MXPA02000531A - Small peptides and methods for downregulation of ige. - Google Patents

Abstract

A method for downregulating IgE levels is described. The method involves administering to a patient an IgE downregulating effective amount of a peptide having the formula f-Met-Leu-X, wherein X is selected from the group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr.

Description

SMALL PEPTIDES AND METHODS TO REDUCE THE REGULATION OF IMMUNOGLOBULIN E DESCRIPTION OF THE INVENTION This invention relates to small peptides, particularly to N-formyl-methionyl peptides, which have down-regulation activity of IgE and to methods for treating indications resulting from responses • mediated by IgE. More particularly, the peptides can be used to replace corticosteroids in any application in which corticosteroids are used. 10 Immunoglobulin E (IgE) is one of five classes of antibodies that occur in man and it has been known for three decades that it is the immunoglobulin responsible for • allergic reactions. IgE is produced and secreted by B cells during the invasion of the allergy. However, IgE 15 constitutes only a small fraction of the total antibodies in human serum - (50-300 ng / ml compared to 10 mg / ml of IgG) and thus, is not present in sufficient quantity to directly neutralize the antigens. Instead, its action is amplified through the receptors 20 cellular targets and produces a wide range of cellular responses to antigens, culminating in inflammation, itching, coughing, lacrimation, bronchoconstriction, mucous secretion, vomiting and diarrhea, all symptoms commonly associated with allergic disorders. 25 Immediate hypersensitive reactions are they shoot through the high affinity IgE receptors (FCeRI) found in mast cells and basophils. The binding of the allergen with the IgE bound to FCeRI causes the cross-linking of the receptor molecules or the cell membrane, which triggers the degranulation of the cell and the subsequent release of histamines and other mediators associated with the immediate phase of the allergic response. These products of degranulation of mast cells cause the activation of inflammatory cells and further induces a low affinity IgE receptor, FceRII, also known as CD23. FceRII / can be found on activated B cells, various inflammatory cells (macrophages, eosinophils, platelets, natural killer cells), T cells, follicular dendritic cells (FDC), Langerhans cells and cells of the bone marrow epithelium and the thymus (Delespesse et al., Adv. Iptmun 49: 149-190, 1991, Delespesse et al., Immunol Rev. 125: 78-97, 1992). On the surface of B cells, FceRII plays a role in the presentation of the IgE-dependent antigen to T cells and also in the cross-linking of B cells. On FDCs, FceRII is expressed in large quantities and is therefore involved in the conscription of B cells to the germinal centers of the secondary follicles in the lymph nodes and the spleen. When expressed on inflammatory cells, it is thought to be responsible for IgE-dependent cytotoxic activities, such as phagocytosis of immune complexes by monocytes. Soluble FceRII (sFceRII) can also initiate cell-mediated humoral and immune responses by triggering the growth and differentiation of precursors of plasma cells, T cells and basophils. The differentiation of B cells into plasma cells that secrete IgE involves a complex signaling cascade of cytokines and surface molecules, thought to occur mainly in the germinal centers of the secondary follicles in the lymph nodes and spleens. Surface molecules are essential to provide the physical interaction of B cells with the T cells and mast cells that is required to trigger the production of IgE. These surface molecules are the ligand CD40 (CD40-L) and FceRII. When T-type 2 (Th2) helper cells are activated during exposure to antigen presenting cells (APC), they express CD40L transiently. CD40L interacts with CD40 in B cells, resulting in the activation of B cells. Activated Th2 cells secrete secrete various cytokines, such as IL-4 and IL-13, which act on activated B cells to switch to IgE production. IL-4 also upregulates the expression of FceRII on B cells and inflammatory cells, providing an additional source of contact stimulation and soluble growth factor.

^ Mast cells and basophils also secrete IL-4 and express CD40L and can thus induce the synthesis of IgE by B cells during physical interaction with B cells in the presence of IL-4, in a similar way as Th2 cells It is likely that IgE synthesis may also occur in the skin, lungs and intestine, in view of flp the tissue distribution of the various cell types involved in the production of IgE. The upregulation of IgE synthesis and the rescue of germinal center B cells from apoptosis is mediated by the cross-linking of IgE bound to the B cell membrane and the complement receptor 2 (CR2), also called CD21 by sFceRII. CR2 is a highly glycosylated membrane protein found in B cells, CDF, and some T cells and basophils. sFceRII can participate in the positive feedback control of IgE synthesis by firing CR2 on B cells to improve IgE synthesis while also promoting the survival of B cells compromised with IgE. 20 The activation of IgE production can lead to two different situations. Acute inflammation due to exposure to the allergen begins with an early phase reaction involving the rapid activation of mast cells, airway macrophages, and bronchial epithelial cells that release proinflammatory mediators that , 2B .fA «a» ».. they include histamines, eicosanoids, factor that activates platelets, free oxygen radicals, neuropeptides, and cytokines. These can induce airway smooth muscle constriction, mucus secretion and vasodilation. The 5 Inflammation of the airways causes increased microvascular leakage that leads to plasma exudation within the air fl ies. It results in thickening of the walls of the airways and narrowing of the lumen of the airways. In the last phase reaction, the cells 10 peripheral blood cells are recruited into the airways to establish a chronic type of inflammation. Such cells include eosinophils, lymphocytes and monocytes and in the recruitment is dependent on cytokines such as IL-5 and factor that stimulates granulocyte-macrophage colonies 15 (GMC-SF). Chemokines such as RANTES and eotaxin also appear to improve the recruitment of eosinophils. At the site of inflammation, these cells are activated and their survival is increased by reduced apoptosis, mediated by factors such as GMC-SF. 20 Treatments for asthma have traditionally been based on the severity and persistence of the disorder. For acute, intermittent symptoms, treatments have usually involved bronchodilators. Bronchodilators include β-adrenergic agonists, 25 methylxanthines, and anticholinergic drugs. These agents * they may improve airway obstruction in patients with asthma but do not appear to be effective in reducing airway inflammation or bronchial hyperreactivity. In more recent years, leukotriene 5 inhibitors have become available for the treatment of mild to moderate asthma. Leukotrienes are generated from arachidonic acid via the metabolic pathway of 5-lipoxygenase and have long been known to possess strong bronchoconstrictive properties. You are so called 10 slowly reacting substances of anaphylaxis ("SRS-A") also induces migration, adhesion, aggregation of various white blood cells to blood vessels and increases • capillary permeability, culminating in interstitial edema, leukocyte chemotaxis, mucus production, 15 mucociliary dysfunction, and bronchospasm in the lungs. Leukotriene D4 (LTD4), in particular, appears to be primarily responsible for this activity in the airways and acts through a specific receptor in the smooth muscle cells of the airways. The leukotrienes, 20 that include cysteinyl leukotrienes, are also released • during degranulation of mast cells mediated by IgE. Leukotriene inhibitors consist of two types: one that blocks the synthesis of leukotrienes by inhibiting the activity of 5-lipoxygenase (5-LO), which is required for 25 the synthesis of leukotriene, and another that blocks i ^^^^^ y ^ fe ^^^? áfcjyÉ competitively the LTD4 receptor in smooth muscle cells. Zileutin is the first of the 5-LO inhibitors that have become available. Zafirlukast is the first LTD4 receptor antagonist to be tested, while 5 others such as monelukast and pranlukast are currently undergoing clinical trials. These leukotriene inhibitors are used or until now for mild persistent asthma treatments but have not yet proven effective for more severe forms of asthma.10 Anti-inflammatory agents are currently used to treat more severe and persistent forms of asthma. The agents categorized as anti-inflammatory agents include theophylline, corticosteroid, cromolyn sodium, and nedocromil sodium. 15 in particular, appear to be more effective in reducing bronchial hyperreactivity and severe exacerbations. They act by suppressing the recruitment of eosinophils by inhibiting cytokine and chemokine production, as well as by inducing eosinophil apoptosis. They also act to 20 cancel the edema and broncorrea of airways and therefore, inhaled corticosteroids are the most common treatment for patients with chronic asthma. Inhaled corticosteroids include beclomethasone, flunisolide, triamcinolone, fluticasone, and budesonide. For chronic asthma, 25 ß2 agonists are ineffective, except that they can improve F temporarily the bronchial obstruction. Thus, the optimal treatment may be combining inhaled corticosteroids and long-acting ß2 agonists. However, the potential side effects of corticosteroids include 5 oropharyngeal candidiasis, dysphonia, adrenal suppression, growth retardation in children, thinning of the skin, flp osteoporosis, glaucoma and cataracts. Furthermore, the relationship between "effective" versus "toxic" doses of these corticosteroids is not clear at the present time. 10 In addition to selecting cases downstream of the IgE signal conduction nerve pathway, some new therapeutic strategies are being developed • to intervene directly with IgE and its synthesis. The central position that IgE plays in the complex network leads to 15 allergic reactions suggesting that that therapy selected to eliminate IgE or to block the binding of IgE to the receptors would indeed prevent the allergic responses altogether. Although still in its early stages, some success has been shown by the use of antibodies 20 monoclonal antibodies directed against IgE. Fahy et al., (Am. J.

• Respir. Crit. Care Med., 155: 1828-1834, 1997) have reported that a humanized murine monoclonal antibody developed against IgE reduced free IgE and was successful in blocking early phase responses. 25 late to the allergen stimulation. Antibodies • Üi üteaA- ».. ~ *? S j? ^ Lr ^? ^^ - *? T ut?? T¡¡rM? T * t! Íí. i IgE that select an IgE region necessary for the FceRI binding not only blocks the IgE binding to its receptor, but also prevents degranulation of mast cells and the anaphylaxis induced by the cross-linking of IgE linked to Fc6RI on basophils and mast cells. Two such anti-IgE antibodies are currently being tested (MacGlashan et al., J I munol 158: 1438-1445, 1997, Corne et al., J. Clin.Invest.99: 879-887, 1997). So far, they appear to reduce serum IgE levels and also lower FceRI levels in basophils, suggesting that IgE-dependent responses can be altered by modulating circulating IgE levels. The treatments to date have typically focused on downstream cases, which result from the activation of IgE. It would therefore be desirable to develop treatments that modulate IgE levels to treat IgE-mediated responses. Chemotactic peptides such as N-formyl-methionyl-leucyl-phenylalanine and pepstatin have been reported to inhibit mast cell degranulation (Inf lammation, Vol. 5, No. 1, pp. 13-16, 1981). The peptides of the present invention down-regulate IgE levels and therefore can be used to modulate a variety of IgE-mediated responses. The present invention provides methods for treating a variety of indications resulting from IgE-mediated responses using pharmaceutical compositions containing a suitable pharmacological carrier in a peptide N-formyl-methionyl-leucyl (?, F-Met-Leu ") having down regulation activity of IgE The 5 particularly useful peptides are those having the formula f-Met-Leu-X wherein X is selected from the group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr The peptides of the present invention can be used to replace corticosteroids in any application in 10 who use corticosteroids. According to the present invention, a method for treating an IgE-mediated response in a mammal • comprises administering to the mammal an effective amount that downregulates IgE of a peptide having the formula f-15 Met-Leu-X wherein X is selected from the group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr. The invention also provides a method for downregulating the membrane bond and the soluble IgE receptors. The method comprises administering 20 patient an effective amount that down regulates the • IgE receptor of a peptide having the formula f-Met-Leu-X wherein X is selected from the group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr. The invention additionally provides a method 25 to inhibit IgE secretion by plasma cells.

The method comprises administering to the patient an effective amount that inhibits the secretion of IgE from a peptide having the formula f-Met-Leu-X wherein X is selected from the group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe- 5 Tyr. According to another embodiment, the invention ^ provides a method to downregulate the expression of CD40L. The method comprises administering to a patient an effective amount that down-regulates CD40L of a peptide Having the formula f-Met-Leu-X wherein X is selected from the group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr. ~ In some preferred embodiments of the present invention, patients may benefit by administering the 15 peptide of the present invention in combination with a second active ingredient. Other active ingredients particularly useful for such a combination according to the present invention are, for example, antileukotrienes, beta2 agonists, corticosteroids and the like. 20 BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 is a log dose response curve illustrating the effects of various dosages of HK-X on serum IgE levels specific for OVA in acute asthmatic mice. 25 FIGURE 2 shows sections of lung of mice i * td? j ii.J aMto »r, ^, _. . . . *. acute asthmatics administered with 50 μg of HK-X. The 'jfei limited cellular infiltrates were present in (A) and (B) and limited mucus accumulation in (C). FIGURE 3 shows lung sections of mice 5 acute asthmatics administered with 10 μg of HK-X. Very few cells were associated with the airways (A) and (B) and the mucus was limited to the surface of the epithelial cell layer of the airways (C). FIGURE 4 shows lung sections of mice 10 acute asthmatics supplied with 1 μg of HK-X. The therapeutic effect decreased, with an increase in cellular infiltrates (A), and increased secretion of mucus in the airways (B) and (C). FIGURE 5 shows lung sections of mice immunized with OVA tested with saline (A) or vehicle (0.05% DMSO) (B). No secretion of mucus was detected in the airways (C). FIGURE 6 is a schematic illustration of the immunization and treatment regimen used to establish a mouse model with chronic asthma. FIGURE 7 is a histogram illustrating the number of granuloma in lungs of chronic asthmatic mice. FIGURE 8 shows the histology of chronic asthmatic lung tissue of immunized mice weekly 25 with OVA for six months and treated with HK-X or solution faith,. A < á * L? & saline. (A) shows the lung histology of control mice, (B) shows the histology of mice treated with HK-X, and (C) shows the histology of untreated but OVA-tested mice. FIGURE 9 shows light micrographs of accumulation of collagen fibrils in lung tissue of chronic asthmatic mouse (A) shows a lung section of a control mouse administered with saline, (B) shows a lung section of a treated mouse with HK-X, and (C) shows a lung section of an untreated mouse but immunized with OVA. FIGURE 10 shows lung sections of mice chronically immunized with OVA and treated with saline. FIGURE 11 shows lung sections of mice chronically immunized with OVA and treated with vehicle (0.5% DMSO). FIGURE 12 is a histogram that illustrates the histomorphometry in chronic asthma. FIGURE 13 is a histogram illustrating the frequency of mucus-containing cells in the airways of chronic asthmatic mice after various treatments. FIGURE 14 is a histogram illustrating the effects of various treatments infiltrated into eosinophil and neutrophil in the lungs of chronic asthmatic mice. FIGURE 15 is a schematic illustration of the immunization protocol and treatment with HK-X and dexamethasone in an acute asthmatic mouse model. 5 FIGURE 16 is a histogram comparing the effects of intranasal administration of dexamethasone and HK-X on specific IgA levels of OVA. FIGURE 17 is a schematic illustration of the immunization protocol and treatment with HK-X and control peptide in an acute asthmatic mouse model. In accordance with the present invention, it has been found that some small peptides having the • formula f-Met-Leu-X wherein X is selected from the group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr have surprising activity to downregulate IgE levels. As a result, such peptides are useful for the treatment of a variety of indications resulting from IgE-mediated responses. The peptides of the present invention can be used to replace corticosteroids f 20 in any application in which corticosteroids are used. Preferred peptides, according to the present invention, reduce IgE levels in blood and block IgE activation of lymphocytes such as, for example, macrophages, monocytes, eosinophils, neutrophils, TNFs and the like. liia4jÉit, ._ fcafefc__i ... _gat.J. ^ ... < * • - »., ..____..__. . "." _ »» ___ »« »** _.». .tKráuMi ,,, .. _j__a _._____.__. «Tafea, i A.

The continued degranulation of mast cells and their release from leukotrienes, histamines, and other cytokines also decreases, or completely ceases in preferred embodiments, after treatment with peptides of the present invention. In accordance with preferred embodiments of the present invention, the peptides can also reduce the • infiltration of eosinophils, basophils and neutrophils into inflammatory tissues. Lymphocytes, eosinophils and neutrophils do not present chemotaxis in response to 10 preferred peptides of the present invention. As a consequence, the chemotactic adhesion, migration and aggregation of lymphocytes, eosinophils and neutrophils in the • site of inflammation is significantly reduced, as is vascular permeability at the site of inflammation.

Additionally, the preferred compounds of the present invention do not exhibit toxicity to vital organs such as heart, liver and lungs. The peptides of this invention can be prepared by conventional small peptide chemistry techniques.

The peptides when used for administration are prepared under aseptic conditions with a pharmaceutically acceptable carrier or diluent. The pharmaceutical compositions can be conveniently presented in unit dosage form and 25 prepare for each type of indication that results from the A AHM ???? *; «W ^^. - ^. . . . t,. M., ",",. "^ ^ J ^, ^," ¿^^^^ IgE-mediated responses that will be treated. The compositions can be prepared by any of the methods 1 well known in the pharmacy technique. The methods V * & * typically include the step of placing the active ingredients of the invention in association with a carrier that constitutes one or more additional ingredients. • For example, the doses of the pharmaceutical compositions will vary depending on the subject, type of indication to be treated, and the particular route of 10 used administration. Active peptide dosages when treating acute IgE-mediated responses can range from 0.1 to 100,000 μg / kg per day, higher • preference 1 at 10,000 μg / kg. The most preferred dosages range from about 1 to 100 μg / kg of weight 15 body, more preferably from about 1 to 20 μg / kg and more preferably from 10 to 20 μg / kg. Active peptide dosages when treating chronic IgE mediated responses may vary from 0.1 to 100,000 μg / kg per day, most preferably from 1 to • 20 10,000 μg / kg. The most preferred dosages range from about 1 to 1,000 μg / kg of body weight, more preferably from about 1 to 100 μg / kg and more preferably from 50-70 μg / kg. The dosages are typically administered once a day to every 4-6 hours depending on the severity of the condition. For acute conditions, it is preferred to administer the peptide every 4-6 hours. For maintenance, it may be preferred to administer only once or twice a day. Preferably, about 0.18 to about 16 mg of peptide per day is administered., depending on the route of administration and the severity of the condition. The desired time intervals for the delivery of multiple doses of a particular composition can be determined by one of ordinary skill in the art employing no more than routine experimentation. Administration routes include oral, parenteral, rectal, intravaginal, local, nasal, ophthalmic, direct injection, etc. In a preferred embodiment, the peptides of this invention are administered to the patient in an effective amount that downregulates IgE. An example of a pharmaceutical composition is an effective amount that modulates the IgE of a peptide according to the present invention that provides an effect that down-regulates IgE, typically included in a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein, and described more fully below, includes one or more compatible solid or liquid filler diluents or encapsulating substances that are suitable for administration to a human or other animal. In the present invention, the term "carrier" denotes a organic or inorganic ingredient, natural or synthetic, with which the molecules of the invention are combined to facilitate the application. The term "effective amount that modulates IgE" is that amount of the present pharmaceutical composition, which produces an effect that downregulates IgE on the particular condition being treated. They can be used • various concentrations to prepare compositions incorporating the same ingredient to provide adjustments in the age of the patient to be treated, the severity of the 10 condition, duration of treatment and mode of administration. The carrier must also be compatible. The term "compatible" as used herein, means that the components of the pharmaceutical compositions are capable of 15 to be mixed with small peptides of the present invention, and each other, in such a form that the desired pharmaceutical efficiency does not decrease substantially. The small peptides of the invention are typically administered per se (net). However, they can 20 administered in the form of a pharmaceutically acceptable salt. Such pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene-sulfonic, tartaric, citric, methanesulfonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzenesulfonic. Also, pharmaceutically acceptable salts can be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium or calcium salts of the carboxylic acid group. Thus, the present invention provides • pharmaceutical compositions, for medical use, comprising peptides of the invention, together with one or more pharmaceutically acceptable carriers thereof and optionally 10 any other therapeutic ingredient. The compositions include those suitable for oral, rectal, intravaginal, local, nasal, • Ophthalmic or parenteral, all of which may be used as administration routes using the materials herein 15 invention. The pharmaceutical compositions containing peptides of the present invention may also contain one or more pharmaceutically acceptable carriers, which may include excipients such as stabilizers (to promote long-term storage, emulsifiers, binding agents, thickening agents, salts, preservatives, solvents, dispersion medium, coatings, antibacterial and antifungal agents, isotonic agents and delaying absorption, and the like The use of such media and agents for active pharmaceutical substances is well 25 known in the art. Except to where any conventional means or agent is incompatible with the peptide of this invention, its use in pharmaceutical preparations is contemplated herein. Supplementary active ingredients may also be incorporated within the compositions of the present invention. The compositions suitable for administration • Oral are typically prepared as an inhalation spray, liquid spray, syrup or tablet. The compositions suitable for local administration are prepared 10 typically as a cream, an ointment, or a solution. To treat an acute IgE-mediated response, the concentrations of the peptide active ingredient in such • compositions are typically less than 1000 μg / ml, more preferably less than 500 μg / ml, and more preferably 15 approximately 200 to 400 μg / ml. To treat a chronic IgE-mediated response, the concentrations of the peptide active ingredient in such compositions is typically less than 3 mg / ml, and more preferably less than 2 mg / ml, and more preferably from about 1 to 1.5 mg / ml . ^^ 20 The compositions of the present invention suitable for administration by inhalation may be presented, for example, as aerosols or solutions for inhalation. An example of a typical aerosol composition for treating acute IgE-mediated responses consists of 25 approximately 0.1 to 100 μg of microcrystalline peptide suspended in a mixture of trichloro-monofluoromethane and dichlorodifluoromethane plus oleic acid, per dose. A more preferable amount of microcrystalline peptide in the composition is 1 to 50 μg, and most preferably 10 to 20 μg per dose of the aerosol composition. An example of a typical aerosol composition for treating responses • mediated by chronic IgE consists of approximately 0.1 to 1000 μg of microcrystalline peptide suspended in a mixture of trichloro-monofluoromethane and dichlorodifluoromethane plus 10 oleic acid, per dose. A more preferable amount of microcritaline peptide in the composition is from 1 to 100 μg, and most preferably is from 50 to 70 μg per dose of the aerosol composition. An example of a typical solution consists of the desired amount of dissolved peptide or 15 suspended in sterile saline (optionally about 5% v / v dimethyl sulfoxide ("DMSO") for solubility), benzalkonium chloride, and sulfuric acid, (to adjust pH). The compositions of the present invention suitable for oral administration may also be presented as discrete units such as capsules. pouches, tablets or pills, each containing a predetermined amount of the peptide of the invention depending on the type of IgE-mediated response to be treated, or which may 25 be contained in liposomes or as a suspension in a S «a .t? MÉ.r? Ík., 3- j-A ... _ .__ »J, < _. _. . __. aqueous liquor or non-aqueous liquid such as a syrup, an elixir, or an emulsion. An example of a tablet formulation base includes corn starch, lactose and stearate "*! - *, * '-"' • magnesium as inactive ingredients. An example of a base 5 for syrup formulation includes citric acid, coloring dye, flavoring agent, hydroxypropylmethylcellulose, saccharin, sodium benzoate, sodium citrate and purified water. The compositions suitable for administration Parenteral agents conveniently comprise a sterile aqueous preparation of the molecule of the invention, which is preferably isotonic with the blood of the recipient. This aqueous preparation can be formulated according to known methods using those dispersing or wetting agents 15 suitable and suspension agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among vehicles and acceptable solvents What can be used is water, Ringer's solution and isotonic sodium chloride solution. In aqueous solutions, up to about 10% v / v of DMSO or Trappsol can be used to maintain the solubility of some peptides. Also, stable oils can be conventionally employed, 25 sterile as a solvent or suspension medium. For this purpose, a number of stable oils including synthetic mono or diglycerides can be employed. In addition, fatty acids (such as oleic acid or neutral fatty acids) can be used in the injectable preparation. In addition, Pluronic block copolymers can be formulated with lipids at 4 ° C for injection of the compound in a base release time of a solid form at 37 ° C for a period of weeks or months. Compositions suitable for topical administration may be presented as a solution of the peptide in Trappsol or DMSO, or in a cream, ointment, or lotion. Typically, about 0.1 to about 2.5% of the active ingredient is incorporated into the base or carrier. An example of a cream formulation base includes purified water, petrolatum, benzyl alcohol, stearic alcohol, propylene glycol, isopropyl myristate, polyoxyl 40 stearate, carbomer 934, sodium lauryl sulfate, acetate disodium, sodium hydroxide, and optionally DMSO. An example of an ointment formulation base includes white petrolatum and optionally mineral oil, sorbitan sesquiolate and DMSO. An example of a lotion formulation base includes carbomer 940, propylene glycol, polysorbate 40, propylene glycol stearate, cholesterol and related sterols, isopropyl myristate, sorbitan palmitate, acetyl alcohol, triethanolamine, ascorbic acid, simethicone, and purified water. In order that the invention described here can be completely understood, the following examples are established. It should be understood that these examples are for purposes of illustration only and are not construed as limiting this invention in any way. • The Mouse Model for Specific Pulmonary Disease of Acute Allergen The in vivo mouse models of asthma have been 10 established that morphological and physiological characteristics are key mimics of human disease (Henderson et al., J. Exp. Med. 184: 1483-1494, 1996). The capacity of • the specific therapeutic reagents also allow the experimental manipulation of the mouse model in a 15 previous systematic, during and after the induction of pulmonary inflammation. A protocol has been developed for the administration of ovalbumin (OVA) as a model allergen to induce specific acute lung disease. 20 allergen in normal Balb / C mice. The protocol involves • intraperitoneal (ip) immunization of mice with 100 μg of ovalbumin (OVA) in alum adjuvants on days 1 and 14, and single intransal doses (in) of 50 to 100 μg of OVA in normal saline on days 14, 25, 26, and 27. The 25 control mice receive only alum by i.p. injections, and normal saline alone by administrations i.n .. On day 28, mice immunized with ova present a disease surprisingly similar to human asthma induced by allergens. This animal model has been used to evaluate the efficiency of drugs in acute allergic lung disease. The Mouse Model for Late Phase Allergen-Specific Chronic Pulmonary Disease. The protocol for the administration of ovalbumin (OVA) as a model allergen to induce chronic late-stage allergen-specific chronic lung disease in normal Balb / C mice includes intraperitoneal (ip) immunization of mice with 100 μg of ovalbumin (OVA) in adjuvant of alum on days 1 and 14, and intranasal (in) single doses of 50 to 100 μg of OVA in normal saline on days 14, 25, 26, and 27 and then weekly in the later for up to 6 months. Control mice receive alum only by i.p injections, and normal saline alone by i.n. administrations. On day 28, mice immunized with OVA exhibit a disease strikingly similar to human asthma induced by allergen. This animal model is also useful for evaluating the efficiency of drugs in chronic allergic lung disease. Materials and Methods Special reagents: OVA Crystalline was obtained from Pierce Chem. Co. (Rockford, IL) and potassium aluminum sulfatop (alum) from Sigma Chemical, St. Louis, MO. The OVA (500ug / ml) was mixed with equal volumes 5 of 10% (weight / volume) of alum in distilled water. The mixture was adjusted to pH 6.5 with 10 N NaOH and incubated for 60 minutes at room temperature. The material was centrifuged at 750 g for 5 minutes; the pellet was resuspended to the original volume in distilled water and used within 1 hour. Immunization protocol: the immunization protocol consisted of the intraperitoneal administration of 100 μg of OVA in alum on day 1 followed by the intraperitoneal administration of 100 μg of OVA in alum 15 combined with the intranasal administration of lOOμg of OVA in saline per day 14. On days 25, 26, and 27 the mice were tested with intranasal OVA (100 μg in saline). For acute asthmatic studies, the animals were euthanized on day 28 and the lungs removed. 20 For chronic asthmatic studies, mice were immunized weekly in the later for up to 6 months. Analysis ELISA protocol for serum IgE: 2 Immulon microtiter plates (Dynex Technologies) were coated with 25 OVA solution in 50 mM carbonate-bicarbonate buffer pH 9.6 at 4 ° C overnight and blocked with 0.1% casein for 2 hr at room temperature (RT). All test sera were diluted 1: 100 in the Tris-NaCL buffer, pH 8.0 containing 0.1% of casein before incubation with the OVA coated plates. A known sample of positive serum containing IgE antibodies to OVA and normal serum samples from non-immunized mice were included in each test as a control. The serum samples were incubated on the plates at room temperature for 2 hours and washed 6X with PBS. An appropriately diluted secondary antibody (Biotin, anti-mouse IgE, sheep (binding site # PB 284, lot # 026917) was added for 2 hr at RT and plates were washed 6X with PBS OPD, Urea and peroxide solution were added. for 30 minutes at RT The reaction was stopped with 2.5 M sulfuric acid The OD was read at 490/630 nm All samples were run in duplicate The inter and intra-sample variation of the positive controls was less than 10% of the media Pulmonary Histology: The lung and trachea were removed and fixed in 10% neutral regulated formalin The tissues were embedded in paraffin and cut into sections of 7 μm After deparaffinization and hydration, the sections were stained with eosinophil staining solution and counter-colorated with methylene blue, Alcian blue, toluidine blue, and acid Schiff stains. periodicals identified mucus within the airways. The tissues were examined by light microscopy. Bronchoalveolar lavage. { BAL): The left lung was tied in the main bronchial tube. The right lung is 5 destroyed with 0.4 ml of normal saline three times, and the fluid was pooled. The number of total cells was determined ^ using a hemocytometer. The remaining cells were pelleted by centrifugation and the cells were placed in a 10% solution of BSA and resuspended. The cells are 10 placed on a microscope slide and stained with an eosinophil staining solution (eosin with methylene blue counterstain). Histomorphometric Lung Analysis: the following parameters of allergic lung disease were measured in the experiments reported here: 1. Airway plug counts were counted as previously reported (Henderson et al., J. Exp. Med. 184: 1483 -1494, 1996). A counting system of + a ++++ was used, which reflects the degree of severity of secretion of the 20 mucus. 2. Total mucus cells were estimated by randomly counting the number of epithelial cells containing mucus per 100 epithelial cells in medium to large airways (diameters of 600 μm to 1,000 μm). HE 25 counted 10 fields in different lung lobes. lA *? á. | »* ftHÍfl I i r» «« ---. »... ..... < »*» .. * .. . , l dt, ^ ^ ti ^? . «A __ ^ 1.M_u« «?? ta ______ ^ 3. The density of cells or infiltrating cells located in the perivascular compartment or in the areas adjacent to the airways (neutrophils, eosinophils, monocytes and lymphocytes) was approximated using a system of counting that varies from oa ++++. A count of + indicates a layer of inflammatory cells of 3 but less than 5 cells; ++ indicates an inflammatory density of 5 cells to 10 cells; +++ indicates an inflammatory density of 10 to 20 cells; and ++++ indicates an inflammatory density of 20 to 40 cells. 4. Numbers of different cell types were quantified by counting the numbers by high sustained increase (1 0X by 40X). 5. The degree of edema was calculated using a counting system where the degree of accumulation of fluid surrounding the blood vessels was estimated. Statistical Analysis of Histomorhometric Data: The 2.0 version of SigmaStat was used to perform statistical analyzes. The differences were analyzed for significance (p <0.05) by ANOVA using the appropriate post-hoc tests for independent media. The 4.0 version of SigmaPlot or GraphPad Prism was used for the construction of graphic representations of the data. EXAMPLE 1: Therapeutic Dosage Response of Acute Asthmatic Mice to HK-X Ideally, an experiment demonstrating that the Therapeutic efficiency correlates with the drug dosage will show three distinct regions of behavior: 1) At low doses, there will be no therapeutic effect; 2) At higher dosage, the therapeutic efficiency will be dose dependent; 3) The third (higher) dose range will not demonstrate therapeutic efficacy greater than that observed in the higher-range dose. The dose response curves are an important source of information for safe dosages for human use. Occasionally, when doses of drugs that exceed the optimal therapeutic dosages are administered, toxic responses may be observed. This is particularly true if the drug is administered in situ, such as intranasally. To establish the therapeutic effectiveness of a dose range of f-met-Leu-Phe-Phe (HK-X) during the acute determinant phase of bronchial asthma on days 25, 26 and 27 induced by repeated immunization with OVA, selected doses of 0.1, 1.0, 10 and 50 μg of intranasal HK-X. HK-X was administered 30 minutes before testing with OVA. The control groups consisted of mice immunized with OVA and tested with OVA as well as animals immunized with alum in saline and tested with saline alone. All the animals were euthanized one day later (day 28) of the final OVA test.

Ij-iAjt-háJ-é-tjt --y_S ____ h_t_.it -., ..... ",., Serum IgE levels were determined and serum and lung tissues were collected for further analysis. To establish first the optimal dose that will effectively regulate serum IgE levels 5 in an acute asthma model, doses of 0.1, 1.0, 10 and 50 μg of HK-X (in 40 μL of saline) were infused. inside of the • lung 15-30 before the antigenic test on days 25, 26, and 27. A dose response curve of serum IgE levels is depicted in Figure 1. 10 The effects of various doses of HK-X on the response of lung tissue to the acute allergic test is shown in Figures 2A to 4C. Fifty micrograms of HK-X • administered intranasally to acute asthmatic mice provided some degree of protection against the 15 effects of acute asthma (Figures 2A-2C). There was limited perivascular and peribronchial accumulation of inflammatory cells (Figures 2A and 2B). Figure 2C shows that mucus accumulation was present but limited. Ten micrograms of HK-X appeared to be the dose 20 more efficient (Figures 3A-3C). Figures 3A and 3B show minimal inflammatory infiltrate surrounding the vessels and airways. The degree of secretion of mucus in the airways is illustrated in Figure 3C. The mucus is confined to the surface of the airway epithelial cells. 25 As the 10-fold dose of HK-X decreased 1 μg, the therapeutic effect decreased. The amount of perivascular inflammation and airways increased (Figure 4A). There was a corresponding increase in mucus secretion from airway epithelial cells (Figure 4B and 4C). For the purposes of contrast and control, the Figures 5A through C illustrate the benign response of immunized mice to the administration of saline or HK-X vehicle (0.05% DMSO). As shown in Figures 5A and 5B, there was little detectable inflammatory infiltrate in the perivascular or airway areas of the lung. Correspondingly, there was no accumulation of mucus in the airway lumen or on the surfaces of the airway epithelial cells (Figure 5C). Of the key histological measurements of the severity of acute asthma, mucus plug, eosinophil numbers and the fraction of airway cells secreting mucus showed a dose-dependent improvement after treatment with 0.1 μg to 10 μg of HK- X. 10 μg of HK-X provided a 70% reduction in the mucus plug count (p <0.05). Interestingly, 50 μg of HK-X provided significantly lower reduction in the mucus plug (p <0.05). This same pattern of sensitivity was observed for the numbers of eosinophils and the fraction of airway cells that secrete mucus. The 10 μg dose of HK-X showed a 57% decline in the number of eosinophils lA.J-il-lii ... - > - »-. ¿?.?,? ÍAi? Íí. ii. »...... ^ ... ^ ,. interstitial, which was significantly greater than the dose effect of 0.1 μg of (p <0.05). The reduction in eosinophils by the 50 μg dose was less than half that provided by 10 μg of HK-X (p <0.05). The fraction of airway cells secreting mucus was also inhibited in a dose-dependent manner from 0.1 μg to 10 μg (37% reduction, p <0.05). The 50 μg dose provided a small amount of reduction (11%) which was not significantly less than the 0.1 μg dose of HK-10 X. The effect of HK-X on the fluid accumulation surrounding the vessels showed a Modest decline at a dose of 10 and 50 μg. However, none of the doses was different from the 0.1 μg dose of HK-X. The dose of HK-X that shows the greatest reduction in the number of inflammatory cells or The accumulation of inflammatory cells was 10 μg. These data demonstrate that the following parameters showed a dose response effect: serum IgE levels and histopathological characteristics (cellular infiltration, mucus plug formation, and 20 total eosinophils in the interstitium). Ten micrograms of HK-X administered intranasally was the most effective dosage compared with lower doses and a higher dose, 50 μg. Compared to the controls, animals treated with 10 μg of HK-X demonstrated a 60% reduction in the 25 levels of serum IgE, 50% reduction in infiltration 4tfcj? Áá-jlr.Í¡__fe «, t, hütrirriii a.,. ..,.« ^ * ** etítee * ^ ** ^? ^ M ^ t? - ^ t? '?, -. - -.- «- ta. ^ ----.

Lung cell, 70% reduction in mucus plug formation and 67% reduction in the number of eosinophils. EXAMPLE 2: Chronic Asthma With and Without the Intervention of HK-X The animal model is also useful for the evaluation of drug efficacy in chronic allergic lung disease. In this study, a 6-month immunization period induced a persistent inflammation that was maintained by weekly intranasal testing with OVA. The mice were treated with saline 8 times over a period of 20 days to assess the changes that occurred in the lungs. Treatments with HK-X from mice with chronic asthma were performed as indicated in Figure 6. 50 μg of HK-X (in 50 μL of saline containing less than 2.5% DMSO) were administered i.n. for a total of 8 dosages delivered during a period of 16 days. The animals were euthanized 4 days after the last saline solution or HK-X dose. The experimental results were compared between mice treated with HK-X and those not treated with HK-X. IgE antibody levels for OVA in the blood of mice tested with or without OVA are shown in Table 1. It is important to note that all animals were immunized with OVA for the first 6 months, however, the group denoted as "solution" saline "were administered saline intranasally but were not tested with OVA during the . «TtiiÉ? ^ .. - ^^ .- ^^ .. ^^^. terminal period of 20 days. Therefore, these IgE levels were moved from the immunization period and used as base values from which all comparisons were corrected. For example, animals treated with saline or DMSO and tested with OVA had a 36% increase in IgE levels compared to a 14% increase in IgE levels in animals treated with HK-X and tested with OVA The amount of suppression of IgE levels by HK-X is 10 calculated to be ~ 60%. TABLE 1: IgE Values in the Serum of Chronic Asthmatic Mice • Note: These values represent the relative IgE levels as OD values from the ELISA test. • 15 One of the important characteristics of chronic asthma in the murine model is the appearance of granulomatous structures in the lung. The effective dose that regulates the low IgE of 50 μg of HK-X significantly reduced (p <0.05) the numbers and sizes of these structures in the lungs of treated animals compared to animals that were allowed to spontaneously reduce collagen deposition [Saline solution or DMSO] (Figure 7). In addition, during the immunization of mice with OVA and the subsequent treatment with HK-X at a dosage of 50 μg for a total of 8 times during a period of 20 days, no adverse reactions or signs of ailments were observed. The mice were active during the experimental period. The examination of the lung tissues of the groups of animals immunized with only OVA revealed severe pulmonary pathological changes consistent with the characteristics of chronic asthma observed in humans. There was a significant infiltration of inflammatory cells in association with the outer limits of the basal lamina (interstitial regions) and blood vessels (Figure 8C).

When the animals were treated with 8 doses of 50 μg of HK-X per treatment for a period of 20 days, the number of inflammatory cells was clearly reduced around the airways and blood vessels (Figure 8B.). of saline by control immunized mice or putative saline solution resulted in obvious airways and blood vessels with normal appearance (Figure 8A). OVA-immunized mice contained increased accumulations with collagen (blue color) around the vessels and airways ^ G3g ^ ^ t ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ 3g ^^^ S ^ £ ^ (Figure 9C). However, the lungs treated with HK-X demonstrated a reduced level of collagen deposition (Figure 9B). In control mice (administered with HK-X in saline) the lung tissue was free of inflammatory cells and fibrotic collagen deposits (Figure 9A). In contrast, in mice sensitized to OVA, and treated • only with saline (Figures 10A-10C) or 0.5% DMSO solution (Figures 11A-11C), the airways contained mucus and had cells that secrete mucus when Alcian blue was used at pH 2.3 to visualize the mucus. The amount of inflammatory airway and perivascular cells, the infiltration was similar. More than 60 percent of the airways of these chronic 6-month-old asthmatic mice were plugged with mucus (Figures 10A-10B and 15 11A-11B, respectively). Changes in lung tissue in chronic asthma were also assessed by morphometric methods to quantify the degree of persistent inflammation, deposition of fibrotic collagen and narrowing of airways with jA > 20 structural changes. In Figures 12 to 14, the responses of the animals immunized with OVA for 6 months and treated with various agents are shown in the left panels of the figures, while the animals supposedly immunized and treated with saline are shown in the panel. law. There was a significant difference (p <0.05) between the degree of mucus plug formation in animals treated with HK-X versus animals that were given only saline during the 20 day period (Figure 12). Animals isolated with the vehicle (0.5% DMSO) for HK-X compared to HK-X also showed no improvement in the mucus plug count. The results were the same for saline treatments. The response patterns for the accumulation of inflammatory cells in and around the airways matched the mucus plug data for the various experimental treatments (Figure 12). When 8 doses of 50 μg each of HK-X were given in 40 μL of vehicle intranasally for a period of 16 days, there was a significant reduction in mucus accumulation and the occurrence of mucus cells in the airways (Figure 13). ). Again, saline treatment alone did not demonstrate therapeutic effects but HK-X significantly reduced the number of mucus-containing cells within the airways (p <0.05). This observation further confirms that there was no spontaneous repair after the establishment of chronic asthma induced with OVA. In the analyzes of the numbers of inflammatory infiltrating cells in association with the airways, the data showed that eosinophils and neutrophils were also reduced per unit area (Figure 14). Animals infused with intranasal saline maintained high eosinophil levels as did the animals treated with DMSO (p> 0.05); however, treatment with HK-X reduced the numbers of eosinophils significantly compared to the two control groups (p <0.05). These studies show that there was little or no spontaneous reduction in airway inflammation or mucus cell secretion in this model of chronic allergen-induced asthma unless HK-X was administered. In this model, the mice were sensitized to OVA and exposed to OVA by the intranasal route weekly for 5 months and treated with HK-X intransally 8 times over a period of 20 days. This allergen immunization and test regimen led to infiltration of chronic airways of theseinophils and other types of inflammatory cells, accumulation of mucus in the airways and hyperplasia of cells that secrete mucus. The administration of an effective dose that regulates the low IgE of 50 μg of HK-X reduced hypersecretion of airways, mucus cell hyperplasia, and the recruitment of eosinophils and neutrophils. These results indicate that by administering an effective amount of HK-X and down-regulating IgE levels, HK-X can also downregulate IgE-mediated responses such as hypersecretion of mucus in the airways and deposition of collagen that occurs in this model of asthma induced by allergen.

EXAMPLE 3: Effects of HK-X and Dexamethasone on Acute Murine Asthma Glucocorticoids are potent inhibitors of inflammatory mediators produced by a variety of 5 cell types, including T cells, mast cells, monocytes, dendritic cells and eosinophils. The • Glucocorticoids are effective in the treatment of human asthma when inhaled or used systemically. They suppress the infiltration of inflammatory cells and have shown that 10 decrease mucus secretion and pulmonary edema. These responses are related to the direct effects of glucocorticoids in bronchial epithelial cells.

• Equally important, steroids reduce bronchial hypersensitivity. Due to its efficiency 15 demonstrated, glucocorticoids represent a first-line therapeutic armament in the treatment of asthma and could be used without reservation were it not for the well-documented cumulative toxicity that limits its value over time. Due to its value as the current standard of 20 efficiency for asthma, the new compounds for the • Asthma treatment should be evaluated in comparison with glucocorticoids. This experiment compares the effectiveness of HK-X with dexamethasone, a widely used glucocorticoid, for 25 modulate mucus release, eosinophil numbers, edema and i * A &A * kM ** A. tth? < to ..- ,, ... ^ .. tté ». ^, ^^^^, ^, ^^^^ ^^^^^. allergen-specific IgE levels in this mouse model. Comparable dosages of 10 μg and 50 μg of HK-X and dexamethasone were used in this study. Intranasal administration was used for both drugs at all doses. Fifty micrograms of HK-X was selected as the high dose based on the previous results of the chronic asthma model. A review of the immunization and treatment protocol is shown in Figure 15. The experimental parameters showed that 10 μg of intranasal HK-X were more effective than 10 μg or 50 μg of dexamethasone to reduce serum IgE levels (Figure 16) . Ten μg of HK-X reduced serum IgE levels by 28%. HK-X was also more effective than dexamethasone in improving two histopathological characteristics: cellular infiltrate and total number of eosinophils in the interstitium. Doses of 10 μg and 50 μg of HK-X significantly reduced the inflammatory infiltrate by 54% compared to the OVA control (p <0.05) and were significantly more effective than 10 μg (13%) and 50 μg (13 μg). %) of dexamethasone (p <0.05). Doses of 10 μg and 50 μg of HK-X were more effective than 10 μg of dexamethasone (p <0.05). Ten μg of HK-X decreased the eosinophil cell count by 57%, while the same dose of dexamethasone decreased the eosinophil cell count by 13%. A high dose of 50 micrograms of HK-X JiA. tAr% go & iat. r. i. i-ji ._______.__,,. -? "? Ttnti -.¡.-A, i .____," _¡ _ (., R - ^, Í..r. ^ R? A ??? ^ go? ¡¡¡!.? SM li h & I.-fc > Í; lj -A administered intranasally was as effective as any dosing of dexamethasone to reduce the following histopathological characteristics: number of eosinophils in bronchoalveolar lavage (BAL), mucus plug formation, by 5 percent of cells that secrete mucus in airways, number of interstitial eosinophils, and edemas. This results • establish the comparative efficiency of HK-X and a glucocorticoid, dexamethasone, in the murine asthma model. EXAMPLE 4: Effects of HK-X and a Control Peptide Related in Acute Murine Asthma This experiment compared the effectiveness of HK-X to • a related member of the peptide family f-Met-Met (referred to as the control peptide) with relation to 15 following measures: mucus release, cellular infiltration, eosinophil numbers, edema and allergen-specific IgE levels in the mouse model of acute asthma. In this study, comparable dosages of 50 μg of HK-X and the control peptide were used. Intranasal administration was used to 20 both compounds. Immunization and the treatment regimen • is reviewed in Figure 17. Although they belong to the same family of chemical compounds and are closely related in molecular size, the control peptide did not present any of the 25 therapeutic properties of HK-X. More significantly, Tifajii 50 μg of HK-X caused a 7% decrease in serum IgE levels in the sera for the allergen, OVA (p> 0.05). 50 μg of the control peptide did not affect serum IgE levels (p> 0.05). In addition, the control peptide delivered in vehicles and administered to control animals promoted well-defined pro-inflammatory increases in the following parameters: mucus plug formation, number of airway cells secreting mucus, and the degree of interstitial inflammatory cells. In previous experiments, HK-X showed no pro-inflammatory change in the measured histological parameters. Therefore, the unique composition of HK-X seems responsible for its efficiency in downregulating IgE levels and IgE-mediated responses.

EXAMPLE 5: Pulmonary Tissue Response to Long-Term Dosage of High Therapeutic Levels of HK-X To determine if there are potential toxic effects of long-term intranasal exposure to HK-X at the upper end of the therapeutic dose range, mice were exposed to weekly doses of 20 μg of intranasal HK-X for 3 months. During the last two weeks, the intranasal dose of HK-X was increased to 50 μg. It was collected lung tissue 24 hours after the last administration of HK-X for histological analysis. Weekly administration of 20 μg of HK-X intranasally for 3 months followed by 2 weeks of 5 administration of 50 μg did not cause pathological alterations of lung tissue. There was no difference (p> 0.05) between the administration of saline and HK-X with respect to the formation of mucus plug and inflammatory infiltrate. Mucus secretion by airway cells rose 10 after administration of HK-X but this is not judged to be biologically significant. A similar phenomenon was observed with respect to the number of interstitial eosinophils.

• While animals treated with HK-X had approximately 2 eosinophils per 2,200 μ, animals 15 treated with saline showed less than 1 per unit area (p <0.05). The livers, spleens, and kidneys were examined for pathological changes. Except for occasional centers of inflammatory cells in the livers of animals of the control and treated groups, they were not observed • 20 pathological changes. These data establish the tolerability of supra-therapeutic doses of HK-X in mice. EXAMPLE 6: Immunogenicity and Antigenicity of HK-X The objective of this study is to determine if HK-X, when administered to mice by various routes 25 different, will produce an immune response as assessed by i-a-t, .ii.í.-A .....- J - "... ^ q ^ -i-i -, - ,. ... ... ... A., .. ^ .. ^^ ,,, ^. ^^^ the production of antibodies. Thus, immunogenicity and antigenicity were evaluated in relation to HK-X in this study. HK-X is a small tetrapeptide. In most 5 of the cases, such small molecules are poorly immunogenic; however, in vivo, small molecules can • be conjugated or absorbed with larger proteins (become haptens) or blood cells (carriers). The allergic responses of penicillin, quinidine and a-methyl-10-dopa are examples of such haptenic responses. Antibodies to haptens can cause anemia and complex immune diseases due to the destruction of red blood cells (carriers). A number of haptens such as dinitrophenol (DNP) 15 or trinitrophenol (TNP) used experimentally are covalently linked to carrier molecules. The more antigenic the carrier molecule, the more likely it is that an immune response to the hapten will occur. Lame Slurried hemocyanin (KLH) is a widely used carrier and generally supports strong antibody responses to haptens similar to DNP or TNP. The use of adjuvants greatly increases the likelihood that a potential immunogen will produce an immune response. The complete Freund's adjuvant (CFA) or 25 bacterial peptidoglycans have been widely used to ^ and £ ...? stimulate immune responses to poorly immunogenic haptens. Therefore, after first determining the availability of antibodies from the normal drug exposure pathways (without anti-HK-X reactivity), the potential immunity, HK-X, was examined when coupled to KLH and administered in a bacterial adjuvant. . These extreme conditions determined whether HK-X could be immunogenic. Immunogenic Materials and Methods of HK-X: HK-X was conjugated with KLH by means of a spacer of 12 to 20 carbons added at the carboxy terminus. The link was completed through lysine residues on the KLH. The United Biochemical, Seattle, WA, prepared the conjugates. Preparation of Immunogens: The conjugate of HK-X-KLH suspended in PBS at 0.1 mg / ml was emulsified in complete Freund's adjuvant (CFA) containing 1.0 mg / ml of bovine Mycobacterium tuberculosis in a ratio of 1: 1. Adjuvant Immunization Protocol: Female Balb / C mice were immunized intradermally with 0.1 ml of emulsion, promoted 4 weeks later and bled at 6 weeks. Soluble Immunization Protocol: Female Balb / C mice were injected intraperitoneally with 100 μg conjugate without adjuvant in a volume of 0.1 ml to 0.2 ml. The mice were bled after 21 days. Normal Route of Drug Exposure: Sera were collected from animals administered with HK-X via the intranasal route in therapeutic studies of asthma. Determination of antibodies: antibodies analyzed by ELISA for conjugated and unconjugated HK-X. 2 Immulon Microtiter plates (Dynex Technologies # of catalog 3455) were coated overnight at 4 ° C with the following HK-X or 10 μg / ml HK-X conjugates in PBS: • HK-X - peptide alone • HK-X - KLH - peptide conjugated with KLH • HK-X LISA - peptide conjugated with BSA • HK-X - Spacer - peptide with linear spacer of 12 carbons. The wells were washed the next day with PBS and then blocked for 30 minutes at room temperature with sample dilution regulator consisting of 0.1 M Tris-0.15M NaCl buffer, pH 8.0, and 0.1% casein (ICN catalog #) 902896, lot 99333). The mouse serum samples were diluted 1: 100 or 1: 200 with the same regulator, added to the wells and incubated 2 hours at room temperature. The wells were then washed with PBS and incubated with goat anti-mouse IgG peroxidase-conjugated secondary secondary (catalog # Cappel 55554, lot # 39714) for 2 hours at room temperature. After t - t-t-jiJ-A. »-« ^. I. _ ti intriiift wash with PBS, the wells were reacted with OPD chromogen (catalog # SIGMA P-9187, lot 18H82111) for 30 minutes at room temperature. The reaction was stopped with 50 μl of 2.5 M sulfuric acid. The ODs were then determined using a BIO-TEK EL800 reader at 490/630. Resulted Determination of HK-X, from the normal drug exposure route: Sera from the following groups of mice were tested for anti-HK-X reactivity: asthma induced with OVA and treated with HK-X, asthma induced with OVA and control treated with DMSO (vehicle), immunized with saline and treated with DMSO (vehicle). Mice were treated intranasally every third day with 50 μg of HK-X or vehicle for 16 days. No IgG reactivity was observed for conjugated HK-X for the 12-C spacer (HK-X + Spacer), KLH (KLH-HK-X) or BSA (BSA-HK-X). IgG reactivity was observed OVA in all mice immunized with OVA and a control mouse immunized with saline and served as a control for the ELISA. IgG reactivity was observed for KLH-HK-X and BSA-HK-X in sera from animals immunized with KLH-HK-X in adjuvant and served as a control for the coating of these antigens on the ELISA plate. Soluble immunized HK-X coupled to a carrier: Mice were immunized with soluble KLH-HK-X and bled after 21 days. The results of the ELISA show that 4/5 of the serum samples reacted with KLH and KLH-HK-X but no reactivity was observed with BSA-HX or HK-X + spacer indicating that no antibodies were generated in 5 against HK -X that was coupled to KLH after immunization with soluble carrier coupled to HK-X. • HK-X immunized-adjuvant coupled to a carrier. To force the generation of antibodies to HK-X, the mice were immunized with KLH or KLH-HK-X in adjuvant from 10 Freund complete, they were boosted once and bled after 6 weeks. The results of the ELISA show that antibodies against KLH were generated. The antibodies are also • generated for HK-X. This was supported by the following: 1) antibody reactivity for KLH-HK-X from 15 sera of KLH-HK-X was 2 times higher than that of only immune KLH sera and 2) sera from mice immunized with HK-X immunized with KLH-HK-X reacted with BSA-HK-X but not with BSA alone. However, no antibody reactivity was observed for the HK-X coupled to the 12C spacer. ^^ 20 From the results of these studies, several conclusions can be made about the immunogenicity and antigenicity of the HK-X peptide. First, the mice did not generate antibodies to HK-X after therapeutic intranasal administration of the peptide for 16 hours. 25 days. Second, the mice did not generate antibodies when they were immunized with soluble peptide conjugated to the immunogenic carrier KLH. Third, mice can be forced under extreme conditions to generate antibodies to HK-X when coupled to KLH and immunized with complete adjuvant. Without However, even in this case, the antibody reactivity is probably generated for neo-epitopes created by the • conjugation of HK-X and KLH since no antibody reactivity could be detected for the HK-X conjugated to the 12C spacer. This conclusion is supported by the 10 observation that the addition of antiserum-free HK-X for at least 30 minutes before incubation with the test antigen, HK-X-KLH, did not reduce the antibody reactivity • for HK-X-KLH. Thus, it seems unlikely that clinically relevant antibodies or other immune responses to HK-X will be produced in the clinical setting. There are five observations that support such a notion. These observations are: 1) The HK-X is only four amino acids of 20 size (less than 600 Daltons), which makes it unlikely that • return immunogenic; 2) All amino acids in HK-X are hydrophobic whose property is not associated with immunogenicity; 3) To become immunogenic, HK-X would have to become covalently or electrostatically associated with a Í? iJLAAt &Jn? ak * tjá., 1tftáln? r ^, - ... ut - * - ^ - * -...-. , _ "I, _tw_¡__ft__t ... - ^ jfet ^ a¡tÉillAAjMi ^ < JjrffcA ÁAAÁ larger and immunogenic carrier in vivo; 4) The antibodies produced for HK-X are probably directed towards an epitope formed by the combination of the carrier and HK-X (neo-antigen); 5) The antibodies directed towards the neo-antigen react only weakly (low affinity) if in any way it releases HK-X. EXAMPLE 7: Primate Toxicology Study of HK-X This study was conducted at BIOSUPPORT, an animal research facility in Redmond, Washington, according to GLP standards. Six adult male and female macaque monkeys obtained from Charles River were studied. Group A considered a control group, consisted of two animals that were given the vehicle (saline regulated with 3% DMSO) IV daily for five days. Blood sampling for CBC and chemistry was performed on days 0-4 and 7. Group B consisted of three animals dosed with 20 μg / kg of HK-X in vehicle (saline regulated with 3% DMSO) IV daily for five days. Blood sampling for CBC and chemistry was performed on days 0-4 and 7. Group C consisted of the three additional animals dosed at 150 μg / kg IV daily in an identical regimen. Group D consisted of the six animals of groups B and C, dosed with 1000 μg / kg IV daily using the same regimen, five days, after the ^ MiJ? At ^ r ^ - ^.? M? LluÍí ^^? ^ T! ^ .. ^ t - £ i r.k-Aíir? U «The animals of group C completed their diet. All animals were observed daily through the study for weight registration and general health and behavior. At the end of the group D regimen, all animals were euthanized, necropsied, and representative tissue samples were obtained from the following organs collected for histological analysis: liver, kidney, spleen, lung, heart, node lymphatic, and brain. Histopathological evaluation was performed by a veterinary pathologist certified by a ministry associated with BIOSUPPORT and independently by a histopathologist associated with Histatek. These dosages of HK-X were selected based on the effective therapeutic dosages of HK-X of 10 and 50 μg / kg in the mouse asthma model. No significant abnormalities of white blood cells, hematocrit / hemoglobin, or platelet counts were noted at any time at any dose level. Likewise, no significant abnormalities of the chemistry values were noted in any of the three dosage levels. No histological abnormality was observed in tissue samples representative of spleen, and lymph nodes obtained from animals that were exposed to 20 or 150 μg / kg of HK-X followed by 1000 μg / kg daily, or in the vehicle control group. Lymphocytic infiltrate was noted t ^ l.l .., iJ --.-- i-l ..

Minimal multifocal tissue samples from liver, kidney, heart, and lung from control and treated animals were therefore judged unrelated to treatment. Mild glomerular lesions, common in aged macaques, were not added according to the treatment and were also considered to be unrelated to the treatment. Other minor histological changes were not considered significant. There was no discernible toxicity observed in the blood or chemical counts obtained from six macaque monkeys exposed to significantly higher HK-X dosage levels than the dosages considered therapeutic. The minor histopathological changes noted in the liver, kidney, spleen, lymph nodes, heart and lung were not added according to the treatment and were considered manifestations of base pathology or artificial change related to euthanasia. This primate study suggests that therapeutic amounts of HK-X may be useful in human treatment without apparent toxicity or side effects. The invention has been described in detail with reference to the preferred embodiments thereof. However, it will be appreciated that, during consideration of the present specification and drawings, those skilled in the art can make modifications and improvements within the spirit and scope of this invention as defined by the claims.

Claims (1)

1. CLAIMS 1. A method for treating an indication that results from an IgE-mediated response in a mammal characterized in that it comprises administering to the mammal an amount 5 which regulates the low IgE of a peptide having the formula f-Met-Leu-X wherein X is selected from the • group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr. 2. The method according to claim 1, characterized in that another active ingredient is administered 10 with the peptide, the active ingredient being selected from the group consisting of anti-leukotrienes, beta2 agonists and corticosteroids. • 3. A method for downregulating an IgE receptor that comprises administering an effective amount that 15 down-regulates the IgE receptor of a peptide having the formula f-Met-Leu-X, characterized in that X is selected from the group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr. 4. The method according to claim 20 3, characterized in that the IgE receptor is selected from the group consisting of FceRI, FceRII, and soluble FceRII. 5. A method to down-regulate the CD40 ligand, thereby preventing further involvement of the 25 same in the production of IgE, including the method l? i?. ", &&.M. _, _, _ > . r u? a &émÍtk? MiilÉÍ t WáÍiis? * r¿? ..... * ju tiMií * ja i¿? t ~ *** j ** ** ú ** '~ administering an effective amount that downregulates the CD40 ligand of a peptide having the formula f-Met-Leu-X characterized in that X is selected from the group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr. 6. A method for inhibiting the secretion of IgE by plasma cells comprising contacting the plasma cells with an effective amount that inhibits IgE secretion of a peptide having the formula f-Met-Leu-X characterized in that X is select from the group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr. t-t jÉUi tAá nr-n- • t? ti? ¡mrwTf ** ^ -'- "> '" * ¿^ * * l *