U.S. patent application number 16/370923 was filed with the patent office on 2020-08-20 for treatment of diseases and conditions caused by increased vascular permeability.
This patent application is currently assigned to Noveome Biotherapeutics, Inc.. The applicant listed for this patent is Noveome Biotherapeutics, Inc.. Invention is credited to Richard A. Banas, Larry R. Brown, Elise M. Gill, Howard C. Wessel.
Application Number | 20200261512 16/370923 |
Document ID | 20200261512 / US20200261512 |
Family ID | 1000004808577 |
Filed Date | 2020-08-20 |
Patent Application | download [pdf] |
United States Patent
Application |
20200261512 |
Kind Code |
A1 |
Brown; Larry R. ; et
al. |
August 20, 2020 |
Treatment of Diseases and Conditions Caused by Increased Vascular
Permeability
Abstract
The invention is directed to methods for the treatment of
diseases and conditions caused by increased vascular permeability.
The invention is also directed to methods for returning vascular
permeability that is a symptom of a disease or condition to a
homeostatic state. Specifically, the invention is directed to
methods for the treatment of diseases and conditions caused by
increased vascular permeability or returning vascular permeability
that is a symptom of a disease or condition to a homeostatic state
by administering to a subject suffering from such diseases and
conditions and symptoms novel cellular factor-containing solution
compositions (referred to herein as "CFS" compositions), including
novel sustained-release cellular factor-containing solution
compositions (referred to herein as "SR-CFS" compositions).
Inventors: |
Brown; Larry R.; (Newton,
MA) ; Banas; Richard A.; (US) ; Wessel; Howard
C.; (Kensington, PA) ; Gill; Elise M.;
(Hagerstown, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Noveome Biotherapeutics, Inc. |
Pittsburgh |
PA |
US |
|
|
Assignee: |
Noveome Biotherapeutics,
Inc.
Pittsburgh
PA
|
Family ID: |
1000004808577 |
Appl. No.: |
16/370923 |
Filed: |
March 30, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15990718 |
May 28, 2018 |
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16370923 |
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15492446 |
Apr 20, 2017 |
9980987 |
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15990718 |
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14717330 |
May 20, 2015 |
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15492446 |
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62001378 |
May 21, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/18 20130101;
A61K 38/1866 20130101; A61K 35/50 20130101; A61K 38/57 20130101;
A61K 38/1891 20130101; A61K 38/19 20130101; A61K 38/191 20130101;
A61K 38/1858 20130101 |
International
Class: |
A61K 35/50 20060101
A61K035/50; A61K 38/18 20060101 A61K038/18; A61K 38/57 20060101
A61K038/57; A61K 38/19 20060101 A61K038/19 |
Claims
1.-15. (canceled)
16. A method for treating a pulmonary disease or condition caused
by or exhibiting increased vascular permeability in a patient in
need thereof comprising administering to the patient a
therapeutically effective amount of Extraembryonic Amnion-derived
Cellular Cytokine Secreting Solution (ECS) cell conditioned medium,
wherein the ECS cell conditioned medium comprises physiological
concentrations of VEGF, TGF.beta., Angiogenin, PDGF, TIMP-1 and
TIMP-2, and wherein the physiological range is .about.5-16 ng/mL
for VEGF, .about.3.5-4.5 ng/mL for Angiogenin, .about.100-165 pg/mL
for PDGF, .about.0.68 .mu.g/mL for TIMP-1 and .about.1.04 .mu.g/mL
for TIMP-2, and wherein the pulmonary disease or condition is
selected from the group consisting of pulmonary edema, pulmonary
fibrosis, and acute respiratory distress syndrome.
17. The method of claim 16 wherein the ECS conditioned medium is
Amnion-derived Cellular Cytokine Solution (ACCS).
18. The method of claim 17 wherein the ACCS is formulated for
parenteral administration.
Description
FIELD OF THE INVENTION
[0001] The field of the invention is directed to methods for the
treatment of diseases and conditions caused by increased vascular
permeability. The field of the invention is also directed to
methods for returning vascular permeability that is a symptom of a
disease or condition to a homeostatic state. Specifically, the
field of the invention is directed to methods for the treatment of
diseases and conditions caused by increased vascular permeability
or returning vascular permeability that is a symptom of a disease
or condition to a homeostatic state by administering to a subject
suffering from such diseases and conditions and symptoms novel
cellular factor-containing solution compositions (referred to
herein as "CFS" compositions), including novel sustained-release
cellular factor-containing solution compositions (referred to
herein as "SR-CFS" compositions).
BACKGROUND OF THE INVENTION
[0002] Vascular permeability, often in the form of capillary
permeability or microvascular permeability, characterizes the
capacity of a blood vessel wall to allow for the flow of small
molecules (i.e., ions, water, nutrients), large molecules (i.e.,
albumin, antibodies, cytokines, nucleic acids, lipids) or even
whole cells (i.e., lymphocytes on their way to a site of
inflammation) in and out of the blood vessel. A single layer of
endothelial cells, called endothelium, line the blood vessel walls
and the heart chambers. Gaps which are located between the
endothelial cells (called cell junctions) are able to open or
close, but are strictly regulated depending on the type and
physiological state of the tissue.
[0003] There are many triggers for vascular permeability. For
example, an increase in vascular permeability occurs at the very
beginning of the inflammatory response and is initially triggered
by agents released by mast cells, which activate endothelial cell
receptors promoting endothelial cell retraction and gap junction
disorganization, leading to gap formation between the endothelial
cells in venules and capillaries. (Garcia Leme, J., Hamamura, L.,
Leite, M. P., Rocha e Silva, M., 1973. Pharmacological analysis of
the acute inflammatory process induced in the rat's paw by local
injection of carrageenan and by heating. Br. J. Pharmacol. 48,
88-96; Holsapple, M. P., Schnur, M., Yim, G. K., 1980.
Pharmacological modulation of edema mediated by prostaglandin,
serotonin and histamine. Agents Actions 10, 368-373.) The
subsequent leakage of macromolecules to the injured tissue is the
main cause of edema formation. A neutrophil-endothelium
interaction, which is necessary for neutrophil migration, will then
contribute to a more persistent increase in vascular permeability
(Kubes, P., Gaboury, J. P., 1996. Rapid mast cell activation causes
leukocyte-dependent and -independent permeability alterations. Am.
J. Physiol. 271, H2438-H2446; Lewis, R. E., Granger, H. J., 1986.
Neutrophil-dependent mediation of microvascular permeability. Fed.
Proc. 45, 109-113).
[0004] In acute inflammation, fluid loss from blood vessels with
increased permeability occurs in distinct phases: (1) an immediate
transient response lasting for 30 minutes or less, mediated mainly
by the actions of histamine and leukotrienes on endothelium; (2) a
delayed response starting at about 2 hours and lasting for about 8
hours, mediated by kinins, complement products, and other factors;
and (3) a prolonged response that is most noticeable after direct
endothelial injury, for example, after burns.
[0005] A critical function of inflammation is to deliver leukocytes
to the site of injury and to activate the leukocytes to perform
their normal functions in host defense. Leukocytes ingest offending
agents, kill bacteria and other microbes, and get rid of necrotic
tissue and foreign substances. However, untoward events manifest
themselves as a result of the defensive potency of leukocytes. For
example, leukocytes may induce tissue damage and prolong
inflammation and leukocyte products that destroy microbes and
necrotic tissues can also injure normal host tissues.
[0006] Increased vascular permeability is also exhibited by certain
viral diseases called viral hemorrhagic fevers (VHFs). VHF refers
to a group of illnesses that are caused by several distinct
families of viruses. In general, the term "viral hemorrhagic fever"
is used to describe a severe multisystem syndrome in which the
overall vascular system is damaged, and the body's ability to
regulate itself is impaired. These symptoms are often accompanied
by hemorrhage; however, the bleeding itself is rarely
life-threatening. While some types of VHFs can cause relatively
mild illnesses, many of these viruses cause severe,
life-threatening disease. Examples include Ebola, Marburg, Lassa
fever, and yellow fever viruses, among others.
[0007] Currently, there is no universal, satisfactory treatment for
many of the VHFs, particularly those that cause life-threatening
disease. Treatments include transfusion of serum from patients that
have recovered from the viral infection as well as aggressive
treatment of symptoms including nausea, vomiting, diarrhea, fever,
and bleeding.
[0008] It is believed that a treatment option that could reduce
abnormal vascular permeability associated with certain diseases and
conditions or which is a symptom of certain diseases and conditions
would vastly benefit patients who otherwise have few treatment
options. In particular, a treatment option that could decrease
vascular permeability could prove very useful in the management of
the notoriously difficult VHF diseases.
[0009] Accordingly, it is an object of the instant invention to
provide such a treatment options.
BRIEF SUMMARY OF THE INVENTION
[0010] The instant invention provides novel cellular
factor-containing solution (CFS) compositions, including
Amnion-derived Cellular Cytokine Solution (ACCS) (see U.S. Pat.
Nos. 8,058,066 and 8,088,732, both of which are incorporated herein
by reference), for use in the described methods for the treatment
of diseases and conditions caused by increased vascular
permeability or returning vascular permeability that is a symptom
of a disease or condition to a homeostatic state, such as VHFs.
Applicant has discovered that ACCS, which contains more than 200
proteins, cytokines, and growth factors, universally modulates
vascular permeability. Applicant has discovered that ACCS modulates
vascular permeability in human umbilical vein endothelial cells
(HUVECs) in tissue culture. Based on these discoveries, Applicant
has further discovered that in vivo, ACCS will result in the
modulation of vascular permeability as assessed using the Miles
Assay (see A. A. Miles and E. M. Miles, Vascular reactions to
histamine, histamine-liberator and leukotaxine in the skin of
guinea-pigs, J. Physiol. (1952) 118, 228-257), the contents of
which is incorporated herein by reference in its entirety.
Applicant has shown that ACCS decreases vascular permeability
stimulated by several different stimulators including VEGF (an
angiogenic protein factor), histamine (an organic nitrogenous
compound involved in local immune responses), bradykinin (a peptide
that causes blood vessels to dilate) and TNF.alpha. (a cell
signaling protein (cytokine) involved in systemic inflammation),
thus demonstrating that ACCS may be used as a universal agent to
treat many diseases or conditions that are characterized by
increases in vascular permeability as well as vascular permeability
that is a symptom of a disease or condition. By way of non-limiting
example, insults resulting from burns, physical injuries,
infections, wounds, radiation exposure, pulmonary edema,
periodontal disease, pulmonary fibrosis, acute respiratory distress
syndrome, severe pustular psoriasis, septic shock,
hyperpermeability triggered by inflammation or ischemia in the
heart, brain, or lung that promotes edema and exacerbates disease
progression and impairs recovery, insect bites, natural or
synthetic chemical irritants, frostbite, toxins, infection by
pathogens (for example, viruses that cause VHFs), immune reactions
due to hypersensitivity, foreign bodies including splinters, dirt
and debris, stress, trauma, alcohol, appendicitis, bursitis,
colitis, cystitis, dermatitis, phlebitis, rhinitis, tendonitis,
tonsillitis, vasculitis, release of prostaglandins (E1, E2,
F1.alpha. and F2.alpha.), histamine, serotonin, bradykinin,
lipopolysaccharides, cytokines or growth factors such as VEGF,
interleukins, TNF-.alpha., etc. resulting in increased vascular
permeability and deviation from homeostatic vascular permeability
could benefit from the methods of the invention.
[0011] Other diseases, disorders and conditions characterized by
undesirable vascular permeability include, for example, edema,
Meigs' syndrome, lung inflammation, nephrotic syndrome, pericardial
effusion, pleural effusion, acute lung injury, inflammatory bowel
disease, ischemia/reperfusion injury in stroke, myocardial
infarction, and infectious and non-infectious diseases that result
in a cytokine storm. Though a cytokine storm is the systemic
expression of a healthy and vigorous immune system, it is an
exaggerated immune response caused by rapidly proliferating and
highly activated T-cells or natural killer (NK) cells and results
in the release of more than 150 inflammatory mediators (including
cytokines, oxygen free radicals, and coagulation factors). Both
pro-inflammatory cytokines (i.e., TNF-.alpha., IFN-.gamma.,
IL-1.beta., IL-2, IL-15 and IL-6) and anti-inflammatory cytokines
(i.e., IL-10, and IL-1ra) are elevated in the serum, and it is the
fierce and often lethal interplay of these cytokines that
characterizes the "cytokine storm."
[0012] Cytokine storms can occur in a number of infectious and
non-infectious diseases including, for example, graft-versus-host
disease (GVHD), adult respiratory distress syndrome (ARDS), sepsis,
avian influenza, smallpox, and systemic inflammatory response
syndrome (SIRS). In the absence of prompt intervention, a cytokine
storm can result in permanent lung damage and, in many cases,
death. Many patients will develop ARDS, which is characterized by
pulmonary edema that is not associated with volume overload or
depressed left ventricular function. The end stage symptoms of a
disease precipitating the cytokine storm may include one or more of
the following: hypotension, tachycardia, dyspnea, fever, ischemia
or insufficient tissue perfusion, uncontrollable hemorrhage, severe
metabolism dysregulation, and multisystem organ failure. Deaths
from infections that precipitate a cytokine storm are often
attributable to the symptoms resulting from the cytokine storm and
are, therefore, not directly caused by the relevant pathogen. For
example, death in severe influenza infections, such as by avian
influenza or "bird flu," is typically the result of ARDS, which
results from a cytokine storm triggered by the viral infection.
[0013] Accordingly, a first aspect of the invention is a method for
treating diseases and conditions caused by increased vascular
permeability in a patient in need thereof comprising administering
to the patient a therapeutically effective amount of a CFS
composition.
[0014] A second aspect of the invention is method for returning
vascular permeability that is a symptom of a disease or condition
to homeostatic state in a patient in need thereof comprising
administering to the patient a therapeutically effective amount of
a CFS composition.
[0015] A third aspect of the invention is a method for treating
viral hemorrhagic fever in a patient in need thereof comprising
administering to the patient a therapeutically effective amount of
a CFS composition.
[0016] Specific embodiments of aspects 1, 2 and 3 of the invention
are ones in which the CFS composition is ACCS; the CFS composition,
including ACCS, is formulated for topical or intranasal
administration; the CFS composition, including ACCS, is formulated
for parenteral administration; the CFS composition, including ACCS,
is formulated for enteral administration.
[0017] In still other embodiments of the invention, the diseases
and conditions caused by increased vascular permeability or
exhibited vascular permeability as a symptom are selected from the
group consisting of burns, physical injuries, infections, wounds,
radiation exposure, pulmonary edema, periodontal disease, pulmonary
fibrosis, acute respiratory distress syndrome, septic shock,
hyperpermeability triggered by inflammation or ischemia in the
heart, brain, or lung, insect bites, natural or synthetic chemical
irritants, frostbite, toxins, infection by pathogens, immune
reactions due to hypersensitivity, foreign bodies, stress, trauma,
alcohol, appendicitis, bursitis, colitis, cystitis, dermatitis,
phlebitis, rhinitis, tendonitis, tonsillitis, vasculitis, release
of prostaglandins (E1, E2, F1.alpha. and F2.alpha.), histamine,
serotonin, bradykinin, cytokine storms, and
lipopolysaccharides.
Definitions
[0018] As defined herein "isolated" refers to material removed from
its original environment and is thus altered "by the hand of man"
from its natural state.
[0019] As used herein, the term "protein marker" means any protein
molecule characteristic of the plasma membrane of a cell or in some
cases of a specific cell type.
[0020] As used herein, "enriched" means to selectively concentrate
or to increase the amount of one or more materials by elimination
of the unwanted materials or selection and separation of desirable
materials from a mixture (i.e., separate cells with specific cell
markers from a heterogeneous cell population in which not all cells
in the population express the marker).
[0021] As used herein, the term "substantially purified" means a
population of cells substantially homogeneous for a particular
marker or combination of markers. By substantially homogeneous is
meant at least 90%, and preferably 95% homogeneous for a particular
marker or combination of markers.
[0022] The term "placenta" as used herein means both preterm and
term placenta.
[0023] As used herein, the term "totipotent cells" shall have the
following meaning. In mammals, totipotent cells have the potential
to become any cell type in the adult body; any cell type(s) of the
extraembryonic membranes (e.g., placenta). Totipotent cells are the
fertilized egg and approximately the first 4 cells produced by its
cleavage.
[0024] As used herein, the term "pluripotent stem cells" shall have
the following meaning. Pluripotent stem cells are true stem cells
with the potential to make any differentiated cell in the body, but
cannot contribute to making the components of the extraembryonic
membranes which are derived from the trophoblast. The amnion
develops from the epiblast, not the trophoblast. Three types of
pluripotent stem cells have been confirmed to date: Embryonic Stem
(ES) Cells (may also be totipotent in primates), Embryonic Germ
(EG) Cells, and Embryonic Carcinoma (EC) Cells. These EC cells can
be isolated from teratocarcinomas, a tumor that occasionally occurs
in the gonad of a fetus. Unlike the other two, they are usually
aneuploid.
[0025] As used herein, the term "multipotent stem cells" are true
stem cells but can only differentiate into a limited number of
types. For example, the bone marrow contains multipotent stem cells
that give rise to all the cells of the blood but may not be able to
differentiate into other cells types.
[0026] As used herein, the term "extraembryonic tissue" means
tissue located outside the embryonic body which is involved with
the embryo's protection, nutrition, waste removal, etc.
Extraembryonic tissue is discarded at birth. Extraembryonic tissue
includes but is not limited to the amnion, chorion (trophoblast and
extraembryonic mesoderm including umbilical cord and vessels), yolk
sac, allantois and amniotic fluid (including all components
contained therein). Extraembryonic tissue and cells derived
therefrom have the same genotype as the developing embryo.
[0027] As used herein, the term "extraembryonic cytokine secreting
cells" or "ECS cells" means a population of cells derived from the
extraembryonic tissue which have the characteristics of secreting a
unique combination of physiologically relevant cytokines in a
physiologically relevant temporal manner into the extracellular
space or into surrounding culture media and which have not been
cultured in the presence of any non-human animal-derived products,
making them and cell products derived from them suitable for human
clinical use. In a preferred embodiment, the ECS cells secrete the
cytokines VEGF, Angiogenin, PDGF and the MMP inhibitors TIMP-1
and/or TIMP-2. The physiological range of the cytokine or cytokines
in the unique combination is as follows: .about.5-16 ng/mL for
VEGF, .about.3.5-4.5 ng/mL for Angiogenin, .about.100-165 pg/mL for
PDGF, .about.0.68 .mu.g/mL for TIMP-1 and .about.1.04 .mu.g/mL for
TIMP-2.
[0028] As used herein, the term "amnion-derived multipotent
progenitor cell" or "AMP cell" means a specific population of ECS
cells that are epithelial cells derived from the amnion. In
addition to the characteristics described above for ECS cells, AMP
cells have the following characteristics. They have not been
cultured in the presence of any non-human animal-derived products,
making them and cell products derived from them suitable for human
clinical use. They grow without feeder layers, do not express the
protein telomerase and are non-tumorigenic. AMP cells do not
express the hematopoietic stem cell marker CD34 protein. The
absence of CD34 positive cells in this population indicates the
isolates are not contaminated with hematopoietic stem cells such as
umbilical cord blood or embryonic fibroblasts. Virtually 100% of
the cells react with antibodies to low molecular weight
cytokeratins, confirming their epithelial nature. Freshly isolated
amnion epithelial cells, from which AMP cells are selected, will
have no reaction with an antibody to the stem/progenitor cell
marker c-kit (CD117), and minimal to no reaction with an antibody
to the stem/progenitor cell marker Thy-1 (CD90). Several procedures
used to obtain cells from full term or pre-term placenta are known
in the art (see, for example, US 2004/0110287; Anker et al., 2005,
Stem Cells 22:1338-1345; Ramkumar et al., 1995, Am. J. Ob. Gyn.
172:493-500). However, the methods described herein provide
improved, novel compositions and populations of cells.
[0029] By the term "animal-free" when referring to certain
compositions, growth conditions, culture media, etc. described
herein, is meant that no non-human animal-derived materials, such
as bovine serum, proteins, lipids, carbohydrates, nucleic acids,
vitamins, etc., are used in the preparation, growth, culturing,
expansion, storage or formulation of the cell, composition or
process. By "no non-human animal-derived materials" is meant that
the materials have never been in or in contact with a non-human
animal body or substance so they are not xeno-contaminated. Only
clinical grade materials, such as recombinantly produced human
proteins, are used in the preparation, growth, culturing,
expansion, storage and/or formulation of such cells, compositions
and/or processes.
[0030] By the term "serum-free" when referring to certain
compositions, growth conditions, culture media, etc., described
herein, is meant that no animal-derived serum (i.e., no non-human)
is used in the preparation, growth, culturing, expansion, storage
or formulation of the cells, composition or process.
[0031] By the term "expanded", in reference to cell compositions,
means that the cell population constitutes a significantly higher
concentration of cells than is obtained using previous methods. For
example, the level of cells per gram of amniotic tissue in expanded
compositions of AMP cells is at least 50 and up to 150 fold higher
than the number of cells in the primary culture after 5 passages,
as compared to about a 20-fold increase in such cells using
previous methods. In another example, the level of cells per gram
of amniotic tissue in expanded compositions of AMP cells is at
least 30 and up to 100 fold higher than the number of cells in the
primary culture after 3 passages. Accordingly, an "expanded"
population has at least a 2 fold, and up to a 10 fold, improvement
in cell numbers per gram of amniotic tissue over previous methods.
The term "expanded" is meant to cover only those situations in
which a person has intervened to elevate the number of the
cells.
[0032] As used herein, "conditioned medium" is a medium in which a
specific cell or population of cells has been cultured, and then
removed. When cells are cultured in a medium, they may secrete
cellular factors that can provide support to or affect the behavior
of other cells. Such factors include, but are not limited to
hormones, cytokines, extracellular matrix (ECM), proteins,
vesicles, antibodies, chemokines, receptors, inhibitors and
granules. The medium containing the cellular factors is the
conditioned medium.
[0033] As used herein, the term "cellular factor-containing
solution" or "CFS" composition means a composition having
physiologic concentrations of one or more protein factors. CFS
compositions include conditioned media derived from ECS cells,
amnion-derived cellular cytokine solution compositions (see
definition below), physiologic cytokine solution compositions (see
definition below), and sustained release formulations of such CFS
compositions.
[0034] As used herein, the term "amnion-derived cellular cytokine
solution" or "ACCS" means conditioned medium that has been derived
from AMP cells or expanded AMP cells.
[0035] As used herein, the term "physiologic cytokine solution" or
"PCS" composition means a composition which is not cell-derived and
which has physiologic concentrations of VEGF, Angiogenin, PDGF and
TGF.beta.2, TIMP-1 and TIMP-2.
[0036] As used herein, the term "suspension" means a liquid
containing dispersed components, i.e., cytokines. The dispersed
components may be fully solubilized, partially solubilized,
suspended or otherwise dispersed in the liquid. Suitable liquids
include, but are not limited to, water, osmotic solutions such as
salt and/or sugar solutions, cell culture media, and other aqueous
or non-aqueous solutions.
[0037] The term "lysate" as used herein refers to the composition
obtained when cells, for example, AMP cells, are lysed and
optionally the cellular debris (e.g., cellular membranes) is
removed. This may be achieved by mechanical means, by freezing and
thawing, by sonication, by use of detergents, such as EDTA, or by
enzymatic digestion using, for example, hyaluronidase, dispase,
proteases, and nucleases. In some instances, it may be desirable to
retain the cellular debris (e.g., cellular membranes) as well.
[0038] The term "physiologic" or "physiological level" as used
herein means the level that a substance in a living system is found
and that is relevant to the proper functioning of a biochemical
and/or biological process.
[0039] As used herein, the term "substrate" means a defined coating
on a surface that cells attach to, grow on, and/or migrate on. As
used herein, the term "matrix" or "scaffold" means a
three-dimensional (3D) structure that cells grow within or on that
may or may not be defined in its components. It may be composed of
biological components, synthetic components, or a combination of
both. Further, it may be naturally constructed by cells (i.e.,
extracellular matrix) or artificially constructed. In addition, the
matrix or scaffold may contain components that have biological
activity under appropriate conditions.
[0040] The term "cell product" or "cell products" as used herein
refers to any and all substances made by and secreted from a cell,
including but not limited to, protein factors (i.e., growth
factors, differentiation factors, engraftment factors, cytokines,
morphogens, proteases (i.e., to promote endogenous cell
delamination, protease inhibitors), extracellular matrix components
(i.e., fibronectin, etc.).
[0041] The term "therapeutically effective amount" means that
amount of a therapeutic agent necessary to achieve a desired
physiological effect (i.e., treating diseases and conditions caused
by increased vascular permeability or treating vascular
permeability that is a symptom of a disease or condition).
[0042] As used herein, the term "pharmaceutically acceptable" means
that the components, in addition to the therapeutic agent,
comprising the formulation, are suitable for administration to the
patient being treated in accordance with the present invention.
[0043] As used herein, the term "therapeutic component" means a
component of the composition which exerts a therapeutic benefit
when the composition is administered to a subject.
[0044] As used herein, the term "therapeutic protein" includes a
wide range of biologically active proteins including, but not
limited to, growth factors, enzymes, hormones, cytokines,
inhibitors of cytokines, blood clotting factors, peptide growth and
differentiation factors.
[0045] As used herein, the term "tissue" refers to an aggregation
of similarly specialized cells united in the performance of a
particular function.
[0046] As used herein, the terms "a" or "an" means one or more; at
least one.
[0047] As used herein, the term "adjunctive" means jointly,
together with, in addition to, in conjunction with, and the
like.
[0048] As used herein, the term "co-administer" can include
simultaneous or sequential administration of two or more
agents.
[0049] As used herein, the term "agent" means an active agent or an
inactive agent. By the term "active agent" is meant an agent that
is capable of having a physiological effect when administered to a
subject. Non-limiting examples of active agents include growth
factors, cytokines, antibiotics, cells, conditioned media from
cells, etc. By the term "inactive agent" is meant an agent that
does not have a physiological effect when administered. Such agents
may alternatively be called "pharmaceutically acceptable
excipients". Non-limiting examples include time release capsules
and the like.
[0050] The terms "parenteral administration" and "administered
parenterally" are art-recognized and refer to modes of
administration other than intranasal, enteral and topical
administration, usually by injection, and includes, without
limitation, intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intra-articulare, subcapsular, subarachnoid, intraspinal, epidural,
intracerebral, intraocular, subdural and intrasternal injection or
infusion.
[0051] As used herein, the term "enteral" administration means any
route of drug administration that involves absorption of the drug
through the gastrointestinal tract. Enteral administration may be
divided into three different categories: oral, gastric, and
rectal.
[0052] As used herein, the term "topical" administration means a
medication that is applied to body surfaces such as the skin or
mucous membranes to treat ailments via a large range of classes
including but not limited to liquids, sprays, creams, foams, gels,
lotions, salves and ointments.
[0053] The term "intranasal" or "intranasal delivery" or
"intranasal administration" as used herein means delivery within or
administered by way of the nasal structures.
[0054] As used herein, the term "aerosol" means a cloud of solid or
liquid particles in a gas.
[0055] The terms "particles", "aerosolized particles", and
"aerosolized particles of formulation" are used interchangeably
herein and shall mean particles of formulation comprised of any
pharmaceutically active ingredient, preferably in combination with
a carrier, (e.g., a pharmaceutically active respiratory drug and
carrier). The particles have a size which is sufficiently small
such that when the particles are formed they remain suspended in
the air or gas for a sufficient amount of time such that a patient
can inhale the particles into the patient's lungs. As used herein,
the term "nebulizer" means a device used to reduce a liquid
medication to extremely fine cloudlike particles (i.e. an aerosol).
A nebulizer is useful in delivering medication to deeper parts of
the respiratory tract. Nebulizers may also be referred to as
atomizers and vaporizers.
[0056] The terms "sustained-release", "extended-release",
"time-release", "controlled-release", or "continuous-release" as
used herein means an agent, typically a therapeutic agent or drug,
that is formulated to dissolve slowly and be released over
time.
[0057] As used herein, the term "vascular permeability" means the
capacity of a blood vessel wall to allow for the flow of small
molecules (such as ions, water, and nutrients), large molecules
(such as albumin, antibodies, cytokines, nucleic acids, and
lipids), or even whole cells (such as lymphocytes, B cells,
neutrophils, mast cells, macrophages, monocytes, eosinophils, and
basophils) in to and out of the blood vessel.
[0058] The term "viral hemorrhagic fever" or "VHF" means any number
of viral diseases including without limitation Ebola, Marburg,
Lassa fever, and yellow fever viruses, among others.
[0059] "Treatment," "treat," or "treating," as used herein covers
any treatment of a disease or condition of a mammal, particularly a
human, and includes: (a) preventing the disease or condition from
occurring in a subject which may be predisposed to the disease or
condition but has not yet been diagnosed as having it; (b)
arresting its development; (c) relieving and or ameliorating the
disease or condition, i.e., causing regression of the disease or
condition; or (d) curing the disease or condition, i.e., stopping
its development or progression. The population of subjects treated
by the methods of the invention includes subjects suffering from
the undesirable condition or disease, as well as subjects at risk
for development of the condition or disease.
[0060] As used herein, a "wound" is any disruption, from whatever
cause, of normal anatomy (internal and/or external anatomy)
including but not limited to traumatic injuries such as mechanical
(i.e. contusion, penetrating), thermal, chemical, electrical,
radiation, concussive and incisional injuries; elective injuries
such as operative surgery and resultant incisional hernias,
fistulas, etc.; acute wounds, chronic wounds, infected wounds, and
sterile wounds, as well as wounds associated with disease states
(i.e., ulcers caused by diabetic neuropathy or ulcers of the
gastrointestinal or genitourinary tract). A wound is dynamic and
the process of healing is a continuum requiring a series of
integrated and interrelated cellular processes that begin at the
time of wounding and proceed beyond initial wound closure through
arrival at a stable scar. These cellular processes are mediated or
modulated by humoral substances including but not limited to
cytokines, lymphokines, growth factors, and hormones. In accordance
with the subject invention, "wound healing" refers to improving, by
some form of intervention, the natural cellular processes and
humoral substances of tissue repair such that healing is faster,
and/or the resulting healed area has less scaring and/or the
wounded area possesses tissue strength that is closer to that of
uninjured tissue and/or the wounded tissue attains some degree of
functional recovery.
[0061] As used herein the term "standard animal model" refers to
any art-accepted animal model in which the compositions of the
invention exhibit efficacy.
DETAILED DESCRIPTION
[0062] In accordance with the present invention there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook et al, 2001, "Molecular Cloning: A Laboratory Manual";
Ausubel, ed., 1994, "Current Protocols in Molecular Biology"
Volumes I-III; Celis, ed., 1994, "Cell Biology: A Laboratory
Handbook" Volumes I-III; Coligan, ed., 1994, "Current Protocols in
Immunology" Volumes I-III; Gait ed., 1984, "Oligonucleotide
Synthesis"; Hames & Higgins eds., 1985, "Nucleic Acid
Hybridization"; Hames & Higgins, eds., 1984, "Transcription And
Translation"; Freshney, ed., 1986, "Animal Cell Culture"; IRL
Press, 1986, "Immobilized Cells And Enzymes"; Perbal, 1984, "A
Practical Guide To Molecular Cloning."
[0063] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range is encompassed within the invention. The
upper and lower limits of these smaller ranges may independently be
included in the smaller ranges is also encompassed within the
invention, subject to any specifically excluded limit in the stated
range. Where the stated range includes one or both of the limits,
ranges excluding either both of those included limits are also
included in the invention.
[0064] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now
described.
[0065] It must be noted that as used herein and in the appended
claims, the singular forms "a," "and" and "the" include plural
references unless the context clearly dictates otherwise.
[0066] Exemplary Therapeutic Applications
[0067] Any insult resulting in increased vascular permeability
could benefit from the methods of the invention. For example, in
septic shock, the rate of loss of albumin to the tissue spaces
rises by more than 300%. In cardiac surgery, it rises by 100%
within 7 hours of the surgery. The transcapillary escape rate in
cachectic cancer patients is twice that of a group of healthy
individuals. Large rate increases in vascular permeability is often
seen in acute and chronic disease.
[0068] Increased vascular permeability contributes to many
diseases, including acute respiratory distress syndrome (ARDS), and
inflammation. Most studies on the vascular barrier function have
focused on soluble regulators, such as tumor-necrosis
factor-.alpha. (TNF-.alpha.). It has been shown that lung vascular
permeability is controlled mechanically by changes in extracellular
matrix structure. Studies reveal that pulmonary vascular leakage
can be increased by altering extracellular matrix compliance in
vitro and by manipulating lysyl oxidase-mediated collagen
crosslinking in vivo. Either decreasing or increasing extracellular
matrix stiffness relative to normal levels disrupts junctional
integrity between endothelial cells and thus increases vascular
leakage. (Akiko Mammoto, Tadanori Mammoto, Mathumai
Kanapathipillai, Chong Wing Yung, Elisabeth Jiang, Amanda Jiang,
Kristopher Lofgren, Elaine P. S. Gee, Donald E. Ingber, Control of
lung vascular permeability and endotoxin-induced pulmonary oedema
by changes in extracellular matrix mechanics, Nature Commun 4:1759.
doi: 10.1038/ncomms2774, 2013).
[0069] Human pathologies such as vascular malformations,
hemorrhagic stroke, and edema are associated with defects in the
organization of endothelial cell junctions. Edema around the
ischemic area extends brain damage in ischemic stroke. Inflammation
is often associated with increases in vascular permeability, which
favors leukocyte diapedesis through the vessel wall and may create
pain and swelling. Edema is often reversible and the control of
vascular permeability may be restored once the triggering cause is
removed.
[0070] There are three microvascular events that characterize acute
inflammation: arteriolar vasodilatation, neutrophil recruitment and
vascular permeability increase. Applicant has previously
demonstrated in various in vivo studies that ACCS decreases
inflammation as seen by reduced neutrophil and leukocyte presence
at the site of injury and that ACCS decreases inflammation in
periodontitis (see U.S. Pat. No. 8,444,417, incorporated herein by
reference in its entirety). The reduction of vascular permeability
to more normal levels by ACCS would likely involve the interruption
of pro-inflammatory cytokine cascades by its multitude of growth
factor and cytokine components.
[0071] There are numerous cytokines, growth factors, and signal
molecules which react with endothelial cell structural components
which control vascular permeability. Interferon-gamma
(IFN-.gamma.), interleukin-1 alpha and beta (IL-1.alpha. and
IL-1.beta.) and tumor necrosis factor-alpha (TNF-.alpha.) have all
been shown to increase endothelial monolayer permeability (Lal B K
et al. (2001) VEGF increases permeability of the endothelial cell
monolayer by activation of PKB/akt, endothelial nitric-oxide
synthase, and MAP kinase pathways. Microvascular Research
62:252-262) VEGF increases permeability of the endothelial cell
monolayer by activation of PKB/akt, endothelial nitric-oxide
synthase, and MAP kinase pathways. Microvascular Research
62:252-262., Burke-Gaffney A et al. (1993) Modulation of human
endothelial cell permeability by combinations of the cytokines
interleukin-1 alpha/beta, tumor necrosis factor-alpha and
interferon-gamma. Immunopharmacology 25:1-9, Marcus B C et al.
(1996) Cytokine-induced increases in endothelial permeability occur
after adhesion molecule expression. Surgery 120:411-417. Campbell W
N et al. (1992) Interleukin-1 alpha and -beta augment pulmonary
artery transendothelial albumin flux in vitro. American Journal of
Physiology 263:128-136). Thrombin stimulation of cytoskeletal
signaling pathways has been shown to manipulate cell permeability.
Lipopolysaccharide (LPS) induces junction barrier loss and cell
detachment by activating protein tyrosine kinases (PTKs) and
caspase cleavage reactions (Bannerman D D et al. (1998) Bacterial
lipopolysaccharide disrupts endothelial monolayer integrity and
survival signaling events through caspase cleavage of adherens
junction proteins. Journal of Biological Chemistry
273:35371-35380.). In contrast, junctional adhesion molecule (JAM)
decreases permeability by initiating cell adhesion (Bazzoni G et
al. (2000) Interaction of junctional adhesion molecule with the
tight junction components ZO-1, cingulin, and occludin. Journal of
Biological Chemistry 275:20520-20526) and angiopoietin-1 (Ang-1)
protects endothelial barrier function through regulation of
junctional molecules (Li X et al. (2008) Basal and
angiopoietin-1-mediated endothelial permeability is regulated by
sphingosine kinase-1. Blood 111:3489-3497, Gamble J R et al. (2000)
Angiopoietin-1 is an anti-permeability and anti-inflammatory agent
in vitro and targets cell junctions. Circulation Research
87:603-607).
[0072] Disruption of the endothelial barrier integrity is
associated with many systemic disease states. Pathological
angiogenic diseases include heart disease, diabetes, stroke,
cancer, hypertension, arthritis, and Alzheimer's (Fu B M. (2001)
Microvessel permeability and its regulation. Recent Advances in
Biomechanics 231-247, Bates D O et al. (2002) Regulation of
microvascular permeability by vascular endothelial growth factors.
Journal of Anatomy 200:581-597, Mooradian A D. (1988) Effect of
aging on the blood-brain barrier. Neurobiological Aging 9:31-39).
In addition, increases in tissue permeability may be caused by any
number of stimuli that affect tight junctions, gap junctions or
matrix organizations.
[0073] One specific example of increased vascular permeability is
in the initial lesion of periodontal disease, in which the gingival
plexus becomes engorged and dilated, allowing large numbers of
neutrophils to extravasate and appear within the junctional
epithelium and underlying connective tissue (see Page, R C;
Schroeder, H E. "Pathogenesis of Inflammatory Periodontal Disease:
A Summary of Current Work." Lab Invest 1976; 34(3):235-249) (the
contents of which is incorporated herein by reference in its
entirety).
[0074] Increased vascular permeability is also exhibited by viral
hemorrhagic fevers (VHFs). VHF refers to a group of illnesses that
are caused by several distinct families of viruses. The term "viral
hemorrhagic fever" is used to describe a severe multisystem
syndrome in which the overall vascular system is damaged, and the
body's ability to regulate itself is impaired. These symptoms are
often accompanied by hemorrhage; however, the bleeding itself is
rarely life-threatening. While some types of VHFs can cause
relatively mild illnesses, many of these viruses cause severe,
life-threatening disease. Examples include Ebola, Marburg, Lassa
fever, and yellow fever viruses, among others.
[0075] Compositions and Methods of Making Compositions
[0076] Detailed information and methods on the preparation of AMP
cell compositions, generation of ACCS, generation of pooled ACCS,
detection of cytokines in non-pooled and pooled ACCS using ELISA,
generation of PCS compositions, and generation of sustained-release
CFS compositions can be found in U.S. Pat. Nos. 8,058,066,
8,088,732, 8,278,095 all of which are incorporated herein by
reference.
[0077] The invention provides for an article of manufacture
comprising packaging material and a pharmaceutical composition of
the invention contained within the packaging material, wherein the
pharmaceutical composition comprises CFS compositions, including
ACCS. The packaging material comprises a label or package insert
which indicates that the CFS compositions, including ACCS,
contained therein can be used for therapeutic applications such as,
for example, treating diseases and conditions caused by increased
vascular permeability or wherein vascular permeability is a symptom
of a disease or condition.
[0078] Formulation, Dosage and Administration of CFS
Compositions
[0079] Compositions comprising CFS compositions may be administered
to a subject to provide various cellular or tissue functions, for
example, treating diseases and conditions caused by increased
vascular permeability. As used herein "subject" may mean either a
human or non-human animal.
[0080] Such compositions may be formulated in any conventional
manner using one or more physiologically acceptable carriers
optionally comprising excipients and auxiliaries. Proper
formulation is dependent upon the route of administration chosen.
For topical administration, the CFS compositions may be formulated
as a spray, liquid, cream, foam, gel, lotion, salve, and ointment,
etc. The compositions may also be administered to the recipient in
one or more physiologically acceptable carriers. Carriers for CFS
compositions may include but are not limited to solutions of normal
saline, phosphate buffered saline (PBS), lactated Ringer's solution
containing a mixture of salts in physiologic concentrations, or
cell culture medium.
[0081] For parenteral administration, the formulation may be
injected intravenously.
[0082] For enteral administration, the formulation may be
administered as an oral liquid, a capsule or tablet designed to
release CFS compositions at a specific portion of the
gastro-intestinal tract.
[0083] For subcutaneous or intramuscular administration, the
formulation may be delivered by needle and syringe, by pen
injectors, by needleless injection devices and the like.
[0084] For intranasal administration, the formulation may be
administered as a nasal spray, a nebulized pulmonary dosage form, a
metered dose inhaler or a dry powder inhaler.
[0085] In addition, one of skill in the art may readily determine
the appropriate dose of the CFS compositions for a particular
purpose. An exemplary topical dose is in the range of about
0.1-to-10,000 milliliters per square centimeter of applied area.
Other exemplary dose ranges are 1.0-to-1,000 milliliters/applied
area. Exemplary intranasal and pulmonary delivery doses range from
about 0.01 milliliters per dose to about 10,000 milliliters per
dose. Exemplary oral doses range from about 0.01 milliliters to
about 10,000 milliliters, or equivalent tableted dosage form.
Exemplary injectable doses may range from about 0.01 milliliters to
about 10,000 milliliters per administration. In a particular
embodiment, it has been found that relatively small amounts of the
CFS compositions are therapeutically useful. One exemplification of
such therapeutic utility is the ability for ACCS (including pooled
ACCS) to accelerate wound healing (for details see U.S. Publication
No. 2006/0222634 and U.S. Pat. No. 8,187,881, both of which are
incorporated herein by reference). One of skill in the art will
also recognize that the number of doses to be administered needs
also to be empirically determined based on, for example, severity
and type of disease, disorder or injury being treated; patient age,
weight, sex, health; other medications and treatments being
administered to the patient; and the like. For example, in a
specific embodiment, one dose is sufficient to have a therapeutic
effect (i.e., treating diseases and conditions caused by increased
vascular permeability or treating vascular permeability that is a
symptom of a disease or condition). Other specific embodiments
contemplate, 2, 3, 4, or more doses for therapeutic effect.
[0086] For VHFs, the administration is typically intravenous. The
timing for administration needs to be empirically determined by the
attending physician as each patient will develop symptoms at
different time points following infection. Optimally, CFS will be
administered as soon as symptoms first appear. This is necessary to
prevent or minimize both the vascular permeability and the
excessive release of cytokines, termed a cytokine storm, which
could eventually causes death.
[0087] One of skill in the art will also recognize that number of
doses (dosing regimen) to be administered needs also to be
empirically determined based on, for example, severity and type of
injury, disorder or condition being treated; patient age, weight,
sex, health; other medications and treatments being administered to
the patient; and the like. In addition, one of skill in the art
recognizes that the frequency of dosing needs to be empirically
determined based on similar criteria. In certain embodiments, one
dose is administered every day for a given number of days (i.e.,
once a day for 7 days, etc.). In other embodiments, multiple doses
may be administered in one day (every 4 hours, etc.). Multiple
doses per day for multiple days are also contemplated by the
invention.
[0088] In further embodiments of the present invention, at least
one additional agent may be combined with the CFS compositions.
Such agents may act synergistically with the CFS compositions of
the invention to enhance the therapeutic effect. Such agents
include but are not limited to growth factors, cytokines,
chemokines, antibodies, inhibitors, antibiotics, immunosuppressive
agents, steroids, anti-fungals, anti-virals or other cell types
(i.e., stem cells or stem-like cells, for example, AMP cells).
Inactive agents include carriers, diluents, stabilizers, gelling
agents, delivery vehicles, ECMs (natural and synthetic), scaffolds,
matrices and the like. When the CFS compositions are administered
conjointly with other pharmaceutically active agents, even less of
the CFS compositions may be needed to be therapeutically
effective.
[0089] CFS compositions may also be inserted into a delivery
device, e.g., a tube, in different forms. For example, the CFS
compositions can be part of a solution contained in such a delivery
device. As used herein, the term "solution" includes a
pharmaceutically acceptable carrier or diluent. Pharmaceutically
acceptable carriers and diluents include saline, aqueous buffer
solutions, solvents and/or dispersion media. The use of such
carriers and diluents is well known in the art. In certain
applications it is be preferable for the solution to be fluid to
the extent that easy syringe loading is possible. Preferably, the
solution is stable under the conditions of manufacture and storage
and may optionally be preserved against the contaminating action of
microorganisms such as bacteria and fungi through the use of, for
example, parabens, chlorobutanol, phenol, ascorbic acid,
thimerosal, and the like. Solutions of the invention can be
prepared by incorporating the CFS compositions in a
pharmaceutically acceptable carrier or diluent and, as required,
other ingredients enumerated above.
[0090] The timing of administration of CFS compositions will depend
upon the type and severity of the disease, disorder, or injury
being treated. In one embodiment, the CFS compositions are
administered as soon as possible after onset of symptoms or
diagnosis. In another embodiment, CFS compositions are administered
more than one time following onset of symptoms or diagnosis.
[0091] Support matrices or scaffolds, including for example
membranes and the like, into which the CFS compositions can be
incorporated or embedded include substances which are
recipient-compatible and which degrade into products which are not
harmful to the recipient. Detailed information on suitable support
matrices, etc. can be found in U.S. Pat. Nos. 8,058,066 and
8,088,732, both of which are incorporated herein by reference.
Other suitable matrices and scaffolds are familiar in the art.
[0092] A "therapeutically effective amount" of a therapeutic agent
within the meaning of the present invention will be determined by a
patient's attending physician or veterinarian. Such amounts are
readily ascertained by one of ordinary skill in the art and will
enable treating diseases and conditions caused by increased
vascular permeability when administered in accordance with the
present invention. Factors which influence what a therapeutically
effective amount will be include, the specific activity of the
therapeutic agent being used, the extent of a wound, the absence or
presence of infection, time elapsed since a surgery, and the age,
physical condition, existence of other disease states, and
nutritional status of the patient. Additionally, other medication
the patient may be receiving will affect the determination of the
therapeutically effective amount of the therapeutic agent to
administer.
EXAMPLES
[0093] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the compositions and methods of
the invention, and are not intended to limit the scope of what the
inventors regard as their invention. Efforts have been made to
ensure accuracy with respect to numbers used (e.g., amounts,
temperature, etc.) but some experimental errors and deviations
should be accounted for. Unless indicated otherwise, parts are
parts by weight, molecular weight is average molecular weight,
temperature is in degrees Celsius, and pressure is at or near
atmospheric.
Example 1: In Vitro Vascular Permeability Assays
[0094] Objective:
[0095] TNF-.alpha. has been shown to increase endothelial monolayer
permeability (Mark, K. S., et al., Life Sciences, 1999,
64(21):1941-1953) In order to test whether ACCS has the ability to
affect vascular permeability, initial experiments were performed to
determine whether ACCS could reduce the level of permeability of
endothelial cells exposed to TNF-.alpha..
[0096] Method:
[0097] The ability of ACCS to modulate vascular permeability was
evaluated using an In Vitro Vascular Permeability Assay (Millipore,
Cat. No. ECM640). In this assay, Human vascular endothelial cells
(HUVEC) were seeded onto collagen or fibrin-coated semi-permeable
membrane inserts and a monolayer of cells was formed which occluded
the membrane pores. The inserts were then placed in a receiver
well. The cell monolayer can be treated with cytokines, growth
factors, or other compounds of interest. In this experiment, the
cells were treated with TNF-.alpha. and a high molecular weight
FITC-labeled Dextran added to the top of the cells. The
FITC-Dextran molecules are able to pass through the endothelial
cell monolayer into the receiver well solution at a rate
proportional to the monolayer's permeability. The extent of
permeability was determined by measuring the fluorescence of the
receiver plate well solution over time. The cells were exposed to
concentrations of TNF-.alpha. ranging from 25 to 200 ng/mL or
varying exposure times to TNF-.alpha. in either ACCS or control
media. The results for a 60 minute time course and 4 different
concentrations of TNF-.alpha. are set forth in Table 1 below and
the results for cumulative fluorescence experiment are set forth in
Table 2 below.
TABLE-US-00001 TABLE 1 60 minutes ACCS Control Media TNF.alpha.
(fluorescence) (fluorescence) 200 ng/mL 335 505 100 ng/mL 283 434
50 ng/mL 248 503 25 ng/mL 304 410
TABLE-US-00002 TABLE 2 Control Media + ACCS + 50 ng/mL TNF.alpha.
50 ng/mL TNF.alpha. Time (fluorescence) (fluorescence) 30 min 168
482 60 min 416 985 180 min 1192 1580 360 min 1638 2303
[0098] Results:
[0099] After 60 minutes of exposure to TNF-.alpha., the ACCS groups
reduced permeability of the endothelial cells at all concentrations
of TNF-.alpha. that were tested. The cumulative fluorescence
demonstrated that, in the presence of ACCS, the endothelial cells
were always less permeable that in cells treated with control
media.
Example 2: Evaluate Whether ACCS can Modulate Increased Vascular
Permeability as a Result of Irradiation
[0100] Objective:
[0101] Radiation is known to increase vascular permeability.
Therefore, a second set of experiments was conducted to determine
whether ACCS could modulate increased vascular permeability as a
result of irradiation from a 5 Gy cesium-137 source.
[0102] Method:
[0103] HUVECs were exposed to a radiation dose of 5 Gy prior to
treatment with ACCS, control media, or endothelial growth media
control.
[0104] Results:
[0105] As shown in Table 3 below, ACCS Lot A-treated cells showed
reduced FITC-Dextran fluorescence compared to endothelial growth
media control, and control media. These results demonstrate that
ACCS is modulating and therefore reducing vascular permeability.
Table 4 shows that radiation exposure to the cells induced an
increase in vascular permeability which was decreased by ACCS Lot A
and Lot B compared to endothelial growth media control, and control
media.
TABLE-US-00003 TABLE 3 Endothelial Growth Media Control ACCS
Control media (fluorescence) (fluorescence) (fluorescence) 5 Gy 797
281 470
TABLE-US-00004 TABLE 4 Endothelial Endothelial Growth Growth ACCS
ACCS ACCS ACCS Control Control Medium Medium Lot A + 5Gy Lot A Lot
B + 5Gy Lot B Media + 5Gy Media Control + 5Gy Control 13.3% 8%
11.8% 5% 21.1% 13% 17.0% 11%
Example 3: The Effect of ACCS on Reduction of Vascular Permeability
in a Setting Wherein Radiation is Combined with TNF-.alpha.
[0106] Objective:
[0107] Increased vascular permeability due to radiation may result
from many stimuli in vivo. Radiation combined with inflammatory
molecules may better simulate multiple inflammatory causes of
permeability in vivo. To further evaluate the effect of ACCS on
reduction of vascular permeability, HUVECs were exposed to both
radiation and TNF-.alpha., in various media.
[0108] Method:
[0109] HUVECs were exposed to 5 Gy radiation and 50 ng/mL
TNF-.alpha. for 4 hours.
[0110] Results:
[0111] Both ACCS Lot A and Lot B showed reduced permeability of
endothelial cells that were exposed to both 5 Gy radiation and 50
ng/mL TNF-.alpha. as compared to endothelial growth media control
and control media.
TABLE-US-00005 TABLE 5 ACCS ACCS Endothelial Control Lot A Lot B
Growth Media Media (fluores- (fluores- Control (fluores- cence)
cence) (fluorescence) cence) 5 Gy + 8.4 9.3 25.9 21.8 50 ng/mL
TNF-.alpha.
Example 4: ACCS Modulates Vascular Permeability In Vivo as Tested
in the Miles Assay
[0112] Objective:
[0113] The purpose of this study was to evaluate whether or not
ACCS can modulate vascular permeability in vivo using the Miles
Assay (A. A. Miles AND E. M. Miles, Vascular reactions to
histamine, histamine-liberator and leukotaxine in the skin of
guinea-pigs, J. Physiol. (I952) 118, 228-257).
[0114] Method:
[0115] Evans Blue Dye (5%) was administered intravenously to male
rats weighing approximately 300 grams. The Evans Blue dye binds
albumin present in the animal's blood stream. Test groups with and
without vascular permeability stimulants were then injected
intradermally on the flank of the animal forming a small bleb.
Changes in vascular permeability were measured by quantifying the
amount of Evans Blue dye present in a skin biopsy taken from each
bleb site. After dye was extracted from the skin biopsies, the
sample absorbance at 630 nm (the Evans Blue peak wavelength) was
normalized to the initial biopsy weight in grams. The vascular
permeability stimulants tested in this manner included histamine,
TNF-.alpha., VEGF and bradykinin. Doses of stimulant were chosen
based on literature references.
[0116] Results:
[0117] Table 6 below shows that compared to saline, ACCS reduced
the Evans Blue signal induced by all stimulants tested. The
reduction of Evans Blue is directly correlated with a reduction in
vascular permeability at the injection site. Reduction in vascular
permeability across all stimulants suggested that ACCS could be
effective in multiple indications involving vascular
permeability.
TABLE-US-00006 TABLE 6 Evans Blue dye Extraction (ABS.sub.630/g
tissue biopsy) 20 .mu.g/mL 20 .mu.g/mL Histamine 2 .mu.g/mL
TNF-.alpha. 4 .mu.g/mL VEGF Bradykinin Saline ACCS Saline ACCS
Saline ACCS Saline ACCS 1.330 0.291 0.505 0.203 1.449 0.519 1.28
0.71
Example 9: Effect of ACCS in an Animal Model of Viral Hemorrhagic
Fever
[0118] ACCS is tested in animal models of viral hemorrhagic fever
(see Badole, S. L. et al., Animal models for some important RNA
viruses of public health concern in SEARO countries: Viral
hemorrhagic fever, J Vector Borne Dis 52, March 2015, pp. 1-10;
Qui, X., et al., Establishment and Characterization of a Lethal
Mouse Model for the Angola Strain of Marburg Virus, November 2014.
Volume 88, Number 21, Journal of Virology p. 12703-12714).
[0119] The present invention may be embodied in other specific
forms without departing from the spirit or essential attributes
thereof. Any equivalent embodiments are intended to be within the
scope of this invention. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description. Such modifications are also intended to fall within
the scope of the appended claims.
[0120] Throughout the specification various publications have been
referred to. It is intended that each publication be incorporated
by reference in its entirety into this specification
* * * * *