurodegenerative disorder, comprising a liquid suspension of
cellular material including from about 10,000 to 10,000,000
apoptotic cells and/or apoptotic bodies per kilogram of patient
body weight.
Description
FIELD OF THE INVENTION
[0001] This invention relates to biochemical and biological
compositions and to the uses thereof in the treatment and/or
prophylaxis of various neurodegenerative and other neurological
disorders in mammalian patients. More particularly, it relates to
treatment and prophylaxis of neurodegenerative and other
neurological disorders by administration of compositions containing
the mammalian cellular materials and fragments thereof, and to the
compositions containing the mammalian cellular materials and
fragments themselves, and to processes for preparing such
compositions.
BACKGROUND OF THE INVENTION
[0002] Two mechanisms of cell death in the body are recognized,
necrosis and apoptosis. Apoptosis is the process of programmed cell
death, described by Kerr et al in 1992 [Kerr J F R, Wyllie A H,
Currie A R (1992). "Apoptosis: a basic biological phenomenon with
wide-ranging implications in tissue kinetics. "British Journal of
Cancer 26: 239-257"], by which steady-state levels of the various
organ systems and tissues in the body are maintained as continuous
cell division and differentiation takes place. Cells undergoing
apoptosis often exhibit distinctive morphological changes such as a
pronounced decrease in cell volume, modification of the
cytoskeletons resulting in pronounced membrane blebbing, a
condensation of the chromatin, and degradation of the DNA into
oligonucleosomal fragments. Following these morphological changes,
an apoptotic cell may break up into a number of small fragments
known as apoptotic bodies, consisting essentially of membrane-bound
bodies containing intact organelles, chromatin, etc. Apoptotic
bodies are normally rapidly removed from the body by phagocytosis
by macrophages, dendritic cells and other antigen presenting cells,
before they can become lysed and release their potentially
pro-inflammatory intracellular contents.
[0003] In simple outline, apoptosis is thought to proceed as
follows. Three phases can be identified in the apoptotic mechanism
of programmed cell death:
[0004] Induction phase;
[0005] Effector phase; and
[0006] Degradation phase.
[0007] The induction phase is dependent, in part, on specific
interactions of death-inducing signals at the cell surface
membrane. One common signal is initiated by the binding of specific
ligands to receptors of the TNF receptor family present on the cell
membrane. One important such receptor is Fas (APO-1, CD95), which
interacts with Fas-ligand to initiate apoptosis.
[0008] The effector phase, activated by the binding of receptors
and ligands of the induction phase, leads to the activation of
caspases, cystinyl-aspartate-requiring proteinases (proteolytic
enzymes), including caspases 1 and 8. This activation may be
associated with a change in the permeability of mitochondria,
allowing the release of cytochrome-c which is involved in caspase
activation. Activated caspases initiate a chain of lethal
proteolytic events culminating in the changes in chromatin and
cytoskeletal components seen in apoptosis.
[0009] Many cells undergoing apoptosis can be identified by a
characteristic `laddering` of DNA seen on agarose gel
electrophoresis, resulting from cleavage of DNA into a series of
fragments. These changes occur a few hours before death of the cell
as defined by the ability of a cell to exclude vital dyes. The
appearance of DNA laddering on agarose gel electrophoresis
following extraction of DNA from cells is one recognised method of
identification of apoptosis in cells [Loo, D. T. and Rillema, J. R.
(1998) "Measurement of Cell Death,"Methods in Cell Biology 57:
251-264], although it is not always sensitive enough to detect
apoptosis. In situ labelling of nuclear DNA fragmentation, for
example, using commercially available terminal dUTP nick end
labelling (TUNEL) assays, are an alternative and more reproducible
measure for the determination of fragmented DNA in apoptotic cells
and cells undergoing apoptosis [Gavrieli Y, Sherman Y, Ben-Sasson S
A (1992)", Identification of programmed cell death in situ via
specific labelling of nuclear DNA fragmentation". Journal of Cell
Biology 119: 493-501].
[0010] During apoptosis, phosphatidylserine becomes exposed
externally on the cell membrane [Fadok V A, Voelker D R, Campbell P
A, Cohen J J, Bratton D L, Henson P M (1992), "Exposure of
phosphatidylserine on the surface of apoptotic lymphocytes triggers
specific recognition and removal by macrophages". Journal of
Immunology 148: 2207-2216] and this exposed phosphatidylserine
binds to specific receptors to mediate the uptake and clearance of
apoptotic cells in mammals [Fadok V A, Bratton D L, Rose D M,
Pearson A, Ezekewitz R A B, Henson P M (2000), "A receptor for
phosphatidylserine-specific clearance of apoptotic cells", Nature
405: 85-90]. The surface expression of phosphatidylserine on cells
is another recognised method of identification of apoptotic
cells.
[0011] Changes in mitochondrial integrity are intimately associated
with apoptosis, resulting in alterations in mitochondrial membrane
permeability and the release of cytochrome-c from the mitochondria
into the cell cytoplasm [Susin, S. A., Lorenzo, H. K., Zamzami, N.,
Marzo, I, Brenner, C., Larochette, N., Prevost, M. C., Alzari, P.
M. and Kroemer, G. (1999) "Mitochondrial Release of Caspase-2 and
-9 during the Apoptotic Process", Journal of Experimental Medicine,
189: 381 -394]. Measurement of changes in mitochondrial membrane
potential, reflecting changes in mitochondrial membrane
permeability, is another recognised method of identification of
apoptotic cells.
[0012] A number of other methods of identification of cells
undergoing apoptosis and of apoptotic cells, many using monoclonal
antibodies against specific markers for apoptotic cells, have also
been described in the scientific literature.
[0013] Necrosis, in contrast, is cell death of a pathological
nature, resulting from injury, bacterial toxin effects,
inflammatory mediators, etc., and involving membrane rupture and
release of intracellular contents to the surrounding tissue, often
with harmful inflammatory consequences. Accordingly, one of the
ways in which necrotic cells may be detected and characterized is
by detection of compromised cell membranes, e.g. by methods of
staining with propidium iodide followed by flow cytometry or
microscopy.
SUMMARY OF THE INVENTION
[0014] This invention is directed, in part, to the novel and
unexpected discovery that administration to a mammal of apoptotic
cells and/or apoptotic bodies previously prepared ex vivo, can be
used in the prophylaxis and/or treatment of neurodegenerative
and/or other neurological disorders in the treated mammal.
[0015] Accordingly, in one of its composition aspects, this
invention is directed to a pharmaceutical composition comprising a
pharmaceutically acceptable excipient and an effective amount of
apoptotic cells and/or apoptotic bodies.
[0016] The pharmaceutical compositions preferably employ an aqueous
based pharmaceutically acceptable excipient although other
excipients can be used.
[0017] As noted above, these compositions are useful in the
prophylaxis and/or treatment of neurodegenerative and/or other
neurological disorders in mammals. Accordingly, in one of its
method aspects, this invention is directed to a method for the
treatment of or prophylaxis against neurodegenerative and other
neurological medical disorders in a mammalian patierit, which
comprises administering to the patient an effective amount of
apoptotic bodies and/or apoptotic cells.
[0018] These methods are preferably accomplished by administering
to the patient the pharmaceutical compositions described
herein.
BRIEF DESCRIPTION OF THE DRAWING
[0019] The FIGURE is a graph showing a comparison of net ear
swelling in mice treated with the compositions of this invention
and a control group.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0020] This invention is directed to the treatment and/or
prophylaxis of neurodegenerative and/or other neurological
disorders by the administration of apoptotic cells and/or
bodies.
[0021] Neurodegenerative disorders, including Down's syndrome,
Alzheimer's disease and Parkinson's disease, are associated with
increased levels of reactive oxygen species (ROS), certain
inflammatory cytokines, including interleukin-1.beta. (IL-1.beta.)
[see Griffin W S T, Stanley L C, Ling C, White L, Macleod V. Perrot
L J, White C L, Araoz C (1989). Brain interleukin 1 and S-100
immunoreactivity are elevated in Down's syndrome and Alzheimer
disease. Proceedings of the National Academy of Sciences USA
867611-7615; Mogi M, Harada M, Narabayashi H, Inagaki H, Minami M,
Nagatsu T (1996). Interleukin (IL)-1 beta, IL-1, IL-4, IL-6 and
transforming growth factor-alpha levels are elevated in ventricular
cerebrospinal fluid in juvenile parkinsonism and Parkinson's
disease. Neuroscience Letters 211:13-16]. It has also been shown
that IL-1.beta. inhibits long-term potentiation in the hippocampus
[Murray C A, Lynch M A (1998). Evidence that increase hippocampal
expression of the cytokine interleukin-1.beta. is a common trigger
for age and stress-induced impairments in long-term potentiation.
Journal of Neuroscience 18:2974-2981]. Long-term potentiation in
the hippocampus is a form of synaptic plasticity and is generally
considered to be an appropriate model for memory and learning
[Bliss T V P, Collinridge G L, (1993). A synaptic model of memory:
long-term potentiation in the hippocampus, Nature 361:31-39]. Thus,
inappropriate cytokine expression in the brain is currently
believed to be involved in the development and progression of
neuroinflammatory diseases.
[0022] Neurodegenerative and other neurological disorders treatable
by the present invention include Down's syndrome, Alzheimer's
disease, Parkinson's disease, senile dementia, depression and the
like. In summary, it can be substantially any neurodegenerative or
other neurological disorder.
[0023] "Apoptotic cells" and "apoptotic bodies," as the terms are
used herein, means cells and cell bodies which exhibit one or more
of the following apoptosis-characterizing features: surface
exposure of phosphatidylserine, as detected by standard, accepted
methods of detection such as Annexin V staining; alterations in
mitochondrial membrane permeability measured by standard, accepted
methods (e.g. Salvioli, S., Ardizzoni, A., Franceschi, C.
Cossarizza, A. (1997) "JC-1, but not DiOC6(3) or Rhodamine 123, is
a Reliable Fluorescent Probe to assess Delta Psi Changes in Intact
Cells: Implications for Studies on Mitochondrial Functionality
during Apoptosis," FEBS Letters 411: 77-82]; evidence of DNA
fragmentation such as the appearance of DNA laddering on agarose
gel electrophoresis following extraction of DNA from the cells
[Teiger, E., Dam, T. V., Richard, L., Wisnewsky, C., Tea, B. S.,
Gaboury, L., Tremblay, J., Schwartz, K. and Hamet, P. (1996)
"Apoptosis in Pressure Overload-induced Heart Hypertrophy in the
Rat," Journal of Clinical Investigation 97; 2891-2897], or by in
situ labeling (see Gavrieli et al., 1992, referenced above).
[0024] The apoptotic cells and/or apoptotic bodies for use in the
present invention preferably comprise not more than about 35 weight
percent of necrotic cells and/or necrotic bodies based on the total
weight of the apoptotic cells/bodies and necrotic cells/bodies;
more preferably, not more than about 20 weight percent; and even
more preferably, not more than about 10 weight percent. At these
levels, the presence of such necrotic cells and/or bodies are
believed not to significantly alter in vivo processes. In its most
preferred embodiment, the apoptotic cells/bodies are substantially
free of necrotic cells and or bodies (i.e., less than about 2
weight percent of necrotic cells/bodies).
[0025] The apoptotic cells and/or apoptotic bodies for use in the
present invention are prepared ex vivo from mammalian cells that
are compatible with those of the mammalian patient. They can be
prepared from substantially any type of mammalian cell including
cultured cell lines. Preferably they are prepared from a cell type
derived from the mammalian patient's own body or from an
established cell line. More preferably they are prepared from white
blood cells of blood compatible with that of the mammalian patient,
more preferably from the patient's own white blood cell and even
more preferably from the patient's own T lymphocytes. Even more
preferably they are prepared from an established cell line. The
apoptotic cells and/or apoptotic bodies are prepared
extracorporeally prior to administration to the patient. Thus, in
one embodiment, an aliquot of the patient's blood may be withdrawn,
e.g. by venipuncture, and at least a portion of the white cells
thereof subjected extracorporeally to apoptosis inducing
conditions.
[0026] A variety of methods of inducing apoptosis in mammalian
cells, so as to create apoptotic cells and apoptotic bodies, are
known in the art and essentially any of these can be adopted in
preparing apoptotic bodies for use in the present invention. One
such method is the subjection of the cells to ionizing radiation
(.DELTA.-rays, x-rays, etc.) and/or non-ionizing electromagnetic
radiation including ultraviolet light. Apoptosis can be induced by
subjecting cells to ultrasound.
[0027] Another method is the treatment of the cells with drugs such
as non-specific protein kinase inhibitors as exemplified by
staurosporine (see Bombeli, Karsan, Tait and Hirlan, (1997)
"Apoptotic Vascular Endothelial Cells Become Procoagulant", Blood,
Vol. 89:2429-2442). Also, certain chemotherapeutic agents used for
the treatment of malignant tumours induce apoptosis, for example
adriamycin, as can statin drugs (3-hydroxy-3methylglutaryl coenzyme
A reductase inhibitors) [Guijarro C, Blanco-Colio L M, Ortego M,
Alonso C, Ortiz A, Plaza J J, Diaz C, Hernandez G, Edigo J (1998),
"3-hydroxy-3methylglutaryl coenzyme A reductase and isoprenylation
inhibitors induce apoptosis of vascular smooth muscle in
culture,"Circulation Research 83: 490-500] and colcicine [Suzuki Y
(1998)", "Cell death, phagocytosis and neurogenesis in mouse
olfactory epithelium and vomeronasal organ after colcicine
treatment," Annals of the New York Academy of Sciences 855:
252-254]. The use of ligands for death receptors on cells, such as
Fas-ligand, will be apparent for inducing apoptosis from the
discussion of apoptosis above.
[0028] Yet another method is the application of oxidative stress to
cells extracorporeally (see for example Buttke and Sandstrom (1994)
"Oxidative Stress as a Mediator of Apoptosis," Immunology Today,
Vol. 15:7-10). This can be achieved by treating the cells, in
suspension, with chemical oxidizing agents such as hydrogen
peroxide, other peroxides and hydroperoxides, ozone, permanganates,
periodates, and the like. Biologically acceptable such oxidizing
agents are preferably used, so as to reduce potential problems
associated with residues and contaminations of the apoptotic cells
and apoptotic bodies so formed.
[0029] The present invention is not restricted to any particular
method of producing apoptotic cells and apoptotic bodies, for use
herein, and any suitable, known process can be used.
[0030] Methods for the detection and quantitation of apoptosis can
be used to determine the presence and level of apoptosis in the
preparation to be administered to the patient in the present
invention. At least one of the methods from those described in the
introduction above should be used to confirm the level of apoptosis
achieved prior to administration. They are suitably purified prior
to use, by methods known in the art, such as differential
centrifugation.
[0031] In preparing the apoptotic cells and/or apoptotic bodies,
care should be taken not to apply excessive levels of oxidative
stress, radiation, drug treatment, etc., since otherwise there is a
significant risk of causing necrosis of at least some of the cells
under treatment. Necrosis causes cell membrane rupture and the
release of cellular contents often with biologically harmful
results, particularly inflammatory events, so that the presence of
necrotic cells and their components along with the apoptotic bodies
is best avoided. Appropriate levels of treatment of the cells to
create apoptotic bodies for use in the present invention depend to
some extent on the nature of the chosen cells and cellular
composition, and the type of treatment chosen to induce apoptosis.
Such appropriate levels are readily determinable by those skilled
in the art, having regard to the available scientific literature on
the subject including the above-reference articles.
[0032] One preferred process according to the present invention
involves the culture of cells from the patient, or a compatible
mammalian cell line. The cultured cells may then be treated to
induce apoptosis and to create apoptotic cells and/or apoptotic
bodies therein. The cells, suspended in the patient's plasma or
another suitable suspension medium, such as saline or a balanced
mammalian cell culture medium, can then be administered as
indicated below. The numbers of apoptotic cells and/or bodies can
be determined by published methods available in the scientific
literature on the subject including the above-reference articles.
The numbers of such apoptotic cells and/or apoptotic bodies
required for administration to the patient to obtain the required
clinical benefit will vary depending on the source of cells, the
patient's condition, the age and weight of the patient and other
relevant factors which are readily determinable by the attending
clinician.
[0033] Thus, an example of a preferred process according to the
present invention accordingly involves extraction of an aliquot of
blood from the patient to be treated, separation of the white cells
therefrom, and treatment of the white cells under apoptosis-causing
conditions, so as to create a cellular composition in which
significant numbers of the white cells therein have been apoptosed
so as to create therein substantial numbers of apoptotic cells or
bodies. Then the treated composition is administered to the
patient. More preferably, T lymphocytes, isolated from the blood by
known means, and suspended as above, may be used as a source of
apoptotic cells and apoptotic bodies.
[0034] The number of viable cells selected for treatment to create
apoptotic cells and/or apoptotic bodies is suitably up to about
4.times.10.sup.9, preferably from about 1,000,000 to about
1,000,000,000 and most preferably from about 50,000,000 to about
150,000,000, for each administration to a human patient. From about
10% to 90%, preferably from about 30% to 70% of the cellular
composition for administration is comprised of apoptotic bodies
and/or apoptotic cells, the balance being viable cells and necrotic
cells. Accordingly, the preferred amounts of apoptotic cells and/or
apoptotic bodies for administration are those produced by
subjecting these numbers of cells to the apoptosing conditions.
When whole blood is used as the source of the cells to be subjected
to the apoptosis inducing conditions, these numbers of white cells
are obtainable in blood aliquots of volume up to about 400 ml,
preferably up to 100 ml. More specifically, 50,000,000-150,000,000
cells is equivalent to the white cells in blood aliquots of volume
10 -30 ml.
[0035] The volume of the aliquot of blood withdrawn from the
patient for treatment to create apoptotic cells and/or apoptotic
bodies therein is suitable up to about 400 ml, preferably from
about 0.1 to about 100 ml and most preferably from about 5 to about
15 ml. Accordingly, the preferred amounts of apoptotic cells and/or
apoptotic bodies for administration are those corresponding to the
numbers derivable from the white blood cells, or isolated T
lymphocytes, contained in such quantities of whole blood, following
subjection to apoptosis-inducing conditions.
[0036] The suspension of treated apoptotic cells and/or bodies for
administration to the patient is prepared in a biologically
acceptable liquid suspending medium, such as the patient's serum or
plasma, saline or balanced mammalian cell culture medium. The
addition of other factors, such as cytokines, hormones, products of
stressed cells or other appropriate biologically active material
may enhance the benefit of the administered apoptotic cellular
materials. The aliquot can be introduced into the patient's body by
any suitable method, most preferably intramuscular injection but
also including subcutaneous injection, mini-grafting, intra
peritoneal injection, intra-arterial injection, intravenous
injection and oral administration. The apoptotic entities can be
delivered to the specific body organ and/or site by using any
appropriate, known delivery system.
[0037] The compositions of this invention may optionally include a
pharmaceutically acceptable excipient. Some examples of suitable
excipients include sterile water, sterile saline, phosphate
buffered saline, and the like.
[0038] When administered, the pharmaceutical compositions comprise
an effective amount of apoptotic bodies/cells to induce a suitable
prophylactic and/or therapeutic response in the patient at risk of
suffering or suffering from a neurodegenerative disease.
Preferably, the composition administered to the mammalian patient
comprises from about 10,000 to 10,000,000 apoptotic cells or bodies
per kilogram of body weight, more preferably from about 500,000 to
5,000,000 and most preferably from about 1,500,000 to about
4,000,000 apoptotic cells and/or bodies per kg body weight. The
specific dose employed will, of course, be dependent upon the age,
weight and severity of the disease in the treated patient all of
which are within the skill of the attending clinician.
[0039] For most effective treatment and/or prophylaxis of mammalian
disorders involving a neurodegenerative or neurological disorder,
the patient may be given a course of treatments with apoptotic
cells and/or bodies according to the invention. Each course of
treatment may involve administration to the patient of from 1 to 6
aliquots of suspended cellular material, as described above. No
more than one such aliquot should be administered per day, and the
maximum rest period between any two consecutive administrations
should be not greater than about 21 days. Booster treatments as
described below may advantageously be used. To maintain the desired
effects, the patient may undergo booster treatments, with a further
course of administration of aliquots of suspended apoptotic cells
and/or apoptotic bodies as described above, at intervals of three
to four months.
[0040] As noted, the present invention is applicable to the
treatment and prophylaxis of a wide variety of mammalian
neurodegenerative and other neurological disorders. These include,
but are not limited to, Down's Syndrome, Alzheimer's disease,
Parkinson's disease, senile dementia, depression, multiple
sclerosis, Huntington's disease, peripheral neuropathies, spinal
cord diseases, neuropathic joint diseases, chronic inflammatory
demyelinating disease (CIPD), neuropathies including
mononeuropathy, polyneuropathy, symmetrical distal sensory
neruopathy, cystic fibrosis, neuromuscular junction disorders and
myasthenias. In summary, it can be substantially any
neurodegenerative or other neurological disorder.
[0041] The invention is further described, for illustrative
purposes, in the following specific examples.
EXAMPLE 1
[0042] Experiments to demonstrate the invention were conducted on
laboratory mice, under approved conditions for conducting such
experiments.
[0043] The effectiveness of the treatment according to a preferred
embodiment of the present invention, on contact hypersensitivity
(CHS), an example of a Th-1-cell inflammatory disorder which is
known to be mediated by inflammatory cytokines, was assessed on
laboratory mice, according to approved animal experimentation
procedures, using the method described by Kondo et. al.,
"Lymphocyte function associated antigen-1 (LFA-1) is required for
maximum elicitation of allergic contact dematitis" Br. J. Dermatol.
131:354-359 (1994), with minor variations. The disclosure thereof
is incorporated herein by reference. Briefly, to induce C H S, the
abdominal skin of each mouse was shaved and painted with
dinitrodifluorobenzene DNFB, the sensitizing chemical, using 25
.mu.l of 0.5% DNFB in 4:1 acetone:olive oil solution. This
sensitization was applied to two groups of Balb/c mice, 10 animals
in total.
[0044] Apoptotic bodies were prepared from murine fibroblasts. The
murine fibroblasts were treated with 50 mM sodium butyrate in RPMI
medium, at confluency for one day, and then the sodium butyrate
medium was changed. To increase the number of apoptotic cells and
bodies, the cells can additionally be irradiated with
UV-light.(e.g. 75 mj). Supernatant containing floating cells is
removed 24 hours following irradiation.
[0045] Apoptotic bodies were quantitated by centrifuging the
supernatant (1200 rpm, 5 minutes), aspirating the supernatant,
washing the resulting cell pellet with PBS and centrifuging again,
as above. The pellet containing the apoptotic bodies was
re-suspended in PBS. The cells were stored in PBS at 4.degree. C.
for the duration of the experiment. The cells to be stained for
quantitation were re-suspended in 1X binding buffer at a
concentration of 1.times.10.sup.6 cells/ml. 100 .mu.l of the cells
were transferred to a 5 m1 tube, and 10 .mu.l of
fluorescein-conjugated annexin V and 10 .mu.l propidium iodide
reagent was added. The cells were gently vortexed and the cell
mixture incubated for 15 minutes at 25.degree. C. in the dark.
Following the incubation, 400 .mu.l of 1X binding buffer is added
to each tube. The sample was analyzed on a flow cytometer over one
hour.
[0046] Of the two groups of sensitized mice, the first, control
group A, received no treatment. The second, test group B, was
treated with an injection of suspended apoptotic bodies prepared as
described above, 50 .mu.l volume containing at least 150,000 bodies
per injection of blood subjected to stressors as described above.
Treatments, each involving intramuscular injection of 50 .mu.l of
the respective liquid, started on the day of sensitization, and
were repeated every day for a total of six days. On the same day as
the last treatment, but after its administration, the animals were
challenged with DNFB, by applying to the right ear of each animal
10.mu.l of 0.2% solution of DNFB in acetone and olive oil. To the
left ear of each animal was applied the acetone/olive oil solvent,
without DNFB. Inflammation due to CHS manifests itself in a
swelling of the right ears. Ear thickness was measured, 24 hours
after challenge, with a Peacock spring-loaded micrometer (Ozaki
Co., Tokyo, Japan). The results were expressed as the thickness and
difference in thickness of the right ears and the left ears of each
animal, at 24 hours after challenge.
[0047] The experiments were repeated, using more sets of two groups
of animals, a sufficient number of times to ensure statistical
significance in the results. A notable and significant reduction in
ear thickness (inflammation) was observed with the animals treated
with the apoptotic cells and apoptotic bodies suspension in
accordance with the invention, as compared with the untreated
group, demonstrating a significant reduction in inflammation. The
results are presented in the following Table, and on the
accompanying Figure, as a bar graph of net ear swelling (difference
between right ear and left ear thickness), for each group, with
"standard deviation" shown by the vertical line at the top of each
column. TABLE-US-00001 TABLE 1 Group Left ear Right ear Difference
A 17 31 14 A 18 39 21 A 17 30 13 A 18 32 14 A 18 31 13 Mean: 15
S.D: 3.391165 B 21 31 10 B 18 18 0 B 17 30 13 B 20 24 4 B 18 22 4
Mean: 6.2 S.D.: 5.215362
[0048] An analysis of the suspension of apoptotic cells and bodies
administered to the animals of test group B indicated the presence
therein of approximately 40% apoptotic cells and bodies, balance
viable cells and minor amounts of necrotic cells (not more than
20%), the presence of which is believed to be insignificant in the
in vivo process.
EXAMPLE 2
[0049] The above test procedure was repeated on similar groups of
animals, a control group and a test group, but using a suspension
of apoptotic cells and bodies on the test group which comprised
about 60% apoptotic cells and bodies, balance viable cells and a
minor amount (not more than 20%) of necrotic cells. Essentially
similar results were obtained.
[0050] The effectiveness of the processes and compositions of the
present invention in preventing and alleviating inflammation due to
CHS indicates that administration of apoptotic cells and bodies as
described up-regulates the in vivo generation of anti-inflammatory
Th-2 derived cytokines such as IL-10 (known to be implicated in
CHS--see Kondo, McKenzie and Sauder, "The Journal of Investigative
Dermatology," Vol. 103, 1994, page 811-814) and/or down-regulates
Th-1 inflammatory cytokines such as TNF.alpha. and IL-6. These
inflammatory cytokines are implicated in inflammation-related
disorders of the brain, namely the neuroinflammatory,
neurodegenerative and neurological disorders such as Alzheimer's
disease, senile dementia, multiple sclerosis, depression, Down's
syndrome, Huntington's disease, peripheral neuropathies, spinal
cord diseases, neuropathic joint diseases, chronic inflammatory
demyelinating disease (CIPD), neuropathies including
mononeuropathy, polyneuropathy, symmetrical distal sensory
neuropathy, cystic fibrosis, neuromuscular junction disorders,
myasthenias and Parkinson's disease.
[0051] Neurodegenerative diseases, including Down's syndrome,
Alzheimer's disease and Parkinson's disease, are associated with
increased levels of certain inflammatory cytokines, including
interleukin-1.beta. (IL-1.beta.) [see Griffin W S T, Stanley L C,
Ling C, White L, Macleod V. Perrot L J, White C L, Araoz C (1989).
Brain interleukin 1 and S-100 immunoreactivity are elevated in Down
syndrome and Alzheimer disease. Proceedings of the National Academy
of Sciences USA 867611-7615; Mogi M, Harada M, Narabayashi H,
Inagaki H, Minami M, Nagatsu T (1996). Interleukin (IL)-1 beta,
IL-1, IL-4, IL-6 and transforming growth factor-alpha levels are
elevated in ventricular cerebrospinal fluid in juvenile
parkinsonism and Parkinson's disease. Neuroscience Letters
211:13-16]. It has also been shown that IL-1.beta., inhibits
long-term potentiation in the hippocampus [Murray C A, Lynch M A
(1998). Evidence that increase hippocampal expression of the
cytokine interleukin-1.beta. is a common trigger for age and
stress-induced impairments in long-term potentiation. Journal of
Neuroscience 18:2974-2981]. Long-term potentiation in the
hippocampus is a form of synaptic plasticity and is generally
considered to be an appropriate model for memory and learning
[Bliss T V P, Collinridge G L, (1993). A synaptic model of memory:
long-term potentiation in the hippocampus, Nature 361:31-39]. Thus,
inappropriate cytokine expression in the brain is currently
believed to be involved in the development and progression of
neurodegenerative diseases. Consequently, the finding of success in
CHS treatment reported in the above Examples, with its attendant
down-regulation of Th-1 inflammatory cytokines, is indicative of
successful use of the process and compositions in the treatment and
prophylaxis of a wide variety of neurological disorders including
those discussed above.
* * * * *