Treatment of atopic dermatitis with polyethylene glycol-modified interleukin 2, glucocorticoid, and hyaluronic acid

Abstract

The present invention discloses a pharmaceutical composition for treating atopic dermatitis, comprising polyethylene glycol-modified interleukin-2, glucocorticoid and low molecular weight hyaluronic acid, and optionally a pharmaceutically acceptable carrier, and provides 10KD PEG-modified IL-2 and its use in the treatment of type I allergic diseases.

Description

[Technical Field]

[0001] The present invention relates to polyethylene glycol-modified interleukin 2 (IL-2 / IL-2), glucocorticoids and hyaluronic acid for treating type I hypersensitivity (allergic) reactions, in particular for treating atopic dermatitis. [Background technology]

[0002] IL-2 is a multi-directional cytokine that predominantly promotes the proliferation of Treg cells at low concentrations, but predominantly promotes the proliferation of Teff cells at high concentrations. Therefore, when used directly for immunomodulatory therapy, it is difficult to control the dose and its effects are unstable [GRAβHOFFH, et al. Low-Dose IL-2 Therapy in Autoimmune and Rheumatic Diseases. Front Immunol, 2021 Apr 1; ​​12: 648408]. Previous studies have shown that the combination of PEG-modified IL-2 and glucocorticoids can effectively promote the proliferation of Treg cells without significantly affecting Teff [WU K, et al. Short-term intratracheal use of PEG-modified IL-2 and glucocorticoid persistently alleviates asthma in a mouse model. Sci Rep, 2016 Aug. 16; 6: 31562; WU Min et al., “Combination of dexamethasone with IL-2 selectively induces the expansion of CD4+ CD25+ FOXP3+ regulatory T cells in vivo and suppresses graft versus host disease”, Chin J Hematol, 2009, 30(11): 726-30; MA J, et al. Alleviating allergic airway diseases by means of short-term administration of IL-2 and dexamethasone. J Allergy Clin Immunol, 2011, 127(6): 1447-56 e6], it is speculated that the change in the spatial structure of PEG-modified IL-2 and its weakened affinity for the IL-2 receptor β subunit significantly reduces the proliferation effect of CD4+CD25- T cells, while only slightly affecting the proliferation effect of CD4+CD25+ Treg cells, ultimately resulting in the dominance of Treg cell proliferation.At the same time, low-affinity PEG-IL-2 promotes FoxO3a phosphorylation in Treg cells with the help of CD25 molecules, enhancing glucocorticoid resistance and inducing apoptosis of immune cells, but has no protective effect on CD4+CD25- T cells.

[0003] Regulatory T cells (Tregs) are important immune monitors that secrete anti-inflammatory cytokines (IL-10, TGF-β, etc.), highly express PD-1 and CTLA-4, and release granule enzymes and perforin to induce apoptosis of effector T cells (Teff). This negatively regulates the immune system, maintains homeostasis of the immune balance in the body, and avoids excessive immune hyperactivation. Immune imbalance resulting from the proliferation of effector T cells or the decline or dysfunction of Treg cells is importantly associated with various allergic and autoimmune diseases, such as allergic asthma, allergic dermatitis, allergic gastroenteritis, systemic lupus erythematosus, and X-linked polyendocrine syndrome [DENG G, et al. Foxp3 Post-translational Modifications and Treg Suppressive Activity. Front Immunol, 2019 Oct. 18, 10:2486; RAFFIN C, et al. T(reg) cell-based therapies: challenges and perspectives. Nat Rev Immunol, 2020, 20(3): 158-72; SHEVYREV D, TERESHCHENKO V. Treg Heterogeneity, Function, and Homeostasis. Front Immunol, 2020 Jan 14; 10:3100; ATTIAS M, et al. Mechanisms of human FoxP3(+) Treg cell development and function in health and disease. Clin Exp Immunol, 2019, 197(1): 36-51]. Such diseases can be effectively treated with regulatory T cells.

[0004] Skin is the largest barrier tissue in the body and is exposed to various stimuli from the external environment for a long period of time. To maintain local immune homeostasis, the proportion of Treg cells in normal skin is higher than that in peripheral blood [ALI N, ROSENBLUM MD. Regulatory T cells in skin. Immunology, 2017, 152(3): 372-81; SCHARSCHMIDT TC, et al. A Wave of Regulatory T Cells into Neonatal Skin Mediates Tolerance to Commensal Microbes. Immunity, 2015, 43(5): 1011-21]. However, in inflamed skin with a compromised barrier, local immunity is overactivated, resulting in a significant increase in pro-inflammatory effector cells and cytokines, and the compensatory increase in Treg cells is insufficient to maintain immune balance [BILLROTH-MACLURG AC, et al. Regulatory T Cell Numbers in Inflamed Skin Are Controlled by Local Inflammatory Cues That Upregulate CD25 and Facilitate Antigen-Driven Local Proliferation. J Immunol, 2016, 197(6): 2208-18]

[0005] Type I hypersensitivity (allergic) reactions have the following characteristics: 1) high prevalence, affecting 20-40% of the population; 2) fatal, such as anaphylactic shock or asthma attacks; 3) delayed healing, recurrence, and troublesome, such as allergic rhinitis and atopic dermatitis (eczema); 4) Type I allergic diseases are a class of diseases with the same pathophysiology, involving three phases: allergen-priming (sensitization), allergen-challenge, and onset of action, involving allergens, allergens (allergen-specific IgE), CD4+ Th2 helper cells, IL-4, IL-13, and IL-5 cytokines, mast cells, and basophils. Type I allergic diseases include: 1) systemic anaphylaxis, including drug anaphylactic shock and serum anaphylactic shock; 2) respiratory allergies, such as allergic rhinitis and allergic asthma; 3) gastrointestinal allergic reactions, such as allergic gastroenteritis; and 4) skin allergic reactions, such as urticaria, atopic dermatitis (eczema), and angioedema.

[0006] Atopic dermatitis (AD) is a skin symptom of type I allergy. Approximately 10-20% of the world's population suffers from AD, and its prevalence is increasing year by year as the sanitation standards of living environments improve. Chronic recurrence of AD severely impacts patients' long-term quality of life. Currently, clinical drug therapies aim to alleviate symptoms such as itching, swelling, and exudation during the acute phase of dermatitis and achieve a certain level of therapeutic efficacy. However, the drug effects are limited and short-lasting, and long-term administration can cause significant side effects. Therefore, there is a need in the art for safe, effective, and long-term treatment and prevention of atopic dermatitis. Summary of the Invention

[0007] In a first aspect, there is provided a pharmaceutical composition for treating atopic dermatitis, the pharmaceutical composition comprising polyethylene glycol-modified interleukin-2 (PEG-IL-2), a glucocorticoid and low molecular weight hyaluronic acid (HA), and optionally a pharmaceutically acceptable carrier.

[0008] In a second aspect, there is provided a method of treating atopic dermatitis in a subject, the method comprising administering to the subject therapeutically effective amounts of PEG-IL-2, a glucocorticoid and low molecular weight HA.

[0009] In a third aspect, there is provided the use of PEG-IL-2, a glucocorticoid and HA in the manufacture of a medicament for treating atopic dermatitis, and PEG-IL-2, a glucocorticoid and HA for use in the treatment of atopic dermatitis, wherein the HA comprises low molecular weight HA, and the PEG-IL-2, glucocorticoid and HA.

[0010] In a fourth aspect, there is provided a kit comprising PEG-IL-2, a glucocorticoid and HA, wherein the HA comprises high molecular weight HA and / or low molecular weight HA.

[0011] In a fifth aspect, there is provided the use of HA in the manufacture of a medicament for enhancing the effect of PEG-IL-2 and / or glucocorticoids in the treatment of atopic dermatitis, and HA for use in enhancing the effect of PEG-IL-2 and / or glucocorticoids in the treatment of atopic dermatitis, wherein the HA comprises low molecular weight HA.

[0012] In a sixth aspect there is provided the use of 10KD PEG-modified IL-2 in the manufacture of a medicament for the treatment of type I allergy.

[0013] In a seventh aspect, there is provided a method of treating type I allergy in a subject, the method comprising administering 10KD PEG-modified IL-2 to the subject.

[0014] In an eighth aspect, there is provided a 10 KD PEG-modified IL-2. [Brief explanation of the drawings]

[0015] [Figure 1] Preparation of polyethylene glycol (PEG)-modified interleukin-2. (a) Recombinant human interleukin-2 before (-) and after (+) polyethylene glycol (PEG) modification was electrophoresed on SDS-PAGE followed by silver staining. (b) Monitoring of PEG-modified IL-2 after purification. M, marker. (c) Activity assay of recombinant human interleukin-2 and polyethylene glycol (PEG)-modified recombinant human interleukin-2 of different molecular weights and structures. (d) Activity assay of polyethylene glycol (PEG)-modified recombinant human interleukin-2 (40 kD-PEG-IL-2) and recombinant human interleukin-2 (IL-2). [Figure 2] OVA-induced atopic dermatitis (AD) mouse model. (A) Photographs of the skin of normal and dermatitis mice show redness, scaling, and lesions in AD skin. (B) H&E staining of the two groups of skin, magnified 40x, reveals significant thickening of the AD skin and the infiltration of large numbers of inflammatory cells. (C) Quantitative RT-PCR mRNA expression profiles of inflammatory factors in the skin. The coordinates represent the 2^(-ΔΔCT) values ​​of qPCR mRNA in experimental and physiological mice. The blank group is the control group (group 1). The expression of IL-4, IL-13, and IL-17 in AD skin was significantly increased, with statistically significant differences. There was no statistical difference in IFN-γ expression. (D) Serum IgE levels measured by ELISA. The serum IgE levels in AD mice were significantly increased, with statistically significant differences. In the figure, ** indicates P<0.01, *** indicates P<0.001, **** indicates P=0.00, and ns indicates P>0.05. [Figure 3](A) Photographs of skin of mice treated with buffer (PBS), 40 kDa PEG-IL-2, budesonide (Bude), or 40 kDa PEG-IL-2 + budesonide 3 days after treatment show alleviated dermatitis in each group. (B) H&E staining of the skin pathology for each group shows comparable levels of inflammatory cell infiltration in the skin (20x magnification). (C) Statistical graph of the dermatitis scores for each group. Only the PBS group showed a statistically significant difference from the budesonide-only group. (D) Flow cytometry analysis of the percentage of skin Tregs in the blank control, AD model, and each treatment group. (E) Statistical graph of the percentage of Tregs. There was a statistically significant difference between the blank control and each group, but no statistically significant difference between the treatment groups. In the figure, * denotes P<0.05, *** denotes P<0.001, and ns denotes P>0.05. [Figure 4] A shows the photographs of the skin of dermatitis mice administered 40kD PEG-IL-2 + budesonide (Bude), HA alone, and 40kD PEG-IL-2 + budesonide + HA. The improvement of skin damage in the last group was significantly better than that in the first two groups. B shows the H&E stained images of the skin pathology of each group. The infiltration of inflammatory cells in the skin of the 40kD PEG-IL-2 + budesonide + HA group was significantly lower than that of the other two groups, by 20 times. C shows the flow cytometry. Figure 1 shows the ratio of CD25+FOXP3+ Treg cells among skin CD4+ cells in each group by cytometry; D shows a statistical chart of dermatitis scores. The 40kD PEG-IL-2 + budesonide + HA treatment group had a lower dermatitis score than the non-HA treatment group and the HA-only treatment group, and the difference was statistically significant; E shows a statistical chart of skin Tregs. The difference between the HA treatment group and the non-HA treatment group or the HA-only treatment group was statistically significant. In the figure, ** indicates P<0.01, *** indicates P<0.001, and ns indicates P>0.05. [Figure 5]A shows the skin photographs of the PBS-treated dermatitis mice group, the group that was exposed to OVA after inducing dermatitis and then treated with HA, PEG-IL-2, and budesonide, and the group that was exposed to OVA after 3 days of treatment on the normal skin of allergen-sensitized mice. The photographs show that because the administration of OVA was only for 3 days during the dermatitis state, the development of dermatitis could be prevented by 7 consecutive days of treatment with the antigen OVA. B shows the H&E stained skin images of each group, enlarged to 20°C, which show a significant reduction in inflammatory cell infiltration in the skin of the group that was administered after the onset of dermatitis. C shows the percentage of CD25+FOXP3+Treg cells among skin CD4+ cells in allergen-sensitized mice that were exposed to OVA for 3 days after administration on the normal skin of allergen-sensitized mice. D shows the statistical chart of dermatitis scores, showing a statistically significant difference between the two drug-treated groups. E shows the statistical chart of skin Tregs, showing a statistically significant difference between the two drug-treated groups. In the figure, *** indicates P<0.001, and ns indicates P>0.05. [Figure 6]A: Photographs of the skin of mice in each group after treatment with 40kD PEG-IL-2, 20kD PEG-IL-2, 10kD PEG-IL-2, branched PEG-IL-2, 10kD unbranched PEG-IL-2, and normal IL-2 in combination with budesonide + HA without any changes; B: Pathological H&E stained images of the skin in each group, magnified 20 times; C: The percentage of Treg in the skin in each group by flow cytometry; D: Statistical chart of the dermatitis score in each group, compared with the 40kD PEG-IL-2 group. No statistically significant differences were observed between the 20kD PEG-IL-2 group, the 10kD PEG-IL-2 group (branched), and the 10kD PEG-IL-2 group (unbranched). There was also no statistically significant difference between the normal IL-2 group and the budesonide-only group. These two groups were statistically significantly different from the 40kD PEG-IL-2 group. Figure E shows the statistical chart of skin Tregs. When comparing the groups, the combination with 10kD PEG-IL-2 induced the greatest Treg activation, and the difference was statistically significant. The normal IL-2 combination group had the lowest Treg percentage, and there was no difference between it and the budesonide-only group. * denotes P<0.05, ** denotes P<0.01, *** denotes P<0.001, and ns denotes P>0.05. [Figure 7]The percentages of FoxP3 and PD-1 cells among CD3+CD4+CD25+ cells were analyzed after 3 days of intraperitoneal injection of PEG-IL-2 modified with unbranched PEG of different molecular weights in physiological model mice. The percentages of CD25+FoxP3+ cells among splenic CD3+CD4+ lymphocytes and the percentages of PD-1+ cells among CD3+CD4+CD25+FoxP3+ cells were analyzed. Figure A shows the percentage of CD4+CD25+FoxP3+ cells, and Figure B shows the percentage of CD4+FoxP3+PD-1+ cells. Figure C shows a flow cytometry image of CD4+CD25+FoxP3+ cells. Figure D shows a flow cytometry image of CD4+CD25+FoxP3+ cells. This study demonstrated that only 10kD PEG-IL-2 significantly upregulated regulatory T cells (Tregs) in a physiological mouse model and induced high expression of PD-1 on Treg cells (PD-1 can enhance the suppressive function of Treg cells). In the figure, *** indicates P<0.001, **** indicates P<0.0001. [Figure 8] (A) Photographs of skin from mice with dermatitis and from mice treated with or without 10 kD PEG-IL-2 and HA; (B) 20x magnification of H&E stained skin histology images for each group; (C) Flow cytometry analysis of the percentage of CD25+FOXP3+ Treg cells among cutaneous CD4+ cells after treatment with 10 kD PEG-IL-2 and HA; (D) Statistical chart of dermatitis scores, showing that the two treatment groups had statistically significant differences compared with the control group, but not between them; (E) Statistical chart of skin Tregs, showing that the two treatment groups had statistically significant differences compared with the control group, but not between them. In the figure, * indicates P<0.05, and ns indicates P>0.05. [Figure 9]AHR analysis of an allergic asthma mouse model and healthy mice using drugs and PBS, and changes in pulmonary (airway) resistance (RI), which is used as an index to evaluate airway responsiveness. * indicates p<0.05 compared with the blank control group and PBS-treated mice. Local administration of 10kD-PEG-IL-2 significantly reduced airway resistance. [Figure 10] A shows the skin photographs of 10kD PEG-IL-2 + budesonide in combination with high molecular weight HA, 2 / 3 high molecular weight HA + 1 / 3 low molecular weight HA, 1 / 2 high molecular weight HA + 1 / 2 low molecular weight HA, 1 / 3 high molecular weight HA + 2 / 3 low molecular weight HA, and low molecular weight HA, respectively; B shows the respective skin pathology H&E stained images, magnified 20x; C shows the percentage of CD25+FOXP3+Treg cells in skin CD4+ cells detected by flow cytometry; D shows the statistical chart of dermatitis scores, comparing the HA (HA) group with the budesonide (Bude) alone group. The 1 / 3 high molecular weight HA + 2 / 3 low molecular weight HA and 2 / 3 high molecular weight HA + 1 / 3 low molecular weight HA groups showed the most significant improvement in dermatitis, followed by the low molecular weight HA group, with statistical differences. There was no statistical difference in the improvement of dermatitis between the high molecular weight HA group and the 1 / 2 high molecular weight HA + 1 / 2 low molecular weight HA groups. Figure E shows the statistical graph of skin Tregs. Compared with the budesonide alone group, the combination of 1 / 3 high molecular weight HA + 2 / 3 low molecular weight HA showed the greatest upregulation of Tregs, with statistical significance, but not statistically significant compared to the low molecular weight HA group (P > 0.05). ns indicates P > 0.05, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. [Figure 11]A shows the skin symptoms after 3 days of treatment with 10 kDa PEG-IL-2 + dexamethasone (DXMS) or budesonide (Bude) + HA (high molecular weight: low molecular weight 1:2); B shows a 20x magnification H&E stained image of the skin pathology; C shows the percentage of CD25+FOXP3+ Treg cells among cutaneous CD4+ cells detected by flow cytometry; D shows the statistical chart of dermatitis scores; there was no statistical difference between the dexamethasone and budesonide groups (P > 0.05); E shows the percentage of cutaneous Treg cells; there was no statistical difference between the dexamethasone and budesonide groups (P > 0.05). [Figure 12] A shows the expression of IL-4, IL-13, IL-17, and IFN-γ in mouse skin in the blank control, dermatitis model, optimal combination group determined in the above experiments, and budesonide (a glucocorticoid, the first-line clinical treatment for atopic dermatitis)-treated group. IL-4, IL-13, and IL-17 were downregulated in the combination group compared to the budesonide group, and the difference was statistically significant, but there was no difference in IFN-γ expression between the groups (P > 0.05). B shows the serum IgE concentration in each group as measured by ELISA. The combination group had lower serum IgE than the budesonide group, and the difference was statistically significant; ns indicates P > 0.05, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. [Figure 13] These images show the detection of OVA patch re-application in the combination and AD control groups after 6 weeks of treatment. Figure A shows photographs taken 4 days after OVA patch re-application in the combination and AD control groups after 6 weeks of OVA isolation; Figure B shows H&E-stained skin histology images magnified 20x; Figure C shows flow cytometry detection of CD25+FOXP3+ Treg cells among skin CD4+ cells in the two groups of mice; Figure D shows the statistical plot of dermatitis scores in the two groups, with a statistical difference between the two groups (P<0.05); Figure E shows the statistical plot of Treg cell ratios, with a statistical difference between the two groups (P<0.05). This study demonstrated that the efficacy of the combination group was maintained for at least 6 weeks. ns indicates P>0.05, * indicates P<0.05, and ** indicates P<0.01.

[0016] Unless otherwise defined, all technical and scientific terms have the same meaning as commonly understood by those skilled in the art. All patents, patent applications, publications, GenBank sequences, websites, and other disclosure materials are incorporated herein by reference unless otherwise specified. For various definitions of the terms of the present invention, this section prevails. When a URL or other identifier or address is mentioned, it should be understood that the identifier may change, specific information on the Internet may be updated from time to time, and related information may be found by Internet search. These disclosures are publicly available.

[0017] As used herein, the terms "protein," "peptide," "polypeptide," and "amino acid sequence" are used interchangeably and refer to polymers of any length, e.g., two or more amino acid residues. The terms encompass both naturally occurring amino acid polymers and amino acid polymers that have been artificially altered, e.g., by disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or other manipulations and modifications, such as labeling or conjugation to a biologically active moiety. Conventional one-letter or three-letter symbols are used herein to represent amino acid residues.

[0018] As used herein, the term "amino acid" or "aa" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids refer to amino acids encoded by the genetic code, as well as amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid (i.e., an α-carbon with a hydrogen, a carboxyl group, an amino group, and an R group). Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid.

[0019] Hyaluronic acid (HA) is a glycosaminoglycan that plays a role in various physiological processes (Laurent TC et al (1992) FASEB J 6:2397-2404). HA is a linear, high-molecular-weight polysaccharide composed of repeating disaccharide units of (1-β-4) D-glucuronic acid and (1-β-3) N-acetyl-D-glucosamine. HA is present in the extracellular matrix of many cells, and is particularly abundant in soft connective tissues. In connective tissues, bound water attached to hyaluronic acid creates interstitial spaces, thereby creating an environment favorable for cell migration and proliferation. Hyaluronic acid plays an important role in biological phenomena related to cell survival, such as rapid development, regeneration, repair, embryogenesis, embryonic development, wound healing, angiogenesis, and tumorigenesis (see, for example, Toole 1991 Cell Biol. Extracell. Matrix, Hay (ed), Plenum Press, New York, 1384-1386; Bertrand et al. 1992 Int. J. Cancer 52:1-6; Knudson et al., 1993 FASEB J. 7:1233-1241). The molecular weight of hyaluronic acid described herein is determined by taking into account the atomic weight of each repeating disaccharide unit that constitutes hyaluronic acid.

[0020] As used herein, high molecular weight HA refers to hyaluronic acid with a molecular weight of approximately 800 KD or greater.

[0021] As used herein, low molecular weight HA refers to hyaluronic acid with a molecular weight of approximately 300 KD or less.

[0022] As used herein, the molecular weight of HA refers to the number average molecular weight of HA.

[0023] The hyaluronic acid used in the present invention can be any suitable form of hyaluronic acid, including, but not limited to, hyaluronic acid salts such as sodium hyaluronate, potassium hyaluronate, and magnesium hyaluronate. In one embodiment, the hyaluronic acid used in the present invention comprises sodium hyaluronate.

[0024] High molecular weight HA aids in the transdermal absorption of drugs and has a relatively weak ability to remain on the skin. However, high molecular weight HA has the immunomodulatory ability to promote the expression of the transcription factor FOXP3 in locally activated Tregs and enhance the immunosuppressive function of Tregs [FALLACARA A, et al. Hyaluronic Acid in the Third Millennium. Polymers (Basel), 2018, 10 (7): 701; GIRISH KS, KEMPARAJU K. The magic glue hyaluronan and its eraser hyaluronidase: a biological overview. Life Sci, 2007, 80(21): 1921-43; ALTMAN RD, et al. The mechanism of action for hyaluronic acid treatment in the osteoarthritic knee: a systematic review. BMC Musculoskelet Disord, 2015, 16: 321; GUPTA RC, et al. Hyaluronic Acid: Molecular Mechanisms and Therapeutic Trajectory. Front Vet Sci, 2019, 6:192].Regarding the interaction between HA and TLRs (Toll-like receptors), low molecular weight HA has pro-inflammatory effects and agonistic activity against TLR-2 and TLR-4, which is unfavorable for allergic dermatitis. On the other hand, high molecular weight HA reduces the receptor-binding ability and forms a dense coating around the cells, covering the receptor surface and reducing the pro-inflammatory effects of low molecular weight HA [ALI N, ROSENBLUM M D. Regulatory T cells in skin. Immunology, 2017, 152(3): 372-81; SCHARSCHMIDT TC, et al. A Wave of Regulatory T Cells into Neonatal Skin Mediates Tolerance to Commensal Microbes. Immunity, 2015, 43(5): 1011-21; BILLROTH-MACLURG AC, et al. Regulatory T Cell Numbers in Inflamed Skin Are Controlled by Local Inflammatory Cues That Upregulate CD25 and Facilitate Antigen-Driven Local Proliferation. J Immunol, 2016, 197(6): 2208-18;GRAβHOFF H, et al. Low-Dose IL-2 Therapy in Autoimmune and Rheumatic Diseases. Front Immunol, 2021, 12:648408].

[0025] Unless the context dictates otherwise, ratios of high molecular weight HA to low molecular weight HA herein are mass ratios.

[0026] Polyethylene glycol (PEG) is a non-toxic, water-soluble, neutral polymer with excellent biocompatibility and blood compatibility, and is used for local administration to the human body, the digestive tract, and intravenously. Protein PEG modification technology involves activating one or two terminal groups on both ends of PEG to confer specific functional groups that are reactive to at least one functional group on the target protein, and then covalently binding PEG to the protein's terminus (N-terminus or C-terminus) or to a specific amino acid. PEG's site of action is universal.

[0027] PEG is a polymer of ethylene glycol and ethylene oxide, also known as carbon wax, and has the following structural formula: CH2(OH)-(CH2CH2O) n -CH2OH

[0028] PEG for modifying drugs includes branched and unbranched types, i.e., linear molecular types (see, for example, EP0593868), and U / Y-branched molecular types (see, for example, EP0809996 and CN1243779C).

[0029] The molecular formula of the U-shaped branched PEG derivative (PEG2-NHS) is as follows: [ka] In the formula, R and R' are each independently a low molecular weight alkyl; n and n' are 600 to 1500; and the average molecular weight of PEG is 26 KD to 66 KD.

[0030] The Y-shaped PEG derivative molecule is shown below: [ka] In the formula, P a and P b are the same or different polyethylene glycol molecules; j is an integer; R iis H, substituted or unsubstituted alkyl, substituted aryl, aralkyl, or heteroalkyl, etc.; X and X are each independently (CH) n , (CH2) n OCO, (CH2) n NHCO and (CH2) n and CO, where n is an integer from 1 to 10, and F is a terminal group that can react with an amino group, a hydroxyl group, or a thiol group on a therapeutic agent or matrix to form a covalent bond, e.g., selected from the group consisting of a hydroxyl group, a carboxyl group, an ester group, an acyl chloride, a hydrazide, a maleimide, and a pyridine disulfide.

[0031] The molecular weight of the PEG used in the present invention is determined by taking into account the atomic weight of each atom constituting the PEG.

[0032] Various methods for determining the molecular weight of a molecule are known in the art. For example, the molecular weight of a molecule can be determined by aligning it with a standard marker using electrophoresis. This is within the capabilities of one skilled in the art.

[0033] As used herein, a "glucocorticoid" can be any synthetic or naturally occurring glucocorticoid. Glucocorticoids that can be used in the present invention are, for example, glucocorticoids useful in the treatment of atopic dermatitis, and examples thereof include, but are not limited to, dexamethasone (Dex), budesonide (Bud), beclomethasone dipropionate (BDP), ciclesonide, hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone, clobetasone butyrate, triamcinolone acetonide, fluocinolone, mometasone furoate, halcinonide, clobetasol propionate, halcinonide, halometasone, diflorasone diacetate, mometasone, loteprednol, etiprednol, triamcinolone, flunisolide, flumoxonide, rofleponide, butixocort, and tipredane.

[0034] In this specification, unless otherwise specified, the term "glucocorticoid" includes all active salts, solvates, or derivatives that can be derived from glucocorticoids. For example, the term "dexamethasone" includes dexamethasone and its active salts, solvates, or derivatives that have the desired activity. Examples of possible salts or derivatives of glucocorticoids include sodium salts, sulfobenzoates, phosphates, isonicotinates, acetates, propionates, dihydrogen phosphates, palmitates, pivalates, fumarates, and pharmaceutically acceptable esters (e.g., C1-C6 alkyl esters). Glucocorticoids and their active salts or derivatives may be solvates, such as hydrates.

[0035] As used herein, "IL-2" refers to IL-2 of any origin, including IL-2 derived from mammals such as humans, mice, rats, primates, and pigs, and encompasses IL-2 derived from natural sources or obtained by recombinant or synthetic techniques, such as recombinant IL-2 polypeptides produced by microbial hosts. IL-2 may be or include a naturally occurring polypeptide sequence, or may be an active variant of a naturally occurring IL-2 polypeptide. Preferably, the IL-2 polypeptide or active variant is of human origin, including recombinant human-derived IL-2, particularly recombinant human-derived IL-2 produced by microbial hosts.

[0036] The IL-2 that can be used in the present invention may be any IL-2 that is clinically useful for treating type I allergic reactions, particularly atopic dermatitis. Those skilled in the art can obtain or determine the IL-2 or its derivatives or variants that can be used in the present invention.

[0037] As used herein, PEG-modified IL-2 refers to IL-2 modified by attaching PEG, such as unbranched PEG or branched PEG, to any suitable site on IL-2.

[0038] As used herein, "treating" a subject suffering from atopic dermatitis refers to partially or completely eliminating the subject's lesions or symptoms, or maintaining a stable condition without progression after treatment.Treatment includes prevention, cure and / or healing.Prevention refers to preventing the occurrence and / or worsening or progression of potential disease, and preventing occurrence includes reducing or eliminating one or more risk factors that cause the occurrence of disease; since it is generally impossible to determine whether a disease has not yet developed, prevention also includes reducing the risk of the occurrence or suffering from disease.

[0039] As used herein, the term "therapeutically effective amount" or "therapeutically effective dose" refers to a dose at least sufficient to produce a therapeutic effect in a subject. For active ingredients of the present invention, specific therapeutically effective amounts can be initially estimated using various techniques known in the art. Suitable dosage ranges for human subjects can be determined, for example, using data from cell culture experiments or other animal studies. Dosage levels and administration protocols can be determined based on known dosages and administration protocols, and, if necessary, can be determined based on known characteristics and / or empirically based on various factors. Such factors include, for example, the subject's weight, general health, age, activity of the specific compound used, sex, diet, timing of administration, drug combinations, severity and course of disease, patient susceptibility to disease, and the physician's judgment. After improvement of the patient's condition, a maintenance dose of the compound or composition may be administered, and the dosage, dosage form, and administration frequency, or a combination thereof, can be modified as needed. The exact dosage and treatment regimen will depend on the physician's discretion and the specific patient's condition.

[0040] The therapeutically effective amount depends largely on the nature of the drug, the condition of the patient, and the nature and severity of the disease to be treated, and ranges from a low dose of 1 ng / kg to a high dose of 10 mg / kg, more specifically 20 ng / kg to 1 mg / kg, of the active ingredient when treating a topical disease such as atopic dermatitis.

[0041] Herein, we induced a typical type I hypersensitivity reaction in sensitized BALB / c mice by intraperitoneally injecting chicken ovalbumin (OVA) three times. OVA exposure at various sites resulted in enhanced skin damage and airway reactivity, elevated serum IgE levels, cutaneous pathological inflammatory cell infiltration, Th2 cell polarization, and high expression of IL-4 and IL-3. OVA-sensitized BALB / c mice, in which type I allergy is induced, are the most common experimental animal model. We first used a mouse model of OVA-induced atopic dermatitis with topical administration of PEG-IL-2 in combination with budesonide and compared the therapeutic efficacy with that of the current clinical first-line treatment protocol (i.e., topical administration of glucocorticoids alone). Both treatment regimens alleviated the skin inflammation symptoms in mice and partially upregulated the proportion of Treg cells in the skin. However, the therapeutic effect of the combination was not superior to that of budesonide alone, and no statistically significant difference in the proportion of Treg cells in the skin was observed. Because the combination therapy included budesonide, the relief of dermatitis was likely due to the action of glucocorticoids.

[0042] The core of this research is to correct the immune imbalance in the skin by increasing the proportion of Treg cells in the skin while enhancing the immunosuppressive ability of Treg cells through drug therapy, thereby treating and preventing atopic dermatitis.

[0043] In normal skin, substances with molecular weights greater than 4 kDa have difficulty penetrating through the stratum corneum into the epidermis and dermis, especially when they are hydrophilic. This study demonstrated that when administered in combination with OVA allergens in the absence of dermatitis (skin lesions) after allergen sensitization, the combination did not inhibit dermatitis induction, suggesting that HA does not promote drug penetration into normal skin. As shown herein, for example, in a mouse model of atopic dermatitis with damaged stratum corneum, the therapeutic effect of budesonide combined with PEG-IL-2 in inflammatory skin was significantly improved, and Treg cells were simultaneously upregulated. This suggests that low-molecular-weight hyaluronic acid can help high-molecular-weight drugs with hydrophilic polarity penetrate or remain deep within the epidermis or dermis (the epidermis or dermis are the immune response layers in atopic dermatitis).The smaller the molecular weight of the combined PEG-IL-2 (10kDa, 20kDa, and 40kDa PEG), the greater the proportion of upregulated Treg cells in the skin and the better the therapeutic effect on dermatitis. The effect of 10kD-IL-2 (molecular weight 25kDa) was superior to that of 20kD-IL-2 (molecular weight 35kDa) and 40kD-IL-2 (molecular weight 55kDa). However, when PEG-IL-2 was replaced with normal interleukin-2 (molecular weight 15kDa) in the combination regimen, the molecular weight was reduced and the hydrophilic polarity of PEG was eliminated (reduced polarity promotes drug penetration). As a result, the increase in induced Treg cells and the improvement of dermatitis were not as good as with PEG-IL-2, and the therapeutic effect was comparable to that of budesonide alone. Therefore, normal interleukin-2 appears unable to penetrate deep into the epidermis of damaged skin due to the low molecular weight of hyaluronic acid. (Madeleine Witting et al. [WARD-HARTSTONGE] KA, KEMP R A. Regulatory T-cell heterogeneity and the cancer immune response. Clin Transl Immunology, 2017, 6(9): e154; DONG L, et al. Programmed death 1 / programmed cell death-ligand 1 pathway participates in gastric surgery-induced imbalance of T-helper 17 / regulatory T cells in mice. J Trauma Acute Care Surg, 2018, 85(3): 549-59] showed that all bovine serum albumins (BSA) with a molecular weight greater than 60 kD can penetrate damaged skin and reach the epidermis.

[0044] The present inventors have demonstrated that topical application of 10 kD PEG-IL-2 (IL-2 modified with such a PEG molecular weight tended to induce Treg cell proliferation) to damaged skin showed therapeutic effects, but the effect was poorer than that of low molecular weight HA combined with 10 kD PEG-IL-2. This suggests that PEG-IL-2 can penetrate the skin even in damaged skin conditions, and therefore non-PEG-modified IL-2 can also enter the skin through the stratum corneum of damaged skin, but is unable to reach or remain in the deeper epidermal or dermal layers where immune responses occur under the influence of HA. On the other hand, under the influence of low molecular weight HA, IL-2 is limited to the superficial layer of the epidermis, but PEG-modified IL-2 can penetrate the deeper epidermal and dermal layers via low molecular weight HA to exert its therapeutic effects. It is believed that the interaction between low molecular weight HA and PEG allows the drug to remain in the active deep layer of the epidermis or dermis layer, resulting in better efficacy. However, in the absence of PEG, low molecular weight HA allows the drug to remain in the superficial layer of the epidermis, but not in the area where the immune response occurs.

[0045] Without being bound by any theory, the experiments presented herein demonstrate that the combination of PEG-modified IL-2 with varying molecular weights within a certain range and glucocorticoids does not significantly affect the therapeutic effect on skin. It is possible that some form of binding between hyaluronic acid and PEG molecules helps PEG-IL-2 penetrate and / or remain stable in the deeper layers of the epidermis or dermis, thereby exerting its therapeutic effect. On the other hand, normal IL-2 not bound to PEG molecules either fails to penetrate the deeper layers of the epidermis or dermis, even with the assistance of HA, or only remains temporarily in the epidermis and disappears before exerting its beneficial effects. The specific binding mode between PEG and HA molecules is unclear, and there have been no reports on their interaction.

[0046] No statistically significant differences were observed between the 10kD branched PEG-IL-2 group and the 10kD unbranched PEG-IL-2 group in terms of skin inflammation, inflammatory cell infiltration, or skin Treg ratio, suggesting that the conjugation of different PEG structures to IL-2 does not significantly affect overall efficacy. This study demonstrated that changes in the molecular weight and structure of PEG have little effect on drug penetration and retention in the deeper layers of the epidermis or dermis.

[0047] Why are the Treg cells induced and the therapeutic effect of 10kD-IL-2 combined with glucocorticoid and low molecular weight HA superior to those of the 20kD or 40kD PEG-IL-2 groups? Without being bound by any theory, it is possible that PEG-modified interleukin-2 has improved skin penetration due to its interaction with hyaluronic acid, and that the function of interleukin-2 may also have been changed by modification with PEG of a specific molecular weight. When BALB / c mice were injected with unbranched PEG-modified interleukin-2 (IL-2) of different molecular weights (10kD, 20kD, and 40kD) and conventional IL-2 under physiological conditions, unexpectedly, only the 10kD PEG-modified interleukin-2 predominantly induced Treg cell proliferation, and PD-1 expression was highly expressed on the induced Treg cells. The highly PD-1-expressing Treg cells enhanced the inhibitory effect of Tregs on effector T cells (Teff) [PARK HJ, PARK JS, JEONG YH, et al. PD-1 upregulated on regulatory T cells during chronic virus infection enhances the suppression of CD8+ T cell immune response via the interaction with PD-L1 expressed on CD8+ T cells [J]. J Immunol, 2015, 194(12): 5801-11].

[0048] Here, we found that 10 kD PEG-IL-2, with or without HA (without glucocorticoids), improved the dermatitis symptoms in mice. The percentage of Treg cells in the skin of treated mice increased to a certain extent compared to that of dermatitis model mice, and the difference was statistically significant. However, 40 kD PEG-IL-2 alone had no therapeutic effect (Figure 3). Therefore, we believe that 10 kD PEG-IL-2 itself is a novel interleukin-2 that can predominantly induce Treg cell proliferation, but that interleukin-2 modified with 20 kD or 40 kD PEG needs to be combined with glucocorticoids to predominantly induce Treg cell proliferation. Clearly, modification with PEG of the appropriate molecular weight can alter the biological activity of interleukin-2, which predominantly induces Treg cell proliferation.

[0049] Furthermore, when 10kD PEG-IL-2 was applied to a mouse asthma model, airway hyperresponsiveness in the mice was significantly reduced. Based on this, and since the addition of HA could not further improve airway hyperresponsiveness, it is believed that 10kD PEG-IL-2 can alleviate type I allergy and alleviate the symptoms of various type I allergy-related diseases, further proving that 10kD PEG-IL-2 is a new IL-2 with immunoregulatory functions.

[0050] Next, when combining 10 kD PEG-IL-2 with budesonide, we varied the molecular weight of HA and the HA ratio. The highest percentage of Tregs in the skin was observed in the group with a high molecular weight / low molecular weight HA ratio of 1:2 and in the group with low molecular weight HA alone. Both high molecular weight and low molecular weight HA are thought to enhance the therapeutic effect of the combination of 10 kD PEG-IL-2 and budesonide. However, low molecular weight HA has a greater auxiliary effect because the combined drug is more likely to be absorbed and retained in the active layer of the skin. As the amount of low molecular weight HA increases, the auxiliary effect becomes stronger and the therapeutic effect becomes stronger. Therefore, if the amount of low molecular weight HA is sufficient to absorb most of the drug into the skin, the therapeutic effect will not be further improved.

[0051] Therefore, it is believed that the best effect can be achieved when the ratio of high molecular weight HA to low molecular weight HA is appropriate.Finally, the combination of 10 kDa PEG-IL-2 + budesonide + 1 / 3 high molecular weight HA + 2 / 3 low molecular weight hyaluronic acid was selected as the most preferable solution in this study. Furthermore, by comparing it with clinical first-line treatment regimens, the optimal solution determined in this study more quickly and effectively alleviated dermatitis symptoms, down-regulated inflammatory factors and serum IgE concentrations, and significantly up-regulated the proportion of Treg cells in the skin, still preventing dermatitis recurrence 6 weeks after administration. The proportion of Treg cells in the skin was also maintained at a higher level. This long-term effectiveness may also be related to the long-term stable residence of Treg cells induced by HA in the skin. As a result, HA interacts with Treg cells, allowing Treg cells to remain in the dermis layer. Treg cells surviving in the dermis layer for a long period of time automatically regulate immune responses in the skin, achieving long-term therapeutic effects, and proving that it can prevent the onset of dermatitis after re-exposure to allergens in allergen-sensitized skin, and can prevent allergies such as seasonal pollen allergies.

[0052] In summary, a 3-day short-term topical application of a combination of 10 kDa PEG-IL-2, budesonide (or dexamethasone), 1 / 3 high molecular weight HA, and 2 / 3 low molecular weight HA rapidly and effectively alleviated and prevented atopic dermatitis in mice, reducing local inflammatory cell infiltration and upregulating the proportion of cutaneous Treg cells, with effects lasting for at least 6 weeks. Unexpectedly, this combination of PEG molecules and low molecular weight HA resolved the problem of hydrophilic, polar, and high molecular weight protein drugs poorly penetrating or remaining in the deep epidermal or dermal layers of the skin. Unexpectedly, modification of interleukin-2 with 10 kDa PEG altered its biological properties, favoring the proliferation of Treg cells (which suppress allergic immunity) and effector cells (which initiate allergic responses), thereby alleviating type I allergic responses. Therefore, 10 kDa PEG-IL-2 is a novel interleukin-2.

[0053] Without being bound by any theory, the possible mechanisms are that 10kD PEG-IL-2 predominantly expands Tregs and promotes their high expression of PD-1, thereby enhancing their immunosuppressive function, while the biased protection of Treg cells resists glucocorticoid-induced apoptosis, while low molecular weight HA aids drug absorption and, by binding to PEG molecules in some way, helps the drug to remain stably in the deep epidermal and dermal layers, and also stabilizes Treg cells to remain in the deep epidermal and dermal layers of the skin, achieving long-term stable local immunity and producing a sustained therapeutic effect, while high molecular weight HA's function of enhancing Treg immunoregulation suppresses the development of allergic immunity.

[0054] Thus, according to a first aspect, there is provided a pharmaceutical composition comprising polyethylene glycol-modified interleukin-2 (PEG-IL-2), a glucocorticoid and low molecular weight hyaluronic acid (HA), and optionally a pharmaceutically acceptable carrier.

[0055] The PEG used to modify the IL-2 can be any suitable type or size of PEG, such as linear or non-linear PEG, and / or be of any suitable size.

[0056] In one embodiment, the polyethylene glycol (PEG) used to modify the IL-2 is a PEG having a molecular weight of about 100, about 90, about 80, about 70, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10, or about 5 KD or less. In one embodiment, the PEG has a molecular weight of about 1 to 100, about 1 to 90, about 1 to 80, about 1 to 70, about 1 to 60, about 1 to 50, about 1 to 40, about 1 to 10, about 1 to 20, about 2 to 100, about 2 to 90, about 2 to 80, about 2 to 70, about 2 to 60, about 2 to 50, about 2 to 40, about 2 to 30, about 2 to 20, about 3 to 100, about 3 to 90, about 3 to 80, about 3 to 70, about 3 to 60, about 3 to 50, about 3 to 40, about 3 to 30, or about 3 to 20. , about 4 to 100, about 4 to 90, about 4 to 80, about 4 to 70, about 4 to 60, about 4 to 50, about 4 to 40, about 4 to 30, about 4 to 20, about 5 to 100, about 5 to 90, about 5 to 80, about 5 to 70, about 5 to 60, about 5 to 50, about 5 to 40, about 5 to 30, about 5 to 20, about 10 to 100, about 10 to 90, about 10 to 80, about 10 to 70, about 10 to 60, about 10 to 50, about 10 to 40, about 10 to 30, or about 10 to 20 KD PEG.

[0057] In one embodiment, the PEG has a molecular weight of about 60 KD, about 55 KD, about 50 KD, about 45 KD, about 40 KD, about 35 KD, about 30 KD, about 25 KD, about 20 KD, about 15 KD, about 10 KD or about 5 KD.

[0058] In one embodiment, the PEG comprises branched or unbranched PEG. For example, the branched PEG can comprise branched PEGs of about 10 KD and 20 KD; unbranched PEG comprises unbranched PEGs of about 10 KD, 20 KD, and 40 KD.

[0059] The PEG modification described herein can be present at any site in IL-2 that is suitable for PEG modification, such as a lysine residue, serine residue, threonine residue, or N-terminal α-amino group of IL-2. In one embodiment, the PEG modification is present at the N-terminal amino acid residue of IL-2, such as the N-terminal lysine residue, serine residue, or threonine residue of IL-2. In one embodiment, the PEG modification is present at the N-terminal α-amino group of IL-2. The PEG modification may be, for example, a single-site modification or multiple-site modification, but is preferably a single-site modification.

[0060] In one embodiment, the IL-2 is from a human or non-human mammal, such as cow, rat, mouse, dog, monkey, goat, sheep, bovine, deer, horse, and cat.

[0061] In one embodiment, human-derived IL-2 or an active variant thereof is used, more preferably recombinantly produced. The nucleotide sequence and amino acid sequence of human-derived IL-2 are disclosed, for example, in Genbank refs 3558 and P60568, respectively. Unless otherwise specified, the IL-2 used is substantially pure, for example, 95% or more pure, more preferably 96%, 97%, 98%, or 99% pure. IL-2 can be used in the form of a monomeric or multimeric protein.

[0062] In one embodiment, IL-2 as used herein comprises the amino acid sequence of SEQ ID NO:1.

[0063] Low molecular weight HA refers to HA having a molecular weight of about 300 KD or less, for example, about 250 KD or less, about 200 KD or less, about 150 KD or less, about 100 KD or less, about 90 KD or less, about 80 KD or less, about 70 KD or less, about 60 KD or less, about 50 KD or less, about 40 KD or less, about 30 KD or less, about 20 KD or less, about 10 KD or less, about 9 KD or less, about 8 KD or less, about 7 KD or less, about 6 KD or less, about 5 KD or less, about 4 KD or less, about 3 KD or less, or about 2 KD or less. In one embodiment, low molecular weight HA refers to HA having a molecular weight of about 2 to 20 KD, about 2 to 15 KD, about 2 to 10 KD, about 2 to 5 KD, about 3 to 20 KD, about 3 to 15 KD, about 3 to 10 KD or about 3 to 5 KD, for example, about 5 KD, about 4 KD, about 3 KD or about 2 KD.

[0064] In one embodiment, the pharmaceutical composition further comprises high molecular weight HA, and the high molecular weight HA and low molecular weight HA can be present in any ratio, but the mass ratio of high molecular weight HA to low molecular weight HA is about 2:1, or the ratio of the mass of high molecular weight HA to the total mass of high molecular weight HA and low molecular weight HA is less than 50%, preferably less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10% or even less. Preferably, the pharmaceutical composition comprises high molecular weight HA to low molecular weight HA in a mass ratio of about 2:1 or about 1:2.

[0065] High molecular weight HA refers to HA having a molecular weight of about 800 KD or greater, e.g., about 900 KD or greater, about 1000 KD or greater, about 1100 KD or greater, about 1200 KD or greater, about 1300 KD or greater, about 1400 KD or greater, about 1500 KD or greater, about 1600 KD or greater, about 1700 KD or greater, about 1800 KD or greater, about 1900 KD or greater, about 2000 KD or greater, or greater. In one embodiment, high molecular weight HA has a molecular weight of about 800 to 2000 KD, about 800 to 1900 KD, about 800 to 1800 KD, about 800 to 1700 KD, about 800 to 1600 KD, about 800 to 1500 KD, about 900 to 2000 KD, about 900 to 1900 KD, about 900 to 18 ... It refers to HA of 1700KD, about 900 to 1600KD, about 900 to 1500KD, about 1000 to 2000KD, about 1000 to 1900KD, about 1000 to 1800KD, about 1000 to 1700KD, about 1000 to 1600KD or about 1000 to 1500KD, and particularly refers to HA of about 800 to 1500KD.

[0066] In one embodiment, the glucocorticoid is selected from the group consisting of dexamethasone, budesonide, beclomethasone dipropionate, ciclesonide, hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone, clobetasone butyrate, triamcinolone acetonide, fluocinolone, mometasone furoate, halcinonide, clobetasol propionate, halcinonide, halometasone, diflorasone diacetate, mometasone, loteprednol, etiprednol, triamcinolone, flunisolide, flumoxonide, rofleponide, butixocort, tipredane, and derivatives thereof. In one embodiment, the glucocorticoid comprises dexamethasone, budesonide, and derivatives thereof.

[0067] In one embodiment, the pharmaceutical composition is used for the treatment of atopic dermatitis.Preferably, the atopic dermatitis is atopic dermatitis accompanied by skin lesions.In this specification, atopic dermatitis accompanied by skin lesions refers to atopic dermatitis in which the structure of the stratum corneum is damaged, for example, the cornified membrane, lipid membrane, and granular layer, which is the final defense layer of the skin barrier, are damaged, and symptoms such as chronic and recurrent severe pruritus, erythema, edema, blisters, serous exudate, etc. appear.

[0068] In one embodiment, 10KD PEG-modified IL-2, dexamethasone or budesonide, and (i) low molecular weight HA, or (ii) HA comprising low molecular weight HA and high molecular weight HA, wherein the mass ratio of high molecular weight HA to low molecular weight HA is about 1:2. A pharmaceutical composition comprising:

[0069] As used herein, "10 KD PEG-modified IL-2" refers to IL-2 modified with PEG having a molecular weight of approximately 10 KD. PEG can be any suitable type of PEG, such as linear or non-linear PEG. "Approximately 10 KD" means that the total atomic weight of the atoms constituting the PEG is 10 KD ± 10%, 10 KD ± 5%, 10 KD ± 4%, 10 KD ± 3%, 10 KD ± 2%, 10 KD ± 1%, 10 KD ± 0.5%, or 10 KD ± 0.1%. In a preferred embodiment, the 10 KD PEG-modified IL-2 is IL-2 (e.g., SEQ ID NO: 1) in which the N-terminus of IL-2 has been modified with PEG having a molecular weight of approximately 10 KD.

[0070] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating agent, antibacterial and antifungal agent, isotonic agent, absorption delaying agent, etc. The use of such media and agents is well known in the art. In this specification, active ingredients such as glucocorticoids and polyethylene glycol-modified interleukin-2 may be mixed with one or more pharmaceutically acceptable additives, diluents, or carriers. Examples of pharmaceutically acceptable carriers include, but are not limited to, lactose, sucrose, dextran, mannitol or glucose starch, talc, magnesium stearate, magnesium oxide, crystalline cellulose, methylcellulose, carboxymethylcellulose, gelatin, glycerol, sodium alginate, saline, and water, and may also include additives such as fillers, binders, humectants, glidants, stabilizers, preservatives, emulsifiers, and additional solvents or solubilizers or preservative-effecting substances.

[0071] Pharmaceutical compositions can be formulated in any form suitable for local administration (e.g., topical or local), such as a powder, spray, foam, solution, ointment, emulsion, etc. Pharmaceutical compositions can be formulated as a solid, liquid, gel, or other form.

[0072] Pharmaceutical compositions may be provided in bulk dose and unit dose forms, as well as in the form of openable or puncturable implants, capsules, blister packs, or cartridges, as are known in the art. Also provided are kits comprising a delivery device, a pharmaceutical composition herein contained in a separate container, and optionally other suitable additives such as other therapeutic compounds, excipients, surfactants (used as therapeutic agents and formulation ingredients), antioxidants, flavors and colorants, fillers, volatile oils, buffers, dispersants, surfactants, antioxidants, flavors, bulking agents, propellants, and preservatives, and instructions for use of the kit components.

[0073] According to a second aspect, there is provided a method of treating atopic dermatitis in a subject, comprising administering to the subject therapeutically effective amounts of PEG-IL-2, a glucocorticoid and low molecular weight HA.

[0074] The PEG-IL-2 administered to a subject can be IL-2 modified with any suitable type or size of PEG, such as linear or non-linear PEG and / or PEG of any suitable size.

[0075] In one embodiment, the PEG-IL-2 administered to a subject is modified with PEG having a molecular weight of about 100 KD, about 90 KD, about 80 KD, about 70 KD, about 60 KD, about 55 KD, about 50 KD, about 45 KD, about 40 KD, about 35 KD, about 30 KD, about 25 KD, about 20 KD, about 15 KD, about 10 KD, or about 5 KD or less. In one embodiment, the PEG has a molecular weight of about 1 to 100 KD, about 1 to 90 KD, about 1 to 80 KD, about 1 to 70 KD, about 1 to 60 KD, about 1 to 50 KD, about 1 to 40 KD, about 1 to 10 KD, about 1 to 20 KD, about 2 to 100 KD, about 2 to 90 KD, about 2 to 80 KD, about 2 to 70 KD, about 2 to 60 KD, about 2 to 50 KD, about 2 to 40 KD, about 2 to 30 KD, about 2 to 20 KD, about 3 to 100 KD, about 3 to 90 KD, about 3 to 80 KD, about 3 to 70 KD, about 3 to 60 KD, about 3 to 50 KD, about 3 to 40 KD, about 3 to 30 KD, or about 3 to 20 KD. and about 4 to 100KD, about 4 to 90KD, about 4 to 80KD, about 4 to 70KD, about 4 to 60KD, about 4 to 50KD, about 4 to 40KD, about 4 to 30KD, about 4 to 20KD, about 5 to 100KD, about 5 to 90KD, about 5 to 80KD, about 5 to 70KD, about 5 to 60KD, about 5 to 50KD, about 5 to 40KD, about 5 to 30KD, about 5 to 20KD, about 10 to 100KD, about 10 to 90KD, about 10 to 80KD, about 10 to 70KD, about 10 to 60KD, about 10 to 50KD, about 10 to 40KD, about 10 to 30KD, or about 10 to 20KD.

[0076] In one embodiment, the PEG has a molecular weight of about 60 KD, about 55 KD, about 50 KD, about 45 KD, about 40 KD, about 35 KD, about 30 KD, about 25 KD, about 20 KD, about 15 KD, about 10 KD or about 5 KD.

[0077] In one embodiment, the PEG comprises branched or unbranched PEG, for example, branched PEG may comprise branched PEG of about 10 KD and about 20 KD, and unbranched PEG may comprise unbranched PEG of about 10 KD, about 20 KD, and about 40 KD.

[0078] In one embodiment, PEG modification is performed on the N-terminal amino acid residue of IL-2, for example, the lysine residue, serine residue, or threonine residue of the N-terminus of IL-2. In one embodiment, PEG modification is performed on the α-amino group of the N-terminus of IL-2. PEG modification may be, for example, a single-site modification or a multi-site modification, with single-site modification being preferred.

[0079] In one embodiment, the IL-2 is human or non-human mammalian IL-2, such as bovine, rat, mouse, dog, monkey, goat, sheep, cow, deer, horse, and cat.

[0080] In one embodiment, IL-2 as used herein comprises the amino acid sequence of SEQ ID NO:1.

[0081] Low-molecular-weight HA refers to HA having a molecular weight of about 300 KD or less, for example, about 250 KD, about 200 KD, about 150 KD, about 100 KD, about 90 KD, about 80 KD, about 70 KD, about 60 KD, about 50 KD, about 40 KD, about 30 KD, about 20 KD, about 10 KD, about 9 KD, about 8 KD, about 7 KD, about 6 KD, about 5 KD, about 4 KD, about 3 KD, or about 2 KD. In one embodiment, low-molecular-weight HA refers to HA having a molecular weight of about 2 to 20 KD, about 2 to 15 KD, about 2 to 10 KD, about 2 to 5 KD, about 3 to 20 KD, about 3 to 15 KD, about 3 to 10 KD, or about 3 to 5 KD, for example, about 5 KD, about 4 KD, about 3 KD, or about 2 KD. In one embodiment, low molecular weight HA refers to HA with a molecular weight of approximately 4 KD.

[0082] In one embodiment, the method of the present invention comprises administering low molecular weight HA having a molecular weight of about 5 KD or less, for example, about 4 KD, about 3 KD, or about 2 KD. In one embodiment, low molecular weight HA refers to HA having a molecular weight of about 4 KD.

[0083] In one embodiment, the method of the present invention comprises administering high molecular weight HA to a subject, and the high molecular weight HA and low molecular weight HA can be administered in any ratio, so long as the mass ratio of the administered high molecular weight HA to the administered low molecular weight HA is about 2:1, or as long as the mass of the administered high molecular weight HA is less than 50%, preferably less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10% of the total mass of the administered high molecular weight HA and low molecular weight HA. Preferably, the HA administered to a subject comprises high molecular weight HA and low molecular weight HA in a mass ratio of about 1:2.

[0084] High molecular weight HA refers to HA having a molecular weight of about 800 KD or more, for example, about 900 KD or more, about 1000 KD or more, about 1100 KD or more, about 1200 KD or more, about 1300 KD or more, about 1400 KD or more, about 1500 KD or more, about 1600 KD or more, about 1700 KD or more, about 1800 KD or more, about 1900 KD or more, or about 2000 KD or more. In one embodiment, high molecular weight HA has a molecular weight of about 800 to 2000 KD, about 800 to 1900 KD, about 800 to 1800 KD, about 800 to 1700 KD, about 800 to 1600 KD, about 800 to 1500 KD, about 900 to 2000 KD, about 900 to 1900 KD, about 900 to 18 ... It refers to HA of 1700KD, about 900 to 1600KD, about 900 to 1500KD, about 1000 to 2000KD, about 1000 to 1900KD, about 1000 to 1800KD, about 1000 to 1700KD, about 1000 to 1600KD or about 1000 to 1500KD, and particularly refers to HA of about 800 to 1500KD.

[0085] In one embodiment, the glucocorticoid administered to the subject is selected from the group consisting of dexamethasone, budesonide, beclomethasone dipropionate, ciclesonide, hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone, clobetasone butyrate, triamcinolone acetonide, fluocinolone, mometasone furoate, halcinonide, clobetasol propionate, halcinonide, halometasone, diflorasone diacetate, mometasone, loteprednol, etiprednol, triamcinolone, flunisolide, flumoxonide, rofleponide, butixocort, tipredane, and derivatives thereof. In one embodiment, the glucocorticoid comprises dexamethasone, budesonide, or a derivative thereof.

[0086] In one embodiment, PEG-IL-2, glucocorticoid, and HA may be applied to the affected area simultaneously or sequentially in any order.

[0087] In one embodiment, the method of the invention comprises administering to a subject a therapeutically effective amount of the pharmaceutical composition of the first aspect.

[0088] As used herein, a subject is a human or non-human subject suffering from atopic dermatitis, non-limiting examples of which include humans and other mammals such as cows, rats, mice, dogs, monkeys, goats, sheep, bovines, deer, horses, cats, and other non-mammals. In some embodiments, the subject is a human.

[0089] In one embodiment, the subject's atopic dermatitis is atopic dermatitis accompanied by skin lesions.

[0090] In one embodiment, the method of the present invention comprises administering to a subject: 10KD PEG-modified IL-2, dexamethasone or budesonide, and (i) low molecular weight HA, or (ii) HA comprising low molecular weight HA and high molecular weight HA, wherein the mass ratio of high molecular weight HA to low molecular weight HA is about 1:2. The method includes administering

[0091] In one embodiment, the method of the present invention comprises controlling: 10KD PEG-modified IL-2, dexamethasone or budesonide, and (i) low molecular weight HA, or (ii) HA comprising low molecular weight HA and high molecular weight HA, wherein the mass ratio of high molecular weight HA to low molecular weight HA is about 1:2. and administering a pharmaceutical composition comprising:

[0092] As used herein, "administration" refers to administering an active or pharmaceutical composition locally (e.g., topically or locally) to the area affected by atopic dermatitis. Administration can be performed using any method known in the art, as well as auxiliary means such as direct application, spray, instillation, patch, etc.

[0093] According to a third aspect, there is provided the use of PEG-IL-2, a glucocorticoid and a low molecular weight HA in the manufacture of a medicament (particularly a topical or externally applied medicament) or kit for treating atopic dermatitis; and PEG-IL-2, a glucocorticoid and a low molecular weight HA for treating atopic dermatitis.

[0094] Low-molecular-weight HA refers to HA having a molecular weight of about 300 KD or less, for example, about 250 KD, about 200 KD, about 150 KD, about 100 KD, about 90 KD, about 80 KD, about 70 KD, about 60 KD, about 50 KD, about 40 KD, about 30 KD, about 20 KD, about 10 KD, about 9 KD, about 8 KD, about 7 KD, about 6 KD, about 5 KD, about 4 KD, about 3 KD, or about 2 KD. In one embodiment, low-molecular-weight HA refers to HA having a molecular weight of about 2 to 20 KD, about 2 to 15 KD, about 2 to 10 KD, about 2 to 5 KD, about 3 to 20 KD, about 3 to 15 KD, about 3 to 10 KD, or about 3 to 5 KD, for example, about 5 KD, about 4 KD, about 3 KD, or about 2 KD. In one embodiment, low molecular weight HA refers to HA with a molecular weight of approximately 4 KD.

[0095] In one embodiment, the PEG-IL-2 is a PEG having a molecular weight of about 100 KD, about 90 KD, about 80 KD, about 70 KD, about 60 KD, about 55 KD, about 50 KD, about 45 KD, about 40 KD, about 35 KD, about 30 KD, about 25 KD, about 20 KD, about 15 KD, about 10 KD, or about 5 KD or less. In one embodiment, the PEG has a molecular weight of about 1 to 100 KD, about 1 to 90 KD, about 1 to 80 KD, about 1 to 70 KD, about 1 to 60 KD, about 1 to 50 KD, about 1 to 40 KD, about 1 to 10 KD, about 1 to 20 KD, about 2 to 100 KD, about 2 to 90 KD, about 2 to 80 KD, about 2 to 70 KD, about 2 to 60 KD, about 2 to 50 KD, about 2 to 40 KD, about 2 to 30 KD, about 2 to 20 KD, about 3 to 100 KD, about 3 to 90 KD, about 3 to 80 KD, about 3 to 70 KD, about 3 to 60 KD, about 3 to 50 KD, about 3 to 40 KD, about 3 to 30 KD, or about 3 to 20 KD. and about 4 to 100KD, about 4 to 90KD, about 4 to 80KD, about 4 to 70KD, about 4 to 60KD, about 4 to 50KD, about 4 to 40KD, about 4 to 30KD, about 4 to 20KD, about 5 to 100KD, about 5 to 90KD, about 5 to 80KD, about 5 to 70KD, about 5 to 60KD, about 5 to 50KD, about 5 to 40KD, about 5 to 30KD, about 5 to 20KD, about 10 to 100KD, about 10 to 90KD, about 10 to 80KD, about 10 to 70KD, about 10 to 60KD, about 10 to 50KD, about 10 to 40KD, about 10 to 30KD, or about 10 to 20KD.

[0096] In one embodiment, the PEG has a molecular weight of about 60 KD, about 55 KD, about 50 KD, about 45 KD, about 40 KD, about 35 KD, about 30 KD, about 25 KD, about 20 KD, about 15 KD, about 10 KD or about 5 KD.

[0097] In one embodiment, the PEG comprises branched or unbranched PEG, for example, branched PEG may comprise branched PEG of about 10 KD and about 20 KD, and unbranched PEG may comprise unbranched PEG of about 10 KD, about 20 KD, and about 40 KD.

[0098] In one embodiment, PEG modification is performed on the N-terminal amino acid residue of IL-2, for example, a lysine residue, serine residue, or threonine residue at the N-terminus of IL-2. In one embodiment, PEG modification is performed on the α-amino group at the N-terminus of IL-2. PEG modification may be, for example, a single-site modification or a multi-site modification, with single-site modification being preferred.

[0099] In one embodiment, the IL-2 is human or non-human mammalian IL-2, for example, bovine, rat, mouse, dog, monkey, goat, sheep, cow, deer, horse, or cat.

[0100] In one embodiment, IL-2 as used herein comprises the amino acid sequence of SEQ ID NO:1.

[0101] In one embodiment, there is further provided the use of PEG-IL-2, glucocorticoid, high molecular weight HA, and low molecular weight HA in the manufacture of a medicament or kit for treating atopic dermatitis, and PEG-IL-2, glucocorticoid, high molecular weight HA, and low molecular weight HA for use in the treatment of atopic dermatitis, wherein the ratio of high molecular weight HA to low molecular weight HA is arbitrary, but the mass ratio of high molecular weight HA to low molecular weight HA is about 2:1, or the ratio of the mass of high molecular weight HA to the total mass of high molecular weight HA and low molecular weight HA is less than 50%, preferably less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10%. Preferably, the mass ratio of high molecular weight HA to low molecular weight HA is about 1:2.

[0102] High molecular weight HA refers to HA having a molecular weight of about 800 KD or greater, e.g., about 900 KD or greater, about 1000 KD or greater, about 1100 KD or greater, about 1200 KD or greater, about 1300 KD or greater, about 1400 KD or greater, about 1500 KD or greater, about 1600 KD or greater, about 1700 KD or greater, about 1800 KD or greater, about 1900 KD or greater, about 2000 KD or greater, or greater. In one embodiment, high molecular weight HA has a molecular weight of about 800 to 2000 KD, about 800 to 1900 KD, about 800 to 1800 KD, about 800 to 1700 KD, about 800 to 1600 KD, about 800 to 1500 KD, about 900 to 2000 KD, about 900 to 1900 KD, about 900 to 18 ... It refers to HA of 1700KD, about 900 to 1600KD, about 900 to 1500KD, about 1000 to 2000KD, about 1000 to 1900KD, about 1000 to 1800KD, about 1000 to 1700KD, about 1000 to 1600KD or about 1000 to 1500KD, and particularly refers to HA of about 800 to 1500KD.

[0103] In one embodiment, the glucocorticoid is selected from the group consisting of dexamethasone, budesonide, beclomethasone dipropionate, ciclesonide, hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone, clobetasone butyrate, triamcinolone acetonide, fluocinolone, mometasone furoate, halcinonide, clobetasol propionate, halcinonide, halometasone, diflorasone diacetate, mometasone, loteprednol, etiprednol, triamcinolone, flunisolide, flumoxonide, rofleponide, butixocort, tipredane, and derivatives thereof. In one embodiment, the glucocorticoid comprises dexamethasone, budesonide, or a derivative thereof.

[0104] In one embodiment, the atopic dermatitis is atopic dermatitis accompanied by skin lesions.

[0105] According to a fourth aspect, there is provided a kit comprising PEG-IL-2, a glucocorticoid and low molecular weight HA.

[0106] The kit may further include other reagents for formulating the included PEG-IL-2, glucocorticoid, and HA into a suitable formulation (eg, solvent, buffer, etc.), and optionally instructions.

[0107] In one embodiment, PEG-IL-2 is IL-2 modified with PEG having a molecular weight of about 100 KD, about 90 KD, about 80 KD, about 70 KD, about 60 KD, about 55 KD, about 50 KD, about 45 KD, about 40 KD, about 35 KD, about 30 KD, about 25 KD, about 20 KD, about 15 KD, about 10 KD, or about 5 KD or less. In one embodiment, the PEG has a molecular weight of about 1 to 100 KD, about 1 to 90 KD, about 1 to 80 KD, about 1 to 70 KD, about 1 to 60 KD, about 1 to 50 KD, about 1 to 40 KD, about 1 to 10 KD, about 1 to 20 KD, about 2 to 100 KD, about 2 to 90 KD, about 2 to 80 KD, about 2 to 70 KD, about 2 to 60 KD, about 2 to 50 KD, about 2 to 40 KD, about 2 to 30 KD, about 2 to 20 KD, about 3 to 100 KD, about 3 to 90 KD, about 3 to 80 KD, about 3 to 70 KD, about 3 to 60 KD, about 3 to 50 KD, about 3 to 40 KD, about 3 to 30 KD, or about 3 to 20 KD. and about 4 to 100KD, about 4 to 90KD, about 4 to 80KD, about 4 to 70KD, about 4 to 60KD, about 4 to 50KD, about 4 to 40KD, about 4 to 30KD, about 4 to 20KD, about 5 to 100KD, about 5 to 90KD, about 5 to 80KD, about 5 to 70KD, about 5 to 60KD, about 5 to 50KD, about 5 to 40KD, about 5 to 30KD, about 5 to 20KD, about 10 to 100KD, about 10 to 90KD, about 10 to 80KD, about 10 to 70KD, about 10 to 60KD, about 10 to 50KD, about 10 to 40KD, about 10 to 30KD, or about 10 to 20KD.

[0108] In one embodiment, the PEG has a molecular weight of about 60 KD, about 55 KD, about 50 KD, about 45 KD, about 40 KD, about 35 KD, about 30 KD, about 25 KD, about 20 KD, about 15 KD, about 10 KD or about 5 KD.

[0109] In one embodiment, the PEG comprises branched or unbranched PEG, for example, branched PEG may comprise branched PEG of about 10 KD and about 20 KD, and unbranched PEG may comprise unbranched PEG of about 10 KD, about 20 KD, and about 40 KD.

[0110] The PEG modification described herein can be performed at any site of IL-2 suitable for PEG modification, such as a lysine residue, serine residue, threonine residue, or N-terminal α-amino group of IL-2. In one embodiment, the PEG modification can be performed at the N-terminal amino acid residue of IL-2, such as a lysine residue, serine residue, or threonine residue at the N-terminus of IL-2. In one embodiment, the PEG modification can be performed at the N-terminal α-amino group of IL-2. The PEG modification can be, for example, a single-site modification or a multiple-site modification, with single-site modification being preferred.

[0111] In one embodiment, the IL-2 is derived from a human or non-human mammal, such as cow, rat, mouse, dog, monkey, goat, sheep, bovine, deer, horse, cat, etc.

[0112] In one embodiment, human-derived IL-2 or an active variant thereof is used, more preferably recombinantly produced. The nucleotide sequence and amino acid sequence of human-derived IL-2 are disclosed, for example, in Genbank refs 3558 and P60568, respectively. Unless otherwise specified, the IL-2 used is substantially pure, for example, 95% or more pure, more preferably 96%, 97%, 98%, or 99% pure. IL-2 can be used in the form of a monomeric or multimeric protein.

[0113] In one embodiment, IL-2 as used herein comprises the amino acid sequence of SEQ ID NO:1.

[0114] Low molecular weight HA refers to HA having a molecular weight of about 300 KD or less, for example, about 250 KD or less, about 200 KD or less, about 150 KD or less, about 100 KD or less, about 90 KD or less, about 80 KD or less, about 70 KD or less, about 60 KD or less, about 50 KD or less, about 40 KD or less, about 30 KD or less, about 20 KD or less, about 10 KD or less, about 9 KD or less, about 8 KD or less, about 7 KD or less, about 6 KD or less, about 5 KD or less, about 4 KD or less, about 3 KD or less, or about 2 KD or less. In one embodiment, low-molecular-weight HA refers to HA with a molecular weight of about 2 to 20 KD, about 2 to 15 KD, about 2 to 10 KD, about 2 to 5 KD, about 3 to 20 KD, about 3 to 15 KD, about 3 to 10 KD, or about 3 to 5 KD, for example, about 5 KD, about 4 KD, about 3 KD, or about 2 KD. In one embodiment, low-molecular-weight HA refers to HA with a molecular weight of about 4 KD.

[0115] In one embodiment, the kit further comprises high molecular weight HA, wherein the high molecular weight HA and low molecular weight HA are present in any ratio, wherein the mass ratio of high molecular weight HA to low molecular weight HA is about 2:1, or the ratio of the mass of high molecular weight HA to the total mass of high molecular weight HA and low molecular weight HA is less than 50%, preferably less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10% or less. Preferably, the kit comprises high molecular weight HA to low molecular weight HA in a mass ratio of about 1:2.

[0116] High molecular weight HA refers to HA having a molecular weight of about 800 KD or greater, e.g., about 900 KD or greater, about 1000 KD or greater, about 1100 KD or greater, about 1200 KD or greater, about 1300 KD or greater, about 1400 KD or greater, about 1500 KD or greater, about 1600 KD or greater, about 1700 KD or greater, about 1800 KD or greater, about 1900 KD or greater, about 2000 KD or greater, or greater. In one embodiment, high molecular weight HA has a molecular weight of about 800 to 2000 KD, about 800 to 1900 KD, about 800 to 1800 KD, about 800 to 1700 KD, about 800 to 1600 KD, about 800 to 1500 KD, about 900 to 2000 KD, about 900 to 1900 KD, about 900 to 18 ... It refers to HA of 1700KD, about 900 to 1600KD, about 900 to 1500KD, about 1000 to 2000KD, about 1000 to 1900KD, about 1000 to 1800KD, about 1000 to 1700KD, about 1000 to 1600KD or about 1000 to 1500KD, and particularly refers to HA of about 800 to 1500KD.

[0117] In one embodiment, the kit comprises: The glucocorticoid may be selected from the group consisting of dexamethasone, budesonide, beclomethasone dipropionate, ciclesonide, hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone, clobetasone butyrate, triamcinolone acetonide, fluocinolone, mometasone furoate, halcinonide, clobetasol propionate, halcinonide, halometasone, diflorasone diacetate, mometasone, loteprednol, etiprednol, triamcinolone, flunisolide, flumoxonide, rofleponide, butixocort, tipredane, and derivatives thereof. In one embodiment, the glucocorticoid is dexamethasone, budesonide, or a derivative thereof.

[0118] According to a fifth aspect, there is provided the use of low molecular weight HA in the manufacture of a medicament for enhancing the effect of PEG-IL-2 and / or glucocorticoids in the treatment of atopic dermatitis, and low molecular weight HA for use in enhancing the effect of PEG-IL-2 and / or glucocorticoids in the treatment of atopic dermatitis.

[0119] Low-molecular-weight HA refers to HA having a molecular weight of about 300 KD or less, for example, about 250 KD, about 200 KD, about 150 KD, about 100 KD, about 90 KD, about 80 KD, about 70 KD, about 60 KD, about 50 KD, about 40 KD, about 30 KD, about 20 KD, about 10 KD, about 9 KD, about 8 KD, about 7 KD, about 6 KD, about 5 KD, about 4 KD, about 3 KD, or about 2 KD. In one embodiment, low-molecular-weight HA refers to HA having a molecular weight of about 2 to 20 KD, about 2 to 15 KD, about 2 to 10 KD, about 2 to 5 KD, about 3 to 20 KD, about 3 to 15 KD, about 3 to 10 KD, or about 3 to 5 KD, for example, about 5 KD, about 4 KD, about 3 KD, or about 2 KD. In one embodiment, low molecular weight HA refers to HA with a molecular weight of approximately 4 KD.

[0120] In one embodiment, there is provided use of high molecular weight HA and low molecular weight HA in the manufacture of a medicament for improving the effects of PEG-IL-2 and / or glucocorticoids in the treatment of atopic dermatitis; and high molecular weight HA and low molecular weight HA for use in improving the effects of PEG-IL-2 and / or glucocorticoids in the treatment of atopic dermatitis, wherein the ratio of high molecular weight HA to low molecular weight HA is arbitrary, but the mass ratio of high molecular weight HA to low molecular weight HA is about 2:1, or the ratio of the mass of high molecular weight HA to the total mass of high molecular weight HA and low molecular weight HA is less than 50%, preferably less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10% or even less, and preferably the mass ratio of high molecular weight HA to low molecular weight HA is about 1:2.

[0121] High molecular weight HA refers to HA having a molecular weight of about 800 KD or greater, e.g., about 900 KD or greater, about 1000 KD or greater, about 1100 KD or greater, about 1200 KD or greater, about 1300 KD or greater, about 1400 KD or greater, about 1500 KD or greater, about 1600 KD or greater, about 1700 KD or greater, about 1800 KD or greater, about 1900 KD or greater, about 2000 KD or greater, or greater. In one embodiment, high molecular weight HA has a molecular weight of about 800 to 2000 KD, about 800 to 1900 KD, about 800 to 1800 KD, about 800 to 1700 KD, about 800 to 1600 KD, about 800 to 1500 KD, about 900 to 2000 KD, about 900 to 1900 KD, about 900 to 1800 KD, about 900 It refers to HA of about 1700KD, about 900 to 1600KD, about 900 to 1500KD, about 1000 to 2000KD, about 1000 to 1900KD, about 1000 to 1800KD, about 1000 to 1700KD, about 1000 to 1600KD or 1000 to 1500KD, and particularly refers to HA of about 800 to 1500KD.

[0122] In one embodiment, the atopic dermatitis is atopic dermatitis accompanied by skin lesions. In particular, the agent for treating atopic dermatitis is a topical or external agent.

[0123] According to a sixth aspect, there is provided the use of 10KD PEG-modified IL-2 in the manufacture of a medicament for treating type I allergy. In particular, the medicament for treating type I allergy is a topical agent.

[0124] In one embodiment, the modified IL-2 is modified with branched or unbranched PEG. In one embodiment, the modified IL-2 is modified with branched PEG. In one embodiment, the modified IL-2 is modified with unbranched PEG.

[0125] In one embodiment, PEG modification is performed on the N-terminal amino acid residue of IL-2, for example, a lysine residue, serine residue, or threonine residue at the N-terminus of IL-2. In one embodiment, PEG modification is performed on the α-amino group at the N-terminus of IL-2. PEG modification may be, for example, a single-site modification or a multi-site modification, with single-site modification being preferred.

[0126] In one embodiment, the IL-2 is from a human or non-human mammal, such as cow, rat, mouse, dog, monkey, goat, sheep, bovine, deer, horse, and cat.

[0127] In one embodiment, the IL-2 comprises the amino acid sequence of SEQ ID NO:1.

[0128] In one embodiment, Type I allergies include: 1) systemic anaphylaxis, including drug anaphylactic shock and serum anaphylactic shock; 2) respiratory allergies, such as allergic rhinitis and allergic asthma; 3) gastrointestinal allergies, such as allergic gastroenteritis; and 4) skin allergies, such as urticaria, atopic dermatitis (eczema), and angioedema.

[0129] According to a seventh aspect, there is provided a method of treating type I allergy in a subject, comprising administering to the subject 10KD PEG-modified IL-2.

[0130] In one embodiment, the method involves administering 10 KD PEG-modified IL-2 modified with about 10 KD branched or unbranched PEG. In one embodiment, the IL-2 is modified with a 10 KD branched PEG. In one embodiment, the IL-2 is modified with a 10 KD unbranched PEG.

[0131] In one embodiment, PEG modification is performed on the N-terminal amino acid residue of IL-2, for example, a lysine residue, serine residue, or threonine residue at the N-terminus of IL-2. In one embodiment, PEG modification is performed on the α-amino group at the N-terminus of IL-2. PEG modification may be, for example, a single-site modification or a multi-site modification, with single-site modification being preferred.

[0132] In one embodiment, the IL-2 is derived from a human or non-human mammal, such as cow, rat, mouse, dog, monkey, goat, sheep, bovine, deer, horse, cat, etc.

[0133] In one embodiment, the IL-2 administered comprises the amino acid sequence of SEQ ID NO:1.

[0134] In one embodiment, Type I allergies include: 1) systemic anaphylaxis, including drug anaphylactic shock and serum anaphylactic shock; 2) respiratory allergies, such as allergic rhinitis and allergic asthma; 3) gastrointestinal allergies, such as allergic gastroenteritis; and 4) skin allergies, such as urticaria, atopic dermatitis (eczema), and angioedema.

[0135] In one embodiment, the subject is a human or non-human subject suffering from atopic dermatitis, including, but not limited to, humans and other mammals such as cows, rats, mice, dogs, monkeys, goats, sheep, bovines, deer, horses, cats, and other non-mammals. In some embodiments, the subject is a human.

[0136] Administration can be by any route suitable for treating allergies, including intravenous, topical, topical, subcutaneous, intraperitoneal, etc. In one embodiment, administration is local, e.g., topical application.

[0137] The medicament may take any suitable dosage form, such as a powder, spray, foam, solution, ointment, emulsion, and / or in the form of a solid, liquid, gel, etc. The medicament may also include adjuvants, such as pharmaceutically acceptable carriers, as described herein above.

[0138] According to an eighth aspect, there is provided a 10 KD PEG-modified IL-2.

[0139] In one embodiment, the IL-2 is IL-2 from a human or non-human mammal, such as a cow, rat, mouse, dog, monkey, goat, sheep, bovine, deer, horse, cat, etc. In particular, the IL-2 comprises the amino acid sequence set forth in SEQ ID NO: 1, and preferably, one site at the N-terminus of the IL-2 is modified with PEG.

[0140] Any suitable type of PEG can be used, such as linear or non-linear PEG. "About 10 KD" means that the total atomic weight of the atoms constituting PEG is 10 KD±10%, 10 KD±5%, 10 KD±4%, 10 KD±3%, 10 KD±2%, 10 KD±1%, 10 KD±0.5%, or 10 KD±0.1%.

[0141] As used herein, the word "or" is intended to include "and" unless the context clearly indicates otherwise.

[0142] As used herein, "optional" or "optionally" means that a described event or circumstance occurs or does not occur, and this description includes both cases where the event or circumstance occurs and cases where the event or circumstance does not occur. For example, "optional step" means that the step is either present or absent, and "optionally a pharmaceutically acceptable carrier" means that a pharmaceutically acceptable carrier is either included or not included.

[0143] As used herein, the term "about" refers to a range of values ​​that includes a particular numerical value, and one of ordinary skill in the art can reasonably assume that the range is close to the particular numerical value. In certain embodiments, the term "about" refers to within a standard error using measurements generally accepted in the art. For example, in certain embodiments, "about" refers to ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, or even ±0.5% of the particular numerical value.

[0144] In this specification, when a specific numerical value or ratio is stated for a feature in the description, any two numerical value or ratio ranges are also included. For example, when the numerical values ​​1, 2, 3, and 4 are stated, ranges of 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, and 3 to 4 are also included. [Example]

[0145] The present invention will be further described by the following examples, but any examples or combinations thereof should not be construed as limiting the scope or embodiments of the present invention. The scope of the present invention is defined by the appended claims and will be apparent to those skilled in the art in light of this specification and common knowledge in the art. Without departing from the spirit and scope of the present invention, those skilled in the art may make any modifications or changes to the technical solutions of the present invention, and these modifications and changes are also encompassed within the scope of the present invention. Unless otherwise specified, the methods used in the following examples are conventional methods.

[0146] Materials and Methods I. Experimental Materials [Table 1]

[0147] (II) Experimental materials and reagents [Table 2] JPEG2026505416000005.jpg54149

[0148] 4. Experimental Animals Six-week-old, SPF-grade female BALB / c mice were purchased from Zhejiang Vital River Laboratory Animal Co., Ltd. and raised at Fudan University. In this experiment, the animals were fed and treated in accordance with the International Standards for the Use of Laboratory Animals, and all animals were quarantined and acclimatized to the experimental environment for one week before starting the animal experiment.

[0149] Example 1: Preparation of polyethylene glycol (PEG)-modified interleukin-2 PEG has two structural types: branched (Y-AALD-10kD, Y-AALD-20kD) and unbranched (M-ALD-10kD, M-ALD-20kD). All PEGs with a molecular weight of 40kD were unbranched (M-ALD-40kD (all purchased from BEIJING JIANKAI TECHNOLOGY CO.,LTD.)).

[0150] IL-2 (the sequence is shown in SEQ ID NO: 1) was mixed in acetic acid-sodium acetate buffer (pH 4-6) as a displacement buffer with M-ALD-20kD (BEIJING JIANKAI TECHNOLOGY CO., LTD.) unbranched PEG in a proportional ratio (mass ratio: 1:2-1:6), reduced with sodium cyanoborohydride, and reacted at 2-10°C for 3-18 hours to obtain a crude IL-2 (PEG) product. The crude IL-2(PEG) was first isolated by cation exchange chromatography in an acetic acid-sodium acetate buffer system, and the target protein peak was collected for reversed-phase chromatography. It was then eluted with a gradient of acetonitrile-trifluoroacetic acid. The target protein peak was collected for cation exchange chromatography, concentrated under an acetic acid-sodium acetate buffer system, and the target protein peak was collected. Finally, the buffer was replaced with sodium acetate by ultrafiltration. IL-2 modified with 10 kDa, 20 kDa, and 40 kDa unbranched PEG and IL-2 modified with 10 kDa and 20 kDa branched PEG were further prepared. This process was consistent with the process for preparing IL-2(PEG) modified with 10 kDa and 20 kDa unbranched PEG.

[0151] The expression patterns of interleukin-2 obtained by modification with PEG of different molecular weights and structures were 10 kD PEG-IL-2, 20 kD PEG-IL-2, and 40 kD PEG-IL-2 for the unbranched form, and 20 kD (branched)-IL-2 and 10 kD PEG (branched)-IL-2 for the branched form, but no other types showed similar electropherograms.

[0152] The primary modification site for the selected PEG type was the N-terminus of the peptide chain. After modification with 20 kD unbranched PEG, the molecular weight of recombinant human IL-2 (normal) increased from 15 kD to 35 kD, and the purity also improved (Figure 1a, b). Activity was detected in a CTLL-2 cell assay, and the specific activity of the PEG-modified IL-2 was confirmed to be equivalent to that of the IL-2 standard (Figure 1c, d).

[0153] The sequence of human IL-2 is shown in SEQ ID NO: 1 (5'-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT-3').

[0154] Example 2: Establishment of a mouse model of atopic (allergic) dermatitis A mouse atopic dermatitis model was established according to the method described previously (JIN H, et al. Animal models of atopic dermatitis. J Invest Dermatol, 2009, 129(1): 31-40; KIM WH, et al. Beneficial effects of melittin on ovalbumin-induced atopic dermatitis in mice. Sci Rep, 2017, 7(1): 17679; WANG G, et al. Repeated epicutaneous exposures to ovalbumin progressively induce atopic dermatitis-like skin lesions in mice. Clin Exp Allergy, 2007, 37(1): 151-61), with some modifications. Chicken ovalbumin (OVA)-sensitized BALB / c mice are a common mouse model of type I allergic disease. Mice were sensitized by intraperitoneal injection of 10 μg of OVA mixed with 200 μL of 2 mg of aluminum hydroxide in phosphate buffered saline (PBS). Each mouse was injected three times weekly (on days 0, 7, and 14), with 200 μL of each injection. On day 14, the mice were anesthetized with intraperitoneal injection of pentobarbital. While under anesthesia, the hair on the back of the mice was removed and cleaned using electric clippers and hair removal paste. Medical adhesive tape was then applied directly to the back skin for a few seconds and then removed, a process repeated about four times to disrupt the stratum corneum. An OVA patch was then applied to the skin of the mice to induce atopic (allergic) dermatitis. The OVA patch measured 1.5 × 1.5 cm. 2Sterile gauze was moistened with 1 mg / mL OVA (dissolved in PBS) solution (approximately 100 μL of OVA solution) and applied to the dorsal skin of the depilated mice using a transparent dressing. The dressing was changed daily for 3 to 7 days to allow the development of dermatitis. On the 4th to 5th day after induction of dermatitis (the day when the dermatitis symptoms were most pronounced), the condition of the dorsal skin of the mice was photographed and the inflammation status of the skin was scored. The mice were then euthanized, and skin tissue from the dermatitis site, spleen, and blood were collected for further examination.

[0155] Example 3: Establishment of an asthma mouse model The procedure for early sensitization in mice was the same as that for the type I allergic disease mouse model. Specifically, 10 μg of OVA and 2 mg of aluminum hydroxide were mixed and dissolved in 200 μL of phosphate buffered saline (PBS). The mixture was injected intraperitoneally into mice once a week (days 0, 7, and 14), for a total of three injections (200 μL per injection). On day 14, mice were intranasally administered 20 μg of 2% OVA in sterile saline. The therapeutic agent was then administered topically into the mouse airways via the tip of a micronebulizer.

[0156] Example 4: Assessment of airway responsiveness in mice Mice were anesthetized with sodium pentobarbital, then tracheotomized and placed on a ventilator. Changes in pulmonary resistance following nebulized inhalation of 3.125 mg / ml, 6.25 mg / ml, and 12.5 mg / ml methacholine were measured using an invasive BUXCO small animal pulmonary function testing system.

[0157] 1. Grouping and Administration Throughout the experiment, mice were randomly divided into 28 groups, each with six mice. Each treatment group in the AD model mice was administered using the patch method described above. Patches were made by wetting 1.5 x 1.5 cm sterile gauze with drug solution (containing approximately 100 μL of drug solution and / or 50 μL of hyaluronic acid). The drugs were dissolved in PBS containing 100,000 IU / mL of regular IL-2 or PEG-IL-2, 20 μg / mL budesonide, 100 μg / mL dexamethasone, and 50 mg / mL hyaluronic acid (HA). Starting on days 4–5 after dermatitis induction, treatment was continued for three consecutive days. The administration protocol was changed daily between groups, except that resting conditions were kept constant. After three days, the mice were euthanized, and skin tissues, spleens, and blood samples from the injection sites were collected for further examination. Specific groupings are as follows (note that 10 kD PEG has two structures, branched and unbranched, and in the following groups, when the molecular weight of the structure is not indicated, both 40 kD PEG and 20 kD PEG are unbranched): 1) To verify the efficacy of drugs for treating dermatitis in mice, the following groups were used: (1) blank control group; (2) atopic (allergic) dermatitis (AD) model group; (3) PBS treatment group; (4) budesonide treatment group (standard control), 20 μg / ml budesonide; (5) 40 kD PEG-IL-2 treatment group; (6) 40 kD PEG-IL-2 + budesonide treatment group; (7) 40 kD PEG-IL-2 + budesonide + low molecular weight HA treatment group; (8) low molecular weight HA treatment group. 2) To investigate the effect of PEG-IL-2 with different molecular weights and PEG-modified IL-2 with different structures on the therapeutic effect, the following groups were used: (9) 10 kD (unbranched) PEG-IL-2 + budesonide + low molecular weight HA treatment group; (10) 10 kD (branched) PEG-IL-2 + budesonide + low molecular weight HA treatment group; (11) 20 kD PEG-IL-2 + budesonide + low molecular weight HA treatment group; (12) regular IL-2 + budesonide + low molecular weight HA treatment group. 3) To investigate the effect of different ratios of high molecular weight HA and low molecular weight HA on the therapeutic effect, the following treatment groups were used: (13) 10kD PEG-IL-2 + budesonide + HA (high molecular weight HA / low molecular weight HA = 1:2) treatment group; (14) 10kD PEG-IL-2 + budesonide + HA (high molecular weight HA / low molecular weight HA = 1:1) treatment group; (15) 10kD PEG-IL-2 + budesonide + HA (high molecular weight HA / low molecular weight HA = 2:1) treatment group; (16) 10kD PEG-IL-2 + budesonide + high molecular weight HA treatment group. 4) To compare the therapeutic effects of different forms of glucocorticoids, the following treatment groups were used: (17) 10 kD PEG-IL-2 + dexamethasone + HA (high molecular weight HA / low molecular weight HA = 1:2) treatment group, in which the dexamethasone concentration was 100 μg / ml, and each time was 80-100 μl (8-10 μg); 5) To investigate the long-term therapeutic effects of drugs, after successfully establishing the original mouse model of atopic (allergic) dermatitis (AD), dermatitis was induced in the control group with an OVA patch without any treatment. After the dermatitis was alleviated after separation from the antigen, the mice were then separated from the OVA environment for 6 weeks. After 6 weeks, dermatitis was again induced with an OVA patch, the mice were euthanized, and the skin, spleen, and blood from the original administration site were collected for further examination. After the experimental group was exposed to OVA to induce dermatitis, the mice were treated with 10 kDa PEG-IL-2 + budesonide + HA (high molecular weight HA / low molecular weight HA = 1:2) and then separated from the OVA environment for 6 weeks. After 6 weeks, dermatitis was again induced with an OVA patch, the mice were euthanized, and the skin, spleen, and blood from the original administration site were collected for further examination. 6) Other control groups: (18) Sensitized mice were first treated with 10kd PEG-IL-2 + budesonide + low molecular weight HA for 3 days, and then dermatitis was induced with OVA; (19) AD model mice were treated with 10kd PEG-IL-2 + HA (low molecular weight) treatment group; 7) Asthma model mice: (20) PBS-treated group; (21) 10kDa PEG-IL-2-treated group: treatment dose was 5000 IU of 10kDa PEG-IL-2 per mouse, once daily for 3 consecutive days; (22) combination treatment group: treatment with 10kDa PEG-IL-2 (5000 IU) + HA (1.25 mg) once daily for 3 consecutive days; (23) blank control group; 8) To investigate the difference in the induction effect of PEG-modified interleukin-2 with different molecular weights on regulatory T cells (Tregs), five groups of physiological model mice were further injected intraperitoneally with PEG-IL-2 modified with unbranched PEG of different molecular weight sizes and regular IL-2 at a dose of 400,000 IU / injection / week.

[0158] 2. Skin inflammation condition score The Skin Inflammation Score (SKS) is a clinical indicator of the severity of dermatitis. The total score ranges from 0 to 12 points, and is based on four dimensions: edema, erythema, scaling, and erosion. Individual scores range from 0 to 3 (0: none, 1: mild, 2: moderate, 3: severe). The final score is the sum of the individual dimensions [SCHWARTZ C, et al. Spontaneous atopic dermatitis in mice with a defective skin barrier is independent of ILC2 and mediated by IL-1β. Allergy, 2019, 74(10): 1920-3].

[0159] 3. Histopathological staining After euthanasia, the mice were euthanized, and the skin samples were collected from their backs. They were fixed in 4% paraformaldehyde fixative at room temperature for at least 24 hours, dehydrated in alcohol, embedded in paraffin, cut longitudinally, and dewaxed. After H&E staining, the pathological condition of the skin was observed under an optical microscope.

[0160] 4. Analysis of the Percentage of Treg Cells in the Skin and Spleen Using Flow Cytometry 1) Sampling: Euthanize the mouse and collect the spleen and the skin (approximately 1.5 x 1.5 cm) from the experimental site on the back. 2) were collected and immersed in PBS solution; 2) Tissue processing: The spleen was crushed and passed through a 70 μm pore size cell strainer to obtain a single cell suspension of the spleen; the skin was first cut into 1 × 1 mm pieces. 2 The tissue was then cut into pieces of 100 mm in size, and digested at 37°C for approximately 50 minutes with HBSS solution containing 2 mg / mL collagenase and 1 mg / mL DAN enzyme, with manual shaking every 5 minutes. After digestion, the tissue was centrifuged briefly to discard the supernatant, washed twice with PBS, crushed, and filtered through a 70 μm filter to obtain a single-cell suspension of skin tissue. 3) Lysis of red blood cells: After transferring each single cell suspension to a centrifuge tube, 3-5 mL of red blood cell lysis solution was added to the tube to resuspend the cells for 2 minutes, and then centrifuged at 500 g and 4°C for 5 minutes. The supernatant was removed, and the cell pellet was washed twice with PBS; 4) Blocking: Count the cells under a microscope and measure approximately 5 x 10 6 The cells were harvested, CD16 / 32 antibody was diluted at a ratio of 1:100, the cell suspension was added, incubated at 4°C for 15 minutes, and washed twice with PBS; 5) Cell surface antigen staining: CD3 antibody, CD4 antibody, and CD25 antibody were diluted at a ratio of 1:100, respectively, added to the cell suspension, incubated in the dark at 4°C for 30 minutes, and washed twice with PBS; 6) Fixation: The supernatant was discarded, and the cells were fixed in the fixation solution of Transcription Factor Fixation / Permeabilization Buffer Set (Biolegend, 424401) in the dark at 4°C for 1 hour; 7) Membrane disruption: the same volume of membrane disruption solution as the fixative was directly added, and the cells were centrifuged at 500g and 4°C for 5 minutes, followed by washing twice with the membrane disruption solution; 8) Intracellular antigen staining: FOXP3 antibody was diluted with membrane disruption solution at a ratio of 1:100, added to the cell suspension, and incubated in the dark at room temperature for 1 hour, followed by washing once with membrane and once with PBS; 9) Assay: Cells were resuspended in PBS and analyzed by flow cytometry and FlowJo software.

[0161] 5. Detection of Skin Cytokine Expression by Real-time PCR 1) Sample collection: The skin on the back of the mouse was collected and cut with ophthalmic scissors on ice, then 1 mL of Trizol reagent was added and homogenized using a tissue homogenizer; 2) RNA extraction: 200 μL of chloroform was added, the mixture was shaken vigorously for 15 seconds, allowed to stand at room temperature for 15 minutes, and centrifuged at 13,000 rpm at 4°C for 15 minutes. The supernatant was transferred to another RNase-free 1.5 mL centrifuge tube, an equal volume of isopropanol was added, the mixture was mixed thoroughly by inversion, allowed to stand at room temperature for 15 minutes, and centrifuged at 12,000 rpm at 4°C for 10 minutes. The supernatant was discarded; 3) Precipitation: 30 μL of pre-chilled 75% ethanol was added for washing, and the supernatant was discarded after centrifugation; 4) Dissolution: 30 μL of DEPC water was added to dissolve the RNA, and the RNA concentration was measured by spectrophotometer; 5) Reverse transcription: According to the measured RNA concentration, 1 μg of cDNA was obtained per tube by reverse transcription, strictly following the procedure instructed by the SYBR Green Reverse Transcription Kit (TIANGEN, FP205-01); 6) RT-PCR filling and detection: A 20 μL reaction system was selected and operated in strict accordance with the instructions of the TR-PCR kit (Takara, RR047A), filled into the PCR machine, and the data was analyzed using Excel software; 7) The primers used in the experiment are as follows: [Table 4]

[0162] 6. Measurement of mouse serum IgE concentrations by ELISA 1) Sample collection: Mice were anesthetized with pentobarbital, and approximately 500 mL of blood was drawn from the heart and left at room temperature for 1 hour. After blood collection, the mice were euthanized. After 1 hour, the blood was centrifuged at 3500 rpm for 15 minutes at room temperature, and serum was collected. 2) Based on the results of the preliminary experiment, the serum was diluted 4-fold or 10-fold, and then the standard samples and serum samples were assayed simultaneously according to the instructions of the Mouse Serum IgE ELISA Kit (NEOBIOSCIENCE, EMC117.48). Each well was set up with duplicate wells, and the color development results were measured using a microplate reader at a detection wavelength of 450 nm; 3) Serum IgE concentrations were calculated according to the standard curve and the curve equation obtained by mapping using Excel.

[0163] III. Data Analysis and Processing Methods The streaming data were compiled using Flowjo software. The data were statistically analyzed using Graphpad Prism 7 software or Excel software. The results are expressed as mean ± standard error, with a P value of <0.05 (*) indicating a difference between the data. The larger the number of *, the smaller the P value, indicating a significant difference between the data.

[0164] result 1. OVA induces dermatitis in mice In this study, we used OVA to create a mouse atopic dermatitis (AD) model, as described above. Compared with the blank control group, the dorsal skin of the AD model mice showed obvious edema, erythema, scaling, and erosion (Figure 2A), and their skin condition scores increased from 0 to an average of 6.99 (Table 2). Histopathological section examination revealed thickened skin, an incomplete stratum corneum, and significant inflammatory cell infiltration in the dermatitis mice (Figure 2B). RT-PCR analysis revealed increased expression of Th2 cytokines IL-4 and IL-13, and Th17 cytokine IL-17 in the skin of AD mice compared with blank control mice, but no significant change in the expression of Th1 cytokine IFN-γ (Figure 2C). ELISA assays also showed a significant increase in serum IgE levels in the AD mice (Figure 2D). Furthermore, the OVA-induced allergic disease model in BALB / c mice was a classic type I allergic disease model.

[0165] Table 5 JPEG2026505416000008.jpg51151

[0166] 2. PEG-IL-2 combined with budesonide has limited therapeutic effect on atopic dermatitis model mice In a previous study, our research team demonstrated that short-term nebulized inhalation therapy using a combination of 40 kDa polyethylene glycol-modified IL-2 (PEG-IL-2) and dexamethasone promoted the proliferation of Treg cells and increased the odds ratio of Treg / Tcon cells in the alveolar lavage fluid of asthma model mice. This significantly reduced airway hyperresponsiveness and suggested that abnormal Th2 cell polarization was corrected [WU K, et al. Short-term intratracheal use of PEG-modified IL-2 and glucocorticoid persistently alleviates asthma in a mouse model. Sci Rep, 2016, 6(31562)]. Therefore, we attempted to treat dermatitis in mice by topical administration using the same protocol in the model group and the control PBS-treated group. Three days after administration, the degree of dermatitis relief in each treatment group varied compared to the AD model mice, and all dermatitis scores were reduced. Compared with the PBS group, the dermatitis score in the budesonide-only group was significantly reduced (P<0.05), whereas the dermatitis scores in the 40kD PEG-IL-2 alone group and the 40kD PEG-IL-2 + budesonide group were not significantly different from the PBS group (Table 2, Figure 3C). Pathological sections showed no significant improvement in skin thickness or inflammatory cell infiltration in the 40kD PEG-IL-2 alone or PBS groups. Flow cytometry showed no significant changes in the proportion of CD4+CD25+FOXP3+ regulatory T cells (Tregs) among CD4+ cells in the skin of mice. However, in the group treated with budesonide alone (currently the clinically standard first-line treatment regimen) and the group treated with 40 kD PEG-IL-2 in combination with budesonide, the thickness of the mouse skin was relatively reduced, as was the infiltration of inflammatory cells. The increase in the proportion of Treg cells in the skin was statistically significant compared to the physiological model control group (blank control), but there was no statistical difference in the proportion of Tregs between the treatment groups (Figure 3).

[0167] 3. HA enhances the therapeutic effect of PEG-IL-2 and budesonide combination on atopic dermatitis in mice These results indicate that the initial drug combination therapy (i.e., the combination of 40kD PEG-IL-2 and glucocorticoids) did not improve efficacy compared to the clinical standard protocol (glucocorticoids). This is likely due to the drug's inability to penetrate the deep epidermal or dermal layers for its intended effect. To promote drug absorption, a separate group of mice with dermatitis was treated with low molecular weight HA for 3 days, based on the initial drug combination therapy (i.e., 40kD PEG-IL-2 + budesonide + low molecular weight HA). Compared with the untreated group, the mice treated with HA showed a significant and rapid alleviation of dermatitis, with skin recovery to normal and a decrease in dermatitis score, with statistically significant differences (Table 2, Figure 4D). Pathological sections showed a significant decrease in skin thickness and inflammatory cell infiltration, and the percentage of Tregs in the skin, as detected by flow cytometry, increased to 19.8%, with statistically significant differences. Furthermore, in the control group given low molecular weight HA alone, although dermatitis symptoms were alleviated to some extent, no upregulation of skin Tregs was observed (Fig. 4). This may be related to the moisturizing effect of HA molecules, and it also demonstrated that low molecular weight HA does not have an immunomodulatory effect.

[0168] 4. The combination of HA adjuvant and drug can prevent recurrence of dermatitis in mice with dermatitis, but application to normal skin alone in sensitized mice does not have such a preventive effect. As described above, after the initial OVA-induced dermatitis, mice were administered a combination of HA, PEG-IL-2, and budesonide for 3 days. We then attempted to reinduce dermatitis with topical OVA application. Mice treated with PBS for 3 days served as controls. Results showed that mice in the treatment group showed no obvious dermatitis symptoms 7 days after topical OVA application, whereas mice in the control group showed dermatitis symptoms, the severity of which was comparable to the initial induction. Concurrently, another group of mice was selected that had been intraperitoneally injected with OVA for sensitization but had not developed local OVA-induced dermatitis. We first applied PEG-IL-2, budesonide, and low-molecular-weight HA topically for 3 consecutive days, followed by topical application of low-molecular-weight HA to induce dermatitis with an OVA patch. The results showed that the OVA patch successfully induced dermatitis for approximately 2–4 days, with no statistically significant differences in the dermatitis score and skin Treg cell percentage compared to AD model mice (Figure 5). If dermatitis (without skin lesions) was not induced after antigen sensitization, it is possible that HA does not facilitate drug penetration into the skin.

[0169] 5. Therapeutic effects of a combination of IL-2 modified with PEG of different molecular weights and structures in AD mice To investigate whether the molecular weight of PEG-IL-2 and the type of PEG modification of IL-2 play an important role in the therapeutic efficacy of the drug combination therapy, we further investigated four treatment groups: 20 kD PEG-IL-2 + budesonide + low molecular weight HA, 10 kD branched PEG-IL-2 + budesonide + low molecular weight HA, 10 kD (unbranched) PEG-IL-2 + budesonide + low molecular weight HA, and regular IL-2 + budesonide + low molecular weight HA. The 40 kD PEG-IL-2 + budesonide + low molecular weight HA treatment group described above served as a control (the 40 kD PEG and 20 kD PEGs with unspecified structures were unbranched). As a result, the treatment groups with PEG-IL-2 of different molecular weight sizes and modification patterns significantly alleviated the symptoms of dermatitis in the mice, significantly reducing the dermatitis scores, and the differences between the treatment groups were not statistically significant (see Table 2 and Figure 6D). However, as the molecular weight of PEG-IL-2 decreased, the proportion of Treg cells in the skin increased, and the differences between the treatment groups were statistically significant; in the case of 10 kDa PEG molecules, there was no statistical difference in the Treg ratio between the unbranched PEG-IL-2 group and the branched PEG-IL-2 group. Interestingly, although the molecular weight of normal IL-2 (15 kDa) was lower than that of PEG-IL-2 (40 / 20 / 10 + 15 kDa), the therapeutic effect of the normal IL-2 + budesonide + low molecular weight HA group was significantly weaker than that of each PEG-IL-2 + budesonide + low molecular weight HA group, and some scaling was still observed in the skin. Compared with the 40 kD PEG-IL-2 + budesonide + low molecular weight HA group, the dermatitis score was higher (average 2.33), statistically significant difference (P < 0.05), and the skin Treg ratio was also the lowest, statistically significant difference (P < 0.05) (Figure 6). The dermatitis score of the normal IL-2 + budesonide + low molecular weight HA group was not significantly different (P < 0.05) from that of the budesonide alone group (P > 0.05). FOXP3 expression in the skin was significantly higher (P < 0.05) than in the normal IL-2 + budesonide + low molecular weight HA group. + CD25 +There was also no difference in T cell induction between these two groups (p > 0.05), indicating that normal interleukin-2 had no effect (see Table 2 and Figures 6D and 6E). Low-molecular-weight HA did not help normal IL-2, with a molecular weight of 15 kDa, penetrate the epidermal layer and penetrate deeper into the skin. However, 10 kD PEG-IL-2 (25 kDa), 20 kD PEG-IL-2 (35 kDa), and 40 kD PEG-IL-2 (55 kDa), which have molecular weights significantly larger than 15 kDa, as well as modified hydrophilic polar PEG, were able to penetrate deeper into the skin and exert their therapeutic effects. Thus, we unexpectedly discovered that HA interacts with PEG to enable ultra-high molecular weight protein drugs to penetrate the stratum corneum and skin surface and remain in the dermis (the immune response layer in atopic dermatitis).

[0170] 6.10kD-PEG-IL-2 promotes high expression of FoxP3 and PD-1 on regulatory T cells (Tregs) Physiological model mice were intraperitoneally administered PEG-IL-2 modified with unbranched PEGs of different molecular weights, and the expression of FoxP3+ regulatory T cells and PD-1 in splenocytes was detected on day 4. Unexpectedly, 10kd-PEG-IL-2 upregulated Treg cells, whereas regular IL-2, 20kd-PEG-IL-2, and 40kd-PEG-IL-2 did not. The level of regulatory T cells in the 10kd-PEG-IL-2 group was higher than that in the regular IL-2, 20kd-PEG-IL-2, and 40kd-PEG-IL-2 groups, and the difference was statistically significant (Figure 7A).

[0171] Furthermore, we examined the expression level of PD-1 on the surface of Treg cells and found that normal IL-2 could promote PD-1 expression by regulatory T cells. The PD-1 expression level on the surface of Treg cells in the 10kd-PEG-IL-2 group was also higher than that in the PBS group (18.1% vs. 13.1%), but the difference was not statistically significant. The PD-1 expression level on the surface of Treg cells in the 10kd-PEG-IL-2 group was similar to that in the normal IL-2 group, and the difference was not statistically significant (Figure 7B). Treg cells express PD-1, which enhances the inhibitory effect of Treg cells on effector T cells (Teff).

[0172] 7.10kD PEG-IL-2 alone upregulates the proportion of cutaneous Treg cells and provides therapeutic benefits for atopic dermatitis. When 10kd PEG-IL-2 + HA was topically applied to treat dermatitis in AD mice, after three days, symptoms such as skin swelling and erosions were alleviated in both groups of mice. However, the skin remained rough and the rash had not completely healed. The dermatitis scores were statistically significantly reduced compared to the original AD model mice. Comparing the two groups, the skin of the mice treated with 10kd PEG-IL-2 + HA was smoother and had lower dermatitis scores. Histopathological sections showed reduced skin thickening and reduced inflammatory cell infiltration in both groups. Compared to the AD model mice, the number of CD4+CD25+FoxP3+Treg cells in the skin of both groups was increased, and the difference was statistically significant, but no statistical difference was observed between the two groups (Figure 8).

[0173] 8.10kD PEG-IL-2 can attenuate airway hyperresponsiveness in asthmatic mice Further studies were conducted on four groups of mice to examine the immunomodulatory effects of 10kd PEG-IL-2 in a mouse asthma model, another classic allergy model. After establishing the mouse asthma model, the mice were divided into three groups and PBS, 10kd PEG-IL-2, and 10kd PEG-IL-2 + HA were nebulized into the trachea using a micronebulizer. Three days later, airway responsiveness was assessed in the mice, using healthy mice as controls.

[0174] As shown in Figure 7, 10kd PEG-IL-2 effectively suppressed changes in airway resistance and reduced airway hyperresponsiveness in asthmatic mice, demonstrating statistically significant differences compared with the blank control and PBS groups. However, hyaluronic acid failed to further enhance the effect of 10kd PEG-IL-2 in alleviating the high airway resistance state in the asthmatic mouse model.

[0175] 9. Effects of different ratios of high molecular weight HA / low molecular weight HA in the combination on the treatment of AD mice As mentioned above, low molecular weight HA has no immunomodulatory function but has a strong drug absorption-promoting effect, while high molecular weight HA, while having relatively low skin permeability, can enhance the immunomodulatory ability of activated Treg cells in the local environment. Therefore, after measuring the immunomodulatory effect of 10 kDa PEG-IL-2, we further investigated the effects of high molecular weight HA / low molecular weight HA and their combination ratios on the treatment of AD mice. Four groups of AD model mice were selected: 10 kDa PEG-IL-2 + budesonide alone, high molecular weight HA, high molecular weight HA2 / 3 + low molecular weight HA1 / 3, high molecular weight HA1 / 2 + low molecular weight HA1 / 2, and high molecular weight HA1 / 3 + low molecular weight HA2 / 3, respectively; and the combination with the above-mentioned low molecular weight HA served as a control. As a result, dermatitis symptoms were clearly and rapidly alleviated in each group, and dermatitis scores also decreased, but the 1 / 2 high molecular weight HA + 1 / 2 low molecular weight HA group and the high molecular weight HA group showed no significant improvement in inflammation compared to the budesonide alone group (Table 2, Figure 10D).Histopathological sections showed that the skin thickness of mice in each group was clearly thinner, and inflammatory cell infiltration was significantly reduced, with the low molecular weight HA group and the 1 / 3 high molecular weight HA + 2 / 3 low molecular weight HA group showing the best effects, followed by the 1 / 3 low molecular weight HA + 2 / 3 high molecular weight HA group. Flow cytometry analysis of mouse skin showed that the 1 / 2 high molecular weight HA + 1 / 2 low molecular weight HA group and the high molecular weight HA group were not statistically significantly different from the budesonide alone group, but the other groups were able to upregulate skin Treg rates. In particular, the low molecular weight HA group and the 1 / 3 high molecular weight HA + 2 / 3 low molecular weight HA group had the highest upregulation rates, which were significantly different from the budesonide group (Figure 10C, E). These results indicate that both high molecular weight and low molecular weight HA can enhance the therapeutic effect of the combination of PEG-IL-2 and budesonide on mouse dermatitis, but the effect of low molecular weight HA is thought to be greater.Low-molecular-weight hyaluronic acid can help the PEGylated protein drug interleukin-2 penetrate and remain in the skin's immune response layer. The interaction between low-molecular-weight hyaluronic acid and PEG is a prerequisite for large interleukin-2 protein drugs to enter the skin. Skin damage may disrupt the structure of the stratum corneum, including the stratum corneum, lipid membrane, and granular layer (the final line of defense of the skin barrier). Whether this is a prerequisite for helping interleukin-2 enter the skin and exert its immune function remains to be determined. Regarding hydrophilic high-molecular-weight HA, considering the immunomodulatory function of high-molecular-weight HA, the administration protocol of 10 kDa PEG-IL-2 + budesonide + 1 / 3 high-molecular-weight HA + 2 / 3 low-molecular-weight HA is currently considered the optimal administration regimen, as it can not only upregulate the proportion of Tregs in the skin but also enhance Treg function.

[0176] 10. The type of glucocorticoid does not significantly affect the efficacy of the drug combination. To investigate the effects of different types of glucocorticoids combined with PEG-IL-2 and HA on treating dermatitis in mice, another group of mice was treated with a combination of 10 kDa PEG-IL-2 + dexamethasone (+HA (high molecular weight:low molecular weight 1:2)) and 10 kDa PEG-IL-2 + budesonide + HA (high molecular weight:low molecular weight 1:2) as a control. The results showed that both groups showed a clear and rapid alleviation of dermatitis symptoms, a decrease in inflammatory cell infiltration, and an upregulation of cutaneous Treg ratios, with no statistically significant difference between the two data groups (see Table 2 and Figure 11).

[0177] 11. Administration of 1 / 3 high molecular weight HA + 2 / 3 low molecular weight HA and 10 kDa PEG-IL-2 in combination with budesonide is significantly more effective than the clinical first-line protocol (topical glucocorticoids). As described above, the treatment protocol combining 10 kDa PEG-IL-2 with budesonide and 1 / 3 high molecular weight HA plus 2 / 3 low molecular weight HA was selected as the best solution and compared with the current clinical first-line treatment protocol (topical glucocorticoids, i.e., budesonide alone). RT-PCR was used to detect changes in the levels of skin inflammatory factors IL-4, IL-13, IL-17, and IFN-γ in the two groups of mice. ELISA was used to detect serum IgE levels in the two groups of mice, using blank and AD model mice as controls. Results showed that skin IL-4, IL-13, and IL-17 expression was significantly reduced in the drug combination group compared with the control group, but IFN-γ expression was not significantly different (P > 0.05). Serum IgE levels also decreased in both groups, with the reduction being particularly significant in the combination group (Figures 12A and 12B). These data indicate that the combination therapy selected by the inventors (10 kDa PEG-IL-2 and budesonide, high molecular weight HA1 / 3, and low molecular weight HA2 / 3) has a significantly superior therapeutic effect than the clinical first-line treatment protocol (budesonide alone).

[0178] 12. The efficacy of budesonide and 10kDa PEG-IL-2 combined with 1 / 3 high molecular weight HA and 2 / 3 low molecular weight HA in treating atopic dermatitis in mice lasted for at least 6 weeks. To investigate whether the effects of this drug combination therapy, combining 10 kDa PEG-IL-2 with budesonide and 1 / 3 high molecular weight HA and 2 / 3 low molecular weight HA, were long-lasting, two groups of AD model mice were randomly selected. One group was transiently treated with this therapy for 3 days, sensitized with OVA before treatment, and then stimulated with an OVA patch to induce atopic dermatitis. The other group of mice was not treated at all. After 3 days, both groups of mice were not further treated and fed under the same conditions. Six weeks later, OVA patches were applied directly to the skin of the two groups of mice, and atopic dermatitis was induced in the mice, using untreated AD mice as controls.

[0179] The results showed that after 6 weeks, dermatitis was still re-induced in the control group of AD mice, and the severity of dermatitis symptoms and skin Treg ratio were the same as those of the initially induced dermatitis, demonstrating that the mice were still OVA-sensitized after 6 weeks, except that the mice were no longer sensitive to OVA after 6 weeks. After 6 weeks, mice treated with 10 kDa PEG-IL-2, budesonide, and 1 / 3 high molecular weight HA + 2 / 3 low molecular weight HA showed no obvious recurrence of atopic dermatitis, their skin remained normal, and their dermatitis scores were low (see Table 2, Figure 13A). Pathological sections showed thickened skin and infiltration of large amounts of inflammatory cells in the AD control group, whereas no obvious thickening was observed in the combination group, with only a small amount of inflammatory cell infiltration (Figure 13B). The proportion of skin Tregs remained at approximately 19.5% (14.2% in the control group), a difference that was statistically significant (Figures 13C, 13D, and 13E). These results demonstrate that a combined administration regimen of 10 kDa PEG-IL-2, budesonide, and 1 / 3 high molecular weight HA plus 2 / 3 low molecular weight HA can not only effectively alleviate the symptoms of atopic dermatitis in mice, but also prevent recurrence of dermatitis, with this effect lasting for at least 6 weeks.

Claims

1. A pharmaceutical composition for treating atopic dermatitis, comprising polyethylene glycol-modified interleukin 2 (PEG-IL-2), a glucocorticoid and low-molecular-weight hyaluronic acid (HA), optionally including a pharmaceutically acceptable carrier, wherein the molecular weight of the low-molecular-weight HA is about 300 KD or less, preferably about 2 to 20 KD, and more preferably about 4 KD.

2. the PEG-IL-2 is modified with polyethylene glycol (PEG) having a molecular weight of about 100 KD or less, 90 KD or less, 80 KD or less, 70 KD or less, 60 KD or less, 50 KD or less, or 40 KD or less; Preferably, the PEG has a molecular weight of about 5-100 KD, 5-90 KD, 5-80 KD, 5-70 KD, 5-60 KD, 5-50 KD, or 10-40 KD; 2. The pharmaceutical composition of claim 1, wherein the IL-2 preferably comprises the amino acid sequence of SEQ ID NO: 1, and more preferably the IL-2 is modified with PEG at one site at the N-terminus.

3. 3. The pharmaceutical composition according to claim 1 or 2, wherein the glucocorticoid is selected from the group consisting of dexamethasone, budesonide, beclomethasone dipropiate, ciclesonide, hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone, clobetasone butyrate, triamcinolone acetonide, fluocinolone, mometasone furoate, halcinonide, clobetasol propionate, halcinonide, halometasone, diflorasone diacetate, mometasone, loteprednol, etiprednol, triamcinolone, flunisolide, flumoxonide, rofleponide, butixocort, tipredane and derivatives thereof, preferably dexamethasone, budesonide and derivatives thereof.

4. The pharmaceutical composition according to any one of claims 1 to 3, further comprising high molecular weight HA, wherein the mass ratio of the high molecular weight HA to the low molecular weight HA is about 2:1, or the ratio of the mass of the high molecular weight HA to the total mass of the high molecular weight HA and the low molecular weight HA is less than 50%, preferably less than 40%, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, more preferably the mass ratio of the high molecular weight HA to the low molecular weight HA is about 1:2 or less, preferably about 1:2, and the molecular weight of the high molecular weight HA is about 800 KD or more, for example, about 800 to 2000 KD, preferably about 800 to 1500 KD.

5. 5. The pharmaceutical composition according to any one of claims 1 to 4, comprising 10 KD PEG-modified IL-2, a glucocorticoid, and HA, wherein the HA comprises (i) low molecular weight HA, or (ii) high molecular weight HA and low molecular weight HA in a mass ratio of about 1:2, and preferably the glucocorticoid is selected from the group consisting of dexamethasone, budesonide, and derivatives thereof.

6. Use of PEG-IL-2, a glucocorticoid, and HA in the manufacture of a medicament or kit for treating atopic dermatitis, wherein the HA comprises low-molecular-weight HA, and the molecular weight of the low-molecular-weight HA is about 300 KD or less, preferably about 2 to 20 KD, and more preferably about 4 KD.

7. the PEG-IL-2 is modified with polyethylene glycol (PEG) having a molecular weight of about 100 KD or less, 90 KD or less, 80 KD or less, 70 KD or less, 60 KD or less, 50 KD or less, or 40 KD or less; Preferably, the PEG has a molecular weight of about 5-100 KD, 5-90 KD, 5-80 KD, 5-70 KD, 5-60 KD, 5-50 KD, or 10-40 KD; The use according to claim 6, wherein the IL-2 preferably comprises the amino acid sequence of SEQ ID NO: 1, and more preferably the IL-2 is modified with PEG at one site at the N-terminus.

8. 8. The use according to claim 6 or 7, wherein the glucocorticoid is selected from the group consisting of dexamethasone, budesonide, beclomethasone dipropiate, ciclesonide, hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone, clobetasone butyrate, triamcinolone acetonide, fluocinolone, mometasone furoate, halcinonide, clobetasol propionate, halcinonide, halometasone, diflorasone diacetate, mometasone, loteprednol, etiprednol, triamcinolone, flunisolide, flumoxonide, rofleponide, butixocort, tipredane and derivatives thereof, preferably dexamethasone, budesonide and derivatives thereof.

9. The use according to any one of claims 6 to 8, wherein the HA further comprises high-molecular-weight HA, and the mass ratio of the high-molecular-weight HA to the low-molecular-weight HA is about 2:1, or the ratio of the mass of the high-molecular-weight HA to the total mass of the high-molecular-weight HA and the low-molecular-weight HA is less than 50%, preferably less than 40%, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, more preferably the mass ratio of the high-molecular-weight HA to the low-molecular-weight HA is about 1:2 or less, preferably about 1:2, and the molecular weight of the high-molecular-weight HA is about 800 KD or more, for example, about 800 to 2000 KD, preferably about 800 to 1500 KD.

10. The use according to any one of claims 6 to 9, wherein the PEG-IL-2 is 10 KD PEG-modified IL-2, the HA (i) comprises low molecular weight HA, or (ii) comprises high molecular weight HA and low molecular weight HA in a mass ratio of about 1:2, and preferably the glucocorticoid is selected from dexamethasone, budesonide, and derivatives thereof.

11. A method for treating atopic dermatitis in a subject, comprising administering to the subject therapeutically effective amounts of PEG-IL-2, a glucocorticoid, and a low-molecular-weight HA, wherein the molecular weight of the low-molecular-weight HA is about 300 KD or less, preferably about 2 to 20 KD, and more preferably about 4 KD.

12. the PEG-IL-2 is modified with polyethylene glycol (PEG) having a molecular weight of about 100 KD or less, 90 KD or less, 80 KD or less, 70 KD or less, 60 KD or less, 50 KD or less, or 40 KD or less; Preferably, the PEG has a molecular weight of about 5-100 KD, 5-90 KD, 5-80 KD, 5-70 KD, 5-60 KD, 5-50 KD, or 10-40 KD; The method of claim 11, wherein the IL-2 preferably comprises the amino acid sequence of SEQ ID NO: 1, and more preferably the IL-2 is modified with PEG at one site at the N-terminus.

13. 13. The method of claim 11 or 12, wherein the glucocorticoid is selected from the group consisting of dexamethasone, budesonide, beclomethasone dipropiate, ciclesonide, hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone, clobetasone butyrate, triamcinolone acetonide, fluocinolone, mometasone furoate, halcinonide, clobetasol propionate, halcinonide, halometasone, diflorasone diacetate, mometasone, loteprednol, etiprednol, triamcinolone, flunisolide, flumoxonide, rofleponide, butixocort, tipredane and derivatives thereof, preferably dexamethasone, budesonide and derivatives thereof.

14. The method according to any one of claims 11 to 13, further comprising administering high molecular weight HA, wherein the mass ratio of the administered high molecular weight HA to the administered low molecular weight HA is about 2:1, or the ratio of the mass of the high molecular weight HA to the total mass of the administered high molecular weight HA and the administered low molecular weight HA is less than 50%, preferably 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, more preferably the mass ratio of the administered high molecular weight HA to the administered low molecular weight HA is about 1:2 or less, preferably about 1:2, and the molecular weight of the high molecular weight HA is about 800 KD or more, for example, about 800 to 2000 KD, preferably about 800 to 1500 KD.

15. The method according to any one of claims 11 to 14, wherein the administered PEG-IL-2 is 10 KD PEG-modified IL-2, the administered HA (i) comprises low molecular weight HA, or (ii) comprises high molecular weight HA and low molecular weight HA in a mass ratio of about 1:2, and preferably the administered glucocorticoid is selected from dexamethasone, budesonide, and derivatives thereof.

16. A kit comprising PEG-IL-2, a glucocorticoid, and a low-molecular-weight HA, wherein the molecular weight of the low-molecular-weight HA is about 300 KD or less, preferably about 2 to 20 KD, and more preferably about 4 KD, and the kit is preferably a kit for treating atopic dermatitis.

17. the PEG-IL-2 is modified with polyethylene glycol (PEG) having a molecular weight of about 100 KD or less, 90 KD or less, 80 KD or less, 70 KD or less, 60 KD or less, 50 KD or less, or 40 KD or less; Preferably, the PEG has a molecular weight of about 5-100 KD, 5-90 KD, 5-80 KD, 5-70 KD, 5-60 KD, 5-50 KD, or 10-40 KD; The kit according to claim 16, wherein the IL-2 preferably comprises the amino acid sequence of SEQ ID NO: 1, and more preferably the IL-2 is modified with PEG at one site at the N-terminus.

18. 18. The kit according to claim 16 or 17, wherein the glucocorticoid is selected from the group consisting of dexamethasone, budesonide, beclomethasone dipropiate, ciclesonide, hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone, clobetasone butyrate, triamcinolone acetonide, fluocinolone, mometasone furoate, halcinonide, clobetasol propionate, halcinonide, halometasone, diflorasone diacetate, mometasone, loteprednol, etiprednol, triamcinolone, flunisolide, flumoxonide, rofleponide, butixocort, tipredane and derivatives thereof, preferably dexamethasone, budesonide and derivatives thereof.

19. The kit according to any one of claims 16 to 18, further comprising high molecular weight HA, wherein the mass ratio of the high molecular weight HA to the low molecular weight HA is about 2:1, or the ratio of the mass of the high molecular weight HA to the total mass of the high molecular weight HA and the low molecular weight HA is less than 50%, preferably less than 40%, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, more preferably the mass ratio of the high molecular weight HA to the low molecular weight HA is about 1:2 or less, preferably about 1:2, and the molecular weight of the high molecular weight HA is about 800 KD or more, for example, about 800 to 2000 KD, preferably about 800 to 1500 KD.

20. The kit according to any one of claims 16 to 19, comprising 10 KD PEG-modified IL-2, a glucocorticoid, and HA, wherein the HA comprises (i) low molecular weight HA, or (ii) high molecular weight HA and low molecular weight HA in a mass ratio of about 1:2, and preferably the glucocorticoid is selected from dexamethasone, budesonide, and derivatives thereof.

21. Use of low-molecular-weight HA in the manufacture of a medicament for improving the effect of PEG-IL-2 and / or glucocorticoid in the treatment of atopic dermatitis, wherein the molecular weight of the low-molecular-weight HA is about 300 KD or less, for example, about 2 to 20 KD, preferably about 4 KD.

22. A use of high molecular weight HA and low molecular weight HA in the manufacture of a medicament for improving the effect of PEG-IL-2 and / or glucocorticoid in the treatment of atopic dermatitis, wherein the mass ratio of the high molecular weight HA to the low molecular weight HA is about 2:1, or the ratio of the mass of the high molecular weight HA to the total mass of the high molecular weight HA and the low molecular weight HA is less than 50%, preferably less than 40%, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, more preferably the mass ratio of the high molecular weight HA to the low molecular weight HA is about 1:2 or less, preferably about 1:2, and the molecular weight of the high molecular weight HA is about 800 KD or more, for example, about 800 to 2000 KD, preferably about 800 to 1500 KD, and the molecular weight of the low molecular weight HA is preferably about 300 KD or less, for example, about 2 to 20 KD, preferably about 4 KD.

23. 1. Use of PEG-IL-2 in the manufacture of a medicament for treating a type I allergic disease in a subject, wherein the PEG-IL-2 is a 10 KD PEG-modified IL-2, preferably the IL-2 comprises the amino acid sequence of SEQ ID NO: 2, more preferably the IL-2 is modified with PEG at one site at the N-terminus, and preferably the type I allergic disease is selected from the group consisting of systemic anaphylaxis including drug anaphylactic shock and serum anaphylactic shock, respiratory allergies such as allergic rhinitis and allergic asthma, gastrointestinal allergic reactions such as allergic gastroenteritis, and skin allergic reactions such as urticaria, atopic dermatitis (eczema), and angioedema.

24. A method for treating a type I allergic reaction in a subject, comprising administering 10 KD PEG-modified IL-2 to the subject, preferably wherein the IL-2 comprises the amino acid sequence of SEQ ID NO: 1, more preferably wherein the IL-2 is modified with PEG at a site at the N-terminus, and preferably wherein the type I allergic disease is selected from the group consisting of systemic anaphylaxis including drug anaphylactic shock and serum anaphylactic shock, respiratory allergies such as allergic rhinitis and allergic asthma, gastrointestinal allergic reactions such as allergic gastroenteritis, and skin allergic reactions such as urticaria, atopic dermatitis (eczema), and angioedema.

25. 10KD PEG-modified IL-2, preferably comprising the amino acid sequence of SEQ ID NO: 1, more preferably modified with PEG at one site at the N-terminus.

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