Note: Descriptions are shown in the official language in which they were submitted.
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s CHEMICALLY-STABILIZED CHLORITE SOLUTIONS FOR TREATING
CANCER AND OTHER DISEASES
The present invention relates to stabilized chlorite matrices as
immunomodulatory agents and anti-cancer agents. The stabilized chlorite
> o matrix inhibits can affect the activation of macrophages in a manner
similar
to interferon gamma but without affecting the release of cytokines that
cause side effects, such as tumor necrosis alpha. The stabilized chlorite
matrix also upregulates the expression of the DCC protein in macrophages,
a protein whose expression is related to neoplastic transformation. By
is activating macrophages, the stabilized chlorite matrix therefore is useful
as
an immunomodulatory agent and in treating cancer.
A feature common to an immune response is the recognition of an
antigen (either foreign or self, but perceived as foreign), and subsequent
processing by the immune system. Typically, antigen is degraded by
2o enzymes in the cytoplasm, endoplasmic reticulum (ER) and lysosomes of
cells, (usually macrophages, dendritic cells and other antigen presenting
cells (APCs)), or in serum. The degraded antigen is presented on the
surface of the APC by MHC class I or II molecules. This presentation of the
antigenic epitope by the MHC molecule, and subsequent binding to the T
2s cell receptor (TCR) of a T cell is known as antigen presentation. Rodgers,
,).R., et al., CLINICAL IMMUNOLOGY, PRINCIPLES AND PRACTICE (RICH):
"Antigens and antigen presentation," Chpt. 7, pp 114-131, Mosby, St. Louis,
MO (1996).
Modulation of the immune response has been achieved by treatment
3o with any of a variety of cytokines or lymphokines. In particular,
interferons
(IFNs) are type of cytokine that plays a complex and central role in the
resistance of mammalian hosts to pathogens and has been found useful in
cancer therapy. Type I interferon (IFN-a and IFN-(3) is secreted by virus
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infected cells, while type II interferon (IFN-x) is secreted by thymus-derived
cells under conditions of activation and by natural killer cells (NK cells).
Among others, IFN-x has an ability to inhibit cell proliferation at much
lower concentrations comparing with IFN-a, and IFN-~. (Rubin, B. Y. et al.
s (1980): Proc. Natl. Acad. Sci. U.S.A., 77, 5928), and also to activate
cells,
including natural killer cell, killer T-cell, K-cell and macrophage, which
have
cancer therapeutic effects J. L. Crane et al., J. Natl. Cancer Inst., 61, 871-
874, 1978; J. E. Blalock et al., Cell Immunol., 49, 390-394, 1980 (reporting
that the antitumor activity of gamma-interferon is 20 to 100 times as high as
to those of alpha- and beta-interferons). U.S. Patent No. 5,268,169 describes
the use of IFN-x for treating ovarian carcinoma. The use of purified
recombinant interferon in cancer therapy and in modulating the immune
response has been of limited success because of the high costs of
producing recombinant material and because IFN-x causes side effects
is which occur from the stimulation of inflammatory and shock related
cytokines such as TNF-a. Furthermore, administration of protein
compounds such as cytokines and elicit a detrimental immune response to
the administered protein.
Aqueous solutions of a chemically stabilized chlorite matrix that are
2o capable of intravenous administration are known. Other chlorine-containing
solutions also are known to have reported medicinal uses. For example,
United States Patent No. 5,019,402 discloses a solution containing chlorine
dioxide or a chlorine dioxide-liberating mixture of a chlorite, a weakly
acidic
buffer and a heat-activated saccharide which can be used for the
2s sterilization of stored blood components with the exception of those which
contain red blood corpuscles, i.e., of leukocytes, blood platelets,
coagulation factors and globulins. In whole blood, a corresponding
disinfecting action does not occur, presumably because the red blood
corpuscles are attacked more quickly by the chlorine dioxide than the
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targeted micro-organisms. Therefore, this agent also is not suitable for a
parenteral administration.
DE-OS 32 13 389, United States Patent No. 4,507,285 and United
States Patent No. 4,296,103, describe chemically-stabilized chlorite
s matrices which are suitable for an external or oral therapeutic use. Besides
various bacterial infections, the external treatment of virus infections, such
as herpes simplex and herpes zoster, may be possible in this manner, but
the documents do not report the use of these chlorite matrices for
intravenous administration for inhibiting an antigen-specific immune
to response.
European Patent EP 0 200 157 and United States Patent No.
4,725,437 further describe solutions of a chemically-stabilized chlorite
matrix for intravenous and perioperative administration. The agent has
proved to be effective in the treatment of Candida albicans infections. From
is EP 0 200 157, it is known to use such stabilized chlorite matrices for
intravenous and/or local administration in cases of infectious conditions
brought about by parasites, fungi, bacteria, viruses and/or mycoplasts. The
action is explained by a phagocyte stimulation which is achieved by a single
effective administration of the chlorite complex shortly after the infection.
2o PCT publication WO 99/17787, which is hereby incorporated by reference
in its entirety, discloses chemically-stabilized chrlorite solutions for
inhibiting
antigen-specific immune responses.
Providing an approach to cancer therapy that is similar to that
achieved by interferon gamma but yet avoids the production of toxic
2s substances such as TNF alpha is needed. Thus, there exists a need to
develop a method of treating cancer and other malignancies using a
substance that has the macrophage activating properties of IFN-x while
lacking the inflammatory effects and toxic shock effects of this molecule
such as occur by the activation of the expression of TNF-a. It is therefore
3o an object of the invention to provide a method of treating cancer by
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administration of an inorganic compound that has immunomodulatory
properties. It is also an object of the invention to provide a method of
treating cancer by activating macrophages from an individual with cancer ex
vivo with a non-protein IFN-x and then administering the activated cells into
s the individual.
In accordance with these and other objects of the invention, there is
provided a method of treating cancer and other malignancies in a animal,
such as a mammal, comprising administering an effective amount of an
aqueous solution comprising a stabilized chlorite matrix. The method
to activates inter alia macrophages resulting in upregulation of the
expression
deleted in colon carcinoma (DCC) protein. An effective amount is 5 to 100
mMol of CIOz per liter of solution.
In one embodiment of the present invention, the cancers for which
the method is used are those cancers characterized by a reduced
is expression of the DCC protein.
In another embodiment of the present invention, the cancer cells to
be treated are selected from the group consisting of colon carcinoma,
gastric carcinoma, esophageal carcinoma, rectal carcinoma, pancreatic
carcinoma, prostate carcinoma, glioma and neuroblastoma.
2o In accordance with an additional object of the present invention,
there is provided methods of treating cancer or other malignancies in an
animal, such as a mammal, wherein the methods comprise (a) removing
macrophages from the mammal; (b) contacting the macrophages with an
effective amount of an aqueous solution comprising a stabilized chlorite
2s matrix under conditions sufficient to increase the expression of DCC on the
macrophages; and (c) introducing the macrophages from step (b) back into
the mammal. An effective amount is 5 to 100 mMol of CIO2 per liter of
solution.
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In one embodiment of the present invention, the cancers for which
the method is used are those cancers characterized by a reduced
expression of the DCC protein.
In another embodiment of the present invention, the cancer cells to
s be treated are selected from the group consisting of colon carcinoma,
gastric carcinoma, esophageal carcinoma, rectal carcinoma, pancreatic
carcinoma, prostate carcinoma, glioma and neuroblastoma.
Further objects, features and advantages of the present invention will
become apparent from the detailed description of preferred embodiments
to that follow.
The chlorite matrix solutions of the present invention can be dosed in
vivo corresponding to the body weight, whereby, because of the continuous
breakdown of the active material in the blood, the agent must be
is administered again at regular intervals. Those skilled in the art are
capable
of varying the concentrations of antigen-presentation-inhibiting solutions
depending on available in vitro data and body weight. Thus, throughout the
specification and claims, the phrase "an effective amount" will be known by
those skilled in the art to mean an amount of solution which, when
2o administered in vivo to subjects of varying weight, will bring about
modulation of the immune response, and consequently, activation of
macrophages and other immune cells. Typically, an inhibition effective
amount of the chlorite matrix solution will vary between about 0.1 ml/kg to
about 1.5 ml/kg, preferably, about 0.5 ml/kg of body weight and at a
2s concentration of about 40 to about 80 mMol CI02 per liter, preferably about
60 mMol CIOZ per liter, respectively.
Preferably, the chlorite matrix solution of the invention is
administered once daily for anywhere from about three to seven days,
preferably five days, followed by a period of rest of from 10 to 20 days,
3o preferably from 14-18 days, and more preferably, 16 days, to constitute one
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cycle of treatment. Preferably, patients are treated with more than one
cycle, more preferably, at least three cycles, and most preferably, at least
five cycles.
An alternative treatment regimen consists of intravenously
s administering the chlorite matrix solution of the invention once daily for a
period of five days, followed by two days of rest (i.e., over the weekend),
followed by five more consecutive days of administration, followed by a
period of rest from anywhere between 1 and 4 weeks to constitute one
cycle. Preferably, patients are treated with more than one cycle, more
to preferably more than three. Skilled artisans are capable of modifying the
administration of the stabilized chlorite matrix of the invention depending on
the disease treated and the size of the patient, using the guidelines
provided herein.
The use of an aqueous solution containing a stabilized chlorite matrix
is for treating wounds and infections was known in the art. United States
Patent Nos. 4,507,285 and 4,725,437 to Kiahne, the disclosures of which is
incorporated by reference herein in their entirety, and EP 0 200 157 to
Kuhne, the disclosure of which also is incorporated by reference herein in
its entirety, describe the use of a stabilized chlorite matrix solution in
2o stimulating the wound healing response in humans, as well as treating
infections caused by parasites, fungi, bacteria, viruses and/or mycoplasma.
Kuhne et al., European Patent No. 200,156, the disclosure of which also is
incorporated by reference herein in its entirety, describes the use of a
stabilized chlorite matrix solution in conjunction with radiation therapy to
aid
2s in repairing damaged irradiated tissue and reducing side effects. The mode
of action in treating damaged andlor infected tissue is described as
amplifying the "oxidative burst" response of phagocytes in the presence of
bioactivators, e.g., heme compounds. Wound healing and treating the
reported infections can be effected in general by activating macrophages
3o present in the body which in turn serve to activate fibroblast cells which
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stimulate _ the wound healing response. The stabilized chlorite matrix
solutions can activate macrophages by complexing with the heme moieties
present in the macrophage membrane. Upon activation, the macrophages
stimulate the fibroblast cells which in turn generate collagen and endothelial
s cells that are useful in repairing damaged tissue caused by the wound or by
the infections. While not intending on being bound by any theory, the
present inventors believe that a macrophage is stimulated by the stabilized
chlorite matrix solution by the following sequence of events. The formula for
the stabilized chlorite matrix can be summarized as (CIOZ )~ x O2, where n is
to between 0.1-0.25, preferably, about 0.21. In the presence of heme
compounds (e.g., hemoglobin, myoglobin, peroxidases, cytochromes, etc.),
which are present in the serum as biological molecules, and they are part of
the cell membrane of phagocytic cells like macrophages, the stabilized
chlorite matrix becomes a secondary oxidant with oxidative properties
is different from chlorite and hydrogen peroxide. Indeed, the stabilized
chlorite
matrix of the invention has shown pharmacological differences when
compared to equimolar chlorite solutions. (Ivankovic et al., 1993).
Macrophage activation brought about by the chlorite matrix occurs
via a mechanism that is different from that of other macrophage-activating
2o agents such as PMA, TNF-a, etc. Macrophages can be activated by a
stabilized chlorite matrix because the matrix acts as an oxidant in a drug-
membrane interaction which is believed to cause an electron deficiency (as
a result of electron-reduction +3C102 + 4e --~ -'CI-) on the surface of the
cell.
The membrane of the macrophage now has a charged surface. The
2s present inventors believe that this charged surface may have an influence
on surrounding particles (e.g., invading bacteria) and/or may trigger a signal
sequence into the cell. The activated macrophage then increases the DNA-
synthesis of fibroblasts which is believed to contribute to its wound healing
and/or parasite, bacteria, etc. infection healing properties.
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The present inventors believe further that the known wound-healing
mechanism via macrophage activation of the chlorite matrix of the invention
also stimulates and enhances the phagocytic activity of the macrophage.
Thus, the activated macrophage is primed to ingest, digest and dispose of
s foreign antigens. The use of the claimed stabilized chlorite matrix to
render
macrophage phagocytic also was known and described in EP 0 200 157 to
Kiahne.
What was not known, however, was that a stabilized chlorite matrix
also can inhibit an antigen-specific immune response, while at the same
io time enhance the activity of phagocytes. While not intending to be bound
by any theory, the present inventors believe that the stabilized chlorite
matrix, when administered to a mammal in need thereof, partially or
completely impedes the antigen presentation of antigen presenting cells
(APCs). Throughout this description, the expression, "antigen presenting
is cells" denotes a cell that is capable of presenting an antigen and
eliciting an
immune response. Useful antigen presenting cells include macrophages
and dendritic cells. That administration of WF-10 prevents and/or impedes
antigen presentation has been confirmed by in vitro data described in the
examples, and is consistent with in vivo data also described in the
2o examples.
A typical immune response involves stimulating a macrophage, the
stimulated macrophages present MHC Class I and II antigens on surface,
which, when coupled with the T cell receptor, will stimulate T cells
(typically
a T cell subset such as CD4 or CD8 cells, and the like) to proliferate and
2s form cytotoxic T-lymphocytes (CTL) cells which in turn kill cells
expressing
the antigen. After antigen presentation and upon coupling with the T cell
receptors, the stimulated APC (macrophage and the like) also secretes
various cytokines that can aid in the proliferation of CTLs. Cytokines, or
growth factors, are hormone-like peptides produced by diverse cells and are
3o capable of modulating the proliferation, maturation and functional
activation
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of particular cell types. Herein, cytokines refer to a diverse array of growth
factors, such as hematopoietic cell growth factors (e.g., erythropoietin,
colony stimulating factors and interleukins), nervous system growth factors
(e.g., glial growth factor and nerve growth factor), mostly mesenchymal
s growth factors (e.g., epidermal growth factor), platelet-derived growth
factor, and fibroblast growth factor I, II and III, including interferons.
It will be appreciated that there may be several cytokines that are
involved in inducing cell differentiation and maturation, and that cytokines
may have other biological functions. In the case of IL-1, there may be
to several forms, such as IL-1-alpha and IL-1-beta, which nevertheless appear
to have a similar spectrum of biological activity. Those cytokines that are
primarily associated with induction of cell differentiation and maturation of
myeloid and possibly other hematopoietic cells include, inter alia, IL-1,
G-CSF, M-CSF, GM-CSF, Multi-CSF (IL-3), and IL-2( T-cell growth factor,
is TCGF). IL-1 appears to have its effect mostly on myeloid cells, IL-2
affects
mostly T-cells, IL-3 affects multiple precursor , G-CSF affects mostly
granulocytes and myeloid cells, M-CSF affects mostly macrophage cells,
GM-CSF affects both granulocytes and macrophage. Other growth factors
affect immature platelet (thrombocyte) cells, erythroid cells, and the like.
2o When an antigen is presented to a patient with a normal, or
uncompromised, immune system, the following sequence of events typically
takes place. The antigen (or foreign body) is enclosed in vesicles in the
macrophage which serves as a type of garbage disposal to break or digest
the foreign matter down into smaller peptides. An MHC class II molecule
2s will transport one of the smaller antigenic peptides to the surface of the
macrophage and present it to a T cell receptor (TCR). Binding with the cell
receptor will trigger the release of activating factors and cytokines such as
IL-1, TNF, etc., which restores the self-defense of the macrophage and
enhances the intracellular killing of the foreign body. If binding does not
30 occur, the activating factors will not be released and the macrophage will
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not know to digest or break the foreign matter down into smaller peptides.
As it is used in this description, the expression "antigen presentation"
therefore denotes the process of presentation of an MHC Class II the
surface of an APC followed by subsequent binding with a TCR.
s The present inventors believe that binding of the T-cell to the
presented antigen is effected not only by recognition of the antigenic
peptide that is presented, but also by the presence of the CD28 antigen on
the surface of the T cell and its binding with B7. Binding of B7 to CD28 is
believed to costimulate T cell proliferation, cytokine stimulation, cytokine
to production and proliferation of cytotoxic T cells (CTL). Rudd, C.E.,
"Upstream-Downstream: CD28 Cosignaling Pathways and T Cell Function,"
Immunity, : 527-34 (1996); Fagnoni, F.F., et al., "Role of B70/B7-2 in CD4+
T-Cell Immune Response Induced by Dendritic Cells," Immunology, : 467-
74 (1995). While not intending on being bound by any theory, the inventors
is believe that one of the mechanisms by which antigen presentation can be
prevented is by preventing binding of B7 and CD28. This can take place by
either preventing presentation of B7, or by increasing the quantity of a
specific subset of T cells -, such as (CD3+, CD8+, CD28~). While there is
evidence showing an increase in CD28- T-cell subset for CD8+ cells in vivo,
2o it is believed that administration of the stabilized chlorite matrix of the
invention also will produce an increase in the CD28- T-cell subset for CD4+
T-cells. Thus, the inventors believe that one of the mechanisms by which
administration of a stabilized chlorite matrix operates to prevent and/or
inhibit antigen presentation is by preventing binding of the antigen
2s presented by the APC to the TCR, and hence, preventing secretion of
cytokines and other activating factors, and preventing proliferation of T
cells.
In contrast to the normal immune response, if no B7 is present, or
the T cell is a subset including CD28~, then the present inventors believe
that an anergic response will ensue. In this context, when the APC
3o presents the MHC Class II antigen, the T cell will not proliferate, nor
will the
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APC release cytokines and other activating factors, even though the TCR
on the T cell recognizes the antigen. While not intending on being bound by
any theory, the present inventors believe that administration of an aqueous
solution of a stabilized chlorite matrix results in an increase in
concentration
s of CD28- T cell subsets and/or a decrease in B7 presentation in APCs. As a
consequence, there is no B7-CD28 interaction between the APC and the
TCR, and therefore no T cell proliferation or cytokine release.
Previously known therapies for preventing T cell proliferation typically
acted on cytotoxic T-cells after cytokine stimulation. For example,
to cyclosporin A is believed to act on the cytotoxic T-Lymphocyte to prevent T-
cell proliferation. At this point, however, the APC already has released
cytokines that might assist CTL proliferation. Accordingly, a significant
amount of these drugs must be administered to prevent the CTL
proliferation. There are no known methods for impeding an immune
is response, however, where the APC or TCR are affected in a manner that
partially or completely interrupts the antigen presentation interaction
between the APC and the T cell.
Patients suffering from autoimmune diseases and diseases caused
by inappropriate immune response such as myasthenia gravis, systemic
20 lupus erythematosis, serum disease, type I diabetes, rheumatoid arthritis,
juvenile rheumatoid arthritis, rheumatic fever, Sjorgen syndrome, systemic
sclerosis, spondylarthropathies, Lyme disease, sarcoidosis, autoimmune
hemolysis, autoimmune hepatitis, autoimmune neutropenia, autoimmune
polyglandular disease, autoimmune thyroid disease, multiple sclerosis,
2s inflammatory bowel disease, colitis, Crohn's disease, chronic fatigue
syndrome, and the like, do so because the immune response is
inappropriate. That is, the patient's body is producing too many CTLs, or
other cytokines which turn against the body's own healthy cells and destroy
them. In transplant or graft patients, an inappropriate immune response
30 occurs because the immune system recognizes the transplanted organ or
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graft's antigens as foreign, and hence, destroys them. Likewise, transplant
and graft patients can develop a graft vs. host response where the
transplanted organ or graft's immune system recognizes the host's antigen
as foreign and destroys them. This results in other inappropriate immune
s responses include excess inflammation, allergic asthma, allergic rhinitis
and
atopic dermatitis.
Conventional therapies for autoimmune disease and transplant
rejection invoke application of cytotoxic agents, particularly those that
affect
the lymphoid system (and therein particularly the T-lymphocytes), to
to depress host immunity in certain autoimmune diseases, e.g., systemic
lupus erythematosis, and in patients receiving organ transplants. These
cytotoxic drugs are similar to those often used in cancer chemotherapy, with
the attendant myeloid and other hematopoietic side effects. In addition to
these drugs, specific antibodies against these lymphoid cells (particularly
is T-cells), e.g., the anti-Tac monoclonal antibody of Uchiyama et al., J.
Immunol. 126:1393 and 1398 (1981), which specifically binds to the human
IL-2 receptor of activated T-cells, can be conjugated to cytotoxic agents,
such as drugs, toxins or radioisotopes, to effect a relatively select killing
of
these cells involved in organ rejection. For example, a T-cell antibody can
2o be conjugated with a beta- or alpha-emitting radioisotope, and this can be
administered to the patient prior to undertaking organ transplantation and, if
needed, also thereafter. In order to effect a high T-cell killing dose without
the concomitant limiting side effects to the hematopoietic system, the
treatment using the aqueous solution containing a stabilized chlorite matrix
2s can be used instead of, or in conjunction with any of the aforementioned
agents.
Administering an aqueous solution containing a stabilized chlorite
matrix to a mammal can therefore inhibit antigen-specific immune
responses, while at the same time not compromise the immune system
3o entirely because the solution also is effective in enhancing phagocytic
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activity. Thus, the invention encompasses methods of treating auto-
immune diseases, preventing transplant organ or graft rejection and septic
shock as a result thereof, and reducing inappropriate immune responses
such as excessive inflammation and allergic reaction. Because there are
s other methods already known to treat these disorders, skilled artisans are
capable of modifying the known techniques by administering an inhibition
effective amount of an aqueous solution containing a stabilized chlorite
matrix, using the guidelines provided herein. For example, skilled artisans
are capable of designing a treatment regimen to treat any of the
o aforementioned disorders using the stabilized chlorite matrix of the
invention
by varying the dosage amount, frequency of administration, or mode of
administration.
A preferred embodiment of the treatment of this invention entails
administration to a mammal in need thereof, an aqueous solution of a
is product that has become known as "tetrachlorodecaoxygen anion complex,"
commonly abbreviated as 'TCDO," which is a product of Example 1 of U.S.
Patent No. 4,507,285. The product is a water clear liquid, miscible with
alcohols, having a melting point of -3°C. The Raman spectrum shows
bands of 403, 802 (chlorite) and 1562 cni' (activated oxygen).
2o The DCC (deleted in colon carcinoma) gene, which contains more
than one million base pairs, is a cell surface molecule receptor that is
considered to function in tumor suppression. Reduction in the expression of
the DCC gene has been observed in Stage 2 and stage 3 carcinomas,
indicating relationship between loss of expression and survival. DCC is
2s related to the neural cell adhesion molecule. Reduced expression in the
level of DCC has been implicated in poor prognosis in gastrointestinal
cancers by Saito et al., Oncology 56(2):134-41 (1999).
The present invention provides a method of treating cancer in a
mammal comprising administering an aqueous solution comprising a
3o stabilized chlorite matrix. This method involves activation of macrophages
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in a manner similar to that of IFN-x, but with release of lower amounts of
inflammatory and shock cytokines such as TNF-a.. The administration of
the stabilized chlorite matrix results in increased expression of the DCC
protein on macrophages.
s The method of treating cancer with stabilized chlorite matrix is useful
in cancers that are characterized by a reduced expression of the DCC
protein. Such cancers include, for example, colon carcinoma, gastric
carcinoma, esophageal carcinoma, rectal carcinoma, pancreatic
carcinoma, prostate carcinoma, glioma and neuroblastoma.
to The present invention also provides a method of treating cancer in a
mammal comprising: (a) removing macrophages from the mammal; (b)
contacting the macrophages with an effective amount of an aqueous
solution comprising a stabilized chlorite matrix under conditions sufficient
to
increase the expression of DCC on the macrophages; and (c) introducing
is the macrophages from step (b) back into the mammal. In this method the
treated macrophages exhibit and activated by the stabilized chlorite matrix
and increase the expression of the DCC antigen on the macrophages.
Administration of such activated macrophages is useful for treating cancers
characterized by a reduced expression of the DCC protein.
2o The invention now will be explained in detail with reference to the
following examples. In the examples, "WF10" denotes an aqueous solution
of TCDO.
Example 1
2s In this example, and the following examples 2-4, details regarding
the methods used in performing these examples can be found in Fagnoni,
F.F., et al., "Role of B70/B7-CD28 in CD4~ T-Cell Immune Response
Induced by Dendritic Cells," Immunology, : 467-74 (1995), the disclosure of
which is incorporated herein by reference in its entirety. This example,
3o together with the following examples 2-4, elucidate the role of WF10 in
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inducing anergy by preventing dendritic cell-mediated costimulation at the
B7/B70-CD28 interface.
Dendritic cells, T cells and monocytes were obtained in the manner
described in Fagnoni et al. To assess the effects of WF10 on DC-
s dependent T cell activation, freshly isolated CD4+-T cells were activated
with allogeneic MLR in the presence or absence of WF10 to DC. Purified
resting CD4+ T cells (5-10 x104/well) were cultured with irradiated (25 Gy)
allogeneic DC in U-bottomed 96-well plates containing 200 ~I of complete
medium. The cultures were carried out at 37°, 8% C02 in humidified air
for
l0 5 days. Cultures were pulsed with 1 ~Ci [3H]thymidine (6-7 Ci/mm, New
England Nuclear, Boston MA) 19 hour before harvest. The [3H]thymidine
incorporation by proliferating cells was measured in a f3-scintillation
counter.
WF10 was added to DC stimulated alto-MLR DC and incubated at 4° for
about 3 minutes before the addition of CD4' T cells. The CD4+ T cell
is response to DC stimulated allogenic MLR was inhibited in a dose-
dependent manner by WF10. The WF10 was administered by adding
WF10 to culture medium at time 0 in doses of 25 pg/ml or 50 ~g/ml. As the
number of dendritic cells (DC) per well was increased, the number of CPM
+ SE (counts per minute + standard error) remained essentially the same,
2o with the greatest degree of inhibition resulting from WF10/1600. The
expression WF101number denotes that dilution of WF10 and designates the
amount of WF10 per ml of solution. For example, WF10/1600 denotes a
diluted solution of WF10 containing 1 ml of WF10 per 1600 ml of solution.
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Example 2
Example 1 was repeated with the exception that Adherent
monocytes, obtained in accordance Fagnoni et al. were used instead of DC.
s Administration of WF10 was effective in inhibiting proliferation of CD4' T-
cells from monocyte stimulated MLR. Indeed, with administration of
WF1/1600, the stabilized chlorite matrix was effective in completely
inhibiting proliferation of CD4+ T-cells from monocytes stimulated with
allogeneic MLR, despite increased concentration of monocytes per well.
to The results of examples 1 and 2 therefore show that WF10 is
effective in inhibiting proliferation of CD4' T cells from DC or monocytes
stimulated with allogeneic MLR.
Example 3
is
Examples 3 and 4 were carried out to determine the effect of WF10
on the inhibition of antigen-induced proliferation of T cells using various
antigens. In this example, purified resting CD4+ T cells (5-10 x104/well)
were cultured with irradiated (25 Gy) autologous DC in U-bottomed 96-well
2o plates containing 200 ~l of complete medium. The cultures were carried out
at 37°, 8% C02 in humidified air for 6 days. Cultures were pulsed with
1 ~Ci
[3H]thymidine (6-7 Cilmm, New England Nuclear, Boston MA) 19 hour
before harvest. The [3H]thymidine incorporation by proliferating cells was
measured in a t3-scintillation counter.
2s Soluble keyhole limpet hemocyanin (KLH) and tetanus toxoid (TT)
were added to autologous DC. Measurements were taken for no addition of
WF10, addition of WF10/200 and WF10/800 (representing administration of
WF10 to the culture medium at time 0 of 0, 1m1/200 ml of solution and 1
m1/800 ml of solution, respectively) to determine the proliferation of CD4+ T
3o cells when no antigen, TT, KLH25 (25 ~glml) and KLH50 (50 p.glml) were
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presented by DC. The number of proliferated T-cells for samples using no
WF10, and for samples using WF10 show significant proliferation of CD4+ T
cells occurred when DC presented the soluble antigens KLH and TT.
Administration of WF10, however, almost completely inhibited the
s proliferation of CD4'" T cells when either KLH or TT were presented by DC.
Example 4
Example 3 was repeated except that monocytes were used instead
to of DC for antigen presentation. In addition, WF10 was administered in the
following increments WF10/200, WF10/400, WF10/800 and WF10/1600.
The results show tha there was significant proliferation of CD4+ T cells when
monocytes presented the soluble antigens KLH and TT. Administration of
WF10, however, almost completely inhibited the proliferation of CD4' T cells
is when either KLH or TT were presented by monocytes.
The results achieved by administration of an aqueous solution
containing a stabilized chlorite matrix reveal that it is capable of
inhibiting an
antigen-specific immune response. It has previously been reported that
administration of an aqueous solution containing a stabilized chlorite matrix
2o is effective in enhancing phagocytic activity. Thus, it now is possible by
administering only one medicament to inhibit one immune response,
(antigen presentation and proliferation of T cells) while at the same time,
enhance another immune response (phagocytosis).
2s Example 5
A phase 2 trial was conducted at San Francisco General Hospital.
The study enrolled 18 patients in an open label pathogenesis study of WF-
10. Patients received one hour infusions of WF-10 for one week, followed
3o by two weeks of rest. On the third week, the patients again received one
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hour infusions of WF-10 daily for one week followed by two weeks of rest.
Parameters studied included measures of macrophage activation/function
immunologic activation and HIV viral load. RBC hemolysis evaluation
studies included 51 Cr-RBC survival studies compared with changes in
s hemoglobin, haptoglobin and reticulocyte values.
There were no side effects noted in any of the 18 patients. Data on
eight of the patients were gathered and the results are tabulated below.
There appeared to be acute increases in the following parameters as
measured by flow cytometry (FACSCAN as recommended by, for example,
to Becton-Dickinson) in relation to drug administration, changes that
generally
returned close to baseline within 2 weeks of drug administration: CD-4, CD-
8, CD14'/CD69+, CD14+ side scatter, CD20/DR' cells. Several values
seemed to generally increase through the study, showing no clear
downward trend by the end of the study and may represent long-term
is changes induced by WF-10. These include an increase in macrophage
phagocytosis index and an increase in the CD3+ICD8+/CD28- subset of T-
cells.
Potential downward trends were noted in the following categories:
macrophage intracellular TNF-a secretion, and a decrease in the number of
2o circulation CD14'/DR' cells. It has been reported that immune paralysis
results when the number of circulating CD14+/DR+ cells decreases to such
an extent to reach a threshold value. No obvious changes were noted in T-
cell PHA activation values or HIV load as measured by the HIV bDNA assay
(most of the patients had no detectable HIV thoughout the study). Results
2s of the RBC survival studies showed no evidence for hemolysis in response
to the treatment.
Administration of WF10 results in an increase in CD14+/CD69+ cells,
with dramatic increases immediately following infusion. A decrease in
CD14~/TNF secretion after administration of WF10 was seen thereby
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indicating that the stabilized chlorite matrix of the invention is effective
in
decreasing secretion of the tumor necrosis factor cytokine.
Administration of WF10 to patients in vivo results in a steady
increase in the number of CD3+/CD8+, as well as a steady increase in the
s number of CD3+/CD8'/CD28- T cells. The in vitro data above shows
inhibition of antigen presentation using CD4' T cells, and an increase in the
number of circulating CD28- T cells (CD3' T cells). Thus, the inventors
believe that one of the possible mechanisms by which the stabilized chlorite
matrix of the invention inhibits and/or prevents antigen presentation may be
to by blockage of the B7/CD28 interaction between the APC and the TCR.
An increase in phagocytosis index is observed upon administration
of WF10. A decrease in immune function upon administration of WF10 by
virtue of the decrease in CD14+/DR+ cells also is observed. The inventors
therefore believe that the stabilized chlorite matrix of the invention is
is capable of up-regulating phagocytosis, while at the same time, down-
regulating or suppressing the cell-mediated and humoral immune response.
The results tabulated below summarize the data from 15 patients
and show the changes in the measured parameter between the 8th day and
the 47th day of treatment. The 8th day represents the first day of WF10
2o administration because the first 7 days of treatment are devoted to patient
evaluation.
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CD3', CD8+, CD28- 0.027 increase
CD14+, TNF- 0.017 decrease
CD14+, DR' 0.032 decrease
CD3+, CD4+, CD38' (MF CD38 Antigen)0.021 decrease
~~i CD3+, CD8+, CD28' (MF CD28 0.010 decrease
Antigen)
~~ CD20+, DR+ (MF DR Antigen) 0.014 decrease
All CD14+ 0.037 decrease
' - One-tailed p-value. Sample size of 15 patients using Wilcoxon rank
statistic.
s
These data show that administration of WF10 in vivo to humans
shows an increase in the production of CD28- subset of CD8+ T-cells. The
data also shows an increase in macrophage activation leading to
phagocytosis. The data also shows no evidence of RBC hemolysis. When
to coupled with the in vitro studies showing the inhibition of antigen
presentation for CD4' cells, it is believed that administration of WF-10 in
vivo will result in inhibition and/or prevention of antigen presentation in
APC,
as well as stimulate macrophage activation resulting in increased
is
phagocytosis.
Example 6
Based on the in vivo data above, administration of WF10 has shown
a consistent down regulation of CD14~/DR+ cells achieving statistical
2o significance. In addition, WF10 administration in vivo has shown overall
reduction of CD3'/CD8~/CD28+ cells, and significant increased levels of
CD3M/CD8+/CD28- cells of long-term duration. These cells are believed by
the inventors to be responsible for antigen specific immune tolerance by
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producing clonal anergy. The in vitro data above also shows that WF10 is
effective in inhibiting and/or preventing antigen presentation and producing
clonal anergy. This reduced antigen presentation may be critical in B-cell
lymphoma and thus, WF10 therapy may be of benefit. One case history
s with B-cell lymphoma responded to WF10 therapy with a notable reduction
of tumor size with no reoccurrence to date.
Adult patients having low grade follicular lymphoma are selected
based on their lack of enrollment in current therapy regimens. Fifteen
patients having lymph nodes > 1 cm in diameter at baseline confirmed by
to CT scan will be enrolled in an open-label, single arm, single center study.
Patients will receive periodic 0.5 ml/kg infusions of WF10 from days 1-5
(week 1 ) and days 8-12 (week 2). After screening evaluations are
completed (about 14 days), eligible patients will attend pre-study visit in
week 0 to acquire the baseline data.
is Screening criteria include the following:
male or female patients greater than 18 years of age;
histologically confirmed follicular lymphoma;
measurable disease defined as having lymph nodes > 1 cm in
diameter as measured by CT;
2o adequate renal function documented by a serum creatinine < 2 times
in institution's ULN;
'- adequate liver function documented by a serum billrubin less than or
equal to 1.5 mg/dl and SGOT (AST) or SGPT (ALT) < 5 times the
institutional upper limit of normal;
2s written informed consent to participate in this study and a willingness
to comply with all procedures and scheduled visits;
hemoglobin > 9.0 g/dl for woman and > 10.0 g/dl for men;
platelet count > 75,000/mm2; and
absolute neutrophil count > 750Immz.
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WF10 will be applied at a dose of 0.5 ml per kg of body weight
diluted into 250 to 500 ml normal saline administered by intravenous
infusion of 1 hour duration. CT measurements will be taken to determine
tumor size at week 0, on day 15, day 30 and day 45. Follow-up period will
s last for a duration of 3 months with final CT measurements on day 90.
CT measurements will reveal that administration of WF10 results in a
reduction of lymph node size. Patients also will exhibit an increase in
CD3+/CD8'/CD28-, an increase in CD14+/DR' and an increase in CD40 T
cell subsets.
to While the invention has been described in detail with reference to the
examples and particularly preferred embodiments, those skilled in the art
will appreciate that various modifications can be made to the invention
without departing from the spirit and scope thereof. All documents referred
to above are incorporated by reference. In particular, the PCT patent
is application WO 99/17787 is incorporated by reference in its entirety
herein.
