U.S. patent application number 09/875429 was filed with the patent office on 2002-06-20 for use of glatiramer acetate (copolymer 1) in the treatment of central nervous system disorders.
Invention is credited to Chabot, Sophie, Yong, V. Wee.
Application Number | 20020077278 09/875429 |
Document ID | / |
Family ID | 26904105 |
Filed Date | 2002-06-20 |
United States Patent
Application |
20020077278 |
Kind Code |
A1 |
Yong, V. Wee ; et
al. |
June 20, 2002 |
Use of glatiramer acetate (copolymer 1) in the treatment of central
nervous system disorders
Abstract
The present invention involves the administration of Copolymer 1
(glatiramer acetate) to treat inflammatory, non-autoimmune central
nervous system (CNS) diseases, alleviate the symptoms thereof,
inhibit the activity of matrix metalloproteinases and suppress
cytokine production by T lymphocytes.
Inventors: |
Yong, V. Wee; (Calgary,
CA) ; Chabot, Sophie; (Montreal, CA) |
Correspondence
Address: |
Cooper & Dunham LLP
1185 Avenue of the Americas
New York
NY
10036
US
|
Family ID: |
26904105 |
Appl. No.: |
09/875429 |
Filed: |
June 5, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60209372 |
Jun 5, 2000 |
|
|
|
Current U.S.
Class: |
424/449 ;
514/17.7; 514/17.8; 514/19.3; 514/2.4; 514/20.8 |
Current CPC
Class: |
A61K 38/02 20130101;
A61K 31/785 20130101 |
Class at
Publication: |
514/2 |
International
Class: |
A61K 038/16 |
Claims
What is claimed is:
1. A method of treating a mammalian subject having an inflammatory,
non-autoimmune central nervous system (CNS) disease, or alleviating
the symptoms of such a disease, comprising administering glatiramer
acetate (Copolymer 1) to the subject in an amount and for a
duration of time effective to treat the inflammatory,
non-autoimmune CNS disease.
2. The method of claim 1, wherein the mammalian subject is
human.
3. The method of claim 1, wherein the disease is Alzheimer's
Disease.
4. The method of claim 1, wherein the disease is Parkinson's
Disease.
5. The method of claim 1, wherein the disease is HIV
encephalopathy.
6. The method of claim 1, wherein the disease is a brain tumor.
7. The method of claim 1, wherein the disease is glaucoma.
8. The method of claim 1, wherein the disease is neuropathy or
dementia.
9. The method of claim 1, wherein the disease is a CNS
infection.
10. The method of claim 9, wherein the CNS infection is a bacterial
infection.
11. The method of claim 10, wherein the bacterial infection is
meningitis.
12. The method of claim 1, wherein the disease results from
stroke.
13. The method of claim 1, wherein the disease results from head
trauma.
14. The method of claim 1, wherein the route of administration is
oral, intravenous, intramuscular, subcutaneous, intraperitoneal,
transdermal, nasal or rectal.
15. The method of claim 14, wherein the route of administration is
oral.
16. The method of claim 14, wherein the route of administration is
subcutaneous injection.
17. The method of claim 14, wherein the dose of glatiramer acetate
(Copolymer 1) administered ranges from about 0.05 mg/kg of body
weight to about 50 mg/kg of body weight.
18. The method of claim 17, wherein the dose of glatiramer acetate
(Copolymer 1) administered ranges from about 0.1 mg/kg of body
weight to about 10 mg/kg of body weight.
19. The method of claim 18, wherein the dose of glatiramer acetate
(Copolymer 1) administered ranges from about 0.1 mg/kg of body
weight to about 1.0 mg/kg of body weight.
20. The method of claim 19, wherein the dose of glatiramer acetate
(Copolymer 1) administered is about 0.3 mg/kg of body weight.
21. The method of claim 14, wherein the dose of glatiramer acetate
(Copolymer 1) administered ranges from about 0.1 mg to about 1000
mg.
22. The method of claim 21, wherein the dose of glatiramer acetate
(Copolymer 1) administered ranges from about 1.0 mg to about 100
mg.
23. The method of claim 22, wherein the dose of glatiramer acetate
(Copolymer 1) administered ranges from about 10 mg to about 30
mg.
24. The method of claim 23, wherein the dose of glatiramer acetate
(Copolymer 1) administered is about 20 mg.
25. The method of claim 14, wherein the dose of glatiramer acetate
(Copolymer 1) is administered at a frequency of about once every 30
days to about once every day.
26. The method of claim 25, wherein the dose of glatiramer acetate
(Copolymer 1) is administered at a frequency of about once every 7
days to about once every day.
27. The method of claim 26, wherein the dose of glatiramer acetate
(Copolymer 1) is administered at a frequency of about once every
day.
28. The method of claim 1, wherein the glatiramer acetate
(Copolymer 1) is administered as part of a therapeutic regimen
during which a cytokine antagonist is also administered to the
subject.
29. A method of treating a mammalian subject at risk of developing
an inflammatory, non-autoimmune CNS disease comprising
administering glatiramer acetate (Copolymer 1) to the subject in an
amount and for a duration of time effective to minimize the
severity of the inflammatory, non-autoimmune CNS disease that may
occur in the subject or prevent its occurrence.
30. The method of claim 29, wherein the subject is human and the
risk is associated with a genetic predisposition to a chronic
medical condition.
31. The method of claim 29, wherein the disease is selected from
the group consisting of Alzheimer's disease, Parkinson's disease,
glaucoma, dementia, neuropathy, stroke, and brain tumor.
32. The method of claim 29, wherein the route of administration is
oral, intravenous, intramuscular, subcutaneous, intraperitoneal,
transdermal, nasal or rectal.
33. A method of inhibiting the activity of a matrix
metalloproteinase comprising contacting the matrix
metalloproteinase with glatiramer acetate (Copolymer 1).
34. The method of claim 33, wherein the matrix metalloproteinase is
MMP-9.
35. The method of claim 34, wherein the matrix metalloproteinase is
in a subject.
36. The method of claim 35, wherein the subject is human.
37. A method of suppressing the production of a cytokine by
activated T lymphocytes comprising contacting the activated T
lymphocytes with glatiramer acetate (Copolymer 1) in an amount
necessary to suppress cytokine production.
38. The method of claim 37, wherein the cytokine is IL-1.
39. The method of claim 37, wherein the cytokine is IL-4.
40. The method of claim 37, wherein the cytokine is IL-6.
41. The method of claim 37, wherein the cytokine is IL-10.
42. The method of claim 37, wherein the cytokine is IL-12.
43. The method of claim 37, wherein the cytokine is IL-13.
44. The method of claim 37, wherein the cytokine is
TNF-.alpha..
45. The method of claim 37, wherein the cytokine is in a
subject.
46. The method of claim 45, wherein the subject is human.
Description
[0001] This application claims benefit of U.S. Provisional
Application No. 60/209,372, filed Jun. 5, 2000.
[0002] Throughout this application, various references are cited,
using arabic numbers within parentheses. Full citations for these
references can be found at the end of the specification,
immediately preceding the claims. These publications, in their
entireties, are hereby incorporated by reference into the
application to more fully describe the state of the art to which
the invention pertains.
FIELD OF THE INVENTION
[0003] The present invention provides methods of treating a
mammalian subject having, or genetically predisposed to, an
inflammatory, non-autoimmune central nervous system (CNS) disease,
or alleviating the symptoms of such a disease.
BACKGROUND
[0004] The nervous system of vertebrates is divided into the
central nervous system, comprised of the brain and spinal cord, and
the peripheral nervous system, consisting of the outlying nerves
(22). The axons of most nerve cells are covered with a myelin
sheath, a stack of specialized plasma membranes. Glial cells that
wrap around the axons produce the myelin sheath. In the CNS, these
glial cells are called oligodendrocytes. Each region of myelin
formed by an individual oligodendrocyte is separated from the next
region by an unmyelinated area called the node of Ranvier (22).
[0005] The myelin sheath acts as an electric insulator of the axon
and all electric activity in axons is confined to the nodes of
Ranvier.
[0006] One of the more common types of CNS diseases among human
adults is multiple sclerosis. This condition is a demyelinating
disease. Multiple sclerosis is a chronic, frequently progressive,
inflammatory CNS disease characterized pathologically by primary
demyelination and axonal injury. In multiple sclerosis patients,
conduction of action potentials by the demyelinated neurons is
slowed (22). Even though the etiology of multiple sclerosis is
unknown, several immunological features of multiple sclerosis, and
its moderate association with certain major histocompatibility
complex alleles, has prompted the speculation that multiple
sclerosis is an immune-mediated disease (17, 33, 55). An autoimmune
hypothesis is supported by the experimental autoimmune (allergic)
encephalomyelitis (EAE) model, where the injection of certain
myelin components into genetically susceptible animals leads to T
lymphocyte-mediated CNS demyelination (58).
[0007] Some researchers view activated T lymphocytes as a trigger
of multiple sclerosis. They postulate that once T lymphocytes
traverse the blood brain barrier (BBB) into the CNS parenchyma,
they are reactivated following antigen presentation by microglia
(53). The entry of leukocytes into tissues is a multi-step process
that includes adhesion onto endothelial cells and transmigration
across the endothelial barrier.
[0008] Recent evidence suggests that the expression of matrix
metalloproteinases (MMPs) by leukocytes is required for T
lymphocytes to enter the CNS parenchyma (42, 77, 88). An MMP is a
proteolytic enzyme that possesses an active site where an invariant
zinc binds to cysteine residues in the propeptide region of the
MMP, keeping the MMP in an inactive state (50, 89). Activating
agents disrupt the cysteine-zinc interaction to expose the active
site so that catalysis can occur.
[0009] The inappropriate expression of MMPs is speculated to be
involved in the pathogenesis of malignant gliomas, stroke and
Alzheimer's disease. Furthermore, several lines of evidence suggest
a role for MMPs in the disease process in multiple sclerosis (37,
91). In this regard, immunohistochemically identified MMPs
(specifically MMP-3, -7, -9 and -12), on microglia, astrocytes and
infiltrating leukocytes, have now been documented by several groups
to be present in the autopsied brains of multiple sclerosis
subjects (4, 18, 21, 47, 57) and in the brains of EAE animals (16,
37). Analyses of serum samples reveal levels of MMP-9 to be
significantly increased in multiple sclerosis patients compared to
healthy controls; within the multiple sclerosis population, serum
MMP-9 levels are higher during clinical relapse relative to periods
of stability (40). In addition, serum MMP-9 levels are correlated
with the number of gadolinium-enhancing lesions detected by
magnetic resonance imaging (MRI) (40, 84).
[0010] In correspondence with a pathogenic role of MMPs, several
hydroxamate-based agents developed to inhibit the activity of MMPs
(e.g. GM6001, Ro31-9790 and BB-1101) were found to alleviate the
incidence and severity of EAE (32, 34, 43, 52).
[0011] A prominent method of microglia activation is believed to be
a non-antigen-specific interaction between these cells and T
lymphocytes, which generates cytokines within the CNS milieu (19,
20, 67). This contention is supported by several findings obtained
in culture studies. For instance, human T lymphocytes and human
microglia interact to generate significant amounts of TNF-A and
IL-10 (14, 15). The effect of T lymphocytes on microglia is
equipotent to that of lipopolysaccharide, a very effective
microglia stimulator. Researchers found additional support in the
observation that the interaction between T lymphocytes and
microglia does not require antigen or MHC restriction. Experiments
have also shown that in a facial nerve resection model in mouse, T
cells infiltrated the CNS and aggregated around microglia, and that
this was correspondent with an increase in IL-1.beta. and
TNF-.alpha. (61). In a graft-versu-shost disease (GVHD) model,
activated microglial cell clusters were invariably intimately
associated with T cell infiltrates (68).
[0012] TNF-.alpha. can influence lymphocyte trafficking across
endothelium by up regulating the expression of various adhesion
molecules involved in this process (76), and is implicated in the
process of demyelination. Indeed, TNF-.alpha. directly induces in
vitro the apoptotic death of the myelin-producing cells in the
brain, the oligodendrocytes (26, 45, 71), and intravitreal
injection of TNF-.alpha. causes demyelination of mouse optic nerve
axons (12). In addition, TNF-.alpha. is pro-inflammatory. The level
of TNF-.alpha. is found to be elevated in the serum, cerebrospinal
fluid, and brain lesions of multiple sclerosis patients, and is
correlated with the disease activity (13, 35, 62, 72). TNF-.alpha.
is also implicated in the pathogenicity of EAE--the administration
of antibodies to TNF-.alpha. or soluble TNF-.alpha. receptors
prevents the transfer of EAE and abrogates autoimmune demyelination
(63, 69, 70).
[0013] IL-12 is another pro-inflammatory cytokine which has a key
role in switching uncommitted T lymphocytes to the pro-inflammatory
Th1 subset which secretes IFN-.gamma. and TNF-.alpha./.beta. (91).
IL-1.beta. also promotes an inflammatory response and has been
associated with multiple sclerosis (66).
[0014] The Th2 subset produces IL-4, IL-10 and IL-13, regulates
humoral immunity and decreases local inflammation. Both IL-4 and
IL-10 can inhibit the differentiation of naive precursors into Th1
cells (91). IL-4 promotes the activation of B lymphocytes and
macrophages and also stimulates class switching of antibodies
(22).
[0015] IL-13 is a Th2 cytokine with important immunomodulating
activities. The best known IL-13 mediated function is its ability
to drive the differentiation of naive CD4+ T cells towards a Th2
phenotype (75). The anti-inflammatory functions of IL-13 include
the suppressive effect on the production of pro-inflammatory
cytokines by activated monocytes or by alveolar macrophages (81),
the induction of 15-lipoxygenase (51) and the inhibition of
prostaglandin E2 (PGE2) formation (27). Functions of IL-13 in the
CNS and on glial cell functions are not well defined (87).
[0016] IL-10 is an anti-inflammatory cytokine produced by a variety
of cells, including monocytes/macrophages, T lymphocytes, and mast
cells. In the CNS, potential sources of IL-10 include microglia
(86) and astrocytes (49). IL-10 has important anti-inflammatory
properties. First, IL-10 inhibits the production of proinflammatory
cytokines by many cell types, including those of the mononuclear
phagocytic lineage; indeed, IL-10 was shown to inhibit the
production of TNF-.alpha. and IL-12 produced by monocytes,
macrophages, and microglia (3, 9, 10, 24, 38). Also, IL-10 plays a
role in causing T lymphocytes to undergo anergy (inactivation or
unresponsiveness) (2). Other anti-inflammatory functions of IL-10
include its inhibitory effect on the process of antigen
presentation. Treatment of macrophages/microglia with IL-10
down-regulated the expression of molecules essential for
presentation of antigens, such as MHC class II (24) and the
costimulatory molecules B7-1 and B7-2 (36). Finally, the role of
IL-10 as an anti-inflammatory molecule is supported by the
phenotype of IL-10-deficient mice; these mice develop chronic
colitis, which appears to be mediated by the proinflammatory Th1
cells (8, 23, 39).
[0017] The afore-mentioned cytokines are inducible, meaning that
they are produced in response to certain stimuli. In contrast,
IL-6, which activates B-lymphocytes (22), is constitutive.
SUMMARY OF THE INVENTION
[0018] The subject invention provides a method of treating a
mammalian subject having an inflammatory, non-autoimmune central
nervous system (CNS) disease, or alleviating the symptoms of such a
disease, comprising administering glatiramer acetate (Copolymer 1)
to the subject in an amount and for a duration of time effective to
treat the inflammatory, non-autoimmune CNS disease. The subject
invention also includes a method of treating a mammalian subject at
risk of developing an inflammatory, non-autoimmune CNS disease
comprising administering glatiramer acetate (Copolymer 1) to the
subject in an amount and for a duration of time effective to
minimize the severity of the inflammatory, non-autoimmune CNS
disease that may occur in the subject or prevent its
occurrence.
[0019] Further encompassed by the subject invention is a method of
inhibiting the activity of a matrix metalloproteinase comprising
contacting the matrix metalloproteinase with glatiramer acetate
(Copolymer 1).
[0020] In addition, the subject invention contains a method of
suppressing the production of a cytokine by activated T lymphocytes
comprising contacting the activated T lymphocytes with glatiramer
acetate (Copolymer 1) in an amount necessary to suppress cytokine
production.
[0021] The subject invention also provides the use of glatiramer
acetate (Copolymer 1) in the preparation of a pharmaceutical
composition for the treatment of an inflammatory, non-autoimmune
central nervous system (CNS) disease, or alleviation of the
symptoms of such a disease, wherein said pharmaceutical composition
is administered to the subject in an amount and for a duration of
time effective to treat the inflammatory, non-autoimmune CNS
disease in a mammalian subject.
[0022] The subject invention further concerns the use of glatiramer
acetate (Copolymer 1) in the preparation of a pharmaceutical
composition for treating a mammalian subject at risk of developing
an inflammatory, non-autoimmune CNS disease, wherein said
pharmaceutical composition is administered to the subject in an
amount and for a duration of time effective to minimize the
severity of the inflammatory, non-autoimmune CNS disease that may
occur in the subject or prevent its occurrence.
[0023] In addition, the subject invention includes the use of
glatiramer acetate (Copolymer 1) in the preparation of a
pharmaceutical composition for inhibiting the activity of a matrix
metalloproteinase.
[0024] The subject invention also provides the use of glatiramer
acetate (Copolymer 1) in the preparation of a pharmaceutical
composition for suppressing the production of a cytokine by
activated T lymphocytes.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1 shows the lack of effect of Copolymer 1 (glatiramer
acetate (GA)) on MMP-9 production by T lymphocytes. AIM-V
conditioned media were collected from anti-CD3 activated T
lyphocytes either in the absence of any drug treatment (control) or
in the presence of 25 .mu.g/ml Copolymer 1 or 1000 IU/ml
recombinant interferon .beta.-1b. Interferon .beta. (IFN-.beta.)
inhibited T lymphocyte production of MMP-9, as previously reported
(77), but Copolymer 1 did not affect MMP-9 protein levels. Similar
results were obtained from monocytes after 1 day of treatment. The
conditioned media at each time point were collected from cultures
with equivalent amounts of T lymphocytes.
[0026] FIG. 2 demonstrates that Copolymer 1 is an inhibitor of the
enzymatic activity of MMP-9. The presence of Copolymer 1 in the
incubation buffers during the development of the zymograms inhibits
the manifestation of MMP activity. In contrast, IFN.beta.-1b is not
a direct MMP activity inhibitor, although it decreases the
production of MMP-9 as demonstrated in FIG. 1. Positive controls,
TIMP-1 and phenanthroline (phe), are shown to inhibit MMP activity
in this assay. FIG. 2-A shows MMP-9 (the strongest band) from Baby
Hamster Kidney (BHK) cells while FIG. 2-B represents MMP-9 produced
by T lymphocytes.
[0027] FIG. 3 reveals that Copolymer 1 does not inhibit T
lymphocyte migration across a fibronectin chamber. FIG. 3-A
demonstrates that 25 .mu.g/ml Copolymer 1 treatment of T
lymphocytes, for 1, 2 or 3 days, did not affect the subsequent
migration of cells across a fibronectin barrier compared to
controls. In contrast, 3 days treatment of cells with IFN.beta.-1b
(1000 IU/ml) decreased migration by 20-50% as reported previously
(77). In FIG. 3-B, 3-day pretreatment of T lymphocytes with various
concentrations of Copolymer 1 similarly did not affect
transmigration. Values are mean.+-.SEM of 3 analyzes each, and have
been expressed as % of controls of the respective dose-response
experiments.
[0028] FIG. 4 shows that Copolymer 1 pretreatment of activated T
lymphocytes suppresses IL-10 and TNF-.alpha. production that is
generated in T lymphocyte--U937 interactions. Activated T
lymphocytes in isolation produce undetectable IL-10 or TNF-.alpha.;
PMA/IFN.gamma.-pretreated U937 cells have detectable IL-10 but
negligible TNF-.alpha. levels. Cytokines are significantly elevated
in co-cultures and this is reduced dose-dependently by Copolymer 1
pretreatment of T lymphocytes. Values are mean.+-.SEM of triplicate
analyzes.
[0029] FIG. 5 reports that Copolymer 1 treatment of activated T
lymphocytes also lead to the suppression of IL-4, IL-12 and IL-13
in T lymphocyte--U937 interactions. Both T lymphocytes and
PMA/IFN.gamma.-pretreated U937 cells do not secrete detectable
amounts of IL-4, IL-12 or IL-13 into their conditioned medium. With
coculture, levels of these cytokines are increased, although the
levels are low in comparison to those for IL-10 or TNF-.alpha.
(FIG. 4); amount of IL-4, IL-12 and IL-13 in T lymphocyte--U937
interactions are, respectively, 13, 12 and 62 pg/ml. These are
dose-dependently reduced by Copolymer 1 pretreatment of T
lymphocytes.
[0030] FIG. 6 demonstrates that cytokine production in co-culture
of T lymphocytes with fetal human microglia is reduced by Copolymer
1. Values are mean.+-.SEM of 3 or 4 analyzes and are expressed as %
of the mean of control T lymphocyte--microglia co-cultures (i.e. 0
.mu.g/ml Copolymer 1). The amount of TNF-.alpha. in control T
lymphocyte--microglia co-culture was 1068.+-.68 pg/ml while that
for IL-10 was 139.+-.4 pg/ml.
[0031] FIG. 7 reflects the effects of Copolymer 1 on cytokine
production in adult human microglia and T lymphocyte co-cultures.
In isolation, neither T lymphocytes nor microglia express IL-1,
IL-10 or TNF-.alpha.. In co-culture of microglia and activated T
lymphocytes, the levels of cytokines induced after 24 h were
1012.+-.86 pg/ml for TNF-.alpha., 18.+-.2 pg/ml for IL-1 and
46.+-.2 pg/ml for IL-10. IL-6 is constitutively expressed in
microglia (1010.+-.215 pg/ml), but not in T lymphocytes. Copolymer
1 pretreatment of T lymphocytes reduced the level of inducible
cytokines in coculture with microglia and also the level of
expression of IL-6. Values are mean+SEM of triplicate cultures.
[0032] FIG. 8 shows the morphology of microglia in T
lymphocyte--microglia co-cultures. FIG. 8-A shows that adult human
microglia are mostly bipolar in morphology in culture. FIG. 8-B
demonstrates that T lymphocytes are present as single cells or
clumps. FIG. 8-C reports that when T lymphocytes are co-cultured
with microglia in the absence of Copolymer 1, bipolar microglia
become rounded/ameboid in morphology (some microglia are shown by
arrows). As FIG. 8-D reflects, this morphological transformation is
prevented by Copolymer 1 pretreatment of T lymphocytes (some
bipolar microglia are indicated by arrows). All figures portray the
same magnification, 400X.
[0033] FIG. 9 reports that Copolymer 1 does not affect CXCR3 and
CXCR4 expression on T lymphocytes. All T lymphocytes were activated
at time 0 with anti-C3 antibody and Copolymer 1 (25 .mu.g/ml) was
added to some cultures at 3 hours. Cells were removed and stained
at 1 (FIGS. 9-A and 9-B), 2 (FIGS. 9C and 9-D) and 3 days (FIGS.
9-E and 9-F). An isotype control antibody was used to stain control
or Copolymer 1-treated T lymphocytes and the fluorescence of these
did not differ. Thus, only the isotype staining for control T
lymphocytes is displayed.
[0034] FIG. 10 demonstrates that Copolymer 1 does not affect CXCR3
and CXCR4 expression on U937 cells. Cells were treated with
Copolymer 1 (25 .mu.g/ml) or left untreated at time 0. Cells were
removed and stained at 1 (FIGS. 10-A and 10-B) or 3 days (FIGS.
10-C and 10-D). An isotype control antibody was used to stain
control and Copolymer 1-treated cells and the fluorsecence of these
did not differ. Thus, only the isotype staining for control U937
cells is displayed.
DETAILED DESCRIPTION OF THE INVENTION
[0035] A non-autoimmune disease refers to a condition characterized
by a lack of significant immune-mediated damage to the diseased
subject.
[0036] Oftentimes, an inflammatory disease is characterized as a
condition in which there is increased blood flow to the affected
areas, resulting in swelling and heightened temperature, which may
produce pain.
[0037] As used in this application, an inflammatory, non-autoimmune
disease includes any disease which impacts the central nervous
system, but does not include an autoimmune component and is
associated with an inflammatory response in the subject afflicted
with the disease. Inflammatory, non-autoimmune diseases include,
inter alia, Alzheimer's disease, Parkinson's disease, HIV
encephalopathy, brain tumor, glaucoma, neuropathy, dementia,
central nervous system infection, central nervous system bacterial
infection, meningitis, stroke, and head trauma.
[0038] The subject invention encompasses a method of treating a
mammalian subject having an inflammatory, non-autoimmune central
nervous system (CNS) disease, or alleviating the symptoms of such a
disease, comprising administering glatiramer acetate (GA or
Copolymer 1) to the subject in an amount and for a duration of time
effective to treat the inflammatory, non-autoimmune CNS
disease.
[0039] In one embodiment, the mammalian subject is human.
[0040] In another embodiment, the disease is Alzheimer's
Disease.
[0041] In a further embodiment, the disease is Parkinson's
Disease.
[0042] In yet another embodiment, the disease is HIV
encephalopathy.
[0043] In a further embodiment, the disease is a brain tumor.
[0044] In another embodiment, the disease is glaucoma.
[0045] In a further embodiment, the disease is neuropathy or
dementia.
[0046] In another embodiment, the disease is a CNS infection.
[0047] In one embodiment, the CNS infection is a bacterial
infection.
[0048] In another embodiment, the bacterial infection is
meningitis.
[0049] In a further embodiment, the disease results from
stroke.
[0050] In yet another embodiment, the disease results from head
trauma.
[0051] The subject invention encompasses embodiments wherein the
route of administration is oral, intravenous, intramuscular,
subcutaneous, intraperitoneal, transdermal, nasal or rectal. The
preferred routes of administration are oral and subcutaneous
injection.
[0052] In one embodiment, the dose of glatiramer acetate (Copolymer
1) administered ranges from about 0.1 mg to about 1000 mg.
[0053] In another embodiment, the dose of glatiramer acetate
(Copolymer 1) administered ranges from about 1 mg to about 100
mg.
[0054] In a further embodiment, the dose of glatiramer acetate
(Copolymer 1) administered ranges from about 5 mg to about 50
mg.
[0055] In yet another embodiment, the dose of glatiramer acetate
(Copolymer 1) administered ranges from about 10 mg to about 30
mg.
[0056] In one embodiment, the dose of glatiramer acetate (Copolymer
1) administered is about 20 mg.
[0057] In another embodiment, the dose of glatiramer acetate
(Copolymer 1) administered ranges from about 0.05 mg/kg of body
weight to about 50 mg/kg of body weight.
[0058] In a further embodiment, the dose of glatiramer acetate
(Copolymer 1) administered ranges from about 0.1 mg/kg of body
weight to about 10 mg/kg of body weight.
[0059] In an additional embodiment, the dose of glatiramer acetate
(Copolymer 1) administered ranges from about 0.1 mg/kg of body
weight to about 1.0 mg/kg of body weight.
[0060] In another embodiment, the dose of glatiramer acetate
(Copolymer 1) administered is about 0.3 mg/kg of body weight.
[0061] In one embodiment, the dose of glatiramer acetate (Copolymer
1) is administered at a frequency of about once every 30 days to
about once every day. In a preferred embodiment, the dose of
glatiramer acetate (Copolymer 1) is administered at a frequency of
about once every 7 days to about once every day. In a more
preferred embodiment, the dose of glatiramer acetate (Copolymer 1)
is administered at a frequency of about once every day.
[0062] In another embodiment, the glatiramer acetate (Copolymer 1)
is administered as part of a therapeutic regimen during which a
cytokine antagonist is also administered to the subject.
[0063] The subject invention also concerns a method of treating a
mammalian subject at risk of developing an inflammatory,
non-autoimmune CNS disease comprising administering glatiramer
acetate (Copolymer 1) to the subject in an amount and for a
duration of time effective to minimize the severity of the
inflammatory, non-autoimmune CNS disease that may occur in the
subject or prevent its occurrence.
[0064] In one embodiment, the subject is human and the risk is
associated with a genetic predisposition to a chronic medical
condition.
[0065] In another embodiment, the disease is selected from the
group consisting of Alzheimer's Disease, Parkinson's Disease,
glaucoma, dementia, neuropathy, stroke, and brain tumor.
[0066] In a further embodiment, the route of administration is
oral, intravenous, intramuscular, subcutaneous, intraperitoneal,
transdermal, nasal or rectal.
[0067] The subject invention additionally includes a method of
inhibiting the activity of a matrix metalloproteinase comprising
contacting the matrix metalloproteinase with glatiramer acetate
(Copolymer 1).
[0068] In one embodiment, the matrix metalloproteinase is
MMP-9.
[0069] In another embodiment, the matrix metalloproteinase is in a
subject.
[0070] In one embodiment, the subject is human.
[0071] The subject invention further provides a method of
suppressing the production of a cytokine by activated T lymphocytes
comprising contacting the activated T lymphocytes with glatiramer
acetate (Copolymer 1) in an amount necessary to suppress the
production of the cytokine.
[0072] In one embodiment, the cytokine is IL-1.
[0073] In another embodiment, the cytokine is IL-4.
[0074] In a further embodiment, the cytokine is IL-6.
[0075] In yet another embodiment, the cytokine is IL-10.
[0076] In a further embodiment, the cytokine is IL-12.
[0077] In another embodiment, the cytokine is IL-13.
[0078] In a further embodiment, the cytokine is TNF-.alpha..
[0079] In one embodiment, the cytokine is in a subject.
[0080] In another embodiment, the subject is human.
[0081] The subject invention also contains the use of glatiramer
acetate (Copolymer 1) in the preparation of a pharmaceutical
composition for the treatment of an inflammatory, non-autoimmune
central nervous system (CNS) disease, or alleviation of the
symptoms of such a disease, wherein said pharmaceutical composition
is administered to the subject in an amount and for a duration of
time effective to treat the inflammatory, non-autoimmune CNS
disease in a mammalian subject.
[0082] In one embodiment of the use, the mammalian subject is
human.
[0083] In another embodiment of the use, the disease is Alzheimer's
Disease.
[0084] In a further embodiment of the use, the disease is
Parkinson's Disease.
[0085] In yet another embodiment of the use, the disease is HIV
encephalopathy.
[0086] In a further embodiment of the use, the disease is a brain
tumor.
[0087] In another embodiment of the use, the disease is
glaucoma.
[0088] In a further embodiment of the use, the disease is
neuropathy or dementia.
[0089] In another embodiment of the use, the disease is a CNS
infection.
[0090] In one embodiment of the use, the CNS infection is a
bacterial infection.
[0091] In another embodiment of the use, the bacterial infection is
meningitis.
[0092] In a further embodiment of the use, the disease results from
stroke.
[0093] In yet another embodiment of the use, the disease results
from head trauma.
[0094] The subject invention encompasses embodiments of the use
wherein the route of administration is oral, intravenous,
intramuscular, subcutaneous, intraperitoneal, transdermal, nasal or
rectal. The preferred routes of administration are oral and
subcutaneous injection.
[0095] In one embodiment of the use, the dose of glatiramer acetate
(Copolymer 1) administered ranges from about 0.1 mg to about 1000
mg.
[0096] In another embodiment of the use, the dose of glatiramer
acetate (Copolymer 1) administered ranges from about 1 mg to about
100 mg.
[0097] In a further embodiment of the use, the dose of glatiramer
acetate (Copolymer 1) administered ranges from about 5 mg to about
50 mg.
[0098] In yet another embodiment of the use, the dose of glatiramer
acetate (Copolymer 1) administered ranges from about 10 mg to about
30 mg.
[0099] In one embodiment of the use, the dose of glatiramer acetate
(Copolymer 1) administered is about 20 mg.
[0100] In another embodiment of the use, the dose of glatiramer
acetate (Copolymer 1) administered ranges from about 0.05 mg/kg of
body weight to about 50 mg/kg of body weight.
[0101] In a further embodiment of the use, the dose of glatiramer
acetate (Copolymer 1) administered ranges from about 0.1 mg/kg of
body weight to about 10 mg/kg of body weight.
[0102] In an additional embodiment of the use, the dose of
glatiramer acetate (Copolymer 1) administered ranges from about 0.1
mg/kg of body weight to about 1.0 mg/kg of body weight.
[0103] In another embodiment of the use, the dose of glatiramer
acetate (Copolymer 1) administered is about 0.3 mg/kg of body
weight.
[0104] In one embodiment of the use, the dose of glatiramer acetate
(Copolymer 1) is administered at a frequency of about once every 30
days to about once every day. In a preferred embodiment of the use,
the dose of glatiramer acetate (Copolymer 1) is administered at a
frequency of about once every 7 days to about once every day. In a
more preferred embodiment of the use, the dose of glatiramer
acetate (Copolymer 1) is administered at a frequency of about once
every day.
[0105] In another embodiment of the use, the glatiramer acetate
(Copolymer 1) is administered as part of a therapeutic regimen
during which a cytokine antagonist is also administered to the
subject.
[0106] The subject invention also concerns the use of glatiramer
acetate (Copolymer 1) in the preparation of a pharmaceutical
composition for treating a mammalian subject at risk of developing
an inflammatory, non-autoimmune CNS disease, wherein said
pharmaceutical composition is administered to the subject in an
amount and for a duration of time effective to minimize the
severity of the inflammatory, non-autoimmune CNS disease that may
occur in the subject or prevent its occurrence.
[0107] In one embodiment of the use, the subject is human and the
risk is associated with a genetic predisposition to a chronic
medical condition.
[0108] In another embodiment of the use, the disease is selected
from the group consisting of Alzheimer's Disease, Parkinson's
Disease, glaucoma, dementia, neuropathy, stroke, and brain
tumor.
[0109] In a further embodiment of the use, the route of
administration is oral, intravenous, intramuscular, subcutaneous,
intraperitoneal, transdermal, nasal or rectal.
[0110] The subject invention additionally includes the use of
glatiramer acetate (Copolymer 1) in the preparation of a
pharmaceutical composition for inhibiting the activity of a matrix
metalloproteinase.
[0111] In one embodiment of the use, the matrix metalloproteinase
is MMP-9.
[0112] In another embodiment of the use, the matrix
metalloproteinase is in a subject.
[0113] In one embodiment of the use, the subject is human.
[0114] The subject invention further provides the use of glatiramer
acetate (Copolymer 1) in the preparation of a pharamcutical
composition for suppressing the production of a cytokine by
activated T lymphocytes.
[0115] In one embodiment of the use, the cytokine is IL-1.
[0116] In another embodiment of the use, the cytokine is IL-4.
[0117] In a further embodiment of the use, the cytokine is
IL-6.
[0118] In yet another embodiment of the use, the cytokine is
IL-10.
[0119] In a further embodiment of the use, the cytokine is
IL-12.
[0120] In another embodiment of the use, the cytokine is IL-13.
[0121] In a further embodiment of the use, the cytokine is
TNF-.alpha..
[0122] In one embodiment of the use, the cytokine is in a
subject.
[0123] In another embodiment of the use, the subject is human.
[0124] COPAXONE.RTM. is the brand name for glatiramer acetate
(formerly known as copolymer-1). Glatiramer acetate, the active
ingredient of COPAXONE.RTM., consists of the acetate salts of
synthetic polypeptides, containing four naturally occurring amino
acids: L-glutamic acid, L-alanine, L-tyrosine, and L-lysine with an
average molar fraction of 0.141, 0.427, 0.095, and 0.338,
respectively. The average molecular weight of glatiramer acetate is
4,700-11,000 daltons. Chemically, glatiramer acetate is designated
L-glutamic acid polymer with L-alanine, L-lysine and L-tyrosine,
acetate (salt). Its structural formula is:
(Glu, Ala, Lys, Tyr).sub.X.multidot.XCH.sub.3COOH
(C.sub.5H.sub.9NO.sub.4=- 19
C.sub.3H.sub.7NO.sub.2.multidot.C.sub.6H.sub.14N.sub.2O.sub.2.multidot.-
C.sub.9H.sub.11NO.sub.3).sub.X.multidot.XC.sub.2H.sub.4O.sub.2
CAS-147245-92-9
[0125] (92).
[0126] Copolymer 1 (Cop-1) can be formulated into pharmaceutical
compositions containing a pharmaceutically acceptable carrier. As
used herein, "pharmaceutically acceptable carrier" includes any and
all solvents, adjuvants, suspending agents, emulsifying agents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, sweeteners, flavor
enhancers and the like. The pharmaceutically acceptable carriers
may be prepared from a wide range of materials including, but not
limited to, flavoring agents, sweetening agents and miscellaneous
materials such as buffers and absorbents that may be needed in
order to prepare a particular oral therapeutic composition. The use
of such media and agents with pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active ingredient, its use in the
therapeutic compositions is contemplated.
[0127] Copolymer 1 can be formulated into any form known in the art
using procedures available to one of skill in the art. Copolymer 1
may be mixed with other food forms and consumed in solid,
semi-solid, suspension or emulsion form. In one embodiment, the
composition is formulated into a capsule or tablet using techniques
available to one of skill in the art. However, the present
compositions may also be formulated in another convenient form,
such as an injectable solution or suspension, a spray solution or
suspension, a lotion, a gum, a lozenge, a food or snack item. Food,
snack, gum or lozenge items can include any ingestible ingredient,
including sweeteners, flavorings, oils, starches, proteins, fruits
or fruit extracts, vegetables or vegetable extracts, grains, animal
fats or proteins. Thus, the present compositions can be formulated
into cereals, snack items such as chips, bars, gum drops, chewable
candies or slowly dissolving lozenges. Copolymer 1 can also be
administered as dry powder or metered dose of solution by
inhalation, or nose-drops and nasal sprays, using appropriate
formulations and metered dosing units.
[0128] One of skill in the art can readily substitute
structurally-related amino acids for Copolymer 1 without deviating
from the spirit of the invention. The present invention includes
polypeptides and peptides which contain amino acids that are
structurally related to tyrosine, glutamic acid, alanine or lysine
and possess the ability to stimulate polyclonal antibody production
upon introduction. Such substitutions retain substantially
equivalent biological activity in their ability to suppress or
alleviate the symptoms of the CNS disease. These substitutions are
structurally-related amino acid substitutions, including those
amino acids which have about the same charge, hydrophobicity and
size as tyrosine, glutamic acid, alanine or lysine. For example
lysine is structurally-related to arginine and histidine; glutamic
acid is structurally-related to aspartic acid; tyrosine is
structurally-related to serine, threonine, phenylalanine and
tryptophan; and alanine is structurally-related to valine, leucine
and isoleucine. These and other conservative substitutions, such as
structurally-related synthetic amino acids, are contemplated by the
present invention.
[0129] Moreover, Copolymer 1 can be composed of 1- or d-amino
acids. As is known by one of skill in the art, 1-amino acids occur
in most natural proteins. However, d-amino acids are commerically
available and can be substituted for some or all of the amino acids
used to make Copolymer 1. The present invention contemplates
Copolymer 1 consisting essentially of 1-amino acids, as well as
Copolymer 1 consisting essentially of d-amino acids.
Experimental Details
[0130] Procedure
[0131] PBMC Isolation
[0132] Heparinized blood was collected from normal volunteers, and
subjected to Ficoll-Hypaque (Pharmacia Biotech, Mississauga,
Ontario) centrifugation to obtain peripheral blood mononuclear
cells (PBMNCs) as described previously (15). After two washes,
cells were suspended at a density of 1-2 million/ml in horizontal
T-25 flasks (Nunc, Becton Dickinson, Mississauga, Ontario) in the
serum free medium, AIM-V (GIBCO/BRL), to which 1 ng/ml of an
anti-CD3 antibody (OKT3, compliments of Jack Antel, Montreal,
Canada) was added. Three hours after the anti-CD3 addition, the
T-25 flasks were stood upright from their horizontal position in
order to kill monocytes that had adhered. Floating cells, which
were mostly lymphocytes, were left for a period of 72 hours at
37.degree. C. Flow cytometry analysis of the MNC population at this
72 hour period indicated that CD3+ cells constituted about 90% of
the total cell population, with approximately 60% CD4+ and 30% CD8+
ratio. B lymphocytes (CD19+) and NK cells (CD56+) consisted of 5-6%
of the total MNC population, and no monocytes (CD14+) were
detected. Henceforth, given that the majority of cells in the MNC
population are T lymphocytes, these will be referred to as T
lymphocytes.
[0133] Copolymer 1 Treatment
[0134] Copolymer 1 (1-50 .mu.g/ml) diluted in phosphate-buffered
saline, was added to cultures 3 hours after the initiation of CD3
ligation, at the time that the T-25 flasks were altered from the
horizontal to upright positions. Cells were left for 69 h at
37.degree. C., then collected, counted and resuspended in fresh
AIM-V at density of 500,000 cells per ml. Following a second
treatment with Copolymer 1, cells were left at 37.degree. C. for an
additional 3 h. Thereafter, 500 .mu.l (250,000 cells) of the
suspension was taken for migration assays described below.
Alternatively, 100 .mu.l (50,000 cells) of cell suspension was
added to each individual well of a 96-well plate already containing
microglia or U937 monocytoid cells (see below). The experiments
involved 72 h treatment of T lymphocytes with Copolymer 1,
administered at 2 time points. The purity of T lymphocytes after 72
h of Copolymer 1 treatment was not different from that of
non-treated controls. In some experiments, recombinant IFN.beta.-1b
was used as a positive control.
[0135] Monocyte-Enriched Culture
[0136] To produce monocyte-enriched cultures, 2.times.10.sup.5 PBMC
were suspended in 100 .mu.l of AIM-V medium and placed into each
well of a 96-well plate. One hour after, floating cells were
removed to leave behind adherent monocytes. Fresh 100 .mu.l of
AIM-V was added per well and cultures were kept at 37.degree. C.
Copolymer 1 was added directly to cells in the 96-well plate.
[0137] MMPs Assay
[0138] To address whether the production of MMPs by cells was
affected by Copolymer 1, the conditioned medium of cells was used,
since MMPs are secreted enzymes. Essentially, for floating T
lymphocytes contained within the T-25 flasks, 1 ml of conditioned
medium was siphoned off and microfuged for 1 min to remove
cells.
[0139] Supernatant was collected, mixed in a 3.1 ratio (v/v) with
4x gel-loading SDS buffer, and used in zymographic assays.
Similarly, for monocytes in wells of 96-well plates, conditioned
medium was collected, microfuged, and mixed with 4x gel-loading SDS
buffer.
[0140] Zymography is essentially SDS-PAGE except that gelatin is
also added to the gel (77, 82). After proteins were resolved based
on molecular weight in SDS-PAGE, the gel was washed in a Triton
X-100 buffer in order to remove SDS which thus allows protein
renaturation. In a calcium-containing "reaction buffer" (77),
gelatinases (MMP-2 and -9) degraded gelatin in their immediate
vicinity (other members of MMPs would also be detected if present
in high amounts). Following staining in Coommasie blue, which binds
all proteins, and destaining , areas containing gelatinases
appeared as clear bands against a dark background, since the
gelatin in its immediate vicinity has been degraded. The size of
the MMP bands was a reflection of the amount of MMP that was
produced by cells, and this was documented by NIH image analysis
software. The molecular weight of the migrated bands revealed the
MMP species, which confirmed previous Western blot analyses and
immunoprecipitations (77, 82).
[0141] The analysis of cell conditioned medium in zymography is a
reflection of the amount of MMPs that was produced by the
particular culture. To determine whether Copolymer 1 was an
inhibitor of the activity of MMPs, independent from its effect on
levels of MMP protein, supernatants from BHK cells, a standard
source of MMPs in many laboratories, or supernatants from T
lymphocytes were resolved by electrophoresis in SDS-PAGE containing
gelatin. During the development of the gelatinolytic activity,
Copolymer 1 was added to the Triton-100 wash, and the "reaction
buffer", described above. An inhibitor of MMP activity prevents
gelatin degradation by gelatinases and the resultant size of the
band of gelatinases would be smaller as compared to that obtained
in the absence of inhibitors.
[0142] Migration Assay
[0143] To address the migration capacity of T lymphocytes,
2.times.10.sup.5 T lymphocytes in 500 .mu.l of 2.5% fetal calf
serum (FCS) containing AIM-V were seeded into the top compartment
of a Boyden chamber (Collaborative Biomedical Products, Bedford,
Md.). This chamber consists of two compartments separated by a
polycarbonate membrane filter (9 mm in diameter, with 3 .mu.m
pores) precoated on its upper surface with fibronectin. Fibronectin
was employed to simulate the basal lamina.
[0144] The bottom compartment of the migration chamber contained
10% FCS-supplemented AIM-V; the higher concentration of FCS in the
bottom chamber served as a directional gradient (77). After 5 h at
37.degree. C., the content of the lower chamber was collected, and
the number of migrated T lymphocytes was counted with the Coulter
Counter Z1.
[0145] Microglial Cells
[0146] Fetal human microglia were isolated from brains obtained at
legal and therapeutic abortions using a protocol described by Lee
et al. (41). Specimens ranged in gestational age from 14 to 20
weeks. 2.5.times.10.sup.4 microglia of over 95% purity were plated
per well of a 96 well plate. Microglia culture medium was minimum
essential medium supplemented with 5% FCS, 0.1% dextrose. For
co-cultures, 100 .mu.l containing 50,000 T lymphocytes in AIM-V (as
described above) was added to individual wells of a 96-well plate
already containing 25,000 microglia (or U937 monocytoid cells see
below) in microglia culture medium. Twenty four hours after,
conditioned medium was collected for cytokine quantifications by
ELISA.
[0147] Adult human microglia were isolated from the resected brain
tissue of patients undergoing surgical resection to treat
intractable epilepsy. Microglia of over 95% purity was obtained
using a previously detailed protocol (90). Cells were used for
interactions with T lymphocytes in a manner identical to that
described for their fetal counter parts.
[0148] A human promonocytoid cell line, U937, was also employed.
Members of this cell line become microglia-like, as assessed by
morphology and expression of cell surface molecules, when treated
sequentially with 50 ng/ml of a protein kinase C activator,
phorbol-12-myristate-13-acetate (PMA) (time 0-48) and 100 U/ml
interferon-.gamma. (IFN.gamma.) (from 48-72 h).
[0149] Cells were used 1-3 days after the IFN.gamma. treatment. As
with microglia, 50,000 T lymphocytes in AIM-V was added to
individual wells of a 96-well plate already containing 25,000
PMA/IFN.gamma.-treated U937 cells, and conditioned medium was
collected after 24 h of co-cultures.
[0150] Cytokine and Chemokine Assays
[0151] Cytokine protein levels in the conditioned medium of
microglia-T lymphocyte co-cultures were measured using
enzyme-linked immunoabsorbent assay (ELISA) kits bought from
BioSource International (Montreal, Canada).
[0152] Chemokine and chemokine receptor expression are currently
recognized to be important mechanisms that regulate the influx of
leukocytes into the CNS. Notable is the up-regulation of the
chemokine IP-10 and its receptor CXCR3 in the lesions of multiple
sclerosis patients (74). Flow cytometry was employed to determine
whether Copolymer 1 affected the expression of the CXCR3 receptor.
Activated T lymphocytes or U937 cells were treated with 25 .mu.g/ml
of Copolymer 1 for 1-3 days.
[0153] Results
[0154] Effect of Copolymer 1 on MMPs
[0155] Activated T lymphocytes secreted MMP-9 into the culture
medium which peaked between 2-3 d of anti-CD3 ligation (FIG. 1).
Treatment of anti-CD3 activated T lymphocytes with Copolymer 1 (25
.mu.g/ml, for 1, 2 or 3 d) did not affect their production of MMP9
as compared to controls (FIG. 1) and neither did a higher
concentration of Copolymer 1 (50 .mu.g/ml). Monocyte production of
MMP-9 was also unaffected by Copolymer 1. In contrast, and in
agreement with a previous report (77), T lymphocytes treated with
IFN.gamma.-1b produced less MMP-9 when compared to control T
lymphocytes (FIG. 1).
[0156] FIG. 2 demonstrates that Copolymer 1 exhibited MMP
inhibitory activity, since the size of the gelatinase band (MMP-9)
was dose-dependently reduced by Copolymer 1 as compared to
controls. In contrast, IFN.gamma.-1b, which inhibits the production
of MMP-9 (FIG. 1), is not a direct inhibitor of MMP enzyme
activity. These results indicate that Copolymer 1 is an inhibitor
of MMP enzyme activity.
[0157] Effect of Copolymer 1 on Cell Migration
[0158] FIG. 3-A shows that the Copolymer 1 did not inhibit the
migration of T lymphocytes when the T lymphocytes were pre-treated
for 1, 2 or 3 days with Copolymer 1 (25 .mu.g/ml) Similarly,
pretreatment of T lymphocytes for 3 days with various
concentrations of Copolymer 1 (1 to 50 .mu.g/ml) did not affect the
trans-migration of activated T lymphocytes (FIG. 3-B).
[0159] In a separate series of experiments, 10 ng/ml monocyte
chemoattractant protein (MCP)-1 or MCP-3 was placed in the bottom
chamber to serve as a chemotactic signal. Under these conditions,
the migration rate of glatiramer pre-treated T lymphocytes also did
not differ from that of controls.
[0160] Effect of Copolymer 1 on T Lymphocyte/Microglia Cultures
[0161] Neither PMA/IFN.gamma.-treated U937 cells nor
anti-CD3-activated T lymphocytes produced detectable levels of
TNF-.gamma. in isolation. However, IL-10was detected in U937 but
not in T lymphocytes conditioned medium (FIG. 4). In co-culture for
24 h, substantial increases in levels of TNF-.gamma. and IL-10 were
obtained. Treatment of T lymphocytes with Copolymer 1 prior to
their encounter with PMA/IFN.gamma.-treated U937 cells reduced, in
a dose-dependent manner, the production of TNF-.gamma. and IL-10
(FIG. 4). The effect of Copolymer 1 appears to be principally on T
lymphocytes since the pretreatment of PMA/IFN.gamma.-treated U937
cells with Copolymer 1 did not influence cytokine production in
subsequent T lymphocyte-U937 interactions.
[0162] Interactions between activated T lymphocyte and
PMA/IFN.gamma.-treated U937 also led to the upregulation of two
Th2-like anti-inflammatory cytokines, IL-4 and IL-13 (FIG. 5),
although the amounts produced were about a log fold lower than
those of IL-10 or TNF-.gamma.. This production of IL-4 and IL-13
was significantly reduced by Copolymer 1 in co-cultures of T
lymphocytes with PMA/IFN.gamma.-treated U937, and this decrease was
also observed for the pro-inflammatory cytokine, IL-12 (FIG. 5). In
summary, Copolymer 1 pretreatment (72 h) of anti-CD3 ligated T
lymphocytes results in the suppression of all inducible cytokines
that were examined in the T lymphocyte-U937 interactions.
[0163] The effect of Copolymer 1 in reducing cytokine production in
T lymphocyte--U937 co-cultures is not a result of a decrease in the
proliferation of T lymphocytes, since Copolymer 1 does not decrease
the number or size of T lymphocyte aggregates that form following
anti-CD3 treatment, indicating that it does not affect the
proliferation of T lymphocytes in any significant manner.
[0164] Indeed, when the total number of cells was counted after 72
h of Copolymer 1, cell numbers were comparable in the control
(24.+-.2.times.10.sup.3) versus Copolymer 1-treated (5, 25 and 50
.mu.g/ml) groups (26.+-.1, 26.+-.2 and 22.+-.1, respectively,
.times.10.sup.3) . Equal numbers of T lymphocytes were added to
microglia or U937 cells in all test situations.
[0165] Fetal human microglia in isolation do not secrete detectable
amounts of IL-10 or TNF-.gamma. into the culture medium; thus, it
appears that these cells are similar to their adult counterparts
(14, 15). In co-culture with activated T lymphocytes, significant
amounts of IL-10 and TNF-.gamma. were produced (139.+-.4 and
1068.+-.68 ng/ml, respectively, FIG. 6). With Copolymer 1
pretreated T lymphocytes, the resultant IL-10 and TNF-.gamma. in T
lymphocyte--microglia co-cultures were significantly reduced (FIG.
6). Indeed, the reduction by Copolymer 1 occurred in a
dose-dependent manner (FIG. 6).
[0166] The interaction of T lymphocytes with adult microglia from
human neural cells led to the upregulation of IL-10 and TNF-.gamma.
from previously undetectable levels, and this was also the case for
IL-1.beta.. IL-6 was constitutively expressed at high levels by
microglia (FIG. 7). The pretreatment of T lymphocytes with
Copolymer 1 resulted in a dose-dependent inhibition of the
inducible cytokines (IL-1.beta., IL-10 and TNF-.alpha.). Similarly,
Copolymer 1 decreased the expression of the constitutive cytokine,
IL-6 (FIG. 7).
[0167] It should be noted that T lymphocytes have to be activated
with anti-CD3 antibody since co-cultures of unactivated T
lymphocytes (even in the presence of 50 U/ml IL-2) with microglia
did not result in increased production of TNF-.gamma.. Moreover, it
is necessary for T lymphocytes to be pretreated with Copolymer 1
since its reducing effect on cytokine production does not occur if
it is added at the time of co-culture.
[0168] FIG. 8 demonstrates that when adult microglia encounter
activated T lymphocytes in vitro, the morphology of microglia
transforms from a ramified/bipolar morphology to an ameoboid
rounded form (14). However, when T lymphocytes were pretreated with
Copolymer 1, the morphological transformation of microglia in T
lymphocyte--microglia co-culture was attenuated. This was also the
case for fetal human microglia or PMA/IFN.gamma.-treated U937 cells
in co-culture with activated T lymphocytes. Overall, the lack of a
morphological transformation of microglia is another indication
that Copolymer 1-pretreatment of T lymphocytes resulted in their
decreased ability to interact with microglia.
[0169] Effect of Copolymer 1 on Chemokines
[0170] FIG. 9 indicates that Copolymer 1 treatment of activated T
lymphocytes did not reduce their expression of CXCR3 after 1-3 days
of treatment; this negative result was also the case for the U937
cell line (FIG. 10). CXCR4 expression was also evaluated and found
to be unresponsive to Copolymer 1 treatment.
[0171] Discussion
[0172] Copolymer 1, or glatiramer acetate (GA), is a heterogeneous
mixture of synthetic random linear copolymers of tyrosine, alanine,
glutamic acid and lysine. This drug is effective in treating
multiple sclerosis.
[0173] The mechanism of action of Copolymer 1 in multiple sclerosis
is based on its capacity to suppress an immune response, which
specifically affects clinical manifestations of the disease. The
subject invention demonstrates that Copolymer 1 has additional
biological activities, such as CNS anti-inflammation and
remyelination. These findings raise the possibility of extending
the potential therapeutic effect of Copolymer 1 in additional
indications other than multiple sclerosis.
[0174] The capability of Copolymer 1 to bind to MHC class II
molecules of various genetic backgrounds, resulting in the
inhibition of T-cell responses, led to the hypothesis that it may
act as a general immunosuppressor.
[0175] Results from in vivo animal models and from Copolymer 1
treated multiple sclerosis patients suggest that, in most cases,
administration of Copolymer 1 in a dosing protocol that affects
multiple sclerosis or EAE does not result in non-specific
immunosuppression. Rather, the beneficial therapeutic effect of
Copolymer 1 in multiple sclerosis and EAE is probably mediated by
antigen-specific suppressor T cells.
[0176] On the other hand, administration of Copolymer 1 using
different protocols (higher doses and/or more frequent
administration) was shown to interfere with alloreactivity in vivo,
prevent experimental GVHD and have a beneficial effect on skin and
thyroid engrafment in mice models. These effects of Copolymer 1 are
most probably mediated by its ability to compete with antigens for
their presentation by MHC molecules on antigen presenting cells,
which has been shown to be dose-dependent.
[0177] Another proof for this postulate is the fact that
D-Copolymer 1--is very active in the mouse GVHD model. In fact--it
is 10 times more active than L-Copolymer 1. Thus, it is reasonable
to predict that using doses of Copolymer 1 higher than those used
for the treatment of multiple sclerosis, will enable it to work as
a general immunosuppressor, and thus to be clinically relevant in
indications other than multiple sclerosis.
[0178] In view of the avid binding of Copolymer 1 to class II
molecules on antigen presenting cells, studies were designed to
examine whether it can interfere with T-cell mediated immune
responses. Copolymer 1 reportedly inhibited in vitro T-cell
responses to MBP in a dose-dependent manner (5, 11, 58, 60, 78,
80). Due to the cross-reactivity between Copolymer 1 and MBP, this
effect can be attributed not only to competition for the binding to
MHC class II molecules, but also to mechanisms related to T-cell
recognition (1, 73). However, the inhibitory capacity of Copolymer
1 was shown to extend also to non cross-reactive T-cell responses
(7, 60, 79, 83). In this case, the inhibitory effect is most
probably due to competition for binding to MHC class II
molecules.
[0179] Copolymer 1 binds to murine and human antigen presenting
cells irrespective of their MHC restriction (promiscuous binding)
in a dose-dependent manner (29, 30). Binding of Copolymer 1 to MHC
class II molecules was demonstrated by its specific inhibition with
anti-class II antibodies (30). Evidence for the direct interaction
of Copolymer 1 with various purified HLA-DR molecules has been
recently reported (28). Furthermore, Copolymer 1 was shown to
compete and even displace antigens already bound to MHC class II
molecules (30, 31, and 79). Binding to MHC class II molecules is
rapid and efficient (30), and no processing seems to be required
for this interaction (29).
[0180] A recent study observed that lymphocytes isolated from
relapsing-remitting multiple sclerosis patients on Copolymer 1 were
significantly reduced in their capacity to transmigrate across a
fibronectin barrier, when compared to cells from untreated multiple
sclerosis patients (59). Fibronectin was used as a model of basal
lamina. Since the transmigration of leukocytes across the
fibronectin barrier is correlated with their expression of matrix
metalloproteineses (42, 77, 88), it was of interest to assess the
effect of Copolymer 1 on MMPs. The subject invention shows that
Copolymer 1 inhibits MMP-9 enzyme activity, but does not decrease
the production of MMP-9 by T lymphocytes or monocytes. This
inhibition does not alter lymphocyte transmigration.
[0181] The findings that Copolymer 1 is an inhibitor of MMP enzyme
activity is relevant to MMP-mediated effects that are unrelated to
leukocyte trafficking. For instance, many cytokines (e.g.
TNF-.gamma. and TGF-.alpha.), cytokine receptors (e.g., TNFRs,
IL-6R.alpha.) and adhesion molecules (e.g., L-selectin, VCAM) are
synthesized in pro-forms that require proteolytic processing to
generate the active agent. While the identity of these
"convertases" or "shedases" remains unresolved, and likely are
members of another group of metalloproteinases (adamalysins), MMPs
have the capacity to convert promolecules (e.g. pro-TNF-.alpha.) to
their active forms (e.g. TNF-.alpha.), hence producing a
proinflammatory environment within the CNS (91). The subject
invention demonstrates the inhibition of MMP enzyme activity by
Copolymer 1, which may decrease the conversion of procytokines to
cytokines, resulting in a non-inflammatory milieu (FIG. 2).
[0182] It has been well documented that many cells are dependent on
attachment to extracellular matrix (ECM) molecules for survival.
Detachment of these cells from their extracellular matrix substrate
results in their apoptosis, a phenomenon that has been refered to
as "anoikis" (64, 65). Because MMPs are the physiological mediators
of extracellular matrix turnover, their aberrant expression and
activity can disrupt the integrity of the extracellular matrix, and
thus result in altered cell adhesion and death (85). By acting as
an inhibitor of MMP activity, glatiramer acetate (Copolymer 1) may
prevent the disruption of the extracellular matrix, and prevent the
detachment of cells from the extacellular matrix. A consequence is
thus decreased neural cell death and thereby the progression of the
disease is slowed. MMPs are also implicated in cell-ECM
interactions that govern processes as diverse as cellular
differentiation, migration and inflammation. In the developing
nervous system, MMP family members regulate angiogenesis, extension
of neuronal growth cones and process formation by oligodendrocytes
(54).
[0183] MMPs may also suppress the production of anti-inflammatory
cytokines, as suggested by the findings that the treatment of EAE
animals with MMP inhibitors led to the increase of the
anti-inflammatory cytokine, IL-4, within the CNS (43); the
mechanism of this activity remains unexplained. Other consequences
of aberrant MMP expression within the CNS can include a direct role
in myelin destruction (48) and the generation of encephalitogenic
fragments of myelin which leads to the propagation of inflammation
(16, 56). The subject finding that Copolymer 1 inhibits MMP enzyme
activity could be relevant to these observations and lead to an
anti-inflammatory milieu in the CNS.
[0184] The encounter of T lymphocytes with microglia is a
significant source of numerous cytokines. Copolymer 1 pretreatment
of T lymphocytes resulted in a substantial diminution of all
cytokines tested in T lymphocyte--microglia (or U937 cell)
interactions.
[0185] Immune deviation is a concept that has gained attention in
recent years. This concept has its origin in the observation that
uncommitted T lymphocytes can differentiate along either the Th1
route, with the production of pro-inflammatory cytokines such as
IFN-.gamma., IL-12 or TNF-.alpha., or into the Th2 pathway with the
production of Th2-like anti-inflammatory cytokines, including IL-4,
IL-10, IL-13 or transforming growth factor-.beta.s (TGF-.beta.s);
cells that produce TGF-.beta.s have also been referred to as Th3
cells. Susceptibility to certain diseases has been attributed to a
predominant Th1 or Th2 response (46).
[0186] The disclosed experiments, using an antigen independent
system, suggest that Copolymer 1 does not have a preferential
effect on Th1 or Th2 type cytokines within the CNS, since all
cytokines, including TNF-.alpha., IL-4, IL-6, IL-10, IL-12 and
IL-13, are suppressed in the T lymphocyte--microglia interactions,
creating a non-inflammatory milieu.
[0187] Although Copolymer 1 lessened the secretion of all cytokines
tested (pro-inflammatory and anti-inflammatory) , treatment of
patients with CNS conditions should result in lessened T
lymphocyte-mediated inflammation. The decreased inflammation should
lessen the severity of the disease. Neuronal and axonal integrity
should also improve in inflammatory CNS disorders since
inflammation is also associated with the destruction of neurons and
axons. Furthermore, individuals at risk for developing CNS diseases
could be treated with Copolymer 1 to prevent the onset of the
disease or lessen its severity. In addition, the lessened
production of TNF-.alpha., a cytokine that is capable of killing
oligodendrocytes (25, 44, 45, 69), would also decrease the degree
of oligodendrocyte loss and demyelination in the CNS of patients on
Copolymer 1. Additionally, Copolymer 1, by inhibition of cytokine
secretion, may decrease the generation of free radicals, which
cause cellular damage and destruction. Therefore, Copolymer 1 would
not only alleviate the symptoms associated with CNS inflammation,
but also slow the progression of the CNS disease itself.
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