U.S. patent application number 11/855092 was filed with the patent office on 2008-03-13 for apoptotic entities for use in treatment of endothelium dysfunction disorders.
This patent application is currently assigned to Vasogen Ireland Limited. Invention is credited to Anthony E. Bolton, Arkady Mandel, Daniel N. Sauder.
Application Number | 20080063631 11/855092 |
Document ID | / |
Family ID | 4166242 |
Filed Date | 2008-03-13 |
United States Patent
Application |
20080063631 |
Kind Code |
A1 |
Bolton; Anthony E. ; et
al. |
March 13, 2008 |
APOPTOTIC ENTITIES FOR USE IN TREATMENT OF ENDOTHELIUM DYSFUNCTION
DISORDERS
Abstract
Treatment and/or prophylaxis of endothelial dysfunction-related
disorders in mammalian patients is effected by administering to the
patient effective amounts of apoptotic bodies and/or apoptotic
cells.
Inventors: |
Bolton; Anthony E.; (Dublin,
IE) ; Mandel; Arkady; (North York, CA) ;
Sauder; Daniel N.; (Princeton, NJ) |
Correspondence
Address: |
FOLEY & LARDNER LLP
975 PAGE MILL ROAD
PALO ALTO
CA
94304
US
|
Assignee: |
Vasogen Ireland Limited
|
Family ID: |
4166242 |
Appl. No.: |
11/855092 |
Filed: |
September 13, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09866569 |
May 25, 2001 |
7279156 |
|
|
11855092 |
Sep 13, 2007 |
|
|
|
Current U.S.
Class: |
424/93.71 |
Current CPC
Class: |
A61P 7/02 20180101; A61P
43/00 20180101; A61K 35/17 20130101; A61P 9/10 20180101; A61P 9/08
20180101; A61P 25/06 20180101; A61P 37/04 20180101; A61P 1/00
20180101; A61P 9/02 20180101; A61P 9/12 20180101; A61P 9/04
20180101; A61P 9/00 20180101 |
Class at
Publication: |
424/093.71 |
International
Class: |
A61K 38/00 20060101
A61K038/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 25, 2000 |
CA |
2,309,417 |
Claims
1-15. (canceled)
16. A method for treatment and/or prophylaxis in mammalian patients
with medical disorders resulting from or involving endothelial
dysfunction, wherein the disorder is selected from the group
consisting of atherosclerosis, peripheral vascular disease, stroke,
myocardial infarction, angina, hypertension, Raynaud's disease,
cardiac syndrome X, migraine, ischemic damage, inflammatory bowel
disease and graft versus host disease, which method comprises
administration to the patient of an effective amount of apoptotic
bodies.
17. The method of claim 16 wherein the apoptotic bodies are in a
liquid suspension along with viable cells.
18. The method of claim 17 wherein the apoptotic bodies comprise
from 10% to 90% of the cellular portion of the suspension.
19. The method of claim 18 wherein the apoptotic bodies comprise
from 30% to 70% of the cellular portion of the suspension.
20. The method of claim 18 wherein the apoptotic bodies are derived
from extracorporeal treatment of blood cells compatible with those
of the mammalian patient.
21. The method of claim 16 wherein the apoptotic bodies are derived
from established cultured cell lines.
22. The method of claim 20 wherein the blood cells are white blood
cells of blood compatible with that of the mammalian patient.
23. The method of claim 22 wherein the blood cells are the
patient's own white blood cells.
24. The method of claim 23 wherein the blood cells are the
patient's own T lymphocytes.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of application Ser. No.
09/866,569, filed May 25, 2001, now pending, which claims priority
under 35 U.S.C. 119 and/or 365 to Serial No. 2,309,417, filed in
Canada on May 25, 2000, entire content of both of which are
incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates to biochemical and biological
compositions and to the uses thereof in the treatment and/or
prophylaxis, in mammalian patients, of various medical disorders
associated with endothelial dysfunction (malfunctioning of the
lining of blood vessels). More particularly, it relates to
treatment and prophylaxis of medical disorders associated with
endothelial dysfunction by administration of compositions
containing 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
[0003] 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 AR (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, comprising 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.
[0004] In simple outline, apoptosis is thought to proceed as
follows. Three phases can be identified in the apoptotic mechanism
of programmed cell death: [0005] Induction phase; [0006] Effector
phase; and [0007] Degradation phase.
[0008] 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.
[0009] 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.
[0010] 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 recognized 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 labeling of nuclear DNA fragmentation, for
example, using commercially available terminal dUTP nick end
labeling (TUNEL) assays, is 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).
[0011] 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 recognized method of identification of apoptotic
cells.
[0012] 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
penneability, is another recognized method of identification of
apoptotic cells.
[0013] 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.
[0014] Methods of quantifying apoptotic cells and apoptotic bodies
in a cellular composition are known and readily practiced by
persons of skill in the art. Techniques include staining of the
treated cell population, with an appropriate, selective dye such as
fluorescein-conjugated annexin V, followed by incubation and
analysis by flow cytometry.
[0015] 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. Necrotic cells may be
detected and characterized by detection of compromised cell
membranes e.g. by methods such as staining with propidium iodide
followed by flow cytometry or microscopy.
SUMMARY OF THE INVENTION
[0016] According to the present invention, the administration of
apoptotic cells and/or apoptotic bodies previously prepared ex
vivo, is used in the prophylaxis and/or treatment of medical
disorders in which there is dysfunction of the cells of the
endothelium, the cellular lining of blood vessels.
[0017] In one of its method aspects, this invention is directed to
a method for the treatment of or prophylaxis against an endothelium
dysfunction disorder in a mammalian patient, 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 endothelium dysfunction disorders by administering
apoptotic cells and/or bodies to a mammalian patient.
[0021] The endothelium is a cellular layer lining the walls of
blood vessels of a mammal. It is a highly specialized interface
between blood and underlying tissues and has a number of functions,
including: control of haemostasis by inhibiting platelet
aggregation (antithrombotic and regulating the coagulation and
fibrolinolytic systems); control of vascular tone, and hence blood
flow; control of blood vessel smooth muscle growth; and selective
permeability to cells and proteins.
[0022] Normally, the endothelium maintains vascular homeostasis by
responding to physiological stimuli, for example, changes in blood
flow, oxygen tension etc., by adaptive alteration of function.
Dysfunctional endothelium has an impaired response to such
physiological stimuli, and can ultimately lead to medical
disorders. A number of subsets of endothelial dysfunction have been
recognized, including Endothelial Activation, and
Endothelial-mediated Vasodilatory Dysfunction (see De Caterina
(2000). "Endothelial dysfunctions: common denominators in vascular
disease". Current Opinions in Lipidology 11:9-23).
[0023] Endothelial activation may lead to the initiation of
atherosclerosis and is a process whereby there is an inappropriate
up-regulation and expression of cell attraction and cell adhesion
molecules on endothelial cells. This particularly involves the
Macrophage Chemoattractant Protein-1 (MCP-1), chemoattractants for
lymphocytes (IP-10, MIG, I-TAG), and the Vascular Cell Adhesion
Molecule-1 (VCAM-1), to which the monocytes and lymphocytes adhere.
Once adherent, the leucocytes enter the artery wall. The monocytes
and lymphocytes are recruited to the intima (sub-endothelial
layers) of the blood vessels by these cell attraction and cell
adhesion molecules of the activated endothelium during the early
stages of atherosclerosis (see Libby, P. (2000) "Changing concepts
of atherogenesis," Journal of Internal Medicine 247:349-358.)
[0024] Endothelial-mediated Vasodilatory Dysfunction is
characterized by a reduction or loss of endothelium-dependent
vasodilation and involves "decreased nitric oxide bioavailability"
(decreased production, increased destruction and/or decreased
sensitivity to nitric oxide). (De Caterina (2000), cited above).
Nitric oxide induces vasodilation by relaxing the smooth muscle
cells of the blood vessel wall. Endothelial-mediated Vasodilatory
Dysfunction can be measured as a reduction in vasodilation in
response to acetylcholine, or as a reduced vasodilatory response
following occlusion of arterial blood flow (reactive hyperaemia)
for example using a sphygmomanometer cuff. As well as leading to a
reduction in vasodilation, decreased endothelial nitric oxide
bioavailability can also result in an increase in the production of
vaso-constriction and hypertension. Platelet aggregation is
inhibited by nitric oxide, hence a decrease in nitric oxide
bioavailability can lead to an increase in platelet aggregation and
consequent thrombosis. These are just a few examples of how
decreased nitric oxide bioavailability resulting from
Endothelial-mediated Vasodilatory Dysfunction can have pathological
consequences.
[0025] The medical disorder resulting from endothelial dysfunction,
and hence treatable in accordance with the present invention, can
be a cardiovascular disorder such as atherosclerosis, peripheral
vascular disease, congestive heart failure, stroke, myocardial
infarction, angina, hypertension and the like. It can be a
vasospastic disorder such as Raynaud's disease, cardiac syndrome X,
migraine and the like. It can be the damage resulting from ischemia
(ischemic injury or ischemia-reperfusion injury). In summary, it
can be substantially any disorder that results from an
inappropriately functioning endothelium.
[0026] "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 penneability 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).
[0027] The compositions of 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).
[0028] 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 cells 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.
[0029] A variety of methods of inducing apoptosis in mammalian
cells, so as to create apoptotic cells and/or 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
(.gamma.-rays, x-rays, etc.) and/or non-ionizing electromagnetic
radiation including ultraviolet light. Apoptosis can be induced by
subjecting cells to ultrasound.
[0030] 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. A further 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, pennanganates,
periodates, and the like. Biologically acceptable oxidizing agents
are preferably used, so as to reduce potential problems associated
with residues and contaminations of the apoptotic cells and/or
apoptotic bodies so formed.
[0031] The present invention is not restricted to any particular
method of producing apoptotic cells and/or apoptotic bodies, for
use herein, and any suitable, known process can be used.
[0032] 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. A method as 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.
[0033] 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.
[0034] 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.
[0035] Another 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, suspension of the white cells in plasma or another
suitable suspension medium, such as saline or a balanced mammalian
cell culture medium and treatment of the white cells under
apoptosis-causing conditions, e.g. with a chemical such as sodium
butyrate, 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 re-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.
[0036] 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
compositon for administration is comprised of apoptotic cells and
apoptotic bodies, the balance being viable cell 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 mls,
preferably up to 100 mls. More specifically, 50,000,000 to
150,000,000 cells is equivalent to the white cells in blood
aliquots of volume 10-30 mls.
[0037] 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.
[0038] 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 re-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.
[0039] 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.
[0040] 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 an endothelial dysfunction related
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
4,000,000 apoptotic cells 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.
[0041] For most effective treatment and prophylaxis of mammalian
disorders involving an endothelial dysfunction, 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.
[0042] As noted, the present invention is applicable to the
treatment and/or prophylaxis of a wide variety of mammalian
disorders that involve endothelial dysfunction. These include, but
are not limited to, cardiovascular disease, such as
atherosclerosis, peripheral vascular disease, congestive heart
failure, stroke, myocardial infarction, angina, hypertension, etc.,
vasospastic disorders such as Raynaud's disease, cardiac syndrome
X, migraine, etc; and the damage resulting from ischemia (ischemic
injury or ischemia-reperfusion injury). In summary it can be
substantially any disorder that results from an inappropriately
functioning endothelium.
[0043] The invention is further described, for illustrative
purposes, in the following specific examples.
EXAMPLE 1
[0044] Experiments to demonstrate the invention were conducted on
laboratory mice, under approved conditions for conducting such
experiments.
[0045] 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 CHS, 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.
[0046] 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.
[0047] 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 1.times. binding buffer at a
concentration of 1.times.10.sup.6 cells/ml. 100 .mu.l of the cells
were transferred to a 5 ml 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 1.times. binding buffer was
added to each tube. The sample was analyzed on a flow cytometer
over one hour.
[0048] 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.
[0049] 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
[0050] 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 not to be significant in
the in vivo process.
EXAMPLE 2
[0051] 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.
[0052] 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.gamma., IL-6 and IL-12.
These inflammatory cytokines are implicated in endothelial
dysfunctions which manifest themselves as cardiovascular disorders,
such as atherosclerosis, peripheal vascular disease, congestive
heart failure, stroke, myocardial infarction, angina, hypertension
and the like; vasospastic disorders such as Raynaud's disease,
cardiac syndrome X, migraine and the like; and damage resulting
from ischemia (ischemic injury or ischemia-reperfusion injury).
Consequently, the finding of success in CHS treatment reported in
the above Examples is indicative of successful use of the process
and compositions in the treatment and prophylaxis of a wide variety
of endothelial dysfunction disorders including those discussed
above.
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