RU2423139C2 - COMPOSITION CONTAINING Actinidia AND METHODS OF THEIR APPLICATION - Google Patents

Abstract

FIELD: medicine, pharmaceutics. ^ SUBSTANCE: claimed group of inventions relates to medicine. Claimed are methods of immune response regulation and reduction of inflammatory symptoms in mammal, with atopic dermatitis. For this purpose introduced is, at least, one medication of frost-resistant plant kiwi and, at least, one corticosteroid. In regulation of immune response in case of atopic dermatitis included is the following treatment period, during which carried out is monotherapy by medication of frost-resistant kiwi without introduction of corticosteroids. Also claimed is composition, which contains, at least, one medication of frost-resistant kiwi and, at least, one corticosteroid. Also claimed is product in form of animal forage, which contains claimed composition. Claimed group of inventions insure efficient regulation of immune response and reduction of inflammatory symptoms in case of atopic dermatitis due to synergetic effect of combination of medications of frost-resistant kiwi and corticosteroids, which makes it possible to reduce need in additional application of corticosteroids in treatment of said pathology and in future carry out monotherapy by medication of frost-resistant kiwi without reduction of treatment efficiency. ^ EFFECT: invention can be applied for regulation of immune response and reduction of , at least, one symptom of inflammation in mammal which has atopic dermatitis. ^ 35 dwg, 5 tbl, 8 ex

Description

APPLICATION AREA

The present invention relates to Actinidia, in particular to species of a frost-resistant kiwi plant, to its various fractions and preparations, as well as compositions containing them, having the ability to prevent and / or treat various diseases in which the regulation of the immune response is effective, including allergic and non-allergic inflammatory diseases , viral infection and cancer. Methods related to the preparation and use of these compositions are also described.

BACKGROUND OF THE INVENTION

Diseases involving inflammation are characterized by the influx of certain types of cells and mediators, the presence of which can lead to tissue damage and sometimes death. Diseases involving inflammation are especially dangerous if they affect certain organs and systems, such as the respiratory system, which can lead to breathing obstruction, hypoxemia, hypercapnia, and damage to the lung tissue. In other diseases or pathological conditions, the development of certain types of inflammation can be an important component of the regulation of the disease, such as a viral infection, although tissue damage in the area of infection is still a risk factor.

Allergic diseases are partially mediated by immunoglobulin E (IgE), whereas it has been shown that type 2 T-helper cells (Th2), mast cells and eosinophils also play an important role in the disease process (Maggi E., Immunotechnology, 3: 233-244 , 1998; Pawankar R., Curr. Opin. Allergy Clin. Immunol., 1: 3-6, 2001; Vercelli D., Clin. Allergy Immunol., 16: 179-196, 2002). Circulating IgE binds to two isoforms of IgE receptors: high affinity IgE receptors (FcεRI) present on the surface of mast cells and basophils, and low affinity IgE receptors (FcεRII or CD23) present on the surface of lymphocytes, eosinophils, platelets and platelets. It is believed that an important factor regulating the pathogenesis of allergic disorders is the cross-linking of mast cell IgE receptors after the detection of the allergen and subsequent mast cell degranulation. Mast cell molecules include histamine, heparin, proteases and free radicals that mediate various biological effects, including vasodilation, intestinal and / or bronchial smooth muscle contraction, mucous secretion, and local proteolysis. After an initial immediate reaction from mast cells, an influx of eosinophils, basophils and lymphocytes is observed 6-24 hours later. This late-phase response can lead to chronic inflammation in tissues that are constantly exposed to antigens.

Degranulation of IgE-dependent mast cells and the accumulation of eosinophils in areas of inflammation are considered as a result of decompensation of hyperactivation of Th2 cells and, therefore, Th2-mediated hyperproduction of IgE (Abbas et al., 1991, Nature 383: 787-93; Vercelli, 2001, Curr Opin Allergy Clin Immunol 2001, 1: 61-5). Typical Th2 cell cytokines, such as IL-4, IL-5, IL-10, and IL-13, are known to play an important role in these reactions. In addition, Th1-mediated cytokines, such as IFN-γ and IL-12, have been reported to negatively regulate Th2 pathways. For example, IFN-γ induces a switch to the IgG2a isotype in B cells, while IL-12, under certain conditions, converts the already established Th2 response to the dominance of the Th1 response (Umetsu and DeKruyff, 1997, J Allergy Clin Immunol 100: 1- 6; Coffman and Carty, 1986, J Immunol 136: 949-54; Gavett et al., 1995, J Exp Med 182: 1527-36). Various cellular transcription factors, such as GATA3 and T-bet, control the differentiation of Th1 and Th2 cells and the production of cytokines in these cells (Lee et al., 2000, J Exp Med 192: 105-15; Ting et al., 1996 , Nature 384: 474-8; Lighvani et al., 2001, Proc Natl Acad Sci USA 98: 15137-42; Szabo et al., 2000, Cell 100: 655-69).

Allergic diseases, such as anaphylaxis, allergic rhinitis, asthma, atopic dermatitis, food allergies and urticaria, affect up to 20% of the population in many countries, and the incidence of these diseases is increasing (Wuthrich B., Int. Arch. Allergy Appl. Immunol. 90: 3-10, 1989).

For example, asthma is a characteristic lung disease that affects millions of people around the world. Asthma is usually characterized by periodic lack of air flow and / or hypersensitivity to various stimuli, which lead to excessive narrowing of the airways. Other characteristics may include airway inflammation, eosinophilia, and airway fibrosis. It is believed that increased airway susceptibility is the result of a combination of an inflammatory cascade involving certain types of cells, including T-lymphocytes and eosinophils. In allergic asthma, Th2 cytokines prevail over Th1 cytokines.

Atopic dermatitis (AD) is a chronic and recurrent inflammatory skin disease characterized by itchy and eczematous skin lesions, along with an increase in IgE levels. The prevalence of AD appears to be increasing worldwide in infants and children. Skin lesions in patients with AD are characterized by inflammatory cell infiltration, including T-lymphocytes, monocytes / macrophages, eosinophils and mast cells. These cells are involved in the pathogenesis and development of AD through the isolation of various cytokines and chemokines, such as IL-4, IL-5, IL-10, IL-13, eotaxin and TARC. Among many cell types, Th2 cells producing IL-4, IL-5, IL-10, and IL-13 play a critical role in the initial phase of disease progression (Leung, 1997, Clin Exp Immunol 107 (suppl. L): 25- thirty). IL-4 and IL-13 act as the main isotype of IgE switching inducers, and IL-5 causes activation of eosinophils that secrete various chemokines, such as eotaxin. IL-10, produced by monocytes / macrophages as well as Th2 cells, increases the production of TARC, which is a Th2-specific chemokine, and is known to be overexpressed in AD lesions. Although Th1-type cytokines, such as IFN-γ, are also found in AD lesions of the skin during the late phase of the disease, it is believed that the development of AD is caused mainly by overproduction of Th2-mediated cytokines / chemokines and IgE, as well as defective IFN-γ production γ and IL-12 (Jonathan et al, 1999, J Clin Invest 103: 1103-11; Christian et al., 1999, J Clin Invest 104: 1097-105; Tomomi et al., 2001, J Allergy Clin Immunol 107: 353-8; Weilie et al., 2002, J Clin Invest 109: 621-8). However, the exact mechanism due to overproduction of IgE and an imbalance of Th1 / Th2 responses has not yet been elucidated.

Since most of the evidence suggests that the Th1 and Th2 types of reactions are mutually regulated in vivo, it is suggested that a change in Th1 / Th2 is a reasonable strategy for developing treatment for allergic diseases (Kato et al., 1999, J Immunol 162 : 7470-9). For example, recombinant cytokines such as IL-12 and IFN-γ, or cytokine receptor antagonists for IL-4 and IL-5, have been tested for their ability to control the balance between Th1 and Th2 responses (Hofstra et al., 1998, J Immunol 161: 5054-60; Tomkinson et al., 2001, J Immunol 166: 5792-800). However, the direct use of these agents often causes undesirable side effects.

Leukotrienes are also associated with various diseases associated with inflammation, in particular with allergic inflammation. Leukotrienes are formed from arachidonic acid, a precursor to prostaglandins. There are two families of leukotrienes. The first group acts mainly in conditions in which the inflammation depends on neutrophils, such as cystic fibrosis, inflammatory bowel disease and psoriasis. The second group (cysteinyl-leukotrienes) mainly affects eosinophil and mast cell-induced bronchospasm in asthma. They bind to highly selective receptors on the bronchial smooth muscles and other tissues of the respiratory tract (O'Byrne et al., Annals of Internal Medicine 1997; 127: 472-80). It is also known that leukotrienes are important in the pathophysiology of allergic rhinitis, chronic urticaria and atopic dermatitis or eczema. Leukotriene antagonists, including both leukotriene synthesis inhibitors and cysteinyl-leukotriene receptor antagonists, are useful for specifically inhibiting the production or effects of these inflammatory mediators.

The suggestion that, by lowering serum IgE levels, can improve allergic symptoms, has been demonstrated through clinical trials using the chimeric anti-IgE antibody (CGP-51901) and recombinant humanized monoclonal antibody (rhuMAB-E25) (Fahy et al., Am. J. Respir. Crit. Care. Med. 155: 1828-1834, 1997). Analogues of diacylbenzimidazole and bacterial polysaccharides that inhibit the synthesis and secretion of IgE have been described in US patent No. 6369091 and publication of US patent No. 200441885, respectively.

Korean Patent Application No. 92-11752 shows an anti-inflammatory, anti-allergic and anti-rheumatic drug containing a biflavonoid, such as 4'-O-methylcnaflavone isolated from Lonicera japonica, which is effective in treating various symptoms caused by allergies or inflammation. Korean Patent Application No. 100744 shows an anti-inflammatory, anti-allergic and anti-rheumatic drug product comprising several biflavonoid compounds isolated from Ginko biloba leaves. Several Asian medical prescriptions containing Siegesbeckia glabrescens have been reported to have IgE-reduced activity (Kim et al., Phytother. Res., 15: 572-576, 2001). In addition, it has been found that many medicinal plants are rich sources of inhibitors releasing histamine or anti-inflammatory compounds.

Other common medications for treating allergic diseases include antihistamines, steroidal or non-steroidal anti-inflammatory drugs, and leukotriene antagonists. These agents, however, have serious hidden side effects, including, but not limited to, increased susceptibility to infection, liver toxicity, drug-induced pulmonary disease, and suppression of bone marrow activity. Thus, such drugs are limited in their clinical use by treating inflammation, in particular allergic inflammation. The use of anti-inflammatory and symptomatic attenuating agents is a serious problem, as their side effects or their insufficient effects cause an inflammatory response. There is an urgent need for less dangerous and more effective agents for treating inflammation. Thus, there is a need for new products with lower side effects, less toxicity and more specificity with respect to the root cause of inflammation.

In conclusion, the identification of an immune response that promotes the activation of Th1-type T cells, IgG2a production and production of Th1-type cytokines (e.g. IFN-γ, IL-6, IL-12, IL-1) contradicts the immune response associated with allergic inflammation discussed above. This type of immune response may have strong, systemic, antitumor and antiviral properties. There is a longstanding need in the art for products with such properties.

SUMMARY OF THE INVENTION

One embodiment of the present invention relates to a method for regulating an immune response in mammals. The method includes the use by mammals of a preparation of a frost-resistant kiwi plant in an amount sufficient to regulate the immune response in a mammal, where the preparation of a frost-resistant kiwi plant is selected from a fresh fruit, crushed fruit, boiled fruit, cooked fruit, squeezed fruit, thickened fruit, dried fruit and concentrate juice of a frost-resistant kiwi plant. In this embodiment, the preparation of the frost-resistant kiwi plant is not extracted.

In one aspect of the aforementioned embodiment, the preparation of a frost-resistant kiwi plant is obtained by a method that includes the step of drying the fetus.

Another embodiment of the present invention relates to a method for regulating an immune response in a mammal, including using a mammal with a preparation of a frost-resistant kiwi plant in an amount sufficient to regulate an immune response in a mammal, wherein the preparation of a frost-resistant kiwi plant is selected from the group consisting of an extract or leaf concentrate and an extract or concentrate bark.

Another embodiment of the present invention relates to a method for regulating an immune response in a mammal. The method includes the use of a mammal in an amount sufficient to regulate the immune response in a mammal, (a) the preparation of a frost-resistant kiwi plant; and (b) a component selected from probiotics; cell walls and bacterial fragments; whey protein; alanine; fatty acids; fatty acid esters; monoglycerides; diglycerides; triglycerides; inositol; turmeric curcumin; methylsulfonylmethane (MSM); ginseng; ginger and proanthocyanidin.

In the aforementioned embodiment, the preparation of a frost-resistant kiwi plant may include, but is not limited to, a fruit extract or concentrate, leaf extract or concentrate, stem extract or concentrate, bark extract or concentrate, root extract or concentrate, fresh fruit, crushed fruit, boiled fruit, cooked fruit, squeezed fruit, thickened fruit, dried fruit, juice concentrate of a frost-resistant kiwi plant, a preparation obtained by extraction from fruit water having a temperature from 0 ° C to about 80 ° C; a preparation obtained by direct extraction of a water-soluble concentrate of a frost-resistant kiwi plant with ethyl acetate, a preparation obtained by extraction of a frost-resistant kiwi plant in distilled water, and a preparation obtained by subsequent extraction of a frost-resistant kiwi plant in water, chloroform and ethyl acetate.

In one aspect of the aforementioned embodiment, the preparation of a frost-resistant kiwi plant is obtained by a method that includes the step of drying the fetus. In another aspect, the preparation of a frost-resistant kiwi plant is obtained by extracting the fetus in water having a temperature of from about 0 ° C to about 25 ° C. In another aspect, the preparation of a frost-resistant kiwi plant is obtained by extracting the fetus in water at room temperature. In yet another aspect, a preparation of a frost-resistant kiwi plant is obtained by direct extraction of a water-soluble concentrate of a frost-resistant kiwi plant with ethyl acetate.

Another embodiment of the present invention relates to a method for regulating an immune response in a mammal, including using a mammal in an amount sufficient to regulate the immune response in a mammal, (a) a preparation of a frost-resistant kiwi plant; and (b) a component selected from steroids, antihistamines, antibodies, antibiotics, cyclosporins, fungicides, respiratory function regulators, analgesics, β-agonists, leukotriene modifiers, cytokines or cytokine receptor antagonists, phosphodiesterase inhibitors, sodium chromochromoglycate, nedochromoglyclate, nedochromoglyclata, nedromokromiliklata, nedromokromiliklata, nedrochromoglykla, theophylline, carbobenzoxy-beta-alanyltaurin and inhibitors of T-cell function.

In the aforementioned embodiment, the preparation of a frost-resistant kiwi plant may include, but is not limited to, an extract or concentrate of the fruit, an extract or concentrate of leaves, an extract or concentrate of the stem, an extract or concentrate of the stem, an extract or concentrate of the root, a fresh fruit, crushed fruit, boiled fruit, cooked fruit, squeezed fruit, thickened fruit, dried fruit, juice concentrate of a frost-resistant kiwi plant, a preparation obtained by extracting fruit water (juice water), having a temperature of 0 ° C approximately about up to 80 ° C; a preparation obtained by direct extraction of a water-soluble concentrate of a frost-resistant kiwi plant with ethyl acetate, a preparation obtained by extraction of a frost-resistant kiwi plant in distilled water, and a preparation obtained by subsequent extraction of a frost-resistant kiwi plant in water, chloroform and ethyl acetate.

In one aspect of the aforementioned embodiment, the preparation of a frost-resistant kiwi plant is obtained by a method that includes the step of drying the fetus. In another aspect, the preparation of a frost-resistant kiwi plant is obtained by extracting the fetus in water having a temperature of from about 0 ° C to about 25 ° C. In another aspect, the preparation of a frost-resistant kiwi plant is obtained by extracting the fetus in room temperature water. In yet another aspect, a preparation of a frost-resistant kiwi plant is obtained by direct extraction of a water-soluble concentrate of a frost-resistant kiwi plant with ethyl acetate.

In any of the above embodiments, a preparation of a kiwi plant can be provided in an amount sufficient to regulate the immune Th2 and Th1 response in a mammal. In one aspect, a qiwi plant preparation is provided in an amount sufficient to control the amount of isotype produced in a mammal of antibodies selected from the group consisting of IgE, IgG2a and IgG1. In another aspect, a kiwi plant preparation is provided in an amount sufficient to reduce the production and / or levels of at least one Th2 cytokine in a mammal or to reduce the level of at least one Th1 cytokine in a mammal. In yet another aspect, a qiwi plant preparation is provided in an amount sufficient to reduce or produce at least one leukotriene in a mammal. In another aspect, a qiwi plant preparation is provided in an amount sufficient to reduce the expression level of a transcription factor selected from the group consisting of GATA-3, T-bet, and NFATc2 in a mammal. In another aspect, the mammal has a pathological condition or is at risk of developing it, in which activation of the Th1 response and / or suppression of the Th2 response is expected. For example, a mammal has or is at risk of developing an allergic disease or a non-allergic inflammatory disease. Such an allergic disease may be a disease that is regulated by leukotrienes. Such allergic diseases include, but are not limited to, asthma and atopic dermatitis. As another example, a mammal may have or be at risk of developing a viral infection or cancer.

In any of the above embodiments, the frost-resistant kiwi plant may include, but are not limited to, Actinidia arguta, Actinidia kolomikta and Actinidia polygama, wherein the Actinidia arguta plant is associated with a preferred embodiment.

In any of the above embodiments, the preparation of a frost-resistant kiwi plant may be present in the composition in an amount of about 0.01% and up to about 95% by weight based on the total weight of the composition.

In one aspect of any of the above embodiments, the use step includes administering to the mammal a preparation of a frost-resistant kiwi plant with a carrier, excipient or solvent. In another aspect, the use step includes administering to the mammal a preparation of a frost-resistant kiwi plant in the form of a tablet, powder, effervescent tablet, effervescent powder, capsule, solution, suspension, granule or syrup. In another aspect, the use step includes administering a preparation of a frost-resistant kiwi plant to a mammal in the form of healthy food. Healthy foods include, but are not limited to, thin baked goods, bread, rolls, cereal breakfast, processed cheese, unprocessed cheese, condiments, dairy products, puddings, sweet jelly, carbonated drink, teas, powdered drink mixes, processed fish products, drinks fruit-based, vegetable-based drinks, chewing gum, caramel, frozen dairy products, meat products, nut pastes, pasta, processed poultry products, gravy and sauces, potato chips, vegetable chips, crisp ofel, chocolate, cookies, sweets, licorice sweets, ice cream, dehydrated foods, cut foods, processed foods, spices, spirits, noodles, fermented foods, soups, soup mixes, soy based products, vegetable oil pastes and Vegetable based drinks. In another aspect, the use step includes administering to the mammal a cosmetic composition comprising a preparation of a frost-resistant kiwi plant. Cosmetic compositions may be used in the form of, including, but not limited to, lotion, cream, essence, tonic, emulsion, mask, soap, shampoo, detergent, cleanser, body wash solution, wash solution, or as a treatment. In one aspect, the use step includes administering to the mammal a preparation of a frost-resistant kiwi plant as part of a nutritional supplement.

In another aspect of any of the above embodiments, the method further comprises administering to the mammal an agent selected from fatty acids; polyketides; organic acids; small organic compounds; aromatic amino acids; phenylpropanoids; terpenoids; steroids; alkaloids; corrins; porphyrins; linear peptides; cyclic peptides; depsipeptides; amino acid derivatives; nucleosides; nucleotides; carbohydrates; proteins; cells; cell fragments; herbal preparations; spices; minerals; sterilizers; seasoning; vitamins; and electrolytes.

In yet another aspect of any of the above embodiments, the method further comprises administering to the mammal an agent selected from probiotics; bacterial cell wall and fragments; whey protein; taurine; alanine; fatty acids; fatty acid esters; monoglycerides; diglycerides; triglycerides; Inositol; turmeric; curcumin; rosemary; rosmarinic acid; methylsulfonylmethane (MSM); ginseng; ginger proanthocyanidins; and β-carotene. Fatty acids include, but are not limited to, conjugated linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, γ-linolenic acid, α-linolenic acid, dihomo-γ-linolenic acid, and stearidonic acid.

In another aspect of any of the above embodiments, the method further comprises administering to mammals formulations of various Actinidia species. Various Actinidia species may include, but are not limited to, A. chenensis, A. deliciosa, A. arguta, A. polygama, and A. kolomikta.

Another embodiment of the present invention relates to a composition for regulating an immune response in a mammal. The composition includes a preparation of a frost-resistant kiwi plant and at least one additional active compound for regulating the immune response in a mammal. In one aspect, the additional active compound is used to treat or prevent an allergic disease in a mammal. In one aspect, the additional active compound is selected from steroids, antihistamines, antibodies, antibiotics, cyclosporins, fungicides, respiratory function regulators, analgesics, β-agonists, leukotriene modifiers, cytokine or cytokine receptor antagonists, phosphodiesterase inhibitors, caffeine chromoglycoside , theophylline, carbobenzoxy-beta-alanyltaurin, and T cell function inhibitors. In yet another aspect, the additional active compound is selected from the group consisting of probiotics; cell walls and bacterial fragments; whey protein; alanine; fatty acids; fatty acid esters; monoglycerides; diglycerides; triglycerides; inositol; turmeric; curcumin; methylsulfonylmethane (MSM); ginseng; ginger and proanthocyanidins. Fatty acids include, but are not limited to, conjugated linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, γ-linolenic acid, α-linolenic acid, dihomo-γ-linolenic acid, and stearidonic acid. Such a composition may include, but is not limited to, a medicament, a healthy food, a nutritional supplement, or a cosmetic.

In the composition described above, a frost-resistant kiwi plant may include, but are not limited to, Actinidia arguta, Actinidia kolomikta, and Actinidia polygama. In one aspect, the preparation of a frost-resistant kiwi plant is an extract or concentrate obtained from a portion of a frost-resistant kiwi plant selected from a fruit, leaf, stem, bark, root, and any combination thereof. In another aspect, the frost-resistant kiwi plant is selected from the group consisting of a fresh fruit, crushed fruit, boiled fruit, cooked fruit, squeezed fruit and thickened fruit. In another aspect, the frost-resistant kiwi plant is a dried fruit. In yet another aspect, a preparation of a frost-resistant kiwi plant is prepared by a method that includes the step of drying the fetus. In another aspect, the preparation of a frost-resistant kiwi plant is a juice concentrate of a frost-resistant kiwi plant. In another aspect, the preparation of a frost-resistant kiwi plant is obtained by extracting the fetus in room temperature water. In yet another aspect, a preparation of a frost-resistant kiwi plant is obtained by direct extraction of a water-soluble concentrate of a frost-resistant kiwi plant with ethyl acetate. In another aspect, the extract is obtained by extraction of a frost-resistant kiwi plant in distilled water. In another aspect, the extract is an ethyl acetate extract of a frost-resistant kiwi plant.

Another embodiment of the invention relates to the use of a frost-resistant kiwi plant or its preparation and agent selected from a steroid, antihistamine, antibody, antibiotic, cyclosporin, fungicide, respiratory function regulators, analgesic, β-agonists, leukotriene modifier, cytokine antagonist, cytokine receptor antagonist , an inhibitor of phosphodiesterase, sodium chromoglycate, nedocrimil, caffeine, theophylline, carbobenzoxy-beta-alanyltaurin and an inhibitor of T-cell function, in the preparation of the composition for treating disease or a pathological condition that is associated with impaired regulation of immune function.

Another embodiment of the present invention relates to the use of a frost-resistant kiwi plant or a preparation thereof and an agent selected from probiotics; cell walls and bacterial fragments; whey protein; alanine; fatty acids; fatty acid esters; monoglycerides; diglycerides; triglycerides; inositol; turmeric curcumin; methylsulfonylmethane (MSM); ginseng; ginger and proanthocyanidins. Fatty acids include, but are not limited to, conjugated linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, γ-linolenic acid, α-linolenic acid, dihomo-γ-linolenic acid, and stearidonic acid.

In one aspect of any of the uses described above, the composition can be used to treat a disease or condition that is associated with the production or activity of leukotrienes. In another aspect of any of the described uses, the disease or condition may include, but is not limited to, atopic dermatitis, asthma, food allergies, allergic rhinitis, and chronic urticaria. In another aspect of any of the uses described, a preparation of a frost-resistant kiwi plant may include, but is not limited to, fruit extract or concentrate, leaf extract or concentrate, stem extract or concentrate, bark extract or concentrate, root extract or concentrate, fresh fruit, crushed fruit, boiled fruit, cooked fruit, squeezed fruit, thickened fruit, dried fruit, juice concentrate of a frost-resistant kiwi plant, a preparation obtained by extracting the fruit in water at room temperature; a preparation obtained by direct extraction of a water-soluble concentrate of a frost-resistant kiwi plant with ethyl acetate, a preparation obtained by extraction of a frost-resistant kiwi plant in distilled water, and a preparation obtained by subsequent extraction in water, chloroform and ethyl acetate.

Another embodiment of the present invention relates to a method for regulating an immune response in a mammal. The method includes the use of a conventional preparation of a kiwi plant in a mammal in an amount sufficient to regulate the immune response in a mammal, where the usual preparation of a kiwi plant is selected from a fruit extract or concentrate, leaf extract or concentrate, stem extract or concentrate, bark extract or concentrate, extract or a root concentrate, fresh fruit, crushed fruit, boiled fruit, cooked fruit, squeezed fruit, thickened fruit, dried fruit, juice concentrate of an ordinary Ki plant and the preparation obtained by extracting the fruit in water at room temperature; a preparation obtained by direct extraction of a water-soluble concentrate of a conventional kiwi plant with ethyl acetate, a preparation obtained by extraction of a conventional kiwi plant in distilled water, and a preparation obtained by sequential extraction in water, chloroform and ethyl acetate.

SHORT DESCRIPTION OF FIGURES

Figure 1 shows the inhibitory effect of various preparations from A. arguta on IgE production in U266B1 cells. The results were calculated as the percentage of IgE produced by U266B1 cells treated only with LPS from three independent experiments.

Figure 2 shows the dose-dependent effect of PG102T and PG102E on the production of IL-4 in OVA-stimulated splenocytes (OVA-egg albumin). The specific activity of PG102T and PG102E was determined from the IC ₅₀ value.

3A-3C depict the effects of PG102T and PG102E on the number of IL-4 or IFN-γ-producing T cells (FIG. 3A) and IgE-producing B cells (FIG. 3B) and IgE biosynthesis in B cells ( figs). Data are average percentages of each population from three independent experiments. *, P <0.05 versus DW-treated mice.

FIGS. 4A-4B show the effect of PG102T and PG102E on the expression of GATA3, T-bet and NFATc2 using Western blot analysis (FIG. 4A) and real-time quantitative PCR (FIG. 4B). Results are presented as mean ± SEM of three independent experiments. *, P <0.05 and **, P <0.01 against DW-treated mice. Β-actin and GAPDH were used as load control.

FIGS. 5A-5B show the effects of PG102T and PG102E on the development of dermatitis in an NC mouse using a dermatitis index (FIG. 5A) and scratching values (FIG. 5B). Values are presented as mean ± SEM for 5-6 animals. *, P <0.05; **, P <0.01, against DW-treated mice.

FIGS. 6A-6C show the effects of PG102T and PG102E on plasma IgE levels (FIG. 6A), IgG1 (FIG. 6B) and IgG2a (FIG. 6C) in NC mice. Values are presented as mean ± SEM over 5 animals. *, P <0.05; **, P <0.01, against DW-treated mice.

FIGS. 7A-7B show the effect of PG102T and PG102E on the number of all white blood cells and eosinophils (FIG. 7A) and the production of eotaxin and TARC (FIG. 7B) in peripheral blood. Values are presented as mean ± SEM over 5 animals. *, P <0.05; **, P <0.01, against DW-treated mice.

On figa-8B shows the effect of PG102T and PG102E on skin lesions in mice NC back skin (figa) and the skin of the facial region (figv). *, P <0.05; **, P <0.01, against DW-treated mice.

On figa-9B shows the effect of PG102T and PG102E on the expression of IL-4, IL-5, eotaxin, TARC, GATA3 and pSTAT6 in skin lesions, measured by ELISA methods (figa) and Western blotting (figv). Values are presented as mean ± SEM over 5 animals. *, P <0.05; **, P <0.01, against DW-treated mice. The amount in the range corresponds to the percentage of activity compared to DW-treated mice.

Figure 10 is a schematic illustration of the method used to produce larger quantities of PG102T; it is a frozen or otherwise dried concentrate of a kiwi plant, also called FD001 (FG refers to the gradation of food carriers).

11 shows the effect of three doses (0.25, 1.0, and 10 mg / ml) of FD001 (PG102T) in relation to the relative degree of cytokine production of IL-4, IL-5, IL-10, IL-13, and IFN-γ γ OVA-stimulated murine splenocytes after 3 days of in vitro exposure. Cytokine levels were measured by ELISA. Each point is represented by an average value, according to data from splenocytes of ten independent mice.

12 shows the effect of three doses (0.25, 1.0, and 10 mg / ml) of ethyl acetate (EtOAc) extract from FD001 (PG102T) on the relative degree of cytokine production of IL-4, IL-5, IL-10, IL -13 and IFN-γ by OVA-stimulated murine splenocytes after 3 days of in vitro exposure. Cytokine levels were measured by ELISA. Each point is represented by an average value, according to data from splenocytes of ten independent mice.

13 shows the effect of three doses (0.25, 1.0, and 10 mg / ml) of A. arguta fetal juice concentrate with respect to the relative degree of cytokine production of IL-4, IL-5, IL-10, IL-13, and IFN-γ OVA-stimulated murine splenocytes after 3 days of in vitro exposure. Cytokine levels were measured by ELISA. Each point is represented by an average value, according to data from splenocytes of ten independent mice.

On figa and 14B shows the effect of three doses of known immunosuppressive compounds on the relative degree of production of IL-13 and IFN-γ OVA-stimulated murine splenocytes after 3 days of exposure in vitro. Cyclosporin was studied at a concentration of 0.0083, 0.083 and 4.15 μM, and dexamethasone was tested at a concentration of 0.01, 0.1 and 1 μM (Fig. 14A), with each point corresponding to the average splenocyte count of ten independent mice. Quercetin was tested at a concentration of 1.0, 10 and 25 μM, where each point corresponds to the average splenocyte value of ten independent mice (Fig.14B). Cytokine levels were measured by ELISA.

FIGS. 15A and 15B show the effect of three doses (1.0, 3.0, and 10 mg / ml) of FD001 (PG102T), ethyl acetate (EtOAc) extract of FD001, and the aqueous residue resulting from this method on the relative degree of IL production -13 (FIG. 15A) and IFN-γ (FIG. 15B) by OVA-stimulated murine splenocytes after 3 days of in vitro exposure. Cytokine levels were measured by ELISA. Each point corresponds to the average splenocyte value of eight independent mice.

On figa and 16B shows the effect of three doses (1.0, 3.0 and 10 mg / ml) FD001 (PG102T) and powder form FD001 (obtained for use in capsules) on the relative degree of production of IL-13 (figa ) and IFN-γ (FIG. 16B) by OVA-stimulated murine splenocytes after 3 days of in vitro exposure. Cytokine levels were measured by ELISA. Each point corresponds to the average splenocyte value of eight independent mice.

On figa and 17B shows the effect of alternative drugs A. arguta on the relative production of IL-13 (figa) and IFN-γ (figv) OVA-stimulated murine splenocytes after 3 days of exposure in vitro. Three doses (1.0, 3.0, and 10 mg / ml) of FD001 (PG102T), a concentrate of fruit juice, an extract obtained by boiling a fresh fetus in water, and an extract of the fetus in water at room temperature were studied. Cytokine levels were measured by ELISA. Each point corresponds to the average splenocyte value of eight independent mice.

FIGS. 18A and 18B show the effect of preparations of alternative plant parts of A. arguta on the relative production of IL-13 (FIG. 18A) and IFN-γ (FIG. 18B) by OVA-stimulated murine splenocytes after 3 days of in vitro exposure. Three doses (1.0, 3.0 and 10 mg / ml) of the fruit juice concentrate, independent extracts obtained by cooking the bark, root or stem in H ₂ O, and FD001 were investigated. Cytokine levels were measured by ELISA. Each point corresponds to the average splenocyte value of eight independent mice.

On figa-19C shows the shift in the changes that occurred between 1 and 14 days of clinical trials in humans, during which the subjects responded positively to the treatment of atopic dermatitis (AD), according to PGA (General Medical Assessment) estimates for AD (classification criteria are shown on figs). Subjects used a placebo or 600 mg FD001 (PG102T) daily (FIGS. 19A and 19B, respectively) and used local steroid treatment at the same time.

DETAILED DESCRIPTION OF THE INVENTION

Previously, the inventors found that a frost-resistant kiwi plant, and in particular some extracts / concentrates derived from it, commonly referred to as PG102 in this document, increase serum levels of Th1-cytokines and IgG2a, reduce serum levels of Th2-cytokines and IgE the release of histamine from mast cells and suppress allergic inflammatory reactions that occur in an allergen-sensitized mouse model of allergic inflammation and hypersensitivity of the respiratory tract, as well as in the study of edema rat paw (see US patent publication №2004 / 0037909, supra). PG102 is active when administered orally.

The inventors of the present invention provided new evidence that two specific extracts derived from Actinidia arguta, designated PG102T and PG102E (described in detail in Examples 1 and 2 below), inhibit IgE production and are also able to regulate selective Th1 and Th2 cytokines. Most likely, this regulation is achieved by regulating cellular transcription factors, GATA-3, T-bet, and NFATc2. The data of the inventors indicate the significant potential of PG102T and PG102E as immunomodulators of Th1 and Th2 pathways, and ultimately, as antiallergic agents. The effectiveness of these extracts is shown herein in vivo in a model for allergic inflammation associated with respiratory diseases and in a model of atopic dermatitis.

The authors of the present invention also described here other preparations of the frost-resistant kiwi plant, including preparations from the whole fruit, stem, root, bark, new extracts, concentrates, juices, dry preparations and unextracted preparations, and showed that such preparations of the frost-resistant kiwi plant have similar properties by their ability to regulate Th1 and Th2 cytokines and, therefore, are believed to have similar antiallergic properties.

In addition, the inventors have shown that preparations of a frost-resistant kiwi plant according to the invention reduce leukotriene levels and weaken the production of leukotrienes in vivo, and therefore, the compositions of the invention can be used to treat leukotriene-mediated diseases, including, but not limited to atopic dermatitis, asthma, food allergies, allergic rhinitis and chronic urticaria.

The identification of the frost-resistant kiwi plant and its preparations as enhancers of the Th1 response makes these agents particularly attractive for the treatment of diseases and pathological conditions in which a positive reaction to such a response is observed, including, but not limited to, viral infection and cancer.

The extraction process used to obtain the total amount of a water-soluble extract and an ethyl acetate extract of a frost-resistant kiwi plant is described in US patent publication No. 2004/0037909 and in examples 1 and 2 of this application. In further embodiments, the inventors have found that from preparations of a frost-resistant kiwi plant obtained by alternative methods of extraction, concentration or processing, it is also possible to obtain compositions with immune regulatory activity, and in particular, with the ability to suppress cytokine production in response to an antigen (e.g. allergen). Such alternative formulations include, but are not limited to, fruit juice concentrate, fresh fruit concentrate, and boiled fresh fruit preparations.

The authors of the present invention in this document also showed that extracts or other preparations from parts of the plant of the frost-resistant kiwi plant, but not the fetus itself, have equivalent or greater immunoregulatory activity compared to water-soluble or ethyl acetate extracts from the fetus. For example, the inventors have now shown the effectiveness of extracts from the stem, root and bark of a frost-resistant kiwi plant in inhibiting the production of cytokines by antigen-stimulated splenocytes in allergen-sensitive mice.

The authors of the present invention also unexpectedly found that the preparations of the frost-resistant kiwi plant, as described herein, can serve as an effective complement to other types of therapy for various atopic conditions, including, but not limited to steroid therapy. For example, the inventors have found that the use of a powdered form of a water-soluble extract of A. arguta, as described herein, by adult patients suffering from moderate atopic dermatitis, significantly reduced the manifestation of characteristic clinical signs. A significant decrease in the specific clinical symptom assessed by the patient himself (redness) was also achieved, and a tendency to a decrease in the severity was observed in relation to other clinical symptoms of the disease in those patients who simultaneously used a local steroid, compared with patients using a steroid, but not receiving extract of a frost-resistant kiwi plant. When the patient stopped using steroids, the effects of the extract of the kiwi plant were no longer so significant in a small experimental study. Therefore, the invention in this embodiment relates to the use of the preparations of the frost-resistant kiwi plant described herein in combination with (or in addition to) other therapeutic agents for treating atopic disease or other diseases associated with immune dysregulation disorders. The inventors believe that the compositions according to the invention can be used to increase the effectiveness of other types of health-improving and diet therapies, especially in patients with atopic conditions. This embodiment of the invention is discussed in detail below.

In yet another embodiment, the inventors unexpectedly discovered that the drying process of a frost-resistant kiwi plant is a previously unappreciated but important element associated with increasing the bioactivity of a frost-resistant kiwi plant. In addition, the inventors have shown here that a dried frost-resistant kiwi plant, extracted exclusively with water at room temperature, exhibits similar bioactivity to extracts of a frost-resistant kiwi plant in hot water or an organic solvent. This can also include a dried frost-resistant kiwi plant, extracted in cool or cold water, thus performing extraction at any temperature from 0 ° C to 80 ° C. The inventors also suggested that a dried frost-resistant kiwi plant that was not extracted (for example, dried slices of a frost-resistant kiwi plant) may have bioactivity of extracts of a frost-resistant kiwi plant. Therefore, the present invention relates to any form of a dried frost-resistant kiwi plant, including extracts obtained from a previously dried frost-resistant kiwi plant, as an agent or to prepare a composition for regulating the immune response in mammals. More specific uses of this agent or composition are described below.

In another embodiment, the invention further relates to the use of any members of the family Actinidiaceae, and especially any members of the genus Actinidia, including, but not limited to, a simple kiwi plant, known as A. chinensis or A. deliciosa, for the preparation of immunoregulatory kiwi plant compositions activity. In one embodiment, the authors, not wishing to be bound by any theory, suggested that by drying other Actinidia plants, a dried Actinidia plant composition having at least several biological activities that have been identified in the frost-resistant kiwi plant described herein can be obtained . In another embodiment, the inventors suggested that other preparations of a simple kiwi plant (e.g., A. chinensis or A. deliciosa), including, but not limited to, preparations from any part of the fetus, whole fruit, stem, foliage, bark or root, including any preparation or extract thereof, including dried preparations, unextracted but processed preparations, fresh fruit, fruit juice, or any extract, concentrate or part thereof, may have at least several biological properties that have been identified as frost-resistant kiwi plants described in this document.

A. arguta, A. polygama, and A. kolomikta, belonging to Actinidiaceae, are wildly distributed in Siberia, northern China, North and South Korea. More than 30 species belonging to Actinidiaceae have been reported. These include the fruit A. chinensis or A. deliciosa, which were called "kiwi" and are common edible fruits. A. arguta and another fetus of the same genus (e.g. A. polygama and A. kolomikta) were used as Chinese medicine materials called 'mihudo' to treat liver disease, gastrointestinal disease and urogenital stone formation without signs of toxicity (Seoul National University Natural Products Science, Tradi-Medi Data Base, dongbang media Co. Ltd. 1999). However, prior to the invention described in US Patent Publication No. 2004/0037909, there were no reports or suggestions regarding the treatment and prevention of allergic disease and non-allergic inflammatory disease using the Actinidia fetus. In addition, prior to the present invention, there were no reports examining the effectiveness of preparations of a frost-resistant kiwi plant, except for the extracts described in US patent publication No. 2004/0037909, considering the effectiveness of extracts obtained from parts of a frost-resistant kiwi plant, with the exception of fruits (berries), considering the importance of the various stages of the preparation process, such as drying, or the effect of the compositions of the frost-resistant kiwi plant on the effectiveness of conventional treatment for atopic disease and or other pathological conditions associated with dysregulation of the mammalian immune system (e.g., steroid therapy).

According to the present invention, a reference to a “frost-resistant kiwi plant” refers to any of A. arguta, A. polygama and A. kolomikta or other related species of the genus Actinidia that have the bioactive properties of A. arguta, A. polygama and A. kolomikta, as described herein, especially with respect to antiallergenic properties and the immune response / cytokine / leukotriene-regulatory properties shown here (for example, see examples).

In preparing any composition or preparation of the invention, including any extract described herein, any one or more parts of a frost-resistant kiwi plant or other Actinidia species can be used, including, but not limited to, their fruits (also called “berries” or “kiwi berries "), leaves, stems, bark and root.

Throughout this document, a general reference to an extract refers to a concentrated preparation from a substrate (for example, a frost-resistant kiwi plant), usually obtained by extracting the active or desired components from it with a suitable solvent, and then by evaporating part, all or almost all of the solvent and adjusting the residual mass or powder to the prescribed standard. The term "concentrate" refers to a form of substrate in which most of its base components, or solvent, are removed. Accordingly, it is obvious that the term "extract" can be used in some embodiments as an equivalent of the term "concentrate". Herein, a reference to a crude extract in one embodiment relates to an extract of a frost-resistant kiwi plant, which is obtained by extracting a preparation of a frost-resistant kiwi plant with water, lower alcohols (e.g. methanol, ethanol, etc.) or mixtures thereof, and preferably, distilled water or 50-90% ethanol, and more preferably 70% ethanol. The non-polar solvent soluble extract can be obtained by further extracting the soluble extract with a non-polar solvent such as hexane, ethyl acetate or a dichloromethane solvent. Specific methods for preparing the crude extract and evaluating the above are described in US Patent Publication No. 2004/0037909, supra, and the like. methods are incorporated herein by reference. Additional bioassays for the regulation, evaluation, or approval of the desired biological activities of the composition of the frost-resistant kiwi plant according to the invention are provided in the examples herein and include both in vitro and in vivo studies.

According to the present invention, the reference to "PG102T" usually refers to the total water-soluble extract (which may also be referred to herein as a concentrate) from the frost-resistant kiwi plant described herein (eg, A. arguta), prepared essentially as described in the patent US publication No. 2004/0037909, above (for example, see example 1 from this publication), or as described in example 1 of this description. In a preferred embodiment, the total water-soluble extract is obtained from A. arguta, although, as is obvious to a person skilled in the art, equivalent total water-soluble extracts can be obtained from another frost-resistant kiwi plant, including, but not limited to, A. polygama and A. kolomikta. If the total water-soluble extract is obtained by a macro process, but using basically the same basic steps as for obtaining PG102T, as described in example 3, then the resulting preparation will be indicated in this document as FD001. Reference to "PG102T" herein refers to the ethyl acetate fraction obtained by sequential extraction of the PG102T preparation described above in chloroform, ethyl acetate and n-butanol using extraction methods known in the art. Specific methods for producing extracts that are PG102T extract and PG102E extract are described in Example 1. In another embodiment, a different ethyl acetate extract was obtained by direct extraction of FD001 (or PG102T) with ethyl acetate (i.e. there was no extraction with chloroform to the ethyl acetate fraction). Again, in a preferred embodiment, an ethyl acetate extract was obtained from A. arguta, although, as is obvious to a person skilled in the art, equivalent extracts can be obtained from another frost-resistant kiwi plant, including, but not limited to, A. polygama and A. kolomikta.

In some embodiments, especially when the fetus is dried as a processing step or to obtain an extract or concentrate or other preparation, extracts or concentrates or other preparations according to the invention can be obtained from any type of Actinidia. Accordingly, this is an object of the present invention, including a medicament, cosmetic composition or composition suitable as or in conjunction with a healthy food, healthy food or drink product, or nutritional supplement (including human and animal (including pet) nutritional supplements) that include, if necessary, such extracts of the frost-resistant kiwi plant or other Actinidia species. Such compositions are intended for use in any method of the present invention, including the selective regulation of Th1 and Th2 immune responses in a patient (i.e., mammals), such as a method of preventing or treating an allergic and non-allergic inflammatory disease or providing an antiviral or anticancer drug or nutraceutical by applying such compositions. Other additives and components from the compositions, as well as the dose and application strategies described herein, also relate to this aspect of the invention.

In the preparation of any of the compositions described herein, in addition to the extracts described herein, the inclusion of extracts / concentrates specifically described above, included in the invention, is the use of whole fruits of a frost-resistant kiwi plant or fruit preparations that process but do not extract, including, not limited to, fresh fruit, crushed fruit (dried or fresh), boiled fruit (dried or fresh), boiled fruit, dried fruit, squeezed fruit, frozen fruit and thickened fruit. Accordingly, this is the purpose of the present invention to obtain a composition, including a medicinal product, a cosmetic composition or a composition suitable as or in conjunction with a healthy food product, healthy food or drink, or a nutritional supplement that contains whole fruits of the frost-resistant kiwi plant or fruit preparations which can be processed in several ways (for example, dried, boiled and others), but which were not necessarily extracted. Therefore, in one embodiment, the present invention relates to preparations of a frost-resistant kiwi plant (and a simple kiwi plant) that have not been extracted. Any such compositions described herein are intended for use in any method of the present invention, including for the selective regulation of Th1 and Th2 immune responses in a patient (i.e., a mammal), such as a method for the prevention or treatment of allergic and non-allergic inflammatory diseases or the provision of antiviral or anticancer pharmaceuticals or nutraceuticals by the use of such compositions. Other additives and components of the compositions, as well as the dosage and application strategies described herein, are also used for the purposes of the present invention.

Furthermore, in preparing any composition described herein, in addition to the extracts described herein, the present invention also includes the use of the juice of a frost-resistant kiwi plant obtained from any part of the frost-resistant kiwi plant and by any suitable method. Juice can be used as obtained directly from the fruit (i.e. undiluted or concentrated), juice can be diluted or concentrated to form a fruit juice concentrate. For example, as described in Example 3, a fresh kiwi plant can be passed through a conventional juice squeezer. A device for squeezing juice can remove the skin from the fruit, which leads to the mixing of seeds, pulp and juice. This mixture can be processed (for example, by centrifugation and pressing) to remove the juice from the solid mass and, if necessary, this juice can be concentrated (for example, by evaporation, distillation, ultrafiltration, etc.) to obtain a concentrated fruit juice (i.e. fruit juice concentrate ) Such compositions are intended for use in any method of the present invention, and especially in a method for the prophylaxis or treatment of an allergic and non-allergic disease or the preparation of antiviral or anti-cancer pharmaceutical preparations or nutraceuticals by the use of such compositions. Other additives and components of the compositions, as well as the dosage and application strategies described herein, are also used for the purposes of the present invention.

In addition, in the preparation of any composition described herein as an alternative to the extracts described in US Patent Publication No. 2004/0037909, the invention also includes the use of other processed products of a frost-resistant kiwi plant, including, but not limited to, fruit extract in water at room temperature, including dried fruit; water extract of a frost-resistant kiwi plant obtained in water at a temperature lower than room temperature; an aqueous extract or other extract of a root, leaf, stem or bark; or an aqueous extract or concentrate or other extract of any fruit, leaves, stem or bark that is not dried prior to extraction (for example, extractable fresh fruit). The authors of this invention suggest in this document that a frost-resistant kiwi plant can be extracted at any water temperature, from 0 ° C to 80 ° C, including room temperature and cooler (for example, from about 0 ° C to about 25 ° C).

According to the present invention, a generic reference to a “dried frost-resistant kiwi plant” or “dried kiwi plant” includes any form of a frost-resistant kiwi plant or other kiwi plant (eg, ordinary kiwi) that is dried by any method. The term “kiwi plant” can be used herein to refer to any member of the genus Actinidia in general, and includes members of the frost-resistant kiwi plant, as discussed above, as well as members of the ordinary kiwi, as discussed above. Therefore, a dried kiwi plant includes any dried part of a kiwi plant (fruit, leaves, stem, root, etc.) and includes dried whole fruits, dried chopped fruit, dried chopped fruit, dried chopped fruit, and dried condensed fruit, and also any extracts of the kiwi plant where the extracted material is first dried and then subjected to extraction. The extracts did not need to be dried or further processed, although this is usually the most useful form of extracts for formulation and storage. Preferred methods for further concentrating the extracts for future use include, but are not limited to, thickening, distillation, ultrafiltration, reverse osmosis, precipitation, adsorption and elution from the stationary phase, and extraction in alternative solvents. Preferred methods for drying extracts or concentrates for later use include, but are not limited to, drying in trays, spray drying and freeze-drying, and with or without using drying apparatus or fillers such as maltodextrin, microcrystalline cellulose and starch. In one embodiment, a preferred dried kiwi plant for use in the present invention was a dried kiwi plant preparation that was not further extracted.

Thus, the compositions and methods described herein are used to use any frost-resistant kiwi plant, including the use of any part of the fruit, stem, leaves, bark or root of the frost-resistant kiwi plant, or any extract or concentrate or fraction thereof, any form of the whole fruit or a processed, but not extracted fruit, fruit juice, or any extract or concentrate or a fraction thereof, and further including parts of the plant of the frost-resistant kiwi plant (parts of the plant, including the fruit, stem, leaves, bark and / Whether root) and preparations of plant parts which had been prepared using a process which includes a drying step.

Thus, it is an object of the present invention to provide a composition comprising a drug, nutraceutical, nutritional supplement, healthy food (including drink or nutrient), or a cosmetic composition comprising essentially consisting or consisting of a frost-resistant kiwi plant described herein (i.e. including any part of the fruit, whole fruit, stem, leaves, bark or root, and including any preparation or extract, or their concentrate, including dried preparations, unextracted but processed preparations, with fresh fruit, fruit juice or any extract, concentrate or their fraction). In one embodiment, the present invention relates to a crude extract, a general water-soluble extract, or an ethyl acetate extract of a frost-resistant kiwi plant. In all cases, the preparation of the frost-resistant kiwi plant is an active ingredient to use to selectively regulate the Th1 and Th2 immune responses in a patient (i.e., a mammal). In particular, the composition has a biological activity selected from at least one of the following activities: (a) reduces the number of IgE-producing B cells in a patient; (b) reduces the amount of IgE produced in a patient (e.g., in serum or plasma); (c) reduces the production and / or levels of at least one Th2 cytokine (e.g., IL-4, IL-5, IL-10); (d) increases the level of at least one Th1 cytokine (e.g., IL-12, IFN-γ); (e) reduces the expression level of the transcription factor, GATA-3; (f) increases the expression level of the transcription factor T-bet; (g) increases the expression level of the transcription factor NFATc2; (h) increases the number of IgG2a-producing B cells in a patient; (i) increases the amount of IgG2a produced in a patient; (j) increases the production or activity of Th1 T lymphocytes (e.g., CD4 +, IFN-γ +), especially in the area of inflammation; (k) reduces the production or activity of Th2 T lymphocytes (e.g., CD4 +, IL-4 +), especially in the area of inflammation; (l) reduces the number of IgG1-producing B cells in a patient; (m) reduces the amount of IgG1 produced in a patient; and / or (n) reduces the level of production of at least one leukotriene in a patient.

The compositions described above that can be used to prevent and / or treat any disease or condition in which the regulation of the immune response by the method described herein would be or are likely to be beneficial to the patient.

Here, the phrase “protected from disease” refers to a reduction in symptoms of the disease; reduction of cases of disease and / or reduction of the risk of disease. Patient protection may relate to the ability of a composition of the invention, when administered to a patient, to prevent the occurrence of a disease and / or to cure or alleviate at least one, and preferably more than one symptom of the disease, symptom or cause. Actually, protecting a patient from a disease includes preventing a disease (preventive treatment), and treating a patient having a disease (therapeutic treatment) to reduce symptoms of the disease. In particular, protecting a patient from a disease or enhancing another type of treatment is carried out by regulating this activity, with a positive effect. The beneficial effect can be easily assessed by an ordinary specialist in the field and / or an experienced clinician who treats a patient. The term "disease" refers to any deviation from the normal state of health of a mammal and includes a condition in which there are symptoms of the disease, as well as conditions in which there are deviations (for example, infection, gene mutation, genetic defect, etc.) but the symptoms are still not showing.

Typically, the biological activity or biological effect of an agent described herein, including a composition of a frost-resistant kiwi plant, an extract of a frost-resistant kiwi plant, or any other preparations thereof, refers to any function exhibited or performed by an agent to which is attributed a natural origin, as defined in vivo (i.e., in natural physiological environment where the agent is used) or in vitro (i.e. in the laboratory). Modifications of the agent, such as changing the processing or preparation of the agent or purifying the agent, can lead to the formation of agents having similar biological activity as a naturally occurring agent, or agents having a reduced or increased biological activity compared to a natural origin agent.

Accordingly, it is an object of the present invention to provide a composition comprising a medicament or nutraceutical (nutritional) composition, including the frost-resistant kiwi plant described herein (i.e., including any part of the fruit, whole fruit, stem, leaves, bark or root, and including any preparation or extract or its concentrate, including dried preparations, not extracted but processed preparations, fresh fruits, fruit juice or any extract or concentrate or their fraction), and in one embodiment, wasp the crude extract, the total water-soluble extract or ethyl acetate extract of the frost-resistant kiwi plant (which can be attributed both to the active components according to the invention and to preparations of the frost-resistant kiwi plant according to the invention), as an active component for regulating the immune response in a mammal, and more specifically , for regulating the immune Th2 and / or Th1 response in a mammal, and even more specifically, for increasing the Th1 response in a mammal and / or suppressing the Th2 response in a mammal. Such active ingredients are useful for the treatment and / or prevention of various pathological conditions and diseases, including, but not limited to, allergic disease, non-allergic inflammatory disease, viral infection and cancer. The composition may further include or be used in combination with additional therapeutic or nutraceutical agents for the prevention, treatment and / or improvement of any of the above conditions or diseases. Dietary uses of the present invention include any use of the invention aimed at providing nutrients and nutritional agents for maintaining, stabilizing, increasing, enhancing or improving the health of an individual or organic process by which the body adapts and uses food and fluids to function, grow and maintenance, and which include the use of nutraceuticals. Therapeutic uses include any use of the invention for the prevention, treatment, management, cure, alleviation and / or healing of a disease or condition, that is, an abnormality in an individual’s state of health. Other uses of the invention include, for example, cosmetic use.

It is also an object of the present invention to provide a frost-resistant kiwi plant described herein (i.e., a preparation of a frost-resistant kiwi plant containing any part of a fruit, whole fruit, stem, leaf, bark or root, and including any preparation or extract or concentrate thereof, including dried non-extracted but processed preparations, fresh fruits, fruit juice or any extract or concentrate or fractions thereof), and in one embodiment, the crude extract, the total water-soluble extract or ethyl a cetate extract of a frost-resistant kiwi plant, to obtain a therapeutic or nutraceutical agent for regulating the immune response in a mammal, in particular for regulating the immune Th2 and / or Th1 response in a mammal, and even more specifically, to increase the Th1 response in a mammal and / or suppression of the Th2 response in a mammal. Such active ingredients are useful for treating and / or preventing a variety of conditions and diseases, including, but not limited to, allergic disease, non-allergic inflammatory disease, viral infection, and cancer. The agent can be used in combination with additional therapeutic or nutraceutical agents to prevent, treat and / or improve any of the above conditions or diseases.

Another objective of the present invention is the provision of dietary food products or nutritional supplements containing a frost-resistant kiwi plant described herein (i.e., a preparation of a frost-resistant kiwi plant, including any part of a fruit, whole fruit, stem, leaf, bark or root, and including any preparation or extract or a concentrate thereof, including dried preparations, not extracted but processed preparations, fresh fruits, fruit juice or any extract or concentrate, or a fraction thereof), and in one embodiment, a raw extract, a general water-soluble extract or ethyl acetate extract of a frost-resistant kiwi plant, together with any suitable additive for regulating the immune response in a mammal, and more specifically for regulating the immune Th2 and / or Th1 response in a mammal, and even more specifically, to increase the Th1 response in a mammal and / or suppress the Th2 response in a mammal. Such dietary foods or dietary supplements are useful for treating and / or preventing various pathological conditions and diseases, including, but not limited to, allergic disease, non-allergic inflammatory disease, viral infection, and cancer.

Another objective of the present invention is the provision of animal food or a feed supplement comprising a frost-resistant kiwi plant described herein (i.e., a preparation of a frost-resistant kiwi plant, including any part of the fruit, the whole fruit, stem, leaves, bark, or root, including any preparation or extract or concentrate thereof, including dried preparations, not extracted but processed preparations, fresh fruits, fruit juice, or any extract or concentrate, or a fraction thereof), and in one embodiment, cheese extract, total water-soluble extract or ethyl acetate extract of the frost-resistant kiwi plant, as an essential component for regulating the immune response in a mammal, in particular for regulating the Th2 and / or Th1 immune response in a mammal, more specifically, to increase the Th1 response in a mammal and / or suppression of the Th2 response in a mammal. Such animal food or animal feed additives are useful for the treatment and / or prevention of various pathological conditions and diseases, including, but not limited to, allergic disease, non-allergic inflammatory disease, viral infection and cancer.

Another objective of the present invention is the provision of a local or cosmetic composition comprising the frost-resistant kiwi plant described herein (i.e., preparation of a frost-resistant kiwi plant, including any part of the fruit, whole fruit, stem, leaves, bark or root, and including any preparation or extract or a concentrate thereof, including dried preparations, not extracted but processed preparations, fresh fruits, fruit juice or any extract or concentrate or fraction thereof), and in one embodiment, a crude extract, sum a water-soluble extract or ethyl acetate extract of a frost-resistant kiwi plant, for regulating the immune response in a mammal, in particular for regulating the Th2 and / or Th1 immune response in a mammal, in particular for increasing the Th1 response in a mammal and / or suppressing the Th2 response in a mammal. Such cosmetic compositions are useful for the treatment and / or prophylaxis of various pathological conditions and diseases, including, but not limited to, allergic disease (including allergic diseases or skin lesions), non-allergic inflammatory disease, viral infection and cancer.

Any compositions, additives or agents described herein may further include at least one conventional carrier, excipient or solvent. For example, the composition according to the invention may include pharmaceutically suitable carriers, excipients or solvents, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , cellulose, methyl cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. The formulations may further include fillers, anti-adhesive agents, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, and the like. Compositions according to the invention can be formulated in such a way as to provide quick, supportive or delayed release of the active component after their administration to the patient.

For example, the compositions of the invention may be dissolved in oils, propylene glycol or other solvents that are commonly used to prepare an injection. Suitable examples of carriers include, but are not limited to, physiological saline, polyethylene glycol, ethanol, vegetable oils, isopropyl myristate, and the like. For topical use, the compounds of the invention can be formulated as ointments and creams.

The compositions or formulations according to the invention can be obtained in any form, such as an oral dosage form (effervescent tablet, effervescent powder, powder, tablet, capsule, harmless capsule, aqueous medicine, syrup, elixir pill, powder, sachet, granule) or topical preparation (cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol, etc.), or an injectable preparation (solution, suspension, emulsion).

The pharmaceutical compositions of the invention may be used alone or in suitable combination with other pharmaceutically active compounds, including anti-inflammatory compounds, anti-allergic compounds, or any other compounds or compositions that can regulate the immune response or may have a beneficial effect on the patient. Compounds that are particularly desirable for use in the compositions and formulations of the present invention are described in detail herein.

The composition according to the invention can also provide as dietary food products, which include the preparation of a frost-resistant kiwi plant according to the invention (for example, various foods, drink, chewing gum, vitamin complex, healthy, improved food, etc.). Dietary foods can provide as a component of food, powder, granule, tablet, chewable tablets, capsules or drink and so on. Baby or infant food is also included in the compositions according to the invention, such as modified milk powder, infant formula and modified infant or infant food.

Appropriate food products in which the composition or agent of the invention can be included to produce a dietary food product include, but are not limited to, thin baked goods, bread and rolls, breakfast cereal, processed and unprocessed cheese, seasonings (ketchup, mayonnaise, and etc.), dairy products (milk, yogurt), puddings and gelatin desserts, carbonated drinks, teas, powdered drinking mixtures, processed fish products, fruit-based drinks (including fruit juices), vegetable-based drinks (including wax juices), chewing gum, hard confectionery, frozen dairy products, processed meat products, nuts and nut pastes, pasta, processed poultry products, gravy and sauces, potato chips and other chips or crisps, chocolate and other confectionery (cookies, sweets, licorice sweets), ice cream, dehydrated foods, sliced or processed foods (e.g. fruits, greens), spices, spirits, noodles, fermented foods, soups and soup mixes, soy products Ukta (milk, drinks, creams, clarifiers), vegetable oil based pastes, and vegetable-based drinks. The composition according to the invention can also be used in food in the form of internal food additives, external coatings or food-miscible additives directly during serving.

The above compositions, in particular cosmetic compositions containing these compositions, can be prepared in any form, such as coating, lotion, cream, essence, tonic, emulsion, mask, soap, shampoo, detergent, cleanser, body wash solutions , washing solutions, treatment, gel, balm, spray solution, etc.

Any of these compositions according to the invention may further include one or more components such as lactose, casein, dextrose, glucose, sucrose and sorbitol.

Any compositions, preparations, additives or agents described herein may further include at least one active agent (i.e., active compound, active component). The additional active agent may be a pharmacologically active agent and / or a nutritionally active agent. Active agents generally contribute to at least one additional desirable, nutritional and / or therapeutic, and / or pharmacological property of the composition, in addition to the kiwi plant preparation described herein. Active agents may be included in any effective amount in a mixture, preparation, supplement or other composition of the invention. An effective amount is an amount sufficient to effect the desired effect imparted by the agent, such as affecting an individual’s health or nutrition (e.g., therapeutic or nutritional effect), taste effect, flavoring effect, visual effect, and the like. The person skilled in the art is able to determine the appropriate amount of additional agents to be added to the composition according to the invention.

For example, any compositions related to or used in the present invention may include one or more natural products as an active agent, including, but not limited to, fatty acids and polyketides; organic acids and mixed small organic compounds; aromatic amino acids and phenylpropanoids; terpenoids and steroids; alkaloids; corrins and porphyrins; linear and cyclic peptides, depsipeptides and other derivatives of amino acids; nucleosides and nucleotides; carbohydrates; proteins, cells and cell fragments; herbal preparations and spices; minerals; sterilizers; seasonings; vitamins; electrolytes and other natural agents.

Other components (compounds or agents) that can be added to the composition according to the invention include synthetic flavoring agents, coloring agent, processing agent, alginic acid or its salt, organic acids, protective colloidal binders, pH controlling agent, stabilizer , a preservative, glycerin, alcohol, aerated agent, or any other necessary food or drink agent of the composition (for the nutritional or therapeutic use of any method, and I).

Particularly preferred components (active agents) for combining with a preparation of a frost-resistant kiwi plant according to the invention or adding to a composition containing such a preparation include, but are not limited to, probiotics; cell walls and bacterial fragments; whey protein; taurine; alanine; fatty acids (e.g. conjugated linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, γ-linolenic acid, α-linolenic acid, dihomo-γ-linolenic acid, stearidonic acid); mono-, di- and triglycerides (making up any compound of these fatty acids); inositol; turmeric; curcumin; rosemary; rosmarinic acid; methylsulfonylmethane (MSM); ginseng; ginger; proanthocyanidins; β-carotene; and any other preparation of various types of kiwi plants when used as the first bioactive component, including any part of Actinidiaceae, especially any parts of a plant of the genus Actinidia, including common kiwi species (e.g. A. chinensis or A. deliciosa) and frost-resistant kiwi plants ( e.g. A. arguta, A. polygama, and A. kolomikta).

Fatty acids and polyketides include, but are not limited to, saturated fatty acids (e.g., α-lipoic acid (R, S or R, S); unsaturated fatty acids (e.g., conjugated linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, γ- linolenic acid, α-linolenic acid, dihomo-γ-linolenic acid, stearidonic acid); fatty acid esters; monoglycerides, diglycerides and triglycerides (made up of any combination of the above fatty acids); acetylene fatty acids; fatty acids with azvetvlennoy chain; prostaglandins, thromboxanes, leukotrienes, aromatic polyketides, macrolides and polyethers; lipoid extracts (e.g., marine lipids, Echium oil, borage oil, olive oil), and lecithin.

Organic acids and mixed small organic compounds include, but are not limited to, citric acid; fumaric acid; guaiacol; methylsulfonylmethane (MSM); and ascorbic acid.

Aromatic amino acids and phenylpropanoids include, but are not limited to, aromatic amino acids and benzoic acids (for example, benzoic acid, gallic acid, gentisic acid, p-hydroxybenzoic acid, protocatechuic acid, vanillic acid, salicylic acid, lilac acid); cinnamic acids (e.g., hydroxytyrosol, curcumin, rosmarinic acid, αr-turmerone, caffeic acid, eugenol, chlorogenic acid, neochlorogenic acid, cinnamic acid, feralic acid, o-coumaric acid, p-coumaric acid); lignans and lignin; phenylpropylene; coumarins; styrylpyrones; flavonoids (for example, anthocyanidins such as dolphinidin; proanthocyanidins; catechins such as catechin, epicatechin and theaflavin; flavonols such as avicularia, hyperosides, quercetrin, isocvercitrin, caempferol, myricetin, rutin such as flavanones; flavonones such as flavanones; phloretin; isoflavones such as vitexin); stilbene; flavonolignans; isoflavonoids; and terpenoidquinines (e.g., vitamins K and tocopherols (Vitamin E) such as tocotrienols).

Terpenoids and steroids include, but are not limited to, monoterpenes (for example, β-pinene, borneol, carvacol, geraniol, thymol, 1,8-cineole, terpineol); iridoids (e.g. monotrophein); β-ionone (for example, a thirteen-carbon precursor for vitamins A); sesquiterpenes (e.g. caryophyllene, farnesol); diterpenes (e.g., vitamins A); sesterpens; triterpenes (e.g., α-amyrin, lipole, ursolic acid); tetraterpenes; carotenoids (e.g. lycopene, β-carotene, lutein, astaxanthin, canthaxanthin); and steroids (e.g. vitamins D, β-sitosterol).

Alkaloids include, but are not limited to, pyrrolidine alkaloids, tropane alkaloids, pyrrolizidine alkaloid, piperidine alkaloids guinolizidinovye alkaloids indolizidinovye alkaloids, pyridine alkaloids, phenylethylamines, tetrahydroisoquinoline alkaloids, galantamine, indole alkaloids, β-carboline alkaloid, terpenoidindolovye alkaloids, quinoline alkaloids , pyrroloindole alkaloids, ergot alkaloids, quinazoline alkaloids, quinoline and acridine alkaloids, imizadol alkaloids, piperis ynoic alkaloids Ephedrines kapsaysiny, piridinmonoterpenovye alkaloids, aconitine, steroid alkaloids, purine alkaloids (e.g., allantoin, caffeine, theophylline).

Corrins and porphyrins include, but are not limited to, B vitamins.

Linear and cyclic proteins, depsipeptides, and other amino acid derivatives include simple amino acids and their derivatives (e.g., L-acetylcarnitine, choline, taurine, alanine), linear peptides, cyclic peptides (e.g., cyclosporins), cyclic depsipeptides, β-lactams, cyanogenic glycosides, glucosinolates, cysteine sulfoxides, but are not limited to.

Carboxylates include, but are not limited to, monosaccharides (e.g., inositol), polysaccharides (e.g., fruit oligosaccharides, such as inulin (of any chain length); galacto-oligosaccharides; chitin and chitosan).

Other natural substances include, but are not limited to, proteins (eg, whey protein and superoxide dismutase); cells and cell fragments (e.g., probiotics, meaning living, untouched microorganisms, such as, for example, Lactobacillus spp., bacterial cells and cell wall fragments, fungal / yeast cells and cell wall fragments); herbal preparations and spices (e.g. ginseng, huang, turmeric, rosemary, ginger); minerals (e.g. K, Mg, Ca, Mn, Fe, Cu, Zn, B, Si, Se). Metabolites and derivatives of any of these compounds are also included in the present invention.

In one embodiment of the invention, the composition of the invention is used as adjunctive therapy for conventional therapy of a pathological condition or disease. For example, the inventors have shown that the preparation of a frost-resistant kiwi plant in accordance with the present invention improves the outcome of the disease in patients with atopic dermatitis when used as an adjunct to local steroid therapy. Thus, the compositions of the invention may include one or more therapeutic agents (eg, drugs), which may also be referred to herein as active agents used to treat a pathological condition or disease that can be treated or improved by regulating immune responses. Such therapeutic agents include, but are not limited to, steroids (including corticosteroids, including oral, inhaled, and injectable), antihistamines (of any type, including systemic, local, inhaled, and including H1 and H2 blockers), antibodies ( e.g. anti-IgE, anti-IL-10), antibiotics, cyclosporins, antimycotics, regulators of respiratory function, analgesics, β-agonists (long or short-term), leukotriene modifiers (receptor inhibitors or antagonists), cytokine or qi antagonists tocin receptor, phosphodiesterase inhibitors, sodium chromoglycate, nedocrimil, theophylline, caffeine, carbobenzoxy-beta-alanyl-taurine, T-cell function inhibitors and other anti-inflammatory agents.

Any of the above compositions may additionally include one or two or more organic acids (e.g., citric acid, fumaric acid, adipic acid, lactic acid, malic acid, ascorbic acid), phosphate (e.g. phosphate, sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate) and / or natural antioxidants (e.g. polyphenol, catechin, α-tocopherol, rosemary extract, vitamin C, green tea extract, licorice root extract, chitosan, tannic acid, phytic acid, etc.).

The compositions of the invention, in particular cosmetic compositions or compositions that are formulated for topical administration, including therapeutic compositions (but not limited to, cosmetic or other topical compositions) may include additional additives, including, but not limited to, a water-soluble vitamin, a fat-soluble vitamin , peptide polymer, polysaccharide polymer, sphingolipid, glycosaminoglycans, B-glycan and seaweed extract. In addition, you can add the compositions and agents according to the invention to existing cosmetics and solutions for washing. Such compositions can be used as creams, lotions, massage masks or oils and solutions for washing the body, soap, shampoos, etc.

Preferred water-soluble vitamins are any vitamins that can be mixed with cosmetics or other topical formulations, however various vitamins such as vitamin B1, B2, B6, pyridoxine, pyridoxine HCl, vitamin B12, pantothenic acid, nicotinic acid, nicotinamide, folic acid , vitamin C, vitamin H and the like, their salts, such as thiamine HCl salt, ascorbic acid Na-salt and the like, or their derivatives, such as Na-salt of ascorbic acid-2-phosphonic acid, Mg the salt of ascorbic acid-2-phosphonic acid are I preferred, and they can be prepared by conventional methods such as conversion processes transform microorganisms, methods of purification from cultures of microorganisms, the enzymatic methods or chemical synthesis methods.

Preferred fat-soluble vitamins are any vitamins that can be mixed with cosmetics or other topical formulations, however various vitamins such as vitamin A, D2, D3, E (dl-α-tocopherol, d-α-tocopherol, d-δ- tocopherol) and their derivatives, such as palmitic acid ascorbate, stearic acid ascorbate, dipalmitic acid ascorbate, acetic acid-dl-α-tocopherol, nicotinic acid-dl-α-tocopherol vitamin E, dl-pantothenyl alcohol, d-pantothenyl alcohol pantothenyl ethyl ester and the like containing fat the soluble vitamin used in the examples of the present invention are preferred, and they can be obtained by conventional methods, such as methods of conversion by microorganisms, methods of purification from a culture of microorganisms, enzymatic methods or methods of chemical synthesis.

Preferred peptide polymers are any polymer that can be mixed with cosmetic or other topical compositions, however collagen, hydrolyzable collagens, gelatin, elastin, hydrolyzable gelatin or keratin and the like, containing the peptide polymer used in the examples of the present invention are preferred.

Preferred polysaccharide polymers are any polymer that can be mixed with cosmetic or other topical compositions, however, hydroxyethyl cellulose, xanthine resin, Na hyaluronic acid, chondroitin sulfate or their salts (Na salt, etc.) and the like are preferred. For example, chondroitin sulfate or similar salts may be used by purification from mammals or fish.

Preferred sphingolipids are any sphingolipid that can be mixed with cosmetic or other topical compositions, however squalane, ceramide, pit-sphingosine, sphingolipopolysaccharide and the like are preferred. Sphingolipids can be obtained by purification from mammals, fish, shellfish, yeast or plants, and the like. using conventional methods.

Preferred seaweed extracts are any extract that can be mixed with cosmetic or other topical compositions, however, an extract from brown algae, red algae, green algae and the like is preferred. or purified carrageenin, alginic acid, arginic acid, Na, K, or glucosaminoglycans isolated from them. Algal extracts can be obtained by purification from seaweed using conventional methods.

Cosmetic and other topical compositions according to the invention can be combined with other components or combined with conventional cosmetic and topical compositions, if necessary, together with the above-described preparations of a frost-resistant kiwi plant. Such other components include, but are not limited to, oil components, humectants, emollients, surfactants, organic or inorganic dyes, organic powders, ultraviolet absorbent material, preservative, antiseptics, antioxidants, plant extract, regulator pH, alcohol, pigments, flavors, antipyretics, antihydrotic, distilled water, etc. Preferred oil components may include ester synthetic oil, mineral oil, silicone oil, fluoride oil, animal oil, plant oil, and the like.

Preferred ester synthetic oils include, but are not limited to, glyceryltri-2-ethylcaproic acid, cetyl-2-ethylcaproic acid, isopropyl myristic acid, butyl myristic acid, isopropyl palmitic acid, ethyl stearic acid, butyloyltin acid, butyl stearic acid, butyloyltin acid, butyric acid, butyric acid , isopropyl linoleic acid, ethyl oleic acid, isoacetyl myristic acid, isostearyl myristic acid, isostearyl palmitic acid, oktildodetsilmiristinovuyu acid izotsetilizostearinovuyu acid dietilsebatsinovuyu acid izopropiladipinovuyu acid izoalkilneopetanovuyu acid glitseriltri (caprylic, caprylic acid), trimetilopropantri-2-etilkaproevuyu acid trimetilopropantriizostearinovuyu acid pentaeritritoltetra-2-etilkaproevuyu acid tsetilkaprilovuyu acid detsillaurinovuyu acid geksillaurinovuyu acid detsilmiristinovuyu acid, myristyl myristic acid, cetyl myristic acid, stearyl stearic acid, decyl oleic acid OTU, tsetillitsinoleinovuyu acid izostearillaurinovuyu acid izotridetsilmiristinovuyu acid izotsetilpalmitinovuyu acid oktilstearinovuyu acid izotsetilstearinovuyu acid izodetsiloleinovuyu acid oktildodetsiloleinovuyu acid oktildodetsillinolevuyu acid izopropilizostearinovuyu acid tsetostearil-2-etilkaproevuyu acid, stearyl-2-etilkaproevuyu acid geksilizostearinovuyu acid etilenglikoldioktanovuyu acid, ethylene glycol dioleic acid, propylene glycol dicapric acid, propylene glycol di ( caprylic, capric acid), propilenglikoldikaprinovuyu acid neopentilglikoldikaprinovuyu acid neopentilglikoldioktanovuyu acid glitseriltrikaprilovuyu acid glitseriltriundekanovuyu acid glitseriltriizopalmitinovuyu acid glitseriltriizostearinovuyu acid oktildodetsilneopentanovuyu acid izosteariloktanovuyu acid oktilizononanovuyu acid geksildetsilneodekanovuyu acid oktildodetsilneodekanovuyu acid izotsetilizostearinovuyu acid izostearilizostearinovuyu acid oktildetsilizostearinovuyu acid polyglycerol ester olenoikovoy acid polyglycerin isostearic acid ester, triizotsetillimonnuyu acid triizoalkillimonnuyu acid triizooktillimonnuyu acid laurilmolochnuyu acid miristilmolochnuyu acid tsetilmolochnuyu acid oktildetsilmolochnuyu acid trietillimonnuyu acid atsetiltrietillimonnuyu acid atsetiltributillimonnuyu acid trioktillimonnuyu acid diizostearilmaleinovuyu acid di- 2-ethylhexylhydroxystearic acid, 2-ethylhexyl succinic acid, diisobutyl adipic acid OTU, diizopropilsebasinovuyu acid dioktilsebatsinovuyu acid holesterilstearinovuyu acid holesterilizostearinovuyu acid holesterilgidroksistearinovuyu acid holesterilgidroksistearinovuyu acid holesteriloleinovuyu acid digidroholesteriloleinovuyu acid pitsterilizostearinovuyu acid pitsteriloleinovuyu acid, isocetyl 12-stealoilgidroksistearinovuyu acid, stearyl 12-stealoilgidroksistearinovuyu acid, isostearyl-12- stealoyl hydroxystearic acid.

The preferred mineral oil described above may include paraffin solution, α-olefin oligomers, isoparaffin, ceresin, paraffin, isoparaffin solution, polybudin, microcrystalline wax, petrolatum, and the like.

Preferred silicone oils may include polymethylsilicon, methylphenylsilicone, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane-methylcetyloxysiloxysiloxane-methyl-oil-stearylmethyl-stearyl copolymer, etc.

A preferred fluoride oil may include perfluoropolyether and the like.

Preferred animal or vegetable oils may include avacado oil, almond oil, olive oil, sesame oil, rice husk oil, safflower oil, soybean oil, corn oil, rapeseed oil, canola oil, coconut oil, palm oil, sunflower oil, cotton seed oil, coconut oil, cucui nut oil, wheat germ oil, rice germ oil, oil from tree oil, two-year-old butterfin oil, macadamia nut oil, menhaden oil and other body oils fish, egg yolk oil, lanolin, hempseed oil, mink oil, hoplostet oil, jojoba oil, carnauba wax, lanolin solution, etc.

Preferred humectants may include water soluble low molecular weight humectants, lipophilic low molecular weight humectants, a water soluble polymer and a low molecular weight polymer.

In particular, preferred water soluble low molecular weight humectants may include cerine, glutamine, sorbitol, mannitol, pyrrolidone carboxylic acid Na, glycerin, propylene glycol, 1,3-butylene glycol, ethylene glycol, polyethylene glycol (polymerization index> 2), polypropylene glycol ( , lactic acid, a salt of lactic acid, and the like.

Preferred fat-soluble low molecular weight humectants may include cholesterol, cholesteryl ester, and the like.

Preferred water-soluble polymers may include a carboxyvinyl polymer, a salt of polyaspartic acid, tragacanth, xanthine resin, HMC (hydroxymethyl cellulose), HEC (hydroxyethyl cellulose), HPC (hydroxypropyl cellulose), carboxymethyl cellulose, water-soluble chitol, water-soluble, chitric, insoluble and the like.

Preferred fat-soluble polymers may include a polyvinylpyrrolidone-eicocene copolymer, a polyvinylpyrrolidone-hexadecene copolymer, nitrocellulose, a fatty acid dextrin ester, a silicone polymer and the like.

Preferred emollients may include long chain acyl glutamic acid cholesteryl ester, cholesteryl hydroxy stearic acid, 12-hydroxystearic acid, Rogic acid, lanolin fatty acid cholesteryl ester and the like.

Preferred surfactants may include nonionic surfactants, anionic surfactants, cationic surfactants, amphivalent surfactants, and the like.

In particular, preferred nonionic surfactants may include monostearic acid self-emulsifiable glycerin, fatty acid propylene glycol ether, fatty acid glycerol ether, polyglycerol fatty acid ester, sorbitan fatty acid ester, polyoxyethylene (POE), sorbitan fatty acid ester, POE, POE sorbitan fatty acid ester, POE glycerol fatty acid ester, POE alkyl ester, POE fatty acid ester, POE solid pimaja oil, POE pimaja oil, POE-POP copolymer, POE-POP alkyl ether, polyesters oxide modified silicone, lauric acid alkanolamide, alkylamine oxides, vodorodprisoedinenny soybean phospholipid and the like

Preferred anionic surfactants may include soapy fatty acids, α-acyl sulfonic acid salt, sulfonic acid alkyl salt, alkylallyl sulfonic acid, alkylnaphthalene sulfonic acid salt, alkyl sulfonic acid salt, POE alkyl ether sulfate salt, alkyl sulfate salt, alkylamide sulfate salt POE alkyl phosphate salt, alkyl amide phosphate salt, alkyl alkyl taurine salt, N-acylamino acid salt, POE salt of the carboxylic acid alkyl ester, alkyl sulfosuccinic acid salt , an alkyl sulfoacetic acid salt, an acylated hydrolyzable collagen peptide salt, perfluoroalkyl phosphate ester and the like.

Preferred cationic surfactants may include the ammonium alkiltrimetilhlorid, steariltrimetilhlorid ammonium steariltrimetilbromid ammonium setosteariltrimetilhlorid ammonium distearildimetilhlorid ammonium stearildimetilbenzilhlorid ammonium feniltrimetilbromid ammonium, benzalkonium, dietilaminoetilamid stearic acid dimetilaminopropilamid stearic acid, lanolin derivatives quaternary ammonium and the like

Preferred ambivalent surfactants may include carboxybetaine type, amide betaine type, hydroxysulfobetaine type, phosphobetaine type, aminocarboxylic acids, imidazoline derivative type, amidamine type, and the like.

Preferred organic and inorganic dyes may include silicic acid, anhydrous silicic acid, magnesium silicic acid, talc, keratite, mica, kaolin, bengal, clay, bentonite, a titanium thin layer mica, bismuth oxychlorin, zirconia, zinc oxide, magnesium oxide titanium, aluminum oxide, calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, chromium oxide, chromium hydroxide, calamine, carbon black and combinations thereof as inorganic dyes; polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenolic resin, fluorine resin, silicon resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, divinylbenzene-styrene copolymer, I silk, yellow powder, yellow powder pigment, orange CI pigment as organic dyes; and their complex and so on.

Preferred organic powders may include a metallic soap, such as calcium stearate; an alkyl metal salt of phosphinic acid such as sodium zinc acetyl acid, zinc lauryl acid, calcium lauryl acid; a polyvalent metal salt of acylamic acid such as calcium N-lauroyl-β-alanine, zinc N-lauroyl-β-alanine, calcium N-lauroylglycine and so on; polyvalent metal salt of sulfonic acid amide, such as calcium N-lauroyl taurine, calcium N-palmitoyl taurine; An N-acyl basic amino acid such as Nε-lauroyl-L-lysine, Nε-palmitoyllysine, Nα-palmitoylornithine, Nα-lauroyl arginine, fortified lanolin, acylarginine fatty acid, and the like; An N-acyl polypeptide such as N-lauroylglycylglycine; α-amino fatty acid such as α-aminocaprylic acid, α-aminolauric acid and the like; polyethylene, polypropylene, nylon, polymethyl methacrylate, polystyrene, divinylbenzene-styrene copolymer, ethylene tetrafluoride and the like.

Preferred ultraviolet light-absorbing agents may include para-aminobenzoic acid, paraaminoetilbenzoat, paraaminoamilbenzoat, paraaminooktilbenzoat, ethylene glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicylate, butylphenyl salicylate, homomenthyl salicylate, benzilkorichnaya acid, paramethoxy 2-etoksietilkorichnaya acid parametoksioktilkorichnaya acid diparametoksi-mono-2-etilgeksanglitserilkorichnaya acid, parametoxyisopropylcinnamic acid, diisopropyldiisopropylcinnamate ester mixture, lesson canoic acid, ethylurocanoic acid, hydroxymethoxybenzophenone, hydroxymethoxybenzophenone sulfonic acid and their salts, dihydroxymethoxybenzophenone, dihydroxymethoxybenzophenone disulfonate Na, dihydroxybenzophenone, tetrahydroxybenzophenone, 4-p-2-t-2-trimethoxybenzene-2-trimethoxybenzene-2-trimethoxybenzene ethylhexyl-1'-hydroxy) -1,3,5-triazine, 2- (2-hydroxy-5-methylphenyl) benzotriazole and the like.

Preferred preservatives may include quinocithiol, trichloric acid, trichlorohydroxydiphenyl ether, chlorhexidine glucuronate, phenoxyethanol, resorcinol, isopropylmethylphenol, azulene, salicylic acid, zinc pilithione, benzalkonium HCl, a photosensitizer, and a sodium psychocide inhibitor.

Preferred antioxidants may include butyl hydroxyanisole, propyl gallate, ellisorbate, and the like.

Preferred pH adjusters may include citric acid, sodium citrate, malic acid, sodium malic acid, fumaric acid, fumaric acid, succinic acid, sodium succinic acid, sodium hydroxide, sodium hydrogen phosphate and the like.

Preferred alcohols may include cetyl alcohol and so on.

In addition, other components may be added to any of the above compositions. In one embodiment, the amount of other components varies from 0.01 to 5%, more preferably from 0.01 to 3%, in this total composition.

The components described above, such as a water-soluble vitamin, a fat-soluble vitamin, a peptide polymer, a polysaccharide polymer, sphingolipid, seaweed extract and other components, can be obtained by the usual methods disclosed in the literature (see, for example, Matsumoto Mithio, Manual for the development of transdermal applied preparations. Seisi Press, 1 ^st Ed., 1985).

The use of preparations of the frost-resistant kiwi plant according to the invention is safe. They are non-toxic to animals and do not cause any significant negative effect.

In accordance with the present invention, any of the foregoing compositions can be combined with a preparation of a frost-resistant kiwi plant according to the invention (including any part of the fruit, whole fruit, stem, leaves, bark or root, and including any preparation or extract thereof, including dried preparations, not extracted, but processed preparations, fresh fruits, fruit juice or any extract or its fraction), the use of any suitable dose or amount of a preparation of a frost-resistant kiwi plant that is sufficiently tvlyaet desired biological activity for the preparation frost kiwi plants, as described above, if the applied one or more times during the corresponding time interval. The respective amounts or doses may vary depending on the task of administration or the pathological condition or treatment of the disease, as well as on the weight of the subject, the severity of the condition, the form of the drug, the course and time of administration and can be selected by a person skilled in the art.

An appropriate amount or dose of a preparation of a frost-resistant kiwi plant according to the invention may include, in one embodiment, an amount of about 0.1 g to about 10 g per kg body weight of the patient, and preferably about 1 to 3 g per kg body weight / day of study a frost-resistant kiwi plant or extract according to the invention. The dose may be administered once a day, several times a day, or at longer intervals (for example, every few days, weekly, monthly, etc.) as required. In the compositions described herein, the amount of the preparation of a frost-resistant kiwi plant according to the invention can be from about 0.01% to 100% by weight, and preferably from about 0.01% to about 95% by weight, and more preferably from 0.5 to 80% by weight based on the total weight of the composition, including any amount from 0.01% to 100%, with a 0.01% increase. In one embodiment, a medicament according to the invention may comprise about 0.01-50% by weight of a preparation of a frost-resistant kiwi plant according to the invention based on the total weight of the composition.

In one embodiment, an extract or other preparation of a frost-resistant kiwi plant according to the invention may be present in any composition from about 20% to 90% of a highly concentrated solution, powder or granule, including any increase between 20% and 90%, at a 1% increase ratio.

The ratio of additional components in the composition may typically vary from about 0 to 20% w / w. 100% w / w. compositions, including any increase between 0 and 100% w / w, at a 1% increase ratio.

In one embodiment, the cosmetic compositions include a preparation of a frost-resistant kiwi plant according to the invention in an amount of about 0.01 to 30%, more preferably 0.01 to 5% by weight based on the total weight of the composition, including any increase between 0, 01% and 30%, at 0.01% increase ratio.

In another embodiment, when a composition comprising a preparation of a frost-resistant kiwi plant according to the invention, which is added to a food, food supplement or drink, can be provided in an amount ranging from about 0.1 to 95% w / w, preferably from 1 to 80% w / w. the total weight of the food, supplement or drink, including any increase from 0.1 to 95% w / w, to 0.1% w / w. an increase, or from about 1 to 30 g per 100 ml, and preferably from 3 to 10 g per 100 ml, including any increase from 1 g per 100 ml and up to 30 g per 100 ml, per 1 g increase factor, healthy composition a drink.

In one embodiment, the dietary food products according to the invention include a preparation of a frost-resistant kiwi plant according to the invention as 0.01 to 80%, preferably 1 to 50% of the total weight of the total weight of the composition, including any increase of 0.01% and 80%, s increment of 0.1%.

In one embodiment, a diet food beverage includes a preparation of a frost-resistant kiwi plant according to the invention in an amount of from about 0.01 to about 20% by weight of the total weight of the composition, including any increase between 0.01% and 20%, incremented by 0.01%. Additional components may include amino acids from 0.001 to 5% by weight, vitamins from 0.001 to 2% by weight, sugars from 0.001 to 20% by weight, organic acids from 0.001 to 10% by weight, sweeteners and flavorings to the required amount. Ensuring that the composition of the healthy drink according to the invention contains the above-described preparation of a frost-resistant kiwi plant according to the invention as an essential component, there is no restriction on other liquid components, where the other component may be various sweeteners and / or intensifiers of taste and aroma, such as be added to a regular drink. Examples of such sweeteners or flavor and aroma enhancers include, but are not limited to, conventional or low calorie sweeteners, including monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; ordinary sugars such as dextrin, cyclodextrin; and sugar alcohols such as xylitol and erythritol and the like. Additional sweeteners include natural sweeteners, such as thaumatin, urticaria raspberry extract, levaudioside A, glycyrrhizin and their derivatives, and low-calorie sweeteners, such as saccharin, sucralose, aspartame and their derivatives. The amount of said sweeteners or flavor and aroma enhancers typically varies from about 1 to 20 g, and preferably from 5 to 12 g, per 100 ml of the beverage composition, including any increase between 1 g and 20 g per 100 ml, 1 g increase.

A nutritional supplement may be added to food by application, spraying or mixing. The amount of additive compared to the total amount of the composition can typically vary from about 0.01 to 20% w / w. 100% w / w. of the present composition, including any increase from about 20% w / w. and up to 100% weight / weight, with an increment of 1% weight / weight. Nutritional supplements may also be mixed with food, such as animal food, in an amount of about 5 to 100 g per 1 kg of weight, based on the total dry weight of the food. , including any increase from 5 g and 100 g for every 1 kg of weight, with an increment of 1 g.

Accordingly, it is an object of the present invention to provide a method for selectively regulating a Th1 and Th2 immune response in a patient by administering or providing a composition, including a drug, nutraceutical composition, nutritional supplement, dietary food products (including drink or nutrient), or cosmetic composition, including, consisting essentially of or consisting of any preparation of the frost-resistant kiwi plant described herein (including any part of the fruit, the whole fruit, banana b, leaves, bark or root, and including any preparation or extract, or their concentrate, including dried preparations, unextracted, but processed, preparations, fresh fruits, fruit juice or any extract, or concentrate, or a fraction thereof), and in one embodiment the implementation of the total extract, the total water-soluble extract or ethyl acetate extract of the frost-resistant kiwi plant as an active ingredient. Compositions according to the invention are described in detail above. In particular, the administration or provision of a composition results in at least one of the following biological activities: (a) a decrease in the number of IgE-producing B cells in a patient; (b) reducing the amount of IgE produced in a patient (e.g., in serum or plasma); (c) reducing the production and / or levels of at least one Th2 cytokine (e.g., IL-4, IL-5, IL-10); (d) increasing the level of at least one Th1 cytokine (e.g., IL-12, IFN-γ); (e) reducing the expression level of transcription factor GATA-3; (f) increasing the expression level of transcription factor T-bet; (g) increasing the expression level of transcription factor NFATc2; (h) increasing the number of IgG2a-producing B cells in a patient; (i) increasing the amount of IgG2a produced by the patient; (j) increasing the production or activity of Th1-T lymphocytes (e.g., CD4 +, IFN-γ +), especially at the site of inflammation; (k) reducing the production or activity of Th2-T lymphocytes (e.g., CD4 +, IL-4 +), especially at the site of inflammation; (1) reducing the number of IgG1-producing B cells in a patient; (m) reducing the amount of IgG1 produced in a patient; and / or (n) reducing the level or production of at least one leukotriene in a patient.

A preferred method according to the invention includes a method for reducing the production of leukotriene in a patient, thereby leading to an improvement in at least one symptom of a condition or disease associated with leukotrienes in a patient. The method includes the use by the aforementioned mammal of an effective amount of any preparation of the frost-resistant kiwi plant described herein (including any part of the fruit, whole fruit, stem, leaves, bark or root, and including any preparation or extract, or concentrate thereof, including dried preparations , non-extracted, but processed, preparations, fresh fruits, fruit juice, or any extract, or concentrate, or a fraction thereof), and in one embodiment, the total extract of the total water-soluble extract or ethyl acetate extract of the frost-resistant kiwi plant, together with their pharmacologically suitable carrier. Preferably, the use of a composition according to the invention leads to a decrease in the production or level of leukotrienes in a patient. Diseases and pathological conditions associated with leukotrienes include, but are not limited to, asthma, food allergies, allergic rhinitis, chronic urticaria, and allergic dermatitis. In this embodiment, preferred routes of administration include oral, respiratory and topical administration, in addition to systemic routes of administration.

The method described above can be used to prevent and / or treat any disease or condition in which the regulation of the immune response described in this document would be favorable for the patient or could be predicted to be favorable.

Thus, it is an object of the present invention to provide a method for treating and / or preventing an allergic disease and non-allergic inflammatory disease in a mammal, comprising using the aforementioned mammal with an effective amount of any preparation of the frost-resistant kiwi plant described herein (including any part of the fruit, whole fruit, stem, leaves, bark or root, and including any preparation, or extract, or their concentrate, including dried preparations, are not extracted But processed, preparations, fresh fruit, fruit juice, or any extract or concentrate, or a fraction thereof), and in one embodiment, the crude extract, the total water-soluble extract of the ethyl acetate extract or frost kiwi plants, together with a pharmacologically suitable carrier.

According to the present invention, allergic diseases may include, but are not limited to, asthma, allergic bronchopulmonary aspergillosis, allergic bronchial bronchiectasis, hypersensitive pneumonitis, allergic sinusitis, anaphylaxis, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, atopic dermatitis, atopic dermatitis, atopic dermatitis, atopic dermatitis, atopic dermatitis, atopic dermatitis, allergy to insects, food allergies and drug allergies.

In one embodiment, the allergic disease is atopic dermatitis. In one aspect of this embodiment, in addition to using a composition comprising substantially or consisting of a preparation of a frost-resistant kiwi plant according to the invention, the patient is simultaneously given conventional therapy for atopic dermatitis, including, but not limited to, treatment with a local steroid. In this embodiment, the preparation of a frost-resistant kiwi plant is used most preferably orally or topically, although the invention is not limited to these routes of administration.

In another embodiment, the allergic disease is asthma. In one aspect of this embodiment, in addition to using a composition comprising, primarily or consisting of a preparation of a frost-resistant kiwi plant of the invention, the patient is simultaneously treated with the usual asthma therapy, including, but not limited to, inhaling the treatment steroid or other asthma regulator. In this embodiment, the preparation of a frost-resistant kiwi plant is most preferably used by oral or local administration, although the invention is not limited to these routes of administration.

According to the present invention, diseases of a non-allergic skin inflammation reaction may include, but are not limited to, various skin diseases caused by inflammation, such as acne, acne, and the like. The cosmetic compositions described above include any preparation of the frost-resistant kiwi plant described herein (including any part of the fruit, whole fruit, stem, leaves, bark or root, and including any preparation or extract, or their concentrate, including dried preparations not extracted but processed, preparations, fresh fruits, fruit juice or any extract, or concentrate, or a fraction thereof), and in one embodiment, a crude extract, a total water-soluble extract, or a frost-resistant ethyl acetate extract Kiwi plants, useful for the prevention, treatment and / or improvement of skin inflammation in a patient.

Other non-allergic inflammatory diseases that can be prevented or treated using the compositions and methods described herein include, but are not limited to, various dermatitis, systemic lupus erythematosus (SLE), retinal inflammation, gastritis, retinopathy, hepatitis, enteritis , pancreatitis, nephritis, etc. conditions, weakening of the Th2-type immune response and / or strengthening of the Th1-type immune response is favorable.

Another objective of the present invention is to provide a method of treating and / or preventing a viral infection in a mammal, comprising administering to said mammal an effective amount of any preparation of a frost-resistant kiwi plant described herein (including any part of the fruit, whole fruit, stem, leaves, bark or root, and including any preparation, or extract, or their concentrate, including dried preparations, unextracted but processed, preparations, fresh fruits, fruit juice or any extract, or concentrate, or a fraction thereof), and, in one embodiment, a crude extract, a total water-soluble extract or an ethyl acetate extract of a frost-resistant kiwi plant, together with a pharmacologically suitable carrier.

Preferred viruses that a mammal is protected against by preventing or treating a viral infection include, but are not limited to, Coxsackie viruses, cytomegaloviruses, Epstein-Barr viruses, flaviviruses, hepatitis viruses, herpes viruses, influenza viruses, influenza viruses, mumps virus, papilloma viruses, parainfluenza viruses, parvovirus, rabies viruses, respiratory syncytial viruses, retroviruses and varicella viruses.

Among these viruses, retroviruses, herpes viruses, and hepatitis viruses are most preferred, with leukemia viruses, lymphotropic viruses, sarcoma viruses and lentiviruses, like other immunodeficiency viruses or tumor viruses, are even more preferred. Particularly preferred are the lymphotropic viruses that the mammal is protected from preventing or treating a viral infection, including T-lymphotropic viruses, such as human T-cell lymphotropic viruses (HTLV, such as HTLV-I and HTLV-II), bovine leukemia viruses ( BLV) and feline leukemia viruses (FLV). Particularly preferred lentiviruses include human (HIV), monkey (SIV), feline (FIV), and canine (CIV) immunodeficiency viruses, with HIV-I and HIV-2 being more preferred.

Another objective of the present invention is to provide a method for the treatment and / or prevention of cancer in a mammal, comprising administering to said mammal an effective amount of any preparation of the frost-resistant kiwi plant described herein (including any part of the fruit, whole fruit, stem, leaves, bark or root and including any preparation or extract thereof, including dried preparations not extracted but processed, preparations, fresh fruits, fruit juice, or any extract, or a fraction thereof), and in one embodiment of the crude extract, the total water-soluble extract of the ethyl acetate extract or frost kiwi plants, together with a pharmacologically suitable carrier.

Cancers for the treatment and prevention using the methods and compositions of the invention include, but are not limited to, melanomas, squamous cell carcinoma, breast cancer, head and neck carcinoma, thyroid cancer, soft tissue sarcoma, bone sarcoma, testicular tumors , prostate cancer, ovarian cancer, bladder cancer, skin tumors, brain tumors, angiosarcoma, hemangiosarcoma, mast cell tumor, primary liver tumors, pulmonary tumors, pancreatic tumors, ventricle non-intestinal tumors, renal cell carcinoma, hematopoietic neoplasia and their metastatic tumors.

In any of the methods of treating or preventing a disease or condition described above, a preparation of a frost-resistant kiwi plant can be used in combination with another type of therapy or composition that is beneficial for treating a particular condition. In these embodiments, the frost-resistant kiwi plant can be considered as a complement to conventional treatment, increasing the chances of improving the treatment or alleviating the symptoms of the patient. Particularly preferred types of commonly used agents or therapies that can be used with a preparation of a frost-resistant kiwi plant according to the invention include, but are not limited to, steroids (including corticosteroids and including oral agents, inhalations and injections), antihistamines (any type including systemic, local, inhaled), antibodies (e.g., anti-IgE, anti-IL-10), antibiotics, cyclosporins, antimycotics, regulators of respiratory function, analgesics, β-agonists (long long or short-term effects), leukotriene modifiers (receptor inhibitors or antagonists), cytokine or cytokine receptor antagonists, phosphodiesterase inhibitors, sodium chromoglycate, nedocrimil, caffeine, theophylline, carbobenzoxy-beta-alanyl taurine, inhibitors and other T-cell inhibitors.

In the method according to the invention, the compositions can be administered to any member of the vertebrate class, mammals, including, but not limited to, primates, rodents, livestock, horses and pets. Preferred patients for protection are pets (e.g. dogs, cats) and humans, with humans being particularly preferred. All methods of administration are acceptable. According to the present invention, the terms “patient,” “object,” and “individual” may be used interchangeably.

Routes of administration include in vivo, in vitro, and ex vivo routes. The ex vivo pathway refers to the implementation of a portion of the regulatory stage outside the patient's body. In vivo routes include, but are not limited to, intravenous administration, intraperitoneal administration, intramuscular administration, intranodal administration, intracoronary administration, intraarterial administration (e.g., to the carotid artery), subcutaneous administration, transdermal delivery, intratracheal administration, intraarticular administration, intraventricular administration , inhalation (e.g., aerosol), intracranial, intraspinal, intraocular, ear, intranasal, oral, pulmonary administration, catheter impregnation, intracutaneous, near spe-, epidural, intracerebroventricular injection and direct injection into the tissue. In one embodiment of the present invention, the composition is administered parenterally (e.g., subcutaneously, intradermally, intravenously, intramuscularly and intraperitoneally). Intravenous, intraperitoneal, intradermal, subcutaneous and intramuscular administration can be performed using standard methods in this field. Ear administration may include ear drops, intranasal administration may include nasal drops or intranasal injections, and intraocular delivery may include eye drops. Aerosol administration (by inhalation) can also be carried out using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad. ScL USA 189: 11277-11281, 1992, which is incorporated herein by reference). For example, in one embodiment, a composition or vaccine according to the invention may be included in a composition suitable for nebulized delivery using an appropriate inhalation device or nebulizer. Oral administration can be performed in a complex manner using the composition according to the invention with a carrier capable of withstanding the degradation of digestive enzymes in the stomach of animals, for example, in tablets or capsules, as well as in food and drink products. Examples of such carriers include synthetic capsules or tablets known in the art. Direct injection methods are especially useful for topical administration of the compound. Oral administration or topical administration are particularly preferred routes of administration or use according to the invention. Routes of administration that modulate mucosal immunity are useful in the treatment of viral infections and certain allergic pathological conditions. Such pathways include bronchial, intracutaneous, intramuscular, intranasal, as well as others - inhalation, rectal, subcutaneous, local, transdermal, vaginal and urethral.

The present invention is more specifically explained in the following examples. However, it should be understood that the present invention is in no way limited to these examples.

EXAMPLES

Example 1

The following examples show that at least two specific extracts derived from A. arguta, designated PG102T and PG102E, have inhibitory activity against IgE production, as well as the ability to regulate selective Th1 and Th2 cytokines.

Materials and methods

The mice. Female BALB / c mice (6 weeks old) were obtained from Daehan Biolink, Co. Ltd. (Korea), kept in an air-conditioned and pathogen-free room and acclimatized for at least 1 week. All of the experimental methods mentioned below were carried out in accordance with the established rules for caring for animals, in accordance with the guidelines of the Animal Experimental Center at Seoul National University.

Obtaining various extracts from A. arguta. The frost-resistant kiwi plants used in this study were obtained from a farm specializing in the cultivation of this fruit (Hurstberry Co. Ltd., Oregon, USA) and their identity was kindly confirmed by Dr. Ella I. Kolbasina (Moscow Branch of Vavilov Plant Cultivation Research Institute, Russia). The dried fruit (10 g) was extracted 3 times by heating in distilled water (DW). It was then concentrated, lyophilized and dissolved in DW to give PG102T in an amount of 100 mg / ml. PG102T dissolved in DW was extracted sequentially with chloroform, ethyl acetate and n-butanol, resulting in the formation of PG102C, PG102E and PG102B, respectively. The remaining aqueous layer was named PG102W. Each solvent-soluble fraction and the resulting aqueous precipitate were filtered, concentrated, lyophilized and dissolved in concentrations of 100 mg / ml. All preparations were stored at -80 ° C until use.

Bioanalysis in U266B1 cells. The inhibitory effects of IgE in LPS-stimulated B cells of human lymphoblastoma, U266B1 (ATCC, Manassas, VA), were measured as described by Kim and others with a slight change (Kim et al, Phytother Res 2001; 15: 572-6). The level of human IgE in the culture supernatant was determined by ELISA for human IgE (total human IgE; AlerChek, Portland, Maine), and cell viability was assessed using an LDH determination kit (Takara Bio, Japan).

In vitro effect of PG102 on cytokine production in response to OVA-stimulated splenocytes. Mice (7 weeks old) were independently immunized and later stimulated with intraperitoneal (ip) injections of 20 μg of egg albumin (OVA; class V; Sigma, St. Louis, Mo) emulsified in 2.25 mg of aluminum hydroxide (ImjectAlum; Pierce, Rockford, IL), at days 0 and 14, respectively. Non-sensitized (intact) mice did not receive any reagent. On day 24, both OVA-sensitized and intact mice were sacrificed (n = 5 / group) and then each spleen was isolated to study cytokine production in splenocytes using a repeated response as described by Shibata et al. (Yoshimi et al., J Immunol 2000; 164; 1314-21). Briefly, the isolated splenocytes were plated on a 24-well culture plate, with a final concentration of 5 × 10 ⁶ cells / ml / well. Splenocytes were incubated with OVA in an amount of 100 μg / ml in the presence of PG102T (1 mg / ml), PG102C, PG102E, PG102B, PG102W (all at 0.1 mg / ml) or medium as a control for 3 days. After incubation, culture supernatants were selected to determine the level of cytokines (IL-4, IL-5, IL-12 and IFN-γ) using ELISA kits (Endogen, Cambridge, MA). In fact, an identical technique was used to determine the specific activity of PG102T and PG102E.

Quantification of cytokines and immunoglobulins in OVA-sensitized mice. Mice were immunized and stimulated as described above. To evaluate the in vivo effects of PG102 preparations on OVA-induced allergic responses, OVA-sensitized mice (n = 10 / group) were orally administered PG102T (15 mg / kg / day) or PG102E (1.5 mg / kg / day) with dexamethasone (DEX, 0.5 mg / kg / day) or DW (100 μl / mouse / day) as a control, once a day, from 14 days to 24 days. Intact mice were orally administered with DW. At day 21, blood was drawn from each mouse, causing eye bleeding, and samples of the isolated plasma were stored at -80 ° C until use. The level of total IgE was determined using a kit for determination of mouse IgE (Shibayagi, Gunma, Japan). The levels of total IgG subtypes and isotypes of OVA-specific Ig were determined by sandwich ELISA (Hirano et al., J Immunol Methods 1989; 119: 145-50). To quantify cytokine production, splenocytes were obtained from animals on day 24, resuspended in culture medium (RPMI-1640 containing 10% FBS), seeded in a 24-well plate (5 × 10 ⁶ cells / ml / well), and incubated only with OVA in an amount of 100 μg / ml for 3 days. Splenocytes isolated from intact mice were cultured in the absence of OVA. The levels of IL-4, IL-5, IL-10, IL-12, IL-13 and IFN-γ in the supernatants were determined by ELISA (Endogen and R&D Systems, Minneapolis, MN).

Immunostaining assay. Splenocytes were exposed to GolgiStop (PharMingen, San Diego, CA) as an inhibitor of intracellular protein transport for 4 hours and prepared to determine IL-4 or IFN-γ-producing cells. Cells were fixed, permeabilized, then they were incubated with PE- or FITC-conjugated antibodies specific for murine CD4, IL-4 or IFN-γ, as described by Kyoko et al. (Kyoko et al., J Derm Science 2002; 29: 19-25). For the analysis of IgE products, cells were incubated with PE-conjugated anti-mouse CD19, and then with FITC-conjugated anti-mouse IgE (all from PharMingen). Cells were then examined by analysis of selective lymphocytes in splenocytes using the FACSort cell sorter and Cell Quest software (Becton Dickinson, San Jose, CA). For confocal microscopy analysis, splenocytes were cultured with OVA on coverslips for 2 days, fixed, permeabilized and stained using FITC-conjugated anti-mouse IgE and PE-conjugated anti-mouse CD19, and finally observations were made on a MRC-1024 confocal laser scanning system images (Bio-Rad Laboratories Inc., Hercules, CA) as described by Semper et al. (Semper et al., J Allergy Clin Immunol 2003; 112: 141-9).

Western blotting. Splenocytes from independent groups of mice were cultured with OVA for 2 days, harvested (10 ⁷ cells / group), and then lysed to obtain protein samples. Immunoblotting was performed using specific mouse antibodies against GATA-3, T-bet, NFATc2 (Santa Cruz Biotechnology, Santa Cruz, CA) or β-actin (Sigma) as a load control.

RNA production and quantitative real-time PCR. Total RNA was isolated from splenocytes cultured with OVA for 2 days using TRIzol Reagent (GIBCOBRL, Carlsbad, CA). The isolated total RNA was used for reverse transcription PCR (RT) -PCR in the AMV RT System (Roche, Mannheim, Germany), then real-time quantitative PCR was performed using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). The forward and reverse primer sets for mouse genes were constructed using the Primer Express Software (Applied Biosystems) computer program to obtain the following nucleotide sequences:

Gata3

straight 5'-CCTCGGCCATTCGTACATG-3 '(SEQ ID NO: 1)

reverse 5'-CGTAGTAGGACGGGACGTGG-3 '(SEQ ID NO: 2)

T-bet

straight 5'-TGTGGATGTGGTCTTGGTGG-3 '(SEQ ID NO: 3)

reverse 5'-ATAAGCGGTTCCCTGGCAT-3 '(SEQ ID NO: 4)

NFATc2

straight 5'-GCACATAAGGCCATCAGCTCA-3 '(SEQ ID NO: 5)

reverse 5'-TCGCCAGAGAGACTGGCAA-3 '(SEQ ID NO: 6)

GAPDH

straight 5'-TGCAGTGGCAAAGTGGAGATT-3 '(SEQ ID NO: 7)

reverse 5'-TTGAATTTGCCGTGAGTGGA-3 '(SEQ ID NO: 8)

Differences in mRNA levels of cell genes between all groups of mice were calculated from recalculation of the quantitative value of ΔCt.

Statistics. Data were expressed as mean ± SEM, and differences between mean values were analyzed using unpaired Student's test. A P value less than 0.05 or 0.01, which was calculated as a one-sided P value, was considered statistically significant.

results

Effects of various preparations from A. arguta in LPS-stimulated cells of the human U266B1 B cell line and OVA-stimulated murine splenocytes. The inventors initially obtained the total water-soluble extract (PG102T) from A. arguta, and subsequently extracted it with chloroform (PG102C), ethyl acetate (PG102E), n-butanol (PG102B) and water (PG102W), to obtain four fractions of different polarity. The effect of PG102T and these four fractions on IgE production was investigated using the human B-lymphoblast cell line U266B1 (FIG. 1). It was found that four drugs, with the exception of PG102W, inhibit the production of IgE from LPS-stimulated U266B1 cells. PG102E showed the highest inhibitory activity, having a 50% inhibitory effect at 25 μg / ml (IC ₅₀ ). PG102T was also active, its quantitative IC ₅₀ value was 126 μg / ml. At all concentrations tested in this study, neither PG102T nor PG102E showed any cytotoxic effects on U266B1 cells.

In addition, the inventors investigated the effects of various preparations from A. arguta on the production of cytokines involved in the Th1 and Th2 pathways in a repeat response model. Mice were immunized with OVA on day 0 and stimulated on day 14. After 10 days, the mice were sacrificed and the spleen was removed to isolate splenocytes. Splenocytes obtained from OVA-sensitized mice stimulated OVA and cultured for 3 days in the presence of five PG102 preparations. As shown in table 1, if the cells were grown in the presence of OVA, then the levels of IFN-γ, IL-4 and IL-5 increased to hundreds of picograms or nanograms. In the case of IL-12, there was a high background level, but OVA stimulation increased the level by approximately 3 times, to 415 ± 48 pg / ml. Treatment with PG102T resulted in an almost 150% increase in IL-12. IFN-γ behaved differently. Splenocytes from non-infected animals produced undetectable IFN-γ levels, but OVA-stimulated splenocytes secreted nearly 2.5 ng / ml. Treatment with PG102T reduced the level of IFN-γ by 40%. If OVA-stimulated splenocytes were grown in the presence of PG102T, IL-4 and IL-5 levels decreased by 62% and 39%, respectively.

Table I In vitro effects of PG102 on cytokine production by splenocytes Treatment IL-12 IFN-γ IL-4 IL-5 Intact 1277 ± 275 (308) Nd 8 ± 1 (2) 15 ± 3 (2) Ova Sensitized Wednesday 415 ± 48 (100) 2525 ± 1474 (100) 324 ± 15 (100) 847 ± 137 (100) PG102T 622 ± 56 (150) * 1478 ± 1214 (59) 124 ± 34 (38) ** 523 ± 30 (61) PG102C 342 ± 12 (82) Nd 90 ± 57 (28) * 84 ± 26 (10) * PG102E 927 ± 218 (223) * 703 ± 406 (28) 70 ± 5 (22) ** 345 ± 166 (41) * PG102B 575 ± 136 (139) 666 ± 366 (26) 290 ± 44 (90) 623 ± 20 (74) PG102W 501 ± 128 (121) 897 ± 592 (36) 280 ± 73 (87) 676 ± 61 (80)

All splenocytes from OVA-sensitized mice were re-stimulated with OVA during cultivation. Quantitative values were expressed as mean ± SEM for five animals. *, P <0.05 and **, P <0.01, against OVA-stimulated splenocytes treated only with medium (Student's T-test). ND = undetectable.

PG102E, which had the highest inhibitory effect on IgE production in this experiment, also reduced the levels of IL-4 and IL-5 by 78% and 59%, although it induced the level of IL-12 by 220%. In contrast, PG102C reduced the level of all cytokines determined in this study due to their cytotoxic effect on splenocytes. Both PG102B and PG102W increased the level of IL-12 and decreased the levels of IFN-γ, IL-4 and IL-5, but not in a statistically significant manner. Taken together, these results showed that PG102T and PG102E may contain compounds (e) that inhibit the production of IgE and control the expression of selective Th1 and Th2 itokines. Based on the above data, the inventors chose two drugs, PG102T and PG102E, for use in further in vivo studies.

Determination of the specific activity of PG102T and PG102E. Based on their pleiotropic action of the immune system, it is believed that PG102T and PG102E, since they are derived from a plant source, contain more than one active compound. Therefore, to quantify the further conduct of the experiments, the inventors developed a reliable bioanalysis based on the inhibitory effect of PG102 on the production of IL-4 in OVA-stimulated splenocytes, as described above. When cells stimulated OVA, the level of IL-4 increased from an undetectable level to several hundred picograms. In the presence of PG102T and PG102E, IL-4 production was inhibited in a dose-dependent manner (FIG. 2). At all concentrations tested in this study, neither PG102T nor PG102E showed any cytotoxic effects. The concentration of PG102T, corresponding to 50% inhibitory activity (IC ₅₀ ), was 806.9 μg / ml, and the concentration of PG102E was 91.8 μg / ml. The IC ₅₀ value of each drug was determined as one unit of activity and the sum of the units was obtained. When calculating the specific activity of PG102T and PG102E using the sum of units and yield, it turned out that the specific activity of PG102T and PG102E is 1.2 units / mg and 10.9 units / mg, respectively. This method was used to control the quality of experimental samples from A arguta.

Effects of PG102T and PG102E with respect to the production of Th1 and Th2 cytokines in an OVA-sensitized mouse model. To confirm the above in vitro data in an animal model, the effects of PG102T and PG102E were investigated in relation to the production of various cytokines involved in the change of Th1 and Th2 pathways using the OVA-sensitized mouse model. Mice were immunized with OVA on day 0 and stimulated on day 14. After stimulation, the animals received an oral dose of PG102T (15 mg / kg / day = 18 units / kg / day) or PG102E (1.5 mg / kg / day = 16.4 units / kg / day), and DEX as a control (0.5 mg / kg / day) or DW, every day, from 14 days to 24 days. Intact mice not treated with OVA orally received DW. The concentrations of PG102T and PG102E used in these experiments correspond to the minimum dosage having maximum activity, according to preliminary dose-response experiments. On day 24, mice were sacrificed and spleens were isolated to obtain splenocytes. Splenocytes from each group of mice were incubated in the presence of OVA for 3 days, and culture supernatants were selected to measure cytokine levels (Table II). When compared with intact splenocytes, it turned out that the level of IL-12 was reduced in DW-treated mice. However, oral administration of PG102T and PG102E increased their level by 1.7 and 2.6 times, respectively. Unlike IL-12, IFN-γ was enhanced by OVA stimulation, and PG102T or PG102E further increased IFN-γ. Unlike PG102T and PG102E, DEX inhibited both IL-12 and IFN-γ.

Table II In vivo effects of PG102 on the production of Th1 and Th2 cytokines by splenocytes Sources IL-12 IFN-γ IL-4 IL-5 IL-10 IL-13 intact 1353 ± 201 (394) Nd Nd Nd 77 ± 54 (2) 23 ± 17 (1.5) OVA-sensitized DW 343 ± 33 (100) 1023 ± 19 (100) 460 ± 42 (100) 1345 ± 68 (100) 3254 ± 298 (100) 1564 ± 132 (100) PG102T 584 ± 136 (170) * 1970 ± 142 (193) ** 258 ± 31 (56) * 913 ± 79 (68) * 1812 ± 296 (56) ** 1220 ± 96 (78) PG102E 889 ± 68 (259) ** 1778 ± 237 (174) * 164 ± 4 (36) * 412 ± 45 (31) ** 1591 ± 114 (49) ** 976 ± 304 (62) Dex 227 ± 44 (66) 224 ± 118 (22) 28 ± 21 (6) * 160 ± 159 (12) * 392 ± 157 (12) ** 912 ± 371 (58)

All splenocytes from OVA-sensitized mice were re-stimulated with OVA. Quantitative values were expressed as mean ± SEM for ten animals. *, P <0.05 and **, P <0.01, versus DW-treated mice (Student T-test). ND = undetectable.

The level of all Th2 cytokines determined in this study increased significantly in OVA-stimulated splenocytes. However, PG102T treatment inhibited OVA-mediated overproduction of IL-4, IL-5, and IL-10 by 44%, 32%, and 44%, respectively. PG102E also inhibited the levels of these three cytokines by 64%, 69% and 51%, respectively. DEX also lowered the concentration of all three cytokines. IL-13 was reduced by either PG102 or DEX, but not in a statistically significant manner. Taken together, these results showed that both PG102T and PG102E can control the production of selective Th1 and Th2 cytokines. Unlike DEX, which suppressed virtually all cytokines in an indistinguishable way, PG102T and PG102E appear to have different biological activities that can modulate the production of Th1 and Th2 cytokines in different ways.

The effects of PG102T and PG102E on plasma levels of immunoglobulin isotypes. The above results showed that PG102T and PG102E can inhibit Th2-mediated IgE hyperproduction in vivo. Therefore, it was determined whether oral administration of PG102T and PG102E to OVA-sensitized mice can control plasma levels of IgE and other immunoglobulins. On day 21 of the same experiment as described above, intact mice produced approximately 140 ng / ml total IgE, but sensitization of OVA increased its level by approximately 20 times. When animals were treated with PG102T and PG102E, the plasma level of total IgE was reduced by about 2 times. The ability of PG102T or PG102E to suppress the level of total IgE in plasma was comparable to that of DEX. When measuring the level of various IgG subtypes, it turned out that both PG102T and PG102E decreased the level of Th2-mediated IgG1, while the level of Th1-mediated IgG2a was significantly increased in a statistically significant way. In all cases, the level of IgG2b did not significantly change (table IIIA).

An almost identical result was obtained when determining the levels of OVA-specific IgE and IgG subtypes (Table IIIB). Oral treatment with PG102T or PG102E reduced the level of OVA-specific IgE and IgG1, while an increase in the level of OVA-specific IgG2a by more than 2 times. These results, along with data on various Th1 and Th2 cytokines, showed that PG102T and PG102E contain compounds that can regulate the balance of Th1 and Th2, ultimately leading to an increase in IgG2a and a decrease in IgE and IgG1.

Table III The effects of PG102 on plasma levels of total and OVA-specific immunoglobulin isotypes A. Sources Total Ig IgE (ng / ml) IgGl (μg / ml) IgG2a (μg / ml) IgG2b (μg / ml) Intact 139 ± 23 (5) 450 ± 81 (26) 46 ± 1 (159) 229 ± 52 (85) OVA Sensitized DW 2783 ± 485 (100) 1700 ± 21 (100) 29 ± 3 (100) 270 ± 36 (100) PG102T 1669 ± 319 (60) * 1009 ± 443 (59) * 67 ± 4 (231) * 325 ± 17 (120) PG102E 1271 ± 247 (46) * 1446 ± 288 (85) 44 ± 4 (152) * 220 ± 65 (81) Dex 1190 ± 317 (43) * 1614 ± 36 (95) 41 ± 7 (141) 313 ± 82 (116)

B. Sources OVA-specific Ig (% control from OD) IgE IgGl IgG2a IgG2b Intact 13 ± 1 5 ± 1 17 ± 1 51 ± 13 OVA Sensitized DW one hundred one hundred one hundred one hundred PG102T 29 ± 7 ** 76 ± 7 * 217 ± 54 * 104 ± 12

PG102E 27 ± 8 ** 69 ± 9 * 304 ± 53 ** 84 ± 9 Dex 27 ± 8 ** 79 ± 11 * 89 ± 25 84 ± 9

Values are mean ± SEM for ten animals. *, P <0.05 and **, P <0.01 against DW-worked mice (Student's T-test). Since OVA-specific antibodies were not available, the relative level of antibodies was calculated as a percentage of the OD control. OD = optical density.

The effects of PG102T and PG102E on a population of T and B cells in splenocytes. To understand the cellular basis of the PG102T and PG102E activities described above, the inventors investigated how the use of PG102 affects the populations of T cells and B cells present in splenocytes. Splenocytes were isolated from OVA-sensitized and intact mice and grown in the presence of OVA for 2 days, followed by FACS and confocal microscopy. As described in FIG. 3A, OVA stimulation increased the ratio of CD4 + IL-4 + cells from 4.7% to 9.3%. The use of PG102T or PG102E reduced the amount by more than 30%. In contrast, CD4 + IFN-γ + cells were slightly increased, from 7.2% to 9.2 or 9.6%, when animals were treated with PG102T or PG102E, although not statistically and significantly. There was a slight change in the number of both types of cells in DEX-treated animals. These results showed that PG102T and PG102E are able to regulate the ratio of IL-4 and IFN-γ-producing T cells.

The inventors then analyzed the effects of PG102T and PG102E on IgE-producing B cells. Compared to DW treatment, oral administration of PG102T or PG102E resulted in an approximately 40% reduction in the number of CD19 + IgE + B cells. DEX reduced the number of such B cells by more than 60% (FIG. 3B). Analysis of confocal microscopy additionally found that there is a significant decrease in the level of IgE production in any given B-cell. IgE signal intensity in B cells increased sharply in OVA-sensitized mice, but decreased significantly if animals were treated with PG102T. It turned out that the effects of PG102E are more potent than PG102T, since the level of fluorescence intensity subsequently decreased. These results show that PG102T and PG102E not only reduce the number of IgE-producing B cells, but also inhibit the production of IgE in the B cells themselves.

The effect of PG102T and PG102E on cellular transcription factors. To understand the molecular mechanisms underlying the biological activities of PG102T and PG102E, it was investigated whether these two drugs have any effects on cellular transcription factors involved in the Th1 and Th2 pathways. This was because the effects of PG102T or PG102E on so many cytokines and IgE could be more easily explained if PG102 directly or indirectly regulates transcription factors. It is well known that transcription factors, including GATA-3, T-bet, and NFATc2, play a major role in balancing Th1 / Th2 responses (Lee et al., J Exp Med 2000; 192: 105-15; Ting et al, Nature 1996; 384: 474-8; Lighvani et al., Proc Natl Acad Sci USA 2001; 98: 15137-42; Szabo et al., Cell 2000; 100: 655-69; Kiani et al., Blood 2001; 98: 1480-8.). Splenocytes isolated from OVA-sensitized mice were cultured for 2 days, as described above, and prepared for immunoblotting. The level of GATA-3 protein was greatly reduced in both PG102T and PG102E, while the level of T-bet and NFATc2 was increased (Fig. 4A). DEX suppressed all of these transcription factors, tested as previously reported (Adcock, Pulm Pharmacol Ther 2001; 14: 211-9).

To check at what level this regulation occurs, the mRNA levels of the corresponding transcription factors were determined using quantitative real-time PCR (Fig. 4B). PG102T treatment reduced the level of GATA-3 mRNA by almost 3 times, while at the same time increasing the levels of T-bet and NFATc2 mRNA by approximately 14 times and 2 times, respectively. It is interesting to note that PG102E, which showed a higher biological activity against IgE inhibition, altered the levels of GATA-3, T-bet, and NFATc2 to a lesser extent than PG102T. DEX lowered the levels of both GATA-3 and NFATc2, and slightly increased the level of T-bet. These results showed that PG102T and PG102E can control the production of Th1 / Th2 cytokines by regulating the level of transcription factors, such as GATA-3 and T-bet, at the RNA level.

Discussion

The biological activities of PG102T and PG102E found in this study convincingly prove that extracts from A. arguta are excellent candidates as a potent and specific anti-allergic agent. Both PG102T and PG102E reduced the number of IgE-producing B cells, as well as the amount of IgE produced by B cells, ultimately leading to a decrease in plasma IgE levels. It turned out that PG102 has such biological activity, changing the balance of Th1 and Th2 by decreasing the level of selective Th2 cytokines and increasing the level of Th1 cytokines.

The effects of PG102T and PG102E on cellular transcription factors may explain how the two drugs work at the molecular level. Both PG102T and PG102E reduced the level of GATA-3, while increasing the level of T-bet and NFATc2. GATA-3 is known as a potent transactivator of the IL-5 promoter and enhancer element of IL-4 (Lee et al., J Exp Med 2000; 192: 105-15; Ting et al., Nature 1996; 384: 474-8). T-bet is involved in committing Th1 cells by inducing IFN-γ synthesis in Th1 cells (Lighvani et al., Proc Natl Acad Sci USA 2001; 98: 15137-42; Szabo et al., Cell 2000; 100: 655- 69). Recent studies have also reported that T-bet regulates the switch to IgG2a isotype and IFN-γ production in B cells (Gerth et al., Int Immunol 2003; 15: 937-44). The data of the inventors are consistent with such well-known functions of the corresponding transcription factors as negative regulation of the effect of the GATA-3-dependent expression of IL-4 and IL-5 and positive regulation of the T-bet-dependent production of IFN-γ and IgG2a in splenocytes.

Unlike T-bet and GATA-3, NFATc2 is a non-selectively expressed antigen-induced transcription factor. Although this protein binds to the IL-4 enhancer and IFN-γ promoter in stimulated Th cells, it is suggested that NFATc2 plays a more important role in promoting intact T cells to effector Th1 cells. In fact, NFATc2 - / - mouse T cells secreted much higher levels of IL-4 than wild-type T cells (Kiani et al., Blood 2001; 98: 1480-8; Erb et al., Infect Immun. 2003; 71 (11): 6641-7). This study showed that PG102T or PG102E increase the level of NFATc2 expression.

The actual molecular mechanism by which PG102T or PG102E promotes Th1 responses and inhibits IgE biosynthesis is still under investigation. One possible option is that PG102T or PG102E acts on antigen-presenting cells that play a key role in Th differentiation by secretion of soluble factors, including IL-12, and expression of costimulatory molecules such as B7-1 (Th1) and B7-2 (Th2) (Kuchroo et al., Cell 1995; 80: 707-18). Another possible option is to directly influence the differentiation of Th precursor cells or the regulation of IgE-producing B cells.

The regulatory effect of PG102T or PG102E on Th1 and Th2 systems may be due to numerous biologically active compounds. To obtain various PG102 preparations in a reliable manner, the inventors developed a bioassay system based on their effect on the production of IL-4, the main inducer of switching to the IgE isotype, in OVA-stimulated splenocytes. This study was sensitive and reproducible enough to calculate the specific activity of PG102 and was used to study various factors affecting the biological activity of PG102, such as harvest time and different geographical locations of A. arguta. This IL-4-based study is currently used to purify the active ingredients as well as the quality control of PG102 reagents used in various experiments.

The toxicity data of the inventors show that PG102T is harmless. In repeated dose toxicity trials, oral administration of PG102T (500, 1000, 2000 mg / kg / day) for 4-12 weeks did not cause any adverse effects in rats. These results are in complete contrast with the data obtained in mice that were injected with DEX (0.5 mg / kg / day), which dramatically reduced body weight and spleen weight (data not shown). These data, describing the different biological activities of PG102T and PG102E, show that A. arguta extracts are harmless natural preparations with minimal risk of side effects and can be used in the treatment of various allergic disorders.

Example 2

The following example shows that at least two specific extracts from A. arguta, designated PG102T and PG102E, have a therapeutic effect on atopic dermatitis.

NC / Nga mice were bred as an inbred line in 1957 based on Japanese decorative mice (Nishiki-Nezumi). The mice remained normal and healthy when they were in a state free of specific pathogens (SPF). However, when they were placed under normal conditions, clinical manifestations began with the initiation of combing behavior from 8 weeks of age, followed by the appearance of an eczematous state. Timely developing eczema is mainly localized on the muzzle, ears, neck and back. The affected mice showed each of the clinical signs individually, including hemorrhage, superficial erosion, severe skin abrasion, peeling, dry skin and growth retardation (Hiroshi et al., Int Immunol 1997; 9 (3): 461-466). In skin lesions, infiltration of numerous CD4 + T cells and eosinophils and an increase in the number of mast cells with degranulation were observed. In addition, plasma IgE levels increased markedly from 8 weeks of age, coinciding with the appearance of skin lesions. Infiltrating cells in skin lesions expressed IL-4, IL-5, and TARC, but little or not IFN-γ, resulting in Th2-dominant immune responses (Masayuki et al., Int Ach Allergy Immunol 2003; 132: 355-63 ; Christian et al., Mol Med Today 2000; 5: 209-10). These findings, similar to individual features, are inherent in AD patients, suggesting that the NC / Nga mouse may be an excellent animal model for human AD, and also that Th1 / Th2 modulation and suppression of IgE biosynthesis may be a therapeutic strategy that can fundamentally improve the clinical symptoms of AD in both humans and mice.

Materials and methods

Animals. Specific Pathogen Free (SPF) female NC / Nga (NC) mice were purchased from SLC (Tokyo, Japan). Mice (5-6 weeks old) were maintained in an SPF environment (mouse SPF NC) and were provided with autoclaved food and water for at least 1 week before use. Seven-week, SPF NC mice were moved to an air-uncontrolled normal room (regular NC mice). Compliant animal experiments are set out by the University Animal Care and Use Committee guidelines at Seoul National University.

Oral administration of PG102T or PG102E. The PG102T and PG102E used in this study were obtained from A. Arguta as previously described (Park et al., J. Allergy Clin. Immunol., 116: 1151-1157, 2005 and Example 1). Conventional NC mice were divided into three groups (n = 6-8 / group) and orally treated with PG102T (50 mg / kg / day), PG102E (5 mg / kg / day), DEX (2.5 mg / kg / day) or distilled water (DW; 100 μl / mouse / day) once a day from 7 weeks to 14 weeks. SPF NC mice, as a negative control, received DW.

Effects of PG102T or PGl02E on the development of dermatitis in NC / Nga mice. The severity of dermatitis in each group of mice was evaluated once a week from 7 weeks to 14 weeks of age by two people who did not pay attention to distribution options, in accordance with a slight change in the criteria described by Leung et al. (Leung et al., J Allergy Clin Immunol 1990; 85: 927-33). The clinical signs and symptoms observed in normal NC mice began with itching, erythema and hemorrhage, followed by edema, superficial erosion, severe skin abrasion, peeling and dry skin, and developmental retardation. A mark was made before skin diseases, the nature of the scratch change was observed for 20 minutes on each mouse, and the itch index was evaluated by measuring the total scratch time for 20 minutes. The total clinical severity of the applied mark of AD-like lesions was determined as the sum of the individual marks made, differentiated as 0 (no), 1 (weak), 2 (moderate) and 3 (strong) for each of the five signals and symptoms (itching, erythema / hemorrhage, edema, skin abrasion / erosion and peeling / dryness).

Measuring the level of plasma immunoglobulins, cytokines and chemokines. A spontaneously induced allergic reaction was observed by measuring the level of plasma immunoglobulins, including IgE and IgG2a, cytokines and chemokines. Blood was collected from a retro-ocular plexus with a glass capillary tube at 7, 10, 12, and 14 weeks old and plasma samples were separated, stored at -80 ° C until use. IgE levels were determined using a mouse IgE detection kit (Shibayagi, Gunma, Japan) and this IgG2a was measured by an ELISA sandwich method as described by Hirano et al. (Hirano et al., J Immunol Methods 1989; 119: 145-50). The detection limit was 1 ng / ml IgE. Murine plasma levels of IL-4, IL-12, eotaxin and TARC were also measured by ELISA kit (Endogen, Cambridge, MA and R&D Systems, Minneapolis, MN) according to factory instructions.

The production of Th1 / Th2 cytokines from splenocytes obtained from NC / Nga mice. At 14 weeks of age, normal NC mice were parenterally administered PG102T, PG102E, DEX or DW, and SPF NC mice were sacrificed by decapitation. To study the production of cytokines from splenocytes, spleens of each group were obtained, and splenocytes isolated from the spleen were resuspended in culture medium (RPMI-1640 includes 10% heat inactivated FBS). A splenocyte suspension was selected for a 24-well culture plate, and the final concentration was 5 × 10 ⁶ cells / ml / well. These cells were then incubated in the absence or presence of ConA at 2 mg / ml for 3 days. ConA concentration was optimized from preliminary effect dose experiments and no cytotoxicity was detected at the concentration used in this experiment. Following incubation, supernatant cultures were collected to determine the level of cytokines (IL-4, IL-5, IL-10, IL-12, IL-13 and IFN-γ) ELISA as described above.

Analyzes of total white blood cells and eosinophils in peripheral blood. At 14 weeks of age, blood was collected from each group of mice. The total leukocyte and eosinophil counts in heparinized blood were counted using a Celldyn hemocytometer (Abbott; Santa Clara, CA).

Histological analysis and measurement from epidermal and skin layers in AD-like skin lesions. For histological examination, small biopsies were obtained from the scalp, neck and back of normal and SPF NC mice at 14 weeks of age. Skin sections were fixed in 10% phosphate-buffered formalin (pH 7.2), fixed in paraffin, cut into 4 μM and soaked with H&E to detect various cells of inflammatory infiltrate. Cells between the epithelium and the stratum corneum were observed under a microscope at 400X magnification. Then the field of view of the microscope was photographed, both layers of the epidermis and dermis were measured as the distance from the stratum corneum of the epidermis to the basement membrane from the dermis. The distance was expressed as the average of three random fields for which 5 measurements were averaged.

Determination of cytokine and chemokine expressed in skin biopsies. Levels of IL-4, IL-5, eotaxin, and TARC in muzzle skin biopsies were measured by ELISA. Briefly, tissue from damaged muzzle skin was excised, homogenized in lysis buffer, and then frozen / thawed, repeating three times. After centrifugation in supernatants containing total cellular protein, quantitatively determined and used to determine the level of cytokines and chemokines. The results led to the total amount of proteins prepared from tissue lysate. Protein lysate samples were prepared from the skin of facial tissue as described above, and were also subjected to Western blotting using specific mouse antibodies GATA-3, pSTAT6 (Santa Cruz Biotechnology, Santa Cruz, CA) or β-actin (Sigma) as a load control.

Statistics. Data were expressed as mean ± SEM, and differences between the quantitative mean values were analyzed by unpaired Student's test. A quantitative P value less than 0.05 or 0.01, which was calculated as a one-sided quantitative P value, was considered statistically significant.

results

Oral administration of PGl02T and PGl02E inhibitors of the development of spontaneous dermatitis in a normal mouse NC. To consider the possible effects of PGl02 in atopic dermatitis, we used NC mice as a model of human atopic dermatitis that showed atopic dermatitis-like skin lesions with age in common diseases. Conventional NC mice were orally administered PG102T (50 mg (60 units) / kg / day), PG102E (5 mg (54.5 units) / kg / day), DEX (2.5 mg / kg / day) or DW (100 μl / mouse / day), based on needs for 7 weeks and the development of atopic dermatitis. The dosage of PG102T and PG102E is based on the concentration that induces therapeutic effects in the OVA-sensitized mouse model used in previous experiments (see Example 1 and Park et al., J. Allergy Clin. Immunol., 116: 1151-1157, 2005). An increase in dermatitis in the severity rating of conventional NC mice treated with DW showed that the development of dermatitis progresses in an age-dependent manner (FIG. 5A). However, oral administration of PG102T or PGl02E significantly reduced the assessment made from 9 weeks of life. The improvement in the severity of dermatitis was accompanied by a decrease in the size of the applied scratch. Treatment with PGl02T significantly reduced the time of scratching by 9 weeks of life. Similar results were observed in the treatment of animals with PG102E (pigv). In addition, these results are comparable with observations made by direct analysis of the complete clinical visual trait of the mouse (data not shown). DEX also reduced the severity of dermatitis and the nature of the measurement of scratching. These data show that PG102T and PG102E can inhibit spontaneously induced dermatitis in this animal model.

PG102T and PG102E decrease the production of IgE and IgG1, while they increase the production of IgG2a in plasma. In addition to visual clinical signs mimicking human atopic dermatitis, normal NC mice also show an increase in plasma IgE after the onset of dermatitis. Therefore, it was investigated whether oral administration of PG102T or PG102E can control the level of plasma Th2-mediated IgE and IgG1 and Th1-mediated IgG2a. From 7 weeks old, animals were orally fed with PG102T, PG102E, DEX or DW, based on needs, and a blood sample was obtained at 7, 10, 12 and 14 weeks of life. On SPF diseases, NC mice normally produced approximately 150 ng / ml total IgE, but when animals were placed on common diseases, IgE levels gradually increased with age, to almost 17 mg / ml at 14 weeks of age. Administration of PG102T or PG102E reduced the level of plasma IgE by 10 weeks of age in a statistically significant manner, resulting in a 5-fold lower level of IgE until the end of the experiment. The IgE-reduced effect of PG102T and PG102E is comparable to DEX using as a positive control (FIG. 6A). At 12 weeks, the level of IgGl, aka Th2-mediated Ig class, was measured. For common diseases, DW-treated mice produced an IgG1 level greater than 5 mg / ml. However, the introduction of PG102T and PG102E reduced this level by 75% and 90%, respectively. The level of IgG2a, which belongs to the Thl-Ig class mediators, increases by approximately 180% in normal NC mice treated with PG102T. PG102E also induces plasma IgG2a levels (Figure 6B). These data show that PG102T and PG102E can inhibit the development of dermatitis by lowering IgE and IgG1 levels and increasing IgG2a.

PG102T or PG102E can regulate the balance of Th1 / Th2 cytokines produced in plasma and splenocytes. The above data show that PG102T and PG102E are effects on the production of Th1 and Th2 cytokines. To understand the activity of PG102T and PG102E at the molecular and cellular levels, IL-4 and IL-12 levels representing the Th2 and Th1 metabolic pathways, respectively, are measured in plasma at 12 weeks of age. Comparison with conventional NC mice treated with DW, IL-4 levels are reduced in mice treated with PG102T or PG102E by 60% and 76%, respectively (Table IVA). Oral administration of PG102T or PG102E increases the level of IL-12 in a statistically significant manner.

The inventors also analyzed the effects of PG102T and PG102E in the production of Th1 and Th2 cytokines in splenocytes isolated from NC mice. Mice were euthanized at 14 weeks of age and isolated splenocytes were obtained from their spleens. Splenocytes from each group of mice were stimulated with a T cell specific mitogen, ConA, on day 3, and various cytokine levels were determined. In the presence of ConA, the level of all Th2 cytokines increased significantly, but treatment with PG102T or PG102E decreased the levels of IL-4, IL-5, and IL-10 from 24% to 78% (Table IVB). DEX also inhibited the levels of all three Th2 cytokines, although the decrease in IL-5 by DEX was not statistically significant. Neither PG102 nor DEX affected the level of IL-13.

Table IV The effect of PG102T and PG102E on the level of Th1 and Th2 cytokines in plasma and splenocyte culture A. Plasma Levels Source IL-4 IL-12 Conventional NC mouse DW 474 ± 31 (100) 859 ± 17 (100) PG102T 189 ± 64 (40) ** 1217 ± 140 (142) * PG102E 113 ± 69 (24) ** 1106 ± 77 (129) * Dex 80 ± 56 (17) ** 236 ± 140 (27) SPF NC mouse 13 ± 5 (3) 1708 ± 116 (199)

B. Spleen levels Source IL-4 IL-5 IL-10 IL-13 IL-12 IFN-γ Conventional NC mouse DW 134 ± 17 (100) 680 ± 98 (100) 782 + 21 (100) 448 ± 1 (100) 656 ± 71 (100) 10407 ± 160 (100) PG102T 30 ± 6 (22) ** 357 ± 52 (53) * 621 ± 1 (79) * 348 ± 7 (78) 957 ± 68 (146) * 15848 ± 2074 (152) * PG102E 54 ± 7 (40) ** 203 ± 6 (30) ** 594 ± 19 (76) * 406 ± 11 (91) 1636 ± 42 (249) ** 18050 ± 1380 (173) ** Dex 54 ± 11 (40) ** 531 ± 283 (78) 416 ± 30 (53) ** 417 ± 2 (93) 149 ± 22 (23) 10096 ± 236 (97) SPF NC mouse 12 ± 2 (9) 33 ± 8 (5) 37 ± 3 (5) Nd 166 ± 1 (25) 390 ± 88 (4)

Plasma samples were isolated from each group of 12-week-old mice. All splenocytes from NC mice were stimulated by ConA during growing. Values are expressed as mean ± SEM for five animals. *, P <0.05 and **, P <0.01, against DW-treated mice (Student T-test). ND = undetectable.

When cells from normal NC mice were grown in the presence of ConA, IL-12 and IFN-γ levels increased. In SPF NC mice, the level of IL-12 was 166 pg / ml, but ConA stimulation increased the level 4-fold. Treatment with PG102T or PG102E further induced an IL-12 level of approximately 150% or 250%, respectively. The level of IFN-γ also dramatically increased in the presence of ConA by almost 11 ng / ml. PG102T administration increased the level of this cytokine by approximately 150%. It turned out that PG102E is more powerful than PG102T. DEX potentially suppressed the level of IL-12, while it did not give such an effect as IFN-γ. In conclusion, PG102T and PG102E increased the level of Th1 cytokines, while a decrease in these selective Th2 cytokines, in contrast to DEX, which randomly inhibits the expression of almost all cytokines measured in this study.

PG102 not only interferes with eosinophils, but also reduces the levels of eotaxin and TARC. Cutaneous inflammatory cell infiltration includes eosinophils, an important feature of atopic dermatitis in NC mice. Because the presence of inflammatory cells in skin lesions may be the result of their mobilization from bone marrow to blood, the inventors were the first to analyze the number of total leukocytes and eosinophils in peripheral blood of 12 weeks of age. As shown in FIG. 7A, the total leukocyte count of normal NC mice increased by the appearance of dermatitis. In particular, the number of eosinophils has increased significantly in DW-treated mice for common diseases resulting in eosinophils. However, PG102T or PG102E administration of a small amount of both common leukocytes and eosinophils may help to prevent eosinophilia.

Changes in the number of circulating eosinophils can be a manifestation of the production of chemokines, leading to chemo-attraction in the inflammation reaction (3-6). Consequently, plasma eotaxin and TARC levels, which are characteristic of chemoattractants from eosinophils and Th2 cells, are determined. In a typical environment, DW-treated mice cause an increase in eotaxin and TARC levels, but their levels decrease in mice treated with PG102T or PG102E by about 25% to 50% (FIG. 7B). There is a slight change in animals using DEX. These results show that PG102T and PG102E inhibited the production of eotaxin and TARC, leading to the prevention of Th2-mediated eosinophilia, which is usually the same as the appearance of dermatitis in NC mice.

Administration of PG102T or PG102E inhibits the infiltration of inflammatory cells into the dermis and thickening of the epidermis and dermis. The improvement in the skin disease clinic and the inhibition of the Th2 response of PG102T and PG102E have also been confirmed by H&E analyzes of stained sections of 14 weeks of age. Mice fed with DW showed a marked thickening of the epidermis and dermis, secreted by hyperkeratosis, inflammatory cell infiltration, and hemorrhage. Morphological studies have shown that these infiltrated cells in the dermis are eosinophils, mast cells and lymphocytes. However, treatment with PG102T or PG102E for 7 weeks inhibited thickening of the epidermis and dermis and the infiltration of inflammatory cells into the dermis, resulting in a histological environment very similar to SPF NC mice (FIG. 8A). The introduction of DEX also gives similar results, but significantly expands the adipocytes of the area. Measurement of the epidermal and dermal layers on the skin of the muzzle and back also showed that both PG102T and PG102E prevented epidermal and dermal hyperplasia in a statistically significant manner (Fig. 8B). These results showed that oral administration of PG102T or PG102E can effectively suppress the development of dermatitis in NC mice with small or non-side effects.

PG102 or PG102E reduce the expression of Th2-mediated cytokines and chemokines that directly suppress the effect of GATAS. To study the effects of PG102T or PG102E in Th2-mediated production of cytokines and chemokines in mouse skin, IL-4, IL-5, eotaxin and TARC levels were measured by ELISA at 14 weeks of birth. In the skin of mouse SPF NC mice, all four proteins were poorly expressed, but their levels were significantly increased in normal NC mice. Administration of PG102T or PG102E lowered the levels of IL-4, eotaxin, and TARC by more than 30%. The effect with pronounced IL-5 is more prominent, with its level there was an increase of almost 90%. Conversely, DEX inhibits the levels of IL-5 and TARC, but not IL-4 and eotaxin (figa).

These findings are based on the expression of a transcription factor, including STAT6 and GATA3, determined by immunoblotting. STAT6 and GATA3 are well known to play a critical role in the differentiation of Th2 cells and the production of Th2-specific cytokines and chemokines (Arakawa et al., Clin Exp Immunol 2004; 135 (3): 505-10; Gunther et al., J Allergy CHn Immunol 2004; 113: 987-94; Konishi et al., Proc Natl Acad Sci 2002; 99 (17): 11340-5). As shown in FIG. 9B, the GATA-3 protein level is reduced for both PG102T and PG102E. The expression of phosphorylated STAT6 (pSTAT6) is also reduced in normal NC mice treated with PG102T, whereas this may not be true with PG102E. DEX suppressed the level of both GATA3 and pSTAT6. These results demonstrate that PG102T and PG102E can suppress the level of Th2-specific cytokines and chemokines that inhibit the expression of GATA3.

Discussion

AD is a large allergic disease that usually begins during early childhood. The characteristic fraction affected individual development of asthma and / or allergic rhinitis in later life (Leung, Clin Exp Immunol 1997; 107 (suppl. L): 25-30). AD occurs as a result of dermal inflammation due to an abnormal immune response, in particular, over-activation of the Th2 metabolic pathway. The inventors found that PG102T and PG102E, water-soluble fractions obtained from A. arguta, regulating the production of selective Th1 and Th2-mediated cytokines and also that IgE in an OVA-sensitized mouse model (see example 1). These data are based on the argument that plant extracts may be useful in treating various allergic diseases (Mayaumi et al., J Allergy Clin Immunol 2000; 106: 159-66; Hisae et al., Phytother Res 2001; 15: 506-10 ) In this example, the inventors examined any of the two PG102, which can give any valid therapeutic effect (s) for atopic dermatitis using NC / Nga mice as a system model. The results show that PG102T and PG102E suppress the development of spontaneously induced dermatitis, which, not being theoretically related, believes in the future to directly control various Th1 and Th2-related factors, namely, inactivation of IL-4, IL-5, IL-10 and IgE as well as increased regulation of IL-12 and IFN-γ. The biological consequence of this kind of biochemical changes in the NC / Nga mouse model includes a significant decrease in the number of eosinophils in the peripheral blood, as well as in skin lesions, suppression of the thickening of the epidermis and dermis, and inhibition of the infiltration of various inflammatory cells. Of particular interest is the reduction in the local expression levels of eotaxin, TARC, IL-4 and IL-5. The levels of these proteins are excessively high in cutaneous lesions. NC / Nga mouse growth is below normal. When animals are orally administered PG102, however, these chemokines and cytokines are actually at a normal level. Eotaxin, together with IL-5, since it is known to be a potent chemoattractant for eosinophils, while TARC is produced by keratinocytes (and also Th2 cells), it is thought to induce Th2 cells and induce a pathological response, mainly due to atopic dermatitis. In this regard, it makes sense to note that the receptors for eotaxin and TARC are CCR3 and CCR4, respectively, which are strongly expressed in Th2 cells (Christian et al., J Clin Invest 1999; 104: 1097-105; Tomomi et al., J Allergy Clin Immunol 2110; 107: 353-8; Weilie et al., J Clin Invest 2002; 109: 621-8; Masayuki et al., IntAch Allergy Immunol 2003; 132: 355-63).

Detailed molecular mechanism (s) and the exact sequence of underlying therapeutic effects of PG102 observed in the NC / Nga mouse model are still being investigated. It is possible that PG102 first affects cellular transcription factors, such as GAT A-3, and subsequently the expression of key cytokine players involved in Th1 and Th2 systems, such as IL-4 and IFN-γ, causing a cascade reaction leading to a decrease in the level of IgE and corresponding chemokines (Zhu et al., Nat Immunol. 2004; 1 1: 1157-65). Alternatively, PG102 may primarily work locally; for example, a decrease in the level of eotaxin and TARC, inhibition of their chemoattractant functions, a decrease in the number of eosinophils at the systemic and local levels, and suppression of histopathological development observed in a normal growing NC / Nga mouse. PG102 activity can be associated with numerous compounds that affect different levels of an allergically linked biochemical metabolic pathway. Full, it turns out that PG102 works in the vein of biological factors associated with atopic dermatitis in this animal model, treating the disease at the rate of its preferred agent, in addition to easily alleviating the symptoms.

PG102T and PG102E are obtained from edible fruits. No toxicity was found in repeated dose toxicity experiments in which 2000 mg / kg / day was 40-fold higher than the concentration used in this study was applied based on daily requirements for 12 weeks. Together with data from previous experiments, including OVA-sensitized mice, results from NC / Nga mice demonstrate that PG102 is a safe and effective reagent for the treatment of various allergic diseases, including atopic dermatitis. Given the frequency of diseases of atopic dermatitis, which are increasing in all large developed countries and do not actually have a reagent for the main treatment of atopic dermatitis, drugs such as those described here represent an achievement in this field.

Example 3

The following example shows a preparation of various agents, including A. arguta, which are used in the following examples.

Plant material

Stems (consisting of reeds and fruitful spurs), roots and bark of Actinidia arguta (Sieb. Et Zucc.) Planch, ex Miq. (Actinidaceae) cultural 'Ananasnaya' collected at Hurst Berry Farm, Sheridan, OR. Control samples (# 518640) certified by Mr. Tim Hogan, Collection Manager, University of Colorado Herbarium, The University of Colorado, Boulder, CO, and set aside in one place. The plant material was air dried for 48 hours and stored at room temperature until extractions or other treatments.

The ripe, ready-to-eat A. arguta fruits were harvested by Hurst Berry Farm, immediately frozen, shipped and stored frozen (-20 ° C) until extraction or other treatments.

Extracts and other preparatory work

A powdery stem (126.6 g), powdery roots (79.0 g) and finely ground bark (126.2 g) were each extracted with distilled water (1 L) at 94 ° C for 4 hours. The mixture was then filtered and the filtrate was concentrated to drying by rotary evaporation to obtain a stem extract (9.9 g), root extract (8.6 g) and bark extract (2.4 g).

Twenty frozen A. arguta berries (154.4 g) were thawed at room temperature, crushed and extracted with distilled water (1 L) 91 ° C for 5 hours. The mixture was filtered, and the filtrate was concentrated to obtain a 'decoction' of fresh fruit extract (12.8 g).

Additionally, a freshly frozen kiwi plant (341.6 g) was thawed and driven away with a juicer. The peel was removed from the fetus with a juicer, and mixtures of seeds, pulp, and juice were obtained. This mixture was centrifuged (30 minutes, -3500 rpm) to obtain 150 ml of juice. This juice was concentrated by drying by rotary evaporation to obtain a fruit juice extract (24.2 g).

In order to produce a large amount of PG102T equivalent extract (as described in Example 1), a large-scale extraction of a kiwi plant was performed (Sungil Bioex Co., Ltd., Bibong, Korea). The frozen kiwi plant (1242 kg) was divided into parts (1/4 "to 3/8" thick) and dried in a convection dryer (65-80 ° C) to a moisture content of 5-20%. A single extraction (Fig. 10) of a dry fetus (239 kg) was performed in a stainless steel reactor enclosed in a casing with an internal strainer supporting the extraction load. An external cooler was an obstacle to the loss of water during extraction. The amount of extraction solution (water) was based on the extraction of 5-10 times the mass of dry fruits. The capacity of the extraction apparatus was heated from 0 to 90 ° C for 2 hours by introducing steam into the jacket of the reactor. Water (90 ° C) was then recycled through biomass using an external recirculation circuit for 4-12 hours. Subsequently, the used biomass was removed for sale and the aqueous extract was filtered through a 10 micron filter. The filtrate was then concentrated in vacuo (-600 mmHg) at 55-65 ° C in a stainless steel stirring reactor equipped with an external cooler and receiving distillate. Once the material was concentrated, it was maintained at 80 ° C for an additional 30 minutes to sterilize the extract. The resulting material (101 kg), equivalent to PG102T, was designated as FD001. Of this material, 3 kg were set aside for further research. The rules for organizing the production and quality control of medicines were used throughout the process.

Creating a powdery material suitable for encapsulation and suitable for clinical use described herein, FD001 concentrate produced as described above (98 kg) was pumped out with a horizontal paddle mixer and mixed with equal weight based on the calculated dry residue from microcrystalline cellulose (MCC). Following this, a homogeneous mixture is transferred to a stainless steel tray, which is located in a forced dryer heated with hot air (70-80 ° C) for 24 hours. The dry, lumpy solid was then ground in a Fitzmill type hammer mill to produce 40 mesh powder (118 kg). This material was encapsulated (GMP Laboratories of America, Inc., Anaheim, CA) in 300 mg or 600 mg capsules, each containing a 1: 1 mixture of FD001 and MCC, for use in canine and human clinical trials.

The dry A. arguta fruit (7.0 g) from the processed-weighed material was divided into parts and dried, as in the initial step described above (but not subjected to a single extraction), turned into a powder and the material was isolated with water (250 ml) 25 ° C for 4 hours. The mixture was filtered and the filtrate was concentrated to dryness by rotary evaporation of the aqueous extract at room temperature from a dry fruit (4.2 g).

FD001 (79.9 g) was mixed with 1.5 L of distilled H ₂ O and this solution was sequentially isolated with four 500 ml portions of ethyl acetate (EtOAc). The combined organic layers and the aqueous layer were concentrated to dryness in vacuo, resulting in an EtOAc extract (7.4 g) and an aqueous residue (41.5 g).

Example 4

The following example describes in vitro testing of immunomodulatory activity in A. arguta preparations.

The purpose of this study is to compare the relative ability of various extracts and preparations made from A. arguta to alter the production of cytokines (IL-4, IL-5, IL-10, IL-13 and IFNγ) in a splenocyte culture derived from egg albumin (OVA , class V, Sigma) -sensitized mice using ELISA (Quantikine kits, R&D systems) analysis. The following samples (prepared as described in Example 3 above) were tested: FD001 (PG102T), fruit juice concentrate, and EtOAc extract.

Isolation and cultivation of splenocytes

Female Balb / c mice (Harlan, Indianapolis, IN) were sensitized with IP injection of 20 μg OVA on days 0 and 14. On day 24, after killing by dislocation of the cervical vertebrae, spleens were aseptically isolated from each mouse and immediately treated to obtain a splenocyte culture in a sterile manner. The spleen was dissociated in the presence of 10 mM HEPES of RPMI-1640 buffered medium, tissues were gently squeezed through the holes of a 70 micron nylon sieve using a piston of 3 cc syringes. Large cell aggregates were removed from the resulting suspension using an FCS gradient. Then the splenocytes were centrifuged (1500 rpm, 5 minutes) and the resulting debris was treated with RBC lysis buffer (10 minutes, RT) to remove contaminating red blood cells. Most of the RBC lysis buffer was removed by centrifugation (1500 rpm, 5 minutes) and the deposited splenocytes were then washed with 3 × 10 mM HEPES buffered with RPMI-1640 medium. After final washing, the precipitated splenocytes were resuspended in a volume of RPMI-1640 containing 10% FCS and Penn / Strep (complete medium), designed to obtain a final cell density of 5 × 10 ⁶ cells / ml. For each analysis, 5 × 10 ⁶ splenocytes were seeded in separate wells of a 24-well plate. On day 3, supernatants from these wells were collected and frozen in the preparation to determine experimental results.

Control splenocyte cultures were also obtained from intact (non-sensitized) mice as described above and plated in separate wells of a 24-well plate to achieve a final cell density of 5 × 10 ⁶ cells / ml. These splenocytes adapted in RPMI-1640 medium containing 10% fetal calf serum, Penn / Strep, and were further processed. On day 3, supernatants from these wells were collected and frozen, using further in the experiment as a negative control.

Stimulation of splenocyte cultures with A. arguta preparations

Ten OVA-sensitized mice were used for each study drug. Splenocytes from each mouse were seeded (5 × 10 ⁶ cells / ml) on 8 separate wells of a 24-well plate in complete RPMI-1640 medium containing 100 μg / ml OVA, 0.5% DMSO and with or without, or the selected concentration of each specific investigated drugs. 6 out of 8 wells were divided into 2 sets of 3 wells. Each well in sets of 3 was treated with A. arguta preparations at a concentration of either 0.25, 1.0, or 10 mg / ml. To serve as a positive control, 7 wells were treated with 2 μM dexamethasone (DEX), a potent anti-inflammatory glucocorticoid. The 8th wells received no further treatment and served as the OVA experimental control only. After 3 days of cultivation, the supernatants of each of the 8 wells for each OVA-sensitized mouse were collected and frozen. These supernatants were used to determine the levels of IL-4, IL-5, IL-10, IL-13 and IFN-γ cytokines present in the culture medium.

Determination of cytokine levels in culture supernatants

The cytokine levels in the culture supernatants obtained from splenocytes treated with A. arguta, splenocytes treated with DEX, splenocytes treated with OVA only, and untreated splenocytes from non-sensitized control mice were determined using ELISA. Two repeating ELISA plate wells were used to determine the level of each cytokine.

results

This in vitro work confirmed the activity of PG102T-equivalent FD001 (FIG. 11), EtOAc extract (FIG. 12), and fruit juice concentrate (FIG. 13) A. arguta. It was observed that three drugs (10 mg / mg) caused significant suppression, to varying degrees, of the cytokines IL-4, IL-5, IL-10, IL-13, and IFNγ. Most of the specific effects were observed on IL-13 and IFNγ for all studied samples comparable to previous in vitro work (Table I; Example 1). Since activity was observed in the EtOAc extract, it is obvious that the active component shown in FD001 is extracted in organic solvents and can be further purified by conventional chromatographic methods. Significantly, the fruit juice concentrate also suppressed the production of cytokines by splenocytes, showing that the extraction of a kiwi plant, as shown in examples 1 and 2, is not an exceptional condition for obtaining active preparations of a frost-resistant kiwi plant. It was found that a relatively low inhibition of cytokines in the fruit juice concentrate was found, suggesting that the drying or heating process used in the preparation of FD001 may be important for enhancing activity.

Example 5

The following example describes an in vitro comparison of the activity of extracts of non-fruit parts of A. arguta, as well as alternative preparations of the fetus A. arguta.

The aim of this study was to evaluate the ability of A. Arguta extracts that were obtained from parts of the plant other than the fetus or from alternative fetal preparations (that is, other than the extracts described in examples 1 and 2) to reduce the production of cytokines (IL-13 and IFNγ) in cultures of splenocytes derived from ovalbumin-sensitized mice using ELISA. The following examples (prepared as described above) were investigated: aqueous extracts of the stem, root, bark of A. arguta, prepared as described in example 3; preparations of “boiled” fresh fruits; fruit juice concentrate prepared as described in example 3; FD001 (the common equivalent of PG102T) obtained as described in example 3; FD001 powder prepared as described in Example 3 (used for the clinical studies described below); room temperature extract of dried A. arguta fruit; EtOAc extract prepared as described in Example 3; and an aqueous residue, also described in Example 3. In addition, the activity of three known immunosuppressive compounds, cyclosporine, dexamethasone and quercetin, was evaluated as controls.

Isolation and cultivation of splenocytes

Obtaining splenocytes was carried out in the same way as described above in example 4.

Stimulation of splenocyte cultures with A. arguta extracts

Splenocyte cells from 8 OVA-sensitized mice (8 replicants) were used to analyze each extract or study drug. 5 × 10 ⁶ splenocyte cells from each mouse were plated on separate wells of a 24-well plate in complete RPMI-1640 medium containing 100 μg / ml OVA and 25 mM HEPES (pH 7.3), 1 ml per well. Kiwi plant preparations were investigated at a concentration of 1.0, 3.0 and 10 mg / ml.

Analysis of cyclosporine, quercetin and dexamethasone

Splenocytes from 8 OVA-sensitized mice (8 replicants) were used to analyze each test compound, with the exception of quercetin, where splenocytes obtained from 2 only OVA-sensitized mice were tested. 5 × 10 ⁶ splenocytes from each mouse were plated on individual wells of a 24-well plate in complete RPMI-1640 medium containing 100 μg / ml OVA and 25 mM HEPES (pH 7.3), 1 ml per well. Cyclosporin was studied at a concentration of 0.0083, 0.083 and 4.15 μM. Dexamethasone was studied at a concentration of 0.01, 0.1 and 1 μM. Quercetin was studied at a concentration of 1, 10, and 25 μM. Wells were treated with 1 μM dexamethasone, a potent anti-inflammatory glucocorticoid serving as positive experimental controls. Wells receiving only complete RPMI-1640 medium containing 100 μg / ml OVA and 25 mM HEPES (pH 7.3) served as OVA experimental controls only. After 3 days of cultivation, supernatants were collected and frozen. These supernatants were used to determine the levels of IL-13 and IFN-γ present in various culture media in the experimental conditions under study.

Determination of cytokine levels in culture supernatants

Cytokine levels in culture supernatants from all treated and control wells were determined by ELISA. Two repeating ELISA plate wells were used to determine the level of each cytokine.

Based on the results of the in vitro study described in example 4, only the expression of IL-13 and IFN-γ was analyzed to assign an estimate of the activity levels presented in the test materials.

results

As shown earlier, a stronger suppression of the studied cytokines was observed, since the concentration of the studied materials A. arguta increased. In general, suppression is more pronounced against IFN-γ. The activity of the prescribed immunosuppressive compounds was similar to A. arguta in this study. The cyclosporin peptide and glucocorticoid steroid dexamethasone showed potent activity (<1 μM), as shown in figa. Flavonoid quercetin showed potent activity in a slightly higher concentration range (1-25 μM, FIG. 14B). Further confirmation of the ability of the EtOAc extract to FD001 activity is shown in figa and 15B. Interestingly, activity was also observed present in aqueous residues, showing that both polar and non-polar components can be responsible for the in vitro immunosuppressive effect. The FD001 powder, which was used in both canine and human clinical trials (described below), was confirmed to be active in this study, as shown in FIGS. 16A and 16B.

As described in Example 4, alternative methods for preparing extracts of the kiwi plant, but not the methods described in Examples 1 and 2, were investigated. All fruit-derived extracts that are dried or fresh, or extruded in hot or room temperature water, exhibiting similar activity in this study, as shown in FIGS. 17A and 17B. Based on this analysis, the inventors of the present invention suggest that there are several competitive alternative methods for preparing a kiwi plant for therapeutic purposes.

Interestingly, hot water extracts prepared from the bark, root and trunk of A. Arguta showed equal or higher activity in this study (FIGS. 18A and 18B) when compared with FD001 (PG102T) and fetal juice concentrate. These results indicate that these other parts of the plant may represent alternative sources of compounds therapeutically interesting for regulating immune markers or suppressing pro-inflammatory cytokines.

Example 6

The following example describes the results of a double-blind, placebo-controlled, outpatient study of the effectiveness of FD001 (PG102T) in adult individuals with moderate atopic dermatitis.

The purpose of this study is to obtain preliminary evidence of the effectiveness of FD001 (PG102T), administered orally for 42 days, in a small number of adult volunteers with atopic dermatitis (AD) of moderate severity. Additional study objectives evaluated tolerance and variability of the response to FD001.

Research plan

Individuals consumed FD001 powder (prepared as described in Example 3) in two 600 mg capsules (600 mg of the total dose of FD001) in the morning, or two placebo capsules consisting of MCC for a period of 42 days starting on day 1 of the study. Individuals were instructed to take half of the steroid medication after 14 days. Blood was used for routine biochemistry and hematology at four time points: on screening for suitability, and on days 1, 14 and 42 of the study. IgE and C-reactive protein levels were measured in blood at days 1, 14 and 42. Urine was collected on all 4 days for routine urinalysis. Performance evaluations were made at each visit after screening. Individuals were either men or women, 19 to 65 years of age, and usually in good health. Individuals had moderate-grade active atopic dermatitis as determined by the Physician's Global Assessment (Feldman and Krueger, Annals of the Rheumatic Diseases 64: ii65-ii68, 2005) with a score of three on a severity scale of 0 to 4. Individuals had AD of at least 10% body surface area (BSA). At that time, individuals used a local steroid to treat AD and were not nursing or pregnant. Safety and tolerance were assessed using an adverse reaction report and standard blood biochemistry, hematology and urinalysis.

Statistical Methods

The primary efficacy variable was a change from baseline at Physician's Global Assessment at day 42, by analysis using the Cochran Mantel-Haenszel criterion (Armitage et al, Statistical Methods in Medical Research, 4 ^th Ed., Blackwell, Oxford, 2002). Secondary variables were changes at 42 days from baseline in symptoms of AD (assessment of erythema severity, induration, secretions / scabs, and itching), and total BSA as analyzed using a two-sample t-test. Descriptive statistics were presented for all initial and subsequent data from the treatment group on days 1, 14, 28, and 42. These statistics include sample size, means, standard deviations, repeatability, percentages, and confidence intervals, as appropriate. The results from individual self-assessment questionnaires were calculated and reported to the treatment group. Any adverse events observed during the study were recorded. Descriptions of adverse events included outbreak data, data on a completed or incomplete event, severity of the event, determination, measures taken, therapy taken and outcome. These data were categorized by the number of individuals reporting adverse events, body system, severity, severity and relationship with the test sample. Comparisons between treatment groups were made by tabulating the repeatability of individuals with one or more adverse events, classified in terms of the MedDRA (Medical Dictionary for Regulatory Activities, http://www.meddramsso.com) during the study.

Descriptive summary statistics of laboratory values and the associated change from baseline (day 1) were determined for all clinical and laboratory assessments. Values outside the normal range have been marked in the data lists. In addition, “shift tables” were compiled showing the number or percentage of individuals who experienced changes in laboratory parameters during the course of the study (for example, a change from normal to high, based on a laboratory normalized range of values).

Table v Group* Number of individuals responding % of the total inappropriate % of the total A Fd001 25 16 64.0 9 36.0 Placebo 26 eleven 42.3 fourteen 53.8 B Fd001 eighteen eleven 61.1 7 38.9 Placebo 24 eleven 45.8 13 54,2 C Fd001 fourteen 8 57.1 6 42.9 Placebo 17 6 35.3 eleven 64.7

* A = all individuals, B = consumers of clobestasol & ultravate removed, C = only consumers of HDRCRT corticoid or triamcinolone.

Performance results

In a preliminary analysis conducted on the first 17 individuals to conduct the study, effective statistical trends were obtained at two secondary endpoints of effectiveness: erythema (p = 0.13, ITT 17) and compaction (p = 0.09, ITT 17). During the preliminary analysis, no statistical discrepancies were found at the primary efficacy endpoints in this small sample.

In the final analysis on day 42, there was no statistical significance shown by the group of patients in the variable primary efficacy (Physician's Global Assessment) or variables of secondary efficacy (change from baseline in symptoms of AD and percentage change in BSA). However, on day 14, a significant distribution shift was observed at the primary efficacy endpoints. This shift, observed between 1 and 14 days, can be seen in FIGS. 19A and 19B for the responses of the placebo recipients and the test sample, respectively (indicated by the PGA classification criterion). When individuals were divided into responding and non-responding groups during the analysis on day 14 (Table V, group A), a significant percentage of responders were observed in the FD001 treatment group, while non-responders showed a significant percentage in the placebo group. These stored results are relatively consistent, if individuals with stronger steroids (group B) are excluded, as well as for individuals with only a weakening to moderate steroid treatment (group C). Additional analysis then revealed statistical significance (p = 0.02) for the primary endpoint of the PGA and patient self-assessment for redness (p = 0.03) on day 14. In addition, effective statistical trends were found in self-assessments at day 14 for pruritus and for evaluating clinical symptoms for discharge / scab (p = 0.08 and p = 0.07, respectively). The observed efficacy of the treatment group compared to the placebo group on day 14 via the PGA endpoint, distributed on day 42. These results indicate that the use of FD001 (PG102T) as an adjunct to topical corticosteroid therapy may be beneficial in the treatment of AD. The results of the laboratory study for 1, 14 and 42 days did not show any harmlessness correlated with the trends for any study presented, including clinical biochemistry, clinical analysis of blood with variations, blood coagulation, indirect bilirubin and macroscopy of urine. No difference was found between the treatment group for IgE, C-reactive protein or the number of eosinophils.

Harmlessness results

No serious adverse events were reported for either treatment with FD001 or placebo. For FD001, 12 non-serious events and 13 for placebo were reported. Of these, for FD001 there were 10 mild to moderate and 2 severe events, and 13 mild to moderate events for placebo. None of the events was considered probable or related to the study of the test sample or placebo.

Conclusions

The planned results of the analysis showed no statistical significance between the groups of patients in the primary and secondary endpoints on day 42. However, an effective distribution shift was detected at the primary endpoint (PGA) on day 14. For this reason, a subsequent analysis was performed for this time point, which showed a statistically significant difference between the groups of patients at the 14-day time point. In addition, several secondary endpoints showed either statistically significant (erythema p = 0.03 for self-esteem) or statistically effective trends (improved pruritus p = 0.08 for self-assessment and excretion / scab p = 0.07 in symptoms of AD assessment). No serious adverse events have been reported in the studies. The number of adverse events reported for the group and the test sample and placebo were comparable and not associated with the test sample. In clinical laboratory values, no hazard factors were found. These data confirm the preclinical studies of small mammals and human studies based on separate examples showing that the extract of A. arguta fetus is harmless and well tolerated by recipients.

Example 7

The following example describes a randomly selected, double-blind, placebo-controlled study to evaluate the use of the extract of a frost-resistant kiwi plant to reduce the CADESI score (Olivry et al, Vet. Dermatol, 13: 77-87, 2002; Hanifm et al, Exp. Dermatol, 10: 11-18, 2001; Kunz et al., Dermatology, 1997, 195, 10-19) of atopic dogs.

The aim of this study was to evaluate the effectiveness of A. arguta fetal extract FD001 (PG102T) as an adjunct therapy for standard steroid treatment for atopic dermatitis (AD) in dogs. Response to treatment was assessed using a worldwide test rating that includes the CADESI scale and manufacturer's ratings of Pruritis. Additional research objectives are presented to evaluate the effectiveness of a study of the effectiveness of a kiwi plant extract as monotherapy, to reduce the need for steroids in the management of clinical symptoms of AD, to evaluate harmlessness.

Research plan

A two-week period with a low dose (0.2 mg / kg) of steroids only (prednisone) was performed to determine steroid sensitivity in dogs, as long as the residual symptoms of AD persisted. All dogs were healthy, excluding non-seasonal AD skin disease. The diagnosis of AD included at least three positive reactions to the skin test of skin allergy. Dogs received flea control treatment (Advantage), and a nutritional study was examined to rule out food allergies as the main cause of symptoms. Dogs were free of concomitant medications such as antihistamines and long-acting steroids and free of any secondary infection. A minimum basic (day 1) rating of 25 on the CADESI scale was required for inclusion.

During an additional treatment (days 14-42), dogs who received oral prednisone (0.2 mg / kg) every other day in addition to the test sample. The test individuals (directed record of 60) ate FD001 powder (prepared as described in Example 4) at a dose of 30 mg / kg once daily, or placebo (MCC), for a period of 28 days. Individuals were randomly selected into groups of the test sample or placebo, in a 1: 1 ratio. Individuals were evaluated using the CADESI scale and producer ratings in weekly diaries. Blood samples were collected to assess the safety and secondary critical points on the first day of the steroid alone and on days 1 and 28 of the study. Assays of these samples included measurement of a clinical blood test, clinical chemistry, total IgE, allergen-specific IgE, and TARC chemokine.

At the end of the follow-up period, any dogs showing an improvement in their overall score will be moved on day 28, an open labeled second study period consisting of the FD001 test sample (30 mg / kg / day) as monotherapy for AD. Individuals who have relapsed in symptoms of AD or require rescue treatment will be recognized second stage treatment disorders and the study is interrupted. Individuals retaining their improvements in stage two will be considered responding to treatment. A CADESI score and Pruritis scores will be done every seven days. Blood samples were collected during stage two on days 14 and 28. The group of laboratory blood tests will be identical to that in the first stage of the study.

Statistical Methods

The primary efficacy analysis will be the ratio of individuals with a positive response to treatment based on a worldwide chi-square test assessment. A temporary analysis may be performed when the first half of those sent by appointment completes the study. p <0.05 seems reliable.

Conclusions

Atopic dermatitis is a secondary most common allergy in dogs, occurring in approximately 10% of the dog population (Scott et al., Small Animal Dermatology, 5 ^th Ed., WB Saunders, 500-518, 1995). Usually, AD in dogs has a tendency to worsen with age. Sick animals suffer from repeated skin and ear infections, which greatly reduce their quality of life. Despite the fact that AD is a very common disease, possible therapeutic options are limited. Systemic treatments like glucocorticoids and cyclosporine may be effective, but have the potential for side effects (Olivry et al., Vet. Dermatol, 13: 77-87, 2002; Ryffel, et al., Arch. Toxicol., 53: 107-141, 1983). Glucocorticoids, which tend to decrease effectiveness in chronic use, oral cyclosporine can be prohibitively costly in most breeding dogs, and the success rate of antihistamines is often reduced (Scott et al., Small Animal Dermatology, 5 ^th Ed., WB Saunders, 500-518 , 1995). Establishing the safety and efficacy of treatment to reduce the symptoms of canine AD is probably a huge benefit. The effectiveness of FD001, either as adjunctive treatment or as monotherapy, may reduce the need for steroids in the management of AD in dogs. It is also envisaged that extracts of the kiwi plant will be used as adjunctive therapy to reduce the activity of the steroids used. It can be assumed that the extract could be used in the form of a capsule, powder mixed with food, or a food component. A dietary supplement of kiwi plants may be effective in maintaining healthy skin in dogs with atopy, when used alone or in combination with a low dose of steroids.

Example 8

The following example describes the use of A. arguta and its extract for regulating the immune response of an allergic and non-allergic inflammatory disease.

The purpose of this study is to provide preliminary evidence of the efficacy of FD001 (PG102T), administered orally for a period of 28 days, in adult volunteers with an allergic disease such as atopic dermatitis (AD), asthma, or mild allergic rhinitis. The second objective of the study is to measure the bioavailability of the product using alternative forms of products (for example, capsules, concentrate added to the drink, emulsion or food component). Another objective of the study is to determine the effect of FD001 on levels of pro-inflammatory blood markers, such as cytokines, chemokines, leukotrienes or antibodies, on the proliferation of myeloid cells (e.g., white blood cells, macrophages, mast cells, etc.) and on the regulated expression of genes and transcription factors.

A double-blind, placebo-controlled, outpatient study is conducted in which the person used FD001 as a test sample or placebo. The study group may include FD001 such as monotherapy or as adjuvant therapy with moderate oral steroids to alter activity. Alternatively, steroid treatment for people with AD may be local. Steroid use may be in the form of an intranasal spray or inhaler for people with asthma and allergic rhinitis, respectively. Human inclusion criteria, assessment effectiveness, safety and tolerance and statistical analyzes are evaluated using methods similar to those described in example 6.

One manifestation of allergies, atopic dermatitis, is a skin disorder in children and is usually observed during the first 6 months of life (Spergel and Paller, J. Allergy Clin. Immunol., 112: S128-S139, 2003). It turns out that the spread of AD is increasing worldwide, as well as other atopic disorders, including asthma (Larsen and Hanikin, Immunology and Allergy Clinics of North America, 22: 1-25, 2002; Wollenberg et al., Clin. Exp. Dermatol, 25 : 530-534, 2000; Mannino et al., Mor Mortal WkIy Rep CDC Surveill Summ., 47: 1-27, 1998; Linneberg et al., Allergy, 55: 767-772, 2000). Serious negative effects of test patients with AD on quality of life and existing treatment options can be a source of adverse side effects and a financial burden for both the family and society. Cutaneous manifestations of atopy often represent the onset of atopy. Based on several lengthy follow-up studies, approximately half of AD patients develop asthma, especially with severe AD, and two-thirds develop allergic rhinitis (Leung et al., J. Clin. Invest., 113: 651-657, 2004; Spergel et al., J. Clin. Invest., 101: 1614-1622, 1998). Identification of safe and effective treatment for AD is probably greatly appreciated. The effectiveness of FD001 can also reduce the amount or activity of steroids or other drugs used in the treatment of AD, asthma, or allergic rhinitis.

The subject of a later consideration of the case may also be the use of extracts of the kiwi plant, concentrates, other drugs, or extracts of other parts of the plant (for example, bark, stem, roots, leaves), for additional treatment or as monotherapy for AD, asthma, allergic rhinitis , or other leukotriene-mediated pathological conditions, such as food allergies and chronic urticaria. As a logical extension of this work, one can further investigate the use of kiwi plant products as a therapy for allergic pathological conditions in small mammals (for example, dogs, see also example 7).

Each link or publication described herein is incorporated by reference in its entirety.

While various embodiments of the invention have been described in detail, it is clear that changes and adaptations of these embodiments may come to mind to those skilled in the art. It should be understood, however, that such changes and adaptations are covered by the present invention, to the extent provided by the subsequent claims.

Claims (31)

1. A method of regulating the immune response in a mammal having atopic dermatitis, comprising administering at least one preparation of a frost-resistant kiwi plant and at least one corticosteroid to a mammal in an amount sufficient to regulate the immune response in a mammal having atopic dermatitis. 2. The method according to claim 1, where the frost-resistant kiwi plant is selected from the group consisting of Actinidia arguta, Actinidia kolomikta and Actinidia polygama. 3. The method according to claim 1, where the frost-resistant kiwi plant is Actinidia arguta. 4. The method according to claim 1, where the preparation of a frost-resistant kiwi plant is an extract of a frost-resistant kiwi plant. 5. The method according to claim 1, where the preparation of a frost-resistant kiwi plant is obtained by extraction in distilled water. 6. The method according to claim 1, where the preparation of a frost-resistant kiwi plant is obtained by extraction in a non-polar solvent. 7. The method according to claim 1, where the preparation of a frost-resistant kiwi plant is obtained by chromatographic purification of an aqueous extract. 8. The method according to claim 1, where the stage of administration comprises administering to the mammal a single preparation of a frost-resistant kiwi plant or a preparation of a frost-resistant kiwi plant and a corticosteroid, with a carrier, adjuvant or diluent. 9. The method according to claim 1, where the stage of administration comprises administering to the mammal one preparation of a frost-resistant kiwi plant or preparation of a frost-resistant kiwi plant and a corticosteroid, in the form of a tablet, powder, effervescent tablet, effervescent powder, capsule, liquid, suspension, granule or syrup. 10. The method according to claim 1, where the introduction stage includes the introduction to the mammal of one preparation of a frost-resistant kiwi plant or a preparation of a frost-resistant kiwi plant and a corticosteroid, as part of one or more healthy foods. 11. The method according to claim 1, where the introduction stage includes the introduction of one preparation of a frost-resistant kiwi plant or a preparation of a frost-resistant kiwi plant and a corticosteroid as part of a local composition. 12. The method according to claim 1, where the preparation of a frost-resistant kiwi plant is part of the same composition as a corticosteroid. 13. The method according to claim 1, where the preparation of a frost-resistant kiwi plant is part of a composition other than a corticosteroid, but it is administered simultaneously with a corticosteroid. 14. The method according to claim 1, where the stage of administration comprises administering to the mammal a single preparation of a frost-resistant kiwi plant or a preparation of a frost-resistant kiwi plant and a corticosteroid, as part of a feed or component of a feed. 15. A method of alleviating at least one symptom of inflammation in a mammal having atopic dermatitis, comprising administering to the mammal a preparation of a frost-resistant kiwi plant and at least one corticosteroid in an amount sufficient to alleviate said symptom of inflammation. 16. The method according to clause 15, where the symptom of inflammation is selected from the group consisting of itching, redness, secretions and education on the skin of a dry crust, thickening and swelling. 17. Composition for regulating the immune response in a mammal having atopic dermatitis, or to alleviate at least one symptom of inflammation in a mammal having atopic dermatitis, containing at least one preparation of a frost-resistant kiwi plant and at least one corticosteroid. 18. The composition according to 17, selected from the group consisting of a pharmaceutical composition, healthy food, food ingredient, animal feed and cosmetic composition. 19. The composition according to 17, where the frost-resistant kiwi plant is selected from the group consisting of Actinidia arguta, Actinidia kolomikta and Actinidia polygama. 20. The composition of claim 17, wherein the frost-resistant kiwi plant is Actinidia arguta. 21. The composition according to 17, where the preparation of a frost-resistant kiwi plant is an extract of a frost-resistant kiwi plant. 22. The composition of claim 17, wherein the preparation of a frost-resistant kiwi plant is obtained by extraction in distilled water. 23. The composition of claim 17, wherein the preparation of a frost-resistant kiwi plant is obtained by extraction in a non-polar solvent. 24. The composition of claim 17, wherein the preparation of a frost-resistant kiwi plant is obtained by chromatographic purification of an aqueous extract. 25. The composition of claim 17, which is formulated for oral administration. 26. The composition of claim 17, which is formulated for topical administration. 27. A product in the form of animal feed containing the composition according to 17. 28. The use of at least one preparation of a frost-resistant kiwi plant and at least one corticosteroid for the preparation of a composition for treating atopic dermatitis associated with impaired immune function in a mammal. 29. The use of at least one preparation of a frost-resistant kiwi plant and at least one corticosteroid to produce a composition for treating atopic dermatitis associated with an increase in at least one symptom of inflammation in a mammal. 30. A method of regulating the immune response in a mammal having atopic dermatitis, comprising administering to the mammal at least one preparation of a frost-resistant kiwi plant and at least one corticosteroid, followed by a period in which the preparation of the frost-resistant kiwi plant is administered to the mammal as monotherapy with lack of corticosteroid administration. 31. The method according to clause 30, in which the period of time is from at least one day to two weeks.

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