KR20010108002A - Small peptides and methods for treatment of asthma and inflammation - Google Patents

Abstract

Methods for treating allergies, contact dermatitis, arthritis, chronic obstructive pulmonary disease and chronic inflammatory bowel disease are described. In addition, the patient's airway inhibits eosinophil invasion, the patient's airway suppresses the release of mucus, the lymphocytes block IgE activation, and the lymphocytes stabilize to increase the inflammatory response to IgE antigen challenge. Methods for preventing involvement in and methods of inhibiting T-cell migration are described. These methods include administering to the patient a therapeutically 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 for treatment of asthma and inflammation

Asthma is a complex disorder. Hereditary and environmental factors-allergies, viral infections, stimuli-are involved in the onset of asthma and its inflammatory exacerbations. More than half of asthma patients (adults and children) have allergies; In fact, allergy to house dust mite feces is a major factor in the development and exacerbation of asthma. Infections of respiratory syncytial viruses during childhood are also highly associated with the development of asthma, and respiratory viral infections often cause acute episodes.

Thirty years ago the introduction of bronchodilator beta ₂ -agents-adrenergic agents selective for receptors-revolutionized the treatment of asthma. These agents have been demonstrated to be potent and longer (4-6 hours) than non-selective adrenergic receptor agonists such as isoproterenol, which stimulate alpha- and beta-adrenergic receptors. Beta ₂ -agonists rapidly relieve symptoms and also protect against acute bronchial contractions caused by stimulation such as exercise and inhalation of cold air. Frequency of use may serve as an indicator of asthma suppression. Recently, the longer acting beta ₂ -agonist salmeterol (lasting up to 12 hours) was introduced to the United States. Salmeterol is so powerful that it can mask inflammatory signals; Therefore, it should be used with anti-inflammatory agents.

Theophylline has a narrow therapeutic limit (control of blood concentration is recommended to avoid toxicity) and drug interaction tendency (competition for liver cytochrome P450 drug-metabolizing enzymes results in plasma concentrations of several important drugs metabolized by the same system). Relatively weak bronchial dilator.

Mild asthma is inhaled daily with anti-inflammatory-corticosteroids or mast cell inhibitors (chromoline sodium or nedocromyl) needed to alleviate breakthrough symptoms or allergen- or exercise-induced asthma, and as needed (1 3-4 times a day) beta ₂ -agent is inhaled for treatment. Chromoline sodium and nedocromil block bronchial spasms and inflammation, but are effective only in asthma and typically young asthma patients, usually associated with allergens or exercise. Inhaled corticosteroids improve inflammation, airways hyperreactivity, and obstruction, and reduce the number of acute exacerbations. However, it takes one month for the effect to be evident and one year for a marked improvement. The most frequent side effects are hoarseness and oral candidiasis. More serious side effects-partial adrenal suppression, growth inhibition, and reduced bone formation-have been reported only with higher doses. Beclomethasone, triamcinolone, and flunisolide probably show similar mg-to-mg potency; Newly approved budesonide and fluticasone have been reported to be more potent and with less systemic side effects.

Even patients with mild disease show airway inflammation including infiltration of activated T-cells, mast cells, and eosinophils into the mucosa and epithelium. T-cells and mast cells release cytokines that promote eosinophil growth and maturation and production of IgE antibodies, which in turn increase microvascular permeability, disrupt the epithelium, stimulate nerve reflexes and mucous glands. As a result, airway responsiveness, bronchial contraction and hypersecretion occur, which are manifested by breathing, coughing and dyspnea.

Traditionally, asthma has been treated with bronchodilators for oral and inhalation. These preparations are helpful for the symptoms of asthma, but not for the underlying inflammation. While the importance of inflammation has been recognized in the etiology of asthma over the past decade, the use of corticosteroids has increased, but many patients continue to suffer from asthma that is not suppressed.

Scientists have determined that leukotrienes (which have A, B, C, D, and E subtypes) play a significant role in asthma. Leukotriene causes airway smooth muscle spasms, increases vascular permeability, causes edema, increases mucus production, reduces mucociliary transport, and causes leukocyte chemotaxis.

Like related prostaglandin compounds, leukotriene is synthesized from arachidonic acid in the cell membrane. In mast cells, eosinophils, macrophages, monocytes, and basophils, arachidonic acid is formed from membrane phospholipids by activating phospholipase A2. After formation, arachidonic acid is metabolized via two major pathways: the cyclooxynase pathway (which produces various prostaglandins and thromboxanes) and the 5-lipoxygenase pathway (which produces leukotriene). A schematic of arachidonic acid metabolism is shown in FIG. 4. Prostaglandins, thromboxanes, and leukotrienes are collectively known as eicosanoids.

Anti-leukotriene is a member of the heterogeneous class of anti-asthma agents with the potential to interfere with early stages in the inflammatory cascade. Leukotriene is an inflammatory substance associated with prostaglandins; Both are produced from arachidonic acid in the cell membrane. After arachidonic acid is formed in mast cells, eosinophils, macrophages, monocytes and basophils, it is metabolized via two major pathways: (1) the cyclooxygenase pathway (produces prostagladins and thromboxane) and (2) 5-lipoxygenase pathway that produces leukotriene in the cytoplasm. Leukotriene is well known in the medical arts as a slow reactant of hypersensitivity (″ SRS-A ″). Leukotriene plays an important role in bronchial inflammation. They induce the migration, adhesion and aggregation of various leukocyte cells (eg, neutrophils, eosinophils, and monocytes) to blood vessels, increase capillary permeability, and cause bronchial and vascular smooth muscle contractions. The results include intestitial edema, leukocyte chemotaxis, mucus production, mucosiliary dysfunction, and bronchial spasms in the lungs. Certain classes of leukotriene, such as cysteinyl leukotriene (LTD ₄ ), are particularly potent bronchial contractors and are approximately 100 to 1,000 times more active than histamine. Leukotriene, including cysteinyl leukotriene, is released from mast cells during degranulation.

A number of anti-leukotrienes that block leukotriene receptors or the enzyme 5-lipoxygenase to prevent leukotriene synthesis are under study and are commercially available. Leukotriene inhibitors are heterogeneous in action: some directly block 5-lipoxygenase, some inhibit proteins that activate 5-lipoxygenase, and some are arachidonic at the arachidonate binding site on the protein. Replace Nate. In contrast, leukotriene agonists block the receptor itself which mediates airway hyperactivity, bronchial contraction and hypersecretion.

Human lung mast cells produce tumor necrosis factor (TNF), IL-4 and IL-5 after IgE stimulation in vitro (Chest 1997; 112: 523-29). Immunohistochemical analysis in bronchial biopsy species confirmed this with IL-6 production. In addition, mast cell counts and TNF are statistically more significant in asthma patients compared to normal groups. TNF and IL-4 can enhance up-regulation of the expression of vascular cell adhesion molecule-1 (VCAM-1)-the adhesion molecule of the immunoglobulin superfamily-in the endothelial layer of bronchial vasculature. have. Eosinophils, basophils and mononuclear cells exhibit very late activating antigen 4 (VLA-4) integrins on their cell surface that interact with VCAM-1. Thus, through interaction VLA-4 / VCAM-1, TNF and IL-4 facilitate the recruitment of circulating white blood cells. The ability of mast cells to release preformed cytokines (TNFs) to IgE-mediated stimuli and the ability to rapidly synthesize others (IL-4, IL-5) may be an early event leading to bronchial inflammation. Indeed, the induction and activation of TH2 clones further facilitate activation and recruitment of eosinophils, which act as terminal effectors of the inflammatory response, further through cytokine production. Again, cytokines produced by leukocytes (particularly TH2 cells) deeply affect the development, activation and priming of mucosal mast cells, thus promoting a positive pre-inflammatory loop. It has recently been shown that human mast cells produce IL-8 and rat lung-derived mast cells express chemokine, monocyte chemotactic protein-1 and macrophage inflammatory protein-1. In addition to cytokines that are taxonomically included in leukocyte recruitment (IL-4, IL-5, TNF), mast cells also act on eosinophils and polymorphonuclear leukocytes (IL-8), producing additional potent chemotactic factors in the airways. do. In addition, because chemokines acting as histamine-releasing factors induce mast cell degranulation, they can also maintain an autocrine activation loop.

Mast cells also play an important role in B-cell growth, which provides the cell contact (such as basophils) required with IL-4 for in vitro IgE synthesis, in which the mast cells are independent of T-cells. Production can be directly regulated and produces an amount of IL-4 sufficient to initiate a local TH2 response upon IgE cross-linking, suggesting that a subset of T-cells play a pivotal role in atopic asthma. Moreover, mast cells can also act as antigen-providing cells on T-lymphocytes, suggesting a greater role of mast cells in the immune network of asthma.

Inhibition of mast cell degranulation by N-formyl-methionyl-leusil-phenylalanine is described in Inflammation , Vol. 5, No. 1, pp. 13-16 (1981). Here, two structurally different chemotactic peptides, pepstatin and N-formyl-methionyl-leucine-phenylalanine, are macromolecules isolated from 40/80, anti-rat IgE serum, or calf lung in rat skin. It has been reported that the anionic permeability factor inhibits the increase in vascular permeability produced by intradermal injection. It has also been reported that these peptides appear to act directly on mast cells.

Due to the importance of treating inflammatory diseases, in particular asthma, arthritis and hypersensitivity in humans, there is a continuing need for new bioactive compounds with fewer side effects. Inhibition of mast cell degranulation by intervening the novel peptides of the invention within the context of the asthmatic inflammatory process is visually depicted in FIG. 4.

The present invention relates to a small peptide having mast cell degranulation inhibitory activity and a method for treating inflammation, in particular allergic, dermatitis such as allergic rhinitis, uticaria, anaphylaxis, drug hypersensitivity, food hypersensitivity, etc. And N-formyl-methionyl peptide useful for the treatment of skin inflammation such as eczema, psoriasis, contact dermatitis, sunburn, aging and the like and osteoarthritis, psoriatic arthritis, lupus, spondyloarthritis and the like. These peptides are also useful for treating chronic obstructive pulmonary disease and chronic inflammatory bowel disease. More particularly, it can be used to replace corticosteroids in all uses of corticosteroids, including immunosuppression and cancer treatment in transplantation.

1 is a log dose response curve illustrating regions of capillary permeability for various concentrations of Compound 48/80.

2 is a dose response curve for inhibition of capillary permeability by various concentrations of f-Met-Leu-Phe.

3 is a dose response curve for inhibition of capillary permeability by various concentrations of preferred peptides of the present invention.

4 is a schematic illustration of the major pathways for arachidonic acid metabolism further illustrating the inhibition of mast cell degranulation.

5A and -5B are schematic illustrations of different protocols of standard 5a and resolution 5b used in the OVA-induced bronchial asthma mouse model.

6A-6D are micrographs illustrating comparative pathology of treatment with compounds of the invention that inhibit OVA-induced asthma in treated and control mice.

7 is a bar graph showing the results of the treatment according to the invention for mucus plug formation in the rat asthma model.

8A-8C show pathology of mouse lung tissue treated according to the present invention following induction of asthma.

9A-9D show pathology of mouse lung tissue of the second group treated with asthma after treatment with the present invention.

10A-10D show pathology of mouse lung tissue of a third group treated with asthma following induction of the present invention.

FIG. 11 is a graph depicting the distribution of inflammatory cells in alveoli regenerated from OVA-induced asthmatic mice.

12 is a graph showing airway plug scores in the airways of OVA-induced asthmatic mice.

FIG. 13 is a graph depicting leukocyte cell migration in airway OVA-induced asthmatic mice.

14 is a graph depicting total cells regenerated from lung lavage of OVA-induced asthmatic mice.

According to the present invention, certain 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, are used to inhibit degranulation of mast cells. It was found to have amazing activity. As a result, these peptides inhibit the release of cytokines (eg TNF) as well as histamine and leukotriene, and for example, allergic rhinitis, uticaria, hypersensitivity, drug hypersensitivity, food hypersensitivity, dermatitis, It is useful for the treatment of skin inflammation such as eczema, psoriasis, contact dermatitis, sunburn, aging and the like, and inflammation that may result from various diseases such as osteoarthritis, psoriatic arthritis, lupus, spondyloarthritis and the like. These peptides are also useful for treating chronic obstructive pulmonary disease and chronic inflammatory bowel disease. The peptides of the present invention can be used to replace corticosteroids in all uses of corticosteroids, including immunosuppression and cancer treatment in transplantation.

After treating the peptides of the invention, subsequent mast cell degranulation and release of histamine, leukotriene, and other cytokines are reduced or stopped entirely in the preferred embodiment. According to a preferred embodiment of the present invention, the peptide can also reduce the penetration of eosinophils, basophils and neutrophils into inflammatory tissue. Lymphocytes, eosinophils, and neutrophils do not exhibit chemotaxis in response to the preferred peptides of the invention. As a result, chemotactic adhesion, migration and aggregation of lymphocytes, eosinophils, and neutrophils are significantly reduced because of vascular permeability at the site of inflammation. In addition, preferred compounds of the present invention are not toxic to extremely important organs such as the heart, liver and lungs.

Preferred peptides according to the present invention provide receptor binding that inhibits IgE activity of lymphocytes such as, for example, macrophages, monocytes, eosinophils, neutrophils, TNF and the like in vitro and in vivo. These peptides stabilize the cell membranes of these lymphocytes while preventing further progression in the increased inflammatory response to IgE antigen challenge. These peptides also block cross-cell IgE binding between mast cells and eosinophils, for example in chronic inflammation.

Peptides of the invention can be prepared by chemical techniques of conventional small peptides. When used for administration, the peptide is prepared with a pharmaceutically acceptable carrier or diluent under sterile conditions.

The dosage of the pharmaceutical composition may vary depending on the subject and the particular route of administration used. The dosage may range from 0.1 to 100,000 μg / kg, more preferably 1 to 10,000 μg / kg per day. The most preferred dosage range is about 1 to 100 μg / kg body weight, more preferably about 1 to 10 μg / kg body weight and most preferably 1.0 to 2.0 μg / kg body weight. Dosages are typically administered once daily to every 4-6 hours, depending on the severity of the condition. In acute situations, it is desirable to administer the peptide every 4-6 hours. In the case of maintenance or treatment, it is preferred to administer only once or twice a day. Preferably, from about 0.18 to about 16 mg of peptide is administered per day, depending on the route of administration and the severity of the condition. Preferred time intervals for the delivery of multiple doses of a particular composition can be determined by one skilled in the art performing only routine experiments.

Routes of administration include oral, parenteral, rectal, vaginal, topical, nose, eye, direct infusion, and the like. In a preferred embodiment, the peptides of the present invention are administered to a patient in an anti-inflammatory effective amount or at a dose that inhibits degranulation of mast cells. Exemplary pharmaceutical compositions are therapeutically effective amounts of peptides according to the invention that typically provide anti-inflammatory effects or inhibit degranulation of mast cells contained in a pharmaceutically acceptable carrier.

The term `` pharmaceutically acceptable carrier '' as used herein and described in more detail below includes one or more compatible solid or liquid filled diluents or encapsulating materials suitable for administration to humans or other animals. In the present invention, the term ″ carrier ″ thus refers to a natural or synthetic organic or inorganic component to which the molecules of the invention are combined to facilitate application. The term ″ therapeutically effective amount ″ is the amount of the present pharmaceutical composition that produces the desired result or exerts the desired effect on the particular condition to be treated. Various concentrations may be used to prepare compositions incorporating the same ingredients to vary the age, severity of symptoms, duration of treatment, and mode of administration of the patient to be treated.

The carrier must also be compatible. As used herein, the term ″ compatibility ″ means that the components of the pharmaceutical composition can be mixed with the small peptides of the present invention and with one another in a manner that does not substantially impair the desired pharmaceutical efficacy.

Small peptides of the invention are typically administered as such (pure). However, they may be administered in the form of pharmaceutically acceptable salts. Such pharmaceutically acceptable salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, bromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluene-sulfonic acid, tartaric acid, Citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Pharmaceutically acceptable salts may also be prepared as alkali metal or alkaline earth metal salts, such as the sodium, potassium or calcium salts of the carboxylic acid groups. Accordingly, the present invention provides a medical pharmaceutical composition comprising a peptide of the invention in combination with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients.

The composition includes compositions suitable for oral, rectal, vaginal, topical, nasal, ocular or parenteral administration that can be used as routes of administration using the materials of the present invention. Pharmaceutical compositions containing peptides of the present invention may also contain stabilizers (which promote long term storage), emulsifiers, binders, thickening agents, salts, preservatives, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorptions. It may also contain one or more pharmaceutically acceptable carriers which may include excipients such as retarders and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the peptides of the invention, its use in pharmaceutical formulations is contemplated. Supplementary active ingredients can also be incorporated into the compositions of the present invention.

Compositions suitable for oral administration are preferred for the treatment of asthma. Typically, such compositions are prepared as inhalation aerosols, nebules, syrups or tablets. Oral compositions may be convenient, but compositions suitable for topical administration are preferred for treating arthritis. Typically, such topical compositions are prepared as creams, ointments or solutions. The concentration of peptide active ingredient in such compositions is typically less than 50 μg / ml, more preferably less than 30 μg / ml, and most preferably about 5 to 10 μg / ml.

The compositions may advantageously be presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. The methods typically comprise the step of bringing into association the active ingredient of the invention with the carrier which constitutes one or more accessory ingredients.

Compositions of the invention suitable for inhalation administration may be provided, for example, as aerosols or inhalation solutions. Examples of typical aerosol compositions consist of the desired amount of microcrystalline peptide suspended in a mixture of trichloro-monofluoromethane and dichlorodifluoromethane plus oleic acid. Examples of typical solutions are the desired amounts dissolved or suspended in sterile saline (optionally about 5% v / v dimethylsulfoxide (″ DMSO ″) for solubility, benzalkonium chloride, and sulfuric acid (for pH adjustment)). It consists of peptides.

Compositions of the invention suitable for oral administration may also be presented in discrete units such as capsules, cachets, tablets or lozenges that contain a predetermined amount of the peptides of the invention or may be contained in liposomes, Or as a suspension in an aqueous or non-aqueous liquid such as syrup, elixir, or emulsion. Examples of tablet formulation bases include corn starch, lactose and magnesium stearate as inert ingredients. Examples of syrup formulation bases include citric acid, coloring dyes, flavoring agents, hydroxypropylmethylcellulose, saccharin, sodium benzoate, sodium citrate and purified water.

Advantageously, a composition suitable for parenteral administration preferably comprises a sterile aqueous formulation of a molecule of the invention that is isotonic with the blood of the receptor. This aqueous formulation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations can be, for example, sterile injectable or suspension in a nontoxic parenterally-acceptable diluent or solvent as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In aqueous solutions, up to about 10% v / v DMSO or Trappsol may be used to maintain solubility of some peptides. In addition, sterile, fixed oils may be advantageously used as a solvent or suspending medium. To this end, a number of fixed oils can be used including synthetic mono- or diglycerides. In addition, fatty acids (such as oleic acid or neutral fatty acids) may be used in the preparation of injections. In addition, Pluronic block copolymers are formulated with lipids at 4 ° C. for compound injection, subject to time release from the solid form over weeks or months at 37 ° C.

Compositions suitable for topical administration may be presented as a peptide solution in trapzol or DMSO or as a cream, ointment, or lotion. Typically, about 0.1 to about 2.5% active ingredient is incorporated into the base or carrier. Examples of cream formulation bases include purified water, waseline, benzyl alcohol, stearyl alcohol, propylene glycol, isopropyl myristate, polyoxyl 40 stearate, carbomer 934, sodium lauryl sulfate, acetate disodium, sodium hydroxide, And optionally DMSO. Examples of ointment preparation bases are white waselin and optionally mineral oil, sorbitan sesquioleate, and DMSO. Examples of lotion preparation bases include carbomo 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.

Rat Skin Model to Measure Inhibition of Mast Cell Degranulation

Allergy-induced asthma is caused by exposure to a substance (allergen) to which the organism is overactive. Exposure to allergens causes degranulation of mast cells in the lung and releases leukotriene and histamine. In response to the release of leukotriene and histamine, capillary permeability dramatically increases and plasma leaks from capillaries into surrounding tissue. Respiratory symptoms resulting from such exposure range from mild symptoms (fastness and sneezing) to potentially fatal symptoms (asthma), including death from hypersensitivity in extreme chronic cases.

To demonstrate this phenomenon experimentally, rat skin was replaced by lungs. In this model, the plasma of experimental rats is labeled with the dye trypan blue. This soluble dye is involved in the bloodstream as a passive marker of plasma itself and is excluded from hepatocytes. Intact blood vessels, including the capillary system, retain this dye under normal circumstances. Compounds that induce degranulation of the mast cells (releasing leukotriene and histamine) are injected into the skin to simulate allergen-induced degranulation. In this experiment, compound 48/80 was used for this purpose. What occurs after leukotriene and histamine release is increased mossel vascular permeability, and blue stained plasma leaks from capillaries to blue stain the skin around the injection site. The area to be blue is a measure of the amount of compound 48/80 injected.

Any compound may be tested for ″ anti-leukotriene ″ and / or ″ anti-histamine ″ activity by mixing with compound 48/80 prior to injection. If the test compound inhibits leukotriene or histamine release, smaller diameter blue areas are observed when compound 48/80 is compared to the injection site in the same rat injected without any test compound. In the case of high anti-leukotriene and anti-histamine activity, greening is almost completely inhibited.

Experiment

Rat skin models were taken and validated. Various peptides were tested at predetermined doses for antileukotriene and / or antihistamine activity. The selected dose allowed a general comparison of f-Met-Leu-Phe, the standard compound for comparison.

A “dose reaction” titration was performed for some compounds and compared to standard compounds. A series of small doses of putative inhibitory compounds were used to observe a series of decreases in the size area of capillary permeability to confirm the inhibition of leukotriene and / or histamine release observed in the initial scheduled dose test.

Materials and methods

Reagents were obtained from Sigma or Aldrich, with the exception of ketamine, a veterinary anesthetic from various veterinary suppliers. Rats used were male Spag-Dolly lineage, 220-240 g, purchased from B & K International.

For rat skin reactions, rats were anesthetized with 0.25 ml 10 mg / ml ketamine. 1.0 ml trypan blue in saline (sterile filtered) was administered to the tail vein and the back of the rat was shaved. Four intradermal injection sites per rat were used for testing and control injection.

Compound 48/80 was prepared as a 1.5 mg / ml stock solution in saline. This material was found to be potentially unstable in aqueous solution. A serial dilution in saline was made at each working level just before injection into each rat.

Peptides were prepared as 23 mM stock solutions in DMSO and stored at −20 ° C. between experiments. When used, the frozen stock solution was thawed and an appropriate aliquot was added to the dilution of compound 48/80 with the appropriate amount of DMSO to obtain a ratio of 5 μL DMSO to 0.1 ml aqueous compound 48/80. This gave a 5% solution of DMSO needed to maintain the solubility of the specific peptide. The effect of 5% DMSO was proven to be zero by control experiments.

For injection, 0.1 ml Compound 40/80, +/- test compounds were injected intradermal into anesthetized, stained and shaved rats. After 15 minutes of incubation, rats were sacrificed by cervical dislocation and the back skin was harvested and placed in a light box. Images of backlit skin were digitized using a CCD video capture camera and compatible hardware / software. Digitized images were analyzed using a scientific graphic analysis software package and the area of capillary permeability (blue light) was integrated to obtain digital values for further analysis.

Dose response curves were prepared using Compound 48/80 at various doses from about 0.01 μg to about 15 μg. The result is shown in FIG. Various variability was noted in the diameter of the capillary permeable area for a given dose of Compound 48/80 based on rat-to-rat changes (eg skin thickness). A dose of 0.15 μg Compound 48/80 was chosen to perform further testing.

Example 1

Dose response curves were prepared for the standard compound, f-Met-Leu-Phe, using selected doses of 0.15 μg Compound 48/80. Dosages from 0 to about 230 nM of f-Met-Leu-Phe are tested and the results are shown in FIG. 2. Inhibition of degranulation induced by compound 48/80 is evident.

Example 2-11

Several f-Met-Leu peptides were tested for inhibition of degranulation induced in a rat skin model using 100 nanomoles of test peptide and a dose of 0.15 μg of compound 48/80. A unique zero-peptide-dose 48/80 control was included for each rat for each experiment and% inhibition was expressed relative to the control (0% inhibition). The percentage of mast cell degranulation produced by 48/80 was also measured. The results are shown in the table below.

Table 1

Example Peptide Inhibition Percent%

Degranulation

2 f-Met-Leu-Phe (prior art) 30 60

3 N-acetyl-Met-Leu-Phe 0 98

4 N-t-BOC-Met-Leu-Phe 0-

5 f-Met-Leu- (iodo) Phe 0-

6 f-Met-Leu-Phe (benzylamide) 0-

7 f-Met-Leu-Phe-Lys 0-

8 f-Met-Leu-Phe (methyl ester) 0-

9 f-Met-Leu-Phe-Phe 100 1-3

10 f-Met-Leu-Tyr 55 30

11 f-Met-Leu-Tyr-Tyr 0-

Example 12

Dose response curves were prepared for f-Met-Leu-Phe-Phe using selected doses of 0.15 μg Compound 48/80. Dosages from 0 to about 230 nM of f-Met-Leu-Phe-Phe were tested and the results are shown in FIG. 3. Surprisingly, a remarkable inhibition of degranulation induced by compound 48/80 has been evident. Inhibition of degranulation induced in the case of f-Met-Leu-Phe-Phe was unexpectedly substantially better than that of the standard compound f-Met-Leu-Phe.

OVA-Induced Bronchial Asthma Mouse Model for Inhibition of Mast Cell Degranulation

Asthma is a complex disease characterized by temporary exacerbation of airway obstruction and persistent bronchial hyperresponsiveness. Chronic infiltration by activated T-lymphocytes, eosinophils and monocytes of macrophages / lower airway mucosa is another established feature. Along with the expression of cytokines, the mechanism of inflammation, and the release of inflammatory mediators, is the etiology of bronchial contraction and bronchial hyperresponsiveness. However, many etiological mechanisms are unclear, for example the mechanisms necessary to control and prevent disease and mechanisms that lead to the persistence of symptoms and chronic inflammation.

It has long been recognized that a single inhaled allergen challenge can cause a sharp increase in airway response in some individual and animal models. However, repeated allergen inhalation shows a superior, sustained, prolonged increase in airway response. This mouse model of long-term repeated inhalation of allergens has been used to study the long-term effects of allergic inhalation in the lung and to describe mediators involved in the induction of cellular, mechanism, molecular, and airway hyperresponsiveness in human lungs.

Materials and methods

reagent; Crystalline OVA Pierce Chem. Co. (Rockford, IL), aluminum potassium sulfate was added to Sigma Chem. Co. (St. Louis, MO), pyrogen-free distilled water from Baxter, Healthcare Corporation (Deerfield, IL), 0.9% sodium chloride (regular saline) from Lymphomed (Deerfield), IL) and TrappsolTM HPB- L 100 (aqueous hydroxypropyl beta cyclodextrin; 45 wt / vol% aqueous solution) was obtained from Cyclodextrin Technologies Development, Inc (Gaitresville, FLA). OVA (500 μg / ml, normal saline) was mixed with 10% (weight / volume) alum of eastern skin in distilled water. After incubation for 60 minutes at room temperature, the mixture (pH 6.5 using 10 N NaOH) was centrifuged at 750 g for 5 minutes; The pellet was resuspended in the original volume in distilled water and used within 1 hour.

Optional 5-lipoxenase inhibitor, Zileuton (N- [1-benzo [b] thien-2-ylethyl] -N-hydroxyurea (J. Pharmacol Exp Ther. 1991; 256; 929) Dr. Bell and George W. Carter Abbot Laboratories, Abbott Part, Ill.). Gilles were dissolved in Trappsol ™, Histatek, Inc. (Seattle, WA) to obtain a mast cell degranulation inhibitor, f-Met-Leu-Phe-Phe (“HK-X”).

Female BALB / c Once (6-8 weeks of age; D and K, Seattle WA) was housed under normal conditions for the study.

Allergen immunization / challenge protocol; Mice were intraperitoneally administered 0.2 ml (100 μg) of OVA with Alum according to different protocols of standard (FIG. 5A) and split (FIG. 5B) (J.Exp Med, 1996; 184; 1483-1494). According to different protocols, mice were given ketamine in normal saline (0.44 mg) before receiving an intranasal dose of 100 μg OVA in 0.05 ml regular saline and 50 μg OVA in 0.05 ml regular saline separately on different days. / ml) / xylazine (6.3 mg / ml) intraperitoneally anesthetized. Two control groups were used. Thus, the first group is administered intranasally with regular saline with alum and intravenously with normal saline without alum; The second group received OVA intraperitoneally with Alum, intranasal OVA without Alum, and regular saline alone.

histology

Trachea and left lung (the right lung was used for bronchoaveolar larvage ("BAL")) were obtained and fixed for 6-15 hours at room temperature in 10% neutral formaldehyde solution. After staining with paraffin, the tissue was cut into 5 μm sections and further treated with different stains or immunolabels. Discombe eosinophil staining was used to count cell numbers by counter staining of methylene blue. The number of cosinophils per unit airway area (2,200 μm ² ) was determined by morphometry (J. Pathol 1992; 166; 395-404; Am Rev Respir Dis, 1993; 147; 448-456). It was. Fibrosis was identified by three color staining of Masson. Airway mucus was identified by reaction with the following staining methods: methylene blue, hematoxylin and eosin, mucicarmine, alcian blue, and alkyan blue / periodic-schiff (PAS) (Troyer, H., "Carbohydrates "in Principles and Techniques of Histochemistry, Little, Brown and Company, Boston, MA, 1980: 89-121; Sheehan, DC, et al.," Carbohydrates "in Theory and Practice of Histotechnology, Battle Press, Columbus, OH, 1980 159-179). Mucin stained with musicarmine solution; Methanyl yellow counter staining was used. Acidic mucus and sulfated mucus material were stained with alcian blue, pH 2.5; Nuclear fast red counter staining was used. Neutral and acidic mucus was identified by alcian blue, pH 2.5, and PAS reactions. The degree of mucous plugging of the airways (0.5-0.8 mm in diameter) was also assessed by biomorphometry. Percentage of airway diameter occlusion by mucus was classified into semiquantative sizes of 0-4 +, as described in the figure description. Histology and biomorphometry analyzes were performed by individuals blinded to the protocol design.

Pulmonary function test

On day 28, 24 hours after the last intranasal administration of regular saline or OVA, the pulmonary kinetics for intravenous infusion of methacholine were determined in mice in vivo by a plethysmorgraphic method modified from that previously described. (10, 1958; 192; 364-368; J. Appl. Physiol. 1998; 64; 2318-2323; J. Exp. Med. 1996; 184; 1483-1494). After completion of the pulmonary function test, each mouse was punctured in the heart to bleed and die, and lung tissue along with treachea was obtained for further analysis.

Bronchoalveolar lavage

The left lung was tied at the main bronchus and the right lung was washed three times with 0.4 ml of normal saline. Bronchoalveolar lavage (BAL) fluid cells from a 0.05 ml aliquot of the collected samples were counted using a hematocytometer and the remaining fluid was centrifuged at 200 g at 4 ° C. for 10 minutes. Supernatants were stored at −70 ° C. until eicosanoid analysis was performed. Cell pellets were resuspended in regular saline containing 10% bovine serum albumin (“BSA”), and then BAL cell applied samples were made on glass slides. To stain eosinophils, the dried slides were placed into a dispensing dilution fluid (0.05% aqueous eosin and 5% acetone (vol. Stain for 8 minutes, rinse with water for 0.5 minutes with water, and counterstain for 2 minutes with 0.07% methylene blue.

Analysis of Airway Mucus Glycoprotein

Mucus glycoproteins in BAL fluids were analyzed by slot blotting and PAS staining (Anal. Biochem. 1989; 182; 160-164; Am, J, Respir. Cell Mol. Biol. 1995; 12: 296-306) . Nitrocellulose membranes (0.2 μm pore size; Schleicher & Schuell, Keene, NH) were wetted in distilled water and then wet in normal saline before being placed in a multiple II 72-well slot blot apparatus (Schleicher & Schuell). Aliquots (0.05-0.75) of BAL fluid samples (0.05 ml) and stock solutions of human respiratory mucin glycoprotein (2 μm / ml) (Am. J. Respir. Cell Mol. Biol. 1991; 5: 71-79) l) was blotted onto the nitro-cellulose membrane by water suction vacuum and the mucoglycoprotein was visualized by PAS reaction. Reflectance measurements were performed to quantify PAS staining. The images were then analyzed by the image processing system described below. The integrated intensity of PAS reactivity of the BAL samples was quantified compared to the standard curve for human respiratory mucus.

Immunocytochemistry

Monoclonal antibodies: CD11c (DAB method) and Mac1 (Beringer Manheim, ABC method with Hitomous Kit, Zymed) were used for inflammatory cell types such as dendric in and around vasculatures, airway and fibrosis areas. ) Cells, macrophages and lymphocytes were used to identify.

Morphometry and Image Analysis

All images were collected and digitized by ScanJet IICX Scanner (Hewlett Packard, Palo Alto, Calif.) With HP DeskScan II software (MicrosoftR WindowsTM version). The system was connected to a Dell Dimension XPS P 90 computer (Dell Corporation, Austin, TX) using Image-ProR Plus, version 1.1 (Media Cybermetics, Silver Spring, MD) for WindowsTM software. Images were evaluated at 256 gray level size using a Dell Ultrascan 17ES monitor with high resolution graphics mode (1.280x1,024 pixels, 78.9-kHz horizontal scanning frequency, 74-Hz vertical scanning frequency).

Leukotriene Inhibitor Study

To assess the role of 5-lipoxygenase product in airway inflammation, a 5-lipoxygenase inhibitor, Gilleuton (35 mg / kg) was administered intraperitoneally 30 minutes prior to each intranasal challenge on days according to FIG. In one set of animals, zileuton was also administered prior to intraperitoneal OVA administration. Gilles at 35 mg / kg inhibited -95% cysteinyl leukotriene release in passively sensitized rats injected with BSA antigen intraperitoneally (J. Pharmacol. Exp. Ther. 1991; 256; 929-937). .

Compound HK-X of the Invention

Compound HK-X was administered at 5 mg / kg and 10 mg / kg using the same procedure as described above.

Statistical analysis

Pulmonary function data was assessed by change analysis (ANOVA) using at least significant difference protected methods (Statview II, Abacus Concepts, Berkeley, Calif.). This method uses multiple t-statistics to evaluate all possible pair comparisons and is applicable for equal and non-equivalent pair sizes. Other data were reported as mean ± SE of the combined experiments. Differences were analyzed for significance (P <0.05) by the Student's two-tailed t test for independent mean values.

1.Eosinophils (Table 2A-2B)

The eosinophil counts in the airways in the 1-, 2- and 3-month group of OVA-treated mice were significantly reduced (P <0.025) from 44.83% to 37.40% and 19.15% (P <0.025), respectively. Eosinophil counts were significantly higher in the OVA treated group than in the other two groups over the same time period (P <0.025), but zileuton was generally able to reduce eosinophils through 1-3 months. However, the HK-X compounds of the present invention reduced eosinophils comparable at 1 month, but much more beneficially at 2 months and 3 months.

Table 2A Airway Inflows of Eosinophils

(%) Brine OVA zileuton HK P value 3 months 1.00 19.15 10.73-<0.0252 months 1.00 37.40 11.66-<0.011 months 1.00 44.83 15.50 14.20 <0.001P Value> 0.05 <0.025 <0.025 <0.025

Table 2B: Percentage of eosinophils in airway tissue

Treatment time Saline OVA Gilleston HK-X 28 Days 1.0 44.8 15.8 14.2

2. other inflammatory cells

Other inflammatory cells exhibit a non-specific inflammatory response following the influx of foreign proteins into the airways. Lymphocytes were recycled to the airways. It was substantially absent in the control group. Neutrophils were recycled following OVA challenge in boot Siamese-sensitized and OVA sensitized mice, although more numbers appeared in the airways of the OVA sensitized group. Unique multinucleated giant cells (fused macrophages) with nucleus crescents around the ends of various cytoplasms were sometimes seen. Langhans giant cells and globulin leukocytes were observed only in animals sensitized with OVA and challenged. They usually exist only in connective tissues connected with large airways. Plasma cells were sometimes seen in the proximity of the airways and in local lymphoid tissue.

3. Airway plug (Table 3)

Mucus: There was no difference in the three groups in the same treatment but different time course (P> 0.05). OVA-treated groups had higher scores than saline, ileuton (P <0.05) and HK-X compound treated.

Table 3: Mucus Plug Scores in Airways

Treatment Time Brine OVA Gilleston HK-X 28 days 0.7 2.8 1.3 1.4 Airway plug%> 5% 55% 16% 19%

Asthma is a chronic inflammation of the airways. In humans, once airway hypersensitivity is established, it can remain stable for years. This persists in the absence of apparent allergen inhalation, detectable airway inflammation or epithelial detachment. Thus, it can be permanent because of irreversibility (or at least slowly reversibility) in the airway hyperstructure.

In mild asthma, these episodes or "attacks" are relatively infrequent and are well treated (reversed) with inhaled bronchodilators. The intensity of latent, characteristic, chronic airway inflammation appears to be associated and linked to more frequent, powerful, long-term attacks that are less reversible by bronchodilators. The reason for this has become increasingly clear in recent years. Inflammation, consisting primarily of Th2-lymphocytes, eosinophils, mast cells, and possibly activated or primed influx of platelets, causes expansion of the perivascular (interstitial) space and release of mediators / growth factors, which thicken the basal membrane , Epithelial damage and shedding, production of viscous mucosa, and hyperplasia, partial contraction of prime and airway smooth muscle. All these results support an increase in airway response, which lowers the threshold for response to environmental stimuli, thus making attacks more frequent and robust.

All these morphological changes will directly and strongly affect lung function. In experiments with acute asthma mouse models and long-term asthma mouse models, significant pathophysiological changes in pulmonary function were observed to support these morphological changes. Allergen inhalation increases eosinophil and mast cell expression in the airway and alveolar endothelium and epithelium, induces E-selective expression only in the airway endothelial, and eosinophil influx, airway response, and other types of inflammatory cells (rangans type globus) By increasing fibroblasts and multinucleated giant cells (fused macrophages), this shows a non-specific inflammatory response in the asthma lung.

Compound HK-X inhibits mucus accumulation in the airways of OVA-treated and control mice. The distribution of mucosal obstruction of the airways is shown in the absence of HK-X treatment from sham-sensitized, saline-infected mice (saline, n = 4), and OVA-sensitive / challenged mice (OVA, n = 4) or in the presence (HK-X / OVA, n = 8). Mucous occlusion of airway diameter was analyzed biomorphologically as follows: 0, no mucus; +,-10% occlusion; ++, 30% occlusion; +++. ˜60% occlusion; ++++, ˜80% occlusion. Ten airways randomly distributed through the lungs of each mouse were evaluated for mucosal occlusion biomorphometrically.

6A-6D provide visual histological evidence of the ability of compound HK-X to inhibit degranulation of mast cells in asthma induced rats using OVA and thereby the effect of treating asthma with compound HK-X. 6A shows the abundance of secreted mucus in the lungs of the airways (AW) of OVA sensitized / challenged mice. 6B shows massive infiltration of interstitial tissue by eosinophils and other inflammatory cells (indicated by arrows). 6C shows that airway mucus release in the airway (AW) lungs is surprisingly reduced when Compound HK-X inhibitors are administered prior to intranasal OVA administration. Infiltration of interstitial tissue by eosinophils is also reduced after compound HK-X treatment as compared to OVA- challenge alone (FIG. C compared to FIGS. 6a and 6b). FIG. 6D shows the absence of mucus and cells in control mice treated with saline-treated airways (AW). The bronchial epithelium is infiltrated by connective tissue cells, but no leukocytes are present in the interstitial space around the bronchus.

Airway macrophages show growth activity signals similar to those reported in macrophages recovered from BAL fluids from allergen-challenged lungs of asthma (Am, Rev. Respir. Dis, 1987; 135: 433-440). Macrophages and dendritic cells can directly or indirectly produce secretions of cytokines that act as antigen-providing cells in the lung and can initiate phenotypic changes in the airway epithelium and its surrounding sites. Stimulation of chronic inflammation of the airways can directly induce proliferation of airway epithelial and fibroblasts, resulting in the deposition of collagen in these areas. Activated macrophages and dendritic cells remain high in the area compared to other inflammatory cells during late stage challenge.

The airway epithelium is thickened, especially due to large airway, but small and terminal bronchioles, mainly due to labeled goblet cell hyperplasia. The ratio of goblet cells to cells with normal, columnar, and cilia was significantly increased compared to the control group. Whereas the control airways (small and large) had only occasional goblet cells, fragments from OVA-infected lungs showed that 100% air and some small airways had goblet cells up to 88% of total airway epithelial cells. Containing. In the unwashed lungs, mucus was found in the high blit cells and in some airways and sometimes completely obstructed the lumen. Cell pieces were caught in these mucus plugs. Goblet cell hyperplasia was not found in the control group and, therefore, cannot be attributed to the "-non-allergic" effect of OVA or intratracheal administration techniques. Some goblet cells in bovine airways are devoid of this feature, possibly indicating an uneven distribution of OVA in the bronchus.

7 is a histogram of the results when treated with Compound HK-X at doses of 5 μg / kg and 10 μg / kg upon formation of mucus plugs in these murine asthma models. Both doses significantly reduce mucus production in the small airways.

Asthma is characterized by a complex inflammatory response of airway eosinophilia, edema, mucus hypersecretion, bronchial epithelial loss and hypersensitivity. Inhaled allergen challenge in allergic asthma causes an immediate airway hyperresponsiveness, an early airway response (EAR), followed by a late phase airway response (LAR) which is frequently delayed after several hours. After recovery from LAR, the acquired airway hyperresponsiveness (AHR) is increased for reagents such as methacholine that can last for several days. Immediately after allergen challenge, EAR development appears to be secondary to the action of bronchial contractile molecules released by human lung mast cells as a result of IgE-mediated degranulation.

IgE-mediated mast cell degranulation is a major event in the pathogenesis of allergic diseases such as allergic rhinitis, asthma and hypersensitivity. In atopic individuals, intradermal infusion of specific allergens causes an immediate wheel and flare response characterized by the release of histamine and the formation of lipid mediators at the skin test site. Initial skin reactions are followed by late skin reactions that occur 6-12 hours later. This late allergic skin response is characterized by an inflammatory response consisting of perivascular edema and cellular infiltration by eosinophils and other inflammatory cells (eg neutrophils, monocytes and basophils). Similar dual phase IgE-dependent reactions such as early and late rhinitis or asthmatic reactions occur in the upper and lower airways of atopic individuals after local challenge with certain allergens. Mast cells are located proximal to the alveolar surface into blood vessels. Mast cells can affect tone and / or promote inflammatory responses, affecting pulmonary vasculature. Mast cells activated by immunological or non-immunological stimuli degranulate and may be mediated by histamine, neutral protease, peroxidase, O ₂ , PAF and eicosanoids (eg, LTB4, LTC4, And release a number of preformed and newly generated mediators such as PDG2, TXA2) and cytokines (eg, IL-4, IL-5, a).

The above mentioned murine models reproduce important features of human asthma. Late-phase allergen-specific lung disease was induced in normal BALB / c mice using ovalbumin (OVA) as the allergen. One protocol includes intraperitoneal administration of OVA to mice 1 and 14 and intranasal administration of OVA on days 14, 25, 26 and 27 to immunize mice. On day 28, OVA-treated mice received (1) increased circulation levels of total and OVA-specific IgE, (2) increased release of LTB4 and LTC4 in BAL fluid, (3) BAL fluid of labeled eosinophils, and Influx into the lung parenchyma, (4) mucosal obstruction of bovine airways, (5) increased expression of T-helper cell type 2 (Th2) cytokines (IL-4, IL-5, and IL-13), and Reduced expression of cytokines (IL-2 and IFN-y) in bronchial lymph node tissue), (6) significantly more rapid in dynamic compliance and airway conductance at increased methacholine doses compared to control mice Disease that is very similar to allergen-induced asthma, including reduction.

Mice are also very water soluble in the development of IgE-mediated allergic airway responses. Late phase influx of eosinophils is reproduced in the murine model where allergic airway disease develops after inhalation of obbualbumin in mice pre-sensitized with intraperitoneal ovalbumin. Increased airway responsiveness to methacholine or acetylcholine challenge also occurs in mice immunized following airway exposure to antigen.

In a mouse model of allergen-induced airway inflammation, the role of mast cells in airway eosinophil infiltration, mucus release, and hyperreactivity to methacholine was investigated. It has been found that bovine peptide HK-X plays an important role in the prevention of mucus release by a mechanism of mast cell degranulation and prevention of eosinophil infiltration into the airways.

The material was used as above. Mice were injected intraperitoneally with 0.2 ml (100 μg) of OVA complexed with aluminum potassium sulfate (alum, alum) on days 0 and 14. On days 14, 25, 26, and 27, mice were given ketamine (0.44 mg / ml) / xylazine (6.34) in normal saline prior to intranasal administration of 100 μg OVA in 0.05 ml normal saline on days 25, 26 and 27. 0.2 ml of mg / ml) was anesthetized by intraperitoneal administration. Lung tissues were obtained 24 hours after the last intranasal challenge on 28 days. The control group received intraperitoneal administration of regular saline with alum on days 0 and 14 and regular saline without alum on days 14, 25, 26 and 27. To assess the inhibitory effect of HK-X on OVA-induced asthma, HK-X (10 μg / ml) was administered 30 minutes prior to intranasal challenge on days 25, 26 and 27, respectively.

After tying the left lung to the main bronchus, the right lung was destroyed three times with 0.4 ml of normal saline. Bronchoalveolar lavage (BAL) fluid cells from 0.05 ml aliquots of pooled samples were counted using a hemocytometer and the remaining fluid was centrifuged at 200 × g at 4 ° C. for 10 minutes. After resuspending the cell pellet in normal saline containing 10% BSA, BAL cell smears were made on slide glass. To stain eosinophils, the dried slides were stained with Discobe's diluting fluid (0.05% aqueous eosin and 5% (v / v) acetone in distilled water) for 5-8 minutes and with water for 0.5 minutes. Wash and counterstain with 0.07 methylene blue for 2 minutes.

Following BAL, the trachea and left lung (upper and lower lobe) were obtained and fixed in 10% buffered formalin solution for 18 hours. After the tissues were treated and sunk in paraffin, the tissues were cut into 5 μm sections and stained with Discombes solution and counterstained with methylene blue or stained with Hematoxylin and Eosin as described above. As described above, the number of eosinophils (2200 μm ² ) per unit area of the airway was measured by morphometry. Airway mucus was identified by various staining methods, namely methyleneblue, hematoxylin and eosin, mucicarmine, toluidine blue, alcian blue, and alkian blue / hypoic acid Schiff (PAS) reactions. Mucin is stained with mucincarmine solution; Methanyl yellow counterstain was used. Mucin and sialic acid-rich non-sulfated mucus were stained heterochromatically using pH4,5, toluidine blue. Acidic mucin and sulfated mucus were stained with pH 2.3, alcian blue; Nuclear fast red counterstain was used. Neutral and acidic mucus was identified by pH 2.5, Alcian blue and PAS reactions. In addition, the degree of mucus plugging of the airways (diameter 0.5-0.8 mm) was evaluated by morphology. The percent closure of airway diameter by mucus was classified into semiquanitative sizes from 0 to ++++++. Histological and morphological analysis protocol design was performed by individual blinded to protocol design.

Mucus glycoproteins in BAL fluids were evaluated by slot blotting and PAS staining as described. Nitrocellulose membranes (pore size: 0.2 μm, Schleicher & Schuell, Keene, NH) are usually brine in distilled water and then normal saline before being placed in a Minifold II 72-well slot blot appratus Wet. BAL fluid samples (0.05 ml) and aliquots (0.05-0.07 ml) of stocks (0.2 μg / ml) of human respiratory mucin glycoproteins were blotted onto the nitrocellulose membrane by water suction vacuum, Mucus glycoproteins were visualized by PAS reaction. Reflective densitometry was performed to measure PAS staining. Images were captured and digitized with ScanJet Hex Scanner (Hewlett packard, Palo Alto, Calif.), Including HP DeskScan II software (Microsoft Windows TMVersion). DellDimension XPS P90 computer using the Image-Pro Plus, Version 1.1 for Windows M software (Media Cybernetics, Silver Spring, MD) (Dell Corporation, Austin, TX). The image was evaluated at 256 gray levels using a Dell UltraScan 17ES monitor with additional high resolution graphics modes (1280 x 1024 pixels, 78.9-kHz horizontal scanning frequency, 74-Hz vertical scanning frequency). . The integrated intensity of PAS reactivity of the BAL samples was measured by comparison with the reference curve for human respiratory mucin.

I.P. using OVA. Immunoassay yielded detectable levels of OVA-specific IgE in BALB / C mice. Indirectly, ELISA was used to measure OVA-specific IgE serum antibody titers. ELISA plates (ICN, Costa Mesa, CA) were coated with OVA (20 mg / ml) diluted in 0.1 M NaHCO, buffer pH 8.3 and incubated at 4 ° C. for 18 hours. After washing three times, the plates were incubated with 3% BSA in PBS at pH 7.4 for 2 hours at 37 ° C. Serial dilutions of serum samples in 1% BSA / PBS buffer were added to the plates and incubated at 4 ° C. for 18 hours before washing again. The wells were bound to rat anti-mouse IgE monoclonal antibodies (Pharmingen, San Diego, Calif.) Diluted in 50% goat serum (Gibco-BRL, Gaithersburg, MD) / PBS buffer (horse radish peroxidase). Incubated at room temperature for 2 hours. Wells were developed with absorbance measured at 610 nm using 3,3 ', 5,5'-tetramethylbenzidine substrate. The internal standard in each assay consisted of congested serum from OVA-immunized BALB / c mice.

Pulmonary action data were assessed by variable analysis (ANOVA) using the protected least significant difference method (Statvies II, Abacus Concepts, Berkeley, Calif.). The comparisons are analyzed in possible pairs and can be applied for pairs of equal and unequal pairs Other data is reported as mean + SE of combined experiments Student's two-tailed t-test on independent method The differences were analyzed for significance (p <0.05) by Student's two-tailed t-test.

result

Allergen-specific IgE products. OVA-specific IgE (12.9 + 0.3 U / mL, n = 5) was detected in the blood of mice receiving ip OVA on day 28, and in the blood of mice receiving alum on days 0 and 14, 25, 26 And 27 days in OVA were detected in the blood of the cormorant. In contrast, anti-OVA IgE could not be detected in control mice treated with ip saline and allum and in saline (n = 6).

Allergen-induced airway inflammation. For histological analysis of allergen-derived airway inflammation, sequentially in OVA challenge three times on days 25, 26, and 27, followed by lung tissue and BAL fluid 24 hours after the last test infection, ie, day 28 Obtained. By light microscopy, eosinophils were noticeably infiltrated into the epilepsy of the trachea (FIG. 8C). Eosinophils were observed in the epithelial mucus layer of the trachea (FIG. 9D) and BAL fluid.

61.0 + 5.0% (n = 10) of BAL fluid cells in OVA-sensitized / challenged mice were eosinophils compared to 0.8 + 0.3% (n = 10) in saline-infected control animals (p = 0.0001).

After tracheal challenge of OVA, mucosal obstruction of the airways occurred in immunized mice (FIG. 10). Airway mucus release in both the lower (Fig. 9b) and the top (Fig. 9c, 9d) discard plots was identified by separate histochemical staining; Mucins are stained with musicarmine, acidic non-sulfated mucus is toludine blue, acidic sulfated mucus is alcian blue (Fig. 10), and neutral and acidic mucilage is alcian blue / PAS It was identified by the reaction. Obstruction of the airway lumen by mucus was greater in the lower airways. i.p. treated with brine (FIG. 8B) or OVA (not shown) containing allum. There was no inflammatory test infection in saline-infected control animals.

HK-X inhibition blocks the infiltration of eosinophils and accumulation of mucus in the airway. Inhibition of inflammation by HK-X significantly reduced the influx of eosinophils into lung tissue and BAL fluid of VOA sensitized / challenged mice and also prevented airway mucus release in these animals (FIGS. 8A, 9A, 10A).

Eosinophil infiltration. In morphometric analysis, the influx of eosinophils to pulmonary epilepsy was reduced to 90% by HK-X treatment (p <0.006, compared to HK-X untreated group) (FIG. 11). SLO Zileuton reduced the number of eosinophils by 82% in BAL fluid (p <0.004, compared to OVA zileuton) (FIG. 11). The number of eosinophils in BAL fluid from OVA-sensitized / challenged mice treated with HK-X was 0.57 + 0.11 × 105 and the number from OVA-immunized / saline-challenged 0.16 + 0.03 × 105 ( Control mice). Similarly, ileuton reduced recovered eosinophils in BAL fluid of OVA-sensitized / challenged mice by 89% (p = 0.0128 compared to zileuton untreated OVA; data not shown). Carrier controls (Trappsol ™ for the Jilleuton study) did not affect lung eositophil infiltration observed in OVA-sensitized / challenged mice.

Mucus accumulation. Cross sections of the upper and lower lobes of the left lung of OVA-treated and control mice were examined by light microscopy for mucus accumulation in the airways. In morphometric analysis, airway mucus release was not seen in the airways of 68% saline treated control mice, and a small amount of mucus layer was observed in the rest. In contrast, OVA-immunized / challenged mice showed morphology for widespread mucus plugging of the airways. The airways of many (74%) OVA-treated mice were closed at least 30% of the airway lumen by mucus; 22% of the mice's airways had mucous obstruction over 80%. When the amount of mucus glycoprotein recovered in BAL fluid was measured (FIG. 12), it was demonstrated that there was a 7-fold increase in airway mucin in OVA-treated mice compared to control mice (p = 0.00001 OVA versus saline). HK-X treatment blocked airway mucus release in OVA-treated mice (FIGS. 8A, 9A, 10A).

8A-8C show histopathology of lung tissue and illustrate the therapeutic efficacy of OVA induced mouse asthma with bovine peptide (HK-X) (n = 8) in each group of animals. 8A is a diagram of HK-X treated mice, showing normal characteristics of the airways (AW) and blood vessels (BV). Very few cells are located around the airway tissue (arrowheads) (H & E staining, X12). 8B is a diagram of mice injected with only saline injection used as normal control. The shape of the airways and blood vessels is normal. Some cells (arrowheads) appeared in airway tissue (H & E staining, X120). FIG. 8C is a diagram of an OVA immunized animal, showing a high potency by the nature of eosinophils and T-cells, monocytes, and macrophage infiltration in airway tissue. The airways were plugged by mucus and cells (arrows) (H & E staining, X120).

9A-9D show histopathology of lung tissue treated with small peptides (HK-X) after mice were induced to suffer from asthma. Data from this group was obtained from another group of mice. The effectiveness of HK-X compounds in the treatment of asthma in mouse models was explained by histopathological evidence. HK-X compound interfered with airway mucus secretion by OVA challenge daily for a total of three days. HK-X also reduced cell infiltration. During asthma episodes, HK-X reduced cell infiltration. On days 1 and 14 the mice were immunized intravenously and intranasally with OVA. After 25, 26, and 27 days, the mice were challenged with OVA or 30 minutes before the mice were challenged with 10 μg HK-X intranasally.

9A is a micrograph of a medium-sized airway (AW) and is representative of a typical HK-X treated mouse lung. In HK-X treated lungs, little pathological conditions were observed in OVA treated animals. The airways were very clear with little or no mucus observed on the lumen or epithelial cell surface. There was little cell penetration into the paraenchyma cells. The thickness of the smooth muscle cell layer is uniform (H & E staining, X150). 9B is a diagram of the lungs of OVA immunized and challenged mice, showing typical characteristics of asthma in humans: prominent features of cell infiltration into the partially plugged airway (AW) lumen (arrow), airway and epilepsy of blood vessels, And edema (arrowheads) associated with blood vessels (H & E staining, X75). 9C is a high magnification of the lung airway of mice with asthma, showing plugged lumen. Many leukocyte cells are located at the base of the airway epithelial cell layer (arrowheads). Many eosinophils appeared in the parenchyma and the thickness of the smooth muscle cell layer was not uniform (H & E staining, X150). FIG. 9D is a diagram of a partially longitudinal cutaway airway of the lung of a mouse with asthma, which shows that the airway lumen was broadly blocked by the released mucus (arrow). Cell penetration is located very densely in the epithelial cell layer (arrow). Smooth muscle cell layers were separated by infiltrating white blood cells and small granuloma was often formed (H & E staining, X150).

10A-10D show histopathological data obtained from another group of mice (n = 9). Additional histopathological and histochemical evidence shows that HK-X compounds interfere with mucosal cell induction by OVA immunization and challenge and reduce the secretion of mucus in the airways. During asthma attacks, mucus secretion and airway constriction increase. Sulfated glulosamine glucan was stained using alcian blue to reveal mucus. 10A is a diagram of a lung of a mouse suffering from HK-X treated asthma, indicating that the lumen of the airway (AW) is empty and no material has been produced in the lumen. Adjacent blood vessels (BV) are also present. In normal form no cell infiltration or edema fluid was seen (H & E staining, X150). FIG. 10B is a continuous section of the same airway shown in FIG. 10A, which was stained with alcian blue at pH 2.4 to locate mucus in epithelial cells. Only a few cells were positive in the lumen (arrowheads). A sporadic thin layer of blue positive material appeared (Alcian blue and neutral red staining, X150). 10C is a diagram of the lungs of OVA immunized and challenged mice, showing contracted airways and intraluminal mucus secretion (arrowheads). Numerous white blood cells have been observed in lung tissue. Many of these are eosinophils (H & F staining, X150). FIG. 10D is a section similar to FIG. 10C, which was stained with alcian blue to show mucus material in the contracted airways. A thick layer of mucus appeared to adhere densely to the epithelial surface (arrowheads). Here, more blue positive cells were seen, indicating a higher percentage of mucous cells appearing in the airway lumen. In addition, longitudinal small airways filled with mucus appeared in the sections (Alcian blue & neutral red staining, X 150).

FIG. 11 shows the distribution of inflammatory cells in lung lungs of lungs obtained from mice with OVA-induced asthma. 12 shows airway plug scores in the airways of mice suffering from OVA-induced asthma. 13 shows white cell migration in the airways of mice suffering from OVA-induced asthma. Figure 14 shows total cells from lung lavage of mice with OVA-induced asthma.

Induced Type II Collagen Arthritis Mouse Model

A mouse model was used to assess the efficacy of the compounds of the invention on histology, radiographs and clinical shapes of induced type II collagen arthritis.

Autoimmune diseases cause significant and chronic pathological conditions and disorders. Many forms of arthritis are a representative family of autoimmune diseases. In the clinical field, rheumatoid arthritis (RA) is the most common form of severe joint dysplasia. All clinicians agree that RA is a progressive disease.

Arthritis lesions occurring in CIA of mice show histopathologically significant similarities to that of RA in human patients. Thus, CIA in mice is an acceptable model for studying potent therapeutics of RA.

Substances and Methods

Mice: 25 gms DBA / 1 (2) male mice (Jackson Laboratories, Bar Harbor, ME or B & K University, Kent WA) were used for this study. Strains of mice are sensitive to CIA by injection of heterologous type II collagen. Bovine collagen (BC), Complete Freun's Adjuvant (CFA) and Incomplete Freun's Adjuvant (ICFA) can be purchased from Sigma Chemical. Antigens for immunization were treated in 0.1 M acetic acid and formulated with CFA or ICFA.

Induction of Arthritis

Immunization Protocol: Mice were injected with 100 μg of Type II collagen in CFA at predetermined time intervals during the study.

Mice were examined for the occurrence of arthritis at scheduled time intervals. Expected symptoms of arthritis include swelling and erythema of at least one toe joint of the anterior and / or posterior foot in two consecutive observations.

Definitive diagnosis of arthritis

Histological examination of joints: To remove, fix, decalcified, sink in paraffin, slice, and usually stain the toe joints of the sacrificed animals at appropriately appropriate time intervals. And cartilage matrix of the pannus of each joint (cartilaginous matrix) was detected. The degree of cellularity and area of inflammation were measured using digitization of histological micrographs as described above and applying standard area and point counting techniques.

Radiographic evaluation of the toe joints was performed to detect the occurrence of joint changes after immunization with type II collagen. A mammography imaging system was modified for this study. The average area of soft tissue (pannus) of the joints was measured by analysis of computer digitized radiographs, with changes in the density of adjacent hard tissue by comparison with internal criteria included with each radiograph. For use as a petrolatum control for the density change of the area of hard tissue and panni, additional mice were used for the same time and the density and area data were compared. Paired T-tests at each time point were used to assess significant differences in density and area for control and experimental mice.

Arthritis evaluation

Animals were observed daily during the onset of arthritis. Arthritis indicators were obtained by grading the severity associated with each foot on a scale of 0-4. Scoring is based on the degree of edema and erythema around the joint and the malformation of the joint. The swelling of the hind limbs was measured by measuring the thickness of the ankle from the mid to lateral astragalus with a constant tension caliper.

Experimental Design

To assess the anti-arthritic efficacy of compound HK-X, the route of administration is considered to be the most appropriate dosing mechanism based on the experience of the human patient.

Administration of HK-X and Prednisolone: Doses representing peptides that were estimated at different and therapeutic levels were placed in the site positioned by both percutaneous (TC) (absorption) and injection by foot. Direct injection into the intrarticular space is very traumatic and therefore easy to manufacture. Thus, drug injection into the footpad adjacent to the intraarticular cavity is the method of choice. Control mice were also injected with prednisolone as a positive control (proven as anti-inflammatory of efficacy in the treatment of experimental and clinical autoimmune diseases).

First, collagen was injected into each group of 10 groups (including controls) daily for 50 days. On days 3 and 18 the mice were injected with 5 or 10 μg / kg of compound HK-X in 0.1 M acetic acid solution at a concentration of 1 mg / ml. On day 50 mice were bled for tissue study.

Subsequently, 8 of 10 groups (A-I) mice were treated according to the specific protocol below.

Group A was immunized and untreated with primary CFA with BC, secondary ICFA with BC (control).

Group B was immunized with primary CFA containing BC, secondary ICFA containing BC and prednisolan was administered for 20 days beginning that day after secondary ICFA containing BC.

Group C was immunized with primary CFA with BC, secondary ICFA with BC and compound HK-X was administered to TC at 4 mg / kg (high dose).

Group D was immunized with primary CFA with BC, secondary ICFA with BC and compound HK-X was administered to TC at 0.4 mg / kg (low dose).

Group E was immunized with primary CFA containing BC, secondary ICFA containing BC, and compound HK-X was administered to TC at 4 mg / kg (high dose) for 20 days beginning on that day after secondary ICFA containing BC. Administered.

Group F was immunized with primary CFA containing BC, secondary ICFA containing BC, and TCV at 0.4 mg / kg (low dose) of compound HK-X for 20 days beginning on that day after secondary ICFA containing BC Was administered.

Group G was immunized with primary CFA with BC, secondary ICFA with BC, and 10 ml of DMSO was administered to TC for 20 days, starting the day after secondary ICFA with BC (control).

Group H was immunized with primary CFA with BC, secondary ICFA with BC and 10 ml of DMSO was administered to FP for 20 days, starting that day after secondary ICFA with BC (control).

Group I was immunized with primary CFA with BC, secondary ICFA with BC and 10 ml of saline was administered to FP for 20 days beginning on the day after the secondary ICFA with BC (control).

Animals from each group were radiated immediately after the second immunization and prior to sacrifice. After sacrifice, the paws were properly removed and treated for histological experiments. Treatment with compound HK-X was found to reduce the degree of arthritis.

The invention has been described in detail with reference to its preferred embodiments. However, during the review of the specification and drawings of the present invention, it will be understood by those skilled in the art that modifications and improvements can be made without departing from the spirit and scope of the invention as defined by the claims.

The present invention provides a novel pharmaceutical composition containing an N-formyl-methionyl-leucine ("f-Met-Leu") peptide having inhibitory activity against mast cell degranulation in a suitable pharmacological carrier. Particularly useful such 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. These peptides are especially used for allergic, dermatitis, eczema, psoriasis, contact dermatitis, sunburn, aging, etc., such as allergic rhinitis, oticaria, hypersensitivity, drug hypersensitivity, food hypersensitivity, and osteoarthritis, psoriatic arthritis, lupus, It is useful for the treatment of arthritis such as spondyloarthritis. These peptides are also useful for treating chronic obstructive pulmonary disease and chronic inflammatory bowel disease. The peptides of the present invention can be used to replace corticosteroids in all uses of corticosteroids, including immunosuppression and cancer treatment in transplantation.

According to the present invention, a method for treating an allergic reaction in a mammal is characterized in that the mammal has an anti- Administering an allergic effective amount of the peptide. Inhalation is the preferred dosage form for treating allergy associated with nasal membranes. Preferred dosage forms for treating contact allergy are topical application using suitable pharmacological carriers. Intradermal infusions or tablets may be used for systemic treatment.

The invention also provides a method of treating dermatitis in a mammal. The method comprises administering to a mammal an anti-inflammatory 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.

The present invention also provides a method for treating arthritis selected from the group consisting of osteoarthritis, psoriatic arthritis, lupus, spondyloarthritis and the like in mammals. The method comprises administering to the mammal an antiarthritis 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.

According to another embodiment, the present invention provides a method for inhibiting eosinophil invasion in a patient's airways. The method comprises administering to said patient an effective amount of an airway eosinophil inhibition of peptides having a formula f-Met-Leu-X, wherein X is selected from the group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr do.

According to another embodiment, the present invention provides a method of inhibiting mucus release in the airways of a patient. The method comprises administering to the patient an effective amount of the inhibitory release of airway mucus release 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 also provides a method of treating chronic obstructive pulmonary disease in a patient. This method involves administering to a patient a therapeutically effective amount of a chronic obstructive pulmonary disease of a peptide having a formula f-Met-Leu-X wherein X is selected from the group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr. Include.

The present invention also provides a method of treating chronic inflammatory bowel disease in a patient. The method comprises administering to a patient a therapeutically effective amount of a chronic inflammatory bowel disease 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 do.

The present invention also provides a method for blocking IgE activation of lymphocytes such as, for example, macrophages, monocytes, eosinophils, neutrophils, TNF and the like. The method comprises contacting the lymphocytes with an IgE activation blocking 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. .

The invention also provides a method for stabilizing the membranes of lymphocytes, such as, for example, macrophages, monocytes, eosinophils, neutrophils, TNF, etc. to prevent further involvement in the increased inflammatory response to IgE antigen challenge. To provide. The method comprises contacting the lymphocytes with a cell stabilizing 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.

The present invention also provides a method of inhibiting migration of T-cells, such as, for example, CD4-cells, to prevent the production and relevance of IL-4 and IL-5 as well as other chemokines. This method comprises contacting said T-cells with an effective amount to inhibit T-cell migration 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. It involves making.

In certain preferred embodiments of the invention, the patient may enjoy the benefit of administering the peptide of the invention in combination with a second active ingredient. Other active ingredients particularly useful for this combination according to the invention are for example antileukotrienes, beta ₂ agonists, corticosteroids and the like.

Claims (20)

A mammal's allergy, comprising administering to the mammal an anti-allergic 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 How to treat a reaction. The method of claim 1, wherein the allergy is selected from the group consisting of allergic rhinitis, uticaria, drug hypersensitivity, and food hypersensitivity. The method of claim 1, wherein another active ingredient selected from the group consisting of an antileukotriene, a beta ₂ agonist, and a corticosteroid is administered with the peptide. Mammalian dermatitis, comprising administering to the mammal an anti-inflammatory 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 How to treat it. 4. The method of claim 3, wherein the dermatitis is selected from the group consisting of dermatitis, eczema, psoriasis, contact dermatitis, sunburn and aging. 5. The method of claim 4, wherein another active ingredient selected from the group consisting of an antileukotriene, a beta ₂ agonist and a corticosteroid is administered with the peptide. Osteoarthritis, psoriatic arthritis, comprising administering to a mammal an antiarthritis effective amount of a peptide having the formula f-Met-Leu-X selected from the group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr A method of treating arthritis selected from the group consisting of lupus and spondyloarthritis. 8. The method of claim 7, wherein another active ingredient selected from the group consisting of an antileukotriene, a beta ₂ agonist and a corticosteroid is administered with the peptide. Chronic obstructive pulmonary disease comprising administering to a patient a therapeutically 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 How to treat a disease. 10. The method of claim 9, wherein another active ingredient selected from the group consisting of an antileukotriene, a beta ₂ agonist and a corticosteroid is administered with the peptide. Chronic inflammatory bowel disease, comprising administering to a patient a therapeutically effective amount of a peptide having the formula f-Met-Leu-X selected from the group consisting of Tyr, Tyr-Phe, Phe-Phe, and Phe-Tyr How to treat it. 12. The method of claim 11, wherein another active ingredient selected from the group consisting of an antileukotriene, a beta ₂ agonist and a corticosteroid is administered with the peptide. A patient's airway comprising administering to the patient an effective amount of an airway eosinophil inhibition of inhibition of a peptide having the formula f-Met-Leu-X selected from the group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr How to suppress eosinophil infiltration into furnace. A patient's airway comprising administering to the patient an effective amount of an airway mucosal release inhibitory peptide of a peptide having the formula f-Met-Leu-X selected from the group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr A method of inhibiting the release of mucus into the furnace. IgE of lymphocytes, comprising contacting lymphocytes with an effective amount of an IgE activation blocking effective amount of a peptide having the formula f-Met-Leu-X selected from the group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr How to block activation. The method of claim 15, wherein the lymphocytes are selected from the group consisting of macrophages, monocytes, eosinophils, neutrophils and TNF. A cell membrane of lymphocytes comprising contacting lymphocytes with a cell stabilizing effective amount of a peptide having the formula f-Met-Leu-X selected from the group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr Stabilization to prevent further involvement in an increased inflammatory response to IgE antigen challenge. 18. The method of claim 17, wherein the lymphocytes are selected from the group consisting of macrophages, monocytes, eosinophils, neutrophils and TNF. Contacting T-cells with a T-cell migration inhibitory effective amount of a peptide having the formula f-Met-Leu-X selected from the group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr , Method of inhibiting migration of T-cells. The method of claim 19, wherein the T-cells are CD4-cells.