Note: Descriptions are shown in the official language in which they were submitted.
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
TITLE OF THE INVENTION
GALECTIN-3 PLASMAPHERESIS THERAPY
Related Applications and Incorporation by Reference
[0001] This application is related to U.S. Provisional Patent Application
No. 61/568,210
filed December 8, 2011 and U.S. Patent Application Serial No. 13/629,932 filed
September
28, 2012, both of which are which is incorporated by reference herein in their
entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] This invention pertains to treatment of disease and biological
conditions mediated
at least in part by one or more galectins. Galcctins are a family of lcctins
(sugar binding
proteins) that are characterized by having at least one carbohydrate
recognition domain
(CRD) with an affinity for beta-galactosides. These proteins were recognized
as a family
only recently, but are found throughout the animal kingdom, and are found in
mammals,
birds, amphibians, fish, sponges, nematodes and even fungi. This application
focuses on
galectins in mammals, and in particular, humans. Although the invention herein
may be
employed with both companion animals (e.g., pets such as dogs and cats) and
commercial
animals (such as cows, pigs and sheep) the methods and subject matter
addressed herein are
particularly focused on the treatment of humans.
[0003] Galectins mediate and modulate a wide variety of intracellular and
extracellular
functions, and thus are both expressed within the cell and frequently targeted
to a specific
cytosolic site, and secreted from the cell, for distribution extra-cellularly,
as a component of
human plasma. Among the many functions that are mediated by extracellular
galectins are
1
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
inflammation, fibrosis formation, cell adhesion, cell proliferation,
metastatic formation,
angiogenesis (cancer) and immunosuppression.
[0004] Galectins are a family of fifteen (15) carbohydrate-binding
proteins (lectins) highly
conserved throughout animal species. Most galectins are widely distributed,
though galectin
-5, -10 and -12 show tissue-specific distribution. While galectins are
variably expressed by
all immune cells, they are upregulated in activated B and T cells,
inflammatory macrophages,
natural killer (NK) cells, and FoxP3 regulatory T cells. Galectins contain a
variety of
structural arrangements, but a relatively conserved carbohydrate recognition
domain (CRD).
The majority of galectins display a single CRD, and are biologically active as
monomers
(galectin-5,-7 and -10), or require homodimerization for functional activity
(galectin-1,-2,-
11,-13,-14 and-15). Alternatively, tandem-repeat-type galectins (galectin-4,-
8,-9, and -12)
contain two CRDs separated by a short linker peptide, while galectin-3
(chimeric type) has a
single CRD fused to a non-lectin domain that can be complexed with other
galectin-3
monomers to form an oligomeric pentamer. Of note, some galectins, such as
galectin-10,
bind to mannose-containing glycans. Among the family of galectins, -1, -3, and
-9 are
particularly important as potential therapeutic targets, and -2,-4,-5,-6,-7,-
8,-10, -11,-12,-13,-
14, and -15 also appear implicated in a variety of biological pathways
associated with
morbidity and mortality.
[0005] Thus, galectin-7 has been implicated in the development of certain
forms of
cancer. St. Pierre et al, Front. Biosci., 1:17,438-50 (2012) and in a variety
of specific
cancers, including gal-2, -4 and -8 in the context of colon and breast cancer,
Barrow et al,
Cl/n. Cancer Res,. 15;17 (22) 7035-46 (2011). Squamous cell carcinoma of the
tongue, Alves
et al., Pathol. Res. Pract. 15;207 (4) 236-40 (2011) has been shown to be
associated with
elevated levels of gal-1, -3 and -7, while cervical squamous carcinoma has
been shown linked
to gal-7 levels, Zhu et al, Int. J. Cancer, (Aug., 2012). A number of
galectins, including gal-
2
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
15, gal-13 and gal-10 have been demonstrated to be linked to implantation and
pregnancy
concerns. See, e.g., Than et al, Eur. I Biochem. 271(6) 1065-78 (2004), Lewis
eta!, Biol.
Reprod 77(6); 1027-36 (2007). A number of galectins, including gal-2, 3, 8 and
others have
been identified as correlating with various autoimmune disorders, such as
lupus. Salwati et
al,J. Infect. Dis. 1;202(1) 117-24 (2010), Pal et al, Biochim. Biophys. Acta.,
1820 (10) 1512-
18 (2012) and Janko eta!, Lupus 21(7) :781-3 (2012). Elevated levels of a
number of
galectins, including gal-3, are associaed with inflammation and fibroses
encountered in
wound healing and the like. Gal eta!, Ada. Hislochem. Cytochem. 26:44(5); 191-
9 (2011).
100061 Quite obviously, mediation of inflammatory and fibrotic pathways
makes galectins
critical elements of a wide variety of disease, injury and trauma related
phenomena. In many
cases, the presence of unwanted concentrations of galectins can aggravate a
disease condition
or trauma situation, or interfere with attempts to treat diseases, such as
cancer or congestive
heart failure. Among the family of galectins recognized as active in humans,
galectin-1,
galectin-3 and galectin-9 are of particular interest. As indicated above,
these proteins are
generally referred to, and referred to herein as, gal-1, gal-3 and gal-9. A
wide variety of
conditions in humans, ranging from problems in conceiving to asthma to chronic
heart failure
to cancer to viral infection to stroke and beyond are mediated or aggravated
by higher than
normal concentrations of galectins. Thus, among other galectins, gal-3 is
particularly
prominent in fibrosis, inflammation and cell proliferation, while gal-1 also
plays a role in the
immunosuppression required for a successful pregnancy. Gal-1 is also thought
to be
involved in the differentiation of nerve cells. Gal-9 has been shown to be
involved in the
control of lesions arising from immuipinflammatory diseases, and is generally
implicated in
inflammation ¨ gal-9 apparently plays a role in eosinophil recruitment in
inflammatory sites.
It also appears to mediate apoptosis in certain activated cells.
3
CA 02893690 2015-06-03
WO 2014/106803
PCT/IB2013/061375
While the discussion herein is applicable to circulating active gal-I, gal-3
and gal-9, and
galectins in general, where elevated circulating galectin levels are
associated with disease or
injury conditions, more has been elucidated about the role of gal-3 in disease
and trauma
progression than any of the other galectins, and so it is exemplified herein.
More
specifically, this invention focuses on the removal of active gal-3 from
mammalian,
particularly human, plasma. Gal-3 has been shown to be involved in a large
number of
biological processes, many of which are related to disease states of various
kinds. Binding
and blocking activity of gal-3 in the circulation, or removal of large amounts
of gal-3 from
circulation may therefore improve existing medical treatments, suppress and/or
reduce
inflammation and fibrosis resulting from others, and make it possible to
intervene in various
disease states not otherwise easily treated. The invention is equally
applicable to the
reduction in circulating levels of other active galectins to address
conditions mediated by
those galectins. By "active" galectins, what is referred to is biologically
active molecules. As
noted, for example, gal-3 can be active, that is, mediate mammalian responses
to various
traumas and conditions, as a monomer and as an oligomer. In any mammal, at any
given
time, significant amounts of gal-3 and other galectins are present in an
inactive state ¨ that is,
they are either tissue bound or ligand bound in such fashion as to inhibit
molecular
interaction. While such galectins molecules may become active, and may be or
become the
target of removal by the invention disclosed herein, when monitoring patient
conditions and
controlling responses, the focus of the invention is the removal of active
galectins from the
blood stream. This invention makes use of plasmapheresis, sometimes referred
to as
therapeutic plasma exchange, to control levels of gal-3, and more specifically
biologically
active galectin, in circulation. PlaL,aa is lead through a fluid pathway and
either intermixed
with a gal-3 binding agent which can be separated from the plasma, or returned
to the body
with blocked inactivated gal-3, or lead past a solid support which binds gal-
3, the plasma
4
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
being subsequently returned to the body with a reduced level of gal-3. Thus,
this invention
can be used to remove bound gal-3 as part of a strategy to reduce total gal-3
content. The
focus, in this application, however, is to remove active or unbound gal-3 as a
therapeutic
measure.
Related Art
[0007] This application is related to U.S. Patent Application Serial No.
13/153,648, filed
June 6, 2011. That application in turn claims priority benefit to U.S. Patent
Application
Serial No. 11/485,955, filed July 6, 2006. The content of both these patent
applications is
expressly incorporated herein-by-reference. In U.S. Patent Application Serial
No.
13/153,648 (U.S. Patent Publication US-2011-0294755 Al) a method of treating
cell
proliferation conditions, inflammation and aggravated fibroses is disclosed
which involves
the administration of an agent that can bind circulating gal-3, such as
modified citrus pectin,
(MCP), a citrus pectin which has a reduced molecular weight of twenty thousand
(20,000)
Daltons or less, preferably ten thousand (10,000) Daltons or so. MCP is
available
commercially from EcoNugenics of Santa Rosa, California and is discussed in
U.S. Patent
Nos. 6,274,566 and 6,462,029.
Back2round of the Technoloev
[0008] Gal-3 is approximately 30 kDa and, like all galectins, contains a
carbohydrate-
recognition-binding domain (CRD) of about one hundred thirty (130) amino acids
that enable
the specific binding of P-galactosides. Gal-3 is encoded by a single gene,
LGALS3, located
on chromosome 14, locus q21¨q22. This protein has been shown to be involved in
a large
number of biological processes. The list set forth herein is exemplary only as
new situations
and roles for gal-3 are continually being revealed. Among the biological
processes at the
cellular level that have been shown to be mediated, at least in part, by gal-
3, are cell
5
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
adhesion, cell migration, cell invasion, cell activation and chemoattraction,
cell growth and
differentiation, cell cycle, angiogenesis and apoptosis.
[0009] Given gal-3's broad biological functionality, it has been
demonstrated to be
involved in a large number of disease states or medical implications. Studies
have also
shown that the expression of gal-3 is implicated in a variety of processes
associated with
heart failure, including myofibroblast proliferation, fibrogenesis, tissue
repair, inflammation,
and ventricular and tissue remodeling. Elevated levels of gal-3 in the blood
have been found
to be significantly associated with increased morbidity and mortality. They
have also been
found to be significantly associated with higher risk of death in both acute
decompensated
heart failure and chronic heart failure populations.
[0010] Various investigations have shown elevated levels of gal-3 to
aggravate a wide
variety of disease conditions associated with cell proliferation. High levels
of gal-3 are
linked to cancer growth and cancer progression to a metastatic stage in a
stunning variety of
cancers. A number of cancers have been specifically linked to or associated
with elevated
gal-3 levels, including liver cancer, kidney cancer, breast cancer, prostate
cancer, colon
cancer, thyroid cancer, cancer of the gallbladder, nasopharyngeal cancer,
lymphocytic
leukemia, lung cancer, melanoma, multiple myeloma, glioblastoma multiforme,
uterine
cancer, ovarian cancer, cervical cancer, brain cancer and others. Elevated gal-
3 levels have
also been shown to interfere with or suppress conventional antineoplastic
regimens, such as
chemotherapeutic treatments like cis-platinum, doxonibicin and related
chemotherapeutics.
100111 Inflammation is a commonly encountered body condition ¨ a natural
response of
the body to a variety of diseases and trauma. As with the other conditions
noted above, gal-3
levels above normal levels are implicated in a wide variety of situations
where harmful
inflammation is encountered. Again, the list of conditions and disease states
is too extensive
to exhaust every possibility, but inflammatory conditions associated with
elevated gal-3
6
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
levels include aggravated inflammation associated with non-degradable
pathogens,
autoimmune reactions, allergies, ionizing radiation exposure, diabetes, heart
disease and
dysfunction, atherosclerosis, bronchial inflammation, intestinal ulcers,
intestinal
inflammation of the bowels, cirrhosis-associated hepatic inflammation,
parasitic infection
associated inflammation, inflammation associated with viral infection,
inflammation
associated with fungal infection, inflammation associated with arthritis, with
multiple
sclerosis, psoriasis, Alzheimer's disease, Parkinson's disease, and
amyotrophic lateral
sclerosis (ALS). Again, while inflammation is a pathway frequently employed by
the body in
responding to any number of challenges, elevated levels of gal-3 have been
found to
aggravate the inflammation, causing lamage and injury leading to morbidity or
mortality in a
wide variety of situations that are otherwise manageable, including
inflammation due to
heavy metal poisoning and similar toxins, stroke and related ischemic
injuries, liver
inflammation due to acetaminophen, a number of T-cell mediated responses
generally
involved in autoimmune diseases and the like. Gal-3 is also involved with
kidney injury and
kidney disease, hepatitis, pulmonary hypertension and fibrosis, diabetes, and
gastrointestinal
inflammatory conditions such as ulcerative colitis, Crohn's disease, celiac
disease, and others.
[0012] As noted, elevated levels of circulating, active gal-3 are
associated with, and
apparently aggravate, a number of inflammatory conditions, including those
contributing to
heart, kidney, lung, brain, and liver disease. Gal-3 is also associated with a
fibrotic
formation, particularly in response to organ damage. Higher levels of
circulating gal-3 are
found to induce pathogenic fibroses in cardiovascular disease,
gastroenterological disease,
cardiovascular trauma, renal tissue trauma, brain trauma, lung trauma, hepatic
tissue trauma,
tissue damage due to radiation therapy and diseases and conditions of
connective tissue and
skin such as systemic sclerosis.
7
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
[0013] Accordingly, the art is replete with observations that elevated
levels of gal-3, as
well as gal-1 and gal-9, can complicate or exacerbate a wide variety of
disease and injury
conditions. It would be of value to find a way to control inflammation and
formation of
fibroses, where the inflammation and fibroses are injurious, particularly in
the environments
described above, and notably in cardiac care and other organ tissue disease
and trauma. By
the same token, it would be of value to control the cellular responses
mediated by gal-3 that
accelerate cell proliferation and transformation, including the formation and
growth of
tumors, the transformation of cancer cells and metastatic spread of cancer.
Another goal in
the art is to avoid the problem posed by the interference in the treatment of
cancer by
conventional agents, like bleomycin, Adriamycin, doxorubicin, cyclophosphamide
and
cyclosporine. Some of the side effects caused by these agents are gal-3
mediated, and can be
addressed and ameliorated by the invention. Elevated gal-3 levels also appear
to interfere
with pharmaceuticals used in other applications, such as the antiarrhythmic
drug amiodarone,
and statin drugs.
[0014] Plasmapheresis is a blood separation technology, where blood is
diverted from the
body through a needle or catheter to a separator which removes blood cells and
returns them
to the body, leaving plasma. This type of technique has been used historically
in the
treatment of autoimmune diseases, where the antibodies at issue are removed by
contacting
the plasma with the ligands to which they bind. The plasma is then augmented
as required,
with anticoagulants, therapeutics and associated elements, and returned to the
body. In prior
art methods employing plasma exchange or replacement therapies generally, as
illustrated in
U.S. patent publication US 2006/0129082, the technology was used to target and
remove
"toxic serum components" such as ammonia, uric acid, and cell growth
inhibitors. The same
reference, at [0009] ¨ [0010] warns against the use of plasma exchange in
general. Similar
warnings are sounded in Kyles et al, Am. J Crit Care, 14, 109¨ 112 (2005)
reviewing the
8
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
use of plasmapheresis for support of immunoglobulin sepsis treatment, noting
that
traditionally, plasmapheresis has been used in treatments to remove pathogenic
autoantibodies and endotoxins in autoimmune disorders and to remove harmful
substances
produced by the infecting organisms causing sepsis. As far as Applicant is
aware, this
invention reflects the first use of plasmapheresis to lower and control the
level of active gal-3,
an essential and relatively low molecular weight molecule, to support patients
in specific
need of hat relief.
100151 An early form of apparatus for plasmapheresis is set forth in U.S.
Patent No.
3,625,212, which describes measures to ensure return of treated plasma, as
well as the
separated blood cells, to the proper donor. U.S. Patent No. 4,531,932
addresses
plasmapheresis by centrifugation, the method used to separate out the red
blood cells, on a
rapid and near-continuous basis. U.S. Patent Nos. 6,245,038 and 6,627,151 each
describe a
variety of methods of separating out plasma contents and returning the treated
plasma to the
patient after first removing red blood cells, in general, to reduce blood
viscosity by removal
of high molecular weight protein. While the invention that is the subject of
this application
focuses on the reduction in galectins circulating levels, such as gal-3
levels, and not high
molecular weight proteins or directly addressing viscosity, the disclosure of
these four (4)
patents is incorporated herein-by-reference for their disclosure of available
plasmapheresis
techniques and apparatus which may generally be employed in this invention.
Advances in
apheresis generally, including plasmapheresis, have demonstrated the
effectiveness of the use
of hemodialysis equipment using a highly permeable membrane like the Plasmaflo
AP-05H
from Asahi Medical and a standard dialysis machine in ultrafiltration mode.
This is similar to
hemoperfusion in application. The use of hemodialysis and single needle
plasmapheresis are
well tolerated, and favored for use in this invention.
9
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
[0016] Prior to the development of this invention, those of skill in the
art had
experimented with the reduction of gal-3 levels in various respects. Thus, the
activity of gal-
3 in aggravating or promoting cancer, as well as the ability of a cancer to
metastasize, is
widely commented on in the literature following 2006. These literature
findings stress
repeatedly the importance of binding or reducing the circulating concentration
or titer of gal-
3, and/or inactivating gal-3 through gal-3 binders such as MCP. See, for
example, Wang et
al, Cell Death and Disease, 1-10 (2010) (gal-3 inhibition promotes treatment)
and Yu et al,
Biol. Chemistry, Vol. 282, 1, pp. 773-781 (2007) establishing that gal-3
interactions may
enhance formation of cancer or transformation of metastatic cancer.
[0017] As disclosed and claimed in U.S. Patent No. 6,274,566, Gal-3 binders
such as
MCP and other compounds can bind to circulating tumor cells (CTC's) and
prevent them
from creating new metastasis. These CTC's are often implicated in mutations
and a more
aggressive disease. Cancer stem cells that may also be circulating and get
stimulated under
conditions of stress and inflammation, provide gal-3 another mechanism for
aggravating
cancer. The method of these prior cases may be used in conjunction with the
invention of
this application. In particular, when there are a high number of gal-3
molecules circulating in
the blood stream it makes it more difficult for the gal-3 binders to target
these CTCs. In this
respect, gal-3 molecules serve as decoy molecules. The decoy prevents, in this
particular
application of the invention, binding of the cancer cells in the circulatory
or lymph system, as
opposed to tissue level gal-3.
100181 As a consequence, reports link acceleration of cancer formation
and transformation
to circulating gal-3 concentrations, and suggest that reducing gal-3
circulating concentrations,
reducing its free expression or otherwise reducing available gal-3 or gal-3
interactions
improves cancer prognosis. Zhao et al, Cancer Res, 69, 6799-6806 (2009), Zhao
et al,
Molecular Cancer 9, 154, 1-12 (2010) and Wang et al, Am. I of Pathology, 174,
4, 1515-
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
1523 (2009) wherein siRNA-induced reduction of gal-3 is shown to slow the
course of
prostate cancer. Similarly, high-risk bladder cancer recurrence and prognosis
is related
indirectly to gal-3 levels. Rodriguez et al, J. Curr. Op/n. Urol. 22(5):415-20
(2012) and
Raspollini et al, App!. Immunohistochem. Mol. Morphol. (July 2012). Clearly,
there is
substantial literature that supports the conclusion that reducing circulating
gal-3, either by
blocking its expression, or by binding it, is important in controlling cancer,
both in tissue and
in circulation.
[0019] Circulating gal-3 is empirically implicated in a wide variety of
biological
conditions, however. Cardiac fibrosis is gaining significant attention as a
complicating risk
factor in cardiac disease, and in particular, chronic heart failure (CHF). Lok
et al, Chn. Res.
Cardiol, 99, 323-328 (2010). DeFillipi et al, US. Cardiology, 7,1, 3-6 (2010)
clearly
indicate that circulating gal-3 is an important factor in fibrosis of many
organs and organ
systems, and that reducing circulating gal-3 may have an important role in
remediating
cardiac injury and progression to heart failure (HF). Similarly, Psarras et
al, Fur. Heart 1,
April 26, 2011 demonstrate that reduction in gal-3 levels in the myocardium
may reduce
fibrosis in the heart and improve outlook. De Boer et al, Ann. Med., 43,1, 60-
68 (2011)
identify gal-3 as a key indicator in cardiac health. Shash et al, Eur J Heart
Fail., 12, 8, 826-
32 (2011) identify gal-3 levels as a key agent in heart failure through
fibrosis. De Boer et al.,
Fur. J. Heart Fail., 11, 9, 811-817 (2009) link an increase in gal-3
expression and presence to
heightened fibrosis, and heart failure. The same article links gal-3 to
inflammation.
Inflammation is the hallmark of arteriosclerosis and therefore gal-3 levels
also contribute to
coronary artery disease, peripheral artery disease, strokes, and vascular
dementia.
[0020] Fibrosis and inflammation, both mediated to some degree by gal-3
(cellular or
circulating) are implicated in a variety of conditions of the mammalian body,
not just cardiac
injury and heart failure. The binding of gal-3 achieved by administration of
low molecular
11
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
weight pectins (at least, as reflected in U.S. Patent Application Serial No.
11/485,955,
10,000-20,000 Daltons molecular weight such as MCP) is effective in reducing
trauma due to
kidney injury. Kolatsi-Jannou et al, PlusOne, 6, 4, e18683 (2011). Reducing
circulating gal-
3 levels may be effective in reducing fibrosis in the lungs and associated
asthma. Cederfur et
al, Biochim. Biophys. Acta. 1820(9):1429-36 (2012). The reduction in
circulating gal-3
levels is also indicated to reduce inflammation associated with type 2
diabetics, and similar
metabolic diseases, as well as obesity. Weigert et al, I Endocrinol. Metab.
95, 3,1404-
1411(2010). Thus, high levels of gal-3 have been linked to thyroid cancer,
Sethi et al,
Exp. Ther. Oncol., 8, 4,341-52 (2010) and reduction of gal-3 expression and
circulation may
delay or reduce tumor cell transformation. Chiu et al, Am I Pathol. 176, 5,
2067-81 (2010).
100211 As noted, gal-3 is implicated in a wide variety of biological
conditions, and a
reduction in gal-3 activity, such as that which can be achieved by gal-3
binding with MCP
and similar low molecular weight pectins may be of value in treating gastric
ulcerative
conditions. Srikanta, Biochimie, 92, 2, 194-203 (2010). Kim et al,
Gastroenterology, 138,
1035-45 (2010) indicate that reducing gal-3 levels may be of therapeutic value
in reducing
gastric cancer progression. By the same methodology, reducing gal-3 levels
sensitizes
gastric cancer cells to conventional chemotherapeutic agents. Cheong et al,
Cancer Sc., 101,
1, 94-102 (2010). Gal-3 is implicated in a wide variety of gastrointestinal
conditions.
Reducing gal-3, by binding for example, may reduce inflammation in the gut
mucosa, making
MCP an important agent for treatment of ulcerative colitis, non-specific
colitis and ileitis,
Crohn's disease, Celiac disease, and gluten sensitivity. Fowler et al, Cell
Microbiol,
81,1,44-54 (2006).
100221 Biliary artesia, a liver disease, is associated with extensive
fibrosis of the liver
linked with elevated gal-3 levels. Honsawek et al, Eur. I Pediatr. Surg,
April, 2011.
Reduction of gal-3 levels resulted in a general improvement in hepatic health,
including
12
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
reducing inflammation, hepatocyte injury and fibrosis. Federici et at, 1
Heptal., 54, 5, 975-
83 (2011). See also, Liu et al, World J. Gastroenterol. 14,48, 7386-91 (2008)
which
reported, following Applicant's teaching in 2005 and 2006 to administer low
molecular
weight MCP, that MCP inhibited liver metastases of colon cancer and reduced
gal-3
concentrations. MCP, or other gal-3 binders, may be used for prevention of
liver
inflammation, liver fibrosis and liver cirrhosis as well as post-disease liver
damage, including
the various viral hepatitis diseases (A, B, C, and others) and may be used as
well in the
treatment of parasitic and chemical hepatitis, chemical liver damage, and
others. Gal-3 levels
are implicated in a wide variety of liver associated ailments. Thus, gal-3 may
be important in
the control of Niemann-Pick disease type C, which is a lysosomal disorder
characterized by
liver disease and progressive neurodegeneration. Cluzeau et at, Hum. Mol.
Geent. 14; 21
(16) 3632-46 (2012). There is increasing evidence that elevated gal-3 levels
are tied to
acetaminophen-induced hepatotoxicity and inflammation. Radosavljeci et at,
Toxicol.
Sci., 127:609-19 (2012). Reduction in gal-3 levels may improve treatments.
Dragomir et al,
Toxicol. Sc!. 127(2):609-19 (2012).
[0023] While administration of MCP, or a similar binding agent, continues
to be a
promising therapy of inhibition of damage, and repair of damage, induced by
gal-3, the
inventor has continued to work to find other methods of providing faster or
more profound
relief. It has now been found that by selective use of certain gal-3 binding
molecules, gal-3
and specifically biologically active gal-3 can be specifically removed from
the plasma in
significant amounts. Return of the plasma with a reduced titer of active gal-3
offers
immediate opportunities for therapy and intervention that may be different
from, or more
profound than, the reduction achieved by administration of binding molecules
to a mammal
in need of same. By removing the circulating gal-3 molecules the invention
removes these
protective but potentially harmful molecules from the circulation. In
addition, it allows
13
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
targeted gal-3 blockers such as MCP, and possibly other oligo-saccharides and
various
pharmaceutical agents to be developed to better attach to the gal-3 on the
cell surface and on
the tissue level. As the expression of gal-3 is increased in the injured and
inflamed tissue,
such as remodeled cardiac muscle or cancer tissue, by removing the circulating
gal-3, the gal-
3 binding agent can more effectively bind to the gal-3 in the target tissue.
SUMMARY OF THE INVENTION
[0024] The invention resides in the removal of biologically active gal-3,
as well as
biologically active problematic galectins, such as gal-1 and gal-9, from a
mammal's
circulation by apheresis of one type or another. The invention is generally
applicable to
removal of any galectin which mediates biological phenomena such as
autoimmunity and cell
proliferation (gal-9 and -1, respectively), such that their removal can
support or improve
therapies already in existence .The mammal may be a human, a primate, a model
such as a rat
or mouse, a commercial animal such as a cow or pig or goat, or a companion
animal such as a
dog or cat. Non-human mammalian animals for treatment include primates, both
as models
and as test beds for treatments and intervention that may benefit from removal
of gal-3 from
circulation. Removal is achieved by plasmapheresis, a process traditionally
developed and
used to remove antibodies from the circulation of those suffering from
autoimmune disorders
and the like.
[0025] Apheresis is defined as a procedure in which one of the components
of blood is
removed. Plasmapheresis of course addresses removal of plasma. Therapeutic
apheresis is a
process in which whole blood is removed from a patient and separated into
components, thus
allowing a single element to be removed or modified while the remaining
components are
returned to the patient. The aim of one form of plasmapheresis, also known as
therapeutic
plasma exchange (TPE), is to remove a large fraction of the patient's plasma
from the body,
14
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
and to exchange this with replaceme, t solutions using automatic devices. This
may be the
patient's plasma following treatment of some type, such as removal of gal-3
from that
plasma, or a plasma substitute that is galectin free.
[0026] TPE differs slightly from donor apheresis, which is used to collect
platelets,
granulocytes, or peripheral blood stem cells from normal individual donors.
The volume of
blood to be exchanged is based on a kinetic model of an isolated one-
compartment
intravascular space, which assumes that the component is neither synthesized
nor degraded
during the procedure and that it remains within the intravascular compartment.
The time
interval between plasma exchanges is generally chosen based on the need to
allow the
component of interest to re-equilibrate into the intravascular space and the
need to minimize
the risk of bleeding as a result of dilutional coagulopathy. In certain
instances such as when
the patient has extremely elevated scrum gal-3 levels, in individuals where
the gal-3 levels
rise quickly between TPE intervals, or individuals with a preexisting
condition such as viral
infections, autoimmune disease, hematologic disorders [hyperviscosity
syndrome,
cryoglobulinemia, porphyria, sickle-cell anaemia, immune complex disease, cold
agglutinin
disease, hemolytic uremic syndrome,. autoimmunehemolytic anemia, autoimmune
thrombocytopenia, autoimmune neutropenia, Clq deficiency, and secondary
immunodeficiency, graft versus host disease (GVHD), etc.], and drug toxicities
as explained,
infra, it may be preferred to perform a plasma exchange introducing amounts of
exogenous
plasma, or exogenous isotonic solutions such as normal saline, with or without
albumin and
other components, to increase benefits of lowering gal-3 in an expeditious
manner and to
reduce complications. Since removing gal-3 more rapidly than it can be renewed
in the body
is of importance, the need to decrease time between intervals of gal-3
lowering plasmaphersis
may also lend itself to more efficacious therapy if the replacement therapy
includes an
amount of replacement plasma in the process to maintain low levels of gal-3 in
the serum.
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
With the inclusion of plasma exchange into the procedure, any circulating
pathogens and
toxins can also be reduced in individnals with these concerns.
[0027] Plasma exchange allows for removal of other components that have
detrimental
effects, and can therefor contribute to the efficacy of the therapy. For
example, immune
inhibitory components that circulate in the plasma. Another example are TNF
alpha receptors
that can be present at elevated amounts in the blood stream of cancer
patients.
100281 In TPE, the patient's plasma is removed from the blood by
centrifugation or a cell
separator/filter and is replaced with saline, albumin, and/or fresh frozen
plasma (FFP), thus
maintaining volume and oncotic equilibrium. (If it's replaced with FFP it is
necessary to pre-
screen or treat the FFP so as to maintain and ensure reduced gal-3 level). An
anti-coagulant
is added to the blood to help avoid clotting. The procedure must be controlled
to ensure that
the patient is kept in fluid balance, maintaining a stable, normal plasma
volume. Before each
TPE for reducing gal-3 serum levels, results of baseline levels of gal-3,
basal complete blood
count, serum protein electrophoresis, coagulation tests and serum electrolytes
must be known.
Additional laboratory testing such as fibrinogen, ESR, PT/PTT (INR) and
comprehensive
metabolic panel may also be used to assess the patient. The efficacy of the
therapy and
treatment interval regimen is assesse: by these values when compared to post
therapy levels.
Depending on the separation chamber configuration of automated TPE and whether
the blood
is batched and processed, or whether separation, removal, and return are
ongoing
simultaneously, these systems can be classified as intermittent (also called
discontinuous) or
continuous flow. As a general rule, high-flow automated devices (1) are primed
to allow for
removal of all air from the circuit and maintenance of the patient's volume,
(2) require
venous access for inflow to the machine and blood return to the patient, (3)
have a significant
extracorporeal volume, which approaches 500 mL for adult configurations, and
(4) are
16
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
programmable with patient sex, height, weight, and hcmatocrit and permit blood
to be
monitored by a marker (e.g. gal-3).
[0029] In plasmapheresis, blood is removed from the patient, and blood
cells are separated
out from the plasma. The blood cells are returned to the body's circulation,
diluted with
fresh plasma or a substitute. Conventional plasmapheresis methods include
medications that
can include blood thinners as necessary. While in typical plasmapheresis, the
plasma is run
over proteins to which the target antibodies bind, in this particular case,
the plasma is
returned to the blood with the antibodies, cytokines, lymphocytes and other
blood
components, after having had gal-3 selectively removed or inactivated by
contact with gal-3
binding molecules. In the case of autoimmune diseases, the removal of gal-3
from the plasma
can be an adjuvant therapy added to the traditional plasmapheresis performed
for such
patients. Other cytokines and plasma components may be removed in the process,
based on
the specific condition and the specific individual.
[0030] This treatment may be used for all the conditions where galectin,
including gal-3,
levels are elevated in the blood or serum or where expression of gal-3 in the
tissues is too
high. Tissues will shed excess gal-3 into the blood stream where it can be
removed through
this invention. Treatment can be varied depending on the patient, the severity
of the
condition and the rate of the mammalian patient's expression of gal-3.
Ordinarily, treatment
every two to four weeks is contemplated until the condition is resolved, but
treatment may be
daily where required, or at any frequency there between. Daily treatment
includes one or
more plasma exchange sessions in a given day, or continuous plasmapheresis
over a multiple
hour period in acute conditions. This may be particularly the case where there
has been a
build-up of gal-3 levels in the tissues of the patient being treated. Initial
removal of gal-3
from the plasma may cause gal-3 from tissue to shift to vascular compartments
rapidly,
necessitating repeat treatments until tissue levels are depleted sufficiently
to reach
17
I
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
equilibrium. Treatment can be administered on an acute or a chronic basis.
Advantageously,
this treatment is combined with the administration of gal-3 blockers and
inhibitors, such as
disclosed in U.S. Patent Application Serial No. 13/153,618. Although MCP is a
target
inhibitor, other gal-3 inhibitors, such as other modified carbohydrates,
including lactulosy1-1-
5 leucine, Zou et al, Carcinogen. (2005), as well as antibodies specific
for gal-3, and other
antagonists like very low molecular weight pectin weighing as low as IKD, GCS-
100,
Demotte et al, Can. Res., 70 (19):476-88 (2010), Streetly et al, Blood,
115(19):3939-48
(published February 26, 2010 as an abstract), may be used. GCS is a
polysaccharide derived
from MCP, as opposed to reduced MCP. A large variety of gal-3 binding
antibodies are
10 commercially available, from suppliers including abeam (ab2473), Novus
Biologics (NB
100-91778) and Abgent (AJ13129). Other galectins-3 specific antibodies may be
used. By
removing large levels of plasma active gal-3 from the blood, the disease and
injury due to
inflammation or fibroses may be reduced, and the progression of cancer may be
impeded.
Similarly, conventional therapeutic treatments may be rendered more effective.
15 [0031] In a preferred embodiment, at the same time active gal-3 is
removed, soluble TNF
receptors, both R-1 and/or R-2 at different ratios based on the condition, are
removed,
through the same process, by running the plasma fluid over a bed of binding
agents of TNF
receptors. TNF can then directly target cancer cells or other targets as an
effective treatment.
The reduction of active gal-3 in both the circulation and the tissue level
will allow TNF to
20 exert its beneficial effects with a reduced amount of inflammation and
fibrosis which limits
its use. Wu et al, Arch. Dermatol. 20:1-7 (2012). The effective removal of
serum gal-3 also
enhances chemotherapy, particularly, but not exclusively, when combined with
TNF receptor
removal. Chemotherapy enhancement will take place by effective removal of
serum gal-3,
reducing drug resistance, even if no TNF receptors were removed from the
circulation.
25 Yamamato-Sugitani et al, PNAS, 18:108(42), 178468-73 (2012). Gal-3
interferes with
18
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
platinum-based chemotherapy and other anti-cancer agents, and increases cell
adhesion, and
angiogenesis. Wu et al, Cell Oncol. 35(3):175-80 (2012). In addition, removal
of gal-3, by
plasmapheresis alone, or together with administration of circulating gal-3
binders like low
molecular weight MCP, may effecti r:ly treat the diseases and conditions
addressed above.
In addition, this can be further enhanced by combining it with other
therapies, one example
being chemotherapy in cancer.
100321 Typical circulating gal-3 level averages for a Caucasian adult
range from 7 on up
to about 20 ng/ml, with a value of 12-15 nanograms of gal-3 per milliliter of
serum being a
representative and reported value. Patients at risk, including those with
advanced illnesses,
exhibit levels, without treatment, that can be much higher than that patient's
average or
normal level. In accordance with the invention, individuals facing serious
illness or
continued disability due to gal-3 mediated fibrosis, gal-3 mediated
inflammation, and cancer
growth, transformation and metastases associated with elevated gal-3 levels
are treated by
plasmapheresis to achieve a significant reduction in circulating gal-3 titer.
100331 By significant reduction in circulating gal-3 levels, inflammation
and/or fibrosis
due to trauma or disease condition can be controlled. Similar reductions in
gal-3 levels can
aid in the control of the growth, spread and the transformation of various
kinds of cancer. In
general, a reduction of circulating gal-3 of at least ten percent (10%) is
necessary to achieve
significant progress in gal-3 mediated fibroses, and even more may be required
in acute
conditions involving inflammation, fibroses due to trauma or aggressive
cancer. In
functional terms, the reduction of gal-3 should be sufficient to reduce or
inhibit the impact of
gal-3 levels on inflammation and fibroses, or cancer growth and
transformation, in said
patient. Reduction in circulating gal-3 of at least twenty percent (20%), and
in some cases at
least forty percent (40%) or even fifty percent (50%), may be required on a
sustained basis.
Severe situations may require reduction in circulating gal-3 levels in a
mammalian patient of
19
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
greater than fifty percent (50%) of that patient's circulating gal-3 titer, on
up to seventy-five
percent (75%) or even more. While some level of gal-3 in circulation is
required for
homeostasis, in acute situations, reductions at least by eighty percent (80%)
of circulating
gal-3, on up to near total removal of gal-3 from serum, may be called for, as
that level is
quickly replenished by the body. Acute situations can be found in all sorts of
individuals, but
a representative example is hepatic inflammation or transformation in an
aggressive cancer
like pancreatic cancer or small cell lung carcinoma.
[0034] The gal-3 levels in races other than Caucasians and subjects may
vary, but the
target is to reduce gal-3 levels below the appropriate normal value. Target
levels can vary
based on the condition, age, gender, and other therapies involved. As a
general matter,
treatment of the patient according to this invention may begin with
plasmapheresis in
conjunction with the absorptive column designed to reduce the patient's gal-3
to a preselected
value consistent with good health and homeostasis in that individual. In some
cases, it may
be necessary to repeat or extend that treatment to achieve even greater
reductions.
[0035] This invention is straightforward in its application. It is
recognizing how many
different indications are served by this technology that is complex and
startling. In the
current invention, blood is removed from the patient according to well
established protocols
generally used for plasmapheresis. See, generally, Samuels et al, editors,
Office Practice of
Neurology, 1996. The removed blood is treated to remove blood cells from the
plasma.
These blood cells, together with an a iditional volume of plasma or plasma
substitute, are
returned directly to the patient. In a single session, two to four liters of
plasma may be
removed, filtered, and replaced. The blood can also be recycled and
recirculated extra
corporally, and filtered as needed, for a number of times (continuously) until
the desired
reduction in serum levels of galecitn-3 is achieved. Different serum levels
can be targeted
for different conditions. The blood cell-depleted plasma is then introduced to
a chamber
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
where gal-3 is removed or inactivated by binding antagonist, possibly creating
a permanent
bond that inactivates the gal-3. One of two alternative measures may be used
to remove gal-3,
although they may be combined. In a first alternative, the plasma is admixed
with a particle
which binds gal-3. Preferably, this is an antibody or similar ligand, or a
polysaccharide
derivative that is most preferably MCP, but any agent that can bind gal-3 can
be used.
Methods of preparing low molecular weight pectins are known in the art, and
set forth in U.S.
Patent Application Serial No. 13/153,648. Alternate adsorbent galectin
affinity columns can
be prepared with matrix linked compounds having multivalent presentation of
galactose,
lactose, poly-N-acetyllactosamine, N acetyl-D-lactosamine, lacto-N-tetraose,
lacto-N-
hexosespecific peptides, aptamers (oligonucleic acid or peptide molecules),
oligo-
saccharides, glycoproteins (such as alpha-2 macroglobulin and haptoglobulin),
antibodies,
engineered Fe (fragment, crystallizable) and Fab (fragment antigen binding)
antibody
fragments, Thomsen¨Friedenreich glycoantigen (TFAg) or with small carbohydrate
(galactose) derivatives.
100361 The binding agent is modified to be complexed with an agent that is
easily
removed. In one embodiment, this is a magnetic particle. After providing for
adequate
circulation time, a magnetic field is applied to the fluid comprising the
plasma and the MCP
complex, and the bound gal-3 can be drawn off. Different filters that
incorporate gal-3
binders can be used in the plasmapheresis process.
[0037] In certain conditions such as cancer, the circulating gal-3 can be
viewed as a sort of
decoy released by the cancer cells. has a protective quality as it doesn't
allow the host,
and doesn't allow gal-3 binders such as MCP to reach the target tissue where
galecin-3 is
over expressed. It also induces inflammation and fibrosis and makes it more
difficult for the
host to bind to the gal-3 in the tissue and cell surface level. Removing the
circulating gal-3
provides both a therapeutic treatment on its own and allows other agents to
bind and
21
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
inactivate the gal-3 in the target tissue level. This is similar to TNF Alpha
and circulating
TNF alpha receptors. Such plasmapheresis can be combined with plasmapheresis
of other
compounds, and can enhance an immune response and an anti-inflammatory
response. The
reduction of circulating gal-3 will allow one of skill in the art, typically a
medical practitioner
with at least five (5) years of experience in the field in addition to
appropriate educational
experience, to more easily neutralize and inactivate the tissue expressed gal-
3, thus allowing
for a local immune response with less inflammation and fibrosis. As such, it
can be
combined with removal of TNF Alpha receptors, both R-1 and R-2. It can also be
combined
with administration of TNF alpha or agents that enhance TNF alpha activity.
[0038] Removing or reducing the level of circulating gal-3 can reduce the
systemic and
unwanted inflammatory process, resulting, as demonstrated in the kidney MCP
study, with
reduction in levels of IL-6, and consequently TNF alpha and TNF kappa beta.
[0039] Accordingly, the invention disclosed herein operates on two (2)
levels:
1. Direct reduction of circulating gal-3; and
2. Ability to better target the gal-3 in the tissue level.
[0038] This has several consequences in terms of treatment effectiveness:
A. By reducing the circulating gal-3, there can be greater shedding of the
tissue gal-3
through greater gradient difference, resulting in reduced inflammation,
fibrosis and
remodeling in the tissue level.
B. Reduction of secondary pro-inflammatory cytokines such as IL-6, TNF alpha,
TNF
kappa beta, and others.
C. It can allow a greater efficacy of circulating various gal-3 blockers in
general, and
specifically modified citrus pectin (MCP) and polyuronides under 40K Dalton.
D. It can increase the efficacy of other therapies that are inhibited by
excessive
circulating gal-3.
22
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
[0039] In an alternative embodiment, the gal-3 comprising plasma may be
run past a solid
phase of immobilized gal-3 binding agents. MCP is one example and gal-3
specific
antibodies, bound to a column or tube, are another. In the preferred
embodiments, these two
approaches to removal of gal-3 from circulation are combined. They can be
combined in
either order, but running the plasma past an immobile phase, followed by
combining the
plasma with an easily removable binding agent is preferred. Alternately the
binding of an
antagonist to gal-3 may be adequate to inactivate the molecule, and thus can
be returned to
the body without the step to remove it from the plasma.
[0040] The binding of gal-3 by a plasmapheresis element that will remove
it from
circulation is an event that will aid medical conditions over a wide variety
of indications. On
a broad scale, the indications are principally associated with inhibiting
tumor growth and
transformation (cancer), inflammation and fibrosis and enhancing innate immune
capacity.
These are implicated in specific indications such as, heart disease, kidney
damage, liver
damage, hepatic and renal disease, bladder disease, thyroid disease, pulmonary
disease,
gastrointestinal disease, immune response, stroke, persistent acute
inflammation due to non-
degradable pathogens, persistent foreign bodies, autoimmune reactions,
hypersensitivities and
allergies, pesticides, environmental toxins, and heavy metals, as well as
heterogenic
conditions such as radiation (examples being medical procedures such as
various radiation
therapies, exposure to ionizing radiation, nuclear radiation, cosmic
radiation, electromagnetic
radiation), chemotherapy damage, and post radiation and chemotherapy induced
inflammation and fibrosis, post-surgery rise in inflammation, acute traumas
such as accidents,
GVHD, and others.
[0041] Elevated circulating gal-3 can change a localized situation, such
as localized
inflammation or fibrosis, and convert it into a larger, systemic problem.
Thus, when gal-3
binds to components in the blood, which also bind toxic agents and the like,
or similarly,
23
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
when localized toxins are bound by gal-3, the damage potentially caused by
these agents
proximate to a localized injury or diseased tissue can become systemic. The
same
phenomenon is observed in connection with potentially metastatic cancer. Gal-3
is a
generally adhesive molecule. Elevated gal-3 levels will accelerate the spread
of cancer from
a localized tumor to a system wide, multi-organ problem. Reducing elevated gal-
3 levels
below 15 or 12 ng/ml, by ten percent (10%) or more, will help to localize
injury and damage,
and maximize the benefit of unrelated therapeutic agents at the local injury
or disease site.
[0042] As noted above, elevated 01-3 levels are associated with growth,
transformation,
angiogenesis and metastatic migration of cancer cells across a wide variety of
cancers,
including liver cancer, kidney cancer, breast cancer, prostate cancer, colon
cancer, thyroid
cancer, cancer of the gallbladder, nasopharyngeal cancer, lymphocytic
leukemia, lung cancer,
melanoma, multiple mycloma, glioblastoma multiformc, uterine cancer, ovarian
cancer,
cervical cancer, and brain cancer among others, as well as reducing
sensitivity in these
cancers to conventional antineoplastic agents.
[0043] Elevated gal-3 levels are also associated with the development and
extension of
fibroses beyond normal and healthy levels, in situations associated with
cardiovascular
disease and heart failure, in tissue injury including brain, lungs, renal,
hepatic, heart and
gastroenterological situations as well as tissue damage due to radiation and
chemotherapy
exposure.
[0044] Above-normal gal-3 levels are also encountered in connection with
inflammation.
This can be disease or trauma associated inflammation, as well as persistent
acute
inflammation due to non-degradable pathogens, persistent foreign bodies, or
autoimmune
reactions, hypersensitivities and allergies, ionizing radiation, nuclear
radiation and
inflammation that may be associated with disease or organ failure modes,
including diabetes
(1 and II), heart disease and dysfunction, atherosclerosis, asthma (bronchial
inflammation),
24
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
gastric and duodenal ulcers, intestinal inflammation in the bowels
(inflammatory bowel
diseases), hepatic inflammation associated with both alcohol and non-alcohol
related
cirrhosis and inflammation, liver infections such as viral hepatitis, among
others. Other
indications associated with inflammation and susceptible to treatment by
plasmapheretic
treatment to reduce gal-3 levels include a variety of parasite-induced
conditions, such as
trypanosomiasis, cerebral malaria, and inflammation and resistance to various
infections
including Paracoccidiosis brasilensis (fungal infection), schistosomiasis,
granulatomatous
bronchopneumonia, Lyme disease, tubercolosis, etc. Reports of elevated gal-3
levels in
connection with infection include Candida albicans, Reales-Calderon et al, J.
Proteomics,
3:75(15) 4734-46 (2012), Schistoma mansoni (a parasitic infection) Brand et
al, Histol.
Histopathol., 27(8) 1109-20 (2012) and many others, including bacterial
infections like
Neisseria meningitides, Quattroni et al, Cell Microbiol., (Jul. 2012). Prion
infection, in CNS
disease, has also been linked to gal-3 elevated levels. Mok et al, Biochem.
Bophys. Res.
Commun. 3:359:672-8 (2007). Elevated gal-3 levels are an important
contributing factor in
inflammation associated with arthritis, multiple sclerosis, Parkinson's, other
neurological
ailments; and other diseases of the skeletomuscular and skin systems,
including inflammation
and fibrosis related conditions such as psoriasis and aging of the skin. See,
for instance,
Ezzat et al, Int. J Rheum. Dis., 14(4):345-52 (2011) (arthritis), Gal et al,
Acta. Histochem.
Cytochem., 44(5):191-9 (2011) and Liu et al, Invest. Dermatol., 10.1038 (2012)
(wound
healing) and Larsen et al, Dermatol. Sci., 64(2):85-91 (2011) (skin diseases).
As noted
above, these conditions may be treated by removal of biologically active,
unbound gal-3 from
circulation by this invention alone, or by removal from circulation combined
with
administration of gal-3 binding agents such as MCP to further address gal-3
mediated
conditions.
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
[0045] Gal-3 has been shown in multiple studies to contribute to the
ability of tumors to
evade the immune system. This can occur via multiple mechanisms: Tumor cellsõ
secrete
gal-3 into the tumor micro environment where the gal-3 via its self-adherent
properties has a
cloaking effect on cancer cells, effectively preventing immune cells from
interaction with the
antigens present on the surface of tumor cells. These immune cells, including
CD4 and CD8
tumor infiltrating T lymphocytes, as well as macrophages, are thus suppressed
in their
capacity to be activated by tumor cell surface antigens; Immune suppression
can occur
because gal-3 also binds directly to immune cells (lymphocytes and
macrophages) or traps
and effectively immobilizes them in a glycoprotein matrix in the tumor
microenvironment.
These immune cells are found in the tumor microenvironment in response to
antigens that are
present on the surface of tumor cells. These immune cells, when properly
stimulated, secrete
a number of factors, such as cytokines, which are cytotoxic to tumor cells. An
elevated gal-3
level in the tumor microenvironment disables their ability to activate in
response to antigens
present on tumor cell surface. Demotte N. et al, Cancer Res. 70(19):7476-88
(2010); van der
Bruggen P., Bull Mem Acad R Med Belg. 164(5-6):183-91 (2009). Similar process
allows for
different infectious agents to evade the immune system, and "hide" from
different therapies
such as antibiotic therapy. This phenomenon is referred to, in popular terms
as Biofilm, and
gal-3 is an integral part of the biofilm. This may be of specific importance
in chronic
infections such as Lyme, Bartobela, Babisia, rickettsia, and other co
infections, as well as
different parasitic and fungal infections.
100461 Activation of the immune system by reduction of gal-3 is a further
aspect of the
invention. Inhibition of gal-3 has been found to enhance the proliferation of
immune cells in
response to antigens presented on tumor cells. Demotte N, et al, Immunity.
28(3):414-24
(2008). Rapid systemic removal of gal-3 could be used in conjunction with oral
gal-3
antagonists to exponentially enhance the effect of freeing the immune system
to effectively
26
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
act on tumor cells. This enhanced response via systemic removal of gal-3 can
be used in
conjunction with a number of immune enhancing therapies currently available or
being
developed.
[0047] Galectins have been documented as main regulators of immune cell
homeostasis
and inflammatory processes. Among these, gal-3 with its anti-apoptotic
activity has been
reported that increased gal-3 expression correlates with defective T-cell
apoptosis in patients
with some immune disorders. The level of gal-3 in patients receiving
allogeneic
hematopoietic stem transplantation (HSCT) in the context of the presence of
acute graft-
versus-host disease (GVHD) has been evaluated. The findings showed the level
of gal-3 was
higher in patients with acute GVHD than those without after HSCT, and also
higher after
transplantation than before or at transplantation day in these patients. These
findings suggests
that the gal-3 might be one of significant molecules in pathogenesis of acute
GVHD, and the
successive evaluation of gal-3 levels might be one of informative tests
predicting the
occurrence of acute GVHD. Min YH et al, Increased level of gal-3 in patients
with acute
graft-versus-host disease after allogeneic haematopoietic stem cell
transplantation. 31st
Annual Meeting of the European Group for Blood and Marrow Transplantation 21st
Meeting
of the EBMT Nurses Group 4th Meeting of the EBMT Data Management Group,
Prague, CZ
(2005).
[0048] Multiple studies have contributed to the understanding of the
immunosuppressive
mechanisms used by mesenchymal stromal cells (MSC). Galectins have recently
been
discovered as a main regulator of MSC immunosuppressive function. It has been
identified
that gal-3 as the first human lectin involved in the modulation of the
immunosuppressive
potential of mesenchymal stem cells (MSC). The double knockdown of galectins-1
and -3
genes have been shown to almost abolish the immunosuppressive capacity of MSC.
The use
of a competitive inhibitor for galectin binding restored alloresponsiveness,
implying an
27
CA 02893690 2015-06-03
WO 2014/106803
PCT/1112013/061375
extracellular mechanism of action of galectins. The published data demonstrate
the
involvement of secreted gal-1 and -3 in MSC-mediated T cell suppression. The
immunosuppression by MSC-secreted galectins should facilitate further
understanding of the
inflammatory reactions such as those seen in GVHD and autoimmune disorders.
Sioud M et
al, Int J Oncol. 38(2):385-90. (2011). In particular, allogenic transplants
frequently give rise
to issues involving inflammatory disorders that ae mediated by Gal-3. Gal-1
and gal-3 are
constitutively expressed and secreted by human bone marrow MSC. Inhibition of
gal-1 and
gal-3 gene expression has cancelled the suppressive effect of MSC on
allogeneic T cells.
This increase in the understanding of MSC suppressor mechanisms offers an
insight into the
use of these cells in human therapy such as the treatment of GVHD, a severe
complication
after haematopoietic stem cell transp"antation. Sioud M. Scand J Immunol.
73(2):79-84.
(2011). Thus, as in other areas and applications, this invention lends itself
to improving the
effectiveness of other therapies by reducing gal-1 and gal-3 mediated
reactions that would
otherwise interfere with the effectiveness, in this case, of transplant
therapies.
[0049] Inflammation mediated at least in part by circulating gal-3 levels
also plays a role
in organic psychiatric and brain disorders. This kind of inflammation has been
associated
with a wide variety of conditions, such as schizophrenia. Muller et al., Adv.
Protein Chem
Struct BioL , 88, 49-68 (2012) and Palladino et al, J. Neuroinflammation,
22;9, 206 (2012).
Thus, reducing elevated gal-3 levels may be one method to assist in the
control of psychiatric
disorders of this type which are difficult to control by therapeutic
intervention alone.
Similarly, a condition receiving increasing attention, attention deficit
hyperactivity disorder
(ADHD) has been shown to be mediated to some degree by gal-3 expression. Wu et
al, Brain
Pathol., 20(6), 1042-54 (2010). Elevated gal-3 expression levels, and the
inflammation
associated therewith, have also been linked to organic tissue damage, as well
as psychiatric
behavioral disorders. Thus, Alzheimer's disease and enhanced Ap, amyloid
deposits have
28
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
been shown to be associated with pro-inflammatory conditions, such as those
mediated by
elevated gal-3 levels. Reale, et al, Curr. Alzheimer Res. 9(4), 447-57 (2012).
Gal-3 has also
been shown to be involved in the proper differentiation of oligodendrocytes
controlling
myelin sheath conditions, Pasquin et al, Cell Death Differ., 18(11), 1746-56
(2012) and
recovery and regrowth following traumatic brain injury. Venkatesan et al,
Neuroinflammation 27(7) 32 (2010). Thus, in addition to being of importance in
the control
of inflammation in disease or injury conditions generally, reduction of
circulating gal-3 levels
through plasmapheresis may be of critical value in controlling for physical
phenomena
associated with disorders of the brain and central nervous system.
[0050] It should be noted that commonly, inflammation and fibrosis can be
induced by
deliberate treatment, not just trauma or disease condition. The removal of
circulating,
unbound gal-3 through this invention can be effective in reducing or
preventing organ
damage induced by chemotherapy and other pharmaceuticals. Some examples
include
bleomycin, which induces lung fibroses, and a wide variety of cardiac drugs
such as
amiodarone. Adriamycin and doxorubicin are widely prescribed and present
cardiac
inflammation and fibroses issues. Bacillus Calmette-Guerin washes to treat
bladder cancer
induce systemic inflammation and cyclophosphamide also induces bladder damage.
Cyclosporine, a widely used immunosuppressant drug, and the active agent in
Restasis'TM,
induces kidney toxicity and inflammation. Studies indicate that the vast array
of organ
damage caused by prescribed pharmaceuticals is mediated, at least in part, by
elevated gal-3
levels, and can be limited if not eliminated by the method of this invention.
100511 Among other specific indications that can be addressed by this
invention are non-
alcoholic steatohepatitis (NASH); Sepsis: (See Bibhuti et al, 2013); post-
trauma nerve
regeneration (Narcisco et al, 2009) (Doverhag et al, 2010; delayed xenograft
rejection
(DXR) Jin, 2006; Chronic allograft injury kidney transplant (Dang et al,
2012); Ischemic-
29
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
reperfusion injury: (IRI) (Fernandes et al, 2008); Ideopathic pulmonary
fibrosis (IPF);
GVHD, and related indications.
[0052] The invention disclosed herein, the use of at least partial donor-
provided apheresis
to reduce active Gal-3 levels in a patient may find particular application in
the treatment of
chronic kidney infection and end stage renal disease. Currently, there are
approximately 26
million adults with CKD and 800,000 individuals with Stage 4 CKD and the
number of these
patients is expected to double by 2020. Patients with Stage 4 CKD have a ¨10%
death rate
and the first-year death rate for ESRD patients is ¨25%.
[0053] In a preferred embodiment, the serum, after having circulating gal-
3 reduced or
removed, as described, is further treated before returning it to the patient's
blood stream.
Specifically, agents that may be more effective in the absence of, or in the
presence of
reduced levels of, galectin-3 are specifically added. This includes a wide
variety of active
agents, but specifically includes agents such as chemotherapeutic drugs,
immune enhancing
therapies, antibiotics and anti microbials in general (anti virals, anti
helmetic, anti parasitic)
and therapeutic agents for the various conditions. For example, an anti-
inflammatory will
work better, cardiac medications, any drugs delivered to address an issue
where gal-3 is a
contributing factor, or prevents effective delivery to the target tissue, will
be enhanced by this
process. These agents will then have the opportunity to work under an
environment of lower
levels of gal-3. Even if just fora few hours, they can exhibit full biological
activity. Once
inflammation, for example, is reduced, naturally less gal-3 is being produced
and expressed
by the target tissue resulting in lower circulating gal-3 on a long term
basis.
[0054] Thus, while in one alternative, the invention involves long term
or chronic
plasmapheresis to maintain reduced gal-3 levels, the invention also
contemplates intervention
on a short term basis, both removing circulating gal-3 and providing agents
otherwise
inhibited by gal-3, to swiftly address inflammation in particular. Gal-3
levels can spike as a
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
transient event, in response to trauma for example, having a technique to
rapidly lower gal-3
levels in the patient, coupled with administration of active agents that are
ordinarily inhibited
to some degree by high levels of gal-3, can offer a lifesaving technique. In
addition, reducing
Gal-3 levels as a means to reduce inflammation can allow other therapeutic
agents, an
example being an anti microbial in acute infections such as sepsis, or others,
to have a better
therapeutic response, resulting in a life saving outcome.
[0055] Although Applicant does not wish to be confined to these few
examples, a large
number of conditions have been shown to be mediated by unbound gal-3, such
that its
removal, by the invention addressed herein, will aid in treatment. It has been
demonstrated
that reducing free gal-3 in humans can prevent renal fibrosis and inflammation
following
kidney injury. Both thyroid cancer and lung cancer treatment has been
demonstrated to
effectively improve by reducing gal-3 concentrations. Enhanced sensitivity to
both radiation
and chemotherapeutic intervention may be achieved by reducing circulating
levels of active
gal-3 through this invention.
[0056] Asthma, and related conditions primarily marked by exaggerated
inflammation
may be avoided or suppressed by removing circulating gal-3 through the process
of this
invention. These include inflammation of the gastrointestinal tract, and
inflammation and
the development of fibroses of the liver, interstitial cystitis, inflammation
associated with
brain and cognitive function, and others. Inflammation associated with
parasite invasion may
also be controlled by removal of gal-3, or reducing its circulating level
through this invention.
Other inflammation-associated diseases, such as diabetes and arthritis are
similarly treated.
These conditions may ideally be targets of this invention as well as
administration of
circulating gal-3 binding agents like MCP, and unrelated therapeutic agents.
[0057] While the present invention has been disclosed both generically,
and with
reference to specific alternatives, those alternatives are not intended to be
limiting unless
31
CA 02893690 2015-06-03
WO 2014/106803
PCT/1B2013/061375
reflected in the claims set forth below. The invention is limited only by the
provisions of the
claims, and their equivalents, as would be recognized by one of skill in the
art to which this
application is directed.
32
