Note: Descriptions are shown in the official language in which they were submitted.
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SUPPRESSION OF CYTOKINE RELEASE AND CYTOKINE STORM
CROSS-REFERENCE TO RELATED APPLICATIONS
100011 This application is a PCT International Application of a continuation-
in-part U.S. Patent
Application Serial No. 16/945,195 filed on July 31, 2020, which claims
priority to U.S.
Provisional Application Serial No. 62/098,898 filed December 31, 2014, U.S.
Provisional
Application Serial No. 62/165,567 filed May 22, 2015, and U.S. Provisional
Application Serial
No. 62/211,450 filed August 28, 2015, the entire contents of which are
incorporated herein by
reference.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates in general to the field of infectious
diseases and disease
conditions that trigger a cytokine cascade, and more particularly, to the use
of compositions that
reduce the cytokine cascade.
STATEMENT OF FEDERALLY FUNDED RESEARCH
[0003] This invention was made with U.S. Government support by the USAMRIID
under
Project No. 1323839. The government has certain rights in this invention.
BACKGROUND OF THE INVENTION
[0004] Without limiting the scope of the invention, its background is
described in connection
with infectious diseases and disease conditions that trigger, e.g., an
anaphylactic cytokine
cascade.
[0005] United States Patent No. 8,354,276, issued to Har-Noy, entitled, "T-
cell compositions
that elicit type I cytokine response", relates to a method of manipulating
allogeneic cells for use
in allogeneic cell therapy protocols is described. The method provides a
composition of highly
activated allogeneic T-cells, which are infused into immunocompetent cancer
patients to elicit a
novel anti-tumor immune mechanism called the "Mirror Effect". The inventors
argue that, in
contrast to current allogeneic cell therapy protocols where T-cells in the
graft mediate the
beneficial graft vs. tumor (GVT) and detrimental graft vs. host (GVH) effects,
the allogeneic cells
of the invention stimulate host T-cells to mediate the "mirror" of these
effects. The highly
activated allogeneic cells of the invention are said to stimulate host
immunity in a complete HLA
mis-matched setting in patients that have not had a prior bone marrow
transplant or received
chemotherapy and/or radiation conditioning regimens.
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[0006] United States Patent No. 8,309,519, issued to Li, et al., is entitled
"Compositions and
methods for inhibiting vascular permeability" and relates to compounds,
compositions and
methods for inhibiting vascular permeability and pathologic angiogenesis.
These inventors teach
methods for producing and screening compounds and compositions capable of
inhibiting vascular
permeability and pathologic angiogenesis. It is said that the compositions
described are useful in,
methods of inhibiting vascular permeability and pathologic angiogenesis,
including methods of
inhibiting vascular permeability and pathologic angiogenesis induced by
specific angiogenic,
permeability and inflammatory factors, such as, for example VEGF, I3FGF and
thrombin.
[0007] United States Patent No. 7,479,498, issued to Keller, is entitled
"Treatments for viral
infections" and relates to improved methods and compositions for treating
viral infections and
other diseases and conditions that induce a cytokine storm. It is further said
that the invention
relates to novel compositions comprising quercetin, and an anti-convulsant,
such as phenytoin, in
combination with multivitamins as an anti-viral composition and methods of use
thereof.
[0008] United States Patent Application No. 20100075329, filed by O'Toole, et
al., is entitled
"Methods For Predicting Production Of Activating Signals By Cross-Linked
Binding Proteins"
and relates to human binding proteins and antigen-binding fragments thereof
that specifically bind
to the human interleukin-21 receptor (IL21R), and uses therefore. The
invention is said to include
methods to predict whether the binding proteins of the invention may take on
agonistic activities
in vivo and produce a cytokine storm. In addition, the invention is said to
provide methods for
determining whether an anti-IL21R binding protein is a neutralizing anti-IL21R
binding protein,
based on the identification of several IL21-responsive genes. Finally, it is
said that the binding
proteins can act as antagonists of IL21R activity, thereby modulating immune
responses in
general, and those mediated by IL21R in particular.
SUMMARY OF THE INVENTION
In one embodiment, the present invention includes a method of ameliorating
symptoms or treating
one or more adverse reactions triggered by a widespread release of cytokines
in a subject
comprising the steps of: identifying the subject in need of amelioration of
symptoms or treatment
of one or more infectious diseases or disease conditions that trigger a
widespread release of
cytokines; and administering one or more pharmaceutical compositions
comprising a
therapeutically effective amount of a lipid dissolved or dispersed in a
suitable aqueous or non-
aqueous medium sufficient to reduce the level of cytokines in the subject. In
one aspect, the
widespread release of cytokines is caused by one or more infectious diseases
selected from at
least one of viral, bacterial, fungal, helminthic, protozoan, or hemorrhagic
infectious agents. In
another aspect, the one or more infectious diseases is selected from at least
one of infection with a
Rhinovirus, Coronavirus, Paramyxoviridae, Orthomyxoviridae, Adenovirus,
Parainfluenza Virus,
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Metapneumovirus, Respiratory Syncytial Virus, Influenza virus, Arenaviridae,
Filoviridae,
Bunyaviridae, Flaviviridae, Rhabdoviridae virus, Ebola, Marburg, Crimean¨Congo
hemorrhagic
fever (CCHF), South American hemorrhagic fever, dengue, yellow fever, Rift
Valley fever, Omsk
hemorrhagic fever virus, Kyasanur Forest, Junin, Machupo, Sabia, Guanarito,
Garissa, Ilesha, or
Lassa fever viruses. In another aspect, the one or more disease conditions is
selected from at least
one of cachexia, septic shock syndrome, a chronic inflammatory response,
septic shock
syndrome, traumatic brain injury, cerebral cytokine storm, graft versus host
disease (GVHD),
autoimmune diseases, multiple sclerosis, acute pancreatitis, or hepatitis. In
another aspect, the one
or more disease conditions is an adverse reaction caused by the treatment with
anti-CD19
Chimeric Antigen Receptor (CAR) T cells or antitumor cell therapy, activated
dendritic cells,
activated macrophages, or activated B cells. In another aspect, the
composition further comprises
a curcumin extract, curcumin, curcuminoids disposed in a lipid, wherein the
curcuminoids are
selected from at least one of Ar-tumerone, methylcurcumin, demethoxy curcumin,
bisdemethoxycurcumin, sodium curcuminate, dibenzoylmethane, acetylcurcumin,
feruloyl
methane, tetrahydrocurcumin, 1,7-bis(4-hydroxy -3-methoxyphenyl) -1,6-
heptadiene -3,5 -dione
(curcumin 1), 1,7-bis (piperony1)-1,6-heptadiene -3,5 -dione (piperonyl
curcumin) 1,7-bis(2-
hydroxy naphthyl)-1,6-heptadiene-2,5-dione (2-hydroxyl naphthyl curcumin) and
1,1-
bis(pheny1)-1,3,8,10 undecatetraene-5,7-dione. In another aspect, the lipid or
the phospholipid is
selected from the group consisting of dimyristoylphosphatidylcholine (DMPC),
dimyristoylphosphatidylglycerol (DMPG), Dipalmitoylphosohatidylcholine (DPPC),
disteroylphosphatidylglycerol (DSPG),
dipalmitoylphosphatidylglycerol (DMPG),
phosphatidylcholine , ly solecithin, ly sophosphatidylethanolamine, ly soDMPC,
ly soDMPG,
lysoDSPG, lysoDPPC, phosphatidy lserine,
phosphatidylinositol, sphingomyelin,
phosphatidylethanolamine, cardiolipin, phosphatidic acid, cerebrosides,
dicetylphosphate,
phosphatidylcholine, and dipalmitoyl-phosphatidylglycerol, stearylamine,
dodecylamine,
hexadecyl-amine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate,
isopropyl myristate,
amphoteric acrylic polymers, fatty acid, fatty acid amides, cholesterol,
cholesterol ester,
diacylglycerol, and diacylglycerolsuccinate. In another aspect, the
therapeutically effective
amount comprises 50 nM/kg, 10 to 100 nM/kg, 25 to 75 nM/kg, 10, 20, 30, 40,
50, 60, 70, 80, 90,
or 100 nM/kg of body weight of the subject. In another aspect, the composition
comprises an
active agent, and has a ratio of lipid phospholipids to active agent of 3:1,
1:1, 0.3:1, and 0.1:1. In
another aspect, the diseases is rheumatoid arthritis, psoriasis, multiple
sclerosis, relapsing
multiple sclerosis, or inflammatory bowel disease.
In another embodiment, the present invention includes a composition for
ameliorating symptoms
or treating one or more adverse reactions triggered by an infectious disease
or a disease condition
that trigger a widespread release of cytokines in a subject comprising a
therapeutically effective
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amount of a lipid or a lysophosphatidyl dissolved or dispersed in a suitable
aqueous or non-
aqueous medium. In one aspect, the one or more infectious diseases are
selected from at least one
of viral, bacterial, fungal, helminthic, protozoan, or hemorrhagic infectious
agents. In another
aspect, the one or more infectious diseases is selected from at least one of
infection with a
Rhinovirus, Coronavirus, Paramyxoviridae, Orthomyxoviridae, Adenovirus,
Parainfluenza Virus,
Metapneumovirus, Respiratory Syncytial Virus, Influenza Virus, Arenaviridae,
Filoviridae,
Bunyaviridae, Flaviviridae, Rhabdoviridae virus, Ebola, Marburg, Crimean¨Congo
hemorrhagic
fever (CCHF), South American hemorrhagic fever, dengue, yellow fever, Rift
Valley fever, Omsk
hemorrhagic fever virus, Kyasanur Forest, Junin, Machupo, Sabia, Guanarito,
Garissa, Ilesha, or
Lassa fever viruses. In another aspect, the one or more disease conditions is
selected from at least
one of cachexia, septic shock syndrome, a chronic inflammatory response,
septic shock
syndrome, traumatic brain injury, cerebral cytokine storm, graft versus host
disease (GVHD),
autoimmune diseases, multiple sclerosis, acute pancreatitis, or hepatitis. In
another aspect, the
curcumin extract, curcuminoids or synthetic curcumin are disposed in a lipid.
In another aspect,
the or the ly sophosphatidyl is selected from the group
consisting of
dimyristoylphosphatidylcholine (DMPC),
dimyristoy 1phosphatidylglycerol (DMPG),
Dipalmitoylphosohatidylcholine (DPPC), disteroylphosphatidylglycerol
(DSPG),
dipalmitoylphosphatidylglycerol (DMPG), phosphatidylcholine,
ly solecithin,
ly sophosphatidylethanolamine, lysoDMPC, ly soDMPG,
lysoDSPG, lysoDPPC,
phosphatidylserine, phosphatidylinositol, sphingomyelin,
phosphatidylethanolamine, cardiolipin,
phosphatidic acid, cerebrosides, dicetylphosphate, phosphatidylcholine, and
dipalmitoyl-
phosphatidylglycerol, stearylamine, dodecylamine, hexadecyl-amine, acetyl
palmitate, glycerol
ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic
polymers, fatty acid, fatty
acid amides, cholesterol, cholesterol ester, diacylglycerol, and
diacylglycerolsuccinate. In another
aspect, the biodegradable polymer is selected from the group consisting of
polyesters,
polylactides, poly glycolides, polycaprolactones, polyanhydrides, polyamides,
polyurethanes,
polyesteramides, polydioxanones, polyacetals, polyketals, polycarbonates,
polyorthocarbonates,
poly orthoe sters, polyphosphoesters, polyphosphazenes,
polyhydroxybutyrates,
polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates,
poly(malic acid),
poly(amino acids), copolymers, terpolymers, and combinations or mixtures
thereof In another
aspect, the composition adapted for intravenous, sub-cutaneous, intramuscular,
or intraperitoneal
injection in the subject. In another aspect, the composition further comprises
a curcumin or
curcuminoids are selected from at least one of Ar-tumerone, methylcurcumin,
demethoxy
curcumin, bisdemethoxycurcumin, sodium curcuminate, dibenzoylmethane,
acetylcurcumin,
feruloyl methane, tetrahydrocurcumin, 1,7-bis(4-hydroxy -3-methoxypheny1)-1,6-
heptadiene -3,5-
dione (curcumin 1), 1,7-bis(piperony1)-1,6-heptadiene-3,5-dione (piperonyl
curcumin) 1,7-bis(2-
hydroxy naphthyl)-1,6-heptadiene-2,5-dione (2-hydroxyl naphthyl curcumin) and
1,1-
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bis(pheny1)-1,3,8,10 undecatetraene-5,7-dione. In another aspect, the
composition comprises an
active agent, and has a ratio of lipid to active agent of 3:1, 1:1, 0.3:1, and
0.1:1.
In another embodiment, the present invention includes a method of determining
if a candidate
drug causes an amelioration of symptoms or treats one or more adverse
reactions triggered by an
infectious disease or a disease condition that trigger a widespread release of
cytokines in a
subject, the method comprising: (a) administering an amount of the candidate
drug in
combination with empty liposomes, and a placebo to a second subset of the
patients, wherein the
candidate drug is provided in an amount effective to reduce or prevent the
overall level of
cytokines in the subject; (b) measuring the level of cytokines in the subject
from the first and
second set of patients; and (c) determining if the candidate drug in
combination with empty
liposomes ameliorates symptoms or treats one or more adverse reactions
triggered by infectious
diseases or disease conditions that trigger a widespread release of cytokines
is statistically
significant as compared to any reduction occurring in the subset of patients
that took the placebo,
wherein a statistically significant reduction indicates that the candidate
drug is useful in treating a
disease state while also reducing or eliminating the overall level of
cytokines in the subject.
In another embodiment, the present invention includes a method of ameliorating
symptoms or
treating a cytokine storm caused by a therapeutic agent in a subject
comprising the steps of:
identifying the subject in need of amelioration of symptoms or treatment of
the cytokine storm
caused by a therapeutic agent; and administering one or more pharmaceutical
compositions
comprising a therapeutically effective amount of a curcumin extract,
curcuminoids or synthetic
curcumin and derivatives thereof, or empty liposomes, dissolved or dispersed
in a suitable
aqueous or non-aqueous medium sufficient to reduce the level of cytokines in
the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] For a more complete understanding of the features and advantages of the
present
invention, reference is now made to the detailed description of the invention
along with the
accompanying figures and in which:
[0010] FIGS. lA and 1B show the percent inhibition and percent viability,
respectively, achieved
with liposomal curcumin in HeLa cells.
[0011] FIGS. 2A and 2B show the percent inhibition and percent viability,
respectively, using
solid 5-curcumin curcumin in HeLa cells.
[0012] FIGS. 3A and 3B show the percent inhibition and percent viability,
respectively,
comparing liposomal curcumin and solid curcumin in HeLa cells.
[0013] FIG. 4A is a graph that shows the effect of liposomal curcumin on liver
function during
sepsis, including asparate aminotransferase (AST) and alanine aminotransferase
(AST) levels.
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The results show the effectiveness of liposomal curcumin compared to the
standard of care in
sepsis fir age matched controls. Liposomal curcumin showed a higher
effectiveness when
compared to the standard of care for AST and ALT.
[0014] FIGS. 4B to 4D are graphs that show the effect of liposomal curcumin
during sepsis on
kidney function measuring Creatine, Neutrophil gelatinase-associated lipocalin
(NGAL) and
Blood Urea Nitrogen (BUN). Liposomal curcumin showed a higher effectiveness is
preserving
kidney function when compared to the standard of care, in particular, and
importantly with NGAL
which measures the progression of chronic kidney disease.
[0015] FIGS. 4E and 4F are graphs that show the effect of liposomal curcumin
during sepsis on
heart function for c-Troponin and the percent ejection fraction. Liposomal
curcumin showed a
decrease in cardiac damage when compared to the standard of care and equaled
the standard of
care in percent ejection fraction.
[0016] FIG. 4G is a graph that shows the effect of liposomal curcumin during
sepsis on overall
survival. Liposomal curcumin showed a higher survival time versus the standard
of care
treatment.
DETAILED DESCRIPTION OF THE INVENTION
[0017] While the making and using of various embodiments of the present
invention are
discussed in detail below, it should be appreciated that the present invention
provides many
applicable inventive concepts that can be embodied in a wide variety of
specific contexts.
The specific embodiments discussed herein are merely illustrative of specific
ways to make and
use the invention and do not delimit the scope of the invention.
[0018] To facilitate the understanding of this invention, a number of terms
are defined below.
Terms defined herein have meanings as commonly understood by a person of
ordinary skill in the
areas relevant to the present invention. Terms such as "a", "an" and "the" are
not intended to
refer to only a singular entity, but include the general class of which a
specific example may be
used for illustration. The terminology herein is used to describe specific
embodiments of the
invention, but their usage does not delimit the invention, except as outlined
in the claims.
[0019] As used herein, the term "cytokine storm" refers to the dysregulated of
pro-inflammatory
cytokines leading to disease has been referred to as a "cytokine storm,"
"cytokine release
syndrome" or "inflammatory cascade". Often, a cytokine storm or cascade is
referred to as being
part of a sequence because one cytokine typically leads to the production of
multiple other
cytokines that can reinforce and amplify the immune response. Generally, these
pro-
inflammatory mediators have been divided into two subgroups: early mediators
and late
mediators. Early mediators, such as e.g., tumor-necrosis factor, interleukin-
1, interleukin-6, are
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not sufficient therapeutic targets for re-establishing homeostatic balance
because they are
resolved within the time frame of a patient's travel to a clinic to receive
medical attention. In
contrast, the so-called "late mediators" have been targeted because it is
during this later
"inflammatory cascade" that the patient realizes that he or she has fallen
ill.
[0020] Infectious diseases commonly associated with a "cytokine storm" include
but at not
limited to, malaria, avian influenza, smallpox, pandemic influenza, adult
respiratory distress
syndrome (ARDS), severe acute respiratory syndrome (SARS). Certain specific
infectious agents
include but are not limited to: infectious diseases is selected from at least
one of Ebola, Marburg,
Crimean¨Congo hemorrhagic fever (CCHF), South American hemorrhagic fever,
dengue, yellow
fever, Rift Valley fever, Omsk hemorrhagic fever virus, Kyasanur Forest,
Junin, Machupo, Sabia,
Guanarito, Garissa, Ilesha, or Lassa fever viruses. Other viruses can include
rhinovirus,
coronavirus, paramyxoviridae, Orthomyxoviridae, adenovirus, parainfluenza
virus,
metapneumovirus, respiratory syncytial virus or influenza virus.
[0021] Disease conditions commonly associated with a "cytokine storm" include
but at not
limited to: sepsis, systemic inflammatory response syndrome (SIRS), cachexia,
septic shock
syndrome, traumatic brain injury (e.g., cerebral cytokine storm), graft versus
host disease
(GVHD), or the result of treatment with activated immune cells, e.g., IL-2
activated T cells, T
cells activated with anti-CD19 Chimeric Antigen Receptor (CAR) T cells.
[0022] Generally, a cytokine storm is a healthy systemic expression of a
vigorous immune
system. The present invention can be used to reduce or eliminate some or most
of an exaggerated
immune response caused by, e.g., rapidly proliferating and highly activated T-
cells or natural
killer (NK) cells that results in the release of the "cytokine storm" that can
include more than 150
inflammatory mediators (cytokines, oxygen free radicals, and coagulation
factors). Both pro-
inflammatory cytokines (such as Tumor Necrosis Factor-a, Interleukin-1, and
Interkeukin-6) and
anti-inflammatory cytokines (such as Interleukin-10, and Interleukin-1
receptor antagonist (IL-
1RA)) become greatly elevated in, e.g., serum. It is this excessive release of
inflammatory
mediators that triggers the "cytokine storm."
[0023] In the absence of prompt intervention, such as that provided by the
present invention, a
cytokine storm can result in permanent lung damage and, in many cases, death.
The end stage
symptoms of the cytokine storm include but are not limited to: hypotension;
tachycardia;
dyspnea; fever; ischemia or insufficient tissue perfusion; uncontrollable
hemorrhage; severe
metabolism dysregulation; and multisystem organ failure. Deaths from
infectious diseases such
as Ebola virus infection are not caused by the virus itself, but rather, the
cytokine storm that
causes uncontrollable hemorrhaging; severe metabolism dysregulation;
hypotension; tachycardia;
dyspnea; fever; ischemia or insufficient tissue perfusion; and multisystem
organ failure.
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[0024] As used herein the term "Curcumin (diferuloyl methane; 1,7-bis(4-
hydroxy-3-
methoxypheny1)-1,6-heptadiene-3,5-dione)" is a naturally occurring compound
which is the main
coloring principle found in the rhizomes of the plant Curcuma longa (U.S. Pat.
No. 5,679,864
(Krackov et al.)). In one aspect, the synthetic curcumin is 85, 86, 87, 88,
89, 90, 91, 92, 93, 94,
95 or 96% pure diferuloylmethane. Non-limiting examples of curcumin and
curcuminoids
include, e.g., Ar-tumerone, methylcurcumin, demethoxy curcumin, bisdemethoxy
curcumin,
sodium curcuminate, dibenzoylmethane, acetylcurcumin, feruloyl methane,
tetrahydrocurcumin,
1, 7-bis (4 -hy droxy -3 -methoxypheny1)-1,6-heptadiene -3,5 -dione
(curcuminl), 1, 7-bis (piperonyl) -
1,6-heptadiene -3,5 -dione (piperonyl curcumin) 1,7-bis(2-hydroxy naphthyl)-
1,6-heptadiene -2,5-
dione (2-hydroxyl naphthyl curcumin) and 1,1-bis(pheny1)-1,3,8,10
undecatetraene -5 , 7-dione .
[0025] The term "liposome" refers to a capsule wherein the wall or membrane
thereof is formed
of lipids, especially phospholipid, with the optional addition therewith of a
sterol, especially
cholesterol. In one specific non-limiting example the liposomes are empty
liposomes and can be
formulated from a single type of phospholipid or combinations of
phospholipids. The empty
liposomes or lipid can further include one or more surface modifications, such
as proteins,
carbohydrates, glycolipids or glycoproteins, and even nucleic acids such as
aptamers, thio-
modified nucleic acids, protein nucleic acid mimics, protein mimics,
stealthing agents, etc. In one
specific, non-limiting example the composition also comprises an active agent
in or about the
liposome or lipid and the composition has a ratio of lipids to active agent of
3:1, 1:1, 0.3:1, and
0.1:1.
[0026] As used herein, the term "lipid" refers to amphiphilic biomolecules
that are soluble in
nonpolar solvents. Lipids are capable of liposome formation, vesicle
formation, micelle
formation, emulsion formation, and are substantially non-toxic when
administrated at the
necessary concentrations as liposomes. The lipid composition of the present
invention can
include, e . g. , dimyristoy 1phosphatidylcholine (DMPC),
dimyristoylphosphatidylglycerol
(DMPG), Dipalmitoylphosohatidy lcholine (DPPC), disteroylphosphatidylglycerol
(D SP G),
dipalmitoylphosphatidylglycerol (DMPG), phosphatidylcholine, ly
solecithin,
ly sophosphatidylethanol-amine, phosphatidylserine, phosphatidylinositol,
sphingomy e lin,
phosphatidylethanolamine, cardiolipin, phosphatidic acid, cerebrosides,
dicetylphosphate,
phosphatidylcholine , and dipalmitoyl-phosphatidylglycerol, stearylamine,
dodecylamine,
hexadecyl-amine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate,
isopropyl myristate,
amphoteric acrylic polymers, fatty acid, fatty acid amides, cholesterol,
cholesterol ester,
diacylglycerol, and diacylglycerolsuccinate.
[0027] As used herein, the term "in vivo" refers to being inside the body. The
term "in vitro" as
used in the present application is to be understood as indicating an operation
carried out in a non-
living system.
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[0028] As used herein, the term "treatment" refers to the treatment of the
conditions mentioned
herein, particularly in a patient who demonstrates symptoms of the disease or
disorder. As used
herein, the term "treating" refers to any administration of a compound of the
present invention
and includes (i) inhibiting the disease in an animal that is experiencing or
displaying the
pathology or symptomatology of the diseased (i.e., arresting further
development of the pathology
and/or symptomatology) or (ii) ameliorating the disease in an animal that is
experiencing or
displaying the pathology or symptomatology of the diseased (i.e., reversing
the pathology and/or
symptomatology). The term "controlling" includes preventing treating,
eradicating, ameliorating
or otherwise reducing the severity of the condition being controlled.
[0029] The terms "effective amount" or "therapeutically effective amount"
described herein
means the amount of the subject compound that will elicit the biological or
medical response of a
tissue, system, animal or human that is being sought by the researcher,
veterinarian, medical
doctor or other clinician. In one example, the therapeutically effective
amount comprises 50
nM/kg, 10 to 100 nM/kg, 25 to 75 nM/kg, 10, 20, 30, 40, 50, 60, 70, 80, 90, or
100 nM/kg of
body weight of the subject.
[0030] The terms "administration of' or "administering a" compound as used
herein should be
understood to mean providing a compound of the invention to the individual in
need of treatment
in a form that can be introduced into that individual's body in a
therapeutically useful form and
therapeutically useful amount, including, but not limited to: oral dosage
forms, such as tablets,
capsules, syrups, suspensions, and the like; injectable dosage forms, such as
intravenous (IV),
intramuscular (IM), or intraperitoneal (IP), and the like; enteral or
parenteral, transdermal dosage
forms, including creams, jellies, powders, or patches; buccal dosage forms;
inhalation powders,
sprays, suspensions, and the like; and rectal suppositories.
[0031] As used herein the term "intravenous administration" includes injection
and other modes
of intravenous administration.
[0032] The term "pharmaceutically acceptable" as used herein to describe a
carrier, diluent or
excipient must be compatible with the other ingredients of the formulation and
not deleterious to
the recipient thereof
[0033] The curcumin formulation of the present invention may comprise one or
more optional
pharmaceutical excipients, diluents, extended or controlled release agents,
lubricants,
preservatives or any combination thereof, and once solubilized may be added to
injectable anti-
diabetic medications or administered in a schedule depending upon the release
kinetics of the
curcumin formulation. A large number of biodegradable polymers may be used in
the
formulation of the present invention. Non-limiting examples of these polymers
include
poly se sters , poly lactide s , poly glycolides, poly caprolactone s poly
anhy drides, poly amide s ,
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polyurethanes, poly e steramide s , poly diaxanone s, poly acetals,
polyketals, poly carbonate s ,
poly orthoc arbonate s , poly orthoesters,
polyphosphoesters, polyphosphazenes,
polyhydroxybuterates, polyhydroxyvalerates, polyalkelene oxalates,
polyalkylene succinates,
poly(malic)acid, poly(amino)acids, copolymers, terpolymers, and combinations
or mixtures
thereof. Specific polymers that may be used include an acrylic acid, a
vinylpyrolidinome, a N-
isopropylacrylamide or combinations and modifications thereof The synthesized
curcumin that
is used includes curcumin, curcumin analogues, curcumin derivatives and any
modifications
thereof
[0034] Treatment of Infectious Diseases. The terminal stage of Ebola and other
viral diseases is
often the onset of cytokine storm, the massive overproduction of cytokines by
the body's immune
system. The present invention includes the treatment of infectious agents that
trigger a cytokine
storm, such as Ebola virus, with curcumin actions to suppress cytokine release
and cytokine
storm.
[0035] It was found that curcumin blocks cytokine release, most importantly
the key pro-
inflammatory cytokines, interleukin-1, interleukin-6 and tumor necrosis factor-
a. Curcumin's
suppression of cytokine release correlates with clinical improvement in
experimental models of
disease conditions where cytokine storm plays a significant role in mortality.
Thus, curcumin can
be used to treat the cytokine storm of patients with Ebola. In certain
examples, intravenous
formulations allow achievement of therapeutic blood levels.
[0036] The high fatality rate in patients infected with the Ebola virus is
thought to be due partly
to the onset of cytokine storm in the advanced stages of the infection1-2.
Cytokine storm can
occur after a wide variety of infectious and non-infectious stimuli. In
cytokine storm, numerous
cytokines, both pro-inflammatory (IL-1, IL-6, TNF-a) and anti-inflammatory (IL-
10), are
released, resulting in hypotension, hemorrhage, and, ultimately, multi-organ
failure. The term
"cytokine storm" is most associated with the 1918 H1N1 influenza pandemic and
the more recent
cases of bird flu H5N1 infection3-5. In these cases, young people, with
presumably healthy
immune systems, died disproportionally from the disease, and aberrant activity
of their immune
systems is thought to be the cause. This syndrome has also been known to occur
in advanced or
terminal cases of SARS6, Epstein-Barr virus-associated hemophagocytic
lymphohistiocytosis7,
gram negative sepsis8, malaria9 and numerous other infectious diseases.
Cytokine storm can
occur from non-infectious causes, such as acute pancreatitisl , severe burns
or trauma" or acute
respiratory distress syndrome secondary to drug use or inhalation of
toxins'''. In a recent phase 1
trial, injection of the monoclonal antibody TGN1412, which binds to the CD28
receptor on T
cells, resulted in severe cases of cytokine storm and multi-organ failure in
the 6 human volunteers
who received this agent. This was despite the fact that the dose of this agent
given was 500 times
lower than had been found to be safe in animals". Other viruses can include
rhinovirus,
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coronavirus, paramyxoviridae, Orthomyxoviridae, adenovirus, parainfluenza
virus,
metapneumovirus, respiratory syncytial virus or influenza virus.
[0037] Curcumin suppression of Cytokines.
[0038] Curcumin has been shown to inhibit the release of numerous cytokines.
Abe et al showed
that curcumin suppresses IL-113, IL-8, TNF-a, monocyte chemoattractant protein-
1 (MCP-1) and
macrophage inflammatory protein-la (MIP-1a) release from monocytes and
macrophages14. Jain
et al., showed that curcumin markedly reduced the release of IL-6, IL-8, TNF-a
and MCP-1 from
monocytes that had been cultured in a high glucose environment15. These same
investigators
studied rats with streptozotocin-induced elevated plasma blood sugar levels
and significantly
elevated levels of IL-6, TNF-a and MCP-1; these levels were markedly reduced
by curcumin15.
Curcumin has been reported to block the release of IL-6 in rheumatoid synovial
fibroblasts16, of
IL-8 in human esophageal epithelial cells17 and alveolar epithelial cells18,
and of IL-1 in bone
marrow stromal cells19, colonic epithelial ce11s2 and human articular
chondrocytes21. Curcumin
also prevents release of IL-222, IL-1222-23, Interferon-722-23 and many other
key cytokines24-26
(Tables 1 and 2).
[0039] EXAMPLE 1: Curcumin Cytokine Suppression Correlates with Clinical
Improvement in
Conditions Associated with Cytokine Storm.
[0040] Curcumin has positive effects on numerous disease conditions in
patients and in animal
systems. Avasarala et al reported on curcumin's effects on cytokine expression
and disease
progression in a mouse model of viral-induced acute respiratory distress
syndrome. Curcumin
reduced the expression of key cytokines IL-6, IL-10, interferon y and MCP-1,
and this correlated
with a marked decrease in inflammation and reduction in fibrosis27. Yu et al
showed curcumin's
suppression of TNF-a levels was associated with decreased pancreatic injury in
an acute
pancreatitis mouse mode128. Cheppudira et al reported that curcumin's
suppression of IL-8 and
GRO-a, and ultimately with NF-KB, correlated with reduction in thermal injury
in a rat mode129.
Curcumin suppression of cytokines also correlates with clinical improvement in
models of severe
viral infection. Song et al showed that curcumin administration reduced
expression of IL-113, IL-6
and TNF-a and ultimately NF-KB, and protected against coxsackie virus-induced
severe
myocardial damage in infected mice30. Curcumin has been shown to have activity
against
numerous viruses, including, Coronavirus, HIV-1, HIV-2, HSV, HPV, HTLV-1, HBV,
HCV, and
Japanese encephalitis virus31. The virus can include rhinovirus, coronavirus,
paramyxoviridae,
Orthomyxoviridae, adenovirus, parainfluenza virus, metapneumovirus,
respiratory syncytial virus
or influenza virus. In addition, curcumin has been shown to have specific
activity against the
H1N1 virus in cu1ture32-33, although cytokine levels were not measured in
these two studies. Most
importantly, curcumin has been shown to stimulate the SOCS proteins34. These
proteins have
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been shown to be crucial in protecting against severe cytokine storm in mice
infected with
influenza virus35.
[0041] Curcumin's activity in suppressing multiple cytokines, and its activity
in experimental
models of diseases and conditions associated with cytokine storm, suggest it
may be useful in the
treatment of patients with Ebola and cytokine storm. Curcumin is poorly
absorbed from the
intestinal tract; however intravenous formulations may allow therapeutic
curcumin blood levels to
be achieved in patients diagnosed with cytokine storm. Clinical status and
levels of important
cytokines, such as IL-113, IL-6 and TNF-a, should be monitored carefully when
patients are
treated with curcumin.
[0042] Table 1: Curcumin Effect on Interleukins
Biomolecule Key Functions
IL-1 Major pro-inflammatory cytokine, hematopoesis, CNS development
IL-2 T-cell lymphocyte differentiation
T IL-4 B-cell proliferation
IL-5 Immunoglobulin secretion, eosinophil function, allergy
IL -6 Major pro-inflammatory cytokine, B-cell differentiation, nerve
cell
differentiation
IL-8 Neutrophil chemotaxis, angiogenesis
T IL-10 Anti-inflammatory cytokine, also has T-cell stimulatory effects
IL-11 Induces acute phase proteins, antigen- antibody reactions, bone
remodeling
IL-12 Defense against intracellular pathogens
IL-13 Induces matrix metalloproteinases, induces IgE
IL-17 Pro-inflammatory cytokine
[0043] Table 2: Curcumin Suppression of Other Key Cytokines
Biomolecules Key Functions
Major pro-inflammatory cytokine, insulin resistance, induces secretion of
TNF-a
corticotropin releasing hormone
Interferon y Macrophage activation, T and B cell activation and
differentiation
MCP-1 (CCL2) Neuroinflammation, monocyte and basophil chemotaxis
MIP-1a (CCL3) Activates granulocytes, induces synthesis of pro-
inflammatory cytokines
GROa (CXCL1) Neutrophil chemoattractant, angiogenesis, wound healing
GROp (CXCL2) Neutrophil and monocyte chemoattractant
Monocyte and macrophage chemoattractant, NK cell chemoattractant,
UP-10 (CXCL10)
angiogenesis
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SDF-1 (CXCL12) Lymphocyte chemoattractant, angiogenesis, suppresses
osteoclastogenesis
[0044] Test results. First study.
[0045] Liposomes and Liposomal-Curcumin were prepared as a 6 mg/ml solutions.
Curcumin
(solid) was solubilized in DMSO at 6mg/ml. All three compounds were tested in
EBOV infection
assay with two cell lines Hela and HFF-1. There were two sets for studies done
with different
dilution strategy.
[0046] Hela cell lines were used in two independent experiments (replica 1 and
rep1ica2) and
HFF-1 in one. 2 hours prior infection Liposome and Curcumin-Liposomal were
diluted in media
from highest concentration of 600 ug/ml (final in assay) to generate 10 points
for dose response
curve with 2 fold step dilution. Sul of each dose was dispensed by PE Janus
384-tip dispenser
into assay wells with cells. The Curcumin (solid) was dispensed by HP D300
directly from the
100% DMSO stock into assay wells with cells. DMSO was normalized in all wells
to final 1%.
Each dose were tested 4 times on the plate n=4.
[0047] Test results. Second study.
[0048] Both cell line were used in one study (replicate 1). 2 hours prior
infection Curcumin-Lip
and Lip. were diluted in Media from highest concentration of 60ug/m1 (final in
assay) to generate
10 points for dose response curve with 2 fold step dilution. In this case
titration was done with
manual mixing and changing tips for each new dose. Curcumin (solid) was
tittered manually in
DMSO and then equal amount for each dose was diluted 1/10 in media with
mixing. Sul of each
dose was dispensed by PE Janus 384-tip dispenser into assay wells with cells.
Each dose were
tested 4 times on the plate n=4. For both studies: Cells were infected with
EBOV(Zaire) at
MOI=0.5 for Hela cells and MOI=3 for HFF-1 and Infection was stopped in 48h by
fixing cells in
formalin solution. To detect infected cells the immunostaining was done using
anti-GP antibody.
Images were taken by PE Opera confocal platform with 10x objective, analyzed
using Acapella
software.
[0049] Signal for GP-staining was converted into % infection. The number of
nuclei per well
was used to determine % viability of cells (in comparison to infected but
untreated controls well
n=16). Data was analyzed using GeneData software and % Infection converted
into % Inhibition
(%INH) using plate controls.
[0050] FIGS. lA and 1B show the percent inhibition and percent viability,
respectively, achieved
with liposomal curcumin in HeLa cells. FIGS. 2A and 2B shows the percent
inhibition and
percent viability, respectively, using solid 5-curcumin curcumin in HeLa
cells. Similar results
were obtained with the same study using HFF-1 cells.
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[0051] FIGS. 3A and 3B show the percent inhibition and percent viability,
respectively,
comparing liposomal curcumin and solid curcumin in HeLa cells. Similar results
were obtained
with the same study using HFF-1 cells.
[0052] The anti-EBOV activity was determined in Hela cells and it was
correlating with
"cytotoxicity". Briefly, the results are as follows: (1) Curcumin-liposomal
EC50=2.5 0.2ug/ml,
safety index=2. Liposomes EC50=3.9 0.2ug/ml, safety index = 4. Curcumin
EC50=6.5
0.5ug/ml, safety index = 1. As such, liposomes show EC50=0.6 2 ug/ml with
very good safety
index ¨50.
[0053] Pro-Inflammatory Cytokines in the Causation of The Prolonged QT
Interval: Role of the
Ceramide and Sphingosine-1 Phosphate Pathways. There is increasing evidence
that excess
levels of pro-inflammatory cytokines play a major role in the pathogenesis of
the prolonged QT
syndrome. In anti-cancer trials, QT prolongation was noted as a side effect of
the cytokine,
interferon y, and QT prolongation has been seen after treatment with
interleukin-18. Patients with
inflammatory diseases, such as rheumatoid arthritis, psoriasis and
inflammatory bowel disease,
have a high incidence of QT prolongation, and die more frequently secondary to
this
complication. It has been shown that the degree of QT prolongation correlates
directly with the
extent of elevation of the key pro-inflammatory cytokines, TNF-a, IL-1I3 and
IL-6. In large-scale
studies of normal populations, it was found that asymptomatic elevations of
these cytokines
correlate with QT prolongation. In trials of tocilizumab, an IL-6 blocker, in
patients with
rheumatoid arthritis, a shortening of the duration of the previously prolonged
QT interval was
noted, and the degree of QT shortening correlated with the decrease in serum
inflammatory
markers. Studies in animal models and in cultured cardiomyocytes have shown
that TNF-a
suppressed IKr, IKs and Ito. This was thought to be due to the stimulation of
reactive oxygen
species (ROS). TNF-a and other cytokines have been shown to cause increased
production of
ROS. The effects of TNF-a could be blocked by administration of an anti-TNF-a
antibody or by
an anti-oxidant. IL-1I3 and IL-6 have been shown to increase the L-type Ca(2)
current (ICaL),
and this effect can be blocked by aspirin or indomethacin. The close link
between
phospholipidosis and prolonged QT provides another hint of the importance of
these cytokines.
77% of the agents that can cause phospholipidosis also are hERG channel
blockers.
Phospholipidotic cells have been shown to secrete large amounts of TNF-a and
IL-6 after LPS
stimulation. It has also been speculated that the mechanism of damage from
drug-induced
phospholipidosis is accumulation of ceramides. Numerous studies have shown
that cytokines
such as interferon y, IL-1I3 and TNF-a increase sphingomyelinase activation,
and increase
production of ceramides, which are known to suppress hERG current. It is also
known that
sphingolipids mediate ROS signaling. Ceramides are metabolized to sphingosine
and fatty acids,
and sphingosine is phosphorylated by sphingosine kinases to form sphingosine-1
phosphate.
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Ceramides and sphingosine-1 phosphate have opposite effects, ceramides causing
apoptosis and
sphingosine-1 phosphate promoting cell survival. Fingolimod, a sphingosine
analogue (which
has both agonist and antagonist effects on the sphingosine-1 phosphate-1
receptor), is used to
treat patients with relapsing multiple sclerosis, causes QT prolongation
through inhibition of the
hERG current, as well as fatal ventricular arrhythmias. Further, studies in
the mouse model of
influenza-induced cytokine storm have shown sphingosine-1 phosphate-1
signaling to be the
primary pathway for activation of the cytokine storm. The cytokine storm was
reversed by a
sphingosine analogue through feedback inhibition of cytokines, with marked
reductions in TNF-
a, IL-la, IL-6, MCP-1, interferon a and MIP-la, and clinical improvement seen.
The survival of
the mice in the study was much higher with the sphingosine analogue than with
anti-viral therapy.
It is also noted that agents found clinically to be suppressors of QT
prolongation, progestins,
statins, liposomal curcumin, resveratrol, anti-oxidants, are also strong
suppressors of these
inflammatory cytokines. Even I3-blockers, the standard treatment for patients
with prolonged QT,
seem to have suppression of cytokines as part of their mechanism of action in
treating these
patients. All these agents shift the balance away from the ceramide pathway
toward the
sphingosine-1 phosphate pathway. Thus, excess levels of pro-inflammatory
cytokines play a
major role in the causation of the prolonged QT syndrome, probably through
stimulation of ROS
and the ceramide pathway.
[0054] By way of explanation, and in no way a limitation of the present
invention, a proposed
mechanism for correction of the previously prolonged QT interval by liposomal
curcumin and
EU8120 is described. There is a great deal of evidence from early phase 2 anti-
cancer trials of
pro-inflammatory cytokines, and from recent studies on patients with
rheumatoid arthritis,
psoriasis and inflammatory bowel disease, suggesting increased levels of pro-
inflammatory
cytokines cause prolongation of the QT interval. These patients have a
markedly increased
incidence of prolonged QT. In addition, in trials of an anti-IL-6-antibody in
patients with
rheumatoid arthritis, shortening of the prolonged QT was noted, which
correlated with the
decrease in cytokines. The correlation of increased cytokine levels and
asymptomatic QT
prolongation is also found in large-scale studies of normal populations. In
animal models and in
ventricular myocytes, TNF-a administration causes a decrease in the rapid
component of the
delayed rectifier potassium current (IKr), in the slow component of the
delayed rectifier current
(IKs) and in the transient outward current (Ito). These effects are thought to
be due to stimulation
of reactive oxygen species (ROS) and can be blocked by administration of an
anti-TNF-a
antibody or by antioxidants. It is known from other studies that TNF-a and
other pro-
inflammatory cytokines stimulate ROS production. IL-1I3 and IL-6 also have QT
prolonging
effects in these models. It is also known from studies in other diseases that
ROS increases
ceramide production, thus shifting the balance from the sphingosine-l-
phosphate (SIP) pathway
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(protective) to the ceramide pathway (destructive). Multiple studies in
experimental models have
shown that ceramides cause suppression of the hERG current. It has been
proposed that statins,
which reduce levels of pro-inflammatory cytokines and cause shortening of the
prolonged QT,
may have as their underlying mechanism the stimulation of this protective S113
pathway. Other
agents that shorten the prolonged QT, including anti-oxidants, such as vitamin
E, reduce levels of
pro-inflammatory cytokines and of ROS, and stimulate Si P. Finally, the
present inventors have
recognized that the list of agents which cause both cytokine suppression and
shortening of the
previously prolonged QT interval is strikingly similar to the list of agents
that have been shown in
animal models to reduce cytokine levels and secondary brain inflammation and
also reduce the
degree of brain damage. Thus, the present invention can be used to target
those diseases that
increase ceramide production, thus shifting the balance from the sphingosine-l-
phosphate (SIP)
pathway (protective), to the ceramide pathway (destructive).
[0055] The present inventors have shown, in both in vitro and in vivo models,
that Liposomal
Curcumin and EU8120 reduce IL-113, IL-6, TNF-a, MCP-1, MIP-1 and Rantes.
Liposomes have
also been shown, in other models, to compete for the enzyme sphingomyelinase
and to reduce
levels of ceramides, thus also shifting the ceramide/S1P balance toward SIP.
[0056] LPS-induced cytokine storm produces QTc prolongation, which is
prevented by an anti-
inflammatory lipid. There is increasing evidence that excess levels of pro-
inflammatory
cytokines play a major role in the pathogenesis of the prolonged QT syndrome.
Inversely,
blockers such as tocilizumab (IL-6), or anti-cytokine antibodies (TNFa)
contribute to a shortening
of the previously-prolonged QT interval.
[0057] In this study, LPS and Kdo2-Lipid-A were used to induce cytokine
release in guinea-pigs
with concomitant ECG monitoring and blood draws, followed by Q-ELISA
measurement of
cytokine production. The guinea pig was selected because it yields reliable
QTc prolongation as a
result of pro-arrhythmic challenge, with consistently visible T-waves on the
ECG. Male adult
guinea pigs received 300 jig/kg LPS at time 0, and had ECGs analyzed at lh,
2h, and 4 hours
post-LPS, with simultaneous blood draw. Animals receiving LPS only exhibited a
8-msec
increase in QTc after lh post-LPS, when TNFa levels were maximal at 5.5-fold
the pre-LPS
values. A 29-msec QTc prolongation 2h post-LPS correlated with 7- and 9- fold
increases in IL-
113 and IL-6, respectively. The QTc prolongation remained (27 msec) after 4
hours post-LPS,
when the animals were euthanized. When 9 mg/kg EU8120 (a lipid blend shown to
prevent IKr-
channel block by a variety of hERG blockers) was given 1 hour prior to LPS-
induction, QTc
prolongation was limited to 5 ms after 2 hours, and completely prevented at 1
and 4 hours post-
LPS. Plasma levels of TNFa, 11113, and IL-6 were significantly lower in EU8120-
administered
animals. This example demonstrates that EU8120 suppresses QTc prolongation via
an anti-
inflammatory cytokine-effect and not by any interaction with the active agent
(LPS).
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[0058] Synthetic Curcumin.
[0059] The present invention can use the compositions to treat the cytokine
storm disorders
using synthetic curcumin (S-curcumin).
[0060] Curcumin is the active principle of the turmeric plant, which has been
synthesized to near
purity (99.2%). It is formulated with liposomes, polymers, or PLGM to render
it capable of being
administered intravenously as a bolus or as a continuous infusion over 1-72
hours in combination
with other active agents. Curcumin has antioxidant and anti-inflammatory
activity, and can block
autonomous intracellular signaling pathways abnormally responsive to
extracellular growth
factors, uncontrolled proliferation of cells and fibrosis-associated and
tissue degenerative
conditions. Specifically, Curcumin reacts negatively with components of key
signaling pathways
commanding proliferation, metabolism, survival and death.
[0061] Oral and topical administration of the extract of the turmeric plant
has been used in
traditional medicine for over two thousand years. While oral administration is
devoid of systemic
toxicity it is also devoid of systemic therapeutic activity. This is due to
blood insolubility, and
intestinal wall and hepatic inactivation, i.e. it has negligible
bioavailability for systemic diseases
by the oral route. To overcome these limitations, parenteral intravenous
curcumin formulations
with liposomes, polymers (n-isopropylacrylamide, N-vinylpyrrolidione and
acrylic acid) and
polylactic glycolic acid copolymer were entered into in pre-clinical drug
development.4
[0062] Curcumin as an extract of turmeric root is available to researchers as
a mixture of three
curcuminoids and to the public as a food supplement or spice according to the
FDA. The extract
is 79.2% curcumin (diferuloylmethane), 18.27% demethoxycurcumin, and 2.53 %
bisdemethoxycurcumin.
[0063] Synthesized curcumin is GMP grade 99.2% pure diferuloylmethane produced
for non-
human experimental study and future Phase I clinical trials. There are obvious
differences
between the C3 three component extract and the single component synthesized 5-
curcumin that
extend to discernable analytic, physicochemical, and biological
characteristics. In certain aspects,
the diferuloylmethane is 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96%
pure diferuloylmethane.
[0064] The present invention relates to synthetic curcumin (S-curcumin) and
compares the
properties and the activity of 5-curcumin with liposomal curcumin, NANOCURC ,
and PLGA-
curcumin (hereinafter C3-complex).
[0065] Liposomal curcumin: The initial studies of liposomal curcumin were done
using material
bought as the complex.6-7 Studies with 5-curcumin are Mach CM, et al (2009)8
and Mach CM et
al (2010)9.
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[0066] NANOCURC : The initial study of Nanocurc0 was done using product bought
as the
complex Savita Bisht et al (2007)10 used a non-sabinsa source. Since then
studies with 5-curcumin
are used in the remainder of Nanocurc0 publications.11-13
[0067] PLGA-curcumin: The initial studies of PLGA-curcumin were done using
product
manufactured as the C3-complex.14-18 Studies included PLGA -curcumin C3
complex and PLGA-
5-curcumin pharmacokinetic studies in rat brains.
[0068] Comparison of PLGA C3- complex-curcumin vs PLGA 5-curcumin indicated
the
following differences. The solubilities of 99.2% S-Curcumin in all four
solvents. Ethanol, Ethyl
acetate, Acetone, and Acetonitrile, differed significantly from the C3 complex
containing 76 %
curcumin. When normalized to equal concentrations, the pure material has
greater solubility.
This confers improved manufacturing capability, and attributes to different
pharmacokinetics and
pharmacodynamics in in vivo settings (Table 3).
[0069] Table 3: Solubilities of 5-curcumin and C3-complex curcumin in
different organic
solvents.
Solvent Wt. of Vol. of solvent Conc. Physical lg of solvent
Curcumin ( L) (mg/mL) appearance of solubilize in
solvent
(mg) solubility (mL)
Batch -C100609, Curcumin=99.2 %
Ethyl acetate 20.8 2000 10.4 Partial 96.2
Ethanol 17.5 4600 3.804 Partial 263
Acetone 46.5 1100 42.3 Fully 23.6
Acetonitrile 17.4 3200 5.44 Partial 184
Batch -C3 complex, Curcumin=76 %
Ethyl acetate 21.5 1800 11.9 Fully 83.7
Ethanol 28.4 6500 4.37 Fully 229
Acetone 39.8 700 56.9 Fully 17.6
Acetonitrile 18.2 2900 6.28 Fully 159
*Weight the sample and transfer into a scintillation vial. Add small volume of
solvent and shake
well repeat for each addition till the solubility.
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[0070] Table 4: A comparison of differences between S-Curcumin and C3-Complex
curcumin.
C3-curcumin S-curcumin
Residue on Ignition 0.03% 0.4%
Loss on drying 0.98% 0.24%
Melting range 172-175 C 179.8-181.9C
Tapped bulk density 0.61g/m1 0.26g/m1
Loose bulk density 0.37g/m1 0.18g/m1
Sieve test
-40 mesh 100% 64.71%
-80 mesh 98.27% 64.0%
HPLC content curcumin 79.2% 99.2%
Total curcuminoids 96.21% 99.2%
Bisdemethoxycurcumin 2.53% 0
Demethoxycurcumin 18.27 0
Heavy Metals
Lead 0.91ppm(ug/g) <0.2ppm
Arsenic 0.54ppm(ug/g) <0.2ppm
Cadmium <0.2ppm(ug/g)
Mercury <0.02ppm(ug/g)
Residual solvents complies complies
Micro ¨total Plate count <100cfug 10cfu/g
Yeast and mold count <10cfu/g 15cfu/g
Escherichia coli neg/10g neg/10g
Salmonella neg/10g neg/10g
Staph. aureus neg/10g neg/10g
Pseudomonas aeruginosa neg/10g neg/10g
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[0071] FIG. 4A is a graph that shows the effect of liposomal curcumin on liver
function during
sepsis, including aspartate aminotransferase (AST) and alanine
aminotransferase (AST) levels in
a mouse model system. The results show the effectiveness of liposomal curcumin
compared to
the standard of care in sepsis for age matched controls. Liposomal curcumin
showed a higher
effectiveness when compared to the standard of care for AST and ALT.
[0072] FIGS. 4B to 4D are graphs that show the effect of liposomal curcumin
during sepsis on
kidney function for glomerular filtration rate (GFR) by measuring Creatine,
Neutrophil
gelatinase-associated lipocalin (NGAL) which measures the progression of
chronic kidney
disease, and Blood Urea Nitrogen which measures the kidney's ability to remove
urea from
blood, respectively. Liposomal curcumin showed a higher effectiveness is
preserving kidney
function when compared to the standard of care, in particular, and importantly
with NGAL which
measures the progression of chronic kidney disease.
[0073] FIGS. 4E and 4F are graphs that show the effect of liposomal curcumin
during sepsis on
heart function for c-Troponin, which measures cardia tissue damage and the
percent ejection
fraction, which measures the efficacy with which the left ventricle (or right
ventricle) pumps
blood with each heartbeat. Liposomal curcumin showed a decrease in cardiac
damage when
compared to the standard of care and equaled the standard of care in percent
ejection fraction.
[0074] FIG. 4G is a graph that shows the effect of liposomal curcumin during
sepsis on overall
survival. Liposomal curcumin showed a higher survival time versus the standard
of care
treatment.
[0075] When compared to the teachings of Yang, et al., Protective effect of
curcumin against
cardiac dysfunction in sepsis rats, Pharmaceutical Biology, 2013; 51(4): 482-
487, the present
invention showed a significant improvement in outcomes without the
cardiotoxicity associated
with curcumin.
[0076] 14:0 Ly sophosphatidylglycerol (Ly so PG)
= 1 0 0
OH
Has H 0"
[0077] Myristoyl monoglyceride
H
0 H
[0078] Myristic acid, a free fatty acid
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..
1 0
1.
OH
.
[0079] EXAMPLE 2
[0080] Brain damage after traumatic brain injury (TBI) is a two-stage process:
the injury caused
by the initial insult is followed by a stage of inflammation where a great
deal of additional
damage may occur. This inflammation begins within minutes of the initial
insult and can
continue for months or years, and results from a complex series of metabolic
processes involving
marked increases in cytokines, particularly the pro-inflammatory cytokines,
interleukin-113,
interleukin-6 and tumor necrosis factor-a. Levels of these cytokines may
increase thousands of
times more than the corresponding levels in serum. Strategies to control the
levels of these pro-
inflammatory cytokines and to reduce the cytokine-induced brain damage are
discussed. There is
extensive evidence from experiments in animal models that suppression of
cytokines is effective
in ameliorating neurologic damage after TBI. However, the efficacy of this
approach remains to
be proven in patient trials.
[0081] It is increasingly recognized that an aberrant immune system and a
massive
overproduction of pro-inflammatory cytokines, a 'cytokine storm', is a major
factor in the disease
progression and the mortality from numerous diseases. Cytokine storm, also
known as 'cytokine
release syndrome,' can occur after infection with malaria [1], SARS [2],
dengue [3], leptospirosis
[4], Lassa fever [5], gram-negative sepsis [6] as well as with numerous other
infectious diseases
(7-10]. Cytokine storm is a major cause of death in patients with Ebola [11-
13]. Patients with
cytokine storm may experience increased vascular permeability, severe
hemorrhage and multi-
organ failure, which may ultimately be the cause of a fatal outcome [8, 13,
141. Marked increases
in systemic cytokine levels, of both pro-inflammatory and anti-inflammatory
cytokines, are seen.
It is thought that this over-production of cytokines by healthy immune systems
is the explanation
for why individuals from 20 to 40 were more likely to die than the elderly
during the 1918 H1N1
pandemic [15, 161. Cytokine storm can occur after severe bums or trauma [17],
with acute
pancreatitis [18], or with ARDS secondary to drug use or inhalation of toxins
[19]. Severe acute
graft vs. host disease can be considered a cytokine storm [20, 211. Cytokine
storm is also a
recognized complication of treatment with the commonly-used antineoplastic
agent rituximab
[22], as well as of treatment with the monoclonal antibodies, tositumomab,
alemtuzumab,
muromonab and blinatumomab [23]. Elevated levels of cytokines are found and
are thought to be
an important cause of the pathology in many neurological conditions, including
Alzheimer's
disease [24], Parkinson's disease [25], autism [26], and multiple sclerosis
[27], as well as in the
acute phase of Guillian-Barre syndrome [28, 291. Increased cytokine levels
have been linked to
exacerbations of psychiatric illnesses [30, 311, and of lupus encephalopathy
[32, 331.
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[0082] TBI represents a major health problem in the United States, with 1.7
million cases, 275
000 hospitalizations and 52 000 deaths each year [34], and neuropsychiatric
sequalae are
common, especially after severe injury [35]. It is now understood that
cerebral damage after
traumatic brain injury occurs in two stages: an initial stage where damage
occurs from the
external mechanical force, and a secondary inflammatory stage where damage can
occur due to a
cascade of processes involving cytokines such as interleukin (IL)-113, IL-6,
and tumour necrosis
factor (TNF)-a [36]. The increases in cytokine levels in the brain can be
massive, especially after
severe TBI. IL-6 is not usually detectable in CSF, or is detectable in only
very low
concentrations (1-23 pg/ml) [37, 381. In one study, CSF levels of IL-6 as high
as 35 500 pg/ml
were seen after severe TBI [38, 391. These IL-6 levels were 40-100x greater
than the
corresponding levels in the serum of these patients [40]. Kushi et al reported
very large increases
in both IL-6 and IL-8, measured on admission, at 24 hours, at 72 hours and at
168 hours after
severe TBI in 22 patients. In the nine fatalities, average IL-6 values at
these times in the CSF
were 15 241, 97 384, 548 225 and 336 500 pg/ml compared to 102, 176, 873, 3
059 pg/ml in the
blood, a 'storm of cytokines mostly localized to the brain. For the 13
survivors, average IL-6
CSF values were lower, but still much greater than in the peripheral blood: 5
376, 3 565, 328 and
764 pg/ml compared to 181, 105, 37 and 26 pg/ml in the blood [41]. Similar
differences were
seen for IL-8. Whereas IL-8 levels in the CSF are normally very low (5-72
pg/ml) [37], Kushi et
al reported CSF IL-8 levels that were consistently elevated thousands of times
more than normal
levels or comparable levels in the peripheral blood [41]. These investigators
also noted that IL-6
and IL-8 blood levels that remained markedly elevated after 72 hours
correlated with a worse
prognosis and high fatality rate. Helmy et al found marked elevations of
multiple cytokines,
including IL-la, IL-113, IL-6, IL-8, IL-10, monocyte chemotactic protein (MCP-
1) and
macrophage inflammatory protein-1a (MIP-1a), in brain extracellular fluid
after severe TBI in 12
patients. These levels were also significantly elevated compared to the
corresponding blood
levels [42]. Other investigators have reported similar results, and have noted
that very high
cytokine levels correlate with a poor prognosis [43, 441. For example, Arand
et al noted that IL-6
levels were eight-fold higher in patients who died compared to those who
survived. In addition,
only patients who died showed increased levels of another pro-inflammatory
cytokine, IL-12 [43].
These data further support, the hypothesis that a cytokine storm is
responsible for increased
neurological damage after TBI. A number of studies suggest that some of these
same cytokines
can have beneficial as well as harmful effects on the brain [45-47]. However,
it has been shown
in numerous studies that blockage of these cytokines, at least in animal
models, can reduce the
cerebral damage after TBI. A list of key cytokines that are elevated in the
brain and CSF after
TBI is given in Table 5.
[0083] Table 5: Key cytokines show marked increases in brain and CSF after TBI
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Cytokine Function
TNF-a Major pro-inflammatory cytokine, insulin resistance,
stimulates apoptosis,
may also have neuroprotective functions
IL-la Pro-inflammatory cytokine, stimulates TNF-a release from
endothelial cells
IL-113 Major pro-inflammatory cytokine, regulates production of IL-
2, IL-6, IL-8 and
interferon-y, stimulation of phagocytosis and programmed cell death, early
CNS development
IL-2 Pro-inflammatory cytokine, T-cell lymphocyte
differentiation
IL-4 Anti-inflammatory cytokine, also stimulates B-cell
proliferation
IL-6 Major pro-inflammatory cytokine, B-cell differentiation,
neurogenesis,
myokine, also has anti-inflammatory functions (suppression of IL-1 and TNF-
a)
IL-8 (CXCL8) Major pro-inflammatory cytokine (chemokine), neutrophil
chemotaxis
IL-10 Major anti-inflammatory cytokine, also has T-cell
stimulatory functions
IL-12 Pro-inflammatory cytokine, T-cell differentiation
Interferon-y Pro-inflammatory cytokine, T and B cell lymphocyte and
macrophage
activation
MCP-1 (CCL2) Pro-inflammatory cytokine, monocyte and basophil chemotaxis
MIP-la (CCL3) Pro-inflammatory cytokine, induces synthesis of pro-inflammatory
cytokines, activates neutrophils
TGF- 3 Anti-inflammatory cytokine, also has pro-inflammatory
functions, may have
long-term harmful effects
[0084] Interleukin-1. The IL-1 family is a group of 11 cytokines which are
intimately involved
in the body's response to injury or infection [48, 491, and which also play a
key role in tumour
angiogenesis [50] and stimulation of cancer stem cells [51]. The most
important cytokines of the
IL-1 group are IL-113, IL-la and the IL-1 receptor antagonist, IL-1RA, but the
IL-1 group also
includes the pro-inflammatory cytokines IL-18, IL-33 and IL-36, as well as
several less well-
studied cytokines. The key cytokine IL-1I3 is a protein produced by activated
macrophages.
Among its most important functions are neutrophil activation, regulation of
production of other
cytokines (IL-2, IL-6, IL-8, interferon-y), regulation of mitosis, stimulation
of phagocytosis,
induction of fever, angiogenesis and induction of programmed cell death [48,
491. Increased
levels of IL-1I3 have been found in the CSF of patients with TBI, and may be
detected within
minutes of acute injury [38, 52, 531. Very high levels in the CSF of TBI
patients have been
associated with a worsening prognosis [54, 551.
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[0085] Studies in animal models have given similar results [56- 601. Kamm et
al showed that
IL-1I3 levels appeared in the rat brain after TBI within the first hour and
peaked at 8 hours, with
no detectable change in IL-1I3 levels in the blood or liver [56]. It has also
been shown, in animal
models, that intraventricular administration of IL-113 significantly worsens
cerebral damage [61].
Most importantly, administration of an IL-1I3 antagonist can prevent the
damage caused by this
cytokine in experimental models. Administration of IL-1RA to rodents has been
shown to reduce
brain damage after TBI. For example, Yang et al showed that the cerebral
damage caused by
middle cerebral artery occlusion in mice was reduced in those animals that
were previously
transfected with an adenoviral vector to induce IL-1RA overexpression [62].
Jones et al showed
that a single intracerebroventricular dose of IL-1RA administered to mice at
the time of TBI
reduced lesion volume, resulted in functional improvement and caused a major
decrease in nitric
oxide synthase-2-positive cells in the lesion [63-Jones]. Sanderson et al
studied the effect of
systemically-administered IL-1RA to Sprague Dawley rats after TBI. No effect
was seen at low
doses. After high-dose administration, the investigators observed decreased
neuronal loss and an
increase in memory and cognitive function in the animals. No improvement was
seen in motor
function, however [64]. Hasturk et al showed IL-1RA reduced tissue IL-1I3
levels and increased
levels of the antioxidant enzymes catalase, superoxide dismutase and
glutathione peroxidase in
rats after TBI [65]. Other groups have reported similar results [66, 671. In
addition, Basu et al
reported that mice lacking the IL-1 receptor experience less brain injury
after a traumatic insult
[68]. The investigators found decreased basal levels of IL-1, IL-6 and COX-2,
as well as fewer
amoeboid microglia/macrophages, suggesting the cycle of brain inflammation was
prevented at
this crucial step. Further, Tehranian et al have shown that transgenic mice
who overexpress
human IL-1RA in astrocytes have decreased levels of IL-113, IL-6 and TNF-a
compared to wild
type mice, and have better neurological recovery after head injury [69].
[0086] These data suggest that the use of IL-1RA might be an effective
strategy in patients with
TBI. Human recombinant IL-1RA has been a standard medication for patients with
rheumatoid
arthritis for several years, and its use has been investigated in a number of
diseases where
increased cytokines play a role in the destructive process, including diabetes
[70], heart failure
[71], multiple myeloma [72] and sepsis [73]. In a randomized phase II trial of
patients with acute
stroke, there was less loss of cognitive function in patients treated with IL-
1RA compared to the
control group [74]. Helmy et al conducted a phase II controlled trial of this
agent in 20 patients
with severe TBI, and were able to conclude that IL-1RA does cross the blood-
brain barrier and is
safe in this population [75]. They were unable to conclude that IL-1 RA
administration resulted in
therapeutic benefit in these patients [75]. While many of these results seem
promising, however,
the efficacy of IL-1RA may be limited, as it directly blocks only one of the
important cytokines
involved in the inflammation (IL-1RA may block other cytokines indirectly
since IL-1 can cause
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increased expression of other cytokines), and this may be part of the
explanation for the failure of
this agent to have a greater than limited success against rheumatoid
arthritis. Further, the use of
IL-1RA in combination with TNF-a blockers is contraindicated, as severe side
effects may result
from their concomitant use [76].
[0087] Tumor necrosis factor-a. A second key pro-inflammatory cytokine is
TNF-a.
This cytokine plays an important role in the body's response to infections and
to cancer. Since
the report on TNF-a by Helson et al in 1975 [77], aberrant TNF-a function has
been reported in
numerous diseases, including conditions as diverse as diabetes [78],
cardiovascular disease [79],
inflammatory bowel disease [80] and Alzheimer's disease [81]. TNF blockers,
such as infliximab,
etanercept, and adalimumab, are standard therapies for patients with
rheumatoid arthritis,
ankylosing spondylitis and psoriasis. As noted, TNF-a is thought to have both
beneficial and
detrimental effects in patients with TBI [46]. However, results in
experimental models suggest
that these effects are mostly detrimental, especially when excessive levels of
this cytokine are
produced. Knoblach et al reported the correlation of TNF levels and the degree
of brain injury
and neurological impairment in rats after experimental TBI, with the highest
levels of TNF at 1-4
hours after injury in rats with the most severe brain injury [82]. In
addition, studies with the
TNF-blocker, etanercept, have consistently shown reduction of brain damage in
these animals
after administration of this agent. Chio et al reported that etanercept, when
given to rats after TBI
reduced ischemia, increased glutamate levels, reduced neuronal and glial
apoptosis and microglial
activation, while also reducing the increased levels of TNF-a [83]. In a later
report, these
investigators concluded etanercept ameliorates brain injury by decreasing the
early expression of
TNF-a by microglia [84]. Ekici et al showed that the combination of etanercept
and lithium
chloride administered one hour after TBI reduced cerebral edema, tissue damage
and TNF levels
[85]. Cheong et al showed that etanercept administered to rats immediately
after TBI resulted in
increased 5-bromodeoxyuridine and doublecortin markers in the injured brain,
suggesting that the
increased TNF-a levels in the brain may be toxic to neural stem cells, thus
interfering with
neurogenesis [86]. Wang et al reported that the early use of this agent after
injury promoted the
survival of transplanted neural stem cells and facilitated neural regeneration
[87]. Other groups
have reported similar results using etanercept or other TNF blockers [88-91].
[0088] Although TNF blockers have been studied extensively in animal models,
little work has
been done to assess the potential efficacy of these agents in patients with
TBI [92]. Tobinick et al
reviewed the medical records of 617 patients with stroke and 12 with TBI who
had been treated
with etanercept. Marked improvement in neurological function was observed,
even for patients
treated more than 10 years after the initial insult. The investigators
concluded that this supported
the view that long-term inflammation, perhaps lasting many years, was a major
cause of
neurological impairment in these patients [93]. However, the small number of
patients in the TBI
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group and the lack of a control group make the data in this report difficult
to interpret, as it is not
clear that TNF blockade was responsible for the observed improvement.
Randomized trials are
needed to prove benefit in TBI patients, and TNF blockers may have substantial
toxicity. In
addition, since TNF blockers target only a single cytokine, and since the use
of these agents is
contradicted in combination with IL-1 antagonists, the use of these blockers
may not be the most
effective strategy in treatment of these patients.
[0089] Interleukin-6. A third major pro-inflammatory cytokine is IL-6. As with
TNF-a,
elevated levels of IL-6 have been thought to have a role in the causation of
numerous diseases,
and like TNF-a, IL-6 is thought to have beneficial as well as harmful effects
after TBI [94].
Indeed, IL-6 appears to have both a beneficial and a deleterious role in a
number of neurological
conditions [95]. IL-6 plays a key role in induction of nerve growth factor by
astrocytes, and thus
in the repair of the injured brain [39]. Ley et al reported that IL-6 knockout
mice demonstrated
reduced neurological function after TBI compared to normal mice, again
suggesting IL-6 is
necessary for neuronal recovery. The IL-6 knockout mice did, however, show
significantly
elevated levels of IL-1I3 [96]. The neuroprotective role of IL-6 was also
suggested in a study of
frontal lobe parenchymal IL-6 levels in patients after severe TBI. Markedly
elevated IL-6 levels
were found in survivors compared to those who died, while levels of IL-1I3
were not different
[97]. However, the numbers in this study were small.
[0090] On the other hand, numerous studies have suggested that IL-6 has
harmful effects after
TBI. Conroy et al showed that IL-6 was toxic to rodent cerebellar granule
neurons in culture
[98]. In another study, intranasal administration of IL-6 to rats was found to
increase the intensity
of seizures, as well as to increase mortality [99]. Similar results were seen
in transgenic mice
with glial fibrillary acidic protein promoter driven-astrocyte IL-6 production
[100]. Yang et al
showed that motor coordination deficits in mice after mild TBI could be
corrected by IL-6
blockade [101]. Similar results were reported in experimental spinal cord
injury. Okada et al
showed that an anti-IL-6 receptor mouse monoclonal antibody could increase
functional spinal
cord recovery in mice after injury [102]. Nakamura et al reported that an
antibody to IL-6R
decreased glial scar formation and increased recovery after spinal injury
[103]. Crack et al
reported that anti-lysophosphatidic acid antibodies markedly reduced brain
damage in mice after
experimental TBI. The investigators attributed this to a dramatic reduction in
IL-6 induced
secondary inflammation. The antibodies had no effect on levels of IL-1I3 or
TNF-a [104]. Suzuki
et al have suggested that the divergent results seen in these studies might be
explained because
IL-6's inflammatory effect seems to dominate in the acute phase after TBI,
while its effect on
neurogenesis may be important later on [105]. Little work has been done to
investigate IL-6
blockers in patients with TBI. An anti-IL-6 antibody, tocilizumab, is
available, and is used for
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treatment of patients with rheumatoid arthritis [106], but this agent has not
been studied in this
population.
[0091] Anti-inflammatory cytokines. Anti-inflammatory cytokines, such as IL-4,
IL-10, IL-11
IL-13 and transforming growth factor (TGF)-13, can also be markedly elevated
in inflammatory
conditions. One of the major functions of these cytokines is to inhibit
synthesis of pro-
inflammatory cytokines [107]. IL-10 is the most important anti-inflammatory
cytokine, and IL-
levels are markedly elevated in the brain and CSF after TBI [54, 1081.
Although IL-10 is
known to also have pro-inflammatory functions [107], its main effect after TBI
appears to be
primarily protective against inflammatory damage. Kumar et al studied cytokine
levels in 87
10 patients with severe TBI over a twelve-month period and found that
patients with an elevated IL-
6/IL-10 ratio at six months had a poor prognosis [109]. Studies in cell
culture and in animal
models seem to confirm the protective effect of IL-10. Bachis et al showed IL-
10 blocks caspase-
3 and reduces neuronal death after exposure of rat cerebellar granule cells in
culture to toxic doses
of glutamate [110]. Knoblach et al showed that either intravenous or
subcutaneous administration
of IL-10 after experimental TBI in rats could reduce synthesis of IL-1 and
enhance neurological
recovery in the animals. Intracerebroventricular administration was not
effective, however [111].
Chen et al showed that mice deficient in IL-10 failed to respond to the
beneficial effects of
hyperbaric oxygen treatment after TBI (112- X. Chen 2013). Bethea et al showed
that IL-10
reduced TNF-a production and improved motor function after spinal cord injury
in rats [113].
Similar neuroprotective effects of IL-10 were also seen in other studies of
experimental spinal
cord injury [114, 1151. This suggests another approach to the treatment of TBI
in patients might
be administration of an anti-inflammatory cytokine like IL-10. Trials of
recombinant human IL-
10 (ilodecakin) have been done in a number of diseases. However, results have
so far been
disappointing [1161.
[0092] Targeting multiple cytokines.
[0093] Progestins. It is well known, from studies in animal systems, that
progestins can reduce
neuronal damage after TBI [117-121]. A major mechanism for the neuroprotection
seen with
progestins is the ability of these agents to suppress pro-inflammatory
cytokines. Cutler et al
showed that progesterone given to aged male rats after TBI reduced brain
levels of IL-6 at 24, 48
and 72 hours. Decreased levels of NF-xl3 and COX-2 were also seen, and the
rats demonstrated
improved motor skills, decreased cerebral edema and decreased mortality (122-
Cutler). He et al
reported that intraperitoneal administration of progesterone could reduce IL-
113 and TNF-a at 3
hours after injury. Similar results were seen after administration of another
progestin,
allopregnanolone [123]. Chen et al reported that progesterone given to rats
following TBI
decreased levels of IL-113, IL-6 and TNF-a in the brain, as well as reducing
apoptosis of brain
tissue [124]. Pan et al showed intraperitoneal administration of progesterone
reduced brain levels
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of TNF-a and NF-KB in rats after experimental TBI. Treated rats also had
better results on the
Neurological Severity Score Test [125]. Unfortunately, these results have not
been confirmed in
patient trials. Xiao et al did report positive results in a randomized trial
of progesterone given
within 8 hours of TBI [126]. However, large multicenter trials have not
confirmed this. A large
phase III trial of progesterone in patients with TBI conducted by The
Neurologic Emergencies
Treatment Trials Network was stopped early because of lack of efficacy [127].
A second major
trial, SYNAPSE, a multinational, placebo-controlled trial of progesterone in
1195 patients with
severe TBI, also showed no efficacy. Among the progesterone group, only 50.4%
showed a
favorable outcome on the Glasgow outcome scale, compared to 50.5% of patients
who received
placebo [128, 1291.
[0094] Statins. These are 3-hydroxy-3-methylglutaryl coenzyme A reductase
inhibitors, which
are used to inhibit cholesterol production in the liver. These drugs are
widely utilized clinically in
patients with hypercholesterolemia. Statins are also known to have marked anti-
inflammatory
effects. Chen et al showed that lovastatin pre-administered to rats with
experimental TBI caused
marked decreases in IL-1I3 and TNF-a in the areas of brain injury at 6 hours
and at 96 hours post-
injury. Treated rats had significantly reduced FJB-positive degenerating
neurons, and better
functional recovery [130]. Simvastatin was shown to decrease brain levels of
IL-1I3 and to reduce
microglial and astrocyte activation in rats after TBI, with functional
improvement on the NCS
score. No change in IL-6 or TNF-a levels was noted, however [131].
Atorvastatin was found to
lower both IL-6 and TNF-a in mice after TBI. Hippocampal degeneration and
functional
neurological deficits were reduced in the treated animals compared to controls
[132]. There is
also evidence that discontinuation of these statins in patients may lead to an
increase in pro-
inflammatory cytokines, including IL-6 [133-135], and that stopping these
medications after TBI
seems to lead to a worse prognosis [136]. Further, a retrospective study
suggests pre-injury statin
use is associated with better outcomes [137]. This has led to the suggestion
that these agents be
studied in patients with TBI. Only a few small trials have been reported.
Tapia-Perez et al
investigated the effect of rosuvastatin in patients with severe TBI and
reported there was a
reduction in amnesia time in the treated patients [138]. However, there was no
difference in
disability at 3 months. Further, this trial included only 8 rosuvastatin
patients and 13 controls,
while 21 of the 43 assessed TBI patients were deemed ineligible. In another
small study, of 19
patients receiving 10 days of rosuvastatin and 17 controls, Sanchez-Aquilar et
al reported that the
rosuvastatin patients had a dramatic decrease in plasma levels of TNF-a
compared to placebo and
an improvement in disability scores. No effect was seen on IL-113, IL-6 or IL-
10 [139]. Rasras et
al investigated the effects of a similar agent, simuvastatin, in a randomized
trial of 66 patients
with severe TBI; however, no difference was found between the treated and the
control groups
[140].
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[0095] Tetracyclines.
Tetracyclines have been shown, in animal models, to suppress
inflammation and better outcomes in several neurological conditions. Bye et al
showed that
minocycline could reduce IL-1I3 and IL-6 expression and microglial and
macrophage activation in
mice after TBI. Neurological functioning was better at day 1 in treated mice,
although there was
no difference between treated mice and controls at day 4 [141]. Later studies
by this same group
did show, however, comparative improvement in the minocycline group by 6 weeks
[142].
Shanchez Mejia et al reported that minocycline given to mice after TBI reduced
IL-1I3 by
inhibiting caspase-1 activation, resulting in improved neurological function
and decreased lesion
volume in the treated animals [143]. Lee et al showed that minocycline given
to rats after spinal
cord injury reduced TNF-a, increased IL-10, reduced neuronal cell death and
improved motor
function [144]. Yrjanheikki reported that either doxycycline or minocycline
could reduce mRNA
induction of IL-1I3 converting enzyme and protect against neuronal death after
ischemic stroke
[145]. Other investigators have also reported positive results with
tetracyclines in animal models
of TBI [146- 1481. However, Turtzo et al could demonstrate no benefit in rats
treated with
minocycline after TBI [149]. Further, in another study, minocycline was found
to cause increased
ischemic brain injury in the neonatal mouse [150, 1511.
[0096] Other anti-inflammatory agents. A number of other agents have shown
anti-
inflammatory activity in animal models of TBI. Melatonin was reported to
decrease TNF-a and
IL-1I3 and increase the number of surviving neurons in mice after TBI. The
investigators felt this
effect was secondary to dephosphorylation of the m-TOR pathway [152]. Other
investigators
have also reported positive results with melatonin in animal models [153-156].
[0097] Zhu et al reported that intraperitoneal administration of curcumin
given to mice 15
minutes after TBI markedly decreased levels of IL-113, IL-6 and MCP-1 and
reduced the number
of TLR4-positive microglia/macrophages, resulting in decreased neuronal
apoptosis [157]. Other
investigators have also reported neuroprotective effects of curcumin in animal
models of TBI
[158-161].
[0098] Cyclosporine is a potent, immunosuppressant drug. Because of its wide-
ranging effects
on cytokines [162-165], and activity in animal models [166], it has been
studied in trials of
patients with TBI. However, a randomized, placebo-controlled, trial of this
agent in patients with
TBI showed no activity [167]. A formulation of cyclosporine (neurostat)
continues to be
investigated in patients with TBI and other neurological conditions, although
a recent report
showed neurostat had no neuroprotective activity in acute ischemic stroke
[168].
[0099] Many other agents which suppress pro-inflammatory cytokines have also
been studied in
animal models. Carprofen, a COX-2 inhibitor, which is currently used to treat
arthritis in dogs
and other animals, was found to markedly reduce IL-1I3 and IL-6, and to
improve neurological
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functioning in mice after TBI [169]. Triptolide, a diterpenoid epoxide, which
has anti-cancer
activity in animal models, was found to suppress IL-113, IL-6 and TNF-a, to
increase IL-10 levels
and to reduce neuronal apoptosis in Sprague-Dawley rats after experimental TBI
[170]. TSG-6
(TNF-a stimulated gene/protein 6) is an anti-inflammatory agent which can
suppress IL-113, IL-6
and other pro-inflammatory cytokines (MIP-la, MCP-1), and stimulate production
of anti-
inflammatory cytokines like IL-4 [171]. Watanabe et al showed that
administration of this agent
to mice after TBI decreased lesion size and improved neurological recovery
[172]. Another
agent, the CNS-penetrating, small molecule, MW151, which is known to suppress
IL-1I3 and
TNF-a, but not to affect anti-inflammatory cytokines like IL-10, has been
tested in mice after
TBI. This agent restored abnormal cytokine levels to normal, reduced glial
activation and caused
improvement in neurologic functioning in the treated animals [173]. None of
these agents have
been tested in patient trials, however.
[0100] The brain damage after TBI may be markedly worsened during a succeeding
phase of
brain inflammation. During this phase, massive increases occur in the levels
of key cytokines,
particularly IL-113, IL-6 and TNF-a, a 'cerebral cytokine storm' where levels
may increase
thousands of times compared to their corresponding levels in serum. Although
some of these
cytokines, such as IL-6 and TNF-a, may have beneficial actions, evidence
suggests excessive
levels are harmful, since numerous studies in animal models have shown
blockade of these
cytokines can reduce brain injury. Thus, suppression of pro-inflammatory
cytokines can limit the
secondary damage caused by neuro-inflammation after TBI.
[0101] It is contemplated that any embodiment discussed in this specification
can be
implemented with respect to any method, kit, reagent, or composition of the
invention, and vice
versa. Furthermore, compositions of the invention can be used to achieve
methods of the
invention.
[0102] It will be understood that particular embodiments described herein are
shown by way of
illustration and not as limitations of the invention. The principal features
of this invention can be
employed in various embodiments without departing from the scope of the
invention. Those
skilled in the art will recognize, or be able to ascertain using no more than
routine
experimentation, numerous equivalents to the specific procedures described
herein.
Such equivalents are considered to be within the scope of this invention and
are covered by the
claims.
[0103] All publications and patent applications mentioned in the specification
are indicative of
the level of skill of those skilled in the art to which this invention
pertains. All publications and
patent applications are herein incorporated by reference to the same extent as
if each individual
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publication or patent application was specifically and individually indicated
to be incorporated by
reference.
[0104] The use of the word "a" or "an" when used in conjunction with the term
"comprising" in
the claims and/or the specification may mean "one," but it is also consistent
with the meaning of
"one or more," "at least one," and "one or more than one." The use of the term
"or" in the claims
is used to mean "and/or" unless explicitly indicated to refer to alternatives
only or the alternatives
are mutually exclusive, although the disclosure supports a definition that
refers to only
alternatives and "and/or." Throughout this application, the term "about" is
used to indicate that a
value includes the inherent variation of error for the device, the method
being employed to
determine the value, or the variation that exists among the study subjects.
[0105] As used in this specification and claim(s), the words "comprising" (and
any form of
comprising, such as "comprise" and "comprises"), "having" (and any form of
having, such as
"have" and "has"), "including" (and any form of including, such as "includes"
and "include") or
containing" (and any form of containing, such as "contains" and "contain") are
inclusive or
open-ended and do not exclude additional, unrecited elements or method steps.
In embodiments
of any of the compositions and methods provided herein, "comprising" may be
replaced with
consisting essentially of' or "consisting of'. As used herein, the phrase
"consisting essentially
of' requires the specified integer(s) or steps as well as those that do not
materially affect the
character or function of the claimed invention. As used herein, the term
"consisting" is used to
.. indicate the presence of the recited integer (e.g., a feature, an element,
a characteristic, a property,
a method/process step or a limitation) or group of integers (e.g., feature(s),
element(s),
characteristic(s), propertie(s), method/process steps or limitation(s)) only.
[0106] The term "or combinations thereof' as used herein refers to all
permutations and
combinations of the listed items preceding the term. For example, "A, B, C, or
combinations
thereof' is intended to include at least one of: A, B, C, AB, AC, BC, or ABC,
and if order is
important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or
CAB. Continuing
with this example, expressly included are combinations that contain repeats of
one or more item
or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The
skilled artisan will understand that typically there is no limit on the number
of items or terms in
.. any combination, unless otherwise apparent from the context.
[0107] As used herein, words of approximation such as, without limitation,
"about",
"substantial" or "substantially" refers to a condition that when so modified
is understood to not
necessarily be absolute or perfect but would be considered close enough to
those of ordinary skill
in the art to warrant designating the condition as being present. The extent
to which the
description may vary will depend on how great a change can be instituted and
still have one of
31
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ordinary skilled in the art recognize the modified feature as still having the
required
characteristics and capabilities of the unmodified feature. In general, but
subject to the preceding
discussion, a numerical value herein that is modified by a word of
approximation such as "about"
may vary from the stated value by at least 1, 2, 3, 4, 5, 6, 7, 10, 12 or
15%.
[0108] Additionally, the section headings herein are provided for consistency
with the
suggestions under 37 CFR 1.77 or otherwise to provide organizational cues.
These headings shall
not limit or characterize the invention(s) set out in any claims that may
issue from this disclosure.
Specifically and by way of example, although the headings refer to a "Field of
Invention," such
claims should not be limited by the language under this heading to describe
the so-called
technical field. Further, a description of technology in the "Background of
the Invention" section
is not to be construed as an admission that technology is prior art to any
invention(s) in this
disclosure. Neither is the "Summary" to be considered a characterization of
the invention(s) set
forth in issued claims. Furthermore, any reference in this disclosure to
"invention" in the singular
should not be used to argue that there is only a single point of novelty in
this disclosure. Multiple
inventions may be set forth according to the limitations of the multiple
claims issuing from this
disclosure, and such claims accordingly define the invention(s), and their
equivalents, that are
protected thereby. In all instances, the scope of such claims shall be
considered on their own
merits in light of this disclosure, but should not be constrained by the
headings set forth herein.
[0109] All of the compositions and/or methods disclosed and claimed herein can
be made and
executed without undue experimentation in light of the present disclosure.
While the
compositions and methods of this invention have been described in terms of
preferred
embodiments, it will be apparent to those of skill in the art that variations
may be applied to the
compositions and/or methods and in the steps or in the sequence of steps of
the method described
herein without departing from the concept, spirit and scope of the invention.
All such similar
substitutes and modifications apparent to those skilled in the art are deemed
to be within the
spirit, scope and concept of the invention as defined by the appended claims.
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