WO2010048731A1

WO2010048731A1 - Aminoacyl trna-synthetase inhibitors as broad-spectrum immunosuppressive agents

Info

Publication number: WO2010048731A1
Application number: PCT/CA2009/001582
Authority: WO
Inventors: Christopher John Ong; Nadya Ogloff
Original assignee: Christopher John Ong; Nadya Ogloff
Priority date: 2008-10-31
Filing date: 2009-11-02
Publication date: 2010-05-06
Also published as: WO2010048731A8

Abstract

Autoimmune disorders, transplant rejection, and inflammatory disorders share a common origin in that the host's immune system is activated such that it attacks otherwise healthy tissue. Aminoacyl t-RNA synthetase inhibitors, such as borrelidin, have now been shown to act as broad spectrum immunosuppressants. Said compounds, and compositions comprising said compounds, are useful in the treatment of the aforementioned diseases.

Description

AMINO ACYL tRNA- SYNTHETASE INHIBITORS AS BROAD-SPECTRUM IMMUNOSUPPRESSIVE AGENTS

TECHNICAL FIELD The present invention relates to the fields of inflammation and immunologic diseases.

BACKGROUND OF THE INVENTION

Autoimmune diseases and inflammation are conditions triggered by aberrant reactions of the human immune system. In autoimmunity, the patient's immune system is activated against the body's own proteins. In inflammatory conditions, it is the overreaction of the immune system, and its subsequent downstream signaling, which causes problems. Transplant rejection occurs when a transplanted organ or tissue fails to be accepted by the body of the transplant recipient. This happens when the immune system of the recipient attacks the transplanted organ or tissue because it recognizes the transplanted organ/tissue as foreign.

Inflammatory Conditions

Inflammation is the body's normal physiological response to injury, infection or foreign substances. The inflammatory response is initiated in response to injury (e.g., and foreign particles) and infection {e,g._r bacterial or viral infection) by multiple events, including chemical mediators (e.g., cytokines and prostaglandins) and inflammatory cells (e.g., leukocytes). Clinically, inflammation is characterized by increased blood flow to the tissue, causing pyrexia, erythema, induration, and pain. Although the ability to mount an inflammatory response is essential for survival, the ability to control inflammation is also necessary for health. The response requires a well-balanced interplay between the humoral and cellular immune elements of the adaptive immune response to eliminate harmful agents and allow for the initiation of repair of damaged tissue. When this delicately balanced interplay is disrupted, the inflammatory response may result in considerable tissue damage and altering the function of affected tissue often causing more harm than the original insult that initiated the reaction. Inflammatory diseases are a group of disorders that are characterized by uncontrolled or excessive inflammatory responses and clinical intervention is needed to prevent tissue damage and organ dysfunction. Diseases such as Crohn's disease, asthma, allergies, septic shock syndrome, atherosclerosis, inflammatory bowel disease among other clinical conditions are characterized by chronic inflammation.

Autoimmune Conditions A substantial proportion of the population suffers from autoimmune disease conditions, which are often chronic, debilitating, and life-threatening. There are more than 60 identified autoimmune disorders that include rheumatoid arthritis, psoriasis, multiple sclerosis, idiopathic thrombocytopenia purpura, vitiligo, systemic lupus erythematosus, Sjogren's syndrome, myasthenia gravis, and uveitis.

Transplant Rejection

Rejection is the consequence of the recipient's alloimmune response to the nonself antigens expressed by donor tissues.

Following transplantation, T cells are activated by the presence of allogeneic human leukocyte antigen (HLA) molecules. The HLA genes involved in the immune response have been classified into class I and II; both class I and II molecules act to present antigens to T cells, a process that initiates the adaptive immune response.

Allogeneic MHC antigens provoke the strongest immunologic responses. These reactions can cause rapid elmύnation of donor cells and graft rejection, thus representing the major hurdle to successful allograft engraftment.

T cells recognize the allogeneic HLA molecules either directly or indirectly. In direct presentation, T cells recognize the determinant peptides on the intact HLA molecules displayed on the surface of the transplanted cells. In indirect presentation,, donor HLA molecules are processed and presented as peptides by the recipient's antigen- presenting cells (APCs). APCs, which include dendritic cells, macrophages, B cells, and endothelial cells, possess co-stimulatory adhesion molecules that serve as ligands for counterreceptors on T cells. APCs also provide essential co-stimulatory signals that promote T-cell proliferation and differentiation into either helper or effector lymphocytes.

In hyperacute rejection, transplant patients are serologically presensitized to allσantigens (i.e., graft antigens are recognized as nonself). Histologically, numerous polymorphonuclear leukocytes (PMNs) exist within the graft vasculature and are associated with widespread microthrombi formation and platelet accumulation. Little or no leukocyte infiltration occurs. Hyperacute rejection manifests within minutes to hours of graft implantation but rejection has become relatively rare since the introduction of routine pretransplantation screening of graft recipients for antidonor antibodies.

In acute rejection, graft antigens are recognized by T cells; the resulting cytokine release eventually leads to tissue distortion, vascular insufficiency, and cell destruction. Histologically, leukocytes are present, dominated by equivalent numbers of macrophages and T cells -within the interstitium. These processes can occur within 24 hours of transplantation and occur over a period of days to weeks.

In chronic rejection, pathologic tissue remodeling results from peritransplant and posttransplant trauma. Cytokines and tissue growth factor induce smooth muscle cells to proliferate, to migrate, and to produce new matrix material. Interstitial fibroblasts are also induced to produce collagen. Histologically, progressive neointimal formation occurs within large and medium arteries and, to a lesser extent, within veins of the graft. Leukocyte infiltration usually is mild or even absent. All these result in reduced blood flow, with subsequent regional tissue ischemia, fibrpsis, and cell death.

Immunosuppression

A number of potent immunosuppressive compounds of microbial origin have been used in the treatment or prevention of transplant rejection, including cyclosporin and the macxolidese, rapamycin and fujimycin. Rapamycin inhibits response to interleuJrin-2 (IL- 2) and thereby blocks activation of T lymphocytes. Cyclosporin is thought to bind to cyclophilin of immunocompetent lymphocytes, including T lymphocytes which inhibits calcineurin thus preventing a transcription of mterlpukin-2. In contrast, fujimycin inhibits production of IL-2. Synthetic immunosuppressants such as mycophenolate and dexamethasone have also been used for transplant rejection. The latter drug is a glucocorticoid exhibiting a broad-spectrum of anti inflammatory and immunosuppressant activities.

Aminoacyl-t-RNA (aa-RS) synthetase inhibitors are agents that inhibit activity of enzyme(s) that catalyze transfer of amino acids to their cognate tRNA molecules. The largest group of such inhibitors function as reaction-intermediate mimics. Examples are mupirocin as well as sulfamoyladenoisine analogues of aa-AMP, the latter group being a form of "universal" aa-RS synthetase inhibitor. Individual inhibitors of particular aa-RS synthetases are known, such as REP8839 (a selective inhibitor of bacterial Met RS), reverornycin (an inhibitor of human He RS) and the macrolide-polyketide, borrelidin which inhibits Thr-RS. Inhibitors of aa-RS synthetases are noted as having antibacterial and antiproliferative activity. For example, borrelidin is known as an antibacterial agent as well having antiangiogenic and antimetastatic properties.

It has been reported that analogues of aa-AMP bearing different amino acids (based on a sulfaraoyladenosine structure) inhibit proliferation of stimulated T-cells (van, de Vijver, P. et αl (2008) J, Med. Chem, 51: 3020-3029).

SUMMARY OF THE EWENTION

This invention is based on. the discovery that aa-RS synthetase inhibitors demonstrate broad-spectrum immunosuppressant activity against immune cells of both myeloid and lymphoid origin, thus being capable of suppressing both innate and adaptive immune responses. Potency and selectivity of such inhibitors with regard to immune cells makes such inhibitors useful in modulating a plurality of immune responses. Such agents can be useful in the treatment of inflammatory diseases or conditions having an immune cell component, autoimmune diseases as well as other conditions and diseases having an immune component (such as asthma and allergic responses). Diseases or conditions that would benefit from a multi-prong suppression of different immune cell participants may be treated. Furthermore, since it is now known that inhibitors of aminoacyl-tRNA synthetase enzymes effect both activation and proliferation of various immune cells, such inhibitors show value for minimizing the onset of adverse immunological responses making such agents particularly useful in situations such as prevention of transplant rejection. For example, such inhibitors may be administered prior to or at the same time as transplantation in an effort to reduce activation of the host's T cells by the presence of allogeneic antigens introduced with or expressed by the donor tissue: The ability of such" inhibitors to affect B cells, mast cells, macrophages and neutrophils contribute to the broad-spectrum effeds of such inhibitors. Reduced effect of such inhibitor on nonimmune cells of a similar lineage (fibroblasts) highlight the selective action of such inhibitors. Various embodiments of the invention provide use of an aminoacyl tRNA synthetase (aa-RS) inhibitor as aa inhibitor of immune cells, providing that when the immune cells to be inhibited are solely T-cells and the inhibitor comprises adenine joined to a sugar molecule as a substituent, the inhibition comprises inhibition of activation of the T-cells.

Various embodiments of the invention provide use of an aminoacyl tRNA synthetase (aa-RS) inhibitor for preparation of a medicament for use as an inhibitor providing that when the immune cells to be inhibited are solely T-cells and the inhibitor comprises adenine joined to a sugar molecule as a substitαent, the inhibition comprises inhibition of activation of the T-cells.

Various embodiments of the invention provide a pharmaceutical composition for use as an inhibitor of immune cells, wherein the composition comprises a pharmaceutically acceptable carrier or excipient and. an aminoacyl tRNA synthetase (aa- RS) inhibitor providing that when the immune cells to be inhibited are solely T-cells and the inhibitor comprises adenine joined to a sugar molecule as a substituent, the inhibition comprises inhibition of activation of the T-cells.

Various embodiments of the invention provide a pharmaceutical formulation for use as an inhibitor of immune cells, wherein the formulation is in oral, topical, parenteral or inhalation foim and comprises an aminoacyl tRNA synthetase (aa-RS) inhibitor providing that when the immune cells to be inhibited are solely T-cells and the inhibitor comprises adenine joined to a sugar molecule as a substituent, the inhibition comprises inhibition of activation of the T-cells. Various embodiments of the invention a method for treatment or prevention of an inflammatory condition or suppression of immune cells in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of an amino acid tRNA synthetase (aa-RS) inhibitor, providing that when immune cells to be inhibited are solely T-cells and the inhibitor comprises adenine joined to a sugar- molecule as a substituent, the inhibition comprises inhibition of activation of the T-cells.

In accordance with one embodiment, there is provided a method of treating inflammation in a subject. The method involves administering to the subject an effective dose of an aminoacyl tRNA synthetase inhibitor. The method may involve treating inflammation which includes an adaptive immune response or an innate immune response. The method may further involve treating inflammation which is associated with an autoimmune disease. The method may further involve treating inflammation which

, includes the activation of any one of the following cell types: a T cell, a B cell, a macrophage, a mast cell, a neutrophil, or a monocyte. The method may further involve treating inflammation which includes tine proliferation of any one of the following cell types: a B cell, a macrophage, a mast cell, a neutrophil, or a monocyte. The method may further involve treating inflammation which is not associated with rheumatoid arthritis or psoriasis. The method may involve treating intlammation with, for example, a threonyϊ tRNA synthetase inhibitor, or a borrelidin compound or borrelidin.

In accordance with another embodiment, there is provided an aminoacyl tRNA synthetase inhibitor for treating inflammation. The inhibitor may be used to treat inflammation which includes an adaptive immune response or an innate immune response, or inflammation which is associated with an autoimmune disease, or inflammation which includes the activation of any one of the following cell types: a T cell, a B cell, a macrophage, a mast cell, a neutrophil, or a monocyte. The inhibitor may be used to treat inflammation which includes the proliferation of any one of the following cell types: a B cell, a macrophage, a mast cell, a neutrophil, or a monocyte. The inhibitor may be used to treat inflammation which is not associated with rheumatoid arthritis or psoriasis. The inhibitor may include, for example, a threonyl tKNA synthetase inhibitor, a borrelidin compound or borrelidin.

In accordance with another embodiment, there is provided a pharmaceutical composition for treating inflammation. The composition includes an aminoacyl tKNA synthetase inhibitor and a pharmaceutically acceptable carrier. The composition may be used to treat inflammation which includes an adaptive immune response or an innate immune response, inflammation which is associated with an autoimmune disease, or inflammation which includes the activation of any one of the following cell types: a T cell, a B cell, a macrophage, a mast cell, a neutrophil, or a monocyte. The composition may be used to treat mflammatkαi which includes the proliferation of any one of the following cell - types: a B cell, a macrophage, a mast cell, a neutrophil, or a monocyte. The composition may be used to treat inflammation which is not associated with rheumatoid arthritis or psoriasis. The composition may include, for example, a threonyl tRNA synthetase inhibitor, or a borrelidin compound or botxelidin.

In accordance with another embodiment, there is provided a use for an aminoacyl tRNA synthetase inhibitor for treating inflammation. Ih accordance with another aspect of the invention, there is provided a use for a aminqacyl tRNA synthetase inhibitor in the manufacture of a medicament for treating inflammation. The uses may involve treating inflammation which includes an adaptive immune response or an innate immune resuonse. Further, the uses may involve treating inflammation which is associated with an autoimmune disease. The uses provided herein may also involve treating inflammation with the inhibitor or medicament which includes the activation of any one of the following cell types: a T cell, a B cell, a macrophage, a mast cell, a neutrophil, or a monocyte. Further, the uses may involve treating inflammation which includes the proliferation of any one of the following cell types: a B cell, a macrophage, a mast cell, a neutrophil, or a monocyte. Further, the uses may involve treating raflammation which is not associated with rheumatoid arthritis or psoriasis. The uses may involve, for example, the use of threonyl tRNA synthetase inhibitors, or borrelidjbα compound or borrelidin. In accordance with another aspect of the invention, a method is described that uses borrelidin alone or in combination with other medication or intervention as an immunosuppressive drug during organ transplantation.

In accordance with another aspect of the invention, there is provided a kit for detecting tumor necrosis factor (TNF) in a sample, the kit comprising at least one antibody specific for TNF.

In accordance with another aspect of the invention there is provided a method for detecting B and T cell proliferation in a subject, the method comprising the use of 3H- Thymidine incorporation into dividing cells as a measure of T or B cell proliferation.

This invention also provides use of aminoacyl tRNA synthetase inhibitors for in vitro application in the inhibition of the immune cells described herein.

This invention also contemplates ex vivo treatment of immune cells which are intended for introduction into a subject.

The present invention is based, in part, on the discovery that aminoacyl tRNA synthetase inhibitors- are -able- to inhibit B cell -proliferation- and function,macrophage. function, and T cell function.

In accordance with one aspect of the invention, a method is described that inhibits B cell proliferation in a subject having a B-cell dependent autoimmune disease condition, through inhibiting aminoacyl tRNA synthetase activity. Such compound may be selected from a group of aminoacyl tRNA synthetase inhibitors. In accordance with another aspect of the invention, a method is described that uses aminoacyl tRNA synthetase alone or in combination with other medication or intervention to treat B cell dependent autoimmune diseases. In accordance with, yet another aspect of the invention, a method is described that inhibits macrophage proliferation in a subject having an inflammatory disease condition, through inhibiting aminoacyl tRNA synthetase activity. Such compound may be selected from a group of aminoacyl tRNA synthetase inhibitors. In accordance with another aspect of the invention, a method is described that uses aminoacyl tRNA synthetase inhibitors alone or in combination with other medication or intervention to treat inflammatory diseases.

In accordance with yet another aspect of the invention, a method is described that inhibits T cell proliferation and/or activation in a subject having an organ transplantation, through inhibiting aminoacyl tRNA synthetase activity. Such compound may be selected from a group of aminoacyl tRNA synthetase inhibitors.

In accordance with another aspect of the invention;, a method is described that uses aminoacyl tRNA synthetase inhibitors alone or in combination with other medication or intervention as an immunosuppressive drug during organ transplantation. In accordance with another aspect of the invention, there is provided a kit. for detecting tumor necrosis factor (TNF) in a sample, the kit comprising at least one antibody specific for TNF.

Other aspects and features of the present invention would become apparent to those ordinarily skilled in the art by reviewing the following description of specific embodiments of the invention in conjunction with accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

In drawings which illustrate embodiments of the invention,

Figure 1 is a graph showing the effect of borrelidin on B-cell proliferation. Figure 2 is a graph showing the effect of borreiidiα on T-cell proliferation. Figure 3 is graph showing the effect of borrelidin on fibroblasts.

Figure 4 is a graph showing the effect of borrelidin on the production of cytokines by T-cells upon stimulation. Figures 5a and 5b show the expression of CD25 and CD69 in borrelidin-treated CD4-positive T-cells.

Figure 6 is a graph showing the effect of L-threonine on cell growth in borrelidin treated primary T-cells. Figure 7 is a photograph of an immunoblot for the C/EBP homologous protein

(CHOP) protein.

Figure 8 is graph showing the effect of borrelidin on LPS-induced TNFα levels in splenocytes.

Figures 9a and 9b are graphs showing the effect of borrelidin on the translation of TNF-α in primary macrophages.

Figure 10 is a graph showing the effect of borrelidin on lipopolysaccharide- induced cytokine production in primary mouse splenocytes.

Figure 11 is a graph showing the effect of borrelidin on apoptosis in macrophages purified from splenocytes, Figure.12 is a graph showing the effect of borrelidin on Annexin V expression and caspase 3 activation in macrophages purified from splenocytes.

Figures 13a and 13b are graphs showing the effect of borrelidin on m RNA transcripts for TNFα and CHOP in primary macrophages.

Figure 14 is a graph showing the effect of borrelidin on TNF -α-release from anti- IgE murine bone marrow derived mast cells.

Figure 15 is a graph showing the effect of borrelidin on myeloperoxidase (MPO) levels in vivo.

Figure 16 is a graph showing the effect of borrelidin on neutrophil recruitment into the conjunctiva.

DETAILED DESCRIPTION

Any terms not directly defined herein shall be understood to have the meanings commonly associated with them as understood within the art of the invention. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the cornppsitions, devices, methods and the like of embodiments of the invention, and how to make or use them. It will be appreciated that the same thing may be said in more than one way. Consequently, alternative language and synonyms maybe used for any one or more of the terms discussed herein. No significance is to be placed upon whether or not a term is elaborated or discussed herein. Some synonyms or substitutable methods, materials and the like are provided. Recital of one or a few synonyms or equivalents does not exclude use of other synonyms or equivalents, unless it is explicitly stated. Use of examples to the specification, including examples of terms, is for illustrative purposes only and does not limit the scope and meaning of the embodiments of the invention herein.

In accordance with another aspect of the invention, a method is described that uses botrelidin alone or in combination with other medication or intervention as an immunosuppressive drug during organ transplantation. In accordance with another aspect of the invention, there is provided a kit for detecting tumor necrosis factor (TNF) in a sample, the kit comprising at least one antibody specific for TNF.

In accordance with another aspect of the invention there is provided a method for detecting B and T cell proliferation in a subject, the method comprising the use of 3H- Thymidine incorporation into dividing cells as a measure of T or B cell proliferation- - .. This invention also provides use of aminoacyl tRNA synthetase inhibitors for in vitro application in the inhibition of the immune cells described herein.

This invention also contemplates ex vivo treatment of immune cells which are intended for introduction into a subject The present invention is based, in part, on the discovery that an aminoacyl tRNA synthetase inhibitors is able to inhibit B cell proliferation and function, macrophage function, and T cell function.

In accordance with one aspect of the invention, a method is described that inhibits B cell proliferation in. a subject haying a B-cell dependent autoimmune disease condition, through inhibiting aminoacyl tRNA synthetase activity. Such compound may be selected from a group of aminoacyl tRNA synthetase inhibitors.

In accordance with another aspect of the invention, a method is described that uses aminoacyl tRNA synthetase alone or in combination with other medication or intervention to treat B cell dependent autoimmune diseases. In accordance with yet another aspect of the invention, a method is described that inhibits macrophage proliferation in a subject having an inflammatory disease condition, through inhibiting aminoacyl tRNA synthetase activity. Such compound may be selected from a group of aminoacyl tRNA synthetase inhibitors. In accordance with another aspect of the invention, a method is described that uses aminoacyl tRNA synthetase inhibitors alone or in combination with other medication or intervention to treat inflammatory diseases.

In accordance with another aspect of the invention, a method is described that uses aminoacyl tRNA synthetase inhibitors alone or in combination with other medication or intervention as an immunosuppressive drug during organ transplantation.

In accordance with another aspect of the invention there is provided a method for . detecting B and. T cell proliferation in a subject, the method- comprising- the use of 3H= Thymidine incorporation into dividing cells as a measure of T or B cell proliferation.

As used herein, "aminoacyl tRNA synthetase" refers to an enzyme that catalyzes the transfer of an amino acid to its cognate tRNA as described below:

ATP + amino acid + cognate tRNA — > AMP + diphosphate + σminoacyl-tRNA.

Briefly, the aminoacyl tRNA synthetase first binds ATF and the corresponding amino acid or its precursor to form an aminoacyl-adenylate and release inorganic pyrophosphate (PPi). The adenylate- aaRS complex then binds the appropriate tRNA molecule, and the amino acid is transferred^" from the aa-AMP to either the 2'- or 3'-OH of^" the last tRNA base (A76) at the 3 '-end. Some synthetases also mediate a proofreading reaction to ensure high fidelity of tRNA charging; if the tRNA is found to be improperly charged, the aminoacyl-tRNA bond is hydrolyzed.

As used herein, the term "aminoacyl tRNA synthetase inhibitor" refers to inhibitors of the reaction involving aminoacyl tRNA synthetase as defined above.

As used herein, a "threonyl tRNA synthetase" (also referred to herein as Thr-RS) is an enzyme that catalyzes the following chemical reaction (aminoacylation):

ATP + L-threonine + tRNAThr →AMP + diphosphate + L-threonyl-tRNAThr Tbreonyl tRNA synthetase is a member of a larger family of aminoacyl tRNA synthetases, defined above.

As used herein, "threonyl tRNA synthetase inhibitor" refers to inhibitors of threonyl tRNA synthetase, described above, that inhibit the reaction shown above by binding to the enzyme and preventing threonine ftøra forming L-threonyl-tRNAThr. The inhibitory activity can be tested by standard assays such as that discussed in Beyer et ah, (2008) "Methods to Assay Inhibitors of tRNA Synthetase Activity" in Methods in Molecular Medicine, Vol. 142: New Antibiotic Targets (Ed, Champney), Humana Press Inc., Totowa, NJ. Briefly, this assay measures the amount of [14C] radiolabeled threonine that is conjugated to uncharged tRNA. [14C]-Aminoacyl-tRNA is separated from [14C] amino acid by ethatiol precipitation followed by filtration through glass fibre filters. Filter-bound radioactivity is detected with a scintillation counter.

As used herein, the term "boixelidm compound" are compounds (which include the specific compound known as borrelidin) that inhibit threonyl tRNA synthetase and have the following core .structure. A variety of substituent groups are present at C2 and C7,- -

A subset of borrelidin compounds has the following structure.

Methods and synthesis of borrelidin compounds are known in the art. Examples of R groups include:

The specific compound known as borrelidin has an. R₁ of CN and an R group of

and is commercially available from numerous commercial sources including Alexis

Biochemicals and AG Scientific.

As used herein, "amino acid" refers to natural amino acids, unnatural amino acids, and amino acid analogs, all in their D and L stereoisomeric forms. Natural amino acids include alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine_* bistidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine. Unnatural amino acids include, but are not limited to azetidinecarboxylic acid, 2-aminoadiρic acid, 3-aminoadipic acid, beta-alanine, amitiopropionic acid, 2-airanobutyric acid, 4-aminρbutyric acid, 6-aminocaρroic acid, 2- amnoheptanoic acid, 2-aminoisobutyric acid, 3-armnoisobutyric acid, 2-aminoρimelic acid, 2,4 diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic - acid, 2,3- diaminopropionic acid, N-ethylglycjne, N-ethylasparagine, hydroxylysine, allo- hydroxylysine, 3-hydroxyproliαe, 4-hydrosyproline, isodesmosine, allo-isoleucine, N- methylglycine, N-methylisoleucine, N-methylvaline, norvaline, norleucine, ornithine and pipecolic acid. Amino acid analogs include the natural and unnatural amino acids which are chemically blocked, reversibly or irreversibly, or modified on their N-terminal amino group or their side-chain groups, as for example, methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteme, S-(carboxymethyl)-cysteine sulfoxide and S- (carboxymethyl)-cysteine sulfone. As used herein a "subject" refers to an animal, such as a mammal. Specific animals include a rodent such as a rat, mouse or rabbit. A subject may further be a human, alternately referred to as a patient. A subject may further be a transgenic animal.

As used herein "antigen" refers to a protein molecule that often protrudes from the surface of a cell that can induce an immune response. The epitope is the part of the molecule that is responsible for this feature, whereas the carrier molecule results in the activity as an antigen.

As used herein "major histocompatibility complex" (MHC) refers to a set of genes that encode cell-surface antigen-presenting proteins. At maturity, MHC molecules are anchored in the cell membrane, where they display short polypeptides to T cells, via the T cell receptors (TCRs). The polypeptides may be "self, that is, originating from a protein created by the organism itself, or they may be foreign ("non-self), originating from bacteria, viruses, pollen, etc. The overarching design of the MHC-TCR interaction is that T cells should ignore self peptides while reacting appropriately to the foreign peptides. . As used herein, the term "allogenic" refers to any two individuals from the same species that differ at the MHC, as defined herein. Rejection of grafted tissue from unrelated donors usually results from T-cell responses to allogenic MHC molecules expressed by the grafted tissue.

As used herein, the term "the half maximal effective concentration" (EC50) refers to the concentration of a drug or antibody which induces a response halfway between the baseline and maximum. The EC50 of a graded dose response curve therefore represents the concentration of a compound where 50% of its maximal effect is observed.

As used herein, the term "effective dose" refers to a dose capable of treating a subject for a disorder, such as an immunologic disease as defined herein, including- a dose - effective to achieve temporary and/or permanent alleviation of some or all of the symptoms associated with such immunologic disease,

As used herein, the term "inflammation" refers to a tissue response to injury, irritation, or infection. Inflammation is typically characterized by pain, redness, swelling, and sometimes loss of function. Inflammation includes, but is not limited to autoimmune inflammation, acute inflammation, catarrhal inflammation, chronic inflammation, exudative inflammation, fibrinous inflammation, granulomatous inflammation, hyperplastic inflammation, interstitial inflammation, parenchymatous inflammation, plastic inflammation, proliferous inflammation, pseudomembranous inflammation, purulent inflammation, serous inflammation, subacute inflammation, suppurative inflammation, and ulcerative inflammation. Inflammation can also be caused by and/or associated with transplant rejection, wound healing, hypertrophic scarring, bums, chemical irritants, frostbite, toxins, infection by pathogens, physical injury, including injury caused by blunt instruments or penetrating injuries, immune reactions due to hypersensitivity, ionizing radiation, foreign bodies, including splinters and dirt, and ultraviolet irradiation, and skin aging.

Immune cells that are associated with inflammation include, but are not limited to: T cells (CD4, CDS, and NKT), B cells, NK cells, mast cells, macrophages, monocytes, neutrophils, and eosinophils.

As used herein, the term "immunologic disease" refers to a disease caused by abnormal or absent immunologic mechanisms, whether humoral, cell-mediated, or both. Autoimmune disease and inflammatory diseases are types of immunological diseases. Inflammatory diseases are often mediated by responses of the innate immune system i.e., an, innate immune -response, mediated ..by,, for example, mast cells, macrophages, . monocytes, neutrophils, eosinophils etc.), whereas autoimmune diseases are mediated by a response from the adaptive immune system (i.e., an adaptive immune response mediated by, for example, T and/or B cells). However, some autoimmune diseases also have an inflammatory component. Immunosuppression typically refers to suppression of the adaptive immunity i.e., suppression of T and B cells whereas anti-inflammatory agents inhibit innate immunity Ie. , macrophages, mast cells etc.

Examples of immunologic diseases include but are not limited to the following: Achlorhydra Autoimmune Active Chronic Hepatitis, Acute disseminated encephalomyelitis. (ADEM), Addison's disease, Allergic Ocular- Conjuctivitis, Alopecia-. areata, Alzheimer's, Amyotrophic Lateral Sclerosis (ALS, Lou Gehrig's Disease), Ankylosing spondylitis, Anti-GBM Nephritis or anti-TBM Nephritis, Antiphospholipid antibody syndrome (APS), AntiphosphoHpid Syndrome, Aplastic Anemia, Artbritϊs, Asthma, Atopic Allergy, Atopic Dermatitis, Atopic Keratoconjunctivitis (AKC)₁ Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune Inner Ear Disease (AIED), Autoimmune Lymphoproliferative Syndrome (ALPS), BaIo Disease, Behcet's Disease, Berger's Disease (IgA Nephropathy), Bullous pemphigoid, Cardiomyopathy, Celiac Disease, Chagas disease, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), Chronic obstructive pulmonary disease, Chronic prostatitis, Churg Strauss Syndrome, Cicatricial Pemphigoid, Cogan's Syndrome, Cold Agglutinin Disease, Colitis, Cranial Arteritis, CREST Syndrome, Crohns Disease (one of two types of idiopathic inflammatory bowel disease "IBD"), Cushing's Syndrome, Dego's Disease, Dermatitis, Dermatomyositis, Devic Disease, Diabetes mellitus type 1, Diabetes, Type 2, Discoid Lupus, Diverticulitis, Dressler's Syndrome, Eczema, Endometriosis, Eosinophilic Fasciitis, Epidermolysis Bullosa Acquisita, Essential Mixed Cryoglobulinemia, Evan's Syndrome, Fibromyalgia, Fibromyositis, Fibrosing Alveolitis, Gastritis, Giant Cell Artertis, Glomerulonephritis, Goodpasture's syndrome, Graft versus host disease, Graves' disease, Guillain-Barre syndrome (GBS)3 Hashimoto's disease, Hemolytic Anemia, Henoch- Schonlein Purpura, Hepatitis, Hidradenitis suppurativa, Host versus graft, Hughes Syndrome, Hypersensitivities, Idiopathic Adrenal Atrophy, Idiopathic Pulmonary Fibrosis, Idiopathic thrombocytopenic purpura, IgA nephropathy, Inflammatory Demylinating Polyneuropathy, Interstitial cystitis, Irritable Bowel Syndrome, Kawasaki's Disease, Lichen Planus, Lou Gehrig's Disease, Lupoid Hepatitis, Lupus erythematosus, Lyme -Disease, Meniere's - Disease, Mixed .Connective.- Tissue Disease, -Morphea, Multiple Myeloma, Multiple sclerosis (MS), Myasthenia Gravis, Myositis, Narcolepsy, Nephritis, Neuromyotonia, Ocular Cicatricial Pemphigoid, Osteoarthritis, Osteoporosis, Parkinsons, Pars Planitis, Pemphigus Vulgaris, Pelvic Inflammatory Diseases, Pernicious anaemia, Polyglandular Autoimmune Syndromes, Polymyalgia Rheumatica (PMR), Polymyositis, Primary biliary cirrhosis, Primary Sclerosing Cholangitis, Psoriatic Arthritis, Raynaud's Phenomenon, Reiter's Syndrome, Reperfusion injury, Rheumatic Fever, Rhinitis, Sarcoidosis, Schizophrenia, Scleritis, Scleroderma, Septic Shock, Sjogren's Syndrome, Sticky Blood Syndrome, Stiff person syndrome, Still's Disease, Sydenham Chorea, SystemicXupus-Erythmatosis (SLE), Takayasu's Arteritis,- Temporal arteritis- .(also known as "giant cell arteritis"). Transplant rejection, Ulcerative Colitis (one of two types of idiopathic inflammatory bowel disease "IBD"), Vasculitis, Vernal Keratoconjunctivitis (VKC), Vitiligo, Wegener's Granulomatosis, and Wilson's Syndrome.

Many immunologic diseases detailed herein are characterized by adverse effects of T cells, or B cells, or neutrophils, or mast cells, or eosinophils, or macrophages, or monocytes. A person skilled in the art would appreciate that various immunologic diseases, as defined herein, are typified by adverse effects of the innate immune system and/or adaptive immune system, and constituent cells thereof. For example, those skilled in the art would appreciate that mast cell activity is associated with atopic diseases such as asthma, allergies, and allergic ocular conjunctivitis. Serving as a further example, those skilled in the art would appreciate that macrophage activity is associated witih. autoimmune diseases, chronic inflammation, chronic prostatitis, glomerulonephritis:, hypersensitivities, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, transplant rejection, and vasculitis. Additionally, inflammation mediated in whole or in part by macrophages can be caused by radiation treatment, bunas, sunburns, UV-exposure, and physical injury.

As used herein, routes of "administering" agents of the invention may vary, and may for example include, but are not limited to, oral, topical, intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, regional, percutaneous, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage, direct injection, and oral administration and formulation. Different ways of preparing an agent for. inhalation are known, (US5,875,776; US5,277,175; US6,325,062 and Remington, infra). Different ways for preparing an agent for topical administration are - known (US5,719,197;.US6,562,363 and. Remington, infra), . .. . - ... . .. .

As referred to herein, "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient or carrier with which an aminoacyl tRNA synthetase inhibitor is administered.

The aminoacyl tRNA synthetase inhibitors of this invention may optionally be combined with one and other for a synergistic effect and/or combined with other immunosuppressants and/or agents that affect the immune system for further synergistic effects.

Inhibition of an immune cell refers to the. inhibition of one or more of the

-following:. (i).-the inhibition of an immune cell's motility, (ii)-the inhibition of-an-immune- cell's biological activity, including but not limited to an immune cell's activation; or (iii) the inhibition of an immune cell's proliferation.

In another aspect of the invention, it has been demonstrated herein that borrelidin at nanomolar doses is effective in inhibiting B cell proliferation, indicating usefulness in treating B-cell dependent autoimmune diseases or other inflammation that is mediated by B cell proliferation. Further, it has been demonstrated herein that borrelidin inhibits TNFα production following LPS stimulation of macrophages/monocytes, therefore indicating usefulness as an aati- inflammatory agent for the treatment of inflammatory diseases (e.g., colitis, asthma, allergy) that involve macrophages/monocytes. Further, it has been demonstrated here that borrelidin inhibits T cell proliferation and activation therefore indicating its potential use as an immunosuppressant (e.g., in bone marrow or organ transplantation)

It is further demonstrated herein that borrelidin exerts its immunosuppressive effects via inhibiting pro-inflammatory mediators via the GCN2 pathway. GCN2, a stress- activated eIF2a kinase regulated indole 2,3-dioxygenase (IDO), an inducible intracellular enzyme which catabolises tryptophan creating nutrient starvation conditions (Munn, D. H. et al (2005) Immunity, 22:633-64). IDO expressing antigen presenting cells (APC) activate the GCN2 pathway in T-CeIIs, ultimately leading to inhibition of protein translation.

Borrelidin selectively inhibits eukaryotic threonine tRNA synthetase, which triggers GCN with the accumulation of uncharged transfer RNA (Munn, D.H et al (2005) Immunity, 22:633-64).

. The phosphorylation of eIF2a inhibits general protein synthesis reducing levels of -eIF2-GTP that are required for binding- of initiator tRNA- to the ribosotne (Homer BM et - al, 2008). Thus as a result, the overall level of protein translation is decreased.

As shown herein, borrelidin leads to suppression of immune cell activation through upregulation of uncharged tRNA that in turn leads to activation of GCN2 kinase that in turn phosphorylates eIF2a that leads to global inhibition of protein translation while upregulating CHOP and the integrated stress response. All amino acyl tRNA synthetase inhibitors will lead to an increase in uncharged tRNA that can activate the GCN2 pathway. .

When immune cells treated with borrelidin are activated by an autoimmune or allergic response, the inventors hypothesized that the inflammatory response will be dampened due to the-suppression, of- protein translation and hence -reduce the release- of- pro-inflammatory mediators. As shown in Figure 5, borrelidin inhibited T cell proliferation via induction of genes in the GCN2 pathway, demonstrating its potential use as an immunosuppressive agent. Compounds as described herein may be in the free form or in the form of a salt thereof. In some embodiments, compounds as described herein may be in the form of a pharmaceutically acceptable salt, which are known in the art (Berge et al. (1977) J. Pharm. Sci. 66: 1-19. Pharmaceutically acceptable salt as used herein includes, for example, salts that have the desired pharmacological activity of the parent compound (salts which retain the biological effectiveness and/or properties of the parent compound and which are not biologically and/or otherwise undesirable). Compounds as described herein having one or more functional groups capable of forming a salt may be, for example, formed as a pharmaceutically acceptable salt. Compounds containing one or more basic functional groups may be capable of forming a pharmaceutically acceptable salt with, for example, a pharmaceutically acceptable organic or inorganic acid. Pharmaceutically acceptable salts may be derived from, for example, and without limitation, acetic acid, adipic acid, alginic acid, aspartic acid, ascorbic acid, benzoic acid, benzenesulfonic acid, butyric acid, cinnamic acid, citric acid, camphoric acid, camphorsulfonic acid, cyclopentanepropioaic acid, diethylacetic acid, digluconic acid, dodecylsulfonic acid, ethanesulfbmc acid, formic acid, fumaric acid, glucoheptanoic acid, gluconic acid, glycerophosphoric acid, glycolic acid, hemisulfonic acid, heptanoic acid, hexanoic acid, hydrochloric acid, hydrobromic acid, hydriodic acid, 2- hydroxyethanesulfonic acid, isonicotinic acid, lactic acid, malic acid, maleic acid, malonic acid, mandelic acid, methanesulfonic acid, 2-naρthalenesulfonic acid, naphthalenedisulphonic acid, p-tohienesulfonic acid, nicotinic acid, nitric acid, oxalic acid, pamoic acid, pectinic acid, 3-phenylproρionic.acid, phosphoric acid, picric acid, pimelic. acid, pivalic acid, propionic acid, pyruvic acid, salicylic acid, succinic acid, sulfuric acid, sulfamic acid, tartaric acid, thiocyanic acid or undecanoic acid. Compounds containing one or more acidic functional groups may be capable of forming pharmaceutically acceptable salts with a pharmaceutically acceptable base, for example, and without limitation, inorganic bases based on alkaline metals or alkaline earth metals or organic bases such as primary amine compounds, secondary amine compounds, tertiary amine compounds, quaternary amine compounds, substituted amines, naturally occurring substituted amines, cyclic amines or basic ion-exchange resins. Pharmaceuticallyacceptable salts may-be- derived from -for-exaraple and without limitation,-a hydroxide,- carbonate, or bicarbonate of a pharmaceutically acceptable metal cation such as ammonium, sodium, potassium, lithium, calcium, magnesium, iron, zinc, copper, manganese or aluminum, ammonia, benzathine, meglumine, methylamine, dimethylamine, trimethylamine, ethylamine, diethylarnine, triethylamine, isopropylamine, tripropylamine, tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2- diethylaininoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, hydrabamine, choline, betaine, ethylenediarnine, glucosamine, glucamine, methyglucamine, theobromine, purines, piperazine, piperidine, procaine, N- eraylpiperidine, theobromine, tetramethylammonium compounds, tetraethylammonium compounds, pyridine, N,N-dimethylaniline, N-methylpiperidine, morpholine, N- methylmorpholine, N-ethylmorpholme, dicyclohexylamine, dibenzylamine, N,N- dibenzylphenemylamine, 1-ephenamine, N,N'-dibenzyiemylenediarnine or polyamrne resins, In some embodiments, compounds as described herein may contain both acidic and basic groups and may be in the form of inner salts or zwitterions, for example, and without limitation, betaiaes. Salts as described herein may be prepared by conventional processes known to a person skilled in the art, for example, and without limitation, by reacting the free form with an organic acid, an inorganic acid, an organic base or an inorganic base, or by anion exchange or cation exchange from other salts. Those skilled in the art will appreciate that preparation of salts may occur in situ during isolation and/or purification of the compounds or preparation of salts may occur by separately reacting an isolated and/or purified compound.

In some embodiments, compounds and all different forms thereof {e.g., free forms, salts, polymorphs, isomeric forms) as described herein may be in the solvent addition form, for-example, solvates. Solvates contain, either stoichiometric. or- non-stoicbiometric amounts of a solvent in physical association with the compound or salt thereof- The solvent may be, for example, and without limitation., a pharmaceutically acceptable solvent. For example, hydrates are formed when the solvent is water or alcoholates are formed when the solvent is an alcohol. In some embodiments, compounds and all different forms thereof (e.g., free forms, salts, solvates, isomeric forms) as described herein may include crystalline and/or amorphous forms, for example, polymorphs, pseudopolymorphs, conformational polymorphs, amorphous forms, or a combination thereof. Polymorphs include different cr-ystal -packing -arr-angements of -the same elemental - composition -of -a- compounds Polymorphs usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability and/or solubility. Those skilled in the art will appreciate that various factors including recrystalhzation solvent, rate of crystallization and storage temperature may cause a single crystal form to dominate. In some embodiments, pharmaceutical compositions in accordance with this invention may comprise a salt of such a compound, preferably a pharmaceutically or physiologically acceptable salt Pharmaceutical preparations will typically comprise one or more carriers, excipients or diluents acceptable for the mode of administration of the preparation, be it by injection, inhalation, topical administration, lavage, or other modes suitable for the selected treatment. Suitable carriers, excipients or diluents include those known in the art for use in such modes of administration.

Suitable pharmaceutical compositions may be formulated by means known in the art and their mode of administration and dose determined by the skilled practitioner. For parenteral administration, a compound may be dissolved in sterile water or saline or a pharmaceutically acceptable vehicle used for administration of non-water soluble compounds such as those used for vitamin K. For enteral administration, the compound may be administered in a tablet, capsule or dissolved in liquid form. The tablet or capsule may be enteric coated, or in a formulation for sustained release. Many suitable formulations are known, including, polymeric or protein rnicroparticles encapsulating a compound to be released, ointments, pastes, gels, hydrogels, or solutions which can be used topically or locally to administer a compound. A sustained release patch or implant may be employed to provide release over a prolonged period of time. Many techniques known .to one of skilLin the art are described in Remington:, the Science & Practice- of Pharmacy by Alfonso Gennaro, 20* ed., Lippencott Williams & Wilkins, (2000) [Remington]. Formulations for parenteral administration may, for example, contain excipients, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for modulatory compounds include ethylene-vinyl acetate copolymer particles, osmotic- pumps, -implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, -for-example, lactose, or -may -be-aqueous- solutions -containing, for. example, ρolyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.

It is understood that it could be potentially beneficial to restrict delivery of the compounds of the invention to the target tissue or cell in, which therapeutic effect is desired. Compositions or methods to facilitate localized tissue delivery would be apparent to one of skill in the art.

Compounds or pharmaceutical compositions in accordance with this invention or for use in this invention may be administered by means of a medical device or appliance such as an implant, graft, prosthesis, stent, etc. Also, implants may be devised which are intended to contain and release such compounds or compositions. An example would be an implant made of a polymeric material adapted to release the compound over a period of time.

An "effective amount" of a pharmaceutical composition according to the invention includes a therapeutically effective amount or a prophylactically effective amount A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as decreased inflammation, increased life span or increased life expectancy. A therapeutically effective amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the compound to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for .periods of time necessary,, to achieve the-desired prophylactic result, such-as- decreased inflammation, increased life span, increased life expectancy or prevention of the progression of an indication.

Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount. It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. The amount ofactive compound(s)- in-the-composition- may-vary-according to-factors such-as- the disease- state, age, sex, and weight of the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.

In some embodiments, compounds and all different forms thereof as described herein may be used, for example, and without limitation, in combination with other treatment methods. For example, compounds and all their different forms as described herein may be used as neoadjuvant (prior), adjunctive (during), and/or adjuvant (after) therapy in combination with other therapies known to one of ordinary skill in the art.

In general, compounds of the invention should be used without causing substantial toxicity. Toxicity of the compounds of the invention can be determined using standard techniques, for example,, by testing in cell cultures or experimental animals and determining the therapeutic index, ie., the ratio between the LD50 (the dose lethal to 50% of the population) and the LD100 (the dose lethal to 100% of the population). In soroe circumstances, however, such as in severe disease conditions;, it may be necessary to administer substantial excesses of the compositions. Some compounds of this invention may be toxic at some concentrations. Titration studies may be used to determine toxic and non-toxic concentrations. Toxicity may be evaluated by examining a particular compound's or composition's specificity across cell lines- Animal studies may also be used to provide an indication if the compound has any effects on other tissues.

Although the foregoing invention has been described in. some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of skill in the art in light of teachings of this invention that changes and modification may be made thereto without departing from the spirit or scope of the invention. AJl patents, patent applications, and other publications referred to herein are hereby uicorporated by reference.

EXAMPLES

Methods and Materials

LPS stimulation of macrophages.

-For the analysis-of lipopolysaccharide (LPS)-stimulated -tumor -necrosis-factor - (TNF) production, spleens were harvested from BALB/C mice and were ground into a single cell suspension followed by purification using a Histopaque column (Sigma). 1x10⁶ splenocytes were seeded per well in RPMI complete with borxehdin for 30 min prior to the addition of lOμg/ml LPS. Supematants were collected 3 hours post LPS stimulation and TNF determination was performed by enzyme-linked immunosorbent assay (ELISA; BD Biosciences, Mississauga, ON, Canada).

B-cell. T-cell and Fibroblast Proliferation Assays Proliferation was monitored by [3H]Thymidine incorporation into growing cells. AU proliferation assays received an 18 hour overnight pulse of the radioactive thymidine. B-cell Proliferation Assay. Spleens were harvested as described above and seeded 5xlθ⁵c/well for B-cells in the presence of borrelidin, Cells were then stimulated with lOμg/ml of anti IgM and were allowed proliferation 24 hours prior to a 3H-Thymidine spike. T-CeIl Proliferation assay. A 2-way mixed lymphocyte reaction was performed using 2x10⁵ c/well of each Balb/c stimulator and CDl responder splenocytes. A titration of borrelidin was added to the cells and they were allowed to incubate for 48 hours prior to a 3H-Thymidine spike. Fibroblast Proliferation Assay. Primary fibroblasts were harvested from human foreskin and cultured in KCMSF media (Gibco). Cells were seeded at 3,000 c/well in the presence of borrelidin and allowed to proliferation for 48 hours before the [3H]Thymidine spike.

Borrelidin formulations. Borrelidin is water soluble. Borrelidin can be foπrωlated, for example, as an eyedrop . formulation as a 2% solution in .tetrahydrozoline hydrochloride or as a topical formulation as 1:2 DMSO/MeOH.

Mouse acute cutaneous anaphylaxis model. BALB/C mice aged 6 to 8 weeks were sensitized by cutaneous application of 25μL

0-5% dinitroflourobenzene (DNFB) in acetone to the hairless abdomen of mice for 2 consecutive days. One week from the first application, test substances dissolved in 1:2

DMSO/Methanol were painted on the right ear while the left ear received vehicle control.

At 30 minutes after drug applicafioh, 0.5% DKFB m 1:4 olive oil:acetone was applied toboth ears to induce mast cell degranulation. A 6 mm punch was taken from the ear and immediately frozen in liquid nitrogen for subsequent determination of neutrophil myeloperoxidase (MPO) activity.

Myeloperoxidase fMPO) Assay Harvested ear tissue was homogenized (Polytron tissue homogenizer) in ImI of

HTAB buffer (0.5g hexadecyltxmethylammonium bromide in 5OmM Potassium Phosphate Buffer pH 6.0) for 1 min at 10500 RPM. The lysates were transferred to 1.5ml eppendorf tubes and microfuged 2x at 10,000 RPM, at 4°C for 10 min. Supernatant was analyzed for MPO activity by the addition of Tetrarnethyl Benzidine (TMB) and monitoring at 650nm.

Microscopic analysis of splenocytes Spleens were harvested from Balb/C mice and ground into a single cell suspension.

They were then pre-incubated in either Borrelidin (at 50, 5 or 0.5μg/ml) or RPMI complete for 30 min. Cells were then activated for 24 hours using immobilized anti CD3 and anti CD2S antibodies and cell morphology was analysed on a light microscope.

Jurkat treatment and Western. Blot Analysis

Jurkat cells were seeded in ImI KPMI (Roswell Park Medical Institute) medium complete at 1x10⁶ cells per well in a 6 well dtsh. 24 hours later, borrelidin (5, 10 20ng/ml ) or DMSO (dimethyl sulfoxide) were added to each well. Tryptophan depleted media was used as positive control for CHOP, as defined herein up regulation, 24 hours after borrelidin addition, cells were harvested and lysed. 40μg of total lysis extracts were analyzed by a 10% SDS-polyacrylamide gel electrophoresis. The proteins were transferred to the nitrocellulose filter. For immunoblotting the membrane was blocked for 1 hour at room temperature in 5%BSA TBST (Tris-Buffered Saline Tween-20) followed by overnight incubation with anti mouse GAD153 sc-7351 at 1:200 in 1%BSA TBST. After washing 3x with TBST, the membrane was incubated with a Dk anti Mu HRP antibody at 1 :2000 for 1 hour at room temperature. The membrane was then wash 3x with TBST and analyzed using ECL.

Western Blot Analysis 10μg of nuclear- extract were analyzed by a 10% SDS- polyacrylamide gel electrophoresis. The proteins were transferred to the nitrocellulose filter. For immunoblotting the membrane was blocked for 1 hour at room temperature in 5%BSA TBST followed by overnight incubation with anti mouse GAD153 sc-7351 at 1 :500 in 1%BSA TBST. After washing 3x with TBST, the membrane was incubated with a Rb anti Mu HRP antibody at 1 :2000 for 1 hour at room temperature. The membrane was then washed 3x with TBST and analyzed using ECL.

The following Examples highlight experiments involving B cells, T cells, macrophages, mast cells, and in vivo allergy models. Example 1. Borrelidin inhibits B-ceU proliferation in a dose dependent manner.

The efficacy of borrelidin on inhibiting B cell proliferation was determined by demonstrating its effects on primary mouse splenocytes in vitro. To monitor B cell proliferation splenocytes were stimulated with ariti-IgM in the presence or absence of borrelidin. The capacity of B cells to undergo proliferation was measured by 3H-

Thymidine incorporation into dividing cells. Splenocytes were harvested from BALB/C mice, treated with borrelidin and then stimulated with 10 μg/ml anti IgM. 24 hours later

3H-Thymidine was spiked into each well at luCi/well. Thymidine incorporation was monitored an addition 24 hours later. Proliferation decreased drastically in B cells treated with borrelidin with an EC₅₀ of 14.5ng/ml (see Figure 1)

Example 2. Borrelidin inhibits T-cell proliferation in a dose dependent manner

Unprimed T-cells can be activated to proliferate upon being cultured with allogenic stimulator lymphocytes. The activation stimulus is the foreign histocompatibility antigen expressed on the allogenic stimulator cells. The capacity of the T-cells to undergo proliferation can be measured by 3H-Thymidine incorporation, into dividing cells. To examine the effect of borrelidin on T cell growth, a two-way mixed lymphocyte reaction (MLR) was performed on BALB/C stimulator and CD1 responder splenocytes. A titration of borrelidin was added to the cells and they were allowed to incubate for 48 hours prior to a 3H-Thymidine spike.

Thymidine incorporation was monitored an addition 24 hours later.

Treatment with boixelidin resulted in a dramatic decrease in T cell proliferation with an EC₅₀ of 1.6ng/ml as shown in Figure 2. ΔCPM represents the change in count per minute at each, borrelidin concentration between stimulated and unstimulated cells. Interestingly fibroblasts showed a lower sensitivity to borrelidin (Figure 3)' than the hematopoietic cells (see Figures 1-2) with an EC₅₀ value of 520ng/ml, 50x times less sensitive than the values observed for splenocytes. With respect to the data presented in Figure 3, primary fibroblasts were harvested from human foreskin and maintained in Keratinocyte SFM media. These cells were incubated with borrelidin for 48 hours before 3H-Thymidine was spiked into each well at lμCi/well.

The morphology of T lymphocytes following treatment with borrelidin was also observed by microscopy. Splenocytes were isolated from BALB/c mice, pre-incubated for 30 minutes in the presence of borrelidin and then stimulated by anti CD3/anti CD28 to expand the T lymphocytes in culture. Controls contained comparable levels of DMSO. When the stimulated cultures were monitored for morphological differences, there was a clear decrease in proliferation of borrelidin treated cells and also an absence of the traditional blasting characteristics of an activated T cell when compared to the control cells

Example 3. Borrelidin decreases cytokine production by T-cetis upon stimulation.

Purified mouse lymphocytes were pretreated with borrelidin (0_? 2, 5, 10 ng/ml) or in the absence of tryptophan for 30 minutes prior to stimulation by plate bound anti- CD3/anti-CD28 crossliriking. Supematants were collected 24 hours later and H-2, IL-4 and IPN-γ levels were determined by enzyme linked immunosorbant assay (ELISA). Results are shown in Figure 4. Levels of IL-2 are shown in the left-most column; levels of IFN-γ are shown in the middle column; and levels of IL-4 are shown in the right-most column of Figure 4.

Example 4. Borrelidin inhibits expression of T cell activation markers, CD69 and CD25 on CD 4 positive T~cell$ upon stimulation.

Purified mouse lymphocytes were pretreated with borrelidin (0,2,5,10 ng/tril) or in the absence of tryptophan for 30 minutes prior to stimulation by plate bound anti- CD3/anti-CD28 crosslinkitig. 24 hours later cells were collected and stained using fluorescently labeled antibodies. The CD4 positive population of stimulated lymphocytes was monitored for CD69 and CD25 expression (Figures 5a and 5b).

Example 5. L-threonine rescues inhibition of cell growth in borrelidin treated primaryT-cells,

Histopaque purified lymphocytes were harvested from BALB/C and C57B1/6 mice spleens and added together in the presence of borxelidin for a two-way mixed lymphocyte reaction (MLR). In some wells, 50units/ml of mouse 11-2 or ImM L-threonine was added to the RPMI media prior to the addition of cells. 48 hours later [³H] -Thymidine was spiked into each well at 1 μCi/well. Thymidine incorporation was monitored an addition 18 hours later ACPM represents change in CPM at each borrelidin concentration between stimulated and unstimtialted cells (Figure 6). Example 6. Borrelidin signals through the GCN2 pathway in T-cells.

Nylon wool enriched primary T-cells from purified spleen lymphocytes were stimulated with 50ng/ral PMA and 500ng/ml Ionomycin at 2x10⁶ cells/ml in 4 ml. After 24 hours, either 0, 10 or 20ng/ml borrelidin were added to the cultures. Cell lysates were made in NP40 lysis buffer at 3 hours after borrelidin addition and immunoblots for eIF2α and CHOP were prepared. Both eIF2α and CHOP appeared to be upregulated in the presence of borrelidin.

Example 7. Borrelidin activates CHOP in the GCN2 pathway. GCN2 is an eIF2a kinase that is activated by stress, and has been previously linked with tumor immunity, autoimmunity and inhibition of protein translation through regulation of indole 2,3-dioxygenase (IDO), an inducible intracellular enzyme which catabolises tryptophan creating nutrient starvation conditions (Munn, D.H. et at (2005) Immunity, 22:633-64). IDO expressing antigen presenting cells (APC) activate the GCN2 pathway in T cells ultimately leading to inhibition of protein translation. To assess whether the effect of borrelidin on T lymphocytes occurred via the GCN2 pathway, Jurkat cells, an immortalized line of T lymphocytes were treated with different dosages of borrelidin or DMSO as a control for 24 hours, and were subjected to western blot analysis for genes involved in the GCN2 pathway. The CHOP gene (also known as gaddl53) was used to monitor GCN2 activation in Jurkat cells, CHOP is a well-accepted marker for GCN2 activation and although it can be induced by many other stress conditions, it is known to be induced by amino acid deprivation and requires and intact GCN2 pathway to be expressed. Jurkat cells grown in Tryptophan depleted media was used as a positive control. As shown in Figure 7, the treatment of borrelidin resulted in an increase in CHOP expression in a dose-dependent manner. CHOP was also_, upregulated when Jurkat cells^' were cultured in the absence of tryptophan. This data indicates that borrelidin is capable of activating the GCN2 pathway in T lymphocytes thus preventing protein translation, and dampening the immune response during an allergic inflammatory condition or during organ transplantation. Specific conditions were as follows: Jurkat cells were seeded in 1ml RPMI complete at 1x10⁶ cells per well in a 6 well dish. 24 hours later, borrelidin (5, 10 20ng/ml) or DMSO were added to each well. Tryptophan depleted media was used as positive control for CHOP up regulation. 24 hours after borrelidin addition, cells were harvested, lysed and subjected to western blot analysis. Briefly, 40μg of total lysis extracts were analyzed by a 10% SDS-polyacrylamide gel electrophoresis. The proteins were transferred to the nitrocellulose filter. For iiranunoblotting the membrane was blocked for 1 hour at room temperature in 5%BSA TBST followed by overnight incubation with anti mouse GAP 153 sc-7351 at 1:200 in 1%BSA TBST. After washing 3x with TBST, the membrane was incubated with a Dk anti Mu HRP antibody at 1 :2000 for 1 hour at room temperature. The membrane was then wash 3x with TBST and analyzed using ECL.

Example 8. BorreUdin inhibits macrophage activation in a dose dependent manner, LPS stimulation of splenocytes is associated with release of pro-inflammatory cytokines such as TNF-α. To examine the effects on. boixeljdm on LPS stimulated BALB/C mouse splenocytes were pretreated for 30 minutes with a titration of borrelidin prior to stimulation with lOμg/ml of LPS and TNFα levels were determined by ELISA. Borrelidin suppressed TNF- α expression in a dose dependent manner with an EC50 of 1 lng/ml (see Figure 8). In the absence of LPS induction, there was no TNF- α production.

Example 9. Borrelidin inhibits translation ofTNFβ in primary macrophages.

Primary macrophages were treated for 1 hour in the presence or absence of GolgiPlug™ (BD) to block gogli export. Macrophages were then treated in the presence (10ng/ml) or absence (Ong/ml) of borrelidin for 30 minutes prior to lipopolysaccaride (LpS) stimulation (0.1μg/ml). At 3 hours superøatnants were collected and cell pellets were lysed useing the non-reducting lysis buffer: 2OmM HEPES pH 7.9, 43OmM NaCl, L5mM MgC12, 0.2mM EDTA, 25% glycerol. TNFα levels from both superaatants and cell lysates were determined using a TNFα ELISA₅ as shown in Figures 9a and 9b, respectively. Levels of TNFd in the presence of GolgiPlug™ are shown in the left-most_. columns for each condition detailed in Figures 9a and 9b. Levels of TNFα in the absence of GolgiPlug™ are shown in the right-most column for each condition detailed in Figures 9a and 9b.

Example 10. Borrelidin inhibits lipopolysaccaride (LPS) induced cytokine/chemokine production in primary mouse splenocytes.

Ficoll- purified splenocytes from Balb/c mice were preincubated in the presence (5.6, 16.7, 50, or 150ng/ml) or absence (Ong/ml) of borrelidin for 30 minutes prior to 0.1μg/ml LPS stimulation. At 3 hours, the supematants were harvested and cytokme/chemokine levels were determined using Bio-Pleχ Mouse Cytokine 23-Plex Panel (BioRad, CA). The results axe shown in Figure 10. With reference to Figure 10, the coltmras represented in Figure 10 for each condition correspond with the following cytokines (from left to right): IL-10, IL- 12, MIP-Io, MlP- lβ, RANTES, and TNF-α.

Example IL Borretidin induces apoptosis as shown by propidum iodine (PI) cell cycle analysis.

Macrophages derived from C57BL/6 splenocytes were preincubated in the presence (2, 5, 10, 20 ng/ml) or absence (0 ng/ml) of borrelidin for 30 minutes prior to O.lμg/ml LPS stimulation. Twenty-four hours later cells were fixed using ice-cold 80%

Ethanol and stained using 50μg/ml PI + 50μg/ml RNase A. The results are shown in

Figure 11 where percent apoptosis is based on the percentage of cells in the Go population.

Example 12. Borretidin induces macrophage apoptosis as shown by Annexin V exppression and activatiøn of Caspase 3.

Macrophages derived from C57BL/6 splenocytes were preincubated ^' in the presence (2, 5, 10, 20 ng/ral) or absence (0 ng/ml) of borrelidin for 30 minutes prior to O.lμg/ml LPS stimulation,. 24 hours later cells were collected and separately stained using fluoresceiitly labeled antibodies for Annexin V or activated Caspase 3. The results are shown in Figure 12. With reference to Figure 12, the levels of Annexin V are shown in the left-most column for each condition tested; the levels of activated Caspase 3 are shown in the right-most column for each condition tested.

Example IM. mRNA transcripts for TNFa and CHOP increase in the presence of - borrelidin.

Relative quantities (RQ) of TNFα and CHOP in primary macrophages were determined by qPCR based on the conditions described in Figures 13a and 13b. Ubiquitin C and RPL32 were used as endogenous controls, respectively.

Example 14. Borrelidin inhibits TNFa-release from anti-IgE stimulated murine bone marrow derived mast cells (BMMQ.

Mature BMMCs were IL-3 starved_^ in IMDM, 10% FCS, 15OuM monotbioglycerol & Penicillian/Streptomycin for 4 hours at 37°C/5% CO2 followed by a 30 ruin borrelidin incubation (0, 10, 100, 250, 500, 1000 ng/ml) in IMDM +0.1% BSA. Cells were stimulated with a final anti IgE Concentration of lμg/ml for 4 hours. Conditioned media was collected and a TNFα ELISA performed for detection of TNFα levels. Results are shown in Figure 14. With reference to Figure 14, the levels of TNFα associated with 1μg/ml of anti-IgE are shown in the left most column for each condition tested; the levels of TNFα associated with Oμg/ml of anti-IgE are shown in the left most column for each condition tested;

Example 15: Borrelidin inhibits cutaneous anaphylaxis in a mouse allergy model in vivo. Borrelidin was tested for its ability to inhibit cutaneous anaphylaxsis, an allergic reaction mediated by allergen-induced degranulation of pre-sensitized mast cells. Mice were pre-sensitized to the haptenizing agent dinitrofluorobenzene (DNFB) by cutaneous application on both ears of mice followed 24 hours later by a second cutaneous application. One week later, vehicle or borrelidin was topically applied to one ear prior to DNFB challenge, and the resulting ear edema or mfiammation was compared in the two ears. A topical formulation of borrelidin was used (1:2 DMSO/MeOH). Ears were harvested and neutrophil-specific myeloperoxidase (MPO) levels were determined (P=0.000004 for borrelidin versus vehicle treated group). As shown in Figure 15, topically applied borxelidάn significantly decreased allergen-induced inflammation compared with the vehicle control-treated ear as demonstrated by a decrease in absorbance at 65 Onm.

Example 16. Borrelidin suppresses neutrophil recruitment into the conjunctiva in an in vivo allergic ocular conjunctivitis mouse model. Mice were immunized on Day 0 with 100μg of ovalbumin in Alum:water (3 : 1 ) and day 6 with 50μg of ovalbumin Alum: water (3:1). On day 18, mice were challenged with 2.5μl of lOOμg/ml ovalbumin after a 30 min pre-treatment in the presence(2μl of lng/μl or lOng/ml of borrelidin in 2% HPMC) or absence (2μl of 2% HPMC) of borrelidin. An eyedrop formulation of borrelidin was prepared in a 2% solution of tetrahydrozoline hydrochloride The left eye receieved borrelidin treatment, the right, vehicle. 20 minutes after the challenge mice were scored on the level of swelling. 24 hours after the challenge, conjunctivas were excised. The levels of myleoid peroxidase (MPO) and total protein were determiiaed on homogenized conjunctiva lysates. Figure 16 compares normalized levels of MPO of the treated eye are to the untreated eye (Mice 1-8 are Black/6; Mice 9-10 are Balb/c; Mice 5 and 6 recieved the higher dose of borrelidin. (20ng/eye); and the remaining mice recieved 2ng/eye).

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of skill in the art in light of the teachings of this invention that changes and modification may be made thereto without departing from the spirit or scope of the appended claims. All patents, patent applications and publications referred to herein are hereby incorporated by reference.

Claims

What is claimed is:

1. Use of an aminoacyl tRNA synthetase (aa-RS) inhibitor as an inhibitor of immune cells, providing that when the immune cells to be inhibited are solely T-cells and the inhibitor comprises adenine joined to a sugar molecule as a substituent, the inhibition comprises mhibition of activation of the T-cells.

2. Use of an aminoacyl tRNA synthetase (aa-RS) inhibitor for preparation of a medicament for use as an inhibitor providing that when the immune cells to be inhibited are solely T-cells and the inhibitor comprises adenine joined to a sugar molecule as a substituent, the inhibition comprises inhibition of activation of the T-cells.

3. A pharmaceutical composition for use as an inhibitor of immune cells, wherein the composition comprises a pharmaceutically acceptable carrier or excipient and an aminoacyl tRNA synthetase (aa-RS) inhibitor providing that when the immune cells to be inhibited are solely T-cells and the inhibitor comprises adenine joined to a sugar molecule as a substituent, the inhibition comprises inhibition of activation of the T-cells,

4. A pharmaceutical formulation for use as an inhibitor of immune cells, wherein the formulation is in oral, topical, parenteral or inhalation form and comprises an aminoacyl tRNA synthetase (aa-RS) inhibitor providing that when the immune cells to be inhibited are solely T-cells and the inhibitor comprises adenine joined to a sugar molecule as a substituent, the inhibition comprises inhibition of activation of the T-cells.

5. The use or composition of any one of claims 1 to 4, wherein, the inhibitor is a threonyl tRNA synthetase inhibitor.

6. The use or composition of claim 5, wherein the inhibitor does not comprise adenine joined to a sugar molecule as a substituent.

7. The use or composition of claim 5 or 6, wherein the threonyl tRKA synthetase inhibitor is borrelidin, a borrelidin compound or a pharmaceutically acceptable salt thereof.

8. The use or composition of claim 5 or 6, wherein the threonyl tRNA synthetase inhibitor is borrelidin or a pharmaceutically acceptable salt thereof.

9. The use or composition of any one of claims 1 to 8 for treatment of inflammation.

10, The use or composition of claim 9, wherein the inflammation is associated with an immunological disease.

11. The use or composition of claims 9 or 10, wherein the inflammation is associated with an adaptive immune response or an, innate immune response.

12. The use or composition of claim 9, wherein the inflammation is associated with an autoimmune disease.

13. The use or composition of any one of claims 9 to 12, wherein the inflammation is associated with activation of one or more of T-cells, B-cells, macrophages, mast cells, neutrophils and monocytes.

14. The use or composition of any one of claims 8 to 12, wherein the inflammation is associated with proliferation of one or more of B-cells, macrophages, neutrophils and- monocytes.

15. The use or composition of any one of claims 9 to 14, wherein the inflammation is not associated with rheumatoid arthritis or psoriasis,

16, The use or composition of any one of claims 9 to 15, wherein the inflammation not associated with microbial infection.

17. The use or composition of any one of claims 9 to 15, wherein the inflammation is not associated with infection at a site of inflammation.

18. The use or composition of any one of claims 9 to 17, wherein the inflammation is not treatable solely by use of antimicrobial therapy.

19. The use or composition of any one of claims 1 to S, wherein the immune cell mhibition comprises prevention of activation of T-cells.

20. The use or composition of claim 19 for treatment of transplant rejection.

21. A method for treatment or prevention of an inflammatory condition or suppression of immune cells in a subject in need thereof, wherein the method comprises admimstering to the subject an effective amount of an amino acid tRNA synthetase (aa-RS) inhibitor, providing that when immune cells to be inhibited are solely T-cells and the inhibitor comprises adenine joined to a sugar molecule as a substituent, the inhibition comprises inhibition of activation of the T-cells.

22. The method of claim 21 for treatment or prevention of inflammation in the patient.

23. The method of claim 21 or 22, wherein said patient does not require the administration solely for treatment of psoriasis or rheumatoid arthritis.

24. The-method of claim 21, wherein the- treatment or prevention is for transplant- rejection.

25. The method of claim 24, wherein the inhibitor is administered prior to transplantation.