COMPOSITIONS AND METHODS FOR MODILATING I UE IMMUNE S\ STI λ1

CROSS REFERENCES TO RELATED APPLICATIONS

[IHHH j This application claims priori i\ from L. S. Prov isional Application No oϋ 74tol 1 entitled "l'ϊi'-l Induced Lffects on PBMC Genome Alone and following F Kposure to

Cl ⁾ 3MA8,'CD28Mλir' filed May 5, 2006 and U.S. Non-Pro\ isiυmii Application No, 1 1 381 ,818 titled Compositions and Methods SIH Modulating the Immune Ssstem illcd Mas 5, 2000, the contents of which are incorporated herein b> reference in their entireties.

BACKGROUND

[1)0021 Mammalian pregnane) is a unique phx sioiogiea! t:\ cnt m which the maternal immune system interacts witb the fetus hi α \ er> efficient manner, bcnctleuil ϊ^r both parties Pregnane > is an immune paradox, chspLmng no graft \s. host or host λ S graft effect i he factors invoKed in this phenomenon are not \ eι tully etucidaicd although the\ ha\ c been cλtensheh studied. The no\ ol erabno-deπ\«ϊ lat tor. preiinplantatioπ factor (Pl! -] ). raa> cause immune tolerance of pregnancy b} creating maternal recognition oi ^" piegnanex ihortl> after lertilization. Synthetic PIF-I replicated the nati\ e peptide's effect and everted potent immune modulatory eilects on activated FBMC proliferation and cvtokmc secretion, acting through no\ol i>iles υn PBMC and hav ing an effect which is distinct liom knov\n immune- supprcssj\« drugs.

J00θ3J There is evidence that se\e?al autoimmune diseases, including multiple sc^erous and rheumatoid arthπtis, undergo ictnissjon during pregnane), supporting the \ ie\* that thoe aie unique protuctne mechanisms operative during that time period fhis is panic u!ari> rcmrukahJc because lhe host mother is simultaiieoush being exposed to a serai- or total allograft {Jonor embrvo^ without advcise trnmunu efi ^' ecis.

[0004J Allogeneic bone marrow transplantation (BMT) is a well-established treatment for malignant and non-malignant hematological diseases, and is performed in tens of thousands ol ^" patients each year. Mature donor T cells within the stem cell graft are the main mediators of the beneficial immune effects, but they are also responsible for the induction of graft-versus-host disease (GVHD), the major cause of morbidity and mortality in BVlT patients. CJV HD occurs

when transplanted donor-derived T cells recognize proteins expressed by recipient antigen- presenting cells. Consequently, this recognition induces dυnor T -ceil activation, proliferation, and differentiation, leading to a cellular and inflammatory attack on recipient target tissues. Acute or chronic GVI lD occurs within a 100-day period post-BMT that leads to dermatitis. enteritis, and hepatitis. The treatment of GVHl) continues to be a challenge. I o eliminate undesirable host-derived hematopoietic elements before BMT, patients have traditionally been treated with myeloabiative conditioning regimens involving high-dose chemotherapy and total- body radiation. Up until now, standard GVI iD prophylaxis and therapy uses immune suppressive drugs (steroids and Cyclosporin A), that place patients in danger of opportunistic infections and tumor relapse. Numerous agents have been evaluated for GVTlD. unfortunately with poor outcome. Ideally, prophylaxis of BMT patients by immune modulation would allow transplant acceptance, while maintaining the ability to protect against pathogens or cancer,

[0005| Type 1 (insulin-dependent) diabetes ( TlDM ) is caused by autoimmune destruction of the insulin-producing pancreatic beta cells. T! DM etiology is multifactorial, complex, and involves a combination of genetic, environmental, and immunological influences. TlDM is a progressive, asymptomatic decline in beta ceil function until hyperglycemia develops. Near total beta-cell destruction may not be universal, and therefore therapeutic measures thai stop destruction and perhaps lead to organ recovery could bring to major advances in TiDM

management. TlDM prevention is currently suboptimal, and most current therapies aira al controlling glucose levels using insulin, or (rarely) by islet transplants. There are also attempts to initiate immune therapies using (anii-CO3 antibodies, and anti- thymocyte globulin) which aim to block the autoimmune cascade when combined with repair/regeneration of beta cells (e.g.. gmiisine, giueagon-like-peptide-1 (OLP- 1), εxtendin-4), and Dia-Pep277 with limited success.

[0006J Multiple sclerosis (MS) is a progressive debilitating autoimmune disease of the central nervous system that has a complex etiology where genetic predisposition may be coupled with early childhood viral exposure. Consequently, there is a gradual destruction of the myelin sheath that causes motor, autonomic, sensory dysfunction that may lead to paralysis. Current therapies are based on limiting the damage by using steroids and interferon, Copaxone and monoclonal antibodies, with limited success. An optima! therapy would reverse the neural damage by blocking the autoimmune cascade while allowing for myelin sheath repair. The experimental autoimmune encephalitis (CAE) model is widely used currently to examine experimental treatments for MS.

(0007) Ulcerative colitis (UC) and Crohn's disease (CD), the primary constituents of inflammatory bowel disease ( IBD), are precipitated by a complex interaction of em ironmentaL genetic, and immunoregubtory factors. Higher rates of ϊBD are seen in northern, industrialized countries. ϊBD's are chronic inflammatory disorders of the gastrointestinal tract. Although the etiology is incompletely understood, initiation and aggravation of the inflammatory process seem to be due to a massive local mucosal immune response. Cytokine-raediated impairment of viability and metabolic function of epithelial cells has been suggested as a possible early pathogenic event in the development of inflammatory bowel disease (IBD) ₁ Among several currently used therapies are azulphidine, steroids and in more serious cases λ/athioprine, 6-

mercaptopurine and methotrexate axe appropriate. When steroids fail, cyclcsporine λ may utilized. IBD's involve both local and systemic alteration of the immune system, In recent years several studies were carried out using peripheral immune cells as welt colonic biopsies to examine the direct effect of possible therapeutic agents on the condition. Data indicates that the milieu of peripheral PBMC is altered and agents that were found to be disease modifiers by in situ testing were considered suitable for clinical application.

(0008] (t has bee!) observed thai PIF has immune modulatory properties and such peptides are useful in the prevention and/or treatment of various immune-mediated diseases, including- but not limited to, autoimmune disorders. Compositions and methods for 'treating and/or preventing immune-mediated disorders are provided herein.

SUMMARY

|0009j Embodimenis of the present invention provide compounds having immune- modulating and/or anti-inflammatory activity, wherein compounds of the invent ton include peptides and peptidomimetics. The invention further provides methods of using iroraune- modulating and/or anti-inflammatory compounds of the invention.

((MIIO] Further embodiments of the present invention provide methods fur treating a disease characterized by an immune disorder or inflammatory response by administering an amount of a PIf peptide or peptidomimetic sufficient to treat, inhibit, or ameliorate the disease. Such compounds are useful for treating diseases characterized by an immune disorder or inflammatory response diseases, e g,, inflammation, arthritis, auto-tmmune diseases, collagen diseases, or allergy. For example, these compounds can be used tυ treat subjects, including mammals such as humans, having or ai risk of having inflammation, arthritis, auto-immune diseases, collagen diseases, or allergy,

[00111 Embodiments of the present invention relate to biological effects induced in viiro and/or m vivo by pre-iinpianiation factor (PIF- ^" ) peptides, peptidomimetics. and compounds derived from pre-implantalion embryos that harbors in part, is identical to. or is homologous to the amino acid sequence of PIF peptides or to the scrambled amino acid sequence o!~ PIF peptides, in particular, the present invention relates tυ use of PIF peptides or peptidυrnimetics to effect changes on the immune system of a patient. More specifically, the addition of PlF peptides creates specific changes both in eeUυlar immunity, as well as in a patient ^' s secreted cytokine profile.

DESCRIPTION OF THE DRAWINGS

[00.12} In part, other aspects, features, benefits and advantages of the embodiments of the present invention will be apparent with regard to the following description, appended ciaims and accompanying drawings where:

JW)13] Figure I. PlF prevents GVlID development following high burden BMT. The number of GVHD+ and GVI-ID- mice at three and live weeks after BMT were evaluated. The differences between control and both 0.1 and hng/kg/day PlF administered for two weeks PlF group and tested one week later were significant. Also, the lmg/kg/day PlF-treated group using

an Alzet© pump at five weeks after BMT provided significant protection, χ ^" : ?<U.00L /'' ^' 0.0I

and P<0.05, respectively.

[0014J Figure 2. PIF-treated mice with a high BMT burden that develop GVHD have a lower score at 30 days post transplant. At 30 days posϊ-BMT, the difference between the control and lhc 0, 1 and i mg/kg/day treated PIF groups using an Alzet® pump, are significant, l-test P<().04 and / ^J <0.04. At 50 days, the effect was not significant.

[0015] Figure 3. Short-term PIF administration to mice with high-burden BMT is associated with long term survival, PIF 1-5 mg/kg/day for two weeks was administered using an Alzetφ) pump and the effect on long-term survival was compared tu control. In lhc lower-dose

PlF-treated group, 7/9 mice survived vs. control 2/10, χ\ / ^! <0.01 , The survival αj ^' the Higher-

close treated group was slightly lower. 5/9.

[0016] Figure 4. PlF treatment prevents diabetes development in NOD male mice, adoptive transfer model. Male mice were injected IV with 250M spleen cells derived from diabetic female NOD mice PIF 0.83-2.73 mg/kg/day was administered using Al/eV-K ⁾ pump for 28 days. This was followed by a 40-day observation period, in low-dose PlF ^' group, no mice developed DM, while in the high-dose group, one mouse became diabetic vs. 6/7 in controls, P<QXϊύl .

[0017] Figure 5. PIF treatment reduces degree of paralysis in EAL: ^' , an acute multiple sclerosis model. SJL mice 7-8 weeks were injected in the tail base with 3 : 1 of 201' μg proteolytic protein peptide (PLP) together with 200 μg CFA and IFA (containing myeobaciermrn tuberculosis}. On the same day and two days later, mice were injected IP with 250 ng pertussis toxin. On the first day of the experiment, PiF- I 0.75 rag/kg/day was administered using a subculancously implanted λlzet® pump. Paralysis scores (0=non to 5 -death) were compared. PlF therapy resulted in overall decreased scores. PlF-Fs effect was compared to a control group by daily monitoring degree of paralysis, up to 40 days, Mann- Whitney non-parametric test,

/*<0.003. In addition, the mean clinical score at day 40 was significantly lower in the PlF- treated group vs. control. / ^J <0.05,

DETAI LED DESCRIPTION

[0018| Before the present compositions and methods are described, ii is so be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols described, as these may vary. It is aiso to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

J0θ19) Unless defined otherwise, all technical and scientific terms used herein have the sarne meanings as commonly understood by one of ordinary skill in the art. .Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. AU publications mentioned herein are incorporated by reference. Nothing herein is to be construed as an admission thai the invention is not entitled to antedate such disclosure by virtue of prior invention,

[002Oj ft must also be noted that as used herein and in the appended claims, the singular forms "V, "an'\ and '"the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to ⁴ "a cell' ^" is a reference to one or more ceils and equivalents thereof known to those skilled in the art, and so forth.

[0021 j As used herein, the term "'about ¹ " means pins or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%~55%.

]0022| "Administering" when used in conjunction with a therapeutic means to administer a therapeutic directly into or onto a target tissue or to administer a therapeutic to a patient whereby ihe therapeutic positively impacts the tissue to which it is targeted. 1 hus. as

used herein, the term "administering ^*1 , when used in conjunction with PIF, can include, but. is noi limited to, providing PlF peptide into or onto the target tissue; providing PlF peptide systernicaϋy to a patient by, e.g., intravenous injection whereby the therapeutic reaches the target; providing PIf peptide in the form of the encoding sequence thereof to the target (e.g., by so-called gene-therapy techniques). "Administering" a composition may be accomplished by parenteral, oral or topical administration.

10023 j As used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials arc capable of administration upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, rash, or gastric upset, (n a preferred embodiment, the therapeutic composition is not immunogenic when administered to a subject for therapeutic purposes. j0024| As used herein, the term "therapeutic" ^* means an agenL utilized to treat, eombai, ameliorate, prevent or improve an unwanted condition or disease of a subject, Jn part, embodiments of the present invention are directed to treating, ameloriating, preventing or improving inflammation and/or an immune-mediate disorder, including auio-irnmunc diseases.

[0025] A ''therapeutically effective amount" or "effective amount" of a composition is a predetermined amount calculated to achieve the desired effect, / t\, to dTuctivdy inhibit or reduce inflammation and/or an immune-mediated disease. Effective amounts of compounds of the present invention can objectively or subjectively reduce or decrease the .severity or frequency of symptoms associated with inflammation and/or immune-mediated disorders. The specific dose of a compound administered according to this invention to obtain therapeutic and'or prophylactic effects will, of course, be determined by the particular circumstances surrounding

the case, including, for example, the compound administered, the route of administration, and the condition being treated. The compounds are effective over a wide dosage range and. for example, dosages per day will normally fall within the range of from about 0.0 i mg/kg to about 10 mg/kg, more preferably about 0.1 rag/kg to about 1 mg/kg. However, it will be understood that the effective amount administered will be determined by the physician in the Sight of the relevant circumstances including the condition to be treated, the choice of compound to be administered, and the chosen route of administration, and therefore the above dosage ranges are not. intended to limit the scope of the invention in any way, A therapeutically effective amount

of compound of this invention is typically an amount such that when it is administered in a physiologically tolerable exeipient composition, it is sufficient to achieve an effective systemic concentration or local concentration in the tissue.

|0026] The terms "treat," "treated," or ''treating" as used herein refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object n to prevent or slow down (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms: diminϊshment of the extent of the condition, disorder or disease, stabilization {i.e.. not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of

the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[0027] '"Disease" or "disorder ^' refers Io an impairment of the norma! function of an organism. λs used herein, a disease may be characterized by, e.g.. an immune disorder or an inflammatory response or a combination of these conditions.

|θO28] "'Immune-modulating ^*' refers to the ability of a compound of the present invention to alter (modulate) one or more aspects of the immune system. The immune system functions to protect the organism from infection and from foreign antigens by cellular and humoral mechanisms involving lymphocytes, macrophages, and other antigen-presenting ceils that regulate each other by means of multiple cell-cell interactions and by elaborating soluble factors, including lymphokines and antibodies, that have autocrine, paracrine, and endocrine effects on immune cells.

[0029 j 'immune disorder ^" ' refers to abnormal functioning of the immune system. Immune disorders can be caused by deficient immune responses (e.g., MIV, AIDS} or overactive immune responses (e g., allergy, auto-immune disorders). Immune disorders car. result in the uncontrolled proliferation of immune cells, uncontrolled response to foreign antigens or organisms leading to allergic or inflammatory diseases, aberrant immune responses directed against host cells leading to auto-immune organ damage and dysfunction, or generalized suppression of the immune response leading to severe and recurrent infections. Immune disorder refers to disorders of the innate immune system (innate immunity) and the adaptive immune system (adaptive immunity), Innate immunity refers to an early system of defense that depends on invariant receptors recognizing common features of pathogens. The innate immune system

provides barriers and mechanisms to inhibit foreign substances, in particular through the action of macrophages and neutrophils. The inflammatory response is considered part of innate immunity, l he innate immune system is involved in initiating adaptive immune responses and

removing pathogens that have been targeted by an adaptive immune response. However, innate immunity can be evaded or overcome by many pathogens, and does not lead to immunological memory. Adaptive immunity refers to the ability to recognize pathogens specifically and to provide enhanced protection against reinfection due to immunological memory based on dona! selection of lymphocytes bearing antige.n-speci.fk receptors, A process of random recombination of variable receptor gene segments and the pairing of different variable chains generates a population of lymphocytes, each bearing a distinct receptor, forming a repertoire of receptors that can recognize virtually any amigcn. If the receptor on a lymphocyte is specific lυr a ubiquitous self antigen, the cell is normally eliminated by encountering the antigen early in its development. Adaptive immunity is normally initiated when an innate immune response fails to eliminate a new infection, and antigen and activated antigen-presenting cells are delivered to draining lymphoid tissues. When a recireulatmg lymphocyte encounters its specific foreign antigen in peripheral lymphoid tissues, it is induced to proliferate and its progeny then differentiate into effector cells that can eliminate the infectious agent. A subset of these proliferating lymphocytes differentiate into memory cells, capable of responding rapidly to the same pathogen if it is encountered again.

[0030| Immune disorders caused by an impaired or immunocompromised immune system can produce a deficient immune response that leaves the body vulnerable to various viral, bacterial, or fungal opportunistic infections. Causes of immune deficiency can include various illnesses such as viruses, chronic illness, or immune system illnesses. Diseases characterized by an impaired immune system include, but are not limited to, H1V/λ1DS and severe combined immunodeficiency syndrome (SClI)S).

|0031| Immune disorders caused by an excessive response by the immune s\ stern. This excessive response can be an excessive response to one or more antigens on a pathogen, or to an antigen that would normally be ignored by the immune system. Diseases characterized by an overactive immune system include, but are not limited ιo, arthritis, allergy, asthma, poHmosis. atopy, and auto-immune diseases,

[0032] "Arthritis" refers to inflammation of the joints that can be caused, inter alia, by wear and tear on joints, or auto-immune attack on connective tissues, or exposure to an allergen, e.g., as in adjuvant-induced arthritis. Arthritis is often associated with, or initiated by, deposition o! ^" antibody-antigen complexes in joint membranes and activation of an inflammatory response. Sometimes the immune response is initiated by cells rather than antibodies, where the cells can produce a deposit in the joint membrane.

[0033] "Allergy" refers to an immune reaction to a normally innocuous environmental antigen (allergen), resulting from the interaction of the antigen with antibodies or primed ^" 1 cells generated by prior exposure to the same antigen. Allergy is characterized by immune and inflammatory aspects, as the allergic reaction is triggered by binding of the antigen to antigen- specific IgF: antibodies bound to a high-affinity IgE receptor on mast cells, which leads to antigen- induced cross-Unking of IgE on mast cell surfaces, causing the release o( large amounts of inflammatory mediators such as histamine. Later events in the allergic response involve leukotrienes, cytokines, and ehemokines, which recruit and activate eosinophils and basophils. The late phase of this response can evolve into chronic inflammation, characterized by the presence of effector 1 ^' ceMs and eosinophils, which is most clearly seen in chronic allergic asthma.

-P.-

[O034J "Asthma ^" ' refers to a chronic inflammatory disorder affecting the bronchial lubes, usually triggered or aggravated by allergens or contaminants. Asthma is characterized by constriction of the bronchial tubes, producing symptoms including, but not limited to, cough, shortness of breath, wheezing, excess production of mucus, and chest constriction

10035] "Atopy ^"' refers to the tendency to develop so-called "classic" allergic diseases such as atopic dermatitis, allergic rhinitis (hay fever), and asthma, and is associated with a capacity to produce an immunoglobulin M (IgE) response to common allergens. .Atopy is often characterized by skin allergies including but not limited to ec/ema, urticaria, and atopic dermatitis. Atopy can be caused or aggravated by inhaled allergens, food allergens, and skin contact with allergens, but an atopic allergic reaction may occur in areas of the body other than where contact with the allergan occurred. A strong genetic (inherited} component of atopy is suggested by the observation that the majority of atopic dermatitis patients have at least one relative who suffers from eczema, asthma, or hay fever.

[00361 "Polfinosis." "hay fever," or "allergic rhinitis," are terms that refer to an allergy characterized by sneezing, itchy and watery eyes, a runny nose and a burning sensation of the palate and throat. Often seasonal poliinosis is usually caused by allergies to airborne substances

such as pollen, and the disease can sometimes be aggravated in an individual by exposure to other allergens to which the individual is allergic.

|0037} "λuto-immune" refers to an adaptive immune response directed at self antigens. "Auto-immune disease" refers to a condition wherein the immune system reacts to a "self" antigen that ii would normally ignore, leading to destruction of normal body tissues. λino- immune disorders are considered to be caused, at least in part, by a hypersensitiv ity reaction similar to allergies, because in both cases the immune system reacts to a substance that it

normally would ignore. Auto-immime disorders include, but are nυt limited to, Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type ϊ (insulin dependent) diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, and Grave's disease, alopecia areata, aiiklosing spondylitis, antiphospholipid syndrome, auto-immunc hemolytic anemia, auto-immune hepatitis, auto-immunc inner ear disease, auio-immune fymphoproliierative syndrome (ALPS), auto-immune thrombocytopenic purpura (ATP). Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue syndrome immune deficiency syndrome (CFIDS), chronic inflammatory demydinatiπg polyneuropathy. eicatrieiaS pemphigoid, eυld agglutinin disease, CRTST syndrome, Crohn's disease, Oegυ's disease, dermatomyositis, dermatomyositis, discoid lupus, essential mixed cryoglobulinemia. fibromyalgia-fibromyosUis, Guillain-Barre syndrome, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (JTP) ₅ IgA nephropathy, juvenile arthritis, Meniere's disease, mixed connective tissue disease, pemphigus vulgaris, polyarteritis nodosa, polychondritis. polygϊancular syndromes, polymyalgia rheumatiea, polymyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, rheumatic fever, sarcoidosis. scleroderma, stiff-man syndrome, Takayasu arteritis, temporal arteritϊs/giani cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis,

[0038 j "Collagen disease ^" or "connective tissue disease" refers to a chronic inflammatory auto-immune disorder in which autoantibodies attack collagen ibund throughout the body. Connective tissues axe composed of two major structural protein molecules, collagen and elasiiπ; in collagen disease, autoantibodies directed against collagen will damage both collagen and elastin due to the resulting inflammation. Collagen diseases include, but are not

limited to. lupus erythematosus, Sjogren's syndrome, scleroderma, dermatomyosiiis. and polyarteritis nodosa. Rheumatoid-collagen disease refers to a disorder affecting the connective tissue, with "rheumatic" symptoms including muscle stiffness, soreness and pain in the joints and associated structures.

|0039j "Inflammatory response" or "inflammation" is a general term for the local accumulation of fluid, plasma proteins, and white blood cells initiated by physical injury. infection, or a local immune response. Inflammation is an aspect of many diseases and disorders, including but not limited to diseases related to immune disorders, viral infection, arthritis, autoimmune diseases, collagen diseases, allergy, asthma, poUinosis. and atopy. Inflammation is characterized by rubor (redness), dolor (pain), caior (heat) and tumor (swelling), reflecting changes in local blood vessels leading to increased local blood How which causes heat and redness, migration of leukocytes into surrounding tissues (extravasation), and the exit of fluid and proteins from the blood and their local accumulation in the inflamed tissue, which results in swelling and pain, as well as the accumulation of plasma proteins that aid in host defense, ^" t hese changes are initiated by cytokines produced by activated macrophages. Inflammation is often accompanied by loss of function due to replacement of parenchyma! tissue with damaged tissue (e.g., in damaged myocardium), reflexive disuse due io pain, and mechanical constraints on function, e.g., when a joint swells during acute inflammation, or when scar tissue bridging an inflamed joint contracts as it matures into a chronic inflammatory lesion. j ⁽ KJ40j "Anti-inflammatory" refers to the ability of a compound of the present ention to prevent or reduce the inflammatory response, or to soothe inflammation by reducing the symptoms of inflammation such as redness, pain, heat, or swelling.

-n-

[1 ⁾ 041 ] Inflammatory responses can be triggered by injury, for example injury to skin, muscle, tendons, or nerves. Inflammatory responses can also be triggered as part of an immune response. Inflammatory responses can also be triggered by infection, where pathogen recognition and tissue damage can initiate an inflammatory response at the site of infection. Generally, infectious agents induce inflammatory responses by activating innate immunity. Inflammation combats infection by delivering additional effector molecules and cells to augment the killing of invading microorganisms by the front-line macrophages, by providing a physical barrier preventing the spread of infection, and by promoting repair of injured tissue.

"Inflammatory disorder" is sometimes used to refer to chronic inflammation due to any cause.

[0042] Diseases characterized by inflammation of the skin, often characterized by skin rashes, include but are not limited to dermatitis, atopic dermatitis {eczema, atopy), contact dermatitis, dermatitis herpetiformis, generalized exfoliative dermatitis, seborrheic dermatitis,

drug rashes, erythema multiforme, erythema nodosum, granuloma annulare, poison ivy. poison oak, toxic epidermal necrolysis and roseacae.

[00431 Inflammation triggered by various kinds of injuries to muscles, tendons or nerves caused by repetitive movement of a part of the body are generally referred Jo as repetitive strain injury (RSl). Diseases characterized by inflammation triggered by RSI include, but are not limited to, bursitis, earpa! tunnel syndrome, Dupuytren's contracture, epicondylitis {e.g. "tennis

elbow"), "ganglion" (inflammation in a cyst that has formed in a tendon sheath, usually occurring on the wrist) rotator cuff syndrome, tendinitis (e.g., inflammation of the Achilles tendon), tenosynovitis, and "trigger finger" (inilammation of the tendon sheaths of lingers or thumb accompanied by tendon swelling).

|0044] It is understood that the terms "immune disorder" and "mflammatoiv lesponse ¹ ' are not exdushe. It JS understood that mans immune disorders include acute ysh,>n kmi ) or cinonie {long term) inOammafion it is also undcrstuod that inflammation can run e immune aspects and non ⁱ mmune aspects l he rolcf sj of immune and nonimmune ecus m a ]\γ1 ^! CULΉ mtlammatorj response ma\ i ary with the t>pc of mflamrrkitoi} response, and oia) \ ar\ uuπny the course <tf an tnilarnmaton response immune aspects of infiammation and diseases i elated to inflammation can uvvυivc boih innate and adaptive inmiumt) Certain diseases ielated to inflammation tepiesem an interpld) t ¹ ! immune and nonimmune cell imeraαiυnv hn c\α;npie intestinal mPammation (Fiotchi et al . 19^7, \m J Ph) MP3 Ciastromtesi ! n er Ph> sjo. 273 G ^* ?6^-G775K pneumonia Uuisg mflammαtion), or glomeiuiυnephritis

|0045| it is lurthci uiuieistoυd that man> diseases dre eharuUeri/ed b\ both an inmune disorder and an mfiammatoiv icsponse. such that the use of discrete terms "immune disorder' oi "tnfidnimatoi> response" JS not intended io Imπt the scope of use or activU} of the coπipoumls o' the present im tmt.on with respect to ticatmg a particular disease For example, arter itis is eonsidered an immune disorder charaeιeii/cd b\ mtlammation of joints, but arthutis is lfki.uj.se considered an milammatun disorder charaeteπ/ed b\ immune attack on ]omt tissue- HH^, the obsenation that a compound of the imention reduces the inilansniation seen m an aiπmal mode! of aithπtis. does not limit the observed aetivit\ oi the compound Iu dnti~inn^mnui<>rs avtϊ\ U> In a disease ha\ ing both immune and mtlammatorj aspects, meiels measunng the eiicUs oi a compound of the present ππ ention on inllanimation docs not exclude the possibility tαat the compound mas also \\n\ c immune-modulating aelivitj m the same disease I ike\u\e. in a disease ha\ mg both immune and jnJlajfnmaton aspects, meieh raeaauπnt' the eifects of a

compound of the present invention on immune responses docs not exclude the possibility that the compound may also have antiinflammatory activity in the same disease.

[0046J As used herein, the terms "peptide," "polypeptide" and ''protein" arc used interchangeably and refer to two or more amino acids cαvaienlly linked by an amide bond or non-amide equivalent. The peptides of the invention can be υf any length, for example, the peptides can have from about two to about 100 or more residues, such as, 5 to 12, 12 to 15. 15 to 18, 18 to 25, 25 to 50, 50 to 75, 75 to 100, or more in length. Preferably, peptides are from about 2 to about I S residues. The peptides of the invention include I- and d-isomers, and combinations of 1- and d~isomers. The peptides can include modifications typically associated with post- translationai processing of proteins, for example, cyclization (e.g., disulfide or amide bond), phosphorylation, giyeosylauon, carboxySatioπ, ubiquitination, royristyiatioπ, or iipidation.

[0047 j Peptides disclosed herein further include compounds having amino acid structural and functional analogues, for example, peptidomimetics having synthetic or non- natural amino acids or amino acid analogues, so long as the mimetic has one or more functions or activities of compounds of the invention. The compounds of the invention therefore include "mimetic" and "peptidomiraelie" forms.

[0048] The terras "mimetic," "peptide mimetic" and "pcptidomiraetie" are used interchangeably herein, and generally refer to a peptide, partial peptide or nort-peptidc molecule that mimics the tertiary binding structure or activity of a selected native peptide or protein functional domain (<?.#,, binding motif or active site). These peptide niimeύcs include recombinant!}' or chemically modified peptides, as well as non-peptide agents such as small molecule drug roimeties. as further described below.

[0049] in one embodiment, the PlP peptides of the invention are modiikϋ to produce peplide mϊmeties by replacement of one or more naturally occurring side chains of the 20 genetically encoded amino acids (or D amino acids) with other side chains, for instance with groups such as alkyi, lower aikyl, cyclic 4-, 5-. 6-, to 7 membered alky], amide, amide lower alkyl, amide di (lower alkyi), lower aikoxy, hydroxy, earboxy and the lower estci derivatives thereof, and with 4-, 5-, 6-, to 7 membered heterocyclics. For example, proline analogs can be made in which the ring size of the proline residue is changed from 5 members io 4, 6. or 7 members. Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or noπaromaiic. Heterocyclic groups can contain one or more nitrogen, oxygen, and/or sulphur heteroatorns. Examples of such groups include the fura/.anyUuryi, imida/.olidinyl iraidazoryi, imidazolinyl. isothiazoly), isoxazolyL moφholinyl (e.g. morphoiino), oxaxolyL piperaziny! (e.g. I-pipera-zinyfh piperidyl {e.g. i-piperidyl. piperidino), pyranyt pvrazinyL pyrazolidinyl. pyrazolinyl, pyrazolyL pyridazinyl, pyridyi. pyrirπjdinyj. pyrrolidinyl (e.g. l -pyrrolidinyl), pyrrolinyl. pyrrolyl. fhiadiazoiy!, ihiazolyL thienyi, thiomorpholinyf (e.g. thiomorphoϋno), and mazolyl These heterocyclic groups can be substituted or unsubtfiiuied. Where a group is substituted, the substituent can be alkyl, aikoxy, halogen, oxygen, or substituted or unsubstuuted phenyl. Peptidαniiraeties may also have amino acid residues that have been chemically modified by phosphorylation, sulfonaiioπ. biotinylation, or the addition or removal of other moieties,

[0050} A variety of techniques are available for constructing peptide mimeiics with the same or similar desired biological activity as the corresponding native but with more favorable activity than the peptide with respect to solubility, stability, and/or susceptibility to hydrolysis or proteolysis (see, e g. , Morgan & Oainor, λtin. Rep. Med. C hem. 24.243-252,198 ^j >). Certain peptidomimetk compounds are based upon the amino acid sequence of the peptides of the

invention. Often, peptidomiπiciic compounds are synthetic compounds having a three- dimensional structure (i.e. a "peptide motif) based upon the three-dimensional structure of a selected peptide. The peptide motif provides the peptidomimetie compound with the desired biological activity, i.e., binding to PlF receptors, wherein the binding activity of she mimetic compound is not substantially reduced, and is often the same as or greater than the activity of the native peptide on which the .mimetic is modeled. Peptklomimetk compounds can have additional characteristics that enhance their therapeutic application, such as increased eel! permeability, greater affinity and/or avidity and prolonged biological half-life,

[00511 Peptidomimetie design strategies are readily available in the art (see. e.g., Ripka & Rich, Curr. Op. Chcm. Biol, 2,441-452.1998; Hruby et a!.. Curr. Op.Chem. Biol. 1 .1 14- 1 19J 997; Hruby & Balsc, Curr.Med. Cheni. 9,945-970,2000). One class of pcptidomimcUcs a backbone that is partially or completely πon-peptide, but mimics the peptide backbone atom-for atom and comprises side groups that likewise mimic ibc functionality of the side groups of the native amino acid residues. Several types of chemical bonds, e.g.. ester, thioester. thiuamide, retϊoarnϊde, reduced carbonyk dimcthylene and keiomcthyiene bonds, are known in the an to be generally useful substitutes for peptide bonds iti the construction of protease-resi stunt peptidomimetics. Another class of peptidomirneties comprises a small non-peptide molecule that binds to another peptide or protein, but which is not necessarily a structural mimetic of the native peptide. Yei another class of peptidomimetics has arisen from combinatorial eheπmiry and the generation of massive chemical libraries. These general Iy comprise novel templates which, though structurally unrelated to the native peptide, possess necessary functional groups positioned on a nonpeptide scaffold to serve as "topographical" mimetics of the original peptide (Ripka & Rich, 1998, supra).

[0052] The iirst natural PIF compound identified, termed nP!F- l _f = ₅ , (SuQ I I) NO; ! }, is

a 15 amino acid peptide, A synthetic version of this peptide. sPII ^< - l _{f i 5} » (SBQ SD NO: 13), showed activity that was similar to the native peptide, nPIF- l _{( 5 ? )} (SKQ !L> NO: 1 ). This peptide is

homologous to a small region of the Circumsporozoite protein, a malaria parasite. The second

PIF peptide, αPIF-2 _{π 3} ) (SEQ ID NO:?), includes 13 amino acids and shares homology with a

short portion of a large protein named thyroid and retinoic acid transcription co-rcprcssor, which

is identified a.s a receptor-interacting factor, (SMRI ^" ); the synthetic version is sPli -2 (Sl- Q (D

NO: 14). The third disiinci peptide, nPIF-3π », (SRQ ID NO: 10). consists of 18 amino acids and

matches a small portion of reverse transcriptase; the synthetic version of this peptide sPi F-3 _{; *} ^,

is (SEQ ID NO: 15). tiPIF-4*, (SEQ ID NO: 12) shares homology with a small portion of reverse transcriptase.

{ ^' 0053 i A list of PiF peptides, both natural and synthetic, are provided below in 1 uhfe 1 .

Antibodies to various PIF peptides and scrambled PlF peptides are also provided.

TT

|0054| Ln one embodiment of the present invention, a PlF peptide is provided. Such PIF peptides may be useful for treating or amciioraiing immune-mediated disorders, such as autoimmune diseases,

[0055] in another embodiment, a pharmaceutical composition comprising a PlF peptide is provided, in preferred embodiments, the pharmaceutical composition comprises an effective amount of a PlF peptide,

[0056| In another embodiment a method of treating or preventing immune -mediated disorders is provided, In a preferred embodiment, the method comprises administering an effective atnouni of a FIf peptide to a subject in need thereof. The methods are particularly useful in treating or preventing immune-mediated disorders, including, but not limited to, graft- versus-host disease, type 1 diabetes, multiple sclerosis, ulcerative colitis, Crohn's disease, rheumatoid arthritis and the ϋke.

[θ057J In a further embodiment, a method for treating or preventing immune-mediated disorders comprising administering an effective amount of a PfF peptide in combination with one or more iramunotherapeutic drugs to a subject in need thereof is provided. Such a combination may enhance the effectiveness of the treatment of either component alone, or may provide less side effects and/or enable a lower dose of either component. fO058j The present data demonstrate thai short-terns exposure to PH -I at low doses is associated with a long-term protection against development of autoimmune disorders of disparate origin. While not wishing to he bound by theory, based on the currently understood aspects of PIF-I ^' s mechanism of action, the peptide appears to act independently of the type of pathophysiological features of the autoimmune disease examined addressing them in an etiology-independent manner. This agrees with the properties of PIF-] following examination of

-^3-

its effects on PBMC. PIF-I was found to have widespread modulatory effects on cellular immunology, as well as on cytokine production and secretion aciing through specific inducible receptors present on subtypes of PBMC. PIF-I appears to affect disparate aspects of immunity since it responds to various mitogen challenge, PHA, CD3Mλb, CS33Mλb/CD28Mλb. and

MLR. PlF-I exposure blocks activated, but not basal PBMC proliferation, In addition while there was some bias towards Tn ^' 2, PIF-I caused an increase in both ^" IVJ and T _{; S} 2 cytokines following mitogen exposure. This may indicate that PlF-I helps to maintain the balance between the two immune modalities, not allowing either extreme inflammation or immune suppression. By blocking activated, but not basaS, immunity the ability Io respond to an immunogenic challenge such as pathogen exposure, and/or maternal rejection is well maintained. This may explain the significant efficacy observed in the current mouse studies. Overall, PIi ^1' -] 's mechanism of action is distinct from other currently used immune suppressive agents.

|Oθ59j The three autoimmune models studied are quite distinct: BMT replicates exposure of the mode! organism to foreign immune cells a_. would be the ease in G Vi II); NOD replicates 1 ype 1 diabetes induced by a specific attack on the pancreas by transplanted foreign T cells; and EAE models MS by stimulating a bacteria! toxin and protein immunogen attack on the nervous tissue of the brain. However, all of the models axe characterized by an induced immune response against the host organism and, consequently, to the aυroiinrnunc-induced destruction of vital organs and, ultimately, death. PIf-I appears to successfully neutralize the initiation of this cascade irrespective of the initiating insult. This can be analogized to pregnancy, in which the embryo is tolerated by the mother, but the mother remains able to respond adequately to pathogens. Pregnancy may aLo leave the mother less susceptible to autoimmunity and malignancy, to some degree. Therefore, recreating an envi.rotime.nt where select ceils are

- ^■ ?4_

tolerated while pathogens are attacked in a non-pregnancy setting may be- the central mechanism of PIF-Ts action in autoimmune model systems.

[0060 j In all three models, efficacy was obtained in Sow doses. 0.1-1 rag/kg/day. By contrast, somewhat lower efficacy is observed at higher dosing in two models (NOD 2.73 mg/kg/day and BMT 5 mg/kg/day). This agrees with in vitro data where maximal PlF-! efficacy was found at 1 -50 riM concentrations, while higher doses were cither less effective or not effective at all. This further suggests a receptor -dependent mechanism ol ^' action that is mostly responsive at a narrow range of concentrations and otherwise may be down-regulated when concentrations are raised beyond optimal levels. These observations strongly indicate that PIF exerts this biphasic effect through physiological and not pharmacological mechanisms,

[00611 While the mechanism for the long-term protective effect of PU- -1 as seen in these autoimmune models is not clear, and without wishing to be bound by theory, it appears that PIF-I initiates, following mitogen exposure, a time-dependent block of proliferation and leads to a cascade of T*jl and T{(2 cytokine secretion, some being secreted earlier while others, later. Such sequential effects may lead to a long-term modification of the immune environment.

(0062) FiIM appears to act through putatively novel receptors that are predominantly expressed on monocytes and macrophages. However, when stimulated by mitogen';, the expression of these receptors becomes .significant on T and B Cells but not NK cells. Differences in the expression pattern of PIF- 1 receptors may explain the differences in the response induced by PII ^' - 1 seen in un-stimulated and stimulated environments. In an unstimulated environment, FlF-I may only have a low level of activity on T and B Oils. However, activation of the immune system in response to an immune system ehaiierrge may lead to the expression of the PIF-I receptors on T and B Cells initiating long term tolerance .

[00631 PIF-I 's action appears to be independent of TCR, calcium-channels or FKC pathways ¹ , mechanisms through which most immune- suppressive agents act. and CD4+/C ^' D25í ceils (T reg) cells that are of relevance in various autoimmune diseases. On the other band. PlF- i 's action may involve NFλT-1 suppression.

[0064] In pregnancy, embryo viability is dependent on maternal tolerance of the embryo, but there is a clear time lag between embryo expulsion by miscarriage and reduction of PIF levels in maternal circulation, In fact. PlF disappears from maternal circulation up to three weeks beibre maternal human chorionic gonadotropin (hCG) levels dropped and miscarriage ensues. Perhaps, a similar mechanism is involved in maintaining tolerance, or protective effects against autoimmunity long term, as is documented in the three model systems tested following cessation of therapy.

Jθ065) In a preliminary study, the effect of PIF- 3 administration using an λlzetw pump for 7 days after mating on implantation rates in mice was examined. λs expected, PiP-I did not exert any adverse effects. Moreover, PlF-I may have actually increased die rates of fetal survival vs. control by day 13 of pregnancy, as documented ai the time of Ceasarcan section. This data combined with the additional six animal trials provides a strong support for the- lack of PIF-I toxicity.

{0066 j We also found that FITC-PIF-I injected IV in mice accumulated in the spleen and was cleared from circulation into the kidney within minutes. This shows that PlF-I specifically targets immune cells of the spleen m vivo, and has a short hall-life in circulation. The long term effect of PH ^; administration may reflect a pharmacodynamic type of mechanism since the peptide has a short, half-life, rather than a pharmacologic effect that is produced while the drug is given and is dependent on the levels of the drug in the circulation.

-?( ^■ >-

|0067| The observations that PIF-I exerts long-term protection after short-term exposure in al! three models tested raises the possibility thai PIl- ^' - 1 therapy could be used for long-term management of patients with autoimmune diseases, perhaps initially using an insulin pump that could replicate the function of an λlzet® pump followed by periodic PIF-I administration, over a long term, Other devices capable of continuous and/or long term administration may also be useful. In addition, it may be possible to develop an increased half- life, modified peptide (such as by PEGyiatiαn) and/or to use transdermal delivery system for long term, but minimally invasive use. Finally, due to PIF-Ts simple structure and small size, in which shorter versions of the peptide are similarly effective (at least in vitro), oral delivery may become possible, which could transform PlF-I into a convenient chronic therapy,

[0068] Ultimately, a novel embryo-derived peptide, PCF. creates a tolerogenic Mate at Sow doses following short-term treatment leading to long-term protection in several distinct severe autoimmune models. This effect is exerted without apparent toxicity.

[θθ69J For therapeutic treatment of the specified indications, a PiF peptide may be administered as such, or can be compounded and formulated into pharmaceutical compositions in unit dosage form for parenteral, transdermal, rectal, nasal, local intravenous administration, or, preferably, oral administration. Such pharmaceutical compositions are prepared in a mariner vveli known in the art and comprise at least one active PIF peptide associated with a pharmaceutically carrier. The terra "active compound", as used throughout this specification. refers to at least one compound selected from compounds of the formulas or pharmaceutically acceptable salts thereof.

10070J In such a composition, the active compound is known as "active ingredient." In making the compositions, the active ingredient vvUl usually be mixed with a carrier or diluted by

a carrier, or enclosed within a carrier thai may be in the form of a capsule, sachet, paper or other container. When the carrier serves as a diluent, it may be a solid, semisolid, or liquid material that acts as a vehicle, excipient σf medium for the active ingredient. Thus, the composition can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, emulsions, solutions, syrups, suspensions, soft and hard gelatin capsules, sterile injectable solutions, and sterile packaged powders.

{007! ] Some examples of suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate alginates, calcium saiicalc mieiυcrystalline cellulose, polyvinylpyrrolidone, cellulose, tragaeanih, gekitin. syrup, methyl cellulose, methyl- and propylhydroxybenzoates. tale, magnesium stearate, water, and mineral oil. The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents. The compositions may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art.

[0072 { For oral administration, a compound tan be admixed with carriers and diluents, molded into tablets, or enclosed in gelatin capsules. The mixtures can alternatively be dissolved in liquids such as 10% aqueous glucose solution, isotonic saline, sterile water, or the like, and administered intravenously or by injection,

|0073] l he local delivery of inhibitory amounts of active compound for the treatment of immune disorders can be by a variety of techniques that administer the compound at or near the targeted site. Examples of local delivery techniques are not intended io be limiting but to be

illustrative of the techniques available. .Examples include local delivery catheters, site specific earners, implants, direct injection, or direct applications, such as topical application.

[0074] ^[ .oca ^] delivery by an implant describes the surgical placement of a matrix that contains the pharmaceutical agent into the affected site. The implanted matrix releases the pharmaceutical agent by diffusion, chemical reaction, or solvent activators.

J0075] For example, in some aspects, the invention is directed to a pharmaceutical composition comprising a PlF peptide, and a pharmaceutically acceptable earner or diluent, or an effective amount of a pharmaceutical composition comprising a PiF peptide.

[0076} The compounds of the present invention can be administered in the conventional manner by any route where they are active. Administration can be systemic, topical, or oral, hor example, administration can be, but is not limited to, parenteral subcutaneous, intra venous. intramuscular, intraperitoneal, transdermal, oral, buccal, ocular routes, intravaginally. by inhalation, by depot injections, or by implants. Thus, modes of administration for the eompimds of the present invention (either alone or in combination with other pharmaceuticals) can be, but are not limited to, sublingual, injectable (including short-acting, depot, implant and pellet forms injected subcutaneous!)' or intramuscularly), or by use of vaginal creams, suppositories, pessaries, vaginal rings, rectal suppositories, intrauterine devices, and transdermal forms such as patches and creams,

[0077 j Specific modes of administration will depend on the indication. The selection of the specific route of administration and the dose regimen is to be adjusted or titrated by the clinician according to methods known to the clinician in order to obtain the optimal clinical response. The amount of compound to be administered is that amount which is therapeutically effective. The dosage to be administered will depend on the characteristics of the subject being

treated, e.g., the particular mammal or human treated, age, weight, health, types of concurrent treatment, if any, arid frequency of treatments, and can be easily determined by one of skill in the ait (e.g., by the clinician).

|0078] Pharmaceutical formulations containing the compounds of the present invention and a suitable carrier can be solid dosage forms which include, but are not limited to, iabiets, capsules, cachets, pellets, pills, powders and granules; topical dosage forms which include, but are not limited to, solutions, powders, fluid emulsions, fluid suspensions, semi-solids, ointments., pastes, creams, gels and jellies, and foams; and parenteral dosage forms which include, but are not limited to, solutions, suspensions, emulsions, and dry powder; comprising an effective amount of a polymer or copolymer of the present invention. St is also known in the art that the active ingredients can be contained in such formulations with pharmaceutically acceptable diluents, fillers, disintegrants, binders, lubricants, surfactants, hydrophobic vehicles, water soluble vehicles, emulsifiers. buffers, humcctants, moisturizers, solubilizers. preservatives and the like. The means and methods for administration are known in the art and an artisan can refer to various pharmacologic references for guidance. For example. Modern Pharmaceutics, Banker & Rhodes, Marcel Dekker, Inc. (1979); and Goodman & Oilman's The Pharmaceutical Basis υ/

Therapeutics. 6th Edition, MacMiilan Publishing Co., New York ( 1980) can be consulted.

[0079] The compounds of the present invention can be formulated for parenteral administration by injection, e g., by bolus injection or continuous infusion The compounds can be administered by continuous infusion subeutaneousiy over a predetermined period of time, formulations for injection can be presented in unit dosage form, e g., in ampoules or in multi- dose containers, with an added preservative. The compositions can take such forms as

suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain ibrmulatory agents such as suspending, stabilizing and/or dispersing agents.

[0080] For oral administration, the compounds can be formulated readily by combining these compounds with pharmaceutically acceptable carriers well known in the art, Such carriers enable the compounds oϊ the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by adding a solid t-xcipietit, optionally grinding the resulting mixture, and processing the mixture ol ^* granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipk-nts include, but are not limited to, fillers such as sugars, including, but not limited to, lactose, sucrose, mannitol. and sorbitol: cellulose preparations such as, but not limited to. maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacaπih, methyl cellulose, hydroxypropylmeirn i-eeilulose. sodium carboxymethyiceiiulose. and poi\vin\lpyrrυlidone (PVP). If desired, disintegrating agents can be added, such as, but not limited to. the cross-linked polyvinyl pyrrolidone. agar, or alginic acid or a salt thereof such as sodium alginate.

[0081] Dragee cores can be provided with suitable coatings l- ^' or this purpose. concentrated sugar solutions can be used, which can optionally contain gum arabie. talc, polyvinyl pyrrolidone, carbopoi gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures, Dye-stuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations ύl ^' active compound doses. f 00821 Pharmaceutical preparations which can be used orally include, but arc not limited to. push-tit capsules- made of gelatin, as well as soft, sealed capsules made of gelatin and

a pkstieizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as, e.g.. lactose, binders such as, e.g., starches, and/or lubricants such as. e.g. _y talc or magnesium st citrate and, optionally, stabilisers. Sn soli capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatt> oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. All formulations for oral administration should be in dosages suitable for such administration.

|0083] For buccal administration, the compositions can take the form of, e g., tablets or lozenges formulated in a conventional manner.

[0D84| For administration by inhalation, the compounds for use according to the present invention arc conveniently delivered in the form of an aerυ^ol spray presentation from pressurized packs or a nebulizer, wiih the use of a suitable propel iant. e g., dichlorodifluorornethane, trichlorofluoromethane, dichlorotetrafluoroεlhane. carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch, fθi)85] The compounds of the present invention can also be ibnnuSated in recta! compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0θ86] In addition to the formulations described previously, the compounds the present invention can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneoαsly or intramuscularly) or by intramuscular injection.

-1?..

|0087J Depot injections can be administered at about 1 Io about 6 months or longer intervals. Thus, for example, the compounds can be formulated with suitable polymeric or h ^y drophobic materials (for example as an emulsion in an acceptable oil} or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0088] In transdermal administration, the compounds of the present im errtiorh for example, can be applied to a plaster, or can be applied by transdermal, therapeutic systems that are consequently supplied to the organism.

{0089 J Pharmaceutical compositions of the compounds also can comprise suitable solid or ge! phase carriers or excipients. Examples of such carriers or excipicnts include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as, e.g., polyethylene glycols.

|θO9O{ The compounds of the present invention can also be administered in combination with other active ingredients, such as, for example, adjuvants, or other compatible drugs or compounds where such combination is seen to be desirable or advantageous in achieving the desired effects of the methods described herein,

{0091 ] This invention and embodiments illustrating the method and materials used may be further understood by reference to the following non-limiting examples.

EXAMPLE ϊ

|0( ⁾ 92] Peptide synthesis: Synthetic PlF-I ₁₅ (MVRIKPG SANKPSDD) was obtained by solid-phase peptide synthesis (Peptide Synthesizer, Applied Biosyslems) employing Fmoc (9- lluorenyimcthoxycarbonyl) chemistry. Final purification is carried out by reverse-phase 1 !PLC and identity is verified by MALDI-TOF mass spectrometry and amino acid analysis and purified to >*>5%, by 1-1 ^' PLC ₅ and documented by mass spectrometry (Biosynthesis, Texas).

-31-

[0093] Mice: C57BL/6{!i-2b) mak and female and (C57BL/6xBλLB/c) M (H-2d/b} male, live- to six-week-old mice (GVlJD studies) and seven- to eight- week old SJl. mice (MS studies) were purchased from Harian (Israel), and male and female seven- io eight- week-old NOD mice were obtained from Jackson Laboratories (Maine). λi! mice were maintained under

conditions approved by the Institutional Animal Care and Use Committee of the Hebrew University in Jerusalem in accordance with the national laws and regulations for protection oi ^' animals.

|0094) GvIlO model; Recipients (C57BI./όxBλLB/c) Fl mice received Mhal whole- body irradiation by a single dose of 10Oϋrad/dose and were reconstiuued with 5-8x 10° C57BL/6 bone marrow (BM) cells and 10-2OxKf spleen cells. BM from C57BU6 donor mice was

collected by flushing of femur, humerus and tibia into 10% FCS/PBS. BM mononuclear cells were isolated frum the interface after centπfugation on a Ficoli-llipaque gradient. Spleens were crushed through 70 μm screens into 10% FCS/PBS. BM cells plus spleen ceils were inoculated intravenously into whole-body irradiated mice otic-day post radiation. PlF-I therapy (0.1-1 mg/kg/day) was administered in three separate animal trials 5-10/group vs. control by implanting under anesthesia an λlzet® pump in the dorsal subcutaneous region at the day of transplant for one to two weeks providing continuous release of Pϊf-I . .H valuation of GvHD model animals was carried out by examining body weight, skin lesions, animal survival, and histological examination. Animal weight was examined every three days following BM transplantation, scoring for skin manifestations of GVIlD was carried out from day ] 2 posl BMT up to four months. Skin and liver samples were fixed in 10% formalin embedded in paraffin and stained with hematoxylin and eosiπ and evaluated for ulcers, in the former and lymphoeyte

infiltration in the latter. Results were evaluated by γj and λNOYA.

|OT95j Assay for ehimerism: Mice were anesthetized and blood taken from the retro- orbital sinus of the eye. WBC {2-8 x lO ^' VsampSe) were separated, directly stained with anti-H- 2K ^b -FITC (IgG ₂₈ ) or anti-H~2K ^d ~FfTC (IgG _2n ) monoclonal antibodies (mAb) (Senπec. USA). and analyzed by I- ^' λCS analysis (FACStar plus, Bectoπ Dickinson, San Jose, CA, USA). Background binding of each H-2K-specific mAb was determined by staining with it the ceils of non-relevant haplotype.

(0096) GVHD Model experiment F Following low-burden BMT, GVHD development was examined following FlF-I therapy (0.1-l mg/kg/day) given lor one week using an implanted

λl/.ct® pump followed by one month observation. Of the PlF- 1 -treated mice. (M or 1 mg/kg/day for one week, all eight mice did not develop GVHU. Four out of live controls developed GVIlD grade IWS! (/^O .01 ). Mice following BMT and short term PlF-I treatment remained completely protected against the development of GVHD at one month alter cessation of therapy, In contrast, in control mice, severe skin ulcerations and weight loss developed (mean mouse weight 21 .9g, high dose PIF-I (N=3), 20.2g low dose PiF- i (N- S), and ] ^c >,.Sg in controls).

[00971 GVHD Mode! experiment II (Figure 1 ). Wc examined whether PIF-I could

prevent GVHD development in a higher-burden BMT (double number of spleen cells transplanted than the low-burden BMT). Following exposure to PIF-I fϋ. l-lmg/kg/day) for two weeks, total protection against GVHD was obtained within three weeks with lhe high dose therapy 7/7 vs. 9/9 in control with GVHD, This protection remained also significant (i ^? <0,04) at thirty days post-BMT in both treatment groups, as evaluated by the GVI ID score in ihosc which developed the disease (Figure 2).

|00981 GVHD Model experiment IO {Figure 3). Wc examined whether short-term treatment can lead to long term survival after cessation of therapy. Following high-burden BMT. PϊF-1 1-5 mg/kg/day for two weeks was administered and mice were followed for an additional three and one-half months without therapy, PiF-I conferred a significant protection as determined by mouse survival at the end of the observation period, Seven of nine of the PiF-! treated mice survived compared to only two out of ten in controls fP<0.02). i ligher-dυse therapy was less effective, although it was still associated with a higher survival rate than controls (5/9 survived). Significant protection from weight loss was also achieved following PIF- I 1 /mg/kg/day exposure vs. controls. This effect became significant alter day 33 from BMT. In control mice, following BMT ^' . GVHD-induced skin ulcerations were observed. Short- term PIF-I therapy prevented the development of such lesions in the long term. Liv er histology also documented that lymphocytic infiltrates., indicating autoimmune response, were noted in control, hut not in PIF- ϊ -treated mice one month after cessation of therapy. The degree of BMT incorporation into the recipient mice was determined . Results show that there was α quasy total incorporation of grafted bone marrow after live weeks after BMT (87.5 ; 2.4). reileeted a very high degree of chimerism.

|0099] AlIo-BMT followed destruction of the host's immune system b> total body radiation. Thereby the BMT ^' recipient is highly vulnerable to immune attack by the irampiantcd foreign immune cells. Low dose (micromolar) PIF-I administration totally prevented GVMD while therapy was administered. More remarkably, long-term protection after cessation of therapy was obtained, as reileeted by the significant prevention of GVHD development and long-term survival for several months vs. control mice. This effect was not associated with any toxicity, as documented by mouse weight skin appearance, and skin and liver histology . This

was also documented by the significant degree of chimcrism (about 90%,) thai developed in the peripheral PSMC within five weeks following BMT ₁ indicating that at that time the great majority of the mice immune system was constituted of the transplanted BM

[00100] PlP-Ts long-term protective effect after cessation of therapy is particularly significant, as other BMT therapies arc effective only during active administration. Funhermore. the current BMT model involved a clear mismatch between the recipient and the donor, and large quantities of cells were transplanted, while clinical settings use closely matched BM donors, which nevertheless often, up to 70% results in various degrees of GVHD.

EXAMPLE 2

JOOJOIj Materials and methods arc die same as Example ! .

100102 J DM (adoptive transfer NOD) model. Male NOD mice were irradiated (650 rad), and injected IV next day with 250 Mil spleen cells collected from female NOD diabetic mice. PIF- I was injected ra two doses 0.83mg/kg/day (N=5) and 2.73mg/kg/day (N=7) for 28 days using an Alzet® pump, implanted subcutaneously providing continuous release of the peptide, followed by a 40-day observation period. Animals were monitored for DM development by determining fasting glucose levels in both blood and urine. Results were evaluated using ANOVλ.

J001 ^Q 3] NOD diabetes model. We examined the effect of PiF-I in a different autoimmune model NOD adoptive transfer, in this model, transfer nf diabetic splenocyu-s from female to male mice leads progressively to the development of diabetes meilitus. iixposure to PIF- 1 0,83-2.73 mg/kg/day for the first 28 days had a long-term protective effect against the destruction of pancreatic cells and the consequent high serum glucose levels. Figure 4 shows a life table analysis of NOD mice following adoptive transfer of spicnocytes from a diabetic female mouse By 70 days, at the conclusion of the experiment, PlF-I was totally protective in

1 1/12 of mice treated with PiF-I while 6/7 in the control group has already developed diabetes. Interestingly, the only PIF-I treated mouse thai developed DM received a higher ireatmenϊ dose. The development of diabetes was documented by increased serum glucose levels: in certain conirol animals it reached >600 mg/di. Table 2, below, shows individual mice glucose levels after cessation of therapy.

[00104] In the control group, most mice developed diabetes by 40 davs. Additionally,

histological examination demonstrated that Pl F treated mice were protected against inflammation, of the pancreas v. control (data not shown).

[00105 J To further document PI K- Ts immune-modulatory effects, we used the NOD

mouse adoptive transfer model, which results in the development of diabetes (reikcted by high

glucose levels) due to the destruction of the recipient's pancreas by transfer of autoreactive

splenoeytcs from a diabetic mouse thai targets specifically that organ, Using this aggressive

model, we documented a long-term protection against DM developrncni using PIF- I therapy.

These results open the possibility of examining young adults that have recently developed I )M in

whom there has not been a total destruction of insulin-producing pancreas cells. Such an early

intervention could lead to a decreased need, for insulin administration, or even allow long-term

oral ami-diabetic therapy. Since we found that PIF-I targets isolated splenocyies thai provides a

rationale for the protective effects thai were observed, in TlDM primed T cells and macrophages directly attack the pancreas which is followed by local increase in Tj ₁ I cytokines (i .e., TMF i

.interferon- Q) that further amplify the auto-destructive process, PIF- I may aci on both of ihese

aspects of autoimmunity by blocking activated immune cells proliferation activation and.

modulating cytokines secretion, towards a T _H 2 pattern (i .e., major increase in IL l O).

EXAMPLE 3

{θ01θ6J Materials and methods are the same as Example 1 .

f SH) .107] MS EAE model: experimental autoimmune encephalomyelitis, SJL mice 7-8

weeks old were injected in the tail base with 1 : 1 of 200 μg proteolytic protein peptide [VL?)

together with 200 μg CFA and IFA {containing mycobacterium tuberculosis). On the same day

and two days later, mice were injected IP with 250 ng pertussis toxin. Within nine days, animals

started developing paralysis, PlF- I was administered using a subcotaneously implanted λizet#

pump at 0.75mg/kg/day for 2$ days and its effect was compared to a control group. Daily monitoring of the degree of paralysis (grade 0/no disease - 5 /dead animal) occurred up to 40 days. PJF-I \s protective effects were calculated using the Mann-Whitney non parametric test.

[00108] MS model. We further examined whether PIF-S therapy could be effective in an additional autoimmune model experimental autoimmune encephalomyelitis ( KAh) in which the majority of the damage occurs in poorly accessible region of the body, the ecinral nervous system. By exposing mice to a combination of a toxic agent (PLP) for she nervous system together with boosting further the inflammatory response with two additional types of bueienal- toxins led to rapid paralysis <10 days. Figure 5 shows thai the exposure to PIl-- 1 at O.?5mg/kg/day for 28 days led to a continuous protection by significantly reducing the paralysis score, as determined by daily observations using a clinical score. The protective effect also lasted for at least two weeks after stopping therapy (/ ^J <0.002).

[0θ109J The experimental myeloencephalitis, RAE, is recognized as a highly relevant and acute model for MS. 1 he exposure to auto-antigens coupled by induction with two bacteria) sraraunogens leads to progressive paralysis within short tern?. We found that PIF-! led to a significant reduction in the paralysis score across the observation period which persisted even twυ weeks after cessation of therapy. This is an indication that autoimmune neurological disorders may be alleviated by PIF-I . Additionally, histological examination demonstrated that PIF treated mice were protected against inflammation of the spinal cord v. control (data not shown).

[00110] MS is believed to be ihe result of a genetic predisposition followed by a viral insult that leads to CD44 autoreactive ceils followed by differentiation Jo the ¹ I ViI phenotype. On the other hand, local damage to central nervous system mav occur by CDH * T cells, and

other elements that are involved in the innate immune system. This leads to altered Ts _f 2 cytokines, regulatory T, and NK cells and IFNn secretion. We have previously shown that several elements of this immune cascade are modulated by PIF-I, consequently, the tolerogenic peptide may be involved in one or more aspects of this immune disorder.

EXAMPLE 4

|00111| To determine maximally tolerated dose of PIF-I in patients who develop GVHD after matched BMT. using an insulin pump. Recipients with grade lϊ GVl IU will be randomized into three groups: (1) continue- conventional therapy {i.e., steroids and cyclosporin λ). as control'. (2) add PlF-I therapy while continuing conventional therapy: am! (3 ϊ stop conventional therapy and use PIF-! alone. Patients will be continuously treated for 4 weeks (using an insulin pump), with 30% dose increments, 15 patients/group. P re-therapy clinical indices, including tumor burden, will be compared to the same indices during/post PJF- 3 exposure, monitoring skin for lesions and testing organ function, including PBMC ability to respond to mitogen challenge.

|00I12| To examine PJF-Fs effectiveness in GVIlD prevention with maintained anticancer effect. Upon successful completion of the first study, BMT recipients will be randomized into three different groups; ( 1 ) conventional therapy (control); (2) conventional prophylaxis of GVl ID combined with Pf F-I ; (3) PlF-I prophylaxis alone. At transplant, patients will begin PIF-I therapy (using an insulin pump) at 30% increments for 12 weeks foil ow ed by 3í months observation, 15 patients/group. The number of patients that develop (JVi U). the degree of the reaction, and response to cancer will be compared between the two test groups.

EXAMPLE 5

{001 13j Assess effect of PIF on PBMC isolated from patients with Chron ^' s disease. Established patients PBMC (N ^::: 20) will be isolated and cultured in die presence of PiF alone using a dose dependent design and in presence of ^■ +/- PHλ, or CD3MAb _' 'CD28Mab, used as

mitogens, using Cloning media, serum iree. After 24 hours of exposure PBMC culture media will be collected and tested for a) cytokine release, both Ti l l and 1112, using the l .uraioex 10 package b) PIF receptor expression exposing to FlTC-PIF and labeled -CO 14, CD4. ClM. _. or CD58, or CD 19MAb followed by flow cytometry) c) in selective eases, rnR?\ ^f A will be exmieted and using an Affyraetrix chip global genome analysis will be carried out. Results vviil be compared with PBMC similarly treated derived from normal volunteers,

[00114J Assess effect of PlF on colon biopsy of patients with €hron ^' s disease. In parallel to obtaining PBMC also biopsies from the same patients will be obtained during

colonoscopy. Biopsy samples will be placed in culture to generate explains usin^ RPM S I 640 medium. Explant cultures will be carried out for 24 hours in the presence of PfI- ¹ 0-2(K) riM. Subsequently, the media will be collected and analyzed for cytokines using Uu* i O multiplex Luminex system (Till and TR2). The tissue itself will be placed in formalin and will be analyzed for cytokine content using HlC as well as immune cell type presence us in ^ flow cytometry and specific CD markers.

fOθϊ iS] Although the present invention has been described in considerable detail with reference to certain preferred embodiments thereof, other versions are possible. Therefore the spirit and scope of the appended claims should .out be limited to the description and the preferred versions contain within this specification.