U.S. patent application number 14/198603 was filed with the patent office on 2014-09-11 for oral composition comprising a tnf antagonist and use thereof.
This patent application is currently assigned to HADASIT MEDICAL RESEARCH SERVICES & DEVELOPMENT LTD.. The applicant listed for this patent is HADASIT MEDICAL RESEARCH SERVICES & DEVELOPMENT LTD.. Invention is credited to Yaron ILAN.
Application Number | 20140255403 14/198603 |
Document ID | / |
Family ID | 51488093 |
Filed Date | 2014-09-11 |
United States Patent
Application |
20140255403 |
Kind Code |
A1 |
ILAN; Yaron |
September 11, 2014 |
ORAL COMPOSITION COMPRISING A TNF ANTAGONIST AND USE THEREOF
Abstract
This application describes a method of delivering a TNF
antagonist molecule, in a biologically active form, to a subject in
need thereof, the method comprising, orally or mucosally
administering to the subject a therapeutically effective amount of
a TNF antagonist molecule. This application further describes a
method of treating, preventing or reducing the severity of obesity,
the method comprising, administering to the subject a
therapeutically effective amount of a TNF antagonist molecule.
Inventors: |
ILAN; Yaron; (Jerusalem,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HADASIT MEDICAL RESEARCH SERVICES & DEVELOPMENT LTD. |
Jerusalem |
|
IL |
|
|
Assignee: |
HADASIT MEDICAL RESEARCH SERVICES
& DEVELOPMENT LTD.
Jerusalem
IL
|
Family ID: |
51488093 |
Appl. No.: |
14/198603 |
Filed: |
March 6, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61773314 |
Mar 6, 2013 |
|
|
|
Current U.S.
Class: |
424/134.1 |
Current CPC
Class: |
Y02A 50/463 20180101;
C07K 2319/30 20130101; A61K 31/00 20130101; C07K 14/7151 20130101;
A61K 38/1793 20130101 |
Class at
Publication: |
424/134.1 |
International
Class: |
C07K 14/715 20060101
C07K014/715 |
Claims
1. A method of delivering a TNF antagonist molecule, in a
biologically active form, to a subject in need thereof, the method
comprising, orally or mucosally administering to the subject a
therapeutically effective amount of a TNF antagonist molecule.
2. A dosage form for delivery of a TNF antagonist to the GI organs
of a subject, the dosage form comprising, a therapeutically
effective amount of the TNF antagonist.
3. A method for treating or preventing or reducing the severity of
a disease in a subject-in-need thereof, the method comprising
orally or mucosally administering to the subject a therapeutically
effective amount of a TNF antagonist thereby treating or preventing
or reducing the severity of a disease.
4. The method of claim 3, wherein the disease is associated with
bacterial translocation.
5. The method of claim 4, wherein the disease is associated with an
inflammatory process that is suppressed by induction of systemic
regulatory cells.
6. The method of claim 3, wherein the disease is inflammatory bowel
disease (IBD), ulcerative colitis (UC) or Crohn's disease (CD).
7. The method of claim 3, wherein the disease is Alzheimer's
disease, hepatic encephalopathy, ADHD, metabolic syndrome, diabetes
both type 1 and type 2, fatty liver disease, atherosclerosis or
chronic fatigue syndrome.
8. The method of claim 3, wherein the disease is Celiac disease,
Behchet disease, vasculitis or Whipples disease.
9. The method of claim 3, wherein the disease is a chronic liver
disease.
10. The method of claim 3, wherein the disease is NASH or
cirrhosis.
11. The method of claim 10, wherein the disease is augmented by
bacterial translocation or derangement of gut flora.
12. A method of treating, preventing or reducing the severity of
obesity, the method comprising, administering to the subject a
therapeutically effective amount of a TNF antagonist molecule.
13. The method of claim 12, wherein the TNF antagonist molecule is
administered parenterally.
14. The method of claim 12, wherein the TNF antagonist molecule is
administered orally or mucosally.
15. A method of treating, reducing or preventing the effect of
medicine that causes liver intoxication by administering to the
subject in need a therapeutically effective amount of TNF
antagonist, thereby treating, reducing or preventing the effect of
the medicine that causes liver intoxication.
16. The method of claim 15, wherein the TNF antagonist may be
administered sequentially or simultaneously or prior to the
addition of the medicine that causes liver intoxication
17. The method of claim 15, wherein, the TNF antagonist is
administered parenterally.
18. The method of claim 15, wherein the medicine that causes liver
intoxication is acetaminophen.
19. A method of inducing an overall weight loss in a subject or
maintaining a constant weight with a diet enriched in carbohydrates
or fats, without changing the eating habits, the method comprising
administering to the subject a therapeutically effective amount of
a TNF antagonist molecule.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 61/773,314, filed Mar. 6, 2013, which is
hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Tumor necrosis factor (TNF), a naturally occurring cytokine,
plays a central role in the inflammatory response and in immune
injury. TNF is formed by the cleavage of a precursor transmembrane
protein, forming soluble molecules, which aggregate to form
trimolecular complexes. These complexes bind to receptors found on
a variety of cells. The binding of TNF causes many pro-inflammatory
effects, including release of other pro-inflammatory cytokines,
such as interleukin IL-6, IL-8, and IL-1; release of matrix
metalloproteinases; and upregulation of the expression of
endothelial adhesion molecules, further amplifying the inflammatory
and immune cascade by attracting leukocytes into extravascular
tissues. TNF was found to have a main role in the pathogenesis of
several immune mediated disorders, among them rheumatoid arthritis
(RA) and Crohn's Disease. TNF alpha may have a role in the gut in
various local inflammatory and infectious disorders. It is known
that high levels of the pro-inflammatory cytokines,
interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha
(TNF-alpha), are present in the gut mucosa of patients suffering
form various diseases, in particularly inflammatory bowel diseases
(IBD).
[0003] Specific TNF inhibitors/antagonists were developed, such as
infliximab, a chimeric anti-TNF monoclonal antibody (mAb), which
showed significant effect in treating Crohn's Disease and RA, as
well as etanercept, a recombinant fusion protein consisting of two
soluble TNF receptors joined by the Fc fragment of a human IgG1
molecule, which was found effective in the treatment of treating RA
and psoriatic Arthritis. Other TNF blockers are pegylated soluble
TNF Receptor Type I (PEGs TNF-R1), other agents containing soluble
TNF receptors, CDP571 (a humanized monoclonal anti-TNF-alpha
antibodies), thalidomide, phosphodiesterase 4 (IV) inhibitor
thalidomide analogues and other phosphodiesterase IV
inhibitors.
[0004] This therapeutic potential of TNF antagonists and anti TNF
alpha receptor is based on the fact that TNF-alpha is the main
mediator of the inflammatory response in many organ systems.
[0005] Various researches showed that all TNF inhibitors are
immunosuppresssants.
[0006] Parenterally administered soluble anti TNF alpha receptor is
being used or was tested in the treatment of various TNF dependent
inflammatory disorders, such as stomatitis, rheumatoid, juvenile
rheumatoid and psoriatic arthritis, plaque psoriasis and ankylosing
spondylitis. Further the effect of TNF antagonists was assessed in
the the following diseases/conditions: demyelinating diseases,
neurodegenerative diseases, trauma, injuries and the like.
[0007] Oral administration, which is non-invasive, provides many
advantages: ease and convenience of use, improved patient
acceptance and compliance, high degree of vascularization,
relatively lengthy retention time, natural disposal of inactive,
non-metabolized ingredients, direct contact with the
gastrointestinal organs.
[0008] Oral administration of anti TNF or of the TNF receptor
fusion protein also carries an advantage of using the unique
ability of the immune system of the gut to induce regulatory
T-Cells (Tregs) or to alter the systemic immune system in specific
ways that are different and more effective than by intravenous
administration. In addition, oral administration may alter the
systemic immune paradigm suppressing inflammation without exposing
the patient to a general immune suppression and therefore to severe
infections or malignancies that are known to be associated with the
intravenous route of treatment of anti TNF compounds.
[0009] Currently, there is no oral TNF antagonist or anti TNF alpha
receptor in the market due to the high acidity and enzymatic
degradation in the stomach that inactivates or destroys the
molecule before reaching the blood circulation. There is a need for
an oral composition comprising a TNF antagonist that overcomes the
above described drawbacks.
[0010] There is further a need to improve the efficacy of TNF
antagonists in the treatment of inflammation, diseases associated
with inflammation and other diseases, by preventing bacterial
translocation and by inducing systematic regulatory cells.
SUMMARY OF THE INVENTION
[0011] In an embodiment of the invention, there is provided a
method of delivering a TNF antagonist molecule, in a biologically
active form, to a subject in need thereof, the method comprising,
orally or mucosally administering to the subject a therapeutically
effective amount of a TNF antagonist molecule.
[0012] In another embodiment, there is provided a dosage form for
delivery of a TNF antagonist to the GI organs of a subject, the
dosage form comprising, a therapeutically effective amount of a TNF
antagonist molecule.
[0013] In some embodiments, there is provided a method for treating
or preventing or reducing the severity of a disease in a
subject-in-need thereof, the method comprising enterally or
mucosally administering to the subject a therapeutically effective
amount a TNF antagonist thereby treating or preventing or reducing
the severity of a disease.
[0014] In some embodiments, the disease is a chronic liver
disease.
[0015] In some embodiments of the invention, there is provided a
method of treating, preventing or reducing the severity of obesity,
the method comprising, administering to the subject a
therapeutically effective amount of a TNF antagonist molecule.
[0016] In some embodiments of the invention, there is provided a
method of inducing an overall weight loss in a subject or
maintaining a constant weight with a diet enriched in carbohydrates
or fats, without changing the eating habits, the method comprising
administering to the subject a therapeutically effective amount of
a TNF antagonist molecule. This is exemplified in Example 1, in
which mice that were treated with ETANERCEPT for nine days showed a
significant loss in the weight in comparison to control mice.
[0017] In some embodiments, there is provided a method of treating,
reducing or preventing the effect of a medicines that causes liver
intoxication, such as for example, acetaminophen on the liver by
administering to the subject in need a therapeutically effective
amount of TNF antagonist, thereby treating, reducing or preventing
the effect of acetaminophen on the liver.
[0018] The TNF antagonist may be administered sequentially or
simultaneously or prior to the addition of the medicine that causes
intoxication.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 is a bar graph describing the weight loss (in grams)
of mice treated with ETANERCEPT in comparison to control mice.
[0020] FIG. 2 is a bar graph showing that administration of 0.1 mg
of ETANERCEPT resulted in about 87% reduction in the serum levels
of ALT, in comparison to the administration of water only
(p<0.007).
[0021] FIG. 3 is a bar graph showing that administration of 0.1 mg
of ETANERCEPT resulted in about 59% reduction in the serum levels
of IFN-.gamma. as compared to the administration of water only
(p<0.055).
[0022] FIG. 4 is a bar graph showing that administration of 0.1 mg
of ETANERCEPT resulted in a decrease of regulatory T cells (Tregs)
in the liver of treated mice, compared to the administration of
water only (significant results were obtained only in
CD8+CD25+Foxp3+ cells, p<0.008).
[0023] FIG. 5 is a bar graph showing that administration of 0.1 mg
of ETANERCEPT resulted in decrease of regulatory T cells (Tregs) in
the spleen of treated mice, compared to the administration of water
only (significant results were obtained only in CD8+CD25+Foxp3+
cells, p<0.008).
DETAILED DESCRIPTION OF THE INVENTION
[0024] In the following detailed description, numerous specific
details are set forth in order to provide a thorough understanding
of the invention. However, it will be understood by those skilled
in the art that the present invention may be practiced without
these specific details. In other instances, well-known methods,
procedures, and components have not been described in detail so as
not to obscure the present invention.
[0025] The invention relates to methods and compositions for the
administration of TNF antagonist, which may be anti TNF alpha
receptor. More particularly, this invention is directed to a form
of the TNF antagonist Etanrecept, that may be administered
enetrally (oral) or via mucosal administration so as to treat or
prevent or reduce the severity of diseases that are associated with
TNF, autoimmune diseases or diseases associated with inflammatory
diseases.
[0026] In some embodiments, the TNF antagonist molecule is anti TNF
alpha receptor. In some embodiments, the anti TNF alpha receptor is
Etanercept.
[0027] The terms "TNF antagonist, TNF inhibitor, TNF blocker, anti
TNF or anti TNF molecule, biomolecule or compound" interchangeably
refer herein to any compound that prevents or inhibits the binding
of TNF to its receptor and includes specific TNF
inhibitors/antagonists, such as infliximab, a chimeric anti-TNF
monoclonal antibody (mAb), which showed significant effect in
treating Crohn's Disease and RA, as well as Etanercept, a
recombinant fusion protein consisting of two soluble TNF receptors
joined by the Fc fragment of a human IgG1 molecule, which was found
effective in the treatment of RA and psoriatic Arthritis. Other TNF
blockers are pegylated soluble TNF Receptor Type I (PEGs TNF-R1),
other agents containing soluble TNF receptors, CDP571 (a humanized
monoclonal anti-TNF-alpha antibodies), thalidomide,
phosphodiesterase 4 (IV) inhibitor thalidomide analogues and other
phosphodiesterase IV inhibitors.
[0028] As used herein, the term "antibody" is meant to refer to
complete, intact antibodies, and Fab fragments, scFv, and
F(ab).sub.2 fragments thereof. Complete, intact antibodies include
monoclonal antibodies such as murine monoclonal antibodies (mAb),
chimeric antibodies, humanized antibodies and human. The production
of antibodies and the protein structures of complete, intact
antibodies, Fab fragments, scFv fragments and F(ab).sub.2 fragments
and the organization of the genetic sequences that encode such
molecules, are well known and are described, for example, in Harlow
et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1988) and Harlow et al.,
USING ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Press,
1999, which are herein incorporated by reference in their
entirety.
[0029] "Epitope" refers to a region on an antigen molecule to which
an antibody or an immunogenic fragment thereof binds specifically.
The epitope can be a three dimensional epitope formed from residues
on different regions of a protein antigen molecule, which, in a
naive state, are closely apposed due to protein folding. "Epitope",
as used herein, can also mean an epitope created by a peptide or
hapten portion of TNF-alpha and not a three dimensional epitope.
Preferred epitopes are those wherein when bound to an immunogen
(antibody, antibody fragment, or immunogenic fusion protein) result
in inhibited or blocked TNF-alpha activity.
[0030] "TNF-alpha blocking" refers to a compound or composition
that blocks, inhibits or prevents the activity of TNF or
TNF-alpha.
[0031] Compounds that possess TNF-alpha inhibitory activity are for
example tetracyclines, (e.g., tetracycline, doxycycline,
lymecycline, oxytetracycline, minocycline), and chemically modified
tetracyclines (e.g., dedimethylamino-tetracycline), hydroxamic acid
compounds, carbocyclic acids and derivatives, thalidomide,
lazaroids, pentoxifylline, napthopyrans, soluble cytokine
receptors, monoclonal antibodies towards INF-alpha, amrinone,
pimobendan, vesnarinone, phosphodiesterase inhibitors, lactoferrin
and lactoferrin derived analogs, melatonin, norfloxacine,
ofloxacine, ciprofloxacine, gatifloxacine, pefloxacine,
lomefloxacine, temafloxacine, TTP and p38 kinase inhibitors.
[0032] Other TNF inhibitors are specific TNF inhibitors Monoclonal
antibodies such as: infliximab, CDP-571 (HUMICADE.TM.), D2E7
(Adalimumab), and CDP-870; Polyclonal antibodies; Soluble cytokine
such as: receptors etanercept, lenercept, pegylated TNF receptor
type 1, and TBP-1; TNF receptor antagonists; Antisense such as:
oligonucleotides.
[0033] In an embodiment of the invention, there is provided a
method of treating, preventing or reducing the severity of obesity
by administering to the subject a therapeutically effective amount
of a TNF antagonist.
[0034] In some embodiments of the invention, there is provided a
method of treating, preventing or reducing the severity of obesity
by parenterally or orally administering to the subject in need a
therapeutically effective a TNF antagonist.
[0035] In some embodiments of the invention, there is provided a
method of treating, preventing or reducing the severity of obesity
by enterally or mucosally administering to the subject a
therapeutically effective amount a TNF antagonist.
[0036] The immune system and the regulation of adipose tissue
metabolism appear to be closely interlinked. Up to fifty percent of
cells within adipose tissues are composed of non-adipose cells,
including many immunocytes. Most of the research in the field has
been focused on the immunological consequences of morbid obesity.
Immunological alterations that are known to exist in obese animals
and humans include reduced DTH and mitogen-stimulated lymphocyte
proliferation responses, impaired phagocyte number and function,
attenuation of insulin induced lymphocyte cytotoxicity, and changes
in the CD4/CD8 ratio, especially during weight loss attempts.
[0037] Adipose cells are known to secrete pro-inflammatory
cytokines including TNF-.beta. and IL6, which are both related to
the level of adiposity. Some of these cytokines are considered to
have metabolic effects such as insulin resistance mediated by
TNF-.beta. and lipoprotein lipase inhibition mediated by IL6.
TNF-.beta. knockout mice have a higher insulin sensitivity and an
improved lipid profile in comparison to their normal littermates.
Other components of the immune system, which are produced by
adipose cells, include the protein adipsin, which is an integral
part of the alternative complement system, and functions
identically to human complement factor D.
[0038] Several recent studies suggest that the immune system may
have an important contributory role in the development of obesity.
Several cytokines are known to act as adipose tissue regulators.
TNF-.beta. suppresses the expression of P3 adreno-receptors on
adipose cells, which are involved in sympathetically mediated
lipolysis, while IL1 stimulates adipose leptin secretion. The
metabolic activity rate of adipose cells has been observed to be
closely correlated to their distance from the closest lymph node
through a mechanism which is partly mediated by IL4, IL6 and
TNF-.beta.. The changes in inflammatory status of adipose tissue
and liver with obesity suggest that obesity represents a state of
chronic low-level inflammation. Various molecular mechanisms have
been implicated in obesity-induced inflammation.
[0039] The use of a TNF antagonist in the treatment of obesity has
not been demonstrated to date.
[0040] However, it is believed that oral delivery of a TNF
antagonist, such as an anti TNF alpha receptor, may be used for the
treatment of obesity by decreasing the inflammatory mechanism
associated with obesity whether in the gut or systemically.
[0041] Further, a treatment of obesity by administrating TNF
antagonists is expected to exert a beneficial therapeutic effect
due to prolonged exposure and better presentation of TNF
antagonists to the gut immune system, by the mechanisms explained
herein.
[0042] The invention is further related to the treatment of
diseases that are associated or augmented by bacterial
translocation and or gut flora derangement, such as, for example,
without being limited, chronic liver diseases and Alzheimer
disease, hepatic encephalopathy, ADHD, metabolic syndrome, diabetes
both type 1 and type 2, atherosclerosis or chronic fatigue
syndrome, NASH, obesity, hepatic encephalopathy and potentially
several immune mediated disorders among them Alopecia Areata,
Lupus, Anlcylosing Spondylitis, Meniere's Disease, Antiphospholipid
Syndrome, Mixed Connective Tissue Disease, Autoimmune Addison's
Disease, Multiple Sclerosis, Autoimmune Hemolytic Anemia,
Myasthenia Gravis, Autoimmune Hepatitis, Pemphigus Vulgaris,
Behcet's Disease, Pernicious Anemia, Bullous Pemphigoid,
Polyarthritis Nodosa, Cardiomyopathy, Polychondritis, Celiac
Sprue-Dermatitis, Polyglandular Syndromes, Chronic Fatigue Syndrome
(CFIDS), Polymyalgia Rheumatica, Chronic Inflammatory
Demyelinating, Polymyositis and Dermatomyositis, Chronic
Inflammatory Polyneuropathy, Primary Agammaglobulinemia,
Churg-Strauss Syndrome, Primary Biliary Cirrhosis, Cicatricial
Pemphigoid, Psoriasis, CREST Syndrome, Raynaud's Phenomenon, Cold
Agglutinin Disease, Reiter's Syndrome, Crohn's Disease, Rheumatic
Fever, Discoid Lupus, Rheumatoid Arthritis, Essential Mixed,
Cryoglobulinemia Sarcoidosis, Fibromyalgia, Scleroderma, Grave's
Disease, Sjogren's Syndrome, Guillain-Barre, Stiff-Man Syndrome,
Hashimoto's Thyroiditis, Takayasu Arteritis, Idiopathic Pulmonary
Fibrosis, Temporal Arteritis/Giant Cell Arteritis, Idiopathic
Thrombocytopenia Purpura (ITP), Ulcerative Colitis, IgA
Nephropathy, Uveitis, Insulin Dependent Diabetes (Type I),
Vasculitis, Lichen Planus, and Vitiligo. The compositions described
herein can be administered to a subject to treat or prevent
disorders associated with an abnormal or unwanted immune response
associated with cell, tissue or organ transplantation, e.g., renal,
hepatic, and cardiac transplantation, e.g., graft versus host
disease (GVHD), or to prevent allograft rejection, by the enetral
or mucosal administration of biologically active TNF
antagonists.
[0043] The invention is further related to the induction of
systemic regulatory cells whether they are CD4+CD25+Foxp3+ or any
other type of regulatory cells or suppressive cell, including, but
not limited to, LAP+ cells, IL10 secreting cells, TGFbeta secreting
cells, by the enetral or mucosal administration of biologically
active TNF antagonists. These systemic regulatory cells may augment
the effect of TNF antagonists in treating diseases associated with
inflammation in comparison to the effect of parenteral TNF
antagonists. The augmentation of the effect can be via the
induction of suppressive cells, or via any other method that
decreases the number or suppresses the function of pro inflammatory
cells, or decreases the secretion of pro inflammatory cytokines, or
acts by altering antigen presentation to professional or non
professional antigen presenting cells. Any of these mechanisms will
further augment the effect of anti TNF when administered orally in
comparison with intravenous administration. In addition, specific
administration of anti TNF alpha antagonists may also work via
specific mechanisms that are promoted by binding to TNF in the gut,
or to TNF expressed on different types of cells in the gut, thereby
altering the systemic immune system by several mechanisms.
[0044] In an embodiment of the invention, there is provided a
dosage form for delivery of a TNF antagonist into the
gastrointestinal (GI) organs of a subject, the dosage form
comprising a TNF antagonist.
[0045] The invention further provides a method for treating or
preventing or reducing the severity of a disease in a subject in
need thereof, the method comprising enterally or mucosally
administering to the subject a therapeutically effective amount of
a TNF antagonist thereby treating or preventing or reducing the
severity of a disease.
[0046] In some embodiments of the invention, the disease is related
to gastrointestinal inflammation and may be inflammatory bowel
disease (IBD), ulcerative colitis (UC) or Crohn's disease (CD).
[0047] Inflammatory bowel disease (IBD), a form of chronic
gastrointestinal inflammation, includes a group of chronic
inflammatory disorders of generally unknown etiology, e.g.,
ulcerative colitis (UC) and Crohn's disease (CD).
[0048] Immunosuppressive and anti-inflammatory agents in high
maintenance doses are the principal drugs used in the therapy of
chronic inflammatory gastrointestinal disorders. Specific anti-TNF
antibodies have also been used for treatment of IBD. About 20-25%
of the patients with UC failed to respond to intensive and optimal
medical therapy. In general, patients with CD are less responsive
to medical therapy and usually do not respond to surgical
treatment. Anti-TNF alpha antibodies have also been introduced to
treat patients with CD with some efficacy, but this treatment is
ineffective in patients with UC.
[0049] Oral administration of anti TNF or of Etanercept may have at
least equally therapeutic effect to that of intravenous
administration, preventing the unwanted side effects of an invasive
procedure. Oral administration may also have a more profound effect
than that of the intravenous route, by inducing any of the above
described mechanisms. In addition, oral administration may alter
the systemic immune paradigm suppressing inflammation without
exposing the patient to a general immune suppression and therefore
to sever infections, or malignancies that are known to be
associated with the intravenous route of treatment of anti TNF
compounds.
[0050] The method of treating, preventing or reducing the severity
of IBD, CD or UC by enterally or mucosally administering to the
subject a therapeutically effective amount of a TNF antagonist is
expected to be beneficial due to the directed administration to the
GI in comparison to parental treatments with TNF antagonists and
with other oral compositions comprising TNF antagonists.
[0051] Oral administration of anti TNF compounds according to the
invention have prolonged exposure and better presentation of the
anti TNF compounds to the gut immune system. It may also have less
systemic side effects.
[0052] The above can be achieved by at least one of the following
potential mechanisms:
Induction of regulatory T cells, which suppress inflammation
without the induction of generalized immune suppression thus
decreasing the unwanted side effects associated with intravenous
administration, among them malignancies and severe infections.
[0053] Oral administration may alter the Th1 Th2 immune
paradigm.
[0054] Oral administration can suppress the secretion of pro
inflammatory cytokines in a more profound way by activating
specific mechanisms in the gut associated immune system.
[0055] Any of the above, as well as other potential mechanism may
also explain why the anti TNF compounds in an oral form, anti TNF
compounds can exert a more profound effect on the immune system for
suppressing inflammation at the target of the disease, but at the
same time, not induce generalized immune suppression state that is
induced by the intravenous route, thereby omitting the unwanted
side effects associated with the intravenous administration of anti
TNF including exposure to malignancies, severe generalized
infections, tuberculosis and others.
[0056] In another embodiment of the invention, there is provided a
method for treating or preventing or reducing the severity of a
disease or disorder that involves an inflammation in the gut
whether infectious or inflammatory, including but is not limited
to, bacterial, viral, fungal infections in a subject-in-need
thereof, the method comprising enterally or mucosally administering
to the subject a therapeutically effective amount of a TNF
antagonist, thereby treating or preventing or reducing the severity
of the disease or the disorder. In an embodiment of the invention
the disease is Celiac disease, Behchet disease, vasculitis,
Whipples disease, NASH, obesity, degenerative neurological
disorders, Alopecia Areata, Lupus, Anlcylosing Spondylitis,
Meniere's Disease, Antiphospholipid Syndrome, Mixed Connective
Tissue Disease, Autoimmune Addison's Disease, Multiple Sclerosis,
Autoimmune Hemolytic Anemia, Myasthenia Gravis, Autoimmune
Hepatitis, Pemphigus Vulgaris, Behcet's Disease, Pernicious Anemia,
Bullous Pemphigoid, Polyarthritis Nodosa, Cardiomyopathy,
Polychondritis, Celiac Sprue-Dermatitis, Polyglandular Syndromes,
Chronic Fatigue Syndrome (CFIDS), Polymyalgia Rheumatica, Chronic
Inflammatory Demyelinating, Polymyositis and Dermatomyositis,
Chronic Inflammatory Polyneuropathy, Primary Agammaglobulinemia,
Churg-Strauss Syndrome, Primary Biliary Cirrhosis, Cicatricial
Pemphigoid, Psoriasis, CREST Syndrome, Raynaud's Phenomenon, Cold
Agglutinin Disease, Reiter's Syndrome, Crohn's Disease, Rheumatic
Fever, Discoid Lupus, Rheumatoid Arthritis, Essential Mixed,
Cryoglobulinemia Sarcoidosis, Fibromyalgia, Scleroderma, Grave's
Disease, Sjogren's Syndrome, Guillain-Barre, Stiff-Man Syndrome,
Hashimoto's Thyroiditis, Takayasu Arteritis, Idiopathic Pulmonary
Fibrosis, Temporal Arteritis/Giant Cell Arteritis, Idiopathic
Thrombocytopenia Purpura (ITP), Ulcerative Colitis, IgA
Nephropathy, Uveitis, Insulin Dependent Diabetes (Type I),
Vasculitis, Lichen Planus, and Vitiligo. The compositions described
herein can be administered to a subject to treat or prevent
disorders associated with an abnormal or unwanted immune response
associated with cell, tissue or organ transplantation, e.g., renal,
hepatic, and cardiac transplantation, e.g., graft versus host
disease (GVHD), or to prevent allograft rejection.
[0057] The gut immune system is challenged to respond to pathogens
while remaining relatively unresponsive to food antigens and the
commensal microflora. The gut immune system can differentiate the
antigenic signals of parasites from the high background noise of
food and bacterial antigens. The tight regulation that is required
to maintain homeostasis and is based on multiple immune factors.
Immune mechanisms involve both innate responses, mediated
particularly by NK cells, NKT cells, dendritic cells, macrophages,
and polymorphonuclear cells, and adaptive cellular and humoral
immunity, mediated by T and B lymphocytes. Lymphocytes that mediate
the adaptive immune response are primarily located in the
epithelium (intraepithelial lymphocytes), lamina propria, and
Peyer's patches. B-cells produce immunoglobulins, primarily of the
IgA type. The unique structure of the gut mucosa immune system
makes it an attractive site for manipulation of the immune system
including for promoting regulatory T-cells (Tregs) as a means of
treating immune-mediated disorders.
[0058] Gut-associated lymphoid tissue (GALT) comprises several
organs and departments: Peyers patches (PPs) (the appendix, and
isolated lymphoid follicles (ILFs), which are considered to be
inductive sites for mucosal B- and T-cells. The occurrence of other
GALT-like elements, such as lymphocyte-filled villi and
cryptopatches, is species-dependent, and these structures do not
appear to be involved in B-cell induction. Any of the above cells
may play a role in the systemic induction of anti inflammatory
state via oral administration of anti TNF compounds.
[0059] Thus, in an embodiment of the invention, there is provided a
method of inducing of regulatory suppressor cells systemically by
enterally or mucosally administering to the subject a
therapeutically effective amount of a TNF antagonist, thereby
inducing regulatory suppressor T cells systemically. Regulatory
cells can be both the classical CD4+CD25+Foxp3+ and non classical
or other suppressor cells including Th1 cells, LAP+ cells, NKT
cells, or any other subsets of T or B cells.
[0060] According to this embodiment, any type of inflammatory
disorder whether associated with TNF antagonist or not can be
treated by the method described herein.
[0061] In an embodiment of the invention, there is provided a
method of treating, preventing or reducing the severity of chronic
liver disease by enterally or mucosally administering to the
subject a therapeutically effective amount of a TNF antagonist,
thereby treating, preventing or reducing the severity of chronic
liver disease. In an embodiment of the invention, the chronic liver
disease is fatty liver disease including non alcoholic
steatohepatitis (NASH).
[0062] In some embodiments of the invention, there is provided a
method of treating, reducing or preventing the a medicine that
causes liver intoxication by administering to the subject in need a
therapeutically effective amount of TNF antagonist, thereby
treating, reducing or preventing the effect of the medicine on the
liver.
[0063] The TNF antagonist may be administered sequentially or
simultaneously or prior to the addition of the medicine that causes
liver intoxication that may be in some embodiments,
acetaminophen.
[0064] In some embodiments, the TNF antagonist is administered
parenterally.
[0065] In some embodiments of the invention, there is provided a
method of treating, reducing or preventing the effect of medicine
that causes liver intoxication by administering to the subject in
need a therapeutically effective amount of a TNF antagonist,
thereby treating, reducing or preventing the effect of the medicine
that causes liver intoxication that maybe in some embodiments
acetaminophen.
[0066] In an embodiment of the invention, there is provided a
method of treating, preventing or reducing the severity of a
disorder related to bacterial translocation (BT) whether it depends
on a TNF antagonist mechanism or not, such as neurological
disorders including Alzheimer disease, hepatic encephalopathy,
ADHD, metabolic syndrome, diabetes both type 1 and type 2,
atherosclerosis or chronic fatigue syndrome by enterally or
mucosally administering to the subject a therapeutically effective
amount of a TNF antagonist, thereby treating, preventing or
reducing the severity of the disorder associated with BT.
[0067] The rational for these embodiments relies on the relation
between bacterial translocation (BT) and chronic liver disease: gut
flora and BT play an important role in the pathogenesis of chronic
liver disease, including cirrhosis and its complication. Pre
clinical and clinical studies over the last decade suggested a role
for BT in the pathogenesis of non alcoholic steatohepatitis (NASH).
BT and derangement of gut flora is of substantial clinical
relevance to patients with chronic liver disease and cirrhosis.
Intestinal bacterial overgrowth and increased bacterial
translocation of gut flora from the intestinal lumen, in
particular, predispose to an increased potential for bacterial
infections and major complication in these patients. Levels of
bacterial lipopolysaccharide (LPS), a component of Gram-negative
bacteria, are increased in the portal and/or systemic circulation
in several types of chronic liver diseases. Impaired gut epithelial
integrity via alterations in tight junction proteins, increased gut
permeability and increase LPS levels were described in patients
with alcoholic and non alcoholic steatohepatitis. Increased serum
LPS levels and activation of the pro-inflammatory cascade may also
be important in disease progression in these settings.
[0068] Thus, prevention of gut flora derangement and BT by the
administration of a TNF antagonist will prevent the development and
or the progress of chronic liver disorder as well as a disorder
related to bacterial translocation (BT) whether it depends on a TNF
antagonist mechanism or not, such as neurological disorders
including Alzheimer disease, hepatic encephalopathy, ADHD,
metabolic syndrome, diabetes both type 1 and type 2,
atherosclerosis or chronic fatigue syndrome.
[0069] Oral administration of the anti TNF compounds in the form of
cells can further increase the effect of the oral route by any of
the following: inducing prolonged presentation, activating
different types of cells in the gut immune system, and altering the
downstream mechanisms associated with the anti TNF.
[0070] Inflammation is a pathogenic component of various types of
immune mediated disorders, and it contributes to progressive damage
in many organs. Systemic inflammation and chronic damage are
mediated by the innate immune response which is regulated by
Toll-like receptors (TLR). Cells of the innate immune system can
both initiate and maintain inflammation in the liver. Lymphocytes
are activated after interacting at the mesenteric lymph nodes
(MLNs) with bacteria translocated from the gut. Systemic activation
of the inflammatory immune system contributes to the progression of
many disorders. BT starts a Th1 immune response in MLNs involving
Th1 polarization and monocyte activation to TNF-alpha production.
The recirculation of these activated effector immune cells into
blood promotes systemic inflammation. This may be applicable to any
of the following disorders whether TNF plays a direct role in their
pathogenesis or not, as the promotion of Tregs, for example, can
suppress inflammation irrespective of the mechanism of the
inflammation.
[0071] Toll-like receptors (TLRs) recognize pathogen-associated
molecular patterns (PAMPs) to detect the presence of pathogens.
TLRs are expressed on both immune cells, Kupffer cells, endothelial
cells, dendritic cells, biliary epithelial cells, hepatic stellate
cells, and hepatocytes. TLR signaling induces potent innate immune
responses in these and other cell types.
[0072] TLRs also play a role in the regulation of inflammation
based on their ability to recognize endogenous TLR ligands termed
damage-associated molecular patterns (DAMPs).
[0073] Oral route of administration of anti TNF may alter the
function of any of the above mechanisms, thereby further enhancing
their effects.
[0074] The systemic effect of oral administration of soluble TNF
antagonist can lead to alleviation of immune mediated disorders,
and to the alleviation of disorders in which the immune system play
some role, whether the TNF antagonist plays a role or not, by
different mechanisms than induction of Tregs, such as alteration of
TLRs downstream pathways, induction of specific cells in the GALT,
of induction of secretion of cytokines or chemokines whether
directly or indirectly.
[0075] Examples of immune mediated disorders include, but are not
limited to, Obesity, NASH, degenerative neurological disorders,
chronic fatigue syndrome, Alopecia Areata, Lupus, Anlcylosing
Spondylitis, Meniere's Disease, Antiphospholipid Syndrome, Mixed
Connective Tissue Disease, Autoimmune Addison's Disease, Multiple
Sclerosis, Autoimmune Hemolytic Anemia, Myasthenia Gravis,
Autoimmune Hepatitis, Pemphigus Vulgaris, Behcet's Disease,
Pernicious Anemia, Bullous Pemphigoid, Polyarthritis Nodosa,
Cardiomyopathy, Polychondritis, Celiac Sprue-Dermatitis,
Polyglandular Syndromes, Chronic Fatigue Syndrome (CFIDS),
Polymyalgia Rheumatica, Chronic Inflammatory Demyelinating,
Polymyositis and Dermatomyositis, Chronic Inflammatory
Polyneuropathy, Primary Agammaglobulinemia, Churg-Strauss Syndrome,
Primary Biliary Cirrhosis, Cicatricial Pemphigoid, Psoriasis, CREST
Syndrome, Raynaud's Phenomenon, Cold Agglutinin Disease, Reiter's
Syndrome, Crohn's Disease, Rheumatic Fever, Discoid Lupus,
Rheumatoid Arthritis, Essential Mixed, Cryoglobulinemia
Sarcoidosis, Fibromyalgia, Scleroderma, Grave's Disease, Sjogren's
Syndrome, Guillain-Barre, Stiff-Man Syndrome, Hashimoto's
Thyroiditis, Takayasu Arteritis, Idiopathic Pulmonary Fibrosis,
Temporal Arteritis/Giant Cell Arteritis, Idiopathic
Thrombocytopenia Purpura (ITP), Ulcerative Colitis, IgA
Nephropathy, Uveitis, Insulin Dependent Diabetes (Type I),
Vasculitis, Lichen Planus, and Vitiligo. The compositions described
herein can be administered to a subject to treat or prevent
disorders associated with an abnormal or unwanted immune response
associated with cell, tissue or organ transplantation, e.g., renal,
hepatic, and cardiac transplantation, e.g., graft versus host
disease (GVHD), or to prevent allograft rejection.
[0076] As used herein, a "pharmaceutical composition" refers to a
preparation of one or more of the active ingredients described
herein with other chemical components such as physiologically
suitable carriers and excipients.
[0077] The pharmaceutical compositions can take the form of
solutions, suspensions, emulsions, tablets, or capsules. The
pharmaceutical compositions can also take the form of powders,
enteric coated, sustained-release formulations and the like. The
compositions can be formulated with traditional binders and
carriers such as triglycerides, microcrystalline cellulose, gum
tragacanth or gelatin. Oral formulations can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Examples of suitable pharmaceutical carriers are
described in: Remington's Pharmaceutical Sciences" by E. W. Martin,
the contents of which are hereby incorporated by reference herein.
Such compositions will contain a therapeutically effective amount
of TNF antagonist, preferably in a substantially purified form,
together with a suitable amount of carrier so as to provide the
form for proper administration to the subject.
[0078] The amount of a source of the TNF antagonist, which will be
effective in the treatment of a particular disorder or condition
will depend on the nature of the disorder or condition and on the
TNF antagonist, and can be determined by standard clinical
techniques known to a person skilled in the art. In addition, in
vitro assays may optionally be employed to help identify optimal
dosage ranges. The precise dose to be employed in the formulation
will also depend on the route of administration, and the nature of
the disease or disorder, and should be decided according to the
judgment of the practitioner and each patient's circumstances.
Effective doses can be extrapolated from dose-response curves
derived from in-vitro or in-vivo animal model test bioassays or
systems.
[0079] It may be desirable to administer the pharmaceutical
composition of the invention locally to the area in need of
treatment; this can be achieved by, for example, and not by way of
limitation, local infusion, topical application, by injection, by
means of a catheter, by means of a suppository, or by means of an
implant, said implant being of a porous, non-porous, or gelatinous
material.
[0080] For oral applications, the pharmaceutical composition may be
in the form of tablets or capsules, which can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose; a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate; or a glidant such as colloidal silicon dioxide.
When the dosage unit form is a capsule, it can contain, in addition
to the ingredients of the above type, a liquid carrier such as
fatty oil. In addition, dosage unit forms can contain various other
materials which modify the physical form of the dosage unit, for
example, coatings of sugar, shellac, or other enteric agents. The
tablets of the invention can further be film coated.
[0081] For enteric coated compositions, the coating composition
typically contains an insoluble matrix polymer and a water soluble
material. An enteric polymer may be used. Suitable water soluble
materials include polymers such as polyethylene glycol,
hydroxypropyl cellulose, hydroxypropyl methyl cellulose,
polyvinylpyrrolidone, polyvinyl alcohol, and monomeric materials
such as sugars (e.g., lactose, sucrose, fructose, mannitol and the
like), salts (e.g., sodium chloride, potassium chloride and the
like), organic acids (e.g., fumaric acid, succinic acid, lactic
acid, and tartaric acid), and mixtures thereof. Suitable enteric
polymers include hydroxypropyl methyl cellulose, acetate succinate,
hydroxypropyl methyl cellulose, phthalate, polyvinyl acetate
phthalate, cellulose acetate phthalate, cellulose acetate
trimellitate, shellac, zein, and polymethacrylates containing
carboxyl groups. Formulations for oral rely on the
co-administration of adjuvants (e.g., resorcinols and nonionic
surfactants such as polyoxyethylene oleyl ether and
n-hexadecylpolyethylene ether) to increase artificially the
permeability of the intestinal walls, as well as the
co-administration of enzymatic inhibitors (e.g., pancreatic trypsin
inhibitors, diisopropylfluorophosphate (DFF) and trasylol) to
inhibit enzymatic degradation. The active constituent compound of
the solid-type dosage form for oral administration can be mixed
with at least one additive, including sucrose, lactose, cellulose,
mannitol, trehalose, raffinose, maltitol, dextran, starches, agar,
arginates, chitins, chitosans, pectins, gum tragacanth, gum arabic,
gelatin, collagen, casein, albumin, synthetic or semisynthetic
polymer, and glyceride. These dosage forms can also contain other
type(s) of additives, e.g., inactive diluting agent, lubricant such
as magnesium stearate, paraben, preserving agent such as sorbic
acid, ascorbic acid, .alpha.-tocopherol, antioxidant such as
cysteine, disintegrator, binder, thickener, buffering agent,
sweetening agent, flavoring agent, perfuming agent, etc.
[0082] Tablets and pills can be further processed into
enteric-coated preparations. The liquid preparations for oral
administration include emulsion, syrup, elixir, suspension and
solution preparations allowable for medical use. These preparations
can contain inactive diluting agents ordinarily used in said field,
e.g., water. Liposomes have also been described as drug delivery
systems for insulin and heparin (U.S. Pat. No. 4,239,754). More
recently, microspheres of artificial polymers of mixed amino acids
(proteinoids) have been used to deliver pharmaceuticals (U.S. Pat.
No. 4,925,673). Furthermore, carrier compounds described in U.S.
Pat. No. 5,879,681 and U.S. Pat. No. 5,871,753 are used to deliver
biologically active agents orally are known in the art.
[0083] Non-enteric-coated time-dependent release polymers include,
for example, one or more polymers that swell in the stomach via the
absorption of water from the gastric fluid, thereby increasing the
size of the particles to create thick coating layer. The
time-dependent release coating generally possesses erosion and/or
diffusion properties that are independent of the pH of the external
aqueous medium. In such preparations, the active ingredient is
slowly released from the particles by diffusion or following slow
erosion of the particles in the stomach.
[0084] Suitable non-enteric time-dependent release coatings are for
example: film-forming compounds such as cellulosic derivatives,
such as methylcellulose, hydroxypropyl methylcellulose (HPMC),
hydroxyethylcellulose, and/or acrylic polymers including the
non-enteric forms of the Eudragit brand polymers. Other
film-forming materials may be used alone or in combination with
each other or with the ones listed above. These other film forming
materials generally include poly(vinylpyrrolidone), Zein,
poly(ethylene glycol), poly(ethylene oxide), poly(vinyl alcohol),
poly(vinyl acetate), and ethyl cellulose, as well as other
pharmaceutically acceptable hydrophilic and hydrophobic
film-forming materials. These film-forming materials may be applied
to the substrate cores using water as the vehicle or,
alternatively, a solvent system. Hydro-alcoholic systems may also
be employed to serve as a vehicle for film formation.
[0085] Various aspects of the invention are described in greater
detail in the following Examples, which represent embodiments of
this invention, and are by no means to be interpreted as limiting
the scope of this invention.
EXAMPLES
Example 1
[0086] Measurement of the effect of ENBREL.RTM. (ETANERCEPT) on
weight loss
Methods:
[0087] Medicine preparation: One ampoule of ETANERCEPT containing
25 mg was dissolved with 1 ml of vehicle (=25 mg/ml, 2500
.mu.g/1000 .mu.l) according to the manufacturer's instructions.
[0088] Animals: Male C57BL/6 (B6) mice (12-13 weeks old) were
purchased from Harlan Laboratories (Jerusalem, Israel). All mice
were maintained in specific pathogen-free conditions. Mice were
maintained in the Animal Core of the Hadassah-Hebrew University
Medical School. All mice were administered standard laboratory chow
and water ad libitum and kept in a 12-hour light/dark cycle.
[0089] Experiment protocol: Four groups (six mice per group) were
included in the following experiment.
Group A: Control mice, per oz. administration of 30 .mu.l water.
Group B: Per oz. (PO) administrations of 1 mg of ETANERCEPT. Group
C: PO administrations of 0.1 mg of ETANERCEPT. Group D: i.p
administrations of 0.1 mg of ETANERCEPT.
[0090] Treatment was for six consecutive days, followed by one day
without treatment after which the mice were treated for another
three consecutive days. Mice were daily weighed and all mice were
sacrificed on the 9.sup.th day.
Weight was calculated as follows: weight differences were
calculated by subtracting the initial weight (one day before
treatment) of every mouse, from its weight on day 9. Then the
average mean of `weight differences` for every group was
calculated. Results are presented in Table 1 and in FIG. 1
TABLE-US-00001 TABLE 1 Effect of ETANERCEPT on mice weight Weight
Weight before after Weight treatment treatment differences Group
(day 0) (day 9) (gr) Control (water) 27.37 .+-. 1.16 27.10 .+-.
1.57 -0.27 PO, 1 mg of 25.05 .+-. 2.3 24.5 .+-. 2.3 -0.55
ETANERCEPT. PO, 0.1 mg of 26.37 .+-. 4.32 25.87 .+-. 4.23 -0.55
ETANERCEPT i.p, 0.1 mg of 28.42 .+-. 1.12 27.93 .+-. 1.35 -0.49
ETANERCEPT
[0091] As can be seen, the pretreatment with ETANERCEPT in
particularly when administered PO caused a dramatic effect on
weight loss in the mice.
Example 2
Effect of ENBREL.RTM. (ETANERCEPT) in Concanvalin A (ConA)
Model
Introduction:
[0092] Several animal models mimicking human liver injury are known
to be applied for exploring the immunopathogenesis in liver
diseases. However, an acceptable in vivo model for hepatitis C
virus HCV does not exist, as HCV is a non cytopathic virus and its
liver damage is immune mediated.
[0093] ConA induced hepatitis. The Concanvalin A (ConA) model is a
widely utilized mouse model that mimics many aspects of human
autoimmune hepatitis. ConA is a bean lectin, which when injected
intravenously to mice, induces activation of lymphocytes in the
liver. ConA induces massive liver necrosis in mice, simultaneously
with the lymphocyte infiltration in the liver, high level of
apoptotic hepatocytes and elevated serum alanine aminotransferase
(ALT) and aspartate aminotransferase (AST). The activated
lymphocytes in the liver injury were later on confirmed to be
Natural Killer T (NKT) cells. Together with Kupffer cells, NKT
cells secrete large amounts of various hepatotoxic cytokines, most
notably IFN-.gamma. and TNF-.alpha.. The immune-mediated liver
injury which develops in the ConA model is very rapid: less than 24
hours. The peak for liver enzymes and proinflammtory cytokines is
less then 15 h and therefore it is an efficient in vivo model for
screening anti inflammatory agents. Here, using the ConA model, the
efficacy of ETANERCEPT in the preventing of hepatic, immunologic
disorder was demonstrated. The results clearly demonstrate an
anti-inflammatory effect of ETANERCEPT.
Methods:
[0094] Medicine preparation: One ampoule of ETANERCEPT containing
25 mg was dissolved with 1 ml of vehicle (=25 mg/ml, 2500
.mu.g/1000 .mu.l) according to the manufacturer's instructions.
[0095] Animals: Male C57BL/6 (B6) mice (12-13 weeks old) were
purchased from Harlan Laboratories (Jerusalem, Israel). All mice
were maintained in specific pathogen-free conditions. Mice were
maintained in the Animal Core of the Hadassah-Hebrew University
Medical School. All mice were administered standard laboratory chow
and water ad libitum and kept in a 12-hour light/dark cycle. Each
group of mice (four mice in a group) was administered with either
0.1 mg ETANERCEPT (treated mice) or 100 .mu.l water (control).
[0096] Experiment protocol: i.p administrations of 0.1 mg of
ETANERCEPT (treated mice) and 100 .mu.l water (control mice) were
performed once a day for three consecutive days prior to ConA
injection. Con A was purchased from MP Biomedicals (Ohio, USA). Con
A (20 mg/kg) was dissolved in 200 .mu.l of 50 mM Tris pH 7, 150 mM
NaCl, 4 mM CaCl.sub.2, and injected (500 .mu.g/mouse) intravenously
to tail vein of all mice. All mice were sacrificed after 14 h. Sera
from individual mice were obtained 14 h after ConA injection. The
serum activities of ALT and AST were determined after 1:20
dilutions, using a Reflovet Plus clinical chemistry analyzer. Serum
levels of IFN-.gamma. were determined by "sandwich" ELISA using
commercial kits (Quantikine, R&D Systems, Minneapolis, Minn.,
US), according to the manufacturer's instructions. After mice were
sacrificed, livers and spleens from all mice were removed for the
use of flow cytometry (FACS) in order to detect changes in the
expression of hepatic and splenic markers (regulatory T cells,
Tregs).
[0097] Isolation of splenocytes and intrahepatic lymphocytes:
Livers and spleens were removed after sacrifice and stored in
RPMI-1640 supplemented with Fetal Calf Serum. Spleens were crushed
through a 70-.mu.m nylon cell strainer (Falcon) and centrifuged
(1250 rpm for 7 min). Red blood cells were lysed in 1 ml of cold
155 mM ammonium chloride lysis buffer. Splenocytes were washed and
resuspended in 1 ml of RPMI supplemented with FCS. The viability of
cells as assessed by trypan blue exclusion exceeded 90%. For
intrahepatic lymphocytes, livers were crushed through a stainless
mesh (size 60, Sigma). Ten milliliters of Lymphoprep (Ficoll,
Axis-Shield PoC AS, Oslo, Norway) was loaded with a similar volume
of the cell suspension in 50-ml tubes. The tubes were centrifuged
at 1800 rpm for 18 min. Cells present in the interface were
collected and centrifuged again at 1800 rpm for 10 min to obtain a
pellet of cells depleted of hepatocytes. Approximately
1.times.10.sup.6 cells/mouse liver were recovered.
Flow cytometry for lymphocyte subsets: Flow cytometry was performed
following splenocyte and hepatic lymphocyte isolation using
1.times.10.sup.6 lymphocytes in 100 .mu.l PBS with 0.1% BSA. For
surface staining, cells were incubated with fluorochrome-conjugated
antibodies to the indicated cell surface markers (eBioscience, San
Diego, Calif., USA) at the recommended dilutions or with isotype
control antibodies for 30 minutes at 4.degree. C. The following
cell surface anti-mouse antibodies were used: CD4-FITC, CD25-PE and
CD8-APC. Cells were then washed in PBS containing 1% BSA and fixed
with fixation buffer (eBiosciences) for another 50 minutes. For
intracellular staining of Foxp3, fixed cells were permeabilized
with Foxp3 staining buffer (eBioscience). Cells were then stained
with PE-Cy7-conjugated antibodies to Foxp3 (eBiosciences), washed
twice and resuspended in 250 .mu.l of PBS containing 1% BSA and
kept at 4.degree. C. One million stained cells in 250 .mu.l of PBS
containing 1% BSA were subsequently analyzed using a FACS LSR II
instrument (Becton Dickinson, San Jose, Calif.) with FCS express
V.3 software (DeNovo software, CA, USA). Only live cells were
counted, and background fluorescence from non-antibody-treated
lymphocytes was subtracted.
[0098] Results
[0099] Table 2 presents the effect of ETANERCEPT on the serum
levels of the liver enzyme ALT.
TABLE-US-00002 TABLE 2 Effect on serum levels of ALT in ConA model
Group ALT (u/L) Cont, water 5680 .+-. 2850 0.1 mg 698 .+-. 792
ETANERCEPT
[0100] As can be seen from the above table, the administration of
0.1 mg of ETANERCEPT resulted in about 87% reduction in the serum
levels of ALT, compared to the administration of water only
(p<0.007).
[0101] These results are also presented in FIG. 2.
TABLE-US-00003 TABLE 3 Effect on serum levels of IFN-.gamma. in
ConA model IFN-g Group (pg/ml) Cont, water 1651.9 .+-. 783 0.1 mg
676.5 .+-. 250 ETANERCEPT
[0102] As can be seen from the above table, administration of 0.1
mg of ETANERCEPT resulted in about 59% reduction in the serum
levels of IFN-.gamma., compared to the administration of water only
(p<0.055). These results are also presented in FIG. 3.
[0103] The effect of ETANERCEPT on hepatic Tregs in ConA model:
ETANERCEPT or water orally administered as described above. Liver
lymphocyes were prepared as described from all mice. One million
cells were analyzed for the expression of CD4, CD8, CD25 and FOXP3.
The numbers of purified CD4+CD25+, CD4+CD25+Foxp3, CD8+CD25+ and
CD8+CD25+Foxp3 cells were calculated. Data are shown in table 4 as
the mean percentage .+-.SD in each group.
TABLE-US-00004 TABLE 4 Effect on regulatory T cells in hepatic
distribution in ConA model Cont, 0.1 mg Treg sub type water
ETANERCEPT CD4+CD25+ 6.6% .+-. 2.6 3.8% .+-. 2.5 CD4+CD25+Foxp3+
2.3% .+-. 1.1 1.5% .+-. 0.9 CD8+CD25+ 3.1% .+-. 1.6 1.8% .+-. 1.1
CD8+CD25+ 13% .+-. 1.8 2.85% .+-. 3.1 Foxp3+
[0104] As can be seen from the above table, administration of 0.1
mg of ETANERCEPT resulted in decrease of regulatory T cells (Tregs)
in the liver of treated mice, compared to the administration of
water only (significant results were obtained only in
CD8+CD25+Foxp3+ cells, p<0.008). These results are also
presented in FIG. 4.
[0105] The effect of ETANERCEPT on splenic Tregs in ConA model:
ETANERCEPT or water orally administered as described above.
Splenocytes were prepared as described from all mice. One million
cells were analyzed for the expression of CD4, CD8, CD25 and FOXP3.
The numbers of purified CD4+CD25+, CD4+CD25+Foxp3, CD8+CD25+ and
CD8+CD25+Foxp3 cells were calculated. Data are shown in Table 5 as
the mean percentage .+-.SD in each group.
TABLE-US-00005 TABLE 5 Effect on regulatory T cells in splenocytes
distribution in ConA model Cont, 0.1 mg Treg sub type water
ETANERCEPT CD4+CD25+ 8.8% .+-. 2.1 5.6% .+-. 0.4 CD4+CD25+Foxp3+
2.6% .+-. 0.7 1.3% .+-. 0.2 CD8+CD25+ 2.7% .+-. 0.6 3.3% .+-. 1.6
CD8+CD25+ 8.0% .+-. 4.9 1.6% .+-. 0.5 Foxp3+
[0106] As can be seen from the above table, the administration of
0.1 mg of ETANERCEPT resulted in decrease of regulatory T cells
(Tregs) in the spleen of treated mice, compared to the
administration of water only. These results are also presented in
FIG. 5.
* * * * *