AU6333796A - Oral tolerance in skin diseases presenting with t-cell media ted inflammation - Google Patents

Description

ORAL TOLERANCE IN SKIN DISEASES PRESENTING

WITH T-CELL MEDIATED INFLAMMATION

FIELD OF THE INVENTION This invention relates to methods and compositions for treatment of skin diseases presenting with T-cell mediated or T-cell dependent inflammation. More particularly, this invention relates to methods and compositions for treating such diseases through orally (or mucosally) induced tolerance resulting in suppression of inflammation.

BACKGROUND OF THE INVENTION

Oral administration of antigen was first shown to induce antigen-specific peripheral tolerance by Wells, H.G. J. Infect. Dis. 8:147-171, 1911 and by Chase, M. Proc. Soc. Exp.

Biol . Med. 61 :257-259, 1946.

Mowat, A.M., Immunol . Today 8:93-98, 1987, described that orally induced tolerance ("oral tolerance") is involved in preventing systemic immune responses to ingested proteins in food. Additionally, oral tolerance to autoantigens and bystander antigens (defined below) has been employed to suppress an autoimmune reaction or response, and -thus to suppress (T-cell mediated) autoimmune disease in both animals and humans: Thompson, H.S.G. et al. Clin. Exp. Immunol . , 64:581-586, 1986; Nagler-Anderson, C. et al. Proc. Nat'l .

Acad. Sci . (USA) 83 : 7443-7446, 1986; Higgins, P. et al. J.

Immunol . 140: 440-445, 1988; Zhang, J.A. et al. Proc. Natl .

Acad. Sci . (USA) 88:10252-10256, 1991; Nussenblatt, R.B. et al. , J. Immunol . , 144 :1689-1695, 1990; Weiner, H.L. et al. Science, 255:1321-1324,

1993; Trentham, D.E. et al., Science, 261, 1727-1730, 1993.

The present inventors and co-workers have several patent applications and patents directed to use of "autoantigens" and "bystander antigens" in inducing oral tolerance to various human autoimmune diseases. Several of these patents and patent applications are listed below. As used in these earlier patents and applications the terms "autoantigen, " "bystander antigen" and "bystander suppression" have the following meanings:

"Autoantigen" is any substance or a portion thereof normally found within a mammal that, in an autoimmune disease, becomes the primary (or a primary) target of attack by the immunoregulatory system. The term also includes antigenic substances that induce conditions having the characteristics of an autoimmune disease when administered to mammals. Additionally, the term includes peptidic subclasses consisting essentially of immunodominant epitopes or immunodominant epitope regions of autoantigens. Immunodominant epitopes or regions in induced autoimmune conditions are fragments of an autoantigen that can be used instead of the entire autoantigen to induce the disease. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens specific to the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.

"Bystander antigen" or "bystander" is a protein, protein fragment, peptide, glycoprotein, or any other immunogenic substance (i.e. a substance capable of eliciting an immune response) that (i) is, or is derived from, a component specific to (or primarily found in) the organ or tissue under autoimmune attack; and (ii) upon oral or enteral administration elicits regulatory (suppressor) T-cells (which can be of the CD4+ or CD8+ type) that are targeted to the organ or tissue under attack where they cause at least one antigen- nonspecific immunosuppressive factor or immunoregulatory cytokine (such as TGF-3, IL-4 or IL-10) to be released and thereby suppress immune attack cells that contribute to autoimmune destruction. The term includes but is not limited to autoantigens and fragments thereof involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction. For example, heatshock proteins, although not specific to a particular tissue, are normally shielded from contact with the immune system.

"Bystander suppression" is suppression at the locus of autoimmune attack of cells that contribute to autoimmune destruction; this suppression is mediated by the release of one or more immunosuppressive factors (including Th2-enhancing cytokines and Thl-inhibiting cytokines) from suppressor T-cells elicited by the ingestion (or inhalation) of a bystander antigen and recruited to the site where cells contributing to autoimmune destruction are found. The resulting downregulation by the secreted cytokines is antigen-nonspecific but locally restricted to the autoimmune responses responsible for tissue destruction.

Methods and compositions useful in suppression of an immune response associated with an autoimmune disease by orally induced tolerance (or by tolerance induced by inhalation) using autoantigens or more generally bystander antigens with and without enhancers have been described in various patents and patent applications by the present inventors and their co- workers: Ser. No. 07/843,752, filed February 28, 1992; Ser. No. 08/202,677, filed February 25, 1994; Ser. No. 08/419,502, filed April 10, 1995; Ser. No. 08/419,505, filed April 10, 1995; Ser. No. 08/235,121, filed April 28, 1994; Pat. No. 5,399,347, issued March 21, 1995; Ser. No. 08/297,395, filed August 11, 1994; Ser. No. 08/046,354, filed April 9, 1993; Ser. No. 08/420,979, filed April 10, 1995; Ser. No. 08/420,980, filed April 10, 1995; Ser. No. 08/105,912, filed August 10, 1993; Ser. No. 08/279,275, filed July 22, 1994; Ser. No. 08/328,562, filed October 24, 1994.

However, the concept of applying bystander suppression to diseases characterized by inflammation and involving the skin (or a component of the skin) as the afflicted organ has not been disclosed or suggested. In particular, application of bystander suppression to treatment of human skin disorders has not been disclosed or suggested. A number of skin disorders of known, unknown or disputed etiology (or symptomatic of other pathologies) are characterized by or associated with an inflammatory response of the skin mediated at least in substantial part by T-cells, more specifically Thl cells. Some of these diseases are believed to be autoimmune: non limiting examples are psoriasis, vitiligo, pemphigus vulgaris, dermatomyositis, alopecia areata, alopecia totalis, and alopecia universalis. Krueger, J., Nature Medicine 1(5) : 442 - 441 , May 1995; Wong, R.I. et al., Jmmunol. Today, 14 (2 ) :69-74, February 1993; Trentham, D.E. et al., Arthritis .Rheum, 24(11) :1363-9, November 1981; Goudie, R.B. et al., Lancet, August 1979, 2(8139) :393-5; Fathman, C.G. et al., N. Engl . J. Med., June 18, 1992, 326 (25) :1693-5; Grunwald, M.H. et al., ∑πt. J^". Dermatol., September 1989, 28(7) :480-l; Fine, R.M., Int. J. Dermatol . , Jan-Feb. 1988, 27(1) :17-8; Carstens, J., Aft. Sinai J. Med. , Oct. 1990, 57(5) :311-4; Reeves, W.H., Semin Dermatol . , Sept. 1991, 10(3) :217-24.

Other skin diseases that present with inflammation are not autoimmune but their incidence is associated with exogenous antigens. Non limiting examples include atopic dermatitis, and contact dermatitis.

The common characteristic of these diseases is that the antigen (whether endogenous or exogenous) is present in the microenvironment of the skin. Inflammation is caused by activated T-cells that recognize the antigen and aggregate at the locus of the antigen.

Although mostly not life-threatening, these T-cell mediated inflammatory skin disorders afflict a large number of people. For example, at least 1% of the United States population suffers from psoriasis. These diseases can be not only physically debilitating but also psychologically damaging (as can be the case, for example with both psoriasis and, especially for dark-skinned people, vitiligo) and often interfere with the victim's productivity, especially when the disease is occupationally related (as is often the case for example, with contact dermatitis) . Thus these diseases add considerably to the economic burden. There are no known cures for these T-cell mediated inflammatory skin disorders. Treatments currently in use are palliative and are accompanied by side-effects that are often at least as serious as the disease they are intended to treat. For example, treatments for psoriasis include ultraviolet B(UVB) radiation, PUVA which involves topical or systemic administration of psoralens (P) combined with ultraviolet A (UVA) radiation, methotrexate administration, topical or systemic use of retinoids, and administration of global immunosuppressants, such as cyclosporin A. Vitiligo is also treated with PUVA or (less effectively) with topical steroids. Bleaching of the skin is also used but it is only effective in masking the depigmentation.

The various types of dermatitis and localized alopecia areata are normally treated with topical steroids. Widespread alopecia areata is treated with cyclosporin A or steroids, systemically administered.

PUVA therapy substantially increases the risk of skin carcinomas (sguamous cell carcinoma, malignant melanoma) and also causes premature (photo)aging.

UVB therapy also has the same disadvantages although both the increase in the risk of malignancies and the photoaging damage are somewhat less pronounced.

Methotrexate (used only for severe psoriasis cases) was developed as a chemotherapeutic drug and its administration has the usual side-effects of chemotherapy, including bone marrow suppression, immune suppression, and liver cirrhosis.

Retinoids are teratogenic and cannot be administered to pregnant women. Moreover, a substantial number of patients have adverse reactions. Some of the most common serious side- effects of retinoids are: increase in blood triglycerides (which is thought to increase the risk of vascular and heart disease) , impaired liver function and benign intracranial hypertension.

Accordingly, there is a need in the field for new methods and compositions for suppressing T-cell mediated inflammation associated with skin disorders and for treating such disorders. More particularly, there is a need in the field for such methods and compositions the use of which is substantially free of serious side-effects such as those enumerated above.

SUMMARY OF THE INVENTION

In one aspect the present invention relates to methods for treating a mammalian (including a human) skin disorder presenting with T-cell mediated (or T-cell dependent) inflammation comprising administering to a mammal in need of treatment via the oral route a "skin-associated bystander antigen" (defined below) in an amount effective to suppress said inflammation.

In another aspect the present invention relates to methods of suppressing T-cell mediated or T-cell dependent inflammation of the skin associated with a skin disorder comprising administering to a mammal in need of treatment a skin-associated bystander antigen in an amount effective to suppress said inflammation. In yet other aspects the invention relates to compositions and dosage forms that: contain at least one skin- associated bystander antigen; are adapted for oral administration; and are useful in the foregoing methods.

The invention is described in detail below by reference to specific preferred embodiments set forth in this specification, claims and drawing.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a plot of the change in footpad thickness (expressed in mm compared to unchallenged controls) for each of three experimental groups of mice: G3 fed Factor XIII (a skin-specific bystander antigen) ; G2 fed hen egg lysozyme (HEL) ; and Gl fed PBS (positive control group) ; immunized in the right flank with Factor XIII and in the left flank with HEL; and then challenged with HEL. DETAILED DESCRIPTION OF THE INVENTION

It is well-known that, in delayed-type hypersensitivity (DTH) experiments, T-cell mediated inflammation is induced by a first (sensitizing) immunization followed by challenge with the same antigen as that used to sensitize the experimental animal. Challenge is usually administered in either the ear or the footpad of the animal, where there is a relative abundance of skin (as opposed to other tissue) and thus inflammation of the skin is induced. The DTH system has been extensively used as a template for assessing the effect of various methods and substances on T- cell mediated inflammation. Ferguson, T.A. et al., PNAS (USA) 88:8072-8076, 1991, Ohmen, J.D. et al. J". Immunol . , 254:1956- 1963, 1995. It is thus particularly useful for assessing the effect of various methods and substances on T-cell mediated inflammation of the skin, such as that associated with the aforementioned skin disorders. The DTH system effectively emulates the presence of an antigen (endogenous or exogenous) in skin and elicits activated T-cells that are reactive with this antigen.

Moreover, the ability to choose (arbitrarily) the antigen that is used to induce inflammation in the DTH system has rendered this system reliable as a template for investigating inflammation (and its suppression) independent of the causative agent. Ahmed, A.R. et al., Arch. Dermatol . , 119(11) :934-945, 1983; MacLean, L.D., Surg. Gynecol . Obstet . , 166: 285 -293 , 1988.

The concept of bystander suppression was first developed by the present inventors and their co-workers during investigations in various autoimmune diseases. It was established that in the case of autoimmune disease, bystander suppression causes regulatory (suppressor) T-cells to be induced in the gut-associated lymphoid tissue (GALT) or, in the case of by-inhalation administration, mucosa associated lymphoid tissue (MALT) . These regulatory cells are released in the blood or lymphatic tissue and then migrate to the organ or tissue afflicted with the autoimmune disease at which location they suppress autoimmune attack of the afflicted organ or tissue. The T-cells elicited by the bystander antigen (which recognize at least one antigenic determinant of the bystander antigen used to elicit them) are targeted to the locus of autoimmune attack where they mediate the local release of certain immunoregulatory factors and cytokines, such as transforming growth factor beta (TGF-0) , interleukin-4 (IL-4) or interleukin-10 (IL-10) . Of these, TGF-/8 (specifically TGF- βl) is an antigen-nonspecific immunosuppressive factor in that it suppresses all immune attack phenomena at the locus of its release regardless of the antigen that triggers these phenomena. (However, because oral tolerization with a bystander antigen causes the release of TGF-S only in the vicinity of autoimmune attack, no systemic immunosuppression ensues.) IL-4 and IL-10 are also antigen-nonspecific immunoregulatory cytokines. IL-4 in particular enhances Th2 response, i.e. acts on T-cell precursors and causes them to differentiate preferentially into Th2 cells. IL-4 also indirectly inhibits Thl exacerbation. IL-10 is a direct inhibitor of Thl responses. After orally tolerizing mammals afflicted with autoimmune disease conditions with bystander antigens in the animal model for multiple sclerosis EAE and in the animal model for Type 1 diabetes (NOD mouse) , the present inventors and their co-workers observed increased levels of TGF-S, IL-4 and IL-10 at the locus of autoimmune attack. See, e.g., Chen, Y. et al., Science, 265: 1237- 1240 , 1994. The secretion of these Thl-suppressive cytokines by antigen-fed animals was therefore used to assess operability of the invention in the experiments that follow. The present inventors have found that oral administration of a skin-associated bystander antigen is effective to suppress (i.e., eliminate or diminish) a T-cell mediated inflammatory immune response associated with T-cell mediated inflammatory skin disorders. "Skin-associatedbystander antigens" that canbe used for this purpose are defined herein to include any skin- associated antigen exposed to the immune system (either normally or as a result of the inflammatory process) that can be recognized by T-cells of the recipient mammal (as assessed for example by proliferation assay) . The definition of "bystander antigens" in the prior applications referenced above is thus also valid here with the additional requirement that the bystander antigen be "skin associated", i . e. , either specifically expressed only in skin or a skin component (e.g. melanocytes in vitiligo) or expressed in substantial quantities in skin (e.g. Collagen Type III) . The skin-associated bystander antigen must come into contact with the immune system either normally, i.e., in the absence of inflammation, or must become exposed to the immune system as a result of inflammation. Also encompassed within this definition are fragments of the naturally occurring skin-associated bystander antigens that contain at least one epitope which would be recognized (in the context of antigen presentation) by the T- cells of a subject to be treated. The fragment thus can be a ligand to a T-cell receptor, or even an epitope that is recognized by autoreactive T-cells. It is important to realize that the bystander antigen need not be the primary or a primary target of the T-cells causing the inflammation. For example, even if the skin-disorder is autoimmune in nature, the bystander antigen need not be an autoantigen.

Non limiting examples of skin-associated bystander antigens that can be used to treat any skin disorder presenting with inflammation include Type I, Type III or Type IV collagen; basal keratins (such as K5 or K14) and superbasal keratins

(such as Kl and K10) ; transglutaminases, such as transglutaminase 1 and transglutaminase 5 (a/k/a Factor XIII) .

In addition, the melanocyte-specific enzyme tyrosinase is useful specifically in treatment of vitiligo, Keratin K16 (specific to hair follicles) is useful specifically in treatment of alopecia areata, totalis, or universalis; and antigen Jo-1 as well as other aminoacyl t-RNA synthetases (Plotz et al. in Manual of Biological Markers of Disease, B6.1:1-118, 1994) useful specifically in the treatment of myositis.

In the treatment of human disorders, it is preferred to employ human-derived skin-associated bystander antigens, either expressed by recombinant techniques or purified from natural sources.

Purification techniques for other skin-associated bystander antigens are well-known in the art. Moreover, the nucleotide and/or amino acid sequences of all of the foregoing examples of skin associated bystander antigens have been elucidated and some of them are commercially available: e.g., Type IV collagen from human placenta, keratin from human epidermis, Factor XIII, tyrosinase (for human tyrosinase see WO 88/02372 which discloses isolation of the gene encoding it as well as its nucleotide and amino acid sequence) . It should be noted that it is not necessary to administer purified skin- associated bystander antigens for the treatment to be effective. Impure constructs or preparations containing one (or more) such antigens can be employed. A nonlimiting example of a class of constructs are synthetic chimeric peptides and polypeptides containing one or more T-cell epitopes of ligands of a skin-associated bystander antigen. The ability to suppress inflammation with oral administration of impure preparations containing mixtures of skin-associated bystander antigens has been seen in experiments by the present inventors using whole keratinocytes as the fed antigen (data not shown) .

Factor XIII can be produced as taught in U.S. Patents

5,395,922; 5,378,687; 5,047506; 5,204,447; 5,164,373 and 4,597,899 (incorporated by reference). Other references teaching how to make and/or purify other skin-associated bystander antigens or revealing the nucleotide or amino acid sequence thereof are: Fuchs, E. et al., Cell 17(3) :573-82, July 1979; Prockop, D.J., J. Biol . Chem. 265 (26 ) -. 15349 - 52 , Sept. 15, 1990; Hudson B.G. et al., J. Biol . Chem. 268 (35) :26033-6, Dec. 15, 1993; Stevens, H.P. et al., Postgrad. Med. J. 70(829) :775-9, Nov. 1994; Slack J.L., et al., Am. J. Med. Genet. 45(2) :140-51, Jan. 15, 1993; Aberdam D. et al., Cell Adhes . Commun . 2(2) :115-29, June 1994; Takami, H. et al., ContriJ . Nephrol . 207:36-46, 1994; Pulkkinen, L. et al., Genomics 24(2) :357-60, Nov. 15, 1994; Pfeiffer, C. et al., Immunol. .Rev. 123:65-84, Oct. 1991; Lee, L.D. et al.. Jut. J. Dermatol . 14 (3 ) :161-71, Apr. 1975; Prockop, D.J. Prog. Clin. Biol . Res . 154:81-8, 1984; Merot, Y., Semin . Dermatol . 7(4) :269-77, Dec. 1988; Steinert, P.M. et al., Trends Genet. 9(8):280-4, Aug. 1993. Keratin 10 sequence is described in U.S. Pat. No. 5,302,511 issued April 12, 1994; an expression system for other human keratins is disclosed in U.S. Pat. No. 4,980,290 issued Dec. 25, 1990.

Furthermore, corresponding skin-associated antigens of mammalian origin (e.g. chicken and bovine collagens, porcine keratin, turkey keratin) ; and guinea pig transglutaminase are commercially available (e.g. from Sigma Chemical Co., St. Louis, Mo.) or have known nucleotide and/or amino acid sequences. If animal-derived skin-associated bystander antigens are used in treating humans, it is preferred to use those animal-derived antigens that are the most closely homologous to the corresponding human antigens.

Effective amounts of the skin-associated bystander antigens are generally within the range of 0.1 mg-100 mg per treatment (dose) per mammalian subject (including a human) . The exact amount may vary with the subject and it may also vary according to the ability of the subject's T-cells to recognize the antigen (e.g., it will be necessary to administer higher amounts of a weak antigen) . Effectiveness in inducing suppression is dose-dependent and there is an optimum effective dose, which is best ascertained by routine experimentation. For collagen I and IV a useful dosage range is 0.1-10 mg/ patient/treatment, preferably 0.1-5 mg. For Factor XIII, an effective dose range for humans is also 0.1-10 mg, preferably 1-10 mg. For keratins, an effective dose range in humans is 0.1-10 mg. Effectiveness of treatment (suppression of inflammation) can be assessed by physical examination of the afflicted area and reduction or disappearance of skin lesions, depigmentation, or inhibition of hair loss, depending on the disease being treated, as further discussed below. The frequency of treatment (for mammals including humans) can vary, e.g. daily, every other day, once or twice a week, once a month. It is also possible to administer multiple daily doses. At the commencement of treatment it is preferred to administer the skin-associated bystander antigen more frequently (e.g. every day or every other day) and the frequency can be tapered (to e.g. once a week or once a month) as treatment progresses. Determination of the optimum frequency of administration is within the skill of the art in light of the present description and will vary with the patient's age and physical condition as well as with the severity of the disorder. It is best determined on an individual basis by the attending physician, using the information given herein and the skill of the art.

The duration of treatment can vary from several days (e.g. ten) to several weeks (e.g. four) to several months (e.g. six) to indefinitely. For humans, treatment is expected to last preferably at least three months and can be continued as long as benefits persist because of the low toxicity of the treatment.

The skin-associated bystander antigens employed in the present invention can be administered in various solid or liquid formulations suitable for oral consumption.

Each oral formulation containing skin-associated bystander antigen according to the present invention may additionally comprise inert constituents including pharmaceutically acceptable carriers, diluents, fillers, solubilizing or emulsifying agents, and salts, as is well-known in the art. For example, tablets may be formulated in accordance with conventional procedures employing solid carriers well-known in the art. Capsules employed in the present invention may be made from any pharmaceutically acceptable material, such as gelatin, or cellulose derivatives. Sustained release oral delivery systems and/or enteric coatings for orally administered dosage forms are also contemplated, such as those described in U.S. Patent No. 4,704,295, issued November 3, 1987; U.S. Patent No. 4,556,552, issued December 3, 1985; U.S. Patent No. 4,309,404, issued January 5, 1982; U.S. Patent No. 4,309,406, issued January 5, 1982; U.S. Patent No. 5,296,236; WO 85/02092; and EP 516,411. Examples of solid carriers include starch, sugar, bentonite, silica, and other commonly used carriers. Further non-limiting examples of carriers and diluents which may be used in the formulations of the present invention include saline, syrup, dextrose, and water. Liquid formulations are preferably solutions; if the skin-associated bystander is insoluble in water, it can be solubilized and/or the pH can be adjusted as is well known in the art; or the antigen can be partially -hydrolyzed or administered as a suspension. It will be appreciated that the unit content of active ingredient or ingredients contained in an individual dose of each dosage form need not in itself constitute an effective amount, since the necessary effective amount can be reached by administration of a plurality of dosage units (such as capsules or tablets or combinations thereof) .

In general, when administered orally, the bystander antigens of the present invention may be administered in single dosage form or in multiple dosage forms.

As stated above, ascertaining the effective dosage range as well as the optimum amount is well within the skill in the art in light of the information given in this specification. For example, dosages for mammals and human dosages can be determined by beginning with a relatively low dose of skin-associated bystander antigen, progressively increasing it (e.g. logarithmically) and measuring a biological reaction to the treatment, for example induction of regulatory cells (CD4⁺ and/or CD8⁺) as described in Chen, Y. et al., Science, 1994, supra, reduction in class II surface markers on circulating T-cells, and/or by assessing the disease severity, according to well-known methods.

Disease severity can be assessed by physical examination by the attending physician. Nonlimiting examples are as follows:

Psoriasis: Size and extent plaques and associated salmon-pink erythema and silvery scale; papules, pustules and nail deformity.

Vitiligo: Surface area (size) and coloration of depigmented macules or patches, hypopigmented lesions, trichrome macules or patches; inflamed borders of depigmented areas, irritation or rash (which often precedes pigment loss) . Contact dermatitis: Standardized patch test used in diagnosis performed according to procedures prescribed by the supplier, skin biopsy, open application of suspected or known causative agent, and prick or scratch skin test; appearance of redness, blisters, cracks, scales, welts, dryness or rash; burning itching or stinging sensation (reported by the patient) , improvement measured by reduction in skin reaction or in skin lesions.

Alopecia Areata: Burning, itching, tingling before hair loss or concurrent with hair growth; location, number and surface area of lesions; microscopic examination of hair; biopsy. Atopic Dermatitis: Extent and severity of lesion characteristics such as excoriation, xerosis, ichthyosis, palmar hyperlinearily, keratosispilaris, erythema, periorbital darkening, pityriasis, alba.

The optimum dosage will be the one having the greatest influence on the biological phenomenon being measured, such as that which causes the greatest decrease in disease symptoms. An effective dosage range will be one that causes at least a statistically or clinically significant attenuation of at least one symptom characteristic of the disease being treated, or a significant change of a marker (such as the frequency of regulatory or activated T-cells) .

Administration of skin-associated bystander antigens can be conjoined to preferably oral administration of one or more enhancers, i.e. substances that enhance the tolerizing effect of the skin-associated bystander antigen, such as LPS, Lipid A (as described in U.S. Application Serial No. 08/202,677) IL-4, IL-10 or Type I interferon (See, e.g. U.S. Application Serial No. 08/420,980 and 08/420,979) . As used in the preceding sentence, "conjoined with" means before, substantially simultaneously with or after administration of these antigens. Naturally, administration of the conjoined substance should not precede nor follow administration of the antigen by so long an interval of time that the relevant effects of the substance administered first have worn off. Therefore, enhancers should usually be administered within about 24 hours before or after the skin-associated bystander antigen and preferably within about one hour. The present invention has been described above primarily by reference to oral induction of tolerance to inflammation associated with skin disorders. It will be understood that "oral" administration includes oral, enteral or intragastric administration; i.e., it includes any administration that brings the skin-associated bystander antigen in contact with gut-associated lymphoid tissue (GALT) .

Furthermore, based on the fact that tolerance has been achieved by placing appropriate antigens in contact with mucosa-associated lymphoid tissue (MALT) an alternative method of achieving tolerance in the present invention is by administration (called mucosal administration and including inhalation, buccal and nasal administration) by which the skin- associated bystander antigen is placed in contact with the buccal, nasal, bronchial or pulmonary mucosa. The amounts of antigen that will be effective in mucosal administration are expected to be somewhat lower than those used in oral administration, i.e., within the lower half of the ranges and preferably within the lower third of the ranges given above.

Formulations useful for mucosal administration include those suitable for administration of polypeptides across the mucosal membrane. For example, U.S. Patent Nos. 4,226,848 and 4,690,683 describe polymeric matrices useful in administering pharmaceuticals into the nasal cavity. U.S. Patent No. 4.952,560 discloses an ointment formulation comprising a water-soluble protein and a monohydric alcohol which may be suitable for use in administering the present invention because it increases absorption of drugs across epithelial barriers. Methods of improving transcutaneous absorption of materials is described in U.S. Patent No. 4,272,516. Each of these formulations and others well known in the art may be used for mucosal delivery of bystander antigen as described in the present invention. Additional suitableformulations includecommercially available vehicles and formulations which may but need not include surface active agents and other skin penetrants as absorption promoters. Specifically, U.S. Patent No.5,407, 911 describes the use of axacycloalkane derivatives as absorption promoters for high molecular weight polypeptides. U.S. Patent No. 5,397,771 describes the use of n-glycofurols in methods of administering pharmaceutical compositions across the mucosal membrane. Additionally, U.S. Patent No. 4,548,922 discloses the use of water-soluble amphophilic steroids to increase absorption. Gel-based compositions, such as those described in Morimoto et al. (Chem. Pharm. Bull . 35(7) :3041-3044) are also suitable for the present invention. It should be noted however that the purpose of mucosal administration of bystander antigens of the present invention is to place them into contact with the immune system at the mucosa associated lymphoid tissue and not to deliver these substances in the bloodstream. Accordingly, the absorption promoters of the foregoing formulations may be decreased or eliminated. Determination of what parameters should be altered is well within the skill of the art.

Formulations administered by inhalation in aerosol form are also contemplated for use with the present invention. General guidance as to formulation of aerosol dosages is given in Barr, Prac. Pharm. 19(11) :675-678. More specifically, U.S. Patent No. 4,352,789 describes an aerosol formulation for the administration of finely divided solid materials which can be used with the present invention. Further, U.S. Patent No. 3,739,951 discloses a method of administering a liquid formulation in an aerosol form. Aerosol delivery of bystander antigens is described in co-pending, commonly assigned U.S. Application 08/419,502. These and other mucosal administration methods well known in the art are contemplated for administration of liquid compositions of the present invention to the MALT. It is preferred that in inhalable compositions the skin-associated bystander antigen be administered as a solution rather than a suspension. Accordingly, the antigens may first be solubilized or hydrolyzed, or fragments of the naturally occurring antigens can be used.

The inhalable pharmaceutical formulations of the present invention may be administered in the form of an aerosol spray using, for example, a nebulizer such as those described in U.S. Patent Nos. 4,624,251 issued November 25, 1986; 3,703,173 issued November 21, 1972; 3,561,444 issued February 9, 1971 and 4,635,627 issued January 13, 1971; 4,548,922 issued Oct. 22, 1985 (it should be noted that use of absorption promoters is not necessary in the practice of the present invention) . The aerosol material is inhaled by the subject to be treated. In the present examples (and for purposes of accuracy) the animals treated with aerosol agents were retained in enclosed (airtight) cages, into which the aerosol was dispensed. Thus, the amount of material per unit of area could be determined and the results quantified in terms of unit of aerosol material per unit volume of cage area.

Other systems of aerosol delivery, such as the pres¬ surized metered dose inhaler (MDI) and the dry powder inhaler as disclosed in Newman, S.P. in Aerosols and the Lung, Clarke, S.W. and Davia, D. eds. pp. 197-224, Butterworths, London, England, 1984; or Laube, B.L. JAMA. 1993, 269:2106-9. can be used when practicing the present invention, provided that amounts of the aerosolized antigen should be adjusted as described herein.

Aerosol delivery systems of the type disclosed herein are available from numerous commercial sources including Fisons Corporation (Bedford, MA) , Schering Corp. (Kenilworth, NJ) and American Pharmoseal Co., (Valencia, CA) . All documents cited herein are incorporated by reference in their entirety. In case of conflicts, the present specification including its definitions controls.

The invention is further illustrated below by the following Examples, which are not intended to limit its scope.

EXPERIMENTAL

Animals: SJL X PLJ (FI) mice (female) six to eight weeks old were divided in groups (n=5/group) and fed various antigens (or PBS) as described below every other day for five times; two days after the last feeding, mice were immunized subcutaneously in the flank with 100 μg antigen in CFA containing 100 μg mycobacterium tuberculosis; on day ten, mice were challenged by subcutaneous injection with the same antigen in the footpad corresponding to the flank of immunization; delayed-type hypersensitivity was measured as the difference in footpad thickness (mm) before and after challenge 24 or 48 hours after challenge.

In vitro T-cell Stimulation: Mice fed and immunized as described in the preceding paragraph were sacrificed 14 days after immunization and their inguinal lymph nodes excised; T- cells were stimulated with antigen in vitro for 72 hours and then pulsed with 1 μCi tritiated thymidine per 5 x 10³ cells and incubated for 16 hours. The cells were then harvested and thymidine uptake was assessed as counts per minute relative to unstimulated controls.

Cvtokine Production: Primary T-cell cultures were stimulated with antigen; supernatants were removed after 40 or 72 hours and assayed by ELISA for IL-4, IL-10, IL-2 and IFN-γ (40-hour supernatant) and TGF-0 (72-hour supernatant) using appropriate antibodies commercially available from Pharmingen, San Diego, CA (for IL-2, IL-4, IL-10 and IFN-γ); and from Celtrix Pharmaceuticals, Santa Clara, CA and R&D, Minneapolis, MN (for TGF-/8) .

EXAMPLE 1: Delayed-Type Hypersensitivity Response is Suppressed by Feeding Skin-Associated Bystander

Antigen Regardless of the Skin-Inflammation Inducing Agent

Three groups of mice were fed as follows: Group 1: phosphate-buffered saline (PBS); Group 2: 3Oμg/mouse/feeding of hen-egg lysozyme (HEL; used as the nonskin-associated antigen); Group 3: 30μg/mouse/feeding of transglutaminase 5 (Factor XIII from Zymogenetics, Palo Alto, CA) according to the aforedescribed schedule. All groups were immunized with 100 μg Factor XIII in CFA in the right flank and with 100 μg HEL in the left flank, and subsequently challenged with the 50 μg of the same antigen in the corresponding footpad. The results (Δ thickness, in mm) are set forth in Fig. 1 for the left footpad.

With respect to the HEL-challenged (left) footpad (Fig. 1) Group 1 (PBS-fed) is the negative control; Group 2 (HEL-fed) is the positive control (because it was fed the same antigen (HEL) as was used to cause hypersensitivity and that antigen is not a skin-associated bystander antigen) ; Group 3 was the experimental group because it was fed a skin-associated bystander antigen which was intended to suppress inflammation

(hypersensitivity) induced by another antigen (HEL) which was only associated with skin artificially (i.e., in the experimental milieu because it was injected subcutaneously) and does not normally occur in skin.

The results in Fig. 1 show that feeding Factor XIII suppressed inflammation induced with HEL as much as feeding HEL. Thus, feeding a skin-associated bystander antigen, i.e. an antigen concentrated in the afflicted tissue but not causally involved in the inflammatory process, causes suppression. This constitutes proof of principle of the operability of the present invention.

Example 2: Dose Response of Fed Skin-Associated Bystander

Antigen

Similar experiments were conducted wherein mice were fed 3, 30 or 300 μg or 1 mg of Factor XIII and immunized and challenged with HEL, as described in Example 1. The dosage of 30 μg Factor XIII produced the strongest suppression, and is a preferred dosage for this antigen and these mammals (data not shown) .

Example 3: Determination of Cytokine Profiles from Animals

Fed with Skin-Associated Bystander Antigens

The cytokine profile of T-cells from animals fed 30 μg of Factor XIII in which DTH responses were induced with HEL as described above, was as follows (compared to that of animals fed PBS) :

Factor XIII-fed PBS-fed

IL-2: 0 pg/ml 22 pg/ml

TGF-0: 3780 pg/ml 1225 pg/ml

IL-10: 7435 pg/ml 0

There was no measurable IL-4 or IFN-γ production in either group.

The foregoing results show that upon stimulation with fed antigen the T-cells of Factor XIII fed animals changed from a Thl-like cytokine profile (production predominantly of IL-2) to a Th2-like profile (production predominantly of TGF-j8 and IL-10) , which means that the Thl response (substantially responsible for inflammation) has been suppressed.

Examples of Humans Treated With

Skin-Associated Bystander Antigens

Example A: Three patients diagnosed with psoriasis and receiving no other treatment will be administered orally 5 mg of Factor XIII (Patient Al) ; 1 mg of Collagen I (Patient A2) , or 5 mg of Keratin 5 (Patient A3) .

The skin-associated bystander antigen will be preferably administered in aqueous suspension or solution. Collagen I can be solubilized by known methods and dissolved in 0.1 M acetic acid. Factor XIII (which may be lyophilized as is well-known) is soluble in water. Keratin 5 is insoluble in water but may be micronized according to known methods and administered in an aqueous suspension, or may be partially hydrolyzed. The frequency of administration will be daily for one month and then depending on the patient's response to the treatment may be continued or its frequency may be progressively reduced to once weekly and then once monthly for the duration of the treatment (anticipated to last 3 months or longer) . Assessment of effectiveness will be made by physical examination. It is anticipated that each patient will experience a substantial decrease in skin symptom(s) .

Example B: Three patients diagnosed with atopic dermatitis and receiving no other treatment will be administered orally 5 mg of Factor XIII (Patient Bl) ; 1 mg of Collagen I (Patient B2) , or 5 mg of Keratin 5 (Patient B3) formulated as in example A.

The frequency of administration will be daily for one month and then reduced to once weekly for at least one more month. Assessment of effectiveness will be made by physical examination. It is anticipated that each patient will experience significant decrease in the number and severity of lesions.

Example C: Three patients diagnosed with vitiligo and receiving no other treatment will be administered orally 5 mg of Factor XIII (Patient Cl) ; 1 mg of Collagen I (Patient C2) , or 5 mg of Keratin 5 (Patient C3) formulated as in Example A. The frequency of administration will be daily for 1-3 months and then reduced to twice weekly for 1-3 months followed by administration once a month for the remainder of therapy. Assessment of effectiveness will be made by physical examination. It is anticipated that each patient will experience a measurable decrease in the area and/or intensity of depigmentation, and or other vitiligo-associated symptoms detailed supr .

The invention has been described above by reference to preferred embodiments. It will be appreciated however that modifications can be made that will be within the scope of the following claims.

Claims (15)

WE CLAIM:

1. A method for treating a mammal suffering from a skin disorder, said disorder presenting with T-cell mediated or T-cell dependent inflammation on the skin of said mammal, the method comprising the step of: administering to said mammal via the oral route a skin-associated bystander antigen; and continuing said treatment for a period of time, wherein the amount of said antigen is effective and said period of time is sufficient to suppress said inflammation.

2. The method of claim 1 wherein said skin- associated antigen is selected from the group consisting of Collagen I, Collagen IV, Keratin 1, Keratin 5, Keratin 10, Keratin 14, Transglutaminase 1, Transglutaminase 5 and combinations of at least two of the foregoing.

3. The method of claim 1 wherein said antigen is formulated in a solid oral dosage form.

4. The method of claim 1 wherein said antigen is formulated in a liquid oral dosage from.

5. The method of claim 1 wherein said mammal is a human.

6. The method of claim 5 wherein said administration is continued on a daily basis at least for a period of time within the range of about 10 days to about 90 days.

7. The method of claim 6 wherein after the expiration of said period of time said administration is continued on a weekly, biweekly or monthly basis for a further period of time of at least about 1-6 months.

8. The method of claim 5 wherein said amount is within the range of about 0.1 to about 10 mg of said antigen per patient per treatment.

9. The method of claim 5 wherein said antigen is selected from the group consisting of Collagen I and Collagen IV and said amount is within the range of 0.1 to 10 mg/patient/treatment.

10. The method of claim 5 wherein said antigen is selected from the group consisting of Keratin 1, Keratin 5, Keratin 10 and Keratin 14 and said amount is within the range of 1 to 10 mg/patient/treatment.

11. The method of claim 5 wherein said antigen is selected from the group consisting of transglutaminase l and 5 and said amount is within the range of 1 to 10 mg/ patient/treatment.

12. The method of claim 1 comprising administering to said mammal an impure but physiologically acceptable preparation of said antigen.

13. A method for treating a mammal afflicted with a T-cell mediated or T-cell dependent inflammation of the skin of a mammal, said inflammatory being associated with a skin disorder comprising orally administering to said mammal a skin- associated bystander antigen in an amount effective to suppress said inflammation.

14. The method of claim 1 wherein said skin disorder is selected from the group consisting of psoriasis, vitiligo, pemphigus vulgaris, myositis, alopecia areata, alopecia totalis, alopecia universalis, contact dermatitis and atopic dermatitis.

15. A method for treating a mammal suffering from a skin disorder said disorder presenting with T-cell mediated or T-cell dependent inflammation on the skin of said mammal, the method comprising the step of: delivering to the mammal's nasal, bronchial or pulmonary mucosa a skin-associated bystander antigen in an amount effective to suppress said inflammation; and continuing said treatment for a period of time sufficient to suppress said inflammation.